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Cell Signaling Technology Inc anti stim1
Anti Stim1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/stim1/pmc13000719-73-6-13?v=Cell+Signaling+Technology+Inc
Average 86 stars, based on 1 article reviews

anti stim1 - by Bioz Stars, 2026-07

86/100 stars

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Effects of STIM1 expression inhibition on MG-induced inflammatory damage, endoplasmic reticulum stress, calcium channel-associated proteins, and calcium ion accumulation in HD-11 cells. (A) Fluorescence microscopy images of HD-11 cells transfected with FAM-labelled siRNA. (B) Knockdown efficiency of three siRNAs at the mRNA level. (C) Knockdown efficiency of three siRNAs at the protein level. (D) Calcium channel-associated protein expression levels markedly decreased following siRNA transfection in HD-11 cells. (E) Validation results via quantitative real-time PCR (qRT-PCR). (F) Western blot analysis. (G) Reactive oxygen species (ROS) levels in HD11 cells across treatment groups. (H) Fluorescence microscopy images of calcium-sensitive dye Fluo-4-AM in each group.

Journal: Poultry Science

Article Title: Mechanism of Scutellaria baicalensis extracellular vesicles in attenuating Mycoplasma gallisepticum -induced inflammation via TRPC1 - STIM1/ORAI1 channel inhibition

doi: 10.1016/j.psj.2026.106773

Figure Lengend Snippet: Effects of STIM1 expression inhibition on MG-induced inflammatory damage, endoplasmic reticulum stress, calcium channel-associated proteins, and calcium ion accumulation in HD-11 cells. (A) Fluorescence microscopy images of HD-11 cells transfected with FAM-labelled siRNA. (B) Knockdown efficiency of three siRNAs at the mRNA level. (C) Knockdown efficiency of three siRNAs at the protein level. (D) Calcium channel-associated protein expression levels markedly decreased following siRNA transfection in HD-11 cells. (E) Validation results via quantitative real-time PCR (qRT-PCR). (F) Western blot analysis. (G) Reactive oxygen species (ROS) levels in HD11 cells across treatment groups. (H) Fluorescence microscopy images of calcium-sensitive dye Fluo-4-AM in each group.

Article Snippet: The antibodies used in this study were as follows: TRPC1 (HuaBio, China), NLRP3 (Wanleibio, China), STIM1 (Wanleibio, China), NF-κB (Abmart, China), ORAI1 (Abclonal, China), CHOP (Bioss, China), and GAPDH (Abmart, China).

Techniques: Expressing, Inhibition, Fluorescence, Microscopy, Transfection, Knockdown, Biomarker Discovery, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Effects of STIM1 overexpression on MG-induced inflammatory damage, endoplasmic reticulum stress, calcium channel-associated proteins, and calcium ion accumulation in HD-11 cells. (A) Fluorescence microscopy images of HD-11 cells transfected with an EGFP-tagged overexpression plasmid. (B) Transfection of HD-11 cells with STIM1 overexpression plasmid resulted in markedly elevated STIM1 protein expression levels. (C) Western blot analysis. (D) Quantitative real-time PCR (qRT-PCR) validation results. (E) Reactive oxygen species (ROS) levels in HD-11 cells across groups. (F) Fluorescence microscopy images of calcium-sensitive dye Fluo-4-AM in cells across groups.

Journal: Poultry Science

Article Title: Mechanism of Scutellaria baicalensis extracellular vesicles in attenuating Mycoplasma gallisepticum -induced inflammation via TRPC1 - STIM1/ORAI1 channel inhibition

doi: 10.1016/j.psj.2026.106773

Figure Lengend Snippet: Effects of STIM1 overexpression on MG-induced inflammatory damage, endoplasmic reticulum stress, calcium channel-associated proteins, and calcium ion accumulation in HD-11 cells. (A) Fluorescence microscopy images of HD-11 cells transfected with an EGFP-tagged overexpression plasmid. (B) Transfection of HD-11 cells with STIM1 overexpression plasmid resulted in markedly elevated STIM1 protein expression levels. (C) Western blot analysis. (D) Quantitative real-time PCR (qRT-PCR) validation results. (E) Reactive oxygen species (ROS) levels in HD-11 cells across groups. (F) Fluorescence microscopy images of calcium-sensitive dye Fluo-4-AM in cells across groups.

Techniques: Over Expression, Fluorescence, Microscopy, Transfection, Plasmid Preparation, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery

(A) Western blot and cumulative quantification data analysis of STIM1, Foxp3, and β-actin expression in lung tissue of normal and IPF Patients. The ratio of STIM1 to Foxp3 expression indicates enhanced STIM1 expression in Treg cells from patients with IPF compared to normal lung tissue (n = 6). (B) Immunofluorescence staining Image of STIM1(green) and Foxp3 (red) in Human Normal (Without IPF) and IPF (With IPF) Lungs, (C) Western blot and cumulative data analysis of STIM1, Foxp3, and β-actin expression in isolated Treg cells from WT (Wild Type, C57BL/6J) mice at 14 days post-phosphate-buffered saline (PBS) or post-bleomycin exposure ( n = 5 ). Data are shown as mean ± SEM, Student two-tailed unpaired t -test applied for (A – C) , ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for Patients Without IPF vs. with IPF, Mouse WT + PBS vs. WT + Bleomycin.

Journal: Redox Biology

Article Title: Deletion of STIM1 in Treg cells protects against lung fibrosis and associated cardiovascular complications in a pre-clinical mouse model

doi: 10.1016/j.redox.2026.104117

Figure Lengend Snippet: (A) Western blot and cumulative quantification data analysis of STIM1, Foxp3, and β-actin expression in lung tissue of normal and IPF Patients. The ratio of STIM1 to Foxp3 expression indicates enhanced STIM1 expression in Treg cells from patients with IPF compared to normal lung tissue (n = 6). (B) Immunofluorescence staining Image of STIM1(green) and Foxp3 (red) in Human Normal (Without IPF) and IPF (With IPF) Lungs, (C) Western blot and cumulative data analysis of STIM1, Foxp3, and β-actin expression in isolated Treg cells from WT (Wild Type, C57BL/6J) mice at 14 days post-phosphate-buffered saline (PBS) or post-bleomycin exposure ( n = 5 ). Data are shown as mean ± SEM, Student two-tailed unpaired t -test applied for (A – C) , ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for Patients Without IPF vs. with IPF, Mouse WT + PBS vs. WT + Bleomycin.

Article Snippet: We bred STIM1 flox/flox mice (Strain #:023350, B6.Cg-STIM1tm1Rao/J, Jackson Laboratory, USA.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Isolation, Saline, Two Tailed Test

(A) Western blot analysis and cumulative data for STIM1, Foxp3, and β-actin on isolated Treg cells from Stim1 flox/flox and Treg Stim1−/- mice, confirming the Treg cells-specific deletion of Stim1 (n = 3). (B) Western blot analysis and cumulative data for STIM1 and β-actin on isolated alveolar epithelial cells from Stim1 flox/flox and Treg Stim1−/- mice. (C) Flow cytometry analysis to show the frequency of CD4 + CD25+FoxP3+ Treg cells in the lung single cell suspension from Stim1 flox/flox and Treg Stim1−/- mice on day 14 following bleomycin administration (n = 3-4). (D) Western blot analysis for cleaved caspase 3 (CC3) in isolated Treg cells from Stim1 flox/flox and Treg Stim1−/- with and without bleomycin (n = 3). Data are shown as mean ± SEM, Student two-tailed unpaired t -test applied for (A – B) , and One-way ANOVA followed by Tukey's multiple comparisons post hoc test for (C – D) . ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for ( A-B) Stim1 flox/flox vs. Treg Stim1−/- , (C – D) Stim1 flox/flox + PBS vs. Treg Stim1−/- + PBS vs. Stim1 flox/flox + bleomycin vs. Treg Stim1−/- + bleomycin.

