Fig. 3: MIR31HG knock-down decreases IL1A translation.

a Western blot for p-RELA, RELA, CEBPB and GAPDH of BJ ER:BRAF cells (Control or siMIR31HG1-2) treated with ethanol (Control) or 1 μM 4-OHT for 72 h. Molecular weight marker is shown in kDa. b Quantification of the p-RELA and CEBPB western blot band intensities relative to GAPDH (n = 3). c Chromatin immunoprecipitation followed by qPCR for CEBPB binding to the IL6 promoter and to an unrelated region in BJ ER:BRAF cells (Control or siMIR31HG1) treated with ethanol (Control) or 1 μM 4-OHT for 48 h. The graph shows the percentage of the input that binds CEBPB (n = 3). d Immunofluorescence analysis for RELA and DAPI of BJ ER:BRAF cells (Control or siMIR31HG1-2) treated with ethanol (Control) or 1 μM 4-OHT for 72 h. Representative images are shown in the figure (n = 3). Scale bar: 20 μm. e Western blot for IL1A in BJ ER:BRAF cells (Control or siMIR31HG1-2) were treated with ethanol (Control) or 1 μM 4-OHT for 72 h. Molecular weight marker is shown in kDa (n = 3). f Quantification of IL1A western blot band intensities relative to Vinculin (n = 3). g Immunofluorescence of IL1A and DAPI in BJ ER:BRAF cells (Control or siMIR31HG1-2) treated with ethanol (Control) or 1 μM 4-OHT for 72 h. Representative merged images are shown in the figure (n = 3). Scale bar: 50 μm. h Relative RNA expression for the indicated SASP factors in BJ ER:BRAF cells transfected with the indicated siRNAs (Control or siMIR31HG1-2), treated with ethanol (Control) or 1 μM 4-OHT for 48 h, in the absence or presence of 10 ng/ml of h-rIL1A for 2 h before RNA extraction (n = 3). i Western blot for IL6 from precipitated protein from the media in the conditions indicated in h in the absence or presence of 10 ng/ml of h-rIL1A for 24 h before harvesting the CM (n = 3). S.e. short exposure, l.e. long exposure. j Polysome profile performed by sucrose gradient separation in senescent BJ ER:BRAF control cells (Sen Control, black) or siMIR31HG cells (Sen siMIR31HG, red) treated with 1 μM 4-OHT for 72 h. Y-axis shows the absorbance at 260 nM and X-axis shows the number of the fractions collected. Red area shows the heavy polysome fractions and grey area shows the light polysome fractions. One representative experiment is shown in the figure (n = 3). k Distribution of the amount of IL1A mRNA in the heavy polysome (red) light polysome (grey) or other fractions (white) from the experiment in g. l Relative amount of IL1A, IL6 and ACTB mRNA present in the heavy polysome fractions in the experiment described in j. m Western blot analysis of newly synthesized IL1A, Vinculin and GAPDH levels in control senescent cells and MIR31HG- depleted senescent cells purified by AHA pulse‐labelling and coupling to biotin followed by streptavidin pull‐down. As control, proliferating cells without AHA labelling is shown. HRP-streptavidin shows the uniform labelling of newly synthesized proteins coupled to biotin. A representative experiment is shown (n = 3). n Quantification of IL1A, Vinculin and GAPDH western blot band intensities from pulldown samples in senescence (Sen, white) and senescence MIR31HG KD cells (Sen-MIR31HG KD, grey) (n = 3). All statistical significances were calculated using two-tailed Student’s t-tests, *p < 0.05; **p < 0.01; ns non-significant. All error bars represent means ± s.d. Source data are provided as a Source Data file.