Fig. 4: Effect of TMEM45A inactivation on DNA damage induction and repair.

SQD9 cells were transduced with lentiviral particles expressing shRNA control (shCTL) or shRNA targeting the mRNA of TMEM45A (shRNA 22). a SQD9 cells were incubated with or without 100 mM of cisplatin in normoxic conditions for 15, 30 min, 1, 2, and 4 h. After the incubation, proteins were extracted and PARP cleavage and gH2AX protein level were assessed by western blot. Actin was used as the loading control. Results are expressed as mean ± SD (n = 3). b SQD9 cells were incubated with or without 100 mM of cisplatin in normoxic conditions for 4 h then the fresh medium without cisplatin was added to the cells for 4 or 20 h. After the incubation, proteins were extracted and PARP cleavage and gH2AX protein level were assessed by western blot. Actin was used as the loading control. Results are expressed as mean ± SD (n = 3). c SQD9 cells were incubated with or without 100 mM of cisplatin in normoxic conditions for 24 h. ATM/ATR substrate phosphorylation profile was assessed by western blot. Actin was used as loading control. Results are expressed as mean ± SD (n = 3). d SQD9 cells were transduced with lentiviral particles expressing shRNA control (shCTL) or shRNA targeting the mRNA of TMEM45A (shRNA 22). The cells were incubated with or without 100 mM of cisplatin in normoxic (N) conditions for 24 h and gene expression level was assessed by RNA sequencing. The transcriptome analysis was performed using Babelomics and GSEA after RNA sequencing. Heatmap of differentially expressed genes produced by using Heatmapper²⁶ (http://www.heatmapper.ca) showed a potential deregulation of DNA damage and apoptosis activation. Genes have been reordered by the method of average clustering. A dendrogram is shown for the clustering of genes. Genes with relatively high expression are marked in red, genes with relatively low expression are marked in blue. The corresponding values are presented in Supplementary Fig. 4. e SQD9 cells were incubated with or without 100 mM of cisplatin in normoxic conditions for 24 h. The expression level of EYA3 was determined by RT-qPCR. Results are expressed as mean ± SD (n = 3). f SQD9 cells were incubated with or without 100 mM of cisplatin in normoxic conditions for 24 h. EYA3 expression level was assessed by western blot. Actin was used as loading control. Results are expressed as mean ± SD (n = 3). N normoxia, NC normoxia + cisplatin. **p < 0.01, ****p < 0.0001.