Journal: Redox Biology

Article Title: Deletion of STIM1 in Treg cells protects against lung fibrosis and associated cardiovascular complications in a pre-clinical mouse model

doi: 10.1016/j.redox.2026.104117

Figure Lengend Snippet: (A) Western blot analysis and cumulative data for STIM1, Foxp3, and β-actin on isolated Treg cells from Stim1 flox/flox and Treg Stim1−/- mice, confirming the Treg cells-specific deletion of Stim1 (n = 3). (B) Western blot analysis and cumulative data for STIM1 and β-actin on isolated alveolar epithelial cells from Stim1 flox/flox and Treg Stim1−/- mice. (C) Flow cytometry analysis to show the frequency of CD4 + CD25+FoxP3+ Treg cells in the lung single cell suspension from Stim1 flox/flox and Treg Stim1−/- mice on day 14 following bleomycin administration (n = 3-4). (D) Western blot analysis for cleaved caspase 3 (CC3) in isolated Treg cells from Stim1 flox/flox and Treg Stim1−/- with and without bleomycin (n = 3). Data are shown as mean ± SEM, Student two-tailed unpaired t -test applied for (A – B) , and One-way ANOVA followed by Tukey's multiple comparisons post hoc test for (C – D) . ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for ( A-B) Stim1 flox/flox vs. Treg Stim1−/- , (C – D) Stim1 flox/flox + PBS vs. Treg Stim1−/- + PBS vs. Stim1 flox/flox + bleomycin vs. Treg Stim1−/- + bleomycin.

Article Snippet: We bred STIM1 flox/flox mice (Strain #:023350, B6.Cg-STIM1tm1Rao/J, Jackson Laboratory, USA.

Techniques: Western Blot, Isolation, Flow Cytometry, Single Cell, Suspension, Two Tailed Test

Stim1 flox/flox and Treg Stim1−/- mice received a single dose of bleomycin via the intratracheal route. The lung and cardiac fibrosis were assessed after 14 days. (A) A representative section of the mouse lung stained with Masson's trichrome staining, (B) corresponding graphs showing Ashcroft fibrosis score, (C) hydroxyproline assay results show lung collagen content, (D) ELISA detection of IL-10, (E) IL-1β, and (F) TGFβ1 levels in the BALF (n = 3-6), (G) A section of mouse heart stained with PSR (Picrosirius red), along with quantitative analysis of fibrotic area (H) , illustrates the protective effect of STIM1 deletion in Treg cells against the bleomycin-induced lung and cardiac fibrosis (n = 3-5). Data are shown as mean ± SEM, One-way ANOVA followed by Tukey's multiple comparisons post hoc test for (A – H) . ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for Stim1 flox/flox + PBS vs. Treg Stim1−/- + PBS vs. Stim1 flox/flox + bleomycin vs. Treg Stim1−/- + bleomycin.

Journal: Redox Biology

Article Title: Deletion of STIM1 in Treg cells protects against lung fibrosis and associated cardiovascular complications in a pre-clinical mouse model

doi: 10.1016/j.redox.2026.104117

Figure Lengend Snippet: Stim1 flox/flox and Treg Stim1−/- mice received a single dose of bleomycin via the intratracheal route. The lung and cardiac fibrosis were assessed after 14 days. (A) A representative section of the mouse lung stained with Masson's trichrome staining, (B) corresponding graphs showing Ashcroft fibrosis score, (C) hydroxyproline assay results show lung collagen content, (D) ELISA detection of IL-10, (E) IL-1β, and (F) TGFβ1 levels in the BALF (n = 3-6), (G) A section of mouse heart stained with PSR (Picrosirius red), along with quantitative analysis of fibrotic area (H) , illustrates the protective effect of STIM1 deletion in Treg cells against the bleomycin-induced lung and cardiac fibrosis (n = 3-5). Data are shown as mean ± SEM, One-way ANOVA followed by Tukey's multiple comparisons post hoc test for (A – H) . ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for Stim1 flox/flox + PBS vs. Treg Stim1−/- + PBS vs. Stim1 flox/flox + bleomycin vs. Treg Stim1−/- + bleomycin.

Article Snippet: We bred STIM1 flox/flox mice (Strain #:023350, B6.Cg-STIM1tm1Rao/J, Jackson Laboratory, USA.

Techniques: Staining, Hydroxyproline Assay, Enzyme-linked Immunosorbent Assay

After bleomycin exposure, lung injury was assessed on day 14. ( A) A representative H&E staining of mouse lung sections, (B) injury score measurement, (C) Lung function parameters of tissue dampening (G), (D) tissue elastance (H), and (E) static pulmonary compliance (Cst) in control (PBS) and bleomycin-treated Stim1 flox/flox and Treg Stim1−/- mice were examined using the FlexiVent system (n = 5). Data are shown as mean ± SEM, One-way ANOVA followed by Tukey's multiple comparisons post hoc test for (A – E) . ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for Stim1 flox/flox + PBS vs. Treg Stim1−/- + PBS vs. Stim1 flox/flox + bleomycin vs. Treg Stim1−/- + bleomycin.

Journal: Redox Biology

Article Title: Deletion of STIM1 in Treg cells protects against lung fibrosis and associated cardiovascular complications in a pre-clinical mouse model

doi: 10.1016/j.redox.2026.104117

Figure Lengend Snippet: After bleomycin exposure, lung injury was assessed on day 14. ( A) A representative H&E staining of mouse lung sections, (B) injury score measurement, (C) Lung function parameters of tissue dampening (G), (D) tissue elastance (H), and (E) static pulmonary compliance (Cst) in control (PBS) and bleomycin-treated Stim1 flox/flox and Treg Stim1−/- mice were examined using the FlexiVent system (n = 5). Data are shown as mean ± SEM, One-way ANOVA followed by Tukey's multiple comparisons post hoc test for (A – E) . ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for Stim1 flox/flox + PBS vs. Treg Stim1−/- + PBS vs. Stim1 flox/flox + bleomycin vs. Treg Stim1−/- + bleomycin.

Article Snippet: We bred STIM1 flox/flox mice (Strain #:023350, B6.Cg-STIM1tm1Rao/J, Jackson Laboratory, USA.

Techniques: Staining, Control

(A) Lung tissues from all groups of mice were immediately placed in cold PSS solution, carefully cleared of connective tissue, and cut into rings (2 mm in length). The lung arteries were mounted in small vessels within a dual-chamber wire myograph to measure isometric tension. After a 30-min equilibration period in PSS solution bubbled with CO 2 at 37 °C (pH 7.4), the arteries were stretched to their optimal lumen diameter for active tension development. After 1 h of incubation, artery rings were pre-constricted with phenylephrine (PE, 10 −8 – 10 −4 M), and when a steady maximal contraction was achieved, cumulative dose-response curves were obtained for acetylcholine (ACh, 10 −8 – 10 −4 M). Pulmonary (lung) artery reactivity, showing contractility in response to sympathetic stimulation (Phenylephrine, PE) and endothelium-dependent and independent relaxation in response to acetylcholine (ACh) in Stim1 flox/flox and Treg Stim1/- with and without bleomycin treated mice (n = 5) and (B) Western blot and cumulative data for phosphorylated nitric-oxide synthase (P-eNOS) in lung tissue from Stim1 flox/flox and Treg Stim1−/- with and without bleomycin treated mice ( n = 5 ). Data are shown as mean ± SEM, One-way ANOVA followed by Tukey's multiple comparisons post hoc test for (A – B) . ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for Stim1 flox/flox + PBS vs. Treg Stim1−/- + PBS vs. Stim1 flox/flox + bleomycin vs. Treg Stim1−/- + bleomycin.

