(A and B) ATR phosphorylates SUN2 at S12. MDA-MB-231 cells expressing empty vector, FLAG-SUN2 WT, or FLAG-SUN2 S12A were subjected to FLAG immunoprecipitation, followed by immunoblotting with a phospho-(Ser/Thr) ATM/ATR substrate antibody. Quantification was performed using a 2-tailed Student’s t test (n = 3). (C and D) Matrix stiffness promotes SUN2 phosphorylation. Cells expressing empty vector or FLAG-SUN2 WT were cultured on substrates of different stiffnesses for 24 hours, followed by FLAG immunoprecipitation and SUN2 phosphorylation analysis. Statistical significance was determined using 2-tailed Student’s t test (n = 3). (E–H) ATR inhibition or knockdown suppresses stiffness-induced SUN2 phosphorylation. SUN2-overexpressing cells cultured on substrates of different stiffnesses were treated with DMSO or VX970 for 18 hours (E and F) or infected with control or ATR-targeting shRNA (G and H). SUN2 phosphorylation was assessed following FLAG or SUN2 immunoprecipitation. Data were analyzed by 2-way ANOVA (n = 3). (I and J) SUN2 S12 phosphorylation is required for MDA-MB-231 invasion. Control, SUN2-knockdown, and SUN2-knockdown cells reconstituted with SUN2 WT or S12A were cultured for 3 days on collagen type I gels of different stiffnesses (150 Pa and 1 kPa). Invasion depth was measured by DAPI staining and analyzed by 2-way ANOVA (n = 3). (K and L) SUN2 knockdown decreases N-cadherin and increases E-cadherin expression. Protein levels were assessed by immunoblotting and analyzed by 2-way ANOVA (n = 3). (M and N) Reexpression of SUN2 WT, but not S12A, rescues the changes in E- and N-cadherin caused by SUN2 knockdown. Quantification was performed using 2-way ANOVA (n = 3). Data are presented as mean ± SD.