Figure 2
From: Endogenous HIF2A reporter systems for high-throughput functional screening
Validation of endogenous HIF2A reporter systems. (a) Schematic of CRISPRi-based inhibition of reporter activity. RNAPol, RNA Polymerase II. KRAB, Kruppel associated box. (b) Relative HIF2A mRNA levels in H2AmC2 and H2AmC3 dCas9 cells transduced with HIF2A-targeting sgRNAs or a non-targeting control. *P < 0.05, **P < 0.01, ***P < 0.005, n.s., non-significant. P-value by Student’s t-test. (c) Western blot analysis of H2AmC2 and H2AmC3 dCas9 cells transduced with HIF2A-targeting sgRNAs or a non-targeting control. B-actin acts as a loading control. Full-length blots are presented in Supplementary Fig. 5. (d) FACS analysis of mCherry fluorescence in H2AmC2 cells transduced with different HIF2A-targeting sgRNA constructs. Untransduced H2AmC2 and UOK101 cells serve as a positive and negative control, respectively. (e) FACS analysis of mCherry fluorescence in H2AmC3 cells transduced with different HIF2A tandem sgRNAs compared to controls. (f) Western blot analysis of VHL-reintroduced H2AmC2 and H2AmC3 cells compared to the empty vector (EV) control. B-actin acts as a loading control. Full-length blots are presented in Supplementary Fig. 5. (g) FACS analysis of mCherry fluorescence in H2AmC2 and H2AmC3 cells transduced with HA-VHL and empty vector.
