Extended Data Fig. 5: HIF1α requires U₃₄ enzymes for proper translation.

a, qPCR products from input and ribosome immunoprecipitation were run in agarose gel. n = 3 independent experiments. b, c, A2058 cells expressing HIF1A^WT–Flag (HIF1α-WT) or HIF1A^Mut–Flag (HIF1α-Mut) constructs were depleted of ELP3 or CTU1 by shRNA. b, The indicated protein levels were detected by western blot. n = 2 independent experiments. c, Cell death was quantified by FACS analysis. n = 4 independent experiments, two-sided t-test, data are mean + s.d. d, e, MM029 short-term primary melanoma cell line (d) and A2058 establish melanoma line (e) expressing the empty vector (Empty), the HIF1A^WT–Flag or HIF1A^Mut–Flag constructs were depleted of ELP3 or CTU1 by shRNA. Measurement of the uptake of 2-deoxy-2-DG by FACS analysis. Data are expressed as fold increase of median fluorescence compared to the corresponding control shRNA condition. n = 3 independent experiments, two-sided t-test, data are mean + s.d. f, g, MM029 short-term primary melanoma cell line (f) and A2058 establish melanoma line (g) expressing the empty vector, the HIF1A^WT–Flag or HIF1A^Mut–Flag constructs were depleted of ELP3 or CTU1 by shRNA. Lactate release in the medium was quantified after 24 h by luminescence assay. Data are expressed as fold increase over the corresponding control shRNA condition. n = 2 independent experiments, data are mean + s.d. h, Protein aggregates were detected in ELP3 and CTU2-depleted primary melanoma cultures after PROTEOSTAT staining using FACS. n = 3 independent experiments, two-sided t-test, data are mean + s.d. Representative plots are shown on the left. i, j, Protein aggregates were isolated in MM029 cells depleted of ELP3 or CTU2 (i) and in B16 cells depleted of Elp3 (j). Indicated protein levels in the total and aggregate compartment were detected by western blot. n = 2 independent experiments. k, B16 cells depleted of Elp3 or Ctu2 by shRNA were treated for 4 h with the indicated concentration of proteasome inhibitor MG132 and lysosome inhibitor chloroquine. The levels of indicated proteins were detected by western blot. n = 2 independent experiments.