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Figure 4. Activation of <t>Stim1</t> in AgRP neurons leads to an obesity-like phenotype (A) Ctrl-AAV and Stim1 <t>D76A-AAV</t> were injected into the Arc nucleus of male adult AgRP-Cre mice. Immunoﬂuorescent staining for Stim1 (red) was then per- formed on brain sections. When Cre recombinase is present, infection of Ctrl-AAV or Stim1-D76A-AAV viruses can lead to EGFP or EYFP expression (see also Figures S4A and S4D). Scale bars, 50 mm. (B–D) AAV viruses were injected into the Arc nucleus of adult male AgRP-Cre mice fed a normal chow. Body weight gain (B), fat mass (C), and lean mass (D) were then assessed. In (B), the box indicates the period of food intake assessment. n = 8 (Ctrl-AAV) or 6 (Stim1 D76A-AAV) mice per group. (E) Representative H&E staining images of mouse tissues harvested 16 weeks after surgery. Scale bars, 100 mm. (F) TC levels of mice at 15 weeks post-surgery. n = 8 mice per group. (G and H) GTT (G) and the AUC of the GTT (H) of mice at 15 weeks post-surgery. n = 7 mice per group.

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Figure 4. Activation of Stim1 in AgRP neurons leads to an obesity-like phenotype (A) Ctrl-AAV and Stim1 D76A-AAV were injected into the Arc nucleus of male adult AgRP-Cre mice. Immunoﬂuorescent staining for Stim1 (red) was then per- formed on brain sections. When Cre recombinase is present, infection of Ctrl-AAV or Stim1-D76A-AAV viruses can lead to EGFP or EYFP expression (see also Figures S4A and S4D). Scale bars, 50 mm. (B–D) AAV viruses were injected into the Arc nucleus of adult male AgRP-Cre mice fed a normal chow. Body weight gain (B), fat mass (C), and lean mass (D) were then assessed. In (B), the box indicates the period of food intake assessment. n = 8 (Ctrl-AAV) or 6 (Stim1 D76A-AAV) mice per group. (E) Representative H&E staining images of mouse tissues harvested 16 weeks after surgery. Scale bars, 100 mm. (F) TC levels of mice at 15 weeks post-surgery. n = 8 mice per group. (G and H) GTT (G) and the AUC of the GTT (H) of mice at 15 weeks post-surgery. n = 7 mice per group.

Journal: Cell reports

Article Title: Deficiency of ER Ca 2+ sensor STIM1 in AgRP neurons confers protection against dietary obesity.

doi: 10.1016/j.celrep.2021.109868

Figure Lengend Snippet: Figure 4. Activation of Stim1 in AgRP neurons leads to an obesity-like phenotype (A) Ctrl-AAV and Stim1 D76A-AAV were injected into the Arc nucleus of male adult AgRP-Cre mice. Immunoﬂuorescent staining for Stim1 (red) was then per- formed on brain sections. When Cre recombinase is present, infection of Ctrl-AAV or Stim1-D76A-AAV viruses can lead to EGFP or EYFP expression (see also Figures S4A and S4D). Scale bars, 50 mm. (B–D) AAV viruses were injected into the Arc nucleus of adult male AgRP-Cre mice fed a normal chow. Body weight gain (B), fat mass (C), and lean mass (D) were then assessed. In (B), the box indicates the period of food intake assessment. n = 8 (Ctrl-AAV) or 6 (Stim1 D76A-AAV) mice per group. (E) Representative H&E staining images of mouse tissues harvested 16 weeks after surgery. Scale bars, 100 mm. (F) TC levels of mice at 15 weeks post-surgery. n = 8 mice per group. (G and H) GTT (G) and the AUC of the GTT (H) of mice at 15 weeks post-surgery. n = 7 mice per group.

