Fig 2: PAF1 regulates the expression of IER5 in CC. a Confocal images showing complementary expression of PAF1 and IER5 in CC cells: overexpression of PAF1 in cells with negligible IER5 expression and low levels of PAF1 expression in cells with highly expressed IER5; the highlighted boxes show the magnified images of single-cell staining. The normal control cervical tissue showed no expression of PAF1 or IER5. DAPI was used for nuclear counterstaining. b, c Western blotting analysis results showing a decrease in the expression of IER5 protein in the PAF1-knockdown cells. GAPDH was used as an internal control protein. All data are expressed as the mean (S.D.): * P < 0.001)

Fig 3: Knocking down PAF1 affects radiosensitivity of CC by regulating IER5. a&b qRT-PCR assays of mRNA expression in radiation-exposed and unexposed Siha and Hela cells showing increased expression of IER5 and reduced PAF1 after radiation. ß-actin served as expression controls. Data are expressed as the mean (S.D.) (n = 3). c&d Western blotting analysis results showing that based on radiation, knocking down PAF1 promoted the expression of IER5, while knocking down IER5 has no significant effect on PAF1. Fold change of intensity is mentioned in the western blotting bands. GAPDH served as expression controls. e&f FCM assay for analyzing the percentage of cells with active caspase-3 showing that PAF1 knockdown resulted in an increased percentage of cell with active caspase-3 and simultaneous PAF1 and IER5 knockdown resulted in little effect on the apoptosis proportion. N = 3 in each group. All data are expressed as the mean (S.D.): * P < 0.05 and ** P < 0.01

Fig 4: Knocking down PAF1 enhances the efficacy of the radiotherapy of CC cells. a CCK-8 assay of Siha and Hela cells treated with radiation showing significantly reduced proliferation rates (Siha cells: P = 0.002, Hela cells: P = 0.019), and knocking down the PAF1 gene enhanced the cell-killing ability upon cell IR (Siha cells: P = 0.004, Hela cells: P = 0.018). b FCM-cell cycle analysis (24 h after IR) with propidium iodide (PI) staining shows PAF1 knockdown and IR resulted in an increased percentage of both Siha and Hela cells blocked in the G2/M cell cycle. c FCM-apoptosis analysis (72 h after cell IR) with PI and annexin V-FITC staining shows an increased percentage of apoptotic PAF1-knockdown Siha and Hela cells (Siha cells: 33.6%, Hela cells: 35.3%) subjected to IR (Siha cells: 3.5 Gy, Hela cells: 4 Gy) compared with that of the control cells (Siha cells: 27.0%, Hela cells: 21.2%), N = 3 in each group. All data are expressed as the mean (S.D.): * P < 0.05 and ** P < 0.01

Fig 5: PAF1 contributes to elevated tumor recycling.a Representative PAF1 immunoreactivity in normal prostate, non-tumor adjacent tissues (NAT), and tumor tissues. Scale bars: 500 µm. b Slides were scanned using an Aperio Digital Pathology Slide Scanner (Leica Biosystems) at 20× magnification. A box plot compares H-scores of PAF1 nuclear staining in 370 tissues. Box plots show median (center), 25th/75th percentiles (box bounds), 10th/90th percentiles (whiskers), and 5th/95th percentiles (outliers as symbols). The values beyond the outliers are not shown in the boxplot. The significance was determined by one-way ANOVA, **p < 0.001. c Five prostate cancer tissues with paired NAT were used. Nuclear extracts of pooled tissues were incubated with PAF1 antibodies to moderately deplete PAF1 protein and then were analyzed by western blot. Histone H3 was used as an endogenous loading control. The graph shows the quantification of PAF1 expression. Values were expressed as the mean ± SEM of three independent experiments. d The transcription recycling assay was performed using pooled tissue nuclear extracts with or without PAF1 depletion. In vitro transcribed RNA products from the 2nd template during the Recycle step were analyzed by reverse transcription followed by qPCR quantification. Values were expressed as the mean ± SEM of three independent experiments. p-values were calculated using two-tailed Student’s t-test, **p < 0.01. Source data are provided as a Source data file.