Journal: bioRxiv

Article Title: Inhibition of a Selective SWI/SNF Function Synergizes with ATR Inhibitors in Cancer Cell Killing

doi: 10.1101/660456

Figure Lengend Snippet: (A) Cell survival data from HCT116 cells infected with shRNA lentivirus targeting ARID2 (blue), ARID1A (green), Control (red), and HCT116(ARID1A −/−) cells (green-dashed). Following lentiviral transduction and selection, cells were exposed to VE-821 or BD98 for 5 continuous days. Error bars represent s.d. of eight technical replicates in three separate experiments in 384-well plates. (B) Gene expression of ARID1A, ARID2, and ARID1B in HCT116 (WT, shARID1A, shARID2). Delta-Delta CT values compared to Gapdh and WT HCT116. (C) Shifting IC50 plot of WT HCT116 cells, ARID1A −/− HCT116 cells, and HCT116 cells infected with shRNA lentivirus targeting ARID2 or ARID1A treated with VE-821 and increasing doses of BD98 for 5 days. (D) Normalized isobologram plots of WT HCT116 cells, ARID1A −/− HCT116 cells, and HCT116 cells infected with shRNA lentivirus targeting ARID2 or ARID1A (as treated in 3C). (E) Average combination indexes for HCT116 cells infected with shRNA lentivirus targeting ARID2, ARID1A, WT, and HCT116 (ARID1A−/−).

Article Snippet: Blots were probed with primary antibodies, rabbit α-Arid1a(D2A8u) (1:1000, Cell Signalling #12354), and mouse α-Arid1b (D2D) (1:1000, Novus Biologicals # H00057492-M01) followed by fluorescence-conjugated secondary antibodies (Li-Cor) and bands were detected using an Odyssey CLX imaging system (Li-Cor).

Techniques: Infection, shRNA, Control, Transduction, Selection, Gene Expression

Journal: bioRxiv

Article Title: Inhibition of a Selective SWI/SNF Function Synergizes with ATR Inhibitors in Cancer Cell Killing

doi: 10.1101/660456

Figure Lengend Snippet: (A) Expression of ARID1A and ARID1B in HCT116, ARID1A −/− HCT116, Cal51 and MCF7 cancer cell lines. (B) Attempted knockdown of ARID1B in Cal51 cell lines. (C) Dose response of Renal cell carcinoma lines to increasing concentrations of ATR inhibitor (VE-821) and BD98. (D) Synergy assessment in SWI/SNF mutant cancer cell lines.

Article Snippet: Blots were probed with primary antibodies, rabbit α-Arid1a(D2A8u) (1:1000, Cell Signalling #12354), and mouse α-Arid1b (D2D) (1:1000, Novus Biologicals # H00057492-M01) followed by fluorescence-conjugated secondary antibodies (Li-Cor) and bands were detected using an Odyssey CLX imaging system (Li-Cor).

Techniques: Expressing, Knockdown, Mutagenesis

Journal: iScience

Article Title: ARID1A loss in pancreas leads to islet developmental defect and metabolic disturbance

doi: 10.1016/j.isci.2022.105881

Figure Lengend Snippet: Pancreas-specific deletion of Arid1a induces DM in mice (A) Body weight was decreased in the Arid1a-depleted PCA L/L mice (n = 5). (B) The pancreas size of the PCA L/L mice was reduced (n = 5). (C) Age-dependent increase of blood sugar in the Arid1a-depleted PCA L/L mice (n = 5). (D) Representative pictures of Arid1a, insulin and glucagon staining in the pancreases of normal and Arid1a-depleted PCA L/L mice. (E) The Arid1a-, insulin- and glucagon-positive areas were expressed as the percentage of total areas measured. Five independent areas were examined in a tissue slide and five tissues were counted in each group. (F) Blood samples were collected from 13-week-old mice and insulin level was measured by ELISA assays. (n = 5). (G) Thirteen-week-old mice (n = 5) were fasted for 6 h in the morning and intraperitoneally injected with 2 g/kg of glucose. Blood glucose levels were continuously measured for 120 min using a glucometer. (H) The survival of control (n = 20) and PCA L/L mice (n = 41). ∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001.

