Fig 2: (A) Phase contrast image of HMLE cells expressing Snail-ER or Twist-ER at 0 and 8 days post 4-OHT treatment. Scale bar = 100 μm. (B) Flow cytometric profiles of CD44 and CD24 on Snail-ER or Twist-ER HMLE cells treated with 4-OHT for the indicated number of days. (C) Relative expression of the mRNAs encoding macroH2A1.1, macroH2A1.2, E-cadherin, vimentin, and Zeb1 in the cells described in (B) as determined by real-time RT-PCR. GAPDH was used as a housekeeping gene control to account for variability in template loading. (D) Western blot analysis of expression of E-cadherin, vimentin, macroH2A1.1 and macroH2A1.2, proteins in the HMLE-Snail-ER cells treated with 4-OHT for the indicated number of days. Histone H3 was used as a loading control. (E) Immunofluorescence images of HMLE-Twist-ER cells treated with 4-OHT for 16 days. Cells were stained using antibodies against macroH2A1.1, macroH2A1.2, E-cadherin, or vimentin. Scale bar = 100 μm.

Fig 3: Functional analysis of macroH2A1.1 in GC cellsA. MacroH2A1.1 levels were detected in HGC-27 cells after treatment with pcDNA-macroH2A1.1 or pcDNA empty vector by immunoblotting; B. Cell proliferation assay of HGC-27 cells after treatment with pcDNA-macroH2A1.1 or pcDNA using CCK-8; C. Flow cytometry was conducted to examine the apoptosis of HGC-27 cells after treatment with pcDNA-macroH2A1.1 or pcDNA and stained with apoptosis markers (FITC-Annexin V and PI). In the apoptosis map, FITC-Annexin V+/PI+ indicates late apoptosis, FITC-Annexin V+/PI− indicates early apoptosis, and FITC-Annexin V−/PI− indicates normal live cells. D. Wound healing assays of HGC-27 cells after treatment with pcDNA-macroH2A1.1 or pcDNA. The relative ratio of wound closure per field is shown in the right; E. Transwell analysis of HGC-27 cells after treatment with pcDNA-macroH2A1.1 or pcDNA. The relative ratio of invasive cells per field is shown in the right. For all qPCR results, the data are presented as the mean ± SEM, and the error bars represent the standard deviation obtained from three independent experiments. * p < 0.05; ** p < 0.01. F. Pathology analysis of tumor sections from GC tissues and adjacent normal tissues. Immunohistochemistry, H&E staining, and labeling with QKI5 and mH2A1.1 was performed. Bars: 20 μm. * p < 0.05; ** p < 0.01. The relative ratio of expression is shown in the right.

Fig 4: (A) Western blot analysis of expression of MDA-MB-231, HMLE-Snail-ER and HMLE-Twist-ER cells transduced by the indicated vectors. Histone H3 was used as a loading control. (B) Phase contrast of MDA-MB-231 cells overexpressing macroH2A1.1, macroH2A1.2 or Zeb1 shRNA. Scale bar = 100 μm. (C) Flow cytometric analysis of cell-surface markers, CD44 and CD24, in the cells described in (B). (D) Relative expression of the mRNAs encoding macroH2A1.1, macroH2A1.2, E-cadherin, vimentin, and Zeb1 in the cells described in (B) as determined by real-time RT-PCR. Normalized to GAPDH mRNA to account for variability in template loading. (E) Flow cytometric analysis of cell-surface markers, CD44 and CD24, in fully mesenchymal HMLE-Snail-ER or HMLE-Twist-ER cells overexpressing macroH2A1.1, macroH2A1.2 or the control vector. (F) Relative expression of the mRNAs encoding macroH2A1.1, macroH2A1.2, E-cadherin, vimentin, and Zeb1 in the cells described in (E) as determined by real-time RT-PCR. Normalized to GAPDH mRNA to account for variability in template loading.

Fig 5: (A) Western blot analysis of expression of macroH2A1.1, macroH2A1.2 proteins in the HMLE-Snail-ER or HMLE-Twist-ER cells transduced with the indicated vectors. Histone H3 was used as a loading control. (B) Phase contrast of HMLE Twist-ER cells overexpressing macroH2A1.1 or macroH2A1.2 after 8-day 4-OHT treatment. Mesenchymal cell morphology was observed in the vector control and macroH2A1.2 cells, but not macroH2A1.1 cells. Scale bar = 100 μm. (C) Flow cytometric analysis of cell-surface markers, CD44 and CD24, in the indicated cells after 8-day 4-OHT treatment. (D) Phase-contrast images of mammospheres formed from HMLE-Snail-ER cells transduced with the indicated macroH2A1 vectors and treated ± 4-OHT for 8 days. Scale bar = 100 μm. (E) Quantification of mammospheres formed in (D). (n = 4 experiments, p = 0.005 (GFP vs. macroH2A1.1)).