Fig 2: Cardiac defects in Carm1 mutants at E18.5. a) Histology of an E18.5 Carm1A296E mutant embryo showing a VSD (arrow). b) Histology of an E18.5 Carm1A296E mutant embryo showing DORV.

Fig 3: B-I09 synergizes with immune checkpoint blockade.a Expression of CARM1 and a loading control ß-actin in UPK10, A1847, and OVCAR3 cells determined by immunoblot. Immunoblots are representative of three biologically independent experiments with similar results. b Schematic of the experimental design for UPK10 syngeneic immunocompetent mouse model. c Mice bearing orthotopic tumors were randomized into four indicated treatment groups. Reproductive tracts with tumors from the indicated treatment groups were dissected at the end of treatment (n = 6 mice per group). d The weights of tumors dissected from the indicated groups were measured as a surrogate for tumor burden. Data represent mean ± SEM. P values were calculated using two-tailed t test. Statistical co-efficiency of drug interaction (CDI) analysis revealed that the CDI for the combination is 0.59 (<1)38, which indicates synergy between B-I09 and anti-PD1 combination. e After stopping the treatment, the mice were followed for survival. Kaplan–Meier survival curves for the indicated groups (n = 7 per group). f Immune cell infiltration in the tumors dissected from the indicated treatment groups were analyzed by flow cytometry. n = 5 mice per group. P values were calculated by two-sided log-rank test.

Fig 4: CARM1 status correlates with response to inhibition of the IRE1a/XBP1s pathway.a Representative images of colonies formed by control and CARM1 knockout A1847 cells treated with or without IRE1a inhibitor B-I09 at the indicated concentrations. b B-I09 dose-responsive curves based on the colony-formation assay for control and CARM1 knockout A1847 cells. c Expression of apoptotic markers cleaved PARP and cleaved Lamin A and a loading control ß-actin in control and CARM1 knockout A1847 cells treated with or without B-I09 at the indicated concentrations determined by immunoblot. Immunoblots are representative of three biologically independent experiments with similar results. d B-I09 dose-responsive curves based on colony-formation assay of the indicated control and CRISPR-mediated activation of endogenous CARM1-expressing COV362 and EF027 cells. e Expression of CARM1, c-MYC, and a loading control ß-actin in a panel of the indicated cell lines determined by immunoblot. Immunoblots are representative of two biologically independent experiments with similar results. f B-I09 dose-responsive curves based on colony-formation assay for the indicated cells. g B-I09 IC50 of CARM1-high (n = 3) and CARM1-low (n = 9) cell lines. h B-I09 dose-responsive curves based on colony-formation assay of the indicated control and shCHOP-expressing A1847 cells. i Expression of apoptotic markers cleaved caspase 3, cleaved PARP p85, and a loading control ß-actin in control and shCHOP-expressing (#1) A1847 cells treated with or without B-I09 (5 µg/ml, 48 h). Immunoblots are representative of two biologically independent experiments with similar results. Data represent mean ± SEM, n = 4 biologically independent experiments unless otherwise stated. P values were calculated using two-tailed t test.

Fig 5: Dysfunctional lipid metabolism and dysregulation of CARM1 in C9orf72-linked ALS patients. (A,B) C9orf72 protein levels in B lymphocytes derived from C9orf72-linked ALS patients and controls were determined by immunoblotting. n = 4. (C,D) C9 ALS patient and control B lymphocytes were cultured in CM or glucose-free medium for 6 h, and C9orf72 protein levels were determined as in A and B. n = 4. (E) FFA levels were determined in two independent pairs of iPSC-derived motor neurons from control and C9 ALS patients. n = 3. (F–J) Protein levels of C9orf72, CARM1, and LAMP1 in spinal cords from three C9 ALS patients and four controls were determined by immunoblotting. n = 3–4. (K) Representative immunoblots for analyzing CARM1, C9orf72, ACC, and LAMP1 in iPSC-derived motor neurons from two C9 ALS patients and two controls. (L) Representative images of iPSC-derived motor neurons from two C9 ALS patients and two controls, including the staining with CHAT (motor neuron marker), BODIPY (LD), and LAMP1 (lysosomal marker). (M,N) Quantitative analyses of BODIPY staining (n = 24–27 fields of view; K) and BODIPY/LAMP1 dual-positive puncta (45–47 cells; L) in iPSC-derived motor neurons from C9 ALS patients and controls. (O–S) Quantitation of the immunoblotting analysis in I for iPSC-derived motor neurons from C9 ALS patients and controls. n = 4. (GS) Glucose starvation. Data are presented as mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.