The Super Elongation Complex mediates phase transition of SPT5 during transcriptional pause release (2022)
Figure 3

Figure 3. The intrinsic disordered regions of SPT5 regulate Pol II pause release.

A Western blot analysis of re-expression of mCherry-SPT5, mCherry-SPT5ΔNTR or mCherry-SPT5ΔCTR after SPT5 depletion. α-Tubulin was used as a loading control. Results are representative of two biological replicates.

B Confocal imaging of nascent RNA labeled by EU in HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion.

C Violin plot showing the intensity of cells in Fig 3B. Results are representative of two biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. ***p < 0.001.

D, E GAPDH/MYC (D) RPL10/RPL29/RPS19 (E) mRNA levels were assessed by RT-qPCR and normalized to U6 snRNA in HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.=not significant. For GAPDH, p value (from left to right, < 0.0001, 0.0002, < 0.0001, 0.0025, 0.3390, 0.8664, 0.4291, 0.7180); For MYC, p value (from left to right, 0.0001, < 0.0001, 0.0002, < 0.0001, 0.9656, 0.3931, 0.7182, 0.4345); For RPL10, p value (from left to right, 0.0028, 0.0039, 0.0024, 0.0098, 0.6381, 0.9173, 0.4390, 0.8740); For RPL29, p value (from left to right, 0.0002, 0.0002, 0.0046, 0.0021, 0.6530, 0.8222, 0.6608, 0.6877); For RPS19, p value (from left to right, 0.0038, 0.0056, 0.0073, 0.0111, 0.4555, 0.5773, 0.8044, 0.8576).

F HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion were heat shocked for 2 hours or not; and HSP70 mRNA levels were assessed by RT-qPCR and normalized to U6. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. **p < 0.01, ***p < 0.001, n.s.=not significant. For non-heat shock, p value (from left to right, 0.0043, 0.0031, 0.0009, 0.0030, 0.3069, 0.4297, 0.7652, 0.5788); For heat shock, p value (from left to right, 0.0016, 0.0011, 0.0004, < 0.0001, 0.0600, 0.3142, 0.1750, 0.1610).

G, H ChIP-qPCR showing the occupancies of Pol II at the promoters and gene bodies of GAPDH (G), and MYC (H) in HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.=not significant. For GAPDH_promoter, p value (from left to right, < 0.0001, < 0.0001, < 0.0001, 0.0044, 0.8873, 0.9310, 0.6855, 0.9615); GAPDH_gene body, p value (from left to right, 0.0370, 0.0214, 0.0574, 0.0256, 0.9536, 0.8038, 0.7623, 0.8773); For MYC_promoter, p value (from left to right, 0.0002, 0.0014, 0.0004, 0.0048, 0.0670, 0.7501, 0.3482, 0.3284); MYC_gene body, p value (from left to right, 0.0004, 0.0013, 0.0081, 0.0299, 0.1332, 0.5839, 0.4404, 0.6170).