Journal: Cell metabolism

Article Title: Identification of a molecular resistor that controls UCP1-independent Ca 2+ cycling thermogenesis in adipose tissue

doi: 10.1016/j.cmet.2025.03.009

Figure Lengend Snippet: (A) FPKM of indicated SERCA-binding peptides in isolated beige adipocytes of mice. n = 3. (B) Relative C4orf3/ALN protein expression levels in microsomes from IngWAT of UCP1 KO mice at the corresponding temperature. Calreticulin was used as a loading control. n = 3. Statistic: one-way ANOVA with Tukey’s post hoc HSD test. (C) Protein interaction between SERCA2b and endogenous C4orf3/ALN protein in beige adipocytes. Immunoprecipitants of a SERCA2 complex (FLAG-tagged) were immunoblotted using a polyclonal antibody for C4orf3. Inputs were included in the immunoblotting. (D) Endogenous C4orf3 protein expression in IngWAT of C4orf3 CRISPRi mice and control male mice. β-actin was used as a loading control. n = 3. (E) Intracellular Ca 2+ flux assay in IngWAT-derived adipocytes from control and C4orf3 CRISPRi mice. Primary adipocytes were differentiated on collagen-coated glass-bottom dishes in which intercellular Ca 2+ levels were determined by using the Fluo-8 dye. Control, n = 50; C4orf3 CRISPRi , n = 50. Statistic: two-way ANOVA with Šídák’s multiple comparisons test. (F) Ca 2+ uptake in isolated microsomes from the inguinal WAT of male C4orf3 CRISPRi mice and control mice at pCa 2+ 6.0. n = 5. (G) SERCA ATP hydrolysis assay in isolated microsomes from (F). (H) The bioenergetic efficiency of SERCA2 was calculated by Ca 2+ uptake per ATP hydrolysis from (E) and (F). (I) Heat rate in isolated microsomes from IngWAT of control and C4orf3 CRISPRi male mice in the presence of ATP and Ca 2+ (pCa 6.0). Thapsigargin was added to calculate SERCA-dependent thermogenesis. n = 5. (J) Relative mRNA levels of C4orf3 in adult dCas9-KRAB male mice 2 weeks after direct injection into IngWAT of AAV expressing scrambled control or gRNA targeting C4orf3 . n = 4. (K) Heat rate in isolated microsomes from the inguinal WAT of dCas9-KRAB male mice 2 weeks after AAV injection into the inguinal WAT. n = 4. Statistic (D and F–K): unpaired t test. Bars represent the mean and error shown as SEM.

Article Snippet: Ing WAT-specific C4orf3 KD mice were generated by injecting AAV expressing gRNA- C4orf3 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, VectorBuilder) directly into Ing WAT, while control mice were injected with AAV8-CAG-EGFP-U6-Scramble.

Techniques: Binding Assay, Isolation, Expressing, Control, Western Blot, Flux Assay, Derivative Assay, Hydrolysis Assay, Injection

Journal: Cell metabolism

Article Title: Identification of a molecular resistor that controls UCP1-independent Ca 2+ cycling thermogenesis in adipose tissue

doi: 10.1016/j.cmet.2025.03.009

Figure Lengend Snippet: (A) Side (left) and top (right) views of the SERCA2b-C4ORF3 complex structure predicted by AlphaFold3. The A-, N-, P-, and TM domains of Ca 2+ -unbound SERCA2b are shown in orange, pink, yellow, and green, respectively. C4ORF3/ALN is shown in blue. (B) Protein interaction interface of SERCA2b to C4orf3 as determined by the Turbo-ID proximity-labeling proteomics. Turbo-tag was fused to the N terminus (the cytoplasmic side) of C4orf3. Orange, TM domain; yellow, actuator domain; pink, nucleotide-binding domain; cyan, P-domain. (C) Cross-linking assays of SERCA2b with C4orf3/ALN W48C in isolated microsomes at indicated SERCA2 catalytic states. The cross-linker BMH was added to induce the SERCA2-C4ORF3 complex formation. The assays were performed in three independent biological samples. Bottom: catalytic cycle of SERCA to transport Ca 2+ into the ER lumen. Indicated compounds were used to induce the specific catalytic states of SERCA. (D) The amino acid sequence alignment of C4ORF3. The lower panel shows the schematics of a C4ORF3 mutant containing only the transmembrane (TM) domain and three mutants (mutants A, B, and C) lacking the evolutionarily conserved domains (shown in red boxes). (E) Ca 2+ uptake in isolated microsomes from cells co-expressing SERCA2b together with empty vector control and indicated C4ORF3 constructs at pCa 2+ 6.0. n = 3. (F) SERCA ATP hydrolysis assay in microsomes from (E). (G) ITC-based thermogenesis assays from microsomes in (E). Statistic (E–G): one-way ANOVA with Tukey’s post hoc HSD test. Bars represent the mean and error shown as SEM.

