PHF8 demethylates H3K9me1/2 and H4K20me1 in endothelial cells.

<p>A: Representative western blot and densitometry for histones and their modifications as indicated from HUVECs with overexpression (A) or knockdown (B) of PHF8 by RNAi. Anti-PHF8 from bethyl was used. Scr = scrambled RNAi, Ctl = pcDNA3-GFP. Numbers above the blots indicate the results of the relative densitometry. n = 3, *p<0.05.</p

PHF8 is expressed in endothelial cells.

<p>A: Expression profile of the KDM7 family members PHF8 and JHDM1D as determined by qRT-PCR normalized to β-Actin, n = 3. B: Representative western blot of PHF8 and RNA-polymerase II (Pol II) from the nucleus of human umbilical vein endothelial cell (HUVEC), human microvascular endothelial cell line (HMEC), human aortic smooth muscle cells (HAoSMC), fibroblast and HEK293. C&D: Western blot from the cytosol and nuclear fraction (C) or only nuclear fraction (D) of HUVECs stained with anti-PHF8 (C: abcam #ab36068, D: bethyl #A301-772A) and transfected with siRNA against PHF8 or scrambled as control M = Protein Ladder.</p

PHF8 maintains endothelial migration in an E2F4-dependent manner.

<p>A: Flow cytometry analyses of HUVEC proliferation with the CFSE dye after RNAi transfection against PHF8 with and without overexpression of PHF8 or E2F or E2F1 or DDK (FLAG tag) as control (Ctl) n = 5. Tube formation (B) Boyden chamber (C) and scratch wound assay (D&E&F&G) each with statistics of HUVECs transfected with control siRNA (siScr) or two different PHF8 siRNAs (siPHF8-1, siPHF8-2) or E2F4 siRNAs (siE2F4-1, siE2F4-2) with and without electroporation of plasmids coding for E2F4 and control (DDK (FLAG tag). n = 3. *p<0.05.</p

PHF8 is required for endothelial proliferation and survival.

<p>A: Proliferation analysis determined by cell counting of HUVECs transduced with control shRNA shScrambled (shScr) or shRNA against PHF8 (shPHF8), n = 5. B&C: Flow cytometry analysis of HUVEC proliferation with the CFSE dye (B) or cell cycle analysis (C) with propidium iodide after 72 h RNAi transfection, n = 5. D: Apoptosis/survival assay for late apoptosis (Q3: annexin V and propidium iodide positive cells), early apoptosis (Q2: only annexin V positive) and annexin V/propidium iodide negative cells (Q1: normal). Tumor necrosis factor alpha (TNFα, 20 ng/ml, 3h) and cycloheximide (CHX, 25 μg/ml, 3h) served as positive control. HUVECs were transduced with control shRNA (shGFP) or shPHF8, n = 3, *p<0.05.</p