Fig. 2

PKCζ phosphorylates YY1 at Thr 398 in response to 3-HAA. A The YY1 phosphorylation was analyzed by immunoblotting and mass spectrometry 2 h after 3-HAA treatment. The YY1 was blotted on the enriched phospho-proteins from SMMC7721 cells. The YY1 modification was analyzed by the mass-spectrometry following YY1 immunoprecipitation. B The T398A but not S247A mutation abolished 3-HAA-induced YY1 phosphorylation. The YY1 was conjugated with HA tag. The YY1 phosphorylation was detected by the T398 phospho-specific antibody. C The mutation of T398E in YY1 promoted DUSP6 expression and apoptosis, whereas the T398A mutation suppressed DUSP6 upregulation. The YY1 was fused with HA tag. D The YY1 mutation of T398A reduces the 3-HAA-induced apoptosis, analyzed by the TUNEL assay. E 3-HAA increased PKCζ binding to YY1, analyzed by co-immunoprecipitation. F 3-HAA increased PKCζ binding to YY1, analyzed by immunoblots following immunoprecipitation. G The kinase screening for T398 phosphorylation of YY1. The kinase candidates were predicted by online tools NetPhos 3.1 (www.cbs.dtu.dk/services/NetPhos) [8] and GPS 5.0 (gps.biocuckoo.cn) [9]. H The effect of kinase inhibitors on 3-HAA-induced YY1 phosphorylation. The YY1 phosphorylation was detected by the T398 phospho-specific antibody. The concentration of 3-HAA was 100 μM. AKT inhibitor, MK2206 (0.5 μM); PKC inhibitor, Go6983 (0.5 μM); mTOR inhibitor, rapamycin (0.1 μM). I The effect of PKC inhibitor Go6983 on the YY1 enrichment at the DUSP6 promoter in SMMC-7721 cells. **: P < 0.01, ***: P < 0.001. J The T398A mutation of YY1 abolished the 3-HAA-inhibited HCC xenografts growth (n = 6). **: P < 0.01. F and G The apoptosis analysis in xenografts by TUNEL assay and the flow cytometry. The histogram presents as mean ± SD (*: P < 0.05; **: P < 0.01.). Note: The dose of 3-HAA used in A was 100 µM. K The proposed 3-HAA binding model with YY1