Article Snippet: Ing WAT-specific C4orf3 KD mice were generated by injecting AAV expressing gRNA- C4orf3 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, VectorBuilder) directly into Ing WAT, while control mice were injected with AAV8-CAG-EGFP-U6-Scramble.

Techniques: Labeling, Binding Assay, Isolation, Sequencing, Mutagenesis, Expressing, Plasmid Preparation, Control, Construct, Hydrolysis Assay

Journal: Cell metabolism

Article Title: Identification of a molecular resistor that controls UCP1-independent Ca 2+ cycling thermogenesis in adipose tissue

doi: 10.1016/j.cmet.2025.03.009

Figure Lengend Snippet: (A) Cold tolerance test of male C4orf3 CRISPRi mice and littermate controls. Mice kept at room temperature were exposed to cold (6°C) for indicated time points. n = 11. Statistic: two-way ANOVA with Šídák’s multiple comparisons test. (B) OCR in iBAT and IngWAT of male C4orf3 CRISPRi mice and control male mice following cold exposure. A subset of isolated tissues was stimulated with NE. n = 12 per group for iBAT; 10 per group for Ing WAT. Statistic: Mann-Whitney U test. (C) Relative mRNA expression of thermogenic genes in IngWAT of male mice following cold exposure. n = 5. (D) Mitochondrial respiration in IngWAT of male mice following cold exposure. J O 2 at indicated states was measured following substrate injection. n = 5. (E) Heat rate in isolated mitochondria (state 4, complexes I and II) from IngWAT of male mice following cold exposure. Data are normalized by mitochondrial protein contents. n = 4. (F) Cold tolerance test of male UCP1 KO mice and DKO mice (UCP1 KO × C4orf3 CRISPRi ). The mice were chronically treated with the β3-adrenergic receptor agonist CL316,243 for 5 days to stimulate beige fat biogenesis in both groups. n = 8 per group. Statistic: two-way ANOVA with Šídák’s multiple comparisons test and individual unpaired t tests. (G) Ca 2+ uptake in isolated microsomes from IngWAT of male DKO mice and control UCP1 KO mice at pCa 2+ 6.0. n = 4. (H) SERCA ATP hydrolysis assay in isolated microsomes from (G). The values were normalized by microsomal protein contents. (I) The bioenergetic efficiency of SERCA2 was calculated by Ca 2+ uptake per ATP hydrolysis from (G) and (H). (J) Heat rate in isolated microsomes from IngWAT of UCP1 KO and DKO male mice at pCa 6.0. Thapsigargin was added to calculate SERCA-dependent thermogenesis. n = 4 per group. Statistic (C–E and G–J): unpaired t test. Bars represent the mean and error shown as SEM.

Article Snippet: Ing WAT-specific C4orf3 KD mice were generated by injecting AAV expressing gRNA- C4orf3 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, VectorBuilder) directly into Ing WAT, while control mice were injected with AAV8-CAG-EGFP-U6-Scramble.

Techniques: Control, Isolation, MANN-WHITNEY, Expressing, Injection, Hydrolysis Assay

Journal: Cell metabolism

Article Title: Identification of a molecular resistor that controls UCP1-independent Ca 2+ cycling thermogenesis in adipose tissue

doi: 10.1016/j.cmet.2025.03.009

Figure Lengend Snippet: (A) GO pathway enrichment analysis for 276 genes that were uniquely upregulated in IngWAT of DKO mice (UCP1 KO × C4orf3 CRISPRi ) relative to other genotypes following cold exposure. n = 6 per group. (B) Relative mRNA levels of indicated genes in IngWAT of WT control, UCP1 KO, C4orf3 CRISPRi , and DKO mice (UCP1 KO × C4orf3 CRISPRi ) following cold exposure at 6°C for 5 h. Data represented as Z score heatmap for each gene in each sample representing quantitated value. GO terms and pathways for each gene are listed on the right. n = 6. (C) The respiratory exchange ratio of mice at 30°C. n = 7 per group. (D) Quantification of respiratory exchange ratio in (C). Statistic: unpaired t test.

Article Snippet: Ing WAT-specific C4orf3 KD mice were generated by injecting AAV expressing gRNA- C4orf3 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, VectorBuilder) directly into Ing WAT, while control mice were injected with AAV8-CAG-EGFP-U6-Scramble.

