Fig 2: METH treatment causes Sigmar1 expression-dependent regulation of CREB-Fis1 expression in cardiomyocytes.a Left panel: representative micrographs of immunohistochemical detection of Sigmar1 (brown) in human non-METH user (n = 4), and METH toxicity (n = 4) LV heart sections. Hematoxylin (blue) was used to counterstain the nuclei. Right panel: fold change of DAB-positive Sigmar1 staining intensity in METH toxicity hearts compared to those of non-METH users. b Left panel: representative micrographs of immunohistochemical detection of Sigmar1 (brown) in the vehicle (n = 4), and METH-treated (n = 4) mouse LV sections. Right panel: fold change of DAB-positive Sigmar1 staining intensity in hearts treated with METH hearts compared with those treated with vehicle. c Left panel: global Sigmar1 knockout (Sigmar1-/-) mouse hearts were used to assess the specificity of the Sigmar1 primary antibody. Right panel: IgG stained slides without primary antibody incubation were used as a negative control. Representative micrographs are from three independent experiments. a–c Scale bar: 50 µm. d Sigmar1 was overexpressed and knocked down in NRC through adenovirus infection (10 MOI) and siRNA (50 nM) transfection, respectively. Right panel: western blot analyses showed that Sigmar1 overexpressed NRC exhibited increased CREB, pCREBSer133, and Fis1 expression, whereas knockdown of Sigmar1 yielded opposite results. Ponceau S staining of the transfer membranes was used to confirm equal loading and transfer across the gel. Left panel: bar graphs represent densitometric quantification of CREB, pCREBSer133, and Fis1 protein bands in cardiomyocytes. ß-Gal adenovirus infection was used as the control for Sigmar1 adenovirus infection, whereas a non-specific siRNA was used as the control for Sigmar1 siRNA transfection. Data are representative of triplicates from two independent experiments. Boxes depict interquartile ranges, lines represent medians, and whiskers represent ranges. P values were determined using a one-way ANOVA, followed by Tukey’s multiple comparisons test. P < 0.05 between groups was considered statistically significant. ß-Gal ß-galactosidase; Adeno adenovirus, siRNA small interfering RNA, Veh, vehicle, METH methamphetamine, NS not significant.

Fig 3: Sigmar1 interacts with SERCA2 and mediates its degradation Relative xbp1 splicing level of WT or Sigmar1 gKO BMMs during osteoclast formation measured by RT‐qPCR (n = 6 biological replicates).Immunoprecipitation of flag‐Sigmar1 interacting protein was visualized by silver gel staining.Western blots showing relative SERCA2 expression in WT and Sigmar1 gKO BMMs.Western blots showing truncated SERCA2 (314–807 aa) expression in HEK‐293T cells transfected with different amounts of Sigmar1 plasmids. The cells were transfected with 1 μg truncated SERCA2 plasmid and Sigmar1 plasmid (0.125, 0.25, 0.5, and 1 μg).Sequence alignment of glutamine residues from SERCA2 orthologs of different species.Western blots showing Q615A mutant full‐length SERCA2 expression in HEK‐293T cells in with different amounts of Sigmar1 plasmids. The cells were transfected with 1 μg Q615A mutant full‐length SERCA2 plasmid and Sigmar1 plasmid (0.125, 0.25, 0.5, and 1 μg).Interactions between Sigmar1 and WT or Q615 mutants of full‐length SERCA2 were detected by Co‐IP assays.Relative mRNA expression of SERCA2 in BMMs treated with PBS or 10 μM dimemorfan for 2 days (n = 6 biological replicates).Western blots showing SERCA2 expression in BMMs treated with PBS or 10 μM dimemorfan for 2 days.Relative SERCA2 activity in BMMs treated with PBS or dimemorfan was detected by the Ca2+‐ATPase assay (n = 6 biological replicates).Knock down of SERCA2 by siRNA in BMMs was verified by western blotting. Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. The unpaired two‐tailed Student’s t‐test (A, H and J) was used for statistical analysis. Source data are available online for this figure.

