Figure 2: The more severe phenotype in TAF10 depleted mESCs is not due to the SAGA assembly defect.

(A) Western blot analyses of SUPT7L, TAF8, TAF10, TAF7 and TBP expression after deletion of Taf7 (-Taf7) or of Taf10 (-Taf10). RT7 and RT10 cells were treated 3 days with 4-OHT and as control, RT7 cells were treated 3 days with EtOH. (B) CRISPR/Cas9 strategy to generate the R26^CreERT2/+;Taf7^f/f; Supt7l^−/− (RT7;Supt7l^−/−) mESCs. The second coding exon (exon 3) of Supt7l was deleted in RT7 mESCs. Deletion of Taf7 in the Supt7l^−/− genetic background is induced by 4-OHT treatment. (C) Western blot analyses of SUPT7L, TAF7, TBP and TAF10 expression in RT7;Supt7l^−/− cells after 2, 4 and 6 days of treatment with EtOH (Control; Supt7l^−/−) or 4-OHT (-Taf7; Supt7l^−/−). As a wild type control, RT7 cells were treated 6 days with EtOH. M; molecular weight marker. (D) Anti-SUPT20H IP-MS analyses on nuclear enriched lysates from RT10 and RT7 cells treated 4 days with EtOH (Control, RT7 and RT10 data merged), RT10 mESCs treated 4 days with 4-OHT (-Taf10) and RT7;Supt7l^−/− cells treated 4 days with EtOH (Control;Supt7l^−/−). For each of the proteins of interest, the XIC values of Control; Supt7l^−/− and -Taf10 lysates were normalized to those of RT7 and RT10 control cells treated with EtOH, respectively. Control; n = 2 biological replicates x 3 technical replicates, -Taf10; n = 1 × 3, Control;Supt7l^−/−; n = 1 × 3. Means ± standard deviation are shown. Kruskal-Wallis test: * <0.05; **<0.01. (E) Total number of cells after 4 and 6 days of treatment. The control condition corresponds to the merging of RT7 and RT10 cells treated with EtOH. The impact on TFIID and SAGA assembly is indicated at the bottom. D4, D6: Ctrl; n = 6, Ctrl;Supt7l^−/−; n = 4, -Taf7;Supt7l^−/−; n = 4, -Taf7; n = 3, -Taf10; n = 3 biological replicates. Means ± standard deviation are shown. Kruskal-Wallis test followed by a Dunn post hoc test: ns; not significant, * <0.05.