Figure 3

α1, αv, β1, and β5 Integrin expression are increased in TRPM2 reconstituted cells. (A) RNA seq analysis of integrin expression in SH-SY5Y cells with TRPM2 deletion (KO-V) compared to the same cells reconstituted with TRPM2 (KO-M2). MA plot (log ratio vs abundance) of RNA seq data is shown. Two biological replicas of each condition were utilized. ITGA1 (α1), ITGA3 (α3), ITGA5 (α5), ITGA9 (α9), ITGAV (αv), and ITGB5 (β5) integrins were significantly increased in KO-M2 cells, as was HIF1A (HIF-1α). These genes with q-value < 0.05 are displayed in red. Positive Log FC indicates genes overexpressed in M2 cells. Degust 4.1.1 software was used for RNA seq analysis and image generation. (B), (D) RT-qPCR of (B) integrins ITGA1, ITGAV, ITGB1, ITGB5, and (D) transcription factors HIF-1α, E2F1, FOXM1, and ARNT mRNA from Scr-V, KO1-V, KO1-M2 or KO1-E960D SH-SY5Y cells. RT-qPCR was performed on cells grown without serum for 24 h. Each experimental group was normalized to Scr. Means + S.E.M. of three (ARNT), four (ITGA1, ITGAV, ITGB1, HIF1A), five (E2F1, FOXM1), or seven (ITGB5) experiments performed with KO clone are shown (KO1 in Fig. 3 B, D and a second clone KO2 in Supplementary Fig. S3B,D). Statistics: one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C), (E) Western blotting was performed on two clones of TRPM2 KO (KO1-V, KO2-V), KO reconstituted with TRPM2 (KO1-M2, KO2-M2) or E960D (KO1-E960D, KO2-E960D), and scrambled SH-SY5Y control cells (Scr1-V, Scr2-V) grown without serum. Blots were probed with antibodies to (C) α1 (ITGA1), αv (ITGAV), β1 (ITGB1), β5 (ITGB5) integrins, or (E) transcription factors HIF-1α, E2F1, FOXM1, and ARNT. Tubulin was probed as a control for loading. Densitometry measurements were from five experiments from each clone for each integrin, eight experiments for transcription factors HIF-1α, E2F1, and FOXM1 for KO1 and five experiments for KO2 clone, and eight experiments for ARNT. Results were standardized to each experiment’s scrambled control, and means + S.E.M. for each group are shown. Statistics: one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Results for KO1 are shown in Fig. 3C,E and for KO2 in Supplementary Fig. S3C,E. Full length gels for Western blots are also shown in Supplementary Fig. S3C,E.