Fig 1: KDM6A/B inhibition reduces the spheroid size and induces apoptosis. (A) Sphere formation assay HeLa, HCT116, and 22Rv1 cell lines were cultured in matrix detachment for six days and simultaneously treated either with GSK J4 (5 µM for HeLa and 22Rv1, 10 µM for HCT116) or with vehicle 0.1% DMSO (control). At the end of the treatment schedule, sphere images were captured using a Nikon inverted light microscope, and images were analyzed for size measurement by ImageJ software N = 8. Histogram plots were plotted for average size in all conditions. (B) HeLa, HCT116, and 22Rv1 cell lines were cultured in matrix detachment for six days and simultaneously treated with different GSK J4 or with vehicle 0.1% DMSO (control). Cell proliferation was measured using WST-1, and IC 50 was calculated. (C) Apoptosis assay HeLa, HCT116, and 22Rv1 cell lines were cultured in matrix detachment for six days and simultaneously treated either with GSK J4 (5 µM for HeLa and 22Rv1,10 µM for HCT116) or with vehicle 0.1% DMSO (control). As mentioned above, a similar treatment strategy was used, and apoptosis assay was performed using Annexin V and pI. The histogram data were plotted for % cells showing live or apoptotic populations. (D) KDM6B/A demethylase activity was measured from the nuclear extract of HeLa, HCT116, and 22Rv1 cell lines and were cultured in matrix detachment for six days and simultaneously treated either with GSK J4 (5 µM for HeLa and 22Rv1,10 µM for HCT116 KDM6B/A) or with vehicle control. The activity was measured using the KDM6 activity assay kit. (E) Histone was extracted from ECM-attached and ECM-detached cells. ECM-detached cells treated with GSK-J4 using a histone extraction kit (ab113476) and 10 µg of histone protein loaded for Western blot; Western blot showing GSK J4 treatment for six days significantly induces the expression of H3K27me2/3 in 22RV1 during matrix detachment conditions. (F) pan H3k27 me2 and me3 was measured from histone extracted as previous conditions mentioned above. p.value **p.value > 0.001, *p.value > 0.01, ***p.value > 0.0001.
Fig 2: Matrix detachment induces the expression of stemness markers that are repressed by the KDM6B-specific inhibitor. (A) mRNA expression of various stemness-related genes (SOX2, SOX9, and CD44) in HeLa, HCT116, and 22Rv1 cell lines during matrix detachment at six days. The values were normalized with the housekeeping gene RPLP0, and other gene expression values were calculated. (B) Flow cytometry–based expression of various surface markers of stemness in HeLa, HCT116, and 22Rv1 cell lines during matrix detachment for six days. (C) Spheroids were treated with GSK-J4 (5 µM for HeLa and 22Rv1,10 µM for HCT116), a specific inhibitor of KDM6 histone demethylases. Moreover, the mRNA expression of various stemness-related genes (SOX2, SOX9, and CD44) in HeLa, HCT116, and 22Rʋ1 cell lines were measured as explained above. (D) Spheroids were treated with GSK-J4 (5 µM for HeLa and 22Rv1,10 µM for HCT116). In addition, the flow cytometry–based expression of various surface markers of stemness was measured. (E) Immunofluorescence for various stemness surface markers was measured in spheroids and spheroids treated with GSK-J4 in HeLa. (F) Enrichment of KDM6B and RNA Pol II on the promoters of SOX2 and CD44 genes in the presence and absence of GSK J4 in ECM-detached (spheroids) cells; nonimmune IgG was used as the input control. **p.value > 0.001, *p.value > 0.01, ***p.value > 0.0001.
Fig 3: Clinical correlation of KDM6B and HIF 1α in various cancer types. Correlation analysis of the gene expression between KDM6B and HIF 1α from metastatic samples. (A) Gene expression in metastatic melanoma samples. (B) Gene expression in metastatic breast samples **p.value > 0.001, *p.value > 0.01, ***p.value > 0.0001.
Fig 4: Matrix detachment induces the hypoxia-regulated KDM6B expression and activity. (A) mRNA expression of various stemness-related genes HIF1α, HIF2 α, and target gene VEGF in HeLa cell lines during matrix detachment for six days. The values were normalized with the housekeeping gene RPLP0, and other gene expression values were calculated. (B) Enrichment of KDM6B on the promoters of HIF 1α both in the presence and absence of GSK J4 (5 µM) in HeLa cells during matrix detachment for six days; nonimmune IgG was used as the input control. **p.value > 0.001, *p.value > 0.01, ***p.value > 0.0001.
Fig 5: CRISPR knockout KDM6B and reduce stemness in ECM-detached cells. (A) Total protein was isolated from +ve control empty CRISPR Cas-transfected cell line and CRISPR KDM6B-knockout cell lines. Western blot was performed as previously mentioned, and the blot was probed with anti-human KDM6B antibodies; the loading control was blot stained with Ponceau staining. (B) Sphere formation assay HeLa + ve Control (non-targeted) and HeLa KDM6B-knockout cell lines were cultured in matrix detachment for six days. At the end of the treatment schedule, sphere images were captured by using a Nikon inverted light microscope. (C) HeLa + ve Control (non-targeted) and HeLa KDM6B-knockout cell lines were cultured in matrix detachment for six days in the end for time duration. The flow cytometry–based expression of various surface markers of stemness was measured.
Supplier Page from Abcam for KDM6A/ KDM6B Activity Quantification Assay Kit (Colorimetric)