Fig 1: ZNF750 facilitates chromatin regulator association with target genes. (A) Re-ChIP-qPCR with ZNF750 and each interacting protein to sites near four differentiation genes and four progenitor genes compared with gene desert-negative control. (B–F) ChIP-qPCR with control or ZNF750 depletion for KLF4 (B), RCOR1 (C), KDM1A (D), CTBP1 (E), or CTBP2 (F). t-test: (*) P < 0.05; (**) P < 0.01, compared with CTLi. (G) Heat map summarizing ChIP-qPCR data from B–F. See also Supplemental Figure S6.
Fig 2: OHL cells express both KLF4 and BLIMP1.H&E analysis (Image: 40X), and immunohistochemistry analysis was performed on a paraffin-embedded, formalin-fixed biopsy of an oral hairy leukoplakia (OHL) lesion using antibodies directed against Z, BMRF1, KLF4 and BLIMP1 as indicated (Images: 100x of region boxed in H & E stain). Examples of OHL cells, positively staining for each of these proteins, are indicated by black arrows.
Fig 3: Identification of functional ZNF750-interacting proteins. (A) Silver stain gel of Flag-HIS ZNF750 or tag-only control (CTL) tandem affinity-purified from differentiated keratinocytes. (B) Network of ZNF750-interacting proteins identified by mass spectrometry, grouped by functional category. Red lines indicate novel ZNF750 interactions, and black lines indicate published interactions. (C) Table of spectral counts and SAINT scores for 13 ZNF750-interacting proteins tested for phenotypic overlap with ZNF750 depletion. (D) Heat map of gene expression changes with depletion of ZNF750-interacting proteins by siRNA in keratinocytes induced to differentiate for 3 d with calcium, shown as number of genes with >1.5-fold expression change by qPCR in the same direction as ZNF750 depletion. Proteins above the dotted line were considered for further analysis. (E) Heat map of gene expression changes with depletion of ZNF750, RCOR1, CTBP1/2, KDM1A, or KLF4 in differentiated keratinocytes by qPCR. (F) Effects of depletion of ZNF750-interacting proteins on differentiation proteins loricrin (LOR; red) and filaggrin (FLG; green) and proliferation (Ki67; red) in regenerated organotypic epidermal tissue. Epidermal basement membrane is noted with dashed lines, and white arrowheads denote suprabasal Ki67-positive cells. Bar, 25µm. See also Supplemental Figure S2.
Fig 4: ZNF750 interacts with KLF4, RCOR1, KDM1A, CTBP1, and CTBP2. (A) PLA in keratinocytes with antibody recognizing ZNF750 or no antibody control in combination with KLF4, RCOR1, KDM1A, CTBP1, or CTBP2 antibodies. Bar, 5 µm. (B) Western blots of co-IPs among ZNF750, KLF4, RCOR1, KDM1A, CTBP1, and CTBP2. Inputs are 1%. (C) Far-Western analysis with recombinant ZNF750 or MBP-negative control on membrane, probed with recombinant proteins and antibodies to KLF4, RCOR1, KDM1A, CTBP1, and CTBP2 or GST as negative control. Ponceau S was used as a loading control. See also Supplemental Figure S3.
Fig 5: KLF4 and BLIMP1 expression is differentiation-dependent in normal tongue tissue.Immunohistochemistry analysis was performed on paraffin-embedded formalin-fixed normal tongue tissue (A), or rafted NOKs-Akata cells (B), using antibodies directed against KLF4 and BLIMP1 as indicated. Examples of positively-staining cells are indicated by red arrows (Images:40x).
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