We cultured MEC-1 cells without any treatment and monitored them for 21 days to assess cell growth, viability, and morphology. This allowed a better understanding of their cell cycle dynamics and overall behavior in culture, as well as documented morphological characteristics that will be important for downstream experimental analysis.

It was successfully maintained in both T-75 and T-25 flasks throughout the 21-day culture period. T75 flasks exhibited higher cell numbers, reaching up to approximately 2.8 × 10⁶ cells/ml, although with greater fluctuations compared to T-25, which showed more stable growth pattern. Cell viability remained consistently high, above 90% at all time points, indicating that cells preserved their health and proliferative capacity under both culture conditions. MEC-1 cells have a reported doubling time of 48 hours and as a result the observed fluctuations in cell density likely reflect differences in seeding density, flask volume, and handling rather than inherent growth limitations. Overall, both systems are suitable for long-term culture, with T-75 favoring higher expansion potential and T-25 offering greater stability.

Microscopic observation of non-treated MEC-1 cell morphology

Morphologically, cells were not affected throughout the culture period. No notable differences were observed between T-25 and T-75 flasks, either before or after passaging, and their appearance remained consistent with reference images from established databases for this cell line (DSMZ Cell Culture) ¹. This confirms that the culture conditions did not alter the characteristic morphology of a MEC-1 cell.

The comparable growth and viability profiles across T-25 and T-75 suggest that the culture conditions were consistent and did not induce stress or differentiation-related effects. The observed minor variations in cell density likely reflect differences in manual handling or local distribution of cells within the flasks rather than biological variability. This consistency validates the robustness of our culturing process and allows us to proceed confidently with subsequent experimental steps (e.g., transfection) using either setup, as both demonstrate equivalent baseline characteristics.

As an initial assessment, we aimed to verify whether BCL-2 is expressed in MEC-1 cells at a level to serve as a therapeutic target and assess in silico selected primers sets for successful and specific amplification of canonical (isomorph A) of BCL-2 gene.

Table 1: Cq values and amplification efficiencies of BCL-2 mRNA in different
non-treated cell lines using the first primer set.

* BR1/BR2: Two biological replicates ≈ BR1P1/ BR2P1 of non-treated Mec-1 cells. Biological replicates performed with different RNA extraction protocols, serving as positive control.

JMP1: Human lymphoblast-like cell line (lymphoma) ²

SUP-M2: Human anaplastic large cell lymphoma cell line ³

This analysis represents relative quantification, indicating that the target gene is present at high levels, consistent with the low Cq values observed. While the measurement remains relative, the signal is clearly detectable. MEC-1 cells exhibit moderate to high expression of the BCL-2 gene, with a Cq value of ~21–22, confirming that MEC-1 expresses it at significant levels compared to the positive controls (BR1P1 and BR2P1). The high amplification efficiency, together with the absence of signal in the non-template control (NTC), further confirms the reliability of these results (Figure 6).

The qPCR run was technically robust, with high efficiency, excellent reproducibility, and no evidence of contamination. BCL-2 mRNA expression levels can be ranked as: BR1P1≈, BR2P1 > MEC1> JMP1 > SUP-M2.

For MEC-1 cells, the melting curve analysis showed a Tm of 85.88°C, indicating a single, specific amplification product without evidence of primer-dimer formation or nonspecific products (Figure 7).

Table 2: Cq values and amplification efficiencies of BCL-2 mRNA in
different non-treated cell lines using the second primer set.

* BR1/BR2: Two biological replicates ≈ BR1P1/ BR2P1 of non-treated Mec-1 cells. Biological replicates performed with different RNA extraction protocols, serving as positive control.

JMP1: Human lymphoblast-like cell line (lymphoma) ²

SUP-M2: Human anaplastic large cell lymphoma cell line ³

The MEC-1 sample shows moderate to high expression of the BCL-2 gene, using the second pair of primers. The Cq value (22) confirms that MEC-1 cells express it at significant levels, compared to the positive controls (BR1P1 and BR2P1). The high amplification efficiency, together with the absence of signal in the non-template control (NTC), further confirms the reliability of these results (Figure 8).