Journal: Redox Biology

Article Title: Deletion of STIM1 in Treg cells protects against lung fibrosis and associated cardiovascular complications in a pre-clinical mouse model

doi: 10.1016/j.redox.2026.104117

Figure Lengend Snippet: (A) Lung tissues from all groups of mice were immediately placed in cold PSS solution, carefully cleared of connective tissue, and cut into rings (2 mm in length). The lung arteries were mounted in small vessels within a dual-chamber wire myograph to measure isometric tension. After a 30-min equilibration period in PSS solution bubbled with CO 2 at 37 °C (pH 7.4), the arteries were stretched to their optimal lumen diameter for active tension development. After 1 h of incubation, artery rings were pre-constricted with phenylephrine (PE, 10 −8 – 10 −4 M), and when a steady maximal contraction was achieved, cumulative dose-response curves were obtained for acetylcholine (ACh, 10 −8 – 10 −4 M). Pulmonary (lung) artery reactivity, showing contractility in response to sympathetic stimulation (Phenylephrine, PE) and endothelium-dependent and independent relaxation in response to acetylcholine (ACh) in Stim1 flox/flox and Treg Stim1/- with and without bleomycin treated mice (n = 5) and (B) Western blot and cumulative data for phosphorylated nitric-oxide synthase (P-eNOS) in lung tissue from Stim1 flox/flox and Treg Stim1−/- with and without bleomycin treated mice ( n = 5 ). Data are shown as mean ± SEM, One-way ANOVA followed by Tukey's multiple comparisons post hoc test for (A – B) . ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for Stim1 flox/flox + PBS vs. Treg Stim1−/- + PBS vs. Stim1 flox/flox + bleomycin vs. Treg Stim1−/- + bleomycin.

Article Snippet: We bred STIM1 flox/flox mice (Strain #:023350, B6.Cg-STIM1tm1Rao/J, Jackson Laboratory, USA.

Techniques: Incubation, Western Blot

(A) Stim1 flox/flox and Treg Stim1−/- mice received three repeated intratracheal bleomycin instillations, administered every two weeks. The mortality was monitored. Kaplan-Meier survival analysis of mice following three repeated intratracheal bleomycin instillations (n = 6). Lung injury and fibrosis were examined two weeks after the third bleomycin instillation. (B) Histology examination of lung sections stained with H&E and (C) Masson's Trichrome staining and corresponding graphs showing (D) lung injury score and (E) Ashcroft fibrosis score in bleomycin-treated Stim1 flox/flox and Treg Stim1−/- mice (n = 5). Data are shown as mean ± SEM, the Mantel-Cox log-rank test was applied for (A) . Student two-tailed unpaired t -test applied for (B – E). ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for Stim1 flox/flox + PBS vs. Treg Stim1−/- + PBS, Stim1 flox/flox + bleomycin vs. Treg Stim1−/- + bleomycin.

Journal: Redox Biology

Article Title: Deletion of STIM1 in Treg cells protects against lung fibrosis and associated cardiovascular complications in a pre-clinical mouse model

doi: 10.1016/j.redox.2026.104117

Figure Lengend Snippet: (A) Stim1 flox/flox and Treg Stim1−/- mice received three repeated intratracheal bleomycin instillations, administered every two weeks. The mortality was monitored. Kaplan-Meier survival analysis of mice following three repeated intratracheal bleomycin instillations (n = 6). Lung injury and fibrosis were examined two weeks after the third bleomycin instillation. (B) Histology examination of lung sections stained with H&E and (C) Masson's Trichrome staining and corresponding graphs showing (D) lung injury score and (E) Ashcroft fibrosis score in bleomycin-treated Stim1 flox/flox and Treg Stim1−/- mice (n = 5). Data are shown as mean ± SEM, the Mantel-Cox log-rank test was applied for (A) . Student two-tailed unpaired t -test applied for (B – E). ns: not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 for Stim1 flox/flox + PBS vs. Treg Stim1−/- + PBS, Stim1 flox/flox + bleomycin vs. Treg Stim1−/- + bleomycin.

Article Snippet: We bred STIM1 flox/flox mice (Strain #:023350, B6.Cg-STIM1tm1Rao/J, Jackson Laboratory, USA.

Techniques: Staining, Two Tailed Test

(A) qPCR analysis of relative mRNA levels of indicated genes in BAT, epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT). n= 2 mice per group (B) Left: Immunoblotting analysis of indicated proteins in total lysates from BAT, eWAT and iWAT from wild-type mice housed at RT. Right: Quantification analysis of the images shown in the left. n= 4 mice per group; One-way ANOVA, p< *0.05, *** p< <0.001. (C) qPCR analysis of relative mRNA levels of indicated genes in BAT, eWAT and iWAT. n= 4 per group; One-way ANOVA **p<0.005. (D, E) qPCR analysis of mRNA levels of indicated genes in iWAT (D) and eWAT (E) from mice exposed to 30°C, 22°C, and 4°C for 24hrs. In D, n= 3 (30°C); n=4 (22°C); n=3 (4°C); One-way ANOVA, * p<0.03. In E n= 4 (30°C); n= 4 (22°C); n=3 (4°C). (F) Left: Immunoblotting analysis of indicated protein in total lysates from BAT of mice housed at 30°C (thermoneutrality), 22°C (RT) or exposed to 4°C for 24 hrs. Right: Quantification analysis of images shown in the left. n= 3 per group (G) Upper panel: Representative bright field images of differentiated brown adipocytes (WT1 cells) control (scramble siRNA) or treated with STIM1/2 siRNA. Bottom panel: qPCR analysis of mRNA levels of indicated genes. n=3 per group. Unpaired Student’s t-test ***p<0.001. (H) Representative bright field images of differentiated primary brown adipocytes (after 7 days differentiation) from STIM1/2 flox and STIM1/2 AdpKO . (I, J) Immunostaining of STIM1 (green) in differentiated brown adipocytes in baseline and in cells treated with 1μM thapsigargin for 30 min in Ca 2+ free media (I) or 1μM or 10 μM Norepinephrine for 30 min in Ca 2+ free media (J). Nucleus are stained in blue.

Journal: bioRxiv

Article Title: Ca 2+ influx through ER-plasma membrane contacts is required for brown fat thermogenesis and metabolic health

doi: 10.64898/2026.03.20.712802

Figure Lengend Snippet: (A) qPCR analysis of relative mRNA levels of indicated genes in BAT, epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT). n= 2 mice per group (B) Left: Immunoblotting analysis of indicated proteins in total lysates from BAT, eWAT and iWAT from wild-type mice housed at RT. Right: Quantification analysis of the images shown in the left. n= 4 mice per group; One-way ANOVA, p< *0.05, *** p< <0.001. (C) qPCR analysis of relative mRNA levels of indicated genes in BAT, eWAT and iWAT. n= 4 per group; One-way ANOVA **p<0.005. (D, E) qPCR analysis of mRNA levels of indicated genes in iWAT (D) and eWAT (E) from mice exposed to 30°C, 22°C, and 4°C for 24hrs. In D, n= 3 (30°C); n=4 (22°C); n=3 (4°C); One-way ANOVA, * p<0.03. In E n= 4 (30°C); n= 4 (22°C); n=3 (4°C). (F) Left: Immunoblotting analysis of indicated protein in total lysates from BAT of mice housed at 30°C (thermoneutrality), 22°C (RT) or exposed to 4°C for 24 hrs. Right: Quantification analysis of images shown in the left. n= 3 per group (G) Upper panel: Representative bright field images of differentiated brown adipocytes (WT1 cells) control (scramble siRNA) or treated with STIM1/2 siRNA. Bottom panel: qPCR analysis of mRNA levels of indicated genes. n=3 per group. Unpaired Student’s t-test ***p<0.001. (H) Representative bright field images of differentiated primary brown adipocytes (after 7 days differentiation) from STIM1/2 flox and STIM1/2 AdpKO . (I, J) Immunostaining of STIM1 (green) in differentiated brown adipocytes in baseline and in cells treated with 1μM thapsigargin for 30 min in Ca 2+ free media (I) or 1μM or 10 μM Norepinephrine for 30 min in Ca 2+ free media (J). Nucleus are stained in blue.