Fig 4: Sigmar1 deletion has no influence on bone mass under steady conditions AMicrocomputed tomography (micro‐CT) images of the proximal femur from 12‐week‐old male WT and Sigmar1 gKO mice. Scale bars, 1 mm.BQuantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp), trabecular thickness (Tb. Th), cortical region BV/TV (Ct. BV/TV), and cortical thickness (Ct. Th, mm) (n = 5 biological replicates).CCoronal images of the fifth lumbar spine. Scale bars, 1 mm.DQuantification of trabecular bone parameters of lumbar spine (n = 5 biological replicates).ETRAP staining of tibias from male WT and Sigmar1 gKO mice. Scale bars, 50 μm.F, GQuantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates).H, IRepresentative images and quantitative analysis of calcein double labeling. Scale bars, 20 μm (n = 6 biological replicates).J, KSerum PINP (procollagen I N‐terminal propeptide) and CTX‐I (C‐terminal telopeptide of type I collagen) concentrations measured by ELISA in male Sigmar1 gKO mice and their WT littermates (n = 6 biological replicates).LMSCs from male WT or Sigmar1 gKO mice underwent osteogenic differentiation for 7 or 21 days and staining for alkaline phosphatase or alizarin red, respectively. Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. The unpaired two‐tailed Student’s t‐test (B, D, F, G, I, J, and K) was used for statistical analysis.

Fig 5: MAM-localized Rac1 inhibits RLR signaling in a protein geranylgeranylation–dependent manner.(A to C) Quantitative RT-PCR analysis of transcript abundance of Ifnb1, Il6, Rac1, and Rac2 in wild-type (WT), Rac1−/−, or Rac2−/− iBMDMs 4 hours after SeV infection. (D) Immunoblot of phospho-IRF3 and phospho-IKK in cell lysates of wild-type or Rac1−/− iBMDMs following SeV infection. Numbers below lanes indicate densitometry of phosphorylated IRF3 or IKK relative to that of total IRF3 or IKK, respectively. (E to H) Quantitative RT-PCR analysis of transcript abundance of Ifnb1, Il6, and SeV-P (E to G) and immunoblot of Rac1 (H) in Rac1−/− iBMDMs reconstituted with wild-type, C189S, G12V, or G12V/C189S of Rac1 4 hours after SeV infection. (I and J) Confocal microscopy of immunofluorescent staining of Rac1 (green) and SIGMA1R (red) in Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs 4 hours after SeV infection. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 10 μm (I). See each single channel in fig. S3J. The percentage of colocalization between Rac1 and SIGMA1R was calculated using the ImageJ software from at least 50 cells randomly selected from the immunostaining slides (J). (K to N) Representative PLA images of Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs (K) or Rac1−/− iBMDMs reconstituted with wild-type, G12V, or C189S of Rac1 (M) 4 hours after SeV infection. The cells were probed with Rac1 and SIGMA1R antibodies. Red spots indicate the association of Rac1 with SIGMA1R. (K and M) Cells were counterstained with DAPI (blue). Scale bars, 10 μm. (L and N) Quantitation of PLA spots per cell in (K) and (M) (n = 20 cells). (O) Schematic display of the C-terminal amino acid sequence of Rac1 highlighting the C178 for palmitoylation and C189 for geranylgeranylation sites. (P) Immunoblots of Rac1 after S-palmitoylated protein assay (acyl-RAC; see Materials and Methods) of captured proteins (Pulldown) and inputs from Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDM cell lysate following infection with or without SeV for 4 hours. (Q) Quantitative RT-PCR of Ifnb1 and Il6 abundance in MEF cells stably expressing wild-type and C178S mutant forms of Rac1 after transfection with poly(I:C) LMW. **P < 0.01, ****P < 0.0001 (two-way ANOVA). Data are representative of three independent experiments.