Fig. 2: JMJD2C is required for survival of JAK2V617F mutated cells.

A UKE1 cells were lentivirally transduced with vectors carrying shRNAs against JMJD2C (sh-2C #1–3) or a scrambled control (scr). Cells were harvested on day 5 for RNA isolation and subsequent analysis by RT-qPCR. B, C Validation of sh-2C #3 in UKE1, SET2 and HEL cells by RT-qPCR (B) and western blotting (C) using an anti-JMJD2C antibody, actin served as a loading control. D–F Western blotting of JMJD2C-depleted or wt UKE1, SET2 and HEL cells with antibodies against H3K9me³ (D), H3K27me³ (E) and H3K36me³ (F). Histone H3 served as a loading control. G–I Proliferation of UKE1 (G), SET2 (H) and HEL (I) cells after introduction of JMJD2C-shRNA (sh-2C) or the scrambled (scr) control. For each condition, 0.25 million cells were seeded and infected on day 1. Cells were counted every second day and used for final analysis on day 9. J–L Detection of apoptotic (AnnexinV+/PI−) and necrotic (AnnexinV+/PI+) cells on day 9 by FACS analysis in UKE1 (J), SET2 (K) and HEL (L) cells. M–O Cell cycle analysis: detection of cells in G1 (DAPI low), S (DAPI medium) and G2/M (DAPI high) phase on day 9 by FACS analysis in UKE1 (M), SET2 (N) and HEL (O) cells. P–R Competitive growth of shRNA (sh-2C or scr) infected UKE1 (P), SET2 (Q) and HEL (R) cells. 0.1 million infected (GFP⁺) cells were seeded at a 1:1 ratio with uninfected cells on day 1. The proportion of GFP⁺ cells was determined daily. G–R Data are represented as mean +/− SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test.