Fig. 2. SET1B depletion impairs the HIF transcriptional response.

(a, b) Cell surface CA9 was measured and quantified in HeLa cells depleted of SET1B by sgRNA after 7-10 days, and then incubated in 21% or 1% overnight. Quantification of geometric mean of cell surface CA9 staining (b). n = 3 biologically independent samples, mean ± SD, *** P ≤ 0.0001, two-way ANOVA. (c-f) A549 (c), HeLa (d), MCF7 (e), and skin fibroblasts (f) stably expressing Cas9 were transduced with an sgRNA against HIF1β or SET1B and after 7-10 days treated with 21% or 1% O₂ for 24 h. Immunoblots representative of 3 biological replicates. (g) Reconstitution of SET1B siRNA depleted cells with overexpressed SET1B. HeLa cells were transfected with a control or a SET1B siRNA, with and without a SET1B overexpression plasmid for 48 h. Cells were then incubated at 21% or 1% O₂ for a further 24 h before lysis. Endogenous SET1B, HIF1α and CA9 levels were detected by immunoblot (representative of 3 biological replicates) (h) Schematic of the SET1A and SET1B containing complexes highlighting core histone methyltransferase complex members (green) and proteins specific for SET1A and SET1B containing complexes (red). (i-k) CRISPR/Cas9-mediated depletion of SET1 complex proteins in HeLa HRE-^ODDGFP Cas9 cells. Cells were transduced with sgRNA targeting SET1A, SET1B, and CFP1. After 7-10 days cells were treated with or without 1 mM DMOG 24 h, and activation of the HIF GFP reporter measured using flow cytometry (i). sgRNA-mediated depletion of SET1 complex proteins was measured by immunoblotting (j, k) (representative of 3 biological replicates). (l) Endogenous SET1B was immunoprecipitated in HeLa cells grown at 21% or 1% O₂ for 6 h. Samples were immunoblotted for SET1 complex members CFP1 and WDR82 (representative of 3 biological replicates).