Fig. 6.

ING4 physically interacts with RARβ. a Western blots of Tsc2 ^−/− MEFs lysed and immunoprecipitated with RARβ, ING4, or rabbit IgG overnight, then incubated with Protein G Sepharose for 2 h. Immunoprecipitated proteins (IP) or whole cell lysates (WCL) were immunoblotted with RARβ, ING4, pS6, or vinculin. Results are representative of three independent experiments. b Tsc2^−/− MEFs stably expressing miR-29b-ZIP were treated with rapamycin (20 nM) for 24 h. Cells were lysed, immunoprecipitated, and immunoblotted as described in a. c Tsc2^−/− MEFs transiently transfected with control vector or RARβ2-Myc-DDK were analyzed by immunoblotting using Myc, ING4 or vinculin antibodies. d Immunoblot analysis of ING4 protein expression in Tsc2^−/− MEFs stably expressing miR-29b-ZIP or Ctrl-ZIP treated with vehicle or rapamycin (20 nM) for 24 h. e ING4 mRNA expression was assessed by RT-qPCR in Tsc2^−/− MEFs stably expressing miR-29b-ZIP or Ctrl-ZIP treated with vehicle or rapamycin (20 nM) for 24 h. Data are presented as mean ± SD relative to vehicle-treated Ctrl-ZIP of n = 3 biological replicates. Data for bar graphs were calculated using two-way ANOVA followed by Bonferroni’s posttest for multiple comparisons. f Tsc2^−/− MEFs transiently transfected with control vector or ING4 were analyzed by immunoblotting using ING4 or vinculin antibodies. g Migration of Tsc2^−/− MEFs transiently transfected with control vector or ING4 towards 10% FBS-containing growth medium. Data are presented as mean ± SD relative to vehicle-treated Ctrl of n = 3 biological replicates. Statistical significance was determined by two-tailed student’s t-test. h Invasion of Tsc2^−/− MEFs overexpressing ING4 or control towards 10% FBS-containing growth medium. Data are presented as mean ± SD relative to vehicle-treated Ctrl of n = 3 biological replicates. Statistical significance was determined by two-tailed student’s t-test. i Tsc2^−/− MEFs transiently transfected with the indicated constructs and/or siRNA were analyzed by immunoblotting using ING4, myc or vinculin antibodies. j Migration of Tsc2^−/− MEFs transiently transfected with the indicated constructs and/or siRNA towards 10% FBS-containing growth medium. Data are presented as mean ± SD relative to vehicle-treated Ctrl siRNA + Ctrl of n = 3 biological replicates. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s posttest for multiple comparisons. k Invasion of Tsc2^−/− MEFs transiently transfected with the indicated constructs and/or siRNA towards 10% FBS-containing growth medium. Data are presented as mean ± SD relative to vehicle-treated Ctrl siRNA + Ctrl of n = 3 biological replicates. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s posttest for multiple comparisons. Scale bar = 85 μm. **P < 0.01; ***P < 0.001; ****P <0.0001