Techniques: Control

Journal: Cell metabolism

Article Title: Identification of a molecular resistor that controls UCP1-independent Ca 2+ cycling thermogenesis in adipose tissue

doi: 10.1016/j.cmet.2025.03.009

Figure Lengend Snippet: (A) Relative mRNA expression of indicated genes in the subcutaneous adipose tissue of people in . Lean: n = 6 female, n = 3 males; overweight: n = 5 female, n = 4 males; obese classes 1 and 2: n = 6 female, n = 3 males; obese class 3: n = 9 female, n = 1 male; obese with T2D: n = 5 female, n = 4 males. Statistic: one-way ANOVA with Tukey’s post hoc HSD test. (B) Fat mass and lean mass of male C4orf3 CRISPRi and littermate control mice at 30°C on a regular chow diet. n = 10. (C) Daily food intake of mice in (B). (D) Body weight of mice in (B) at 20 weeks old. (E) Indicated tissue weight of male mice on a regular diet at 30°C. n = 11. (F) H&E staining and adipocyte size of IngWAT ( n = 917 cells for control, 623 cells for C4orf3 CRISPRi ) and epididymal WAT (n = 699 cells for control, 364 for C4orf3 CRISPRi ). Male mice at 30°C on a regular chow diet ( n = 3 per group). Statistic: unpaired t test with Welch’s correction. (G) Insulin tolerance test of male C4orf3 CRISPRi mice and controls after 4 h fasting (0.5 U per kg −1 ). n = 8. (H) Fasting insulin levels in male C4orf3 CRISPRi mice and control mice. n = 6. (I) Blood triglyceride (TG) levels in male C4orf3 CRISPRi mice and littermate control mice. n = 6. Statistic (C–E and G–I): unpaired t test. Bars represent the mean and error shown as SEM.

Article Snippet: Ing WAT-specific C4orf3 KD mice were generated by injecting AAV expressing gRNA- C4orf3 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, VectorBuilder) directly into Ing WAT, while control mice were injected with AAV8-CAG-EGFP-U6-Scramble.

Techniques: Expressing, Control, Staining

Journal: Neuron

Article Title: Neurovascular coupling in the dentate gyrus regulates adult hippocampal neurogenesis

doi: 10.1016/j.neuron.2019.05.045

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: AAV8-GFP-U6-m-NOS1-shRNA , Vector Biolabs , Cat# shAAV-279053.

Techniques: Virus, Plasmid Preparation, Recombinant

Journal: Cell genomics

Article Title: Genome-scale CRISPR screening in a single mouse liver

doi: 10.1016/j.xgen.2022.100217

Figure Lengend Snippet: (A) Lentiviral vectors for U6-driven expression of an sgRNA and hepatocyte-specific expression of a fluorescent reporter (mCherry or mTurq2). (B) Images of endogenous mCherry and mTurq2 fluorescence in livers from mice 4 days after injection with an equal mixture of sgAAVS1-mCherry and sgAAVS1-mTurq2 lentiviruses. Livers were counterstained with phalloidin (green) to label actin. Scale bars, 100 μm. (C) Percentage of mCherry-, mTurq2-, and double-positive hepatocytes in livers from mice 4 days after injection with an equal mixture of sgAAVS1-mCherry and sgAAVS1-mTurq2 lentiviruses. Error bars indicate standard deviation. n = 3 mice per dose and 200 hepatocytes per mouse. See also .

Article Snippet: To deliver AAV-shRNA, a stock solution of AAV8-mCherry-U6-scrmb-shRNA (AAV-shScramble, Vector Biolabs) and/or AAV8-GFP-U6-mNDST1-shRNA (AAV-shNDST1, Vector Biolabs) was diluted in PBS to a total volume of 20 μL and injected intraperitoneally into postnatal day five mice.