Article Snippet: N/A N/A Recombinant DNA AAV-EF1a-DIO-EGFP Vigene Biosciences Cat# AV1091 AAV-EF1a-DIO-hM3Dq-mCherry Addgene Cat# 50460; RRID: Addgene_50460 AAV-EF1a-DIO-Stim1 D76A-EYFP This paper N/A AAV-EF1a-DIO-Oas3-mCherry This paper N/A AAV-EF1a-DIO-ns2-mCherry This paper N/A rAAV-EF1a-DIO-GCaMP6m-WPRE-hGHpolyA BrainVTA Cat# PT-0283 pAAV-hSyn-GCaMP6f-WPRE Obio Technology Cat# E1272 Lentilox 3.7 Addgene Cat# 112284; RRID: Addgene_112284 Lentilox Stim1 shRNA This paper N/A pEYFP-C1 Clontech Cat# 6005-1 pEYFP-C1-Stim1 D76A Dr. YoujunWang (Beijing Normal University) N/A pGP-CMV-NES-jRGECO1a Addgene Cat# 61563, RRID: Addgene_61563 (Continued on next page) Cell Reports 37, 109868, October 19, 2021 e1

Techniques: Activation Assay, Injection, Staining, Infection, Expressing

Figure 5. SOCE and Stim1 regulate protein synthesis in neuronal cells (A–C) The Neuro2a neuronal cells were incubated with vehicle (control) or BTP2 (A), or were trans- fected with Lentilox 3.7 (shCtrl) and Lentilox Stim1 shRNA (shStim1) (B) or pEYFP-C1 (Ctrl) and pEYFP- C1-Stim1 D76A (S D76A or Stim1 D76A) (C) plas- mids at 37C for 96 h. Subsequently, puromycin was supplemented to the cell culture medium for 1 h. Cells were then harvested, and total proteins were prepared. Western blot for puromycin (A–C, left panel) and densitometry of the blots (A–C, right panel) are shown. n = 4 cell cultures per group. (D and E) Neuro2a cells were incubated with vehicle (control) or BTP2 at 37C for 96 h. Total RNAs were then extracted, and the relative mRNA (D) and rRNA (E) levels of the indicated genes were assessed. n = 6 cell cultures per group. (F and G) Neuro2a cells were transfected with Lentilox 3.7 (shCtrl) or Lentilox Stim1 shRNA (shStim1) plasmids at 37C for 96 h. Total RNAs were then extracted, and the relative mRNA (F) and rRNA (G) levels of genes were assessed. n = 6 cell cultures per group. (H and I) Neuro2a cells were transfected with pEYFP-C1 (Ctrl) or pEYFP-C1-Stim1 D76A (Stim1 D76A) plasmids at 37C for 96 h. Total RNAs were prepared, and the relative mRNA (H) and rRNA (I) levels of genes were assessed. n = 4 (H) or 6 (I) cell cultures per group. Data are presented as means ± SEMs. *p < 0.05, **p < 0.01, 2-tailed Student’s t test (A)–(I). See also Figure S5.

Journal: Cell reports

Article Title: Deficiency of ER Ca 2+ sensor STIM1 in AgRP neurons confers protection against dietary obesity.

doi: 10.1016/j.celrep.2021.109868

Figure Lengend Snippet: Figure 5. SOCE and Stim1 regulate protein synthesis in neuronal cells (A–C) The Neuro2a neuronal cells were incubated with vehicle (control) or BTP2 (A), or were trans- fected with Lentilox 3.7 (shCtrl) and Lentilox Stim1 shRNA (shStim1) (B) or pEYFP-C1 (Ctrl) and pEYFP- C1-Stim1 D76A (S D76A or Stim1 D76A) (C) plas- mids at 37C for 96 h. Subsequently, puromycin was supplemented to the cell culture medium for 1 h. Cells were then harvested, and total proteins were prepared. Western blot for puromycin (A–C, left panel) and densitometry of the blots (A–C, right panel) are shown. n = 4 cell cultures per group. (D and E) Neuro2a cells were incubated with vehicle (control) or BTP2 at 37C for 96 h. Total RNAs were then extracted, and the relative mRNA (D) and rRNA (E) levels of the indicated genes were assessed. n = 6 cell cultures per group. (F and G) Neuro2a cells were transfected with Lentilox 3.7 (shCtrl) or Lentilox Stim1 shRNA (shStim1) plasmids at 37C for 96 h. Total RNAs were then extracted, and the relative mRNA (F) and rRNA (G) levels of genes were assessed. n = 6 cell cultures per group. (H and I) Neuro2a cells were transfected with pEYFP-C1 (Ctrl) or pEYFP-C1-Stim1 D76A (Stim1 D76A) plasmids at 37C for 96 h. Total RNAs were prepared, and the relative mRNA (H) and rRNA (I) levels of genes were assessed. n = 4 (H) or 6 (I) cell cultures per group. Data are presented as means ± SEMs. *p < 0.05, **p < 0.01, 2-tailed Student’s t test (A)–(I). See also Figure S5.

