Fig 2: (A) MV-4-11 cell lines were treated for 18 h with 1 µM GSK-3326595, and compound 20 showed stabilization of PRMT5 in cellular thermal shift analysis in whole-cell lysates. (B) The relative strength of PRMT5 signal on the Western blots was measured by densitometry. The melting temperature for PRMT5 was shifted by 5.5 °C and 7.2 °C. All data were analyzed using a Boltzmann sigmoidal fit. Each point plotted represents the mean of three replicates for each temperature; error bars denote ±SD. (C) The binding of 20 inhibits the degradation of PRMT5 at 58 °C in a dose-dependent manner.

Fig 3: Loss of PRMT5 resulted in invasive gastric cancer. H&E staining and IHC of E-cadherin and tdTomato in the invasive gastric cancer of SP-A-Cre;Prmt5fl/fl;Rosa26tdTomato mice at 8 months of age. A black dotted line demarcated two regions for the mucosa and submucosa. The red and green solid-line boxes were magnified on the right, respectively. Scale bar, 100 µm.

Fig 4: Slug cooperates with PRMT5 and LSD1 to regulate the transcription of E-cadherin and vimentin. (A) ChIP and Re-ChIP experiments were done in MDA-MB-231 cells with the indicated antibodies. (B) MDA-MB-231 cells were infected with lentiviruses bearing the indicated shRNAs, and the association of Slug, PRMT5, LSD1 at the promoter of E-cadherin and vimentin was analyzed with the qChIP assay. (C and D) MCF10A cells were infected with lentiviruses carrying the indicated shRNAs together with Slug expression constructs or empty vector. The mRNA or protein level of indicated genes was monitored by qPCR (C) or western blotting (D). (E) MDA-MB-231 cells were infected with lentiviruses carrying the indicated shRNAs, and the association of H4R3me2s, H3R2me2s, H3K9me2 and H3K4me2 at the promoter of E-cadherin and vimentin was analyzed with the qChIP assay. (F and G) MDA-MB-231 cells were infected with lentiviruses carrying shNTC, shLSD1, shPRMT5 and shLSD1 + shPRMT5. The mRNA or protein level of indicated genes was measured by qPCR (F) or western blotting (G). (H and I) SUM159 cells were infected with lentiviruses carrying shNTC, shLSD1, shPRMT5 and shLSD1 + shPRMT5. The mRNA or protein level of indicated genes was measured by qPCR (H) or western blotting (I). (J) MCF10A cells were infected with lentiviruses carrying the indicated shRNAs together with Slug expression constructs or empty vector. The invasiveness of these cells was analyzed with a matrigel-coated chamber invasion assay. (K and L) MDA-MB-231 (K) or SUM159 (L) cells were infected with lentiviruses carrying shNTC, shLSD1, shPRMT5 and shLSD1 + shPRMT5. (M) MDA-MB-231 cells transfected with shSlug and the expression plasmids for PRMT5 or LSD1 for cell invasion assay. For J, K, L and M, representative photomicrographs are shown in the left. The histograms show the mean ± SD of the fold changes of numbers of invasive cells in each of samples, relative to that of control sample from three separate experiments. ***P < 0.001, as determined by Student’s t test. For B, C, E, F and H, the data represent the mean ± SD from three independent experiments (**p < 0.01, ***p < 0.001, Student’s t-test)

Fig 5: Slug interacts with PRMT5 and LSD1. (A) Immunoaffinity purification and mass spectrometry analysis of Slug-binding proteins. Extracts from HEK293T cells bearing Flag (Vector) or Flag-Slug were immunopurified with anti-Flag affinity columns and eluted with Flag peptide. The eluates were resolved by SDS-PAGE and visualized by silver staining. The protein bands on the gel were excised and identified by mass spectrometry. Representative peptide fragments of PRMT5 and LSD1 are indicated on the right. (B) Representative peptide coverage of the indicated proteins is shown in the table. (C) The purified fractions were analysed by western blotting with antibodies against indicated proteins. (D) Cell lysates from MDA-MB-231, SUM159 or Hs578T cells were immunoprecipitated with antibodies against indicated proteins followed by immunoblotting with various antibodies indicated. The arrows denote the light chains of IgG and Slug antibody. The asterisks indicate a nonspecific band. (E) Flag-PRMT5, HA-Slug and HA-LSD1 (Left), Flag-Slug, HA-PRMT5 and HA-LSD1 (Middle) or Flag-LSD1, HA-Slug and HA-PRMT5 (Right) were co-expressed in HEK293T cells, respectively. After immunoprecipitation with appropriate antibodies, bound proteins (e.g., Slug, PRMT5 or LSD1) were examined by western blotting. (F) GST-fused Slug, LSD1 or PRMT5 were incubated with the Flag-tagged PRMT5, Slug or LSD1 purified from HEK293T cells. The binding proteins by GST pull-down assays were examined by western blotting with indicated antibodies. Coomassie brilliant blue staining of the GST-fused proteins was shown below