Techniques: Expressing, Fluorescence, Injection, Standard Deviation

Journal: Cell genomics

Article Title: Genome-scale CRISPR screening in a single mouse liver

doi: 10.1016/j.xgen.2022.100217

Figure Lengend Snippet: (A) KEGG gene sets exhibiting significant depletion (FDR q < 0.05) at the endpoint of the screen ranked by FDR q-value (−log10). Bars extending to the end of the plot indicate an FDR q-value of 0. (B) KEGG gene sets exhibiting significant depletion (FDR q < 0.05) in our screen relative to screens in either mouse ESCs (dark gray bars) or human hepatocellular carcinoma (HCC) cell lines (light gray bars) ranked by FDR q-value (−log10) for our screen relative to mouse ESCs. Bars extending to the end of the plot indicate an FDR q-value of 0. (C) Median fold change (log2)for genes in the KEGG gene set for antigen processing and presentation in quantile-normalized ESC screens, HCC cell line screens, and our screen. Genes uniquely depleted in our screen are highlighted in red. The bounds of the box indicate the first and third quartiles, and the whiskers extend to the furthest data point that is within 1.5 times the interquartile range. (D) Median fold change (log2) for genes in the KEGG gene set for glycosaminoglycan biosynthesis and heparan sulfate in quantile-normalized ESC screens, HCC cell line screens, and our screen. Genes uniquely depleted in our screen are highlighted in red. The bounds of the box indicate the first and third quartiles, and the whiskers extend to the furthest data point that is within 1.5 times the interquartile range. (E) Median fold change (log2) for genes in the heparan sulfate interactome in our screen. The bounds of the box indicate the first and third quartiles, and the whiskers extend to the furthest data point that is within 1.5 times the interquartile range. (F) Scheme for determining effects of NDST1 knockdown on hepatocyte proliferation (top panel). Image of liver from postnatal day 15 mouse injected with 1 × 10 10 genome copies (GC) of AAV-shNDST1 and AAV-shScramble on postnatal day 5 immunostained for Ki67 (white), GFP (green), and mCherry (magenta) and counterstained for Hoechst (blue) (left panel). Scale bar, 25 μm. Quantification of proliferation as inferred by Ki67 positivity in shNDST1 hepatocytes relative to shScramble hepatocytes (right panel). Bar and whiskers indicate mean and standard deviation across mice, respectively, and closed and open circles represent values from male and female mice, respectively. n = 2 male and 2 female mice and 200 cells per shRNA per mouse. **p = 0.0023 by two-tailed Fisher’s exact test. See also .

Article Snippet: To deliver AAV-shRNA, a stock solution of AAV8-mCherry-U6-scrmb-shRNA (AAV-shScramble, Vector Biolabs) and/or AAV8-GFP-U6-mNDST1-shRNA (AAV-shNDST1, Vector Biolabs) was diluted in PBS to a total volume of 20 μL and injected intraperitoneally into postnatal day five mice.

Techniques: Injection, Standard Deviation, shRNA, Two Tailed Test

Journal: Cell genomics

Article Title: Genome-scale CRISPR screening in a single mouse liver

doi: 10.1016/j.xgen.2022.100217

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To deliver AAV-shRNA, a stock solution of AAV8-mCherry-U6-scrmb-shRNA (AAV-shScramble, Vector Biolabs) and/or AAV8-GFP-U6-mNDST1-shRNA (AAV-shNDST1, Vector Biolabs) was diluted in PBS to a total volume of 20 μL and injected intraperitoneally into postnatal day five mice.

Techniques: Plasmid Preparation, RNA Sequencing Assay, CRISPR, Sequencing, Clone Assay, Amplification, Recombinant, Knock-Out, Software

Journal: Nature

Article Title: BCAA catabolism in brown fat controls energy homeostasis through SLC25A44

doi: 10.1038/s41586-019-1503-x

Figure Lengend Snippet: a. Expression profile of SLC25A family members in human supraclavicular BAT and abdominal subcutaneous WAT from the same individual at 27°C and 19°C 5 . b. Correlation of SLC25A44 mRNA expression with UCP1 or BCKDHA in human BAT. Expressions at TN (red) and cold (blue) from 6 biological independent subjects. Pearson’s correlation coefficient was calculated. c. Protein expression of SLC25A44 in indicated tissues of mice. GAPDH as a loading control. Representative result from two independent experiments. Gel source data are in . d-e. Mitochondrial uptakes of indicated molecules in control and Slc25a44 KO brown adipocytes ( d ) or in Neuro2a cells expressing Slc25a44 or an empty vector ( e ). n = 3/group, biologically independent samples. f. [U- 14 C 6 ] Leu transport into mitochondrial-liposomes from Slc25a44 KO brown adipocytes expressing an empty vector (KO+vector) or Slc25a44 (KO+ Slc25a44 ). n = 3/group, technically independent samples. Representative result from two independent experiments. Mean ± s.e.m.; two-sided P- values by unpaired Student’s t -test ( d , e ) or two-way ANOVA ( f ).

Article Snippet: BAT-specific Slc25a44 KD mice ( Slc25a44 BAT KD) were generated by injecting AAV expressing gRNA- Slc25a44 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, Vector Biolabs) or control GFP (AAV8-CAG-EGFP) into interscapular BAT following the published protocol .

