Fig 1: GRWD1 knockdown reduces levels of H3K4me3 marks. (A) Western blot assays showed that the signals of H3K4me3 marks were decreased after GRWD1 knockdown in MM and KMM cells. (B) Heatmaps showing alterations of H3K4me3 marks after GRWD1 knockdown in MM and KMM cells. (C) Differential alterations of H3K4me3 marks between MM and KMM cells after GRWD1 knockdown. (D and E) ChIP-qPCR validation of reduction of H3K4me3 peaks at specific gene loci in MM (D) and KMM (E) cells. The same H3K4me3 peaks were also examined in other cells for comparison. P values are from comparisons between each of the shRNA-treated groups (sh1 and sh3) and the scrambled control (Ctl). ns, not significant. (F and G) Tracks of H3K4me3 peaks in the promoters of the four candidate GRWD1 targets in MM (F) and KMM (G) cells. Primer positions were labeled with arrows.
Fig 2: GRWD1 directly interacts with WDR5. (A) Confocal images showing the colocalization of GRWD1 and WDR5 in MM and KMM cells. (B) FLAG-GRWD1 immunoprecipitated endogenous WDR5 in 293T cells. (C) FLAG-WDR5 immunoprecipitated endogenous GRWD1 in 293T cells. (D) Purified recombinant GST-GRWD1 but not GST physically pulled down purified WDR5 in vitro. (E) WDR5 knockdown reduced the levels of total H3K4me3 in MM and KMM cells.
Fig 3: Epigenetic factors that are essential for KSHV-transformed cells identified by CRISPR-Cas9 screening. (A and B) Heatmaps of H3K4me3 (A) and H3K27me3 (B) peaks of MM and KMM cells. (C) Distribution of epigenetic factors in different groups identified by CRISPR-Cas9 screening of MM and KMM cells (21). (D) Functional classification of epigenetic factors by CRISPR-Cas9 screening of MM and KMM cells (21). TF, transcription factor; N/A, not available. (E) Top 9 epigenetic factors with the largest differences of CRISPR score between MM and KMM cells in group 8 identified by CRISPR-Cas9 screening. CRISPR score is defined as the average [log2(final sgRNA abundance/initial sgRNA abundance)] of 3 single guide RNAs (sgRNAs) (21). (F) CRISPR scores at days 4, 11, and 21 of the top 9 epigenetic factors with the largest differences of CRISPR score between MM and KMM cells. P values are from comparisons between day 4, 11, or 21 and day 1 for MM (blue) and KMM (red) cells, respectively. (G) Survival analysis of GRWD1 expression in brain lower-grade glioma (LGG), sarcoma (SARC), and skin cutaneous melanoma (SKCM). H, L, and M, high, low, and medium, respectively.
Fig 4: GRWD1 interacts with multiple methyltransferases, and MLL2 knockdown shares the same phenotype of GRWD1 knockdown. (A) FLAG-GRWD1 immunoprecipitated endogenous methyltransferases MLL1, MLL2, and SET1A in 293T cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) FLAG-MLL2 immunoprecipitated endogenous GRWD1 in 293T cells. (C) MLL2 knockdown reduced the level of H3K4me3 in MM and KMM cells. (D to F) The effects of MLL2 knockdown on cell proliferation (C), cell cycle progression (D), and apoptosis (F) in MM and KMM cells. P values are from comparisons between each of the shRNA-treated groups (sh1, sh2, and sh3) and the scrambled control (Ctl). (G) MLL2 knockdown reduced the efficiency of colony formation on soft agar of KMM cells.
Fig 5: GRWD1 is essential for KSHV-induced cell proliferation and cellular transformation. (A and B) Knockdown efficiencies of GRWD1 shRNAs examined by RT-qPCR (A) and Western blotting (B). (C to E) The effects of GRWD1 knockdown on cell proliferation (C), cell cycle progression (D), and apoptosis (E) of MM and KMM cells. (F) GRWD1 knockdown reduced the efficiency of colony formation on soft agar of KMM cells. (G) GRWD1 knockdown inhibited the progression of KMM tumors in nude mice. Mice from different groups terminated before the final day of the experiment were labeled with the matched color stars. (H) At the endpoint, week 21, GRWD1 knockdown reduced the volume of KMM tumors in nude mice. P values are from comparisons between each of the shRNA-treated groups (sh1, sh2, and sh3) and the scrambled control (Ctl).
Supplier Page from Abcam for Recombinant Human GRWD1 protein (GST tag N-Terminus)