Article Snippet: Cellular proteins were resolved by the 10% Bis-Tris gradient gel (Invitrogen), transferred to the polyvinylidene difluoride membrane, blocked in 5% non-fat milk in PBS/Tween-20, and probed by the following primary antibodies: anti-insulin (Cell Signaling Technology, Cat# 3014); anti-ARID1A (Abnova, MAB15809); anti-Neurogenin3 (Santa Cruz Biotechnology, sc-374442), and anti-MafA (Santa Cruz Biotechnology, sc-390491).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Injection, Control

Journal: iScience

Article Title: ARID1A loss in pancreas leads to islet developmental defect and metabolic disturbance

doi: 10.1016/j.isci.2022.105881

Figure Lengend Snippet: Arid1a depletion impairs islet development (A) Islet isolation was performed as described in Materials and Methods. The cultured islets were imaged by light microscope and the islet size was calculated. The average diameters of the islets were expressed as Mean ± SD (n = 20). (B) The conditioned media of control and isolated PCA L/L islets were collected at 72 h after culture and the levels of insulin were measured by ELISA assays. (C) Gene expression of isolated control and PCA L/L islets was investigated by RNA sequencing and the expression profile was presented by heatmap. (D) The pathways enriched in the PCA L/L islets studied by KEGG pathway analysis. (E) GSEA analysis suggested β cell dysfunction in the isolated PCA L/L islets. (F) Activation of interferon α signaling was found in the PCA L/L islets. (G) Total RNAs were extracted from the isolated islets and the expression of cytokines and chemokines was studied by RT-PCR analysis. (H) Pancreatic tissues collected from the control and PCA L/L mice. Infiltration of T cells in the pancreas was studied by IHC staining. (n = 5). ∗∗∗p<0.001.

Article Snippet: Cellular proteins were resolved by the 10% Bis-Tris gradient gel (Invitrogen), transferred to the polyvinylidene difluoride membrane, blocked in 5% non-fat milk in PBS/Tween-20, and probed by the following primary antibodies: anti-insulin (Cell Signaling Technology, Cat# 3014); anti-ARID1A (Abnova, MAB15809); anti-Neurogenin3 (Santa Cruz Biotechnology, sc-374442), and anti-MafA (Santa Cruz Biotechnology, sc-390491).

Techniques: Isolation, Cell Culture, Light Microscopy, Control, Enzyme-linked Immunosorbent Assay, Expressing, RNA Sequencing Assay, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry

Journal: iScience

Article Title: ARID1A loss in pancreas leads to islet developmental defect and metabolic disturbance

doi: 10.1016/j.isci.2022.105881

Figure Lengend Snippet: Metabolic alteration in the islets of the PCA L/L mice (A) The metabolite profiles of the islets isolated from the control and PCA L/L mice were compared by principal component analysis. (B) Differentially expressed metabolites were demonstrated by heatmap. (C) Top 10 upregulated metabolites in the isolated PCA L/L islets were shown as fold change (Arid1a/control). (D) Top 10 downregulated metabolites in the isolated PCA L/L islets were shown as fold change (Arid1a/control). (E) Alteration of carbohydrate metabolism and increase of glycolytic intermediates in the isolated PCA L/L islets. The metabolite levels in the control islets (red color) and PCA L/L islets (blue color) were presented. (F) The levels of various amino acids in the control islets (red color) and PCA L/L islets (blue color) were shown and the increase of alanine, aspartic acid, asparagine, glutamate, proline was found. ∗p<0.05; ∗∗p<0.01.