Techniques: Incubation, Control, shRNA, Cell Culture, Western Blot, Transfection

Figure 6. SOCE and Stim1 regulate Oas3 expression in neuronal cells (A–C) Neuro2a cells were incubated with vehicle (control) or BTP2 at 37C for 96 h, and the relative mRNA levels of Oas3 and RNase L were then as- sessed (A). In (B) and (C), western blots for Oas3 and RNase L were performed (B), and the densi- tometry of the blots is shown (C). Glyceraldehyde 3- phosphate dehydrogenase (GAPDH) was used as a loading control. n = 6 (A) or 3 (C) cell cultures per group. (D–F) Neuro2a cells were transfected with Lentilox 3.7 (shCtrl) or Lentilox Stim1 shRNA (shStim1) at 37C for 96 h, and the relative mRNA levels of Oas3 and RNase L were then assessed (D). In (E) and (F), western blots were performed (E), and the quanti- ﬁcation of the blots is shown (F). GAPDH was used as a loading control. n = 6 (D) or 3 (F) cell cultures per group. (G–I) Neuro2a cells were transfected with pEYFP- C1 (Ctrl) or pEYFP-C1-Stim1 D76A (Stim1 D76A) at 37C for 96 h, and the relative mRNA levels of Oas3 and RNase L were then assessed (G). In (H) and (I), western blots (H) and densitometry (I) of the blots are shown. GAPDH was used as a loading control. n = 6 (G) or 3 (I) cell cultures per group. (J and K) Neuro2a cells were transfected with the indicated plasmids at 37C for 96 h. Total RNAs were then extracted, and relative mRNA (J) and rRNA (K) levels of the indicated genes are pre- sented. shCtrl, Lentilox 3.7; shStim1, Lentilox Stim1 shRNA; ns2, ns2-AAV; Cre, pcGlobin2-Cre. n = 6 cell cultures per group. Data are presented as means ± SEMs. *p < 0.05, 2- tailed Student’s t test (A–I), 1-way ANOVA with Turkey’s post hoc test (J and K). See also Figures S5 and S6.

Journal: Cell reports

Article Title: Deficiency of ER Ca 2+ sensor STIM1 in AgRP neurons confers protection against dietary obesity.

doi: 10.1016/j.celrep.2021.109868

Figure Lengend Snippet: Figure 6. SOCE and Stim1 regulate Oas3 expression in neuronal cells (A–C) Neuro2a cells were incubated with vehicle (control) or BTP2 at 37C for 96 h, and the relative mRNA levels of Oas3 and RNase L were then as- sessed (A). In (B) and (C), western blots for Oas3 and RNase L were performed (B), and the densi- tometry of the blots is shown (C). Glyceraldehyde 3- phosphate dehydrogenase (GAPDH) was used as a loading control. n = 6 (A) or 3 (C) cell cultures per group. (D–F) Neuro2a cells were transfected with Lentilox 3.7 (shCtrl) or Lentilox Stim1 shRNA (shStim1) at 37C for 96 h, and the relative mRNA levels of Oas3 and RNase L were then assessed (D). In (E) and (F), western blots were performed (E), and the quanti- ﬁcation of the blots is shown (F). GAPDH was used as a loading control. n = 6 (D) or 3 (F) cell cultures per group. (G–I) Neuro2a cells were transfected with pEYFP- C1 (Ctrl) or pEYFP-C1-Stim1 D76A (Stim1 D76A) at 37C for 96 h, and the relative mRNA levels of Oas3 and RNase L were then assessed (G). In (H) and (I), western blots (H) and densitometry (I) of the blots are shown. GAPDH was used as a loading control. n = 6 (G) or 3 (I) cell cultures per group. (J and K) Neuro2a cells were transfected with the indicated plasmids at 37C for 96 h. Total RNAs were then extracted, and relative mRNA (J) and rRNA (K) levels of the indicated genes are pre- sented. shCtrl, Lentilox 3.7; shStim1, Lentilox Stim1 shRNA; ns2, ns2-AAV; Cre, pcGlobin2-Cre. n = 6 cell cultures per group. Data are presented as means ± SEMs. *p < 0.05, 2- tailed Student’s t test (A–I), 1-way ANOVA with Turkey’s post hoc test (J and K). See also Figures S5 and S6.

Techniques: Expressing, Incubation, Control, Western Blot, Transfection, shRNA

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