The qPCR run was technically robust, with high efficiency, excellent reproducibility, and no evidence of contamination. Expression levels can be ranked as: BR1P1 ≈, BR2P1 > MEC1> JMP1 > SUP-M2.

For MEC-1 cells, the melting curve analysis showed a Tm of 85.88°C, indicating a single, specific amplification product without evidence of primer-dimer formation or nonspecific products (Figure 9).

Both primer pairs performed consistently across all samples, producing nearly identical Cq values within each condition. This indicates high primer specificity, efficiency, and reproducibility, with no evidence of primer-dimer formation or non-specific amplification (as confirmed by NTC) (Figure 10).

The detection of moderate to high expression in MEC-1 provides a critical foundation for downstream validation. This successfully establishes the first step of our proof-of-concept, enabling subsequent siRNA-based functional experiments. Consequently, MEC-1 cells are confirmed as a suitable model for downstream experiments and reliably validate our literature-based findings.

In our work, we employed GSK126 before siRNA experiments in MEC-1 cells, not only to examine how our model responds to a well-characterized epigenetic inhibitor, but also to establish proof-of-principle control for dose-dependent cytostatic or cytotoxic effect. Given that GSK126 is a selective inhibitor of EZH2 (Enhancer of Zeste Homolog 2), an epigenetic modifier associated with transcriptional repression ⁴, the cellular effects will be assessed.

A clear decreasing trend in cell number was observed with increasing concentrations of GSK126, with the most pronounced reduction detected at 40 μM. The 10 μM and 20 μM concentrations resulted in comparable low cell numbers, both lower than those observed at 5 μM. All tested concentrations exhibited reduced cell numbers compared to the control (Figure 11).

Cell viability remained high at the lower concentrations of the compound, but showed a decreasing trend from 20 μM onwards, dropping below 80%. At 40 μM, viability was drastically reduced, reaching as low as ~5% (Figure 12).

A clear decreasing trend in cell number remained at 48h with increasing concentrations of GSK126, with the most pronounced reduction detected at 40 μM. The 5 μΜ, 10 μM and 20 μM concentrations resulted in comparable low cell numbers. All tested concentrations exhibited reduced cell numbers compared to the control (Figure 13).

Cell viability remained high at the lower concentrations of the compound, but showed a decreasing trend from 20 μM onwards, dropping below 60%. At 40 μM, viability was drastically reduced, reaching as low as ~5% (Figure 14).

Cell viability revealed a clear dose-dependent effect of GSK126. At both 24 h and 48 h, treatment with 5 μM and 10 μM maintained viability near control levels. A noticeable decline was observed at 20 μM, where viability dropped below 80%. At the highest tested concentration of 40 μM, viability was drastically reduced to nearly 5%. The reduction was more severe at 48 h, highlighting a combined effect of concentration and exposure time (Figure 15).

Analysis of relative cell growth demonstrated a strong dose-dependent inhibitory effect of GSK126. At 24 h, growth decreased progressively with concentration, reaching ~40% of control at 10 μM and 20 μM, and was nearly abolished at 40 μM. At 48 h, the inhibitory effect was more evident, with a marked decline already at 5 μM, a transient plateau at 10 μM, followed by further reduction at 20 μM, and complete suppression at 40 μM. These findings highlight both the dose- and time-dependent growth inhibition effect of GSK126 (Figure 16).

Overall, treatment with GSK126 across a range of increasing concentrations revealed a distinct dose-dependent inhibitory effect on MEC-1 cells. Progressive escalation in concentration resulted in a consistent reduction of cell proliferation, indicating that GSK126 exerts its biological activity in a concentration-dependent manner. At 5 μM and 10 μM, GSK126 primarily exerted a cytostatic effect, consistent with cell cycle arrest rather than cell death, whereas at 20 μM and 40 μM, a clear cytotoxic effect was observed. The response was dose-dependent, and at 20 μM, there was also an indication of time-dependent enhancement of cytotoxicity. This aligns with literature reports that lower concentrations of GSK126 (5 μM and 10 μM) often require prolonged exposure (≥72 h) to manifest significant effects, with the nature of the response—cytostatic or cytotoxic—being cell-type dependent ^{4 5}. The requirement for higher concentrations or longer duration to observe effects in our system compared to some literature reports underscores the cell-type-specific sensitivity to EZH2 inhibition.

