Fig. 9: pTINCR promotes differentiation through the SUMOylation and activation of CDC42. | Nature Communications

Fig. 9: pTINCR promotes differentiation through the SUMOylation and activation of CDC42.

From: pTINCR microprotein promotes epithelial differentiation and suppresses tumor growth through CDC42 SUMOylation and activation

Fig. 9: pTINCR promotes differentiation through the SUMOylation and activation of CDC42.The alternative text for this image may have been generated using AI.

a Analysis of the pTINCR interactome using CRAPome algorithm. b GSEA analysis of pTINCR-induced genes in hSCC10 cells versus the indicated gene signatures. c. Interaction of pTINCR with CDC42 analyzed by co-immunoprecipitation assay. d CDC42 activation assay in hSCC10 cells after 4 days of pTINCR expression. pTINCR-HA syORF was used. e Representative CDC42 SUMOylation assay in U2OS cells transduced with pTINCR-HA, Flag-CDC42 and His6-SUMO2. SUMOylated CDC42 is indicated with arrowheads. f Quantification of 4 independent CDC42 SUMOylation assays performed in U2OS cells. Values of CDC42 SUMOylation are normalized to total CDC42 and relative to the control. Multiple T-TEST corrected for multiple comparison was performed. g Representative SUMOylation assay of endogenous p53 and B23 proteins in U2OS cells transfected with His6-SUMO2 and pTINCR-HA. SUMOylation of p53 and B23 is indicated with arrowheads. h CDC42 activation assay in HaCaT cells after 16 h of ML-792 treatment and/or 24 h of pTINCR syORF overexpression. i CDC42 activation assay in WT, pTINCR-KO or pTINCR-KO overexpressing pTINCR-HA syORF (pTINCR KO + OE) HaCaT cells during calcium-induced differentiation. INVOLUCRIN is shown as a marker of cell differentiation. j Expression of the indicated differentiation markers after 24 h of calcium-induced-differentiation or pTINCR-HA syORF overexpression in HaCaT cells, treated or not with the CDC42 inhibitors CASIN or ML-141. Error bars represent the mean ± SD of N = 3 technical replicates from a representative experiment performed 3 times independently with similar results. We show statistical differences within each experimental condition and between basal control cells and differentiated control cells or pTINCR-overexpressing cells (shown in brackets). n.s: not significant. 2-way ANOVA test with multiple comparison was performed. k–l Immunostainings of F-actin, E-cadherin and β-catenin upon pTINCR- (k) or calcium-(l) induced differentiation for 24 h (actin and E-cadherin) or 4 days (β-catenin), in basal HaCaT cells treated or not with CASIN (actin and E-cadherin) or ML-141 (β-catenin). pTINCR-HA syORF was used. Nuclei are counterstained with DAPI. Total percentage of cells showing membranous staining is indicated. Scale bars correspond to 50 µm. Source data are provided as a Source Data file.

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