Article Snippet: Cellular proteins were resolved by the 10% Bis-Tris gradient gel (Invitrogen), transferred to the polyvinylidene difluoride membrane, blocked in 5% non-fat milk in PBS/Tween-20, and probed by the following primary antibodies: anti-insulin (Cell Signaling Technology, Cat# 3014); anti-ARID1A (Abnova, MAB15809); anti-Neurogenin3 (Santa Cruz Biotechnology, sc-374442), and anti-MafA (Santa Cruz Biotechnology, sc-390491).

Techniques: Isolation, Control

Journal: iScience

Article Title: ARID1A loss in pancreas leads to islet developmental defect and metabolic disturbance

doi: 10.1016/j.isci.2022.105881

Figure Lengend Snippet: Ngn3 is a direct target of Arid1a (A) The islets were collected from the control and PCA L/L mice and the expression of endocrine developmental lineage genes was studied by RT-PCR. ∗p<0.05; ∗∗p<0.01. (B) Genomic DNAs were collected from the control and PCA L/L islets and ChIP assay was performed to study the binding of Arid1a to the predicted regions (marked from region 1 to 4 upstream of the transcriptional start site) in the Ngn3 gene promoter. (C) Arid1a-depleted islet cells were infected with control (left) or Ngn3+Arid1a viral expression vectors. After 48 h, the conditioned media were collected and the insulin levels were determined by ELISA assay. (n = 5). ∗p<0.05. (D) Mouse NIT-1 insulinoma cells were transfected with Arid1a shRNA and the protein levels of Arid1a, insulin and Ngn3 were analyzed by Western blotting. The expression levels of E-cadherin (E-Cad) and MafA were also examined. (E) Human 1.2B4 insulin-secreting hybrid cells were transfected with Arid1a shRNA and the protein levels of ARID1A, Insulin and NGN3 were analyzed by Western blotting. The expression levels of HDAC6 and MafA were also examined. The insulin level in the conditioned media of cells transfected with control or shArid1a was determined by ELISA assay. (n = 3). ∗p<0.05.

Article Snippet: Cellular proteins were resolved by the 10% Bis-Tris gradient gel (Invitrogen), transferred to the polyvinylidene difluoride membrane, blocked in 5% non-fat milk in PBS/Tween-20, and probed by the following primary antibodies: anti-insulin (Cell Signaling Technology, Cat# 3014); anti-ARID1A (Abnova, MAB15809); anti-Neurogenin3 (Santa Cruz Biotechnology, sc-374442), and anti-MafA (Santa Cruz Biotechnology, sc-390491).

Techniques: Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Infection, Enzyme-linked Immunosorbent Assay, Transfection, shRNA, Western Blot

Journal: iScience

Article Title: ARID1A loss in pancreas leads to islet developmental defect and metabolic disturbance

doi: 10.1016/j.isci.2022.105881

Figure Lengend Snippet: Upregulation of HDACs expression in Arid1a depletion islet (A) GSEA analysis revealed the upregulation of HDAC pathway in the PCA L/L islets. (B) Islets were isolated from the pancreas of the control and PCA L/L mice and the protein levels of HDACs were determined by Western blotting. (C) ChIP-sequencing was performed to identify genome-wide binding of HDAC6 in control and PCAL/L mice. Distribution figure of peaks on various functional elements of genes. (D) Differential volcano plot (Red dots represent peaks that are significantly up-regulated and blue dots represent those that are significantly down-regulated. X axis:log2 fold change of peaks. Y axis: statistical significance of the differential in log10(q-value). (E) The most significant motif sequence. The abscissa is motif sequence. The ordinate is percentage of each base. (F) GO enrichment histogram (X axis: number of differential peak-associated genes in this GO category. Color code is to distinguish the categories - biological processes, cellular components and molecular functions. (G) KEGG enrichment histogram (X axis: gene number. Y axis: pathway term). (H) Scatterplot of peak-associated genes KEGG enrichment (X axis: RichFactor. Y axis specify KEGG pathways. The size of the dot is positively correlated with the number of peak-associated genes in the pathway. Color code is to indicate Q-value ranges. (I) Depicted are the top30 gene targets with minimum p-values identified by ChIP-Seq peak calling analysis. (J) Genomic DNAs were collected from the control and PCAL/L islets and the binding of HDAC6 to the Arid1a-occupied region 3 (Ngn3-3) in the Ngn3 gene promoter was studied by ChIP assay. Acetylation of histone H3 around the Ngn3-3 region in the Ngn3 gene promoter was also studied by ChIP assay.