Transfection of BCL-2–targeting siRNAs into MEC-1 cells enables the specific silencing of BCL-2 gene expression. This approach allows us to evaluate the consequent effects on cell growth and viability.

At 24 h post-transfection, both cell proliferation and viability were reduced across all conditions compared to the control, with the greatest decrease observed in the condition containing only INTERFERin (5 µL), as well as in the groups treated with 50 nM siRNA + INTERFERin (5µL) and 20 nM siRNA + INTERFERin (5µL). Cell viability in these groups dropped to approximately 65–70%, showing the same pattern as the reduction in cell numbers.

At 48 h, proliferation remained lower than the control across all conditions but showed an overall increase compared to 24 h, except for the 50 nM scrambled siRNA group, which did not recover to the same extent. Cell viability at 48 h was high across all conditions, showing an increase relative to 24 h and a stable trend thereafter.

At 72 h, proliferation continued to be lower than the control but showed a substantial increase compared to 24 h and 48 h, while viability remained high and stable across all conditions. Finally, at 96 h, proliferation in all experimental groups surpassed that of the control, with cell numbers markedly higher than at earlier time points, particularly in the higher concentration and INTERFERin-only conditions. Viability at 96 h remained relatively high in all conditions, although slightly decreased compared to 72 h, with the lowest values observed in the 50 nM scrambled siRNA group (Figure 17-18).

Across the tested concentrations (5–50 nM), a consistent pattern was observed at 24 h, 48 h, and 72 h post-transfection, with cell growth decreasing progressively as siRNA concentration increased. At 48 h, a ~40% reduction in cell growth was noted in the 20 nM condition compared to the control, highlighting a dose-dependent effect. By 72 h, the same pattern remained, although the magnitude of reduction was lower than at earlier timepoints, indicating partial recovery of the cells. The condition containing only INTERFERin showed the strongest inhibitory effect at 24 h, with cell growth reduced by ~60% compared to the control. However, by 48 h and 72 h the reduction in this group was smaller, stabilizing around ~35%. At 96 h, overall growth increased across conditions, suggesting that MEC-1 cells progressively adapt and recover over time following transfection (Figure 19).

Relative cell viability remained largely stable across all conditions, showing that the treatments did not cause significant cytotoxicity. At 20 nM, a slight decrease was observed at 24 h, 48 h, and 72 h, while the strongest effect appeared at 50 nM, with only a ~20% reduction at 24 h. The INTERFERin-only condition did not display any notable toxicity at any timepoint. By 96 h, full recovery of cell viability was observed across all conditions, confirming that the treatments primarily affected proliferation rather than cell survival (Figure 20).

Microscopic observation of MEC-1 cell morphology following transfection with siRNAs

In both the transfected conditions (+INTERFERin) and the INTERFERin-only condition, similar morphological changes were observed. At 5 h and 24 h post-transfection, cells appeared stressed, showing aggregation and clustering, while the overall number of viable cells under the microscope seemed noticeably reduced compared to the control. These early points indicate that INTERFERin exposure induces transient cellular stress, regardless of whether siRNA is present, which is confirmed by cell growth and viability across all conditions at 24h post-transfection. By 72 h post-transfection, cells showed signs of recovery, with morphology returning closer to normal and cell numbers visibly increasing. At 96 h, the cultures appeared fully recovered, with healthy morphology and growth restored across all conditions, suggesting that the observed effects were temporary and reversible. As well as conditions with siRNA alone even though with increased concentration (50nM) manifest morphologically the same features as control group, while conditions transfected with maximum volume of INTERFERin showed the same morphological pattern as INTERFERin alone condition (Figure 21).

