Fig. 6

SETD2 has reduced Pol II dependency at high expression levels. a Western blot of whole-cell lysates probed with the depicted antibodies. setd2Δ 293T (KO) cells were transfected with Halo-vector control (VC), SETD2 full-length (FL) or SETD2 C (C), and the lysates were prepared 72 h post-transfection. b Western blot of whole-cell lysates of 293T cells expressing Halo-SETD2 constructs, under the control of either CMV or CMVD2, probed with the depicted antibodies. The expected bands are marked by arrows. *Non-specific. c Bar graph showing H3 normalized H3K36me3 signal intensity of data depicted in b. The data plotted are from three independent biological replicates. d Halo purification was performed from extracts of 293T cells expressing Halo-SETD2 C. Input and eluted samples were resolved on a gel and probed with the depicted antibodies. e RNA was isolated from 293T cells expressing SETD2 from different vectors and RT-PCR was performed to check transcript levels. GAPDH was used as a normalization control. f Plates showing the results of the colony formation assay to test the effect of introducing full-length SETD2 (FL) or SETD2 C under the control of a CMV promoter on the cell proliferation of setd2Δ 293T cells. g, h Quantification of the number and size of colonies obtained in the colony formation assay (n = 6). Unpaired t test was used for statistical analysis. P value is < 0.05