Article Snippet: Cellular proteins were resolved by the 10% Bis-Tris gradient gel (Invitrogen), transferred to the polyvinylidene difluoride membrane, blocked in 5% non-fat milk in PBS/Tween-20, and probed by the following primary antibodies: anti-insulin (Cell Signaling Technology, Cat# 3014); anti-ARID1A (Abnova, MAB15809); anti-Neurogenin3 (Santa Cruz Biotechnology, sc-374442), and anti-MafA (Santa Cruz Biotechnology, sc-390491).

Techniques: Expressing, Isolation, Control, Western Blot, ChIP-sequencing, Genome Wide, Binding Assay, Functional Assay, Sequencing

Journal: iScience

Article Title: ARID1A loss in pancreas leads to islet developmental defect and metabolic disturbance

doi: 10.1016/j.isci.2022.105881

Figure Lengend Snippet: HDAC inhibitor suppresses the onset of DM induced by Arid1a depletion (A) Seven-week-old PCA L/L mice were administered twice weekly by intraperitoneal injections of 50 mg/kg of SAHA or PBS vehicle for 4 weeks (n = 5, per group). Mice were sacrificed, and the weights of pancreases were measured. In addition, blood sugar levels were measured at different time points before animal sacrifice. (B) Blood glucose levels in the PCA L/L mice treated with or without SAHA were measured every 14 days (n = 5). (C) Blood insulin levels in the PCA L/L mice treated with or without SAHA were measured by ELISA assays. (n = 5). (D) The pancreases of vehicle- or SAHA-treated PCA L/L mice were harvested for IHC staining to detect the expression of insulin, glucagon and neurogenin3 (Ngn3). Five independent areas were examined in a tissue slide and five tissues were counted in each group. (E) Areas of positive insulin, glucagon and neurogenin3 immunostaining were quantified by ImageJ software. (F) Infiltration of CD3 + T cells and F4/80 + macrophages in the pancreas of vehicle- or SAHA-treated PCA L/L mice was studied by IHC staining. (G) Areas of positive CD3 + and F4/80 + immunostaining were quantified by ImageJ software. ∗p<0.05; ∗∗p<0.01.

Article Snippet: Cellular proteins were resolved by the 10% Bis-Tris gradient gel (Invitrogen), transferred to the polyvinylidene difluoride membrane, blocked in 5% non-fat milk in PBS/Tween-20, and probed by the following primary antibodies: anti-insulin (Cell Signaling Technology, Cat# 3014); anti-ARID1A (Abnova, MAB15809); anti-Neurogenin3 (Santa Cruz Biotechnology, sc-374442), and anti-MafA (Santa Cruz Biotechnology, sc-390491).

Techniques: Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Expressing, Immunostaining, Software

Journal: iScience

Article Title: ARID1A loss in pancreas leads to islet developmental defect and metabolic disturbance

doi: 10.1016/j.isci.2022.105881

Figure Lengend Snippet:

Article Snippet: Cellular proteins were resolved by the 10% Bis-Tris gradient gel (Invitrogen), transferred to the polyvinylidene difluoride membrane, blocked in 5% non-fat milk in PBS/Tween-20, and probed by the following primary antibodies: anti-insulin (Cell Signaling Technology, Cat# 3014); anti-ARID1A (Abnova, MAB15809); anti-Neurogenin3 (Santa Cruz Biotechnology, sc-374442), and anti-MafA (Santa Cruz Biotechnology, sc-390491).

Techniques: Recombinant, Transfection, Protease Inhibitor, Staining, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Software