Our results highlight a transient toxicity with INTERFERin exposure, at higher concentrations, manifesting an initial reduction in cell number and viability. However, this effect appears to be reversible, as the cells progressively recover over time, regaining both growth capacity and viability. This suggests that while INTERFERin at higher concentrations exerts a temporary cytostatic effect immediately after transfection, MEC-1 cells can adapt and restore normal proliferation dynamics at later timepoints. It is important to note that in the absence of direct siRNA uptake validation, the observed phenotypes cannot be definitively attributed to specific BCL-2 silencing at these early timepoints.

In our initial experiments, we observed signs of cytotoxicity, most likely arising due to the increased volume of INTERFERin required for transfection. Since no data was available on the cellular uptake of the siRNAs under our specific conditions, the next experiment included performing a titration of INTERFERin volumes, in parallel with adjusting the cell seeding density. Specifically, we compared two setups: 300,000 cells/mL, as in our previous experiment, and a lower density of 150,000 cells/mL, which is closer to the manufacturer’s recommendations. This approach allowed us to evaluate how MEC-1 cells respond to different levels of transfection stress under varying cell densities, providing more reliable insights into the balance between transfection efficiency and cell viability.

Cell number decreases with increasing volumes of INTERFERin, reflecting the dose-dependent toxic effect of the reagent. Cell numbers are higher at 300.000 cell density seeding set up rather than lower cell density across all conditions. At lower volume of INTERFERin cell proliferation is less affected by INTERFERin (Figure 22).

Cell viability decreases with increasing volumes of INTERFERin, while it remains high at smaller volumes. Between the two seeding densities tested, relatively higher viability was observed in the 300,000 cells/mL setup compared to the lower-density condition. At 5 µL of INTERFERin viability dropped to 50–60% in both seeding setups, indicating that toxicity is directly associated with the volume of transfection reagent used rather than cell density alone (Figure 23).

Cell number decreases with increasing volumes of INTERFERin, reflecting the dose-dependent toxic effect of the reagent. Cell numbers are higher at 300.000 cell density seeding set up rather than lower cell density across all conditions. At lower volume of INTERFERin cell proliferation is less affected by INTERFERin.

Nevertheless, across nearly all conditions, after 48 h the cultures showed an approximate two-fold increase in cell number compared to the initial seeding and transfection, indicating that despite the early impact of INTERFERin, MEC-1 cells retain the capacity to proliferate and partially recover over time (Figure 24).

Cell viability decreases with increasing volumes of INTERFERin, while it remains high at smaller volumes. Between the two seeding densities tested, slightly higher viability was observed in the 300,000 cells/mL setup compared to the lower-density condition. At 5 µL of INTERFERin viability remained relatively high >80% in both seeding setups, indicating that MEC-1 cells retain the capacity to proliferate and partially recover over time (Figure 25).

At 48 h, cell viability remained like at 24 h in the Control and 1 μL INTERFERin conditions, indicating stable survival at low reagent volumes. In contrast, at higher volumes (2–5 μL), viability significantly increased at 48 h compared to 24 h, suggesting a recovery effect over time. The most pronounced improvement was seen at 5 μL, where viability reached from approximately 50% at 24 h to nearly 80% at 48 h, demonstrating that MEC-1 cells can partially overcome the initial toxic effect of INTERFERin (Figure 26).

At 24 h post-transfection, cell numbers across all conditions were relatively similar, ranging from approximately 120,000 to 300,000 cells. By 48 h, however, cell numbers had increased substantially in all conditions, highlighting a clear proliferation trend compared to the earlier timepoint (Figure 27).

At 48 h, across all conditions cell viability increased at 48 h compared to 24 h, suggesting a recovery effect over time. The most pronounced improvement was seen at 5 μL, where viability reached from approximately 65% at 24 h to nearly 90% at 48 h, demonstrating that MEC-1 cells can partially overcome the initial toxic effect of INTERFERin, (Figure 28).

At 24 h post-transfection, cell numbers across all conditions were relatively similar, ranging from approximately 250,000 to 600,000 cells. By 48 h, however, cell numbers had significantly increased in all conditions, highlighting a clear proliferation trend compared to the earlier timepoint, (Figure 29).

