ai cell confluency module labscope software zeiss axiovert 5 digital microscope  (Carl Zeiss)

Journal: bioRxiv

Article Title: SRRM1 coordinates an alternative splicing program that promotes expression of oncogenic protein isoforms

doi: 10.1101/2025.08.20.671097

Figure Lengend Snippet: (A) Immunoblots demonstrated relative SRRM1 expression in HCT116 cell lines expressing stet-inducible short hairpin targeting SRRM1 (464/611/960) or a non-target control (NTC) following induction with tetracycline for 48hrs. Numbers under blots denote quantification of relative band intensity normalized to NTC control. (B) Representative images from colony formation assays conducted on tetracycline-inducible HCT116 SRRM1 short hairpin cell lines (shSRRM1 611/960) relative to a non-target control line (NTC) seeded at either 2500, 1250 and 675 cells per well and cultured for two weeks under tetracycline induction before crystal violet staining and imaging. (C) Quantification of colony formation assays (C) across 3 biological replicates normalized to NTC control. Significant differences are denoted with a p-values < 0.05 = *, p-values < 0.01 = ** according to a paired t-test. (D) Quantification of proliferation assays conducted on HCT116 short hairpin cell lines measuring phase area confluence using an Incucyte live cell imaging system. Cells were cultured at low confluency under tetracycline induction and allowed to grow for 96 hours. (E) Immunoblots demonstrating total p38 and phospho-p38 (Thr180/Tyr182) expression in DLD1 cell lines treated with siRNA targeting SRRM1 or SRSF11 or non-target control. Numbers under blots denote quantification of relative band intensity utilizing Bio-Rad imaging software normalized to NTC control. (F) Real-time quantitative PCR measuring COX2 expression in DLD1, A549, and MDA-MB-468 cells treated with SRRM1 or SRSF11 siRNA relative to non-target control (NTC) treatment across 3 biological replicates. Relative fold change expression values were normalized to NTC control. Significant differences with a p-values < 0.05 = *, p-values < 0.01 = **. (G) Volcano plot demonstrating −log10(FDR adjusted p-value) vs Log2fold change of gene expression in SRRM1 knockdown cells identified by RNA-seq Deseq2 analysis. Only genes that exhibited p-adjusted > 0.05 and Log2Fold change > 1 or < −1 were considered hits. Labeled downregulated oncogenic genes and upregulated tumor suppressive genes and potential targets of alternative spliced transcription factors (FOXM1, TBX3, TCF7L2) are shown. (H) Predicted targets of FOXM1, TBX3 and TCF7L2 transcription factors predicted through ChEA3 ( https://maayanlab.cloud/chea3/ ), which exhibited significant expression changes in response to SRRM1 knockdown. Red nodes denote reduced expression, and blue nodes denote increased expression in response to SRRM1si treatment. (I) Real-time quantitative PCR measuring PRDX2, DDB2, CCND1 and CDKN2B gene expression in DLD1, HCT116, A549 and MDA-MB-468 cells treated with SRRM1 siRNA relative to non-target control treatment across 3 biological replicates. Relative fold change expression values were normalized to NTC control (red dotted line FC =1.0). Significant differences with a p-values < 0.05 = *, p-values < 0.01 = **, p-values < 0.001 = ***.

Article Snippet: Cell confluence was evaluated in a minimum of eight wells using the Incucyte® AI confluence and basic fluorescence analysis software following Sartorius Incucyte guidelines.

Techniques: Western Blot, Expressing, Control, Cell Culture, Staining, Imaging, Live Cell Imaging, Software, Real-time Polymerase Chain Reaction, Gene Expression, Knockdown, RNA Sequencing, Labeling