Figure 4.
CDK7 Activity Is Required for Transfer of SPT5 from MYC to Pol II
(A and B) Immunofluorescence images of PLAs between SPT5 and MYC (A) or SPT5 and total Pol II (B) in cells treated with CDK inhibitors (DRB, THZ1, LDC4297, LDC067) or DMSO vehicle.
(C) Quantification of PLAs shown in (A). The number of proximity pairs upon inhibitor treatment was quantified in independent experiments and normalized to the DMSO condition.
(D) Quantification of PLAs shown in (B). The number of proximity pairs upon inhibitor treatment was quantified in independent experiments and normalized to the DMSO condition.
(E) Immunoblots of endogenous SPT5 precipitated from HEK293 cells treated with LDC4297 or DMSO. Co-precipitated total Pol II and SPT4 were analyzed by immunoblotting. Beads coupled to non-specific IgG were used as controls.
(F) Immunoblots of immunoprecipitation experiments. FLAG-SPT5 and HA-MYC were overexpressed by transient transfection in HEK293 cells. HA-MYC was immunoprecipitated and incubated with recombinant CDK7, and co-precipitating FLAG-SPT5 was analyzed by immunoblotting. Cells not expressing HA-MYC were used as control.
(G) Immunoblot of TFIIEβ depleted cells. U2OS cells were treated with an siRNA against TFIIEβ or a non-targeting control (Vinculin: loading control).
(H and I) Immunofluorescence images (H) and quantification (I) of PLAs between SPT5 and total Pol II. U2OS cells were treated with LDC4297 or an siRNA against CDK7 after depletion of TFIIEβ and in control cells. The number of proximity pairs was quantified and its change in TFIIE-depleted cells to control cells was calculated as fold change (FC).
For (A), (B), and (H): yellow dots: intensity centers of proximity pairs; blue: Hoechst stained nuclei; magenta: Phalloidin staining; scale bar: 5 μm. See also Figure S4.