The comparison between the two seeding densities (150,000 vs 300,000 cells/mL) showed very similar patterns, suggesting that the observed effects are primarily volume-dependent rather than cell density–dependent. Cell viability gradually decreased with increasing volumes of INTERFERin, reaching a reduction of approximately 40% at 5 μL compared to the control. Similarly, cell growth displayed a clear dose-dependent decline, with growth at 5 μL reduced to less than half of the control, confirming the strong inhibitory effect of higher reagent volumes (Figure 30-31).

The cell density effect showed that higher seeding density (300,000 cells/mL) supported slightly better growth compared to 150,000 cells/mL, particularly at higher INTERFERin volumes. Viability remained relatively stable (~85–95% of control) across conditions, with only a slight dip at 3 μL followed by recovery at 5 μL. In contrast, cell growth displayed a clear dose-dependent decrease, with proliferation at 5 μL reduced to ~45–60% of the control, highlighting the strong inhibitory effect of higher INTERFERin volume (Figure 32-33).

Microscopic observation of MEC-1 cell morphology following transfection with INTERFERin (titration)

With increasing volumes of INTERFERin, cells displayed clear signs of stress and aggregation at both 24 h and 48 h post-transfection. However, at 48 h, in both cell density setups, the extent of cell stress and aggregate formation was reduced compared to 24 h, indicating a partial recovery of the culture over time (Figure 34).

High volumes of INTERFERin primarily induce a transient inhibition of proliferation, confirming a temporary arrest of the cell cycle, while overall viability remained largely unaffected.

When comparing cultures seeded at 150,000 cells/mL versus 300,000 cells/mL, a generally similar trend was observed, though the effect of INTERFERin was slightly less pronounced at the higher density. In denser cultures (300,000 cells/mL), the dose of toxic lipid complexes per cell is effectively lower, meaning that each cell internalizes and processes less INTERFERin. In addition, a denser cell population can secrete more survival factors and create a more resilient microenvironment, helping the overall culture withstand the stress caused by INTERFERin. Cell–cell contacts may also strengthen pro-survival signaling pathways. As a result, with a lower effective dose per cell and a more “supportive” cellular environment, cultures at higher density experience slightly less stress, leading to a milder inhibition of growth compared to lower-density cultures. Furthermore, higher-density cultures provide a greater number of cells available for downstream analyses and further experimentation.

Concluding, INTERFERin exhibits cytotoxic/cytostatic effects at high doses. Its main negative impact at larger volumes (3–5 µL) is an inducible and reversible arrest of the cell cycle, rather than induction of cell death. This suggests that the observed effects are likely non-permanent and that MEC-1 cells can recover once the stress is alleviated.

Transfection of BCL2-targeting siRNAs into MEC-1 cells enables the specific silencing of BCL2 gene expression through the RNA interference pathway. This experimental setup was designed based on the results of previous experiments and was repeated with optimized parameters, to assess the efficiency of siRNA uptake by MEC-1 cells, evaluate the downregulation of BCL2 mRNA levels, and determine the effects of BCL2 silencing on cell growth and viability.

At 20 nM, a distinct early fluorescence peak was observed at 6 h, indicating rapid uptake through active endocytosis. This signal gradually decreased and stabilized by 24–48 h, suggesting that the siRNA was being processed intracellularly. The decrease in fluorescence following the initial peak is a positive indication that the siRNA is being directed to its site of action, reflecting the normal pattern of siRNA action and being processed and used for target gene silencing, target gene.

At 50 nM, fluorescence levels increased over time (6 h, 24 h, 48 h), but the pattern did not show the same sharp uptake and subsequent stabilization seen at 20 nM. Instead, the sustained high signal suggests that the system becomes overloaded, with siRNA likely trapped in vesicles rather than being efficiently processed for gene silencing.

Other conditions, including scrambled siRNA and INTERFERin alone, show no significant fluorescence changes and, as a result, no cellular uptake (Figure 35-36).

