Journal: iScience

Article Title: C5aR1-positive adipocytes mediate non-shivering thermogenesis in neonatal mice

doi: 10.1016/j.isci.2024.111261

Figure Lengend Snippet: In vitro differentiated CD45 + adipocytes were distinct from classical brown adipocytes (A) Oil red O staining of adipogenic differentiation of isolated CD45 − and CD45 + ASCs in vitro . Scale bars, 20μm. (B) mRNA expression of the adipogenic differentiation of isolated CD45 − and CD45 + ASCs in vitro . ( n = 3). (C) mRNA expression of the indicated genes in differentiated adipogenic cells in (A) and white adipocytes ( n = 3). (D) Immunoblotting for UCP1 and PPARγ in differentiated cells in (A). (E) RNA-seq of CD45 − and CD45 + adipocytes. Volcano plot of the global gene expression profile ( n = 3). (F) Top 10 genes upregulated and downregulated. (G) (Top) upregulated KEGG pathways and typical genes. (Bottom) downregulated KEGG pathways and typical genes. (H) Expression of C5ar1 mRNA in CD45 − and CD45 + ASC and adipogenic differentiated CD45 − and CD45 + adipocytes. (I) Immunoblotting for C5aR1 in adipogenic differentiated CD45 − and CD45 + adipocytes. Statistical significance was assessed by two-tailed Student’s t test. Data are represented as mean ± SEM. ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.005.

Article Snippet: Anti-rabbit IgG (ABclonal, AS014) was used as the second antibody for UCP1, C5aR1, PPARγ, β-ACTIN and HSP90.

Techniques: In Vitro, Staining, Isolation, Expressing, Western Blot, RNA Sequencing, Gene Expression, Two Tailed Test

Journal: iScience

Article Title: C5aR1-positive adipocytes mediate non-shivering thermogenesis in neonatal mice

doi: 10.1016/j.isci.2024.111261

Figure Lengend Snippet: The loss of the C5ar1 gene in adipocytes during the perinatal period decreased the thermogenesis ability of newborn mice (A) Administration of shRNA- C5ar1 in CD45 + adipocytes significantly suppressed C5ar1 expression compared to the control group. (Left) RT-qPCR for C5ar1 in shNC and sh C5ar1 CD45 + adipocytes. (Right) Immunoblotting for C5aR1 in shNC and sh C5ar1 CD45 + adipocytes ( n = 3). (B) Oil red O staining of shNC and sh C5ar1 CD45 + adipocytes. Scale bars, 20μm. (C) The mRNA expression of the indicated genes of C5ar1 knockdown differentiated CD45 + adipocytes ( n = 3). (D) Immunoblotting for UCP1 and PPARγ of C5ar1 knockdown differentiated CD45 + adipocytes ( n = 3). (E) C5ar1 AKO mice ( C5ar1 flox/flox ; Adipoq Cre + ) were generated by breeding C5ar1 flox/flox mice with Adiponectin -Cre mice. (F) Immunofluorescence staining of UCP1 and C5aR1 in the BAT of the control and C5ar1 AKO neonatal mice. Scale bars, 50μm. (G) (left)Infrared thermo-imaging 5 min after the beginning of cold challenge. (Right) back skin temperature during cold challenge (22° C) (control, n = 8 AKO, n = 7). (H) Body weight and BAT/body weight ratio in control and C5ar1 AKO neonatal mice. (I) Gross appearance of the interscapular BAT of the control and C5ar1 AKO neonatal mice. Scale bars, 1mm. (J) H&E (Top) and UCP1 immunohistochemical staining (bottom). Scale bars, 20μm. BAT adipocyte sizes of control and C5ar1 AKO neonatal mice (right). (K) Relative mRNA expression of indicated genes of BAT from control and C5ar1 AKO neonatal mice ( n = 8). (L) Immunoblotting for UCP1 of BAT from control and C5ar1 AKO neonatal mice ( n = 6). (M) RNA-seq of BAT from control and C5ar1 AKO neonatal mice. Volcano plot of the global gene expression profile ( n = 3). (N) Upregulated KEGG pathways and downregulated KEGG pathways. Statistical significance was assessed by two-tailed Student’s t test. Data are represented as mean ± SEM ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.005.

Article Snippet: Anti-rabbit IgG (ABclonal, AS014) was used as the second antibody for UCP1, C5aR1, PPARγ, β-ACTIN and HSP90.

Techniques: shRNA, Expressing, Control, Quantitative RT-PCR, Western Blot, Staining, Knockdown, Generated, Immunofluorescence, Imaging, Immunohistochemical staining, RNA Sequencing, Gene Expression, Two Tailed Test

Journal: iScience

Article Title: C5aR1-positive adipocytes mediate non-shivering thermogenesis in neonatal mice

doi: 10.1016/j.isci.2024.111261

Figure Lengend Snippet: C5ar1 knockdown in CD45 + brown adipocytes promote PF4 release to inhibit brown adipocyte maturation (A) Relative mRNA expression of the cytokines genes ( Pf4 , Ccl3 , Ccl4 , Ccl12 ) of C5ar1 knockdown differentiated CD45 + adipocytes compared to the control group ( n = 6). (B) The concentration of PF4 in the supernatant of C5ar1 knockdown differentiated CD45 + adipocytes compared to the control group ( n = 3). (C) Pf4 mRNA expression in differentiated CD45 − and CD45 + adipocytes ( n = 6). (D) Concentration of PF4 in the supernatant of differentiated CD45 − and CD45 + adipocytes ( n = 3). (E) Pf4 mRNA expression of BAT from Control and C5ar1 AKO neonatal mice ( n = 8). (F) Concentration of PF4 in BAT of Control and C5ar1 AKO neonatal mice ( n = 6). (G) Relative mRNA expression of the indicated genes of adipocyte differentiation of CD45 − ASCs cultured without or with 20 ng/mL PF4 ( n = 6). (H) Immunoblotting for UCP1 and PPARγ of adipocyte differentiation of CD45 − ASCs cultured without or with 20 ng/mL PF4 ( n = 3). (I) Relative mRNA expression of the indicated genes from adipocyte differentiation of CD45 − ASCs cultured in conditioned media from shNC, sh C5ar1 , sh Pf4 or sh C5ar1 + shPf4 CD45 + adipocytes ( n = 3). (J) Immunoblotting for UCP1 and PPARγ of adipocyte differentiation of CD45 − ASCs cultured in conditioned media from shNC, sh C5ar1 , sh Pf4 or sh C5ar1 + shPf4 CD45 + adipocytes ( n = 3). (K) Graphical abstract of this study: The loss of C5ar1 in CD45 + adipocytes increased Pf4 mRNA level and increased the secretion of PF4. PF4 inhibited the maturity and thermogenesis ability of both CD45 + and CD45 − adipocytes. Statistical significance was assessed by two-tailed Student’s t test (A–G) or one-way ANOVA (I). Data are represented as mean ± SEM ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.005.

Article Snippet: Anti-rabbit IgG (ABclonal, AS014) was used as the second antibody for UCP1, C5aR1, PPARγ, β-ACTIN and HSP90.

Techniques: Knockdown, Expressing, Control, Concentration Assay, Cell Culture, Western Blot, Two Tailed Test

Journal: iScience

Article Title: C5aR1-positive adipocytes mediate non-shivering thermogenesis in neonatal mice

doi: 10.1016/j.isci.2024.111261

Figure Lengend Snippet:

Article Snippet: Anti-rabbit IgG (ABclonal, AS014) was used as the second antibody for UCP1, C5aR1, PPARγ, β-ACTIN and HSP90.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, SYBR Green Assay, Sequencing, Software

Journal: Cell reports

Article Title: Streptozotocin-induced hyperglycemia alters the cecal metabolome and exacerbates antibiotic-induced dysbiosis

doi: 10.1016/j.celrep.2021.110113

Figure Lengend Snippet:

Article Snippet: We prepared metagenomic libraries from DNA (100 ng) using the NEBNext® Ultra II FS DNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA) and the > 100 ng input protocol as per the manufacturer’s instructions, which generated a pool of fragments whose average size was between 250 and 500 bp.

Techniques: Recombinant, Staining, Lysis, Enzyme-linked Immunosorbent Assay, Glucose Assay, Purification, RNA Sequencing Assay, Sensitive Assay, Software, Flow Cytometry, Imaging