Fig 2: A new Arid1a conditional allele to explore ARID1A tumor suppressor function in vivo(A) Targeting scheme to insert two loxP sites flanking exon 5 and 6 of mouse ARID1A. (B) Representative Southern blot showing properly targeted ES cell clones. Insertion of loxP sites generates 5.6 kb EcoRV and 8.5 kb ApaI genomic fragments. (C) PCR genotyping and quantitative, dual-color fluorescent western blot of ARID1A (red) and ßACTIN (green) protein expression in whole-embryo lysates from four Arid1afl/+ and four Arid1afl/+;Sox2Cre0/+ embryos. (D) Graph of quantitative western blot results depicting normalized protein levels. Significant differences based on the average normalized protein expression ±SD of four independent embryos were calculated using a two-tailed Student’s t test (p-values <0.05 were considered significant). (E,F) Representative LacZ-stained, control (uninjected) or intrabursal AdCRE-injected (Gt)Rosa26lacZ ovary showing patterns of CRE-mediated recombination in the ovarian surface epithelium. (F, inset) Histological section of LacZ-stained AdCRE-injected (Gt)Rosa26lacZ ovary. (G-I) Live-luminescence overlaid whole-mount images of an intrabursal AdCRE injected, (Gt)Rosa26luciferase female mouse. (I) Excised reproductive tract. Asterisks in H indicate wound staple.

Fig 3: Colocalization of Arid1a and Gli1+ cells and their progeny in developing roots(A–H) H&E staining (A–D) and Arid1a immunofluorescence (red) (E–H) of the first mandibular molar of a wild-type (WT) mouse from PN4.5 to PN21.5. Mes, dental mesenchyme; epi, dental epithelium. (I–L) Co-immunofluorescence of Arid1a (green) and Gli1 (stained by ß-gal in red) in the first mandibular molar of a Gli1-LacZ mouse at PN4.5. Box in (I) is shown at higher magnification at the right. Arrows indicate positive signals. White dashed lines outline Hertwig’s epithelial root sheath (HERS).(M–P) Arid1a immunofluorescence (green) and visualization of tdTomato (red) of the first mandibular molar of a Gli1-CreER;tdTomato mouse at PN18.5 after induction at PN3.5. The progeny of the Gli1+ lineage show red signal. Arrows indicate positive signals. TdT, tdTomato; DPC, dental pulp cell; OD, odontoblast; PDL, periodontal ligament. Schematic at the bottom indicates induction protocol. TAM, tamoxifen. Scale bars: 100 µm.

Fig 4: Loss of Arid1a in Gli1+ root progenitor cells leads to downregulated Hh signaling activity(A) Heatmap hierarchical clustering showing the gene expression profiles in the apical regions of control and Gli1-CreER;Arid1afl/fl mouse molars at PN7.5.(B–M) RNAscope in situ hybridization (red) of Gli1 (B–E), Ptch1 (F–I), and immunofluorescence of Ccnd1 (red, J–M) of first mandibular molars of control and Gli1-CreER;Arid1afl/fl mice at PN7.5. The boxed areas are enlarged on the right. Arrows indicate positive signals in control group; arrowheads indicate reduced signal in targeted region of Gli1-CreER;Arid1afl/fl mouse molars.Schematic at the bottom indicates induction protocol. Scale bars: 100 µm. See also Figures S4 and S5.

Fig 5: Loss of ARID1A results in selective sensitivity to mitochondrial inhibition in 2D. (A) Table of compounds with mitochondrial inhibitions properties in clinical trials. Compound names and conditions for which they are used are indicated [57]. (B) Dose-dependent sensitivity of scramble (scr.) and ARID1A KD (KD) ES-2 WT cells to the panel of mitochondrial inhibitors. Results are expressed as residual colony formation as compared to control. Drug treatment was conducted over a period of 7 days. (C) Western blot analysis for expression levels of ARID1A in scramble and ARID1A KD RMG1 cells obtained by lentiviral-mediated delivery of shRNA-ARID1A. Actin was used as a loading control. (D) Residual colony formation in scramble and ARID1A KD RMG1 cells exposed to 10 nM of the mitochondrial inhibitor IACS-010759 over a period of 10 days. (E) Real-time oxygen consumption rate (OCR) in scramble versus ARID1A KD RMG1 cells exposed to increasing concentrations of the mitochondrial inhibitor IACS-010759. The injection order: a. IACS-010759, b. oligomycin, c. FCCP, and d. rotenone and antimycin. Inset shows the OCR alterations after IACS-010759 injection during the first 100 min. (F) ATP-linked respiration in ARID1A in scramble (scr.) versus ARID1A knockdown (KD) ARID1A WT RMG1-2 cells exposed to increasing concentrations on the mitochondrial inhibitor IACS-010759. (G) Maximal respiration in ARID1A in scramble (scr.) versus ARID1A knockdown (KD) ARID1A WT RMG1-2 cells exposed to increasing concentrations on the mitochondrial inhibitor IACS-010759.