Cell growth across all conditions showed a clear increase from 24 h to 48 h, indicating that cells remained viable and able to proliferate. The increase observed at 20nM is higher than at 50nM comparable to control levels. Analysis of the fluorescence uptake and cell growth data provides a consistent picture of how MEC-1 cells respond to different siRNA concentrations. Especially at 20 nM, siRNA is efficiently internalized and processed, leading to effective activity while cells remain viable and proliferative within timepoints (24h-48h). At 50 nM, the system becomes overloaded (Figure 37), resulting in cellular stress and a slight growth inhibition.

Lower concentrations (5–10 nM) overall showed modest fluorescence uptake (Figure 35-36) and relative moderate growth, while controls (scrambled siRNA, INTERFERin alone) demonstrated that part of the observed reduction in proliferation originates from the delivery reagent itself rather than siRNA activity (Figure 37).

Cell viability remained consistently high (>85–90%) across conditions, indicating that no significant cytotoxic effects were observed. This suggests that the treatments primarily influenced cell proliferation rather than inducing cell death (Figure 38).

For both primer sets, at 6h post-transfection, minimal differences were observed among 5nM-10nM conditions compared to control, indicating that early silencing effects had not yet fully manifested at low and medium concentrations, along with fluorescence results which showed at those conditions not cellular uptake. Despite manifesting a distinct early fluorescence peak at 20nM, Cq value is comparable to control levels.

At 50 nM manifests Cq lower to control, but not statistically significant, along with low signal in cellular uptake at that early timepoint. At 20nM, alone Cq is lower to statistically insignificant control, as well. At INTERFERin and 20 nM scrambled conditions manifest Cq very high (>30) compared to control at both primer sets. For the first primer set, certain high Cq values resulted from single replicate amplification (only one Cq value was recorded, while the other failed to give a value) rather than biological differences. For the second primer set, high Cq values across all three biological replicates were observed at INTERFERin and 20 nM scrambled statistically significant conditions (p < 0.05) and as a result amplification is unexpectedly not observed.

For both primer sets, at 24h post-transfection, minimal differences were observed among 5nM-10nM conditions compared to control, indicating that early silencing effects had not yet fully manifested at low and medium concentrations, along with fluorescence results, which showed that at those conditions, no cellular uptake occurred. At 20nM, the Cq value is comparable to control levels, while the signal is gradually decreased and stabilized by 24h.

The increase at 50 nM might suggest reduced BCL-2 transcript abundance due to siRNA-mediated degradation of mRNA, with cellular uptake to increase at 24h post-transfection. At 20nM alone, Cq has unexpectedly increased as well. For the second primer set, high Cq values across all three biological replicates were observed at 20 nM alone and 20 nM scrambled conditions, which are statistically significant (p < 0.05) and, as a result, amplification is unexpectedly not observed (Figures 39-40).

However, unexpectedly high Cq values were also observed in some groups (INTERFERin-only, 20 nM scrambled siRNA, and siRNA alone), possibly reflecting technical variability or RNA quality issues. To further verify these results, agarose gel electrophoresis was performed both for total RNA samples and for some qPCR products at 6h, confirming the integrity of RNA and the specificity of amplification.

At the 6 h time point, RNA quantification revealed a lower absorbance compared to the 24 h samples, indicating a smaller total RNA yield. This difference is validated in the agarose gel, where RNA bands corresponding to the 6 h samples are markedly less intense than those at 24 h. The reduced band brightness reflects the lower RNA concentration and overall limited template availability for qPCR. Lower RNA input might have contributed to the increased Cq values observed in qPCR at 6 h, particularly in INTERFERin-only and 20nM scrambled siRNA conditions. The observed elevation in Cq at early time points and INTERFERin-only and 20nM scrambled siRNA conditions primarily stems from technical limitations related to low template concentration and low-quality RNA, rather than from biological factors.

In contrast, at 24 h, the increased RNA yield, which aligns with gel electrophoresis observations, enabled robust amplification and reduced variability between replicates and improved qPCR performance. RNA extraction quality and yield improve significantly between 6 h and 24 h post-transfection. Low RNA yield at 6 h contributes to technical elevation of Cq values in qPCR while at 24 h samples provide more reliable data for evaluating siRNA uptake and BCL-2 silencing (Figure 39-40).

