Fig 1: Identification of KDM5A mutations in patients with ASD.(A) Pedigrees of seven families with KDM5A mutations in nine probands with ASD. Double lines: first cousin status; circles: females; squares: males; shaded symbols: affected individuals. (B) A schematic of KDM5A domains and location of the identified mutations. De novo and recessive mutations are indicated in red and blue, respectively. The Selbst mutation is a cysteine to premature stop codon substitution at position 322 of the protein. ARID, A-T rich interaction domain; JmjC, Jumonji C; JmjN, Jumonji N; PHD, plant homeodomain; PLU1, putative DNA/chromatin binding motif; Zn, zinc finger. (C) Western blot analysis of lymphoblastoid cell line lysates from affected individuals (KD-1–3, KD-2–4, KD-5–3, and KD-6–3) and unaffected family members (KD-1–1, KD-2–1, KD-2–2, KD-5–1, KD-5–2, KD-5–4, KD-6–1, and KD-6–2) showed reduced KDM5A protein in affected individuals KD-1–3, KD-5–3, and KD-6–3, and a truncated KDM5A protein in affected individual KD-2–4. Western blot analysis of HEK293T cells with knock-in of the splice site mutation present in affected individuals KD-4–3 and KD-4–4 (Var) and HEK293T cells which underwent transfection but kept the reference sequence (Ctrl 1 and Ctrl 2), showed a decrease in KDM5A protein level in the targeted cells compared to control cells. β-actin and vinculin were used as loading controls. The black arrowhead points to the KDM5A band (196 kDa) and the white arrowhead points to the truncated KDM5A band (174 kDa).
Fig 2: miR-181d directly regulates RBP2 expression. (A, B) qRT-PCR and Western blot analysis of RBP2 mRNA and protein levels after transfection with miR-181d mimics or inhibitor. (C) Schematic illustration of the predicted miR-181d binding sites in RBP2 3′-UTR. (D–G) RBP2 wild type 3′UTR and its mutated activity following miR-181d mimics/inhibitor transfection in HL60 and HEK-293 cells. Luciferase activities were determined at 48 h and normalized by Renilla luciferase activity. The results are from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not statistically significant.
Fig 3: p65 is directly and epigenetically downregulated by RBP2. (A, B) qRT-PCR of RBP2 and p65 mRNA level after transfection with RBP2 wild-type or RBP2-mutant (defective in demethylase activity, RBP2 H483A) plasmids in K562 and HL60 cells. (C) Western blot analysis of RBP2 and p65 protein expression levels after transfection with RBP2 wild-type or RBP2 H483A plasmids in K562 and HL60 cells. (D) Schematic illustration of the predicted RBP2 binding sites in the p65 promoter. (E, F) Regulation of p65 wild-type promoter by RBP2, including the first RBP2 binding site (p65 pro1) and the second RBP2 binding site (p65 pro2). HL60 and HEK-293 cells were transfected with p65 wild-type reporters, together with RBP2 expression plasmids, followed by luciferase activity assessment 48 h post-transfection. (G, H) p65 wild-type pro1 and its mutated activity following RBP2 wild-type plasmids transfection in HL60 and HEK-293 cells. (I, J) ChIP assay for RBP2 binding to the p65 promoter in K562 and HL60 cells. (K, L) The binding of RBP2 and H3K4me3/2 to p65 promoter after RBP2 plasmid transfection in K562 and HL60 cells. The results are from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not statistically significant.
Fig 4: Depletion of KDM5A suppressed the proliferative, migrative, invasive and angiogenic capacities of HCC cells. A, silencing efficiency of independent KDM5A siRNAs in Hep3B and MHCC97H cells determined with RT‐qPCR. B, VEGF expression in the supernatant of Hep3B and MHCC97H cells measured by ELISA. C, effect of KDM5A silencing on the migrative capacities of Hep3B and MHCC97H cells determined by scratch assay. D, effect of KDM5A silencing on the cell viability of Hep3B and MHCC97H cells determined by MTS. E, effect of KDM5A silencing on the invasion capacities of Hep3B and MHCC97H cells determined by transwell assay. F, effect of KDM5A silencing on proliferation determined by EDU assay. G, CD31 expression levels in HCC biopsy specimens determined by IHC. H, the effect of media from KDM5A silenced Hep3B and MHCC97H cells on the angiogenesis of HUVECs determined by pseudo‐tube formation assay. *P < .05; **P < .01, compared to si‐NC. Data were shown as the mean ± standard deviation. Statistical comparisons were performed by Tukey's test‐corrected one‐way ANOVA when more than two groups were compared. The experiment was repeated 3 times
Fig 5: Depletion of KDM5A up‐regulated miR‐433 to suppress HCC angiogenesis and progression. A, the expression levels of miR‐433 and KDM5A after restoration of miR‐433 in KDM5A‐silenced Hep3B and MHCC97H cells determined by RT‐qPCR. B, the effect of miR‐433 restoration on the migrative capacity of KDM5A silenced Hep3B and MHCC97H cells determined by scratch assay. C, the effect of miR‐433 restoration on invasive capacity of KDM5A silenced Hep3B and MHCC97H cells determined by transwell assay. D, the effect of miR‐433 restoration on proliferative capacity of KDM5A silenced Hep3B and MHCC97H cells determined by EDU assay. E, the effect of miR‐433 restoration on angiogenesis of KDM5A silenced Hep3B and MHCC97H cells determined by pseudo‐tube formation assay. *P < .05; **P < .01, compared to si‐NC+ inhibitor‐NC. #P < .05; ##P < .01, compared to si‐KDM5A+ inhibitor‐NC. Data were shown as the mean ± standard deviation. Statistical comparisons were performed by Tukey's test‐corrected one‐way ANOVA when more than two groups were compared. The experiment was repeated 3 times
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