Techniques: Expressing, Control, Plasmid Preparation, Liposomes

Journal: Nature

Article Title: BCAA catabolism in brown fat controls energy homeostasis through SLC25A44

doi: 10.1038/s41586-019-1503-x

Figure Lengend Snippet: a. Expression profile of Slc25a family members in the inguinal WAT of mice acclimated to 23°C or 12°C for one week. n = 3/group. b. mRNA expression of UCP1 , SLC25A44 , and SLC25A39 normalized to TBP levels in the supraclavicular BAT from the same individuals (6 pairs) at thermoneutrality (TN, 27°C) and cold temperature (19°C). c. Mitochondrial localization of SLC25A44 protein in differentiated mouse beige adipocytes. TOM20 as a mitochondrial marker. d. Immunoblotting for SLC25A44 in BAT and liver of control and Slc25a44 KD mice. GAPDH as a loading control. Red arrows indicate specific bands whose intensities were decreased in Slc25a44 KD mice. e. mRNA expression of Slc25a44 and indicated genes normalized by 36B4 levels during mouse brown adipogenesis. n = 4/group. f. Protein expression of SLC25A44 in mouse beige preadipocytes and differentiated adipocytes. β-actin as a loading control. g. Protein expression of UCP1 and SLC25A44 in immortalized human brown preadipocytes and differentiated adipocytes. β-actin as a loading control. a , b , e , biologically independent samples. Means ± s.e.m.; one-sided P -values by paired t -test ( b ) and two-sided P- values by unpaired Student’s t -test ( a ). c , d , f , g , representative results from two independent experiments. Uncropped images are available in .

Article Snippet: BAT-specific Slc25a44 KD mice ( Slc25a44 BAT KD) were generated by injecting AAV expressing gRNA- Slc25a44 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, Vector Biolabs) or control GFP (AAV8-CAG-EGFP) into interscapular BAT following the published protocol .

Techniques: Expressing, Marker, Western Blot, Control

Journal: Nature

Article Title: BCAA catabolism in brown fat controls energy homeostasis through SLC25A44

doi: 10.1038/s41586-019-1503-x

Figure Lengend Snippet: a. Genomic Slc25a44 sequence and amino acid sequence of Slc25a44 KO brown cell line. Homozygous mutation in the Slc25a44 gene by CRISPR-Cas9 results in a premature stop codon in KO cells. b. Scheme of mitochondrial BCAA uptake assay. Isolated mitochondria from differentiated brown adipocytes were incubated with [U- 14 C 5 ] Val. Mitochondrial uptake was quantified by a scintillation counter. c. Validation of mitochondrial Val uptake assay in differentiated brown adipocytes. Note that addition of excess non-labeled Val (20 mM) abolished [U- 14 C 5 ] Val uptake into the mitochondria. d. mRNA expression of Slc25a44 and Slc25a39 in differentiated mouse brown adipocytes expressing a scrambled control shRNA (Scr) and shRNAs targeting Slc25a44 (shRNA #1, #2), Slc25a39 , or both Slc25a44 shRNA #1 and Slc25a39 shRNA (double knockdown). n = 4/group for all group except n = 6 for Scr control. e. Mitochondria uptake of [U- 14 C 5 ] Val (left) and [U- 14 C 6 ] Leu (right) in brown adipocytes in ( d ). n = 3/group. f. mRNA and protein expression of Slc25a44 in mitochondria of Neuro2a cells expressing an empty vector or Slc25a44 . COX-IV as a loading control. n = 3/group. g. Immunoblotting for SLC25A44 in the isolated mitochondria from differentiated Slc25a44 KO brown adipocytes expressing an empty vector or Slc25a44 . TOM20 as a loading control. h. Immunoblotting of SLC25A44 in the mitochondria-fused liposome. Mitochondrial membrane isolated from Slc25a44 KO brown adipocytes expressing an empty vector or Slc25a44 was fused with liposome. TOM20 as a loading control. i. [U- 14 C 6 ] Leu uptake rate in the liposome in ( h ). n = 3/group. j. [U- 14 C 5 ] Glutamate (Glu) uptake rate in the liposome in ( h ). n = 3/group. k. Coomassie-blue staining of purified SLC25A44 protein from HEK293S cells overexpressing Slc25a44 . l. Immunoblotting of SLC25A44 in liposomes reconstituted with purified SLC25A44 (proteo-liposome) and liposomes reconstituted without SLC25A44 (empty liposome). m. Left: [U- 14 C 6 ] Leu transport into proteo-liposome in ( l ). Right: Leucine uptake rate. n = 3/group. d-f , biologically independent samples. i , j , m , technically independent samples. f-m , representative result from two independent experiments. Means ± s.e.m.; two-sided P- values by unpaired Student’s t -test ( f , i , j , m ) or one-way ANOVA followed by Tukey’s post-hoc test ( d , e ). f-h , k - l , uncropped images are available in .

Article Snippet: BAT-specific Slc25a44 KD mice ( Slc25a44 BAT KD) were generated by injecting AAV expressing gRNA- Slc25a44 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, Vector Biolabs) or control GFP (AAV8-CAG-EGFP) into interscapular BAT following the published protocol .

Techniques: Sequencing, Mutagenesis, CRISPR, Isolation, Incubation, Biomarker Discovery, Labeling, Expressing, Control, shRNA, Knockdown, Plasmid Preparation, Western Blot, Membrane, Staining, Purification, Liposomes

Journal: Nature

Article Title: BCAA catabolism in brown fat controls energy homeostasis through SLC25A44

doi: 10.1038/s41586-019-1503-x

Figure Lengend Snippet: a. DNA constructs used in the generation of dCas9-KRAB mice. The dCas9-KRAB construct was inserted into the Hipp11 (H11) gene locus by the site-specific PhiC31 integrase. b. Experimental procedure of gRNA screening. MEFs from dCas9-KRAB mice were used to identify gRNA that effectively deplete Slc25a44 . The right graph: Slc25a44 knockdown efficiency for six independent gRNAs in the dCas9-KRAB-derived MEFs ( n = 2/group). gRNA- Slc25a44 #1 (indicated by a red arrow) was used for generation of gRNA Tg mouse. c. Schematics of BAT-specific Slc25a44 KD mice ( Slc25a44 BAT KD) by using the dCas9-KRAB system. AAV8-CAG-EGFP-U6-gRNA-tracr targeting Slc25a44 was injected into the interscapular BAT of mice expressing dCas9-KRAB on the H11 locus (dCas9-KRAB mouse). AAV8-CAG-EGFP without gRNA was used as a control. d. mRNA expression of Gfp normalized by 36B4 in the indicated tissues of dCas9-KRAB mice in ( c ). n = 4/group. e. H&E staining of inguinal WAT and liver of control and Slc25a44 BAT KD mice. b , d , biologically independent samples. Means ± s.e.m.; two-sided P -values by unpaired Student’s t -test ( d ).

Article Snippet: BAT-specific Slc25a44 KD mice ( Slc25a44 BAT KD) were generated by injecting AAV expressing gRNA- Slc25a44 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, Vector Biolabs) or control GFP (AAV8-CAG-EGFP) into interscapular BAT following the published protocol .

Techniques: Construct, Knockdown, Derivative Assay, Injection, Expressing, Control, Staining

Journal: Nature

Article Title: BCAA catabolism in brown fat controls energy homeostasis through SLC25A44

doi: 10.1038/s41586-019-1503-x

Figure Lengend Snippet: a. Slc25a44 mRNA expression in indicated tissues of Slc25a44 BAT KD and control mice. n = 4/group. b. Immunoblotting of SLC25A44 in BAT of mice in ( a ). β-actin as a loading control. Representative result from two independent experiments. Gel source data are in . c . Morphology, H&E staining, and immunofluorescent GFP staining of BAT in ( a ). DAPI was used for counter staining. Scale bar: 100 μm. Representative result from two independent mice. d. Tissue temperature of BAT and muscle in ( a ) following NE treatment (arrows). n = 5 (control), n = 7 ( Slc25a44 BAT KD). e. Rectal core-body temperature of Slc25a44 KD ( n = 6) and controls ( n = 7) following cold exposure at 8°C. f. Valine oxidation in indicated tissues normalized by tissue mass. n = 4/group. g. Plasma BCAA levels in ( e ) following 8-hour cold temperature at 8°C. n = 6/group. h. NE-induced OCR normalized by total protein in control and Slc25a44 KO brown adipocytes. n = 9/group (Ctrl+Val, KO+Val+KIV), n = 10/group (KO+Val, KO+Val+succinate). i. Valine oxidation in inguinal WAT-derived white adipocytes expressing an empty vector or Slc25a44 after NE treatment. n = 5 (vehicle), n = 6 (NE). j. A proposed model of BCAA catabolism in thermogenic adipose cells. Cold stimuli activate BCAA uptake and oxidation in the mitochondria of thermogenic adipocytes. Mitochondrial BCAA oxidation promotes BAT thermogenesis. This process requires SCL25A44, the mitochondrial BCAA transporter. SLC7A5, L-amino acid transporter 1. Norepinephrine, NE. a , d-i , biologically independent samples. Mean ± s.e.m.; two-sided P- values by unpaired Student’s t -test ( a,f ), one-way factorial ( h ) or two-way repeated measures ANOVA ( d,e,g ) followed by post-hoc paired/unpaired t -test with Bonferroni’s correction ( g ) or Tukey’s test ( h,i ).

Article Snippet: BAT-specific Slc25a44 KD mice ( Slc25a44 BAT KD) were generated by injecting AAV expressing gRNA- Slc25a44 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, Vector Biolabs) or control GFP (AAV8-CAG-EGFP) into interscapular BAT following the published protocol .

Techniques: Expressing, Control, Western Blot, Staining, Clinical Proteomics, Derivative Assay, Plasmid Preparation

Journal: Nature

Article Title: BCAA catabolism in brown fat controls energy homeostasis through SLC25A44

doi: 10.1038/s41586-019-1503-x

Figure Lengend Snippet: a. Generation of Slc25a44 KD mice by the dCas9-KRAB system. The dCas9-KRAB mouse was crossed with transgenic mouse expressing gRNA targeting Slc25a44 to generate SLC25A44 deficient mice. b. Slc25a44 mRNA expression normalized by 36B4 levels and protein expression in the BAT of mice in ( a ). β-actin as a loading control. n = 5 (control), and 4 ( Slc25a44 KD). c. Expression profile of Slc25a family members in BAT in ( a ) by RNA-seq analysis. The color scale shows Z-scored fold change in FPKM ( Slc25a44 KD vs control). n = 3/group. d. mRNA expression of Slc25a families normalized by 36B4 levels in Slc25a44 KO and control brown adipocytes. n = 6/group. e. H&E staining of BAT, inguinal WAT, liver, and gastrocnemius muscle from mice in ( a ). f. Triglyceride (TG) content in the interscapular BAT of Slc25a44 KD and control mice. n = 4/group. g. Expression profile of fatty acid synthesis- and oxidation-related genes in BAT of mice in ( a ) by RNA-seq analysis. The color scale shows Z-scored fold change in FPKM ( Slc25a44 KD vs control). n = 3/group. h. Oleic acid oxidation normalized by tissue mass (mg) in BAT of Slc25a44 KD and control mice acclimated to thermoneutral 30°C or cold temperature (12°C) for one week. n = 4/group. i. EMG measurement of muscle shivering in control mice and Slc25a44 KD mice at 30 ºC or 8 ºC. The lower graph shows the quantitative root mean square (RMS) of the EMG. n = 6/group. j. Tissue temperature in indicated tissues of control and Slc25a44 KD mice following NE treatment (indicated by red arrows). n = 4/group. b-d , f-j , biologically independent samples. Means ± s.e.m.; two-sided P -values by unpaired Student’s t -test ( b-d , f - g ), two-way factorial ANOVA followed by Tukey’s post-hoc test ( h ), or two-way repeated measures ANOVA ( i,j ) followed by post hoc paired/unpaired t -test with Bonferroni’s correction ( i ). b , e , representative results from two independent experiments. Uncropped immunoblot images are available in .

Article Snippet: BAT-specific Slc25a44 KD mice ( Slc25a44 BAT KD) were generated by injecting AAV expressing gRNA- Slc25a44 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, Vector Biolabs) or control GFP (AAV8-CAG-EGFP) into interscapular BAT following the published protocol .

Techniques: Transgenic Assay, Expressing, Control, RNA Sequencing, Staining, Western Blot

Journal: Nature

Article Title: BCAA catabolism in brown fat controls energy homeostasis through SLC25A44