Gel electrophoresis in qPCR products at 6h confirms the absence of major degradation or nonspecific artifacts, while validating the consistency with the high Cq values and the low amount and quality of initial RNA and except for INTERFERin, which produced a faint, barely detectable band (Figure 41).

Consequently, it appears that the low initial number of cells used at the beginning of the experiment led to a reduced total RNA yield, as well as lower levels of high-quality RNA. This limitation was subsequently reflected in the qPCR results of certain samples, particularly at the 6 h time point, where higher Cq values were observed due to insufficient RNA input. In contrast, at 24 h, a clear recovery in cell growth and an increase in total cell number were evident, resulting in higher RNA concentrations and improved amplification performance.

For both primer sets, the 5 nM and 10 nM siRNA conditions, as well as the 20 nM siRNA-alone condition, exhibited a ~5-fold and above overexpression of BCL-2 compared to the control at 6 h post-transfection. By 24 h, BCL-2 levels under these conditions were reduced compared to control.

For the 20 nM siRNA condition, both timepoints showed a similar tendency, with BCL-2 expression slightly reduced relative to control (20% reduction).

At 50 nM, both timepoints showed a consistent reduction to about half of the control levels (40-50% reduction), with the strongest downregulation observed at 24 h, along with increased cellular uptake.

At 6 h post-transfection, both the INTERFERin-only and 20nM scrambled siRNA conditions showed a slight apparent increase in BCL2 expression compared to the control, which is attributed to the low RNA input and minor technical variability observed at this early timepoint.

At 24 h, BCL2 mRNA levels in both conditions returned close to baseline (control) values, with no statistically significant differences detected (Figure 42-43).

During the experiments, an unexpected amplification in the negative control of T the reference gene (YWHAZ), was observed, probably due to the formation of primer dimers. Consequently, to investigate this anomaly, new qPCR reactions will be performed, including both biological replicates and additional reference gene controls to further investigate this observation and clarify its underlying cause.

Microscopic observation of MEC-1 cell morphology following transfection with siRNAs

At 6 h post-transfection, morphological examination revealed signs of cellular stress, characterized by the presence of cell aggregates and clustering. From 24 h onwards, and more prominently at 48 h, a recovery was observed, with MEC-1 cells regaining a more typical and physiological morphology.

The cellular response observed at 10 nM - 50 nM siRNA concentrations was morphologically similar across all time points. At 6 h, pronounced cellular stress was evident, with cell aggregates indicating a strong response. This effect persisted at 24 h, while by 48 h a recovery was observed, with cells progressively regaining their typical morphology (Figure 44).

Our initial assessment indicates that BCL-2 siRNA is efficiently internalized at 20 nM and 50 nM without inducing cytotoxicity. The 20 nM condition showed rapid siRNA uptake followed by a decrease in fluorescence, indicating efficient initial possible gene silencing without detectable cytotoxicity. In contrast, the 50 nM condition exhibited stronger uptake without signs of cytotoxicity, as well. To confirm these observations, the investigation of target mRNA downregulation was made by qPCR, where while a corresponding trend of mRNA downregulation was observed (~30% and ~50%, respectively), the data were variable and not statistically significant. Specifically, at 20 nM, a partial reduction in BCL2 mRNA levels was observed at both 6 h and 24 h, amounting to approximately 30% and at 50 nM, expression levels were reduced by about half (~50%), corresponding to changes that did not reach statistical significance. Given the variability and the persistence of certain technical limitations, these findings should be interpreted with caution. Consequently, from these experiments, it became evident that scaling up the experimental setup is necessary to increase the number of seeded cells and thereby obtain higher RNA yields. This will allow for more reliable and consistent downstream analyses, such as qPCR and related molecular assays, by ensuring both the quality and the quantity of extracted RNA, for reliable analysis.

Furthermore, the observed instability of the YWHAZ reference gene in some negative control groups mandates its re-evaluation under our specific transfection conditions. Additional validation will include both biological replicates and protein-level assessments to confirm whether YWHAZ remains a stable normalizer under our experimental conditions.