Extended Data Fig. 3: Loss of U₃₄ enzymes correlates with decreased HIF1α protein levels and functionality.

a–c, B16 melanoma cells were depleted of Elp3 by shRNA. RNA and RPF libraries were prepared and deep sequencing was performed. Differential expression analysis between control (shCtrl) and Elp3 shRNA (shElp3) RNA and RPF libraries was performed. Results of one-sided Mann–Whitney U-tests assessing the difference between the distribution of residuals between groups of genes. Top 1%, n = 137; top 5%, n = 683; top 10%, n = 1,366; top 20%, n = 2,732. a, For none of the sets of randomly selected mRNAs is the average as high or as low as the one observed for the top 5% richest or poorest mRNAs, respectively. b, Volcano plot of control and Elp3 shRNA RNA libraries is shown. n = 6 independent samples, Wald tests assessing whether the log₂ fold-change coefficient (control versus Elp3 shRNA versus; paired design) fitted for each gene in a negative binomial generalized linear model comparing control (shCtrl) and Elp3 shRNA (paired design) are significantly different from zero. Correction for multiple tests was performed using the Benjamini–Hochberg procedure. c, GSEA analysis was used to analyse for significant protein signatures of genes. Datasets of the target genes of transcription factors were used. n = 17,128 genes. d–f, Indicated melanoma lines were depleted of ELP3 and CTU1 by shRNA. mRNA expression (e) and protein levels (d and f) of indicated genes were assessed by qPCR (data are mean + s.d.) and western blot, respectively. n = 2 independent experiments. g, h, B16 melanoma cells were depleted of Elp3 or Ctu2 by shRNA. Indicated protein (g) and mRNA (h) levels were quantified by western blot and qPCR, respectively. n = 2 independent experiments, data are mean + s.d. i, j, A2058 melanoma cells were depleted of ELP3 or CTU1 by shRNA. Indicated protein (i) and mRNA (j) levels were quantified by western blot and qPCR, respectively. n = 2 independent experiments, data are mean + s.d.