doi: 10.1038/s41586-019-1503-x

Figure Lengend Snippet: a. Immunoblotting of SLC25A44 in human brown adipocytes expressing a scrambled control shRNA (Scr) and shRNAs targeting SLC25A44 (#1, #2). β-actin as a loading control. b. mRNA expression of SLC25A44 normalized by TBP levels in ( a ). n = 3/group. c. NE-induced OCR normalized by total protein (μg) in the presence and absence of Val supplementation in ( a ). Differentiated human brown adipocytes in the BCAA-free medium were supplemented with Val or vehicle, and subsequently treated with NE. n = 9/group (Scr control, sh- Slc25a44 #1), n = 10/group (sh- Slc25a44 #2). d. Means NE-induced OCR in ( c ). e. Illustration of Val metabolism in the mitochondria. f. Immunoblotting of mitochondrial proteins (as indicated) in the interscapular BAT of control and Slc25a44 KD mice. GAPDH as a loading control. g. ETC activity of BAT mitochondria. Isolated mitochondria from BAT of control mice and Slc25a44 KD mice were treated with rotenone (2 μM), succinate (10 mM), AA (5 μM), and TMPD (100 μM) with ascorbate (Asc, 10mM). n = 5/group. h. mRNA expression of Slc25a44 normalized by 36B4 in mouse beige adipocytes expressing an empty vector ( n = 3) or Slc25a44 ( n = 4). i. Mitochondrial Val uptake in beige adipocytes in ( h ). n = 3/group. j. NE-induced OCR in ( h ). Differentiated adipocytes in the BCAA-free medium were supplemented with Val or vehicle, and subsequently stimulated with NE. Vector: n = 20 (vehicle) and 16 (Val). Slc25a44 : n = 13 (vehicle) and 16 (Val). k. Immunoblotting of SLC25A44 in C2C12 myotubes expressing an empty vector or Slc25a44 . β-actin as a loading control. l. Valine oxidation normalized by total protein (μg) in C2C12 myotubes in ( k ). n = 6/group. m. OCR normalized by total protein (μg) in C2C12 myotubes in ( k ). n = 9/group. b-d , g-j , l-m , biologically independent samples. Means ± s.e.m; two-sided P -values by unpaired Student’s t -test ( h , i , l , m ), one-way ( b ) or two-way ( d , j ) factorial ANOVA followed by Tukey’s post-hoc test, or two-way repeated measures ANOVA ( c , g ). a , f , k , representative results from two independent experiments. Uncropped immunoblot images are available in .

Article Snippet: BAT-specific Slc25a44 KD mice ( Slc25a44 BAT KD) were generated by injecting AAV expressing gRNA- Slc25a44 (AAV8-CAG-EGFP-U6-gRNA-long tracr; custom order, Vector Biolabs) or control GFP (AAV8-CAG-EGFP) into interscapular BAT following the published protocol .

Techniques: Western Blot, Expressing, Control, shRNA, Activity Assay, Isolation, Plasmid Preparation

Journal: Neuron

Article Title: Neurovascular coupling in the dentate gyrus regulates adult hippocampal neurogenesis

doi: 10.1016/j.neuron.2019.05.045

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rat anti-BrdU Abcam Cat# ab6326, RRID: AB_305426 Goat anti-doublecortin Santa Cruz Cat# sc-8066, RRID: AB_2088494 Mouse anti-NOS1 Santa Cruz Cat# sc-5302, RRID: AB_626757 Rabbit anti-parvalbumin Abcam Cat#: 11427, RRID: AB_298032 Mouse anti-NeuN MilliporeSigma Cat# MAB377, RRID: AB_2298772 Rat anti-CD31 BD Biosciences Cat# 557355, RRID: AB_396660 Rabbit anti-Cleaved Caspase-3 Cell Signaling Cat# 9664, RRID: AB_2070042 Bacterial and Virus Strains AAV9.CamKII.GCaMP6f.WPRE.SV40 Penn Vector Core/Addgene Cat# 100834-AAV9 AAV8-CaMKIIa-EGFP Addgene Cat# 50469-AAV8 AAV8-CaMKIIa-hM3Dq-mCherry Addgene Cat# 50476-AAV8 AAV8-CaMKIIa-hM4Di-mCherry Addgene Cat# 50477-AAV8 AAV8-hSyn-DIO-hM3Dq-mCherry Addgene Cat# 44361-AAV8 AAV8-hSyn-DIO-hM4Di-mCherry Addgene Cat# 44362-AAV8 AAV8-hSyn-DIO-mCherry Addgene Cat# 50459-AAV8 AAV8-GFP-U6-m-NOS1-shRNA Vector Biolabs Cat# shAAV-279053 AAV8-GFP-U6-scrmb-shRNA Vector Biolabs Cat# AAV8-GFP-U6-scrmb-shRNA Chemicals, Peptides, and Recombinant Proteins 5-bromo-2′-deoxyuridine (BrdU) Sigma Cat# B5002 Clozapine-N-oxide (CNO) Enzo Life Sciences Cat# BML-NS105-0005 N omega-nitro-L-arginine methyl ester (L-NAME) Sigma Cat# N5751 Picropodophyllotoxin (PPP) Cayman Chemical Company Cat# 17329 Experimental Models: Organisms/Strains Mouse: PV-Cre Jackson Laboratory stock# 017320 Open in a separate window KEY RESOURCES TABLE Hippocampus-engaged exploration induces functional hyperemia in the dentate gyrus Functional hyperemia is critical to exploration-induced hippocampal neurogenesis Parvalbumin expressing neurons increase blood flow via nitric oxide signaling Functional hyperemia elevates IGF-1 pathway activity in the dentate gyrus

Techniques: Virus, Plasmid Preparation, Recombinant