Article Snippet: Antibodies used were STIM1 (Cell Signaling, 4916), STIM2 (Cell Signaling, 4917), α-Tubulin (Proteintech, 66031-1-Ig), β-Tubulin (Cell Signaling, 2146S), GAPDH (Cell Signaling, 5174S), UCP1 (Cell Signaling, 14670); Junctophilin (JH2) (Proteintech, 51054-2-AP) ; pHSL (Cell Signaling, 4139) ; HSL (Cell Signaling, 4107) ; Oxphos cocktail (Abcam, ab110413); pDRP1 ser616 (Cell Signaling, 4494); DRP1 (Cell Signaling, 8570), anti-rabbit IgG (Cell Signaling, 7074S), and anti-mouse IgG (Cell Signaling, 7076S).

Techniques: Western Blot, Control, Immunostaining, Staining

(A) Left: Immunoblotting analysis of indicated proteins in total lysates from BAT of mice maintained at 30°C (thermoneutrality), 22°C (RT) or exposed to 4°C (cold) for 24hrs. Right: Quantification analysis of images shown in A; n= 3 mice per group. One-way ANOVA with Tukey’s multiple comparisons test, *p<0.05, *** p<0.001. (B) mRNA levels of indicated genes in BAT from mice exposed to 30°C, 22°C, n= 4 and 4°C for 24hrs, n=3. One-way ANOVA with Tukey’s multiple comparisons test, * p<0.05, ** p<0.01, *** p<0.005. (C) Upper panel: Immunostaining of STIM1 (green) in differentiated brown adipocytes in baseline and in cells treated with 1μM thapsigargin (Tg) for 10 min in Ca 2+ free media; lipid droplets (LD) are stained in blue. Bottom: Quantification of the ratio of fluorescent intensity in the plasma membrane per intensity in the adjacent cytosolic area. n= 16 cells baseline, 10 cells Tg. Representative of 2 independent experiments; unpaired Student t-test ****p< <0.0001. (D) Left: Fluo-4 based cytosolic Ca 2+ measurements in differentiated primary brown adipocytes from STIM1/2 flox (control) and STIM1/2 AdpKO mice. Cells were first treated with 1μM Tg in Ca 2+ free media. Subsequently, 2mM Ca 2+ was added in the indicated time point. The graph shows a representative run of 2 independent experiments. n=15 cells for STIM1/2 flox and n=13 cells for STIM1/2 AdpKO . Right: Quantification of the cytosolic peak after Tg and Ca 2+ addition. Unpaired Student t-test *p<0.05. (E) Upper panel: Immunostaining of STIM1 (green) in differentiated brown adipocytes in baseline and in cells treated with 10μM norepinephrine (NE) for 30 min in Ca 2+ free media. Lipid droplets are stained in blue. Bottom: Quantification of the ratio of fluorescent intensity in the plasma membrane per intensity in the adjacent cytosolic area. n= 11 cells baseline, 8 cells NE. Representative of 3 independent experiments; Unpaired Student t-test ****p<0.0001. (F) Fluo-4-based cytosolic Ca 2+ measurements in differentiated primary brown adipocytes from STIM1/2 flox (control) and STIM1/2 AdpKO mice. Cells were first treated with 10μM NE in Ca 2+ free media. Subsequently, 2mM Ca 2+ was added in the indicated time point. n= 10 cells per group; Representative of 3 independent experiments. (G) Left: Fluo-4-based cytosolic Ca 2+ measurements in differentiated primary brown adipocytes from STIM1/2 flox and STIM1/2 AdpKO mice. Cells were treated with 10μM NE in the presence of 2mM Ca 2+ in the media. The graph shows a representative run of 2 independent experiments. n=23 for STIM1/2 flox , n=19 for STIM1/2 AdpKO . Right: Area under the curve of the graph shown on the left. Unpaired Student t- test *** p<0.001. (H) Rhod-2-based mitochondria Ca 2+ measurements in differentiated brown adipocytes transfected with scramble siRNA or with siRNA against STIM1/2. Cells were treated with 10μM NE in the presence of Ca 2+ in the media. Representative of 2 independent experiments.

Journal: bioRxiv

Article Title: Ca 2+ influx through ER-plasma membrane contacts is required for brown fat thermogenesis and metabolic health

doi: 10.64898/2026.03.20.712802

Figure Lengend Snippet: (A) Left: Immunoblotting analysis of indicated proteins in total lysates from BAT of mice maintained at 30°C (thermoneutrality), 22°C (RT) or exposed to 4°C (cold) for 24hrs. Right: Quantification analysis of images shown in A; n= 3 mice per group. One-way ANOVA with Tukey’s multiple comparisons test, *p<0.05, *** p<0.001. (B) mRNA levels of indicated genes in BAT from mice exposed to 30°C, 22°C, n= 4 and 4°C for 24hrs, n=3. One-way ANOVA with Tukey’s multiple comparisons test, * p<0.05, ** p<0.01, *** p<0.005. (C) Upper panel: Immunostaining of STIM1 (green) in differentiated brown adipocytes in baseline and in cells treated with 1μM thapsigargin (Tg) for 10 min in Ca 2+ free media; lipid droplets (LD) are stained in blue. Bottom: Quantification of the ratio of fluorescent intensity in the plasma membrane per intensity in the adjacent cytosolic area. n= 16 cells baseline, 10 cells Tg. Representative of 2 independent experiments; unpaired Student t-test ****p< <0.0001. (D) Left: Fluo-4 based cytosolic Ca 2+ measurements in differentiated primary brown adipocytes from STIM1/2 flox (control) and STIM1/2 AdpKO mice. Cells were first treated with 1μM Tg in Ca 2+ free media. Subsequently, 2mM Ca 2+ was added in the indicated time point. The graph shows a representative run of 2 independent experiments. n=15 cells for STIM1/2 flox and n=13 cells for STIM1/2 AdpKO . Right: Quantification of the cytosolic peak after Tg and Ca 2+ addition. Unpaired Student t-test *p<0.05. (E) Upper panel: Immunostaining of STIM1 (green) in differentiated brown adipocytes in baseline and in cells treated with 10μM norepinephrine (NE) for 30 min in Ca 2+ free media. Lipid droplets are stained in blue. Bottom: Quantification of the ratio of fluorescent intensity in the plasma membrane per intensity in the adjacent cytosolic area. n= 11 cells baseline, 8 cells NE. Representative of 3 independent experiments; Unpaired Student t-test ****p<0.0001. (F) Fluo-4-based cytosolic Ca 2+ measurements in differentiated primary brown adipocytes from STIM1/2 flox (control) and STIM1/2 AdpKO mice. Cells were first treated with 10μM NE in Ca 2+ free media. Subsequently, 2mM Ca 2+ was added in the indicated time point. n= 10 cells per group; Representative of 3 independent experiments. (G) Left: Fluo-4-based cytosolic Ca 2+ measurements in differentiated primary brown adipocytes from STIM1/2 flox and STIM1/2 AdpKO mice. Cells were treated with 10μM NE in the presence of 2mM Ca 2+ in the media. The graph shows a representative run of 2 independent experiments. n=23 for STIM1/2 flox , n=19 for STIM1/2 AdpKO . Right: Area under the curve of the graph shown on the left. Unpaired Student t- test *** p<0.001. (H) Rhod-2-based mitochondria Ca 2+ measurements in differentiated brown adipocytes transfected with scramble siRNA or with siRNA against STIM1/2. Cells were treated with 10μM NE in the presence of Ca 2+ in the media. Representative of 2 independent experiments.

Techniques: Western Blot, Immunostaining, Staining, Clinical Proteomics, Membrane, Control, Transfection

(A) Schematic depicting the genetic strategy used to generate mice with adipose tissue specific deletion of STIM1/2 (STIM1/2 AdpKO ). AdpCre- adiponectin Cre. (B) Upper panel: Immunoblotting analysis of indicated proteins in total lysates from iWAT of STIM1/2 flox and STIM1/2 AdpCre mice. Bottom panel: Quantification analysis of images shown in upper panel; n= 5 mice per group. Unpaired Student’s t-test, **p< 0.01. (C) Upper panel: Immunoblotting analysis of indicated proteins in total lysates from eWAT of STIM1/2 flox and STIM1/2 AdpKO mice. Bottom panel: Quantification analysis of images shown in upper panel; n= 5 mice per group. Unpaired Student’s t-test, * p<0.05, **p<0.002. (D) Body weight gain curves of STIM1/2 flox and STIM1/2 AdpKO mice fed a chow diet. n = 6 mice per group. (E) Schematic depicting the strategy used to generate mice with brown adipose tissue specific deletion of STIM1/2. (F) Immunoblotting analysis of Cre recombinase in the indicated tissues from mice injected with AAV-Rec2-UPC1-Cre-miR122. (G) Upper panel: Immunoblotting analysis of indicated proteins in in total lysates from BAT, eWAT and iWAT. Bottom panel: Quantification analysis of images shown in upper panel; n= 3 mice per group. Unpaired Student’s t-test, **p< 0.01, *p<0.05. (H) Top: Representative H&E-stained sections of BAT from STIM1/2 flox and STIM1/2 AdpKO mice housed at RT. Bottom: Quantification analysis of area of LD and frequence distribution.

Journal: bioRxiv

Article Title: Ca 2+ influx through ER-plasma membrane contacts is required for brown fat thermogenesis and metabolic health

doi: 10.64898/2026.03.20.712802

Figure Lengend Snippet: (A) Schematic depicting the genetic strategy used to generate mice with adipose tissue specific deletion of STIM1/2 (STIM1/2 AdpKO ). AdpCre- adiponectin Cre. (B) Upper panel: Immunoblotting analysis of indicated proteins in total lysates from iWAT of STIM1/2 flox and STIM1/2 AdpCre mice. Bottom panel: Quantification analysis of images shown in upper panel; n= 5 mice per group. Unpaired Student’s t-test, **p< 0.01. (C) Upper panel: Immunoblotting analysis of indicated proteins in total lysates from eWAT of STIM1/2 flox and STIM1/2 AdpKO mice. Bottom panel: Quantification analysis of images shown in upper panel; n= 5 mice per group. Unpaired Student’s t-test, * p<0.05, **p<0.002. (D) Body weight gain curves of STIM1/2 flox and STIM1/2 AdpKO mice fed a chow diet. n = 6 mice per group. (E) Schematic depicting the strategy used to generate mice with brown adipose tissue specific deletion of STIM1/2. (F) Immunoblotting analysis of Cre recombinase in the indicated tissues from mice injected with AAV-Rec2-UPC1-Cre-miR122. (G) Upper panel: Immunoblotting analysis of indicated proteins in in total lysates from BAT, eWAT and iWAT. Bottom panel: Quantification analysis of images shown in upper panel; n= 3 mice per group. Unpaired Student’s t-test, **p< 0.01, *p<0.05. (H) Top: Representative H&E-stained sections of BAT from STIM1/2 flox and STIM1/2 AdpKO mice housed at RT. Bottom: Quantification analysis of area of LD and frequence distribution.

Techniques: Western Blot, Injection, Staining

(A) Left: Immunoblotting analysis of indicated proteins in total lysates of BAT from STIM1/2 flox (controls) and STIM1/2 AdpKO . Right: Quantification of the images shown in the left. n=7 mice per group. Unpaired Student t-test, ****p<0.0001, *** p<0.005. (B, C) Body temperature over time of male (B) and female (C) mice exposed to 4°C in the absence of food. n =12 male mice per group and n =5 female mice per group. Two-way ANOVA with Šídák’s multiple comparisons test ***p<0.001,****p<0.0001. (D) Left: thermographic images from FLIR E5 infrared camera of STIM1/2 flox and STIM1/2 AdpKO mice exposed to 4°C for 4-6 hrs. Right: Quantification of images shown in the left. n= 6 mice per group. Unpaired Student t-test, *p<0.05. (E) Left: Norepinephrine-stimulated O2 consumption in anesthetized STIM1/2 flox and STIM1/2 AdpKO female mice. Right: Area under the curve of graph in the left. n= 6 mice STIM1/2 flox and n=5 mice STIM1/2 AdpKO . Unpaired Student’s t-test ** p<0.001. (F) Body temperature over time of male STIM1/2 flox mice expressing AAV-Rec2-UCP1-Null AAV-Rec2-UCP1-Cre recombinase exposed to 4°C in the absence of food. n=4 Null mice and n=3 Cre mice per group. Two-way ANOVA with Šídák’s multiple comparisons test * p<0.05, ** p< 0.01, *** p=.0001. (G) Left: Representative Hematoxylin and eosin (H&E) stained sections of BAT from STIM1/2 flox and STIM1/2 AdpKO mice exposed to cold for 6hrs. Right: Quantification analysis of lipid droplet (LD) number and size of images shown in the left. n=5 STIM1/2 flox , n=7 for STIM1/2 AdpKO . Unpaired Student t-test * p<0.05. (H) Left: Immunoblotting analysis of indicated proteins in total lysates of BAT from STIM1/2 flox and STIM1/2 AdpKO maintained at RT or exposed to cold (4°C) for 2hrs. Right: Quantification of the images shown in the left. n=3 per group. One-way ANOVA with Tukey’s multiple comparisons test. * p<0.05, ** p< 0.005.

Journal: bioRxiv

Article Title: Ca 2+ influx through ER-plasma membrane contacts is required for brown fat thermogenesis and metabolic health

doi: 10.64898/2026.03.20.712802

Figure Lengend Snippet: (A) Left: Immunoblotting analysis of indicated proteins in total lysates of BAT from STIM1/2 flox (controls) and STIM1/2 AdpKO . Right: Quantification of the images shown in the left. n=7 mice per group. Unpaired Student t-test, ****p<0.0001, *** p<0.005. (B, C) Body temperature over time of male (B) and female (C) mice exposed to 4°C in the absence of food. n =12 male mice per group and n =5 female mice per group. Two-way ANOVA with Šídák’s multiple comparisons test ***p<0.001,****p<0.0001. (D) Left: thermographic images from FLIR E5 infrared camera of STIM1/2 flox and STIM1/2 AdpKO mice exposed to 4°C for 4-6 hrs. Right: Quantification of images shown in the left. n= 6 mice per group. Unpaired Student t-test, *p<0.05. (E) Left: Norepinephrine-stimulated O2 consumption in anesthetized STIM1/2 flox and STIM1/2 AdpKO female mice. Right: Area under the curve of graph in the left. n= 6 mice STIM1/2 flox and n=5 mice STIM1/2 AdpKO . Unpaired Student’s t-test ** p<0.001. (F) Body temperature over time of male STIM1/2 flox mice expressing AAV-Rec2-UCP1-Null AAV-Rec2-UCP1-Cre recombinase exposed to 4°C in the absence of food. n=4 Null mice and n=3 Cre mice per group. Two-way ANOVA with Šídák’s multiple comparisons test * p<0.05, ** p< 0.01, *** p=.0001. (G) Left: Representative Hematoxylin and eosin (H&E) stained sections of BAT from STIM1/2 flox and STIM1/2 AdpKO mice exposed to cold for 6hrs. Right: Quantification analysis of lipid droplet (LD) number and size of images shown in the left. n=5 STIM1/2 flox , n=7 for STIM1/2 AdpKO . Unpaired Student t-test * p<0.05. (H) Left: Immunoblotting analysis of indicated proteins in total lysates of BAT from STIM1/2 flox and STIM1/2 AdpKO maintained at RT or exposed to cold (4°C) for 2hrs. Right: Quantification of the images shown in the left. n=3 per group. One-way ANOVA with Tukey’s multiple comparisons test. * p<0.05, ** p< 0.005.

Techniques: Western Blot, Expressing, Staining

(A) Heat map of RNA-seq analysis of mRNA expression of BAT from STIM1/2 flox and STIM1/2 AdpKO at RT or exposed to cold (4°C) for 6hrs; n= 2 mice for STIM1/2 flox RT; n=3 mice for STIM1/2 AdpKO RT; n= 4 mice for STIM1/2 flox and STIM1/2 AdpKO cold (B) Volcano plots showing differentially expressed genes comparing STIM1/2 flox and STIM1/2 AdpKO exposed to cold. Red dots show the significant regulated genes (p<0.05), Unpaired Student’s t-test. (C, E) Ingenuity pathway analysis showing pathways predicted to be decreased (C) or increased (E) in STIM1/2 AdpKO compared to STIM1/2 flox exposed to cold (4°C). p-value was calculated using Benjamin-Hochberg analysis. (D, F) qPCR-based analysis of mRNA expression of indicated genes in BAT from STIM1/2 flox and STIM1/2 AdpKO at RT or exposed to 4°C for 6h; n= 2 mice STIM1/2 flox RT; n = 4 mice STIM1/2 AdpKO RT; n = 5 mice STIM1/2 flox cold; n= 8 mice STIM1/2 AdpKO cold. Unpaired Student’s t-test *p<0.05, **p<0.01, ***p<0.0005. (G) Relative activation of ATF6, XBP1s and PERK gene sets from RNA seq analysis shown in A. These analyses were performed as previously described . Differential activation of the ATF6, XBP1s, and PERK gene-sets was assessed by one-way ANOVA and significance of pairwise comparison confirmed by unpaired t-test. (H, I) SEM images of BAT tissue from STIM1/2 flox (H) and STIM1/2 AdpKO (I) mice exposed to cold (4°C) for 12hrs. White boxes highlight the ER/ aggregated membranes. (J) Segmentation and 3D reconstruction of FIB-SEM images of BAT from STIM1/2 flox and STIM1/2 AdpKO exposed to cold for 12hrs.

Journal: bioRxiv

Article Title: Ca 2+ influx through ER-plasma membrane contacts is required for brown fat thermogenesis and metabolic health

doi: 10.64898/2026.03.20.712802

Figure Lengend Snippet: (A) Heat map of RNA-seq analysis of mRNA expression of BAT from STIM1/2 flox and STIM1/2 AdpKO at RT or exposed to cold (4°C) for 6hrs; n= 2 mice for STIM1/2 flox RT; n=3 mice for STIM1/2 AdpKO RT; n= 4 mice for STIM1/2 flox and STIM1/2 AdpKO cold (B) Volcano plots showing differentially expressed genes comparing STIM1/2 flox and STIM1/2 AdpKO exposed to cold. Red dots show the significant regulated genes (p<0.05), Unpaired Student’s t-test. (C, E) Ingenuity pathway analysis showing pathways predicted to be decreased (C) or increased (E) in STIM1/2 AdpKO compared to STIM1/2 flox exposed to cold (4°C). p-value was calculated using Benjamin-Hochberg analysis. (D, F) qPCR-based analysis of mRNA expression of indicated genes in BAT from STIM1/2 flox and STIM1/2 AdpKO at RT or exposed to 4°C for 6h; n= 2 mice STIM1/2 flox RT; n = 4 mice STIM1/2 AdpKO RT; n = 5 mice STIM1/2 flox cold; n= 8 mice STIM1/2 AdpKO cold. Unpaired Student’s t-test *p<0.05, **p<0.01, ***p<0.0005. (G) Relative activation of ATF6, XBP1s and PERK gene sets from RNA seq analysis shown in A. These analyses were performed as previously described . Differential activation of the ATF6, XBP1s, and PERK gene-sets was assessed by one-way ANOVA and significance of pairwise comparison confirmed by unpaired t-test. (H, I) SEM images of BAT tissue from STIM1/2 flox (H) and STIM1/2 AdpKO (I) mice exposed to cold (4°C) for 12hrs. White boxes highlight the ER/ aggregated membranes. (J) Segmentation and 3D reconstruction of FIB-SEM images of BAT from STIM1/2 flox and STIM1/2 AdpKO exposed to cold for 12hrs.

Techniques: RNA Sequencing, Expressing, Activation Assay, Comparison

(A, B) Representative TEM images of BAT from STIM1/2 flox (A) and STIM1/2 AdpKO (B) mice maintained at RT. (C, D) TEM images of BAT from STIM1/2 AdpKO exposed to cold highlighting the aggregated membranes from two different mice. (E) Representative TEM image of BAT from mice treated with tunicamycin (0.5mg/kg) for 6 hrs.

Journal: bioRxiv

Article Title: Ca 2+ influx through ER-plasma membrane contacts is required for brown fat thermogenesis and metabolic health

doi: 10.64898/2026.03.20.712802

Figure Lengend Snippet: (A, B) Representative TEM images of BAT from STIM1/2 flox (A) and STIM1/2 AdpKO (B) mice maintained at RT. (C, D) TEM images of BAT from STIM1/2 AdpKO exposed to cold highlighting the aggregated membranes from two different mice. (E) Representative TEM image of BAT from mice treated with tunicamycin (0.5mg/kg) for 6 hrs.

Techniques:

(A, B) Reconstruction of individual mitochondria from FIB-SEM data of BAT from STIM1/2 flox (A) and STIM1/2 AdpKO mice (B). 3D reconstructions were performed using Zeiss Arivis Pro software. (C) Quantification of mitochondria volume from data shown in A and B. n = 886 and 445 mitochondria, respectively. Unpaired Student’s t-test ***p<0.001 (D) Quantification of mitochondria sphericity and frequence distribution from data shown in A and B. n = 624 mitochondria in STIM1/2 flox and n=337 mitochondria in STIM1/2 AdpKO . Unpaired Student’s t-test ****p<0.0001. (E) Mitochondria complexity index calculated according to . Unpaired Student’s t-test ****p<0.0001. (F) Confocal images from differentiated primary adipocytes derived from STIM1/2 flox and STIM1/2 AdpKO mice at baseline or treated with 10uM norepinephrine (NE) for 50 min. Mitochondria (green) was staining with OXPHOs antibody, LD (blue) and Nucleus (magenta). (G,H) Quantification of mitochondria aspect ratio (G) and mitochondria area (H) of images shown in F. STIM1/2 flox NT= 1689 mitochondria, NE=861 mitochondria; STIM1/2 AdpKO NT=1185 mitochondria, NE=1618 mitochondria from > 5 images. Representative of 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test **** p<0.0001. (I) Upper panel: Immunoblotting analysis of indicated proteins in total lysates of BAT from STIM1/2 flox and STIM1/2 AdpKO mice maintained at RT or exposed to cold (4°C) for 2hrs. Bottom panel: Quantification analysis of images shown in the upper panel. n=3 mice per group for RT conditions and n=4 mice per group for cold conditions. One-way ANOVA with Tukey’s multiple comparisons test. **p< 0.005. (J) Seahorse based oxygen consumption rate of mitochondria isolated from STIM1/2 flox and STIM1/2 AdpKO exposed to cold (4°C) for 12 hrs. Arrows depicts the addition of 20mM Pyruvate + 20mM Malate; 2μg/mL Oligomycin, 9μM FCCP and 4 µM rotenone and 2 µM antimycin. n=10 wells per group. Representative of 3 independent experiments. (K) Quantification of basal and maximal respiration. Unpaired Student t-test ** p<0.001, **** p<0.0001.

Journal: bioRxiv

Article Title: Ca 2+ influx through ER-plasma membrane contacts is required for brown fat thermogenesis and metabolic health

doi: 10.64898/2026.03.20.712802

Figure Lengend Snippet: (A, B) Reconstruction of individual mitochondria from FIB-SEM data of BAT from STIM1/2 flox (A) and STIM1/2 AdpKO mice (B). 3D reconstructions were performed using Zeiss Arivis Pro software. (C) Quantification of mitochondria volume from data shown in A and B. n = 886 and 445 mitochondria, respectively. Unpaired Student’s t-test ***p<0.001 (D) Quantification of mitochondria sphericity and frequence distribution from data shown in A and B. n = 624 mitochondria in STIM1/2 flox and n=337 mitochondria in STIM1/2 AdpKO . Unpaired Student’s t-test ****p<0.0001. (E) Mitochondria complexity index calculated according to . Unpaired Student’s t-test ****p<0.0001. (F) Confocal images from differentiated primary adipocytes derived from STIM1/2 flox and STIM1/2 AdpKO mice at baseline or treated with 10uM norepinephrine (NE) for 50 min. Mitochondria (green) was staining with OXPHOs antibody, LD (blue) and Nucleus (magenta). (G,H) Quantification of mitochondria aspect ratio (G) and mitochondria area (H) of images shown in F. STIM1/2 flox NT= 1689 mitochondria, NE=861 mitochondria; STIM1/2 AdpKO NT=1185 mitochondria, NE=1618 mitochondria from > 5 images. Representative of 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test **** p<0.0001. (I) Upper panel: Immunoblotting analysis of indicated proteins in total lysates of BAT from STIM1/2 flox and STIM1/2 AdpKO mice maintained at RT or exposed to cold (4°C) for 2hrs. Bottom panel: Quantification analysis of images shown in the upper panel. n=3 mice per group for RT conditions and n=4 mice per group for cold conditions. One-way ANOVA with Tukey’s multiple comparisons test. **p< 0.005. (J) Seahorse based oxygen consumption rate of mitochondria isolated from STIM1/2 flox and STIM1/2 AdpKO exposed to cold (4°C) for 12 hrs. Arrows depicts the addition of 20mM Pyruvate + 20mM Malate; 2μg/mL Oligomycin, 9μM FCCP and 4 µM rotenone and 2 µM antimycin. n=10 wells per group. Representative of 3 independent experiments. (K) Quantification of basal and maximal respiration. Unpaired Student t-test ** p<0.001, **** p<0.0001.

Techniques: Software, Derivative Assay, Staining, Western Blot, Isolation

(A) Representative TEM images of BAT from STIM1/2 flox and STIM1/2 AdpKO exposed to cold (4°C). (B) Left: Immunoblotting analysis of indicated proteins in isolated mitochondria from BAT of STIM1/2 flox and STIM1/2 AdpCre mice exposed to cold for 12hrs. Right: Quantification analysis of images shown in left panel; n= 3 mice per group. (C) Quantification strategy for images shown in . (D) Left: Immunoblotting analysis of indicated proteins in total lysates of BAT from STIM1/2 flox and STIM1/2 AdpKO mice maintained at RT or exposed to cold (4°C) for 2hrs. Right: Quantification analysis of images shown in the upper panel. n=3 mice per group for RT conditions and n=4 mice per group for cold conditions. One-way ANOVA with Tukey’s multiple comparisons test.

Journal: bioRxiv

Article Title: Ca 2+ influx through ER-plasma membrane contacts is required for brown fat thermogenesis and metabolic health

doi: 10.64898/2026.03.20.712802

Figure Lengend Snippet: (A) Representative TEM images of BAT from STIM1/2 flox and STIM1/2 AdpKO exposed to cold (4°C). (B) Left: Immunoblotting analysis of indicated proteins in isolated mitochondria from BAT of STIM1/2 flox and STIM1/2 AdpCre mice exposed to cold for 12hrs. Right: Quantification analysis of images shown in left panel; n= 3 mice per group. (C) Quantification strategy for images shown in . (D) Left: Immunoblotting analysis of indicated proteins in total lysates of BAT from STIM1/2 flox and STIM1/2 AdpKO mice maintained at RT or exposed to cold (4°C) for 2hrs. Right: Quantification analysis of images shown in the upper panel. n=3 mice per group for RT conditions and n=4 mice per group for cold conditions. One-way ANOVA with Tukey’s multiple comparisons test.

Techniques: Western Blot, Isolation

(A) Left: Immunoblotting analysis of indicated proteins in total lysates from BAT of mice fed chow diet of high fat diet for 16 weeks. Right: Quantification analysis of the images shown in A; n= 6 mice for chow diet, n=6 mice for HFD group. Unpaired Student’s t-test, *p<0.05 (B) Body weight gain of STIM1/2 flox and STIM1/2 AdpKO mice fed a HFD over the indicated time. n=16 STIM1/2 flox mice, n=10 STIM1/2 AdpKO mice. (C) Insulin tolerance test, n =15 STIM1/2 flox mice, n=10 STIM1/2 AdpKO mice fed a HFD for 10 weeks. Two-way ANOVA **p < 0.01. (D) Oral glucose tolerance test, n=16 STIM1/2 flox mice, n=10 STIM1/2 AdpKO mice fed a HFD for 11 weeks (E) Insulin levels during the oral glucose tolerance test shown in D, Two-way ANOVA *p <0.05. (F) Representative hematoxylin and eosin-stained histology sections of BAT from STIM1/2 flox and STIM1/2 AdpKO mice fed HFD for 14 weeks. (G) Schematic depicting protocol of high fat diet (HFD) feeding and AAV-Rec2-UCP1-Null or - AAV-Rec2-UCP1-Cre injection. (H) Left: Immunoblotting analysis of indicated proteins in total lysates from BAT of mice fed HFD infected with AAV-Rec2-UCP1-Null or - AAV-Rec2-UCP1-Cre. Right: Quantification analysis of the images shown in A; n= 5 mice per group. Unpaired Student’s t-test, **p<0.01. (I) Insulin tolerance test, n= 4 Null mice and n=5 Cre mice; Two-way ANOVA *p <0.05. (J) Left: Oral glucose tolerance test, n =5 mice in each group. Two-way ANOVA *p <0.05. Right: Area under the curve from the graph shown on the left. Unpaired Student’s t-test *p<0.05

Journal: bioRxiv

Article Title: Ca 2+ influx through ER-plasma membrane contacts is required for brown fat thermogenesis and metabolic health

doi: 10.64898/2026.03.20.712802

Figure Lengend Snippet: (A) Left: Immunoblotting analysis of indicated proteins in total lysates from BAT of mice fed chow diet of high fat diet for 16 weeks. Right: Quantification analysis of the images shown in A; n= 6 mice for chow diet, n=6 mice for HFD group. Unpaired Student’s t-test, *p<0.05 (B) Body weight gain of STIM1/2 flox and STIM1/2 AdpKO mice fed a HFD over the indicated time. n=16 STIM1/2 flox mice, n=10 STIM1/2 AdpKO mice. (C) Insulin tolerance test, n =15 STIM1/2 flox mice, n=10 STIM1/2 AdpKO mice fed a HFD for 10 weeks. Two-way ANOVA **p < 0.01. (D) Oral glucose tolerance test, n=16 STIM1/2 flox mice, n=10 STIM1/2 AdpKO mice fed a HFD for 11 weeks (E) Insulin levels during the oral glucose tolerance test shown in D, Two-way ANOVA *p <0.05. (F) Representative hematoxylin and eosin-stained histology sections of BAT from STIM1/2 flox and STIM1/2 AdpKO mice fed HFD for 14 weeks. (G) Schematic depicting protocol of high fat diet (HFD) feeding and AAV-Rec2-UCP1-Null or - AAV-Rec2-UCP1-Cre injection. (H) Left: Immunoblotting analysis of indicated proteins in total lysates from BAT of mice fed HFD infected with AAV-Rec2-UCP1-Null or - AAV-Rec2-UCP1-Cre. Right: Quantification analysis of the images shown in A; n= 5 mice per group. Unpaired Student’s t-test, **p<0.01. (I) Insulin tolerance test, n= 4 Null mice and n=5 Cre mice; Two-way ANOVA *p <0.05. (J) Left: Oral glucose tolerance test, n =5 mice in each group. Two-way ANOVA *p <0.05. Right: Area under the curve from the graph shown on the left. Unpaired Student’s t-test *p<0.05

Techniques: Western Blot, Staining, Injection, Infection

Calcium image analysis in PQ-injured A549 cells compared to normal cells ( A ) and PQ-injured A549 cells w/wo lysine supplementation ( B ) with thapsigargin (TG) or ionomycin (IONO) stimulation. Mean ± SEM. C NFAT luciferase expression in A549 cells treated w/wo 800 μM PQ, together with 0-, 1-, 2.5-, or 5-fold lysine as in the culture medium as indicated, for 24 h. Mean ± SD, *** P < 0.001; Two-Way ANOVA. n = 3. NS not significant. D WB analysis of E-Cadherin, ZO-1, EPCAM and acetyl-α-Tubulin in PQ-injured A549 cells treated w/wo 5 μM SKF-96365 (SKF). E Co-IP analysis of STIM1 association with ORAI1 or TRPC1 in PQ-injured A549 cells with indicated concentration, together w/wo 5 mM lysine. F Immunofluorescence analysis of Myc and ARL13B in PQ-injured TRPC1-Myc stably expressed A549 cells w/wo lysine supplementation. Scale bar, 10 μm.

Journal: Cell Death Discovery

Article Title: Lysine attenuates acute lung injury by restoring α-tubulin acetylation and ciliary activity

doi: 10.1038/s41420-026-03025-x

Figure Lengend Snippet: Calcium image analysis in PQ-injured A549 cells compared to normal cells ( A ) and PQ-injured A549 cells w/wo lysine supplementation ( B ) with thapsigargin (TG) or ionomycin (IONO) stimulation. Mean ± SEM. C NFAT luciferase expression in A549 cells treated w/wo 800 μM PQ, together with 0-, 1-, 2.5-, or 5-fold lysine as in the culture medium as indicated, for 24 h. Mean ± SD, *** P < 0.001; Two-Way ANOVA. n = 3. NS not significant. D WB analysis of E-Cadherin, ZO-1, EPCAM and acetyl-α-Tubulin in PQ-injured A549 cells treated w/wo 5 μM SKF-96365 (SKF). E Co-IP analysis of STIM1 association with ORAI1 or TRPC1 in PQ-injured A549 cells with indicated concentration, together w/wo 5 mM lysine. F Immunofluorescence analysis of Myc and ARL13B in PQ-injured TRPC1-Myc stably expressed A549 cells w/wo lysine supplementation. Scale bar, 10 μm.

Article Snippet: The lysates were quantified by the Bicinchoninic acid (BCA) method and incubated with STIM1 antibody (Cell Signaling Technology, 5668, 1 μl/sample) overnight, followed by incubation with 20 μl protein A/G beads (Proteintech, PR40025) for another 3 h at 4 °C.

Techniques: Luciferase, Expressing, Co-Immunoprecipitation Assay, Concentration Assay, Immunofluorescence, Stable Transfection

A GSEA analysis uncovered top-ranked molecular functions enhanced in PQ-injured lung tissues with lysine supplementation compared to the untreated group. B KEGG enrichment of the top 20 upregulated pathways in PQ-injured lung tissues with lysine supplementation compared to the untreated group. C Quantification of NADP and NADPH (left) or NAD and NADH (right) in PQ-injured A549 cells w/wo lysine supplementation. D ELISA analysis of acetyl coenzyme A (Acetyl-CoA) in PQ-injured A549 cells w/wo lysine supplementation. E WB analysis of E-Cadherin and acetyl-α-Tubulin in PQ-injured A549 cells treated w/wo 1 mM sodium acetate (SA). F WB analysis of ZO-1, E-Cadherin, acetyl-α-Tubulin and EPCAM in PQ-injured A549 cells w/wo lysine supplementation, together w/wo 0.5 μM GM-90257 (GM). G Calcium image analysis in PQ-injured A549 cells w/wo lysine supplementation, together w/wo 0.5 μM GM-90257 treatment. The cells were transiently stimulated with thapsigargin (TG) or ionomycin (IONO). Mean ± SEM. H Co-IP analysis of STIM1 association with TRPC1 in PQ-injured A549 cells w/wo lysine supplementation, together w/wo GM-90257 treatment. Immunofluorescence analysis of ARL13B, SFTPC, and HOPX in A549 cells ( I ) or normal lung tissues ( J ). Scale bar, 10 μm. Mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001; Two-Way ANOVA. NS not significant.

Journal: Cell Death Discovery

Article Title: Lysine attenuates acute lung injury by restoring α-tubulin acetylation and ciliary activity

doi: 10.1038/s41420-026-03025-x

Figure Lengend Snippet: A GSEA analysis uncovered top-ranked molecular functions enhanced in PQ-injured lung tissues with lysine supplementation compared to the untreated group. B KEGG enrichment of the top 20 upregulated pathways in PQ-injured lung tissues with lysine supplementation compared to the untreated group. C Quantification of NADP and NADPH (left) or NAD and NADH (right) in PQ-injured A549 cells w/wo lysine supplementation. D ELISA analysis of acetyl coenzyme A (Acetyl-CoA) in PQ-injured A549 cells w/wo lysine supplementation. E WB analysis of E-Cadherin and acetyl-α-Tubulin in PQ-injured A549 cells treated w/wo 1 mM sodium acetate (SA). F WB analysis of ZO-1, E-Cadherin, acetyl-α-Tubulin and EPCAM in PQ-injured A549 cells w/wo lysine supplementation, together w/wo 0.5 μM GM-90257 (GM). G Calcium image analysis in PQ-injured A549 cells w/wo lysine supplementation, together w/wo 0.5 μM GM-90257 treatment. The cells were transiently stimulated with thapsigargin (TG) or ionomycin (IONO). Mean ± SEM. H Co-IP analysis of STIM1 association with TRPC1 in PQ-injured A549 cells w/wo lysine supplementation, together w/wo GM-90257 treatment. Immunofluorescence analysis of ARL13B, SFTPC, and HOPX in A549 cells ( I ) or normal lung tissues ( J ). Scale bar, 10 μm. Mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001; Two-Way ANOVA. NS not significant.

Techniques: Enzyme-linked Immunosorbent Assay, Co-Immunoprecipitation Assay, Immunofluorescence

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