Fig. 3

PTPN20 activates p65/NF-κB signaling pathway in TNBC cells. (A-B) Cignal finder signal transduction 45-pathway reporter array showing that PTPN20 overexpression significantly activated NF-κB signaling in MDA-MB-231 cells. (C-D) Relative NF-κB-driven luciferase activity was analyzed in the indicated cells. (E-F) Real-time PCR analysis of downstream NF-κB pathway target genes mRNA levels in the indicated cells. (G) Western blotting analysis of downstream NF-κB pathway target genes protein levels in the indicated cells. GAPDH was used as a loading control. (H) Western blotting analysis of levels of p-IKKα/β, IKKα, IKKβ, p-IκBα, IκBα, p-p65 (Ser468), p-p65 (Ser536) and total p65 in the indicated cells. GAPDH was used as a loading control. (I) Western blotting analysis of level of p-p65 (Ser468) in the cells treansfected with PTPN20 plasmid (0 µg, 0.5 µg, 1 µg and 2 µg). GAPDH was used as a loading control. (J) Western blotting analysis of level of nuclear p65 and total p65 in the indicated cells. H3, Histone 3. GAPDH was used as a loading control. (K) Total cell lysates from MDA-MB-231 cells and SUM159PT cells were immunoprecipitated with anti-Flag antibody in the indicated cells. The immunoprecipitates were subjected to immunoblotting analysis with anti-p65 or anti-PTPN20 to detect interaction. (L) Total cell lysates were immunoprecipitated with anti-HA antibody in the indicated cells. The immunoprecipitates were subjected to immunoblotting analysis with anti-Ub, anti-Flag and anti-HA. (M) Total cell lysates from MDA-MB-231 cells were immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to immunoblotting analysis with indicated antibodies to detect interaction between COMMD1 and p65. (N) Hypothetical model illustrating that PTPN20 dephosphorylates and stablizes p65 by inhibiting the combination between p65 and COMMD1, leading to NF-κB activation (Created in BioRender.com). In (C), (D), (E) and (F), error bars represent the mean ± SD of three independent experiments. In (C), (E) and (F), Mann-Whitney U test was used. In (D), Kruskal-Wallis test was used. ***P < 0.001, **P < 0.01, *P < 0.05