Fig. 1: Experimental approach to measure local and global DRB run-off of elongating RNAPII.
From: Hierarchical mechanisms control the clearance of DNA lesion–stalled RNA polymerase II
a Schematic of local DRB run-off assay by immunofluorescence imaging. Cells were treated with 100 μM DRB, preventing new RNAPII molecules from going into elongation, immediately after local UV-C (100 J/m2), then fixed after 0, 1, or 2 h and stained by immunofluorescence. b Representative images of (a), stained for RNAPII-S2P (Abcam, Ab5095) in RPE1 WT cells at the indicated time points after local UV-C, marked by CPD (Cosmo Bio, CAC-NM-DND-001). Scale bar, 10 μm. c Quantification of (b). RNAPII-S2P mean pixel intensity inside local damage was divided by that outside local damage in the same cells to correct for the general RNAPII run-off caused by DRB. The experiment was performed three times (n = 3). Each transparent colored circle represents one cell, each outlined solid circle the mean of two technical replicates ( > 50 cells / technical replicate). Black lines represent the means of three independent experiments. d Schematic of global DRB run-off assay by western blotting. RNAPII was detected by western blot from whole cell lysates of RPE1 WT cells at 0, 1, and 2 h after global UV-C (30 J/m2) and incubation with 100 μM DRB. e Representative image of (d). The antibody against RNAPII-S2P (Millipore, 04-1571 (3E10)) detects only elongating RNAPII (upper blot). The antibody against the N-terminal domain of RNAPII (Cell Signaling, 14958 (D8L4Y)) detects both initiating (RNAPIIa) and elongating (RNAPIIo) forms (middle blot). HSPA4 was used as loading control. f Quantification of blots in (e) RNAPII-S2P mean pixel intensity was divided by that of HSPA4 and normalized to 0 h. g RNAPII-S2P + UV / -UV ratio of quantification in (f). The value of each UV-treated time point in (f) was normalized to its respective unirradiated condition. h Representative images of local DRB run-off with and without proteasome inhibitor (MG132; 20 µM; 1 h pretreatment and the indicated incubation times after UV-C). i Quantification of (h) as described in (c). j Representative image of global DRB run-off assay with and without proteasome inhibitor as described for (h). HDAC1 was used as loading control. RNAPII antibodies are as in (e). k Quantification of (j) as described in (f) and (g).
