Journal: Frontiers in Immunology

Article Title: Male Macrophages and Fibroblasts from C57/BL6J Mice Are More Susceptible to Inflammatory Stimuli

doi: 10.3389/fimmu.2021.758767

Figure Lengend Snippet: Characterization of bone marrow derived macrophages (BMMs) and cardiac fibroblasts. (A) Flow cytometric analysis of the F4/80 and CD11b expression in BMMs. Representative images of BMMs stained against (B, C) F4/80 and (D, E) CD11b. Representative images of cardiac fibroblasts stained against (F, G) vimentin, (H, I) CD31 (endothelial marker; negative control) and (J, K) desmin (smooth muscle marker; negative control). Nuclei were stained with DAPI (C, E, G, I, K) Magnification: 40x; scale bar: 50 µm. Data are representative of 3 independent experiments with similar results.

Article Snippet: Fibroblasts were activated using 20 ng/ml TNF-α ( ) (PeproTech, Germany), 10 ng/ml TGF-β (PeproTech, Germany) ( ) or 10 ng/ml LPS (Sigma, Germany) for 24 h in fibroblast starvation medium (with 2.5% charcoal-stripped FCS, Biochrom, Germany).

Techniques: Derivative Assay, Expressing, Staining, Marker, Negative Control

Journal: Frontiers in Immunology

Article Title: Male Macrophages and Fibroblasts from C57/BL6J Mice Are More Susceptible to Inflammatory Stimuli

doi: 10.3389/fimmu.2021.758767

Figure Lengend Snippet: TNF-α, but not TGF-β, induces pro-fibrotic phenotype in mouse cardiac fibroblasts. Real-time PCR analyses of (A) MCP-1, (B) TGF-β, (C) IL-1β, and (D) Col1A1 performed with lysates from male and female mice cardiac fibroblasts treated with 20 ng/ml TNF-α for 24 h. Real-time PCR analyses of (E) MCP-1 and (F) Col1A1 performed with lysates from male and female mice cardiac fibroblasts treated with 10 ng/ml TGF-β for 24 h. Data are shown as means ± SEM (n = 6; independent experiments with technical duplicates). Data are normalized to the male untreated group. *p < 0.05, **p < 0.01, untreated vs. treated.

Article Snippet: Fibroblasts were activated using 20 ng/ml TNF-α ( ) (PeproTech, Germany), 10 ng/ml TGF-β (PeproTech, Germany) ( ) or 10 ng/ml LPS (Sigma, Germany) for 24 h in fibroblast starvation medium (with 2.5% charcoal-stripped FCS, Biochrom, Germany).

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Male Macrophages and Fibroblasts from C57/BL6J Mice Are More Susceptible to Inflammatory Stimuli

doi: 10.3389/fimmu.2021.758767

Figure Lengend Snippet: Pro-inflammatory environment promotes a pro-inflammatory and pro-fibrotic fibroblast phenotype. Real-time PCR analyses of (A) MCP-1, (B) TNF-α, (C) NFκB, and (D) IL-1β performed with lysates from male and female mice cardiac fibroblasts cultivated with conditioned medium from M1 polarized BMMs for 24 h. Data are shown as means ± SEM (n = 6; independent experiments with technical duplicates). Data are normalized to the male untreated group. **p < 0.01, ***p < 0.001, untreated vs. treated; ## p < 0.01, male vs. female.

Article Snippet: Fibroblasts were activated using 20 ng/ml TNF-α ( ) (PeproTech, Germany), 10 ng/ml TGF-β (PeproTech, Germany) ( ) or 10 ng/ml LPS (Sigma, Germany) for 24 h in fibroblast starvation medium (with 2.5% charcoal-stripped FCS, Biochrom, Germany).

Techniques: Real-time Polymerase Chain Reaction

Journal: Nature cell biology

Article Title: The lysosomal GPCR-like protein GPR137B regulates Rag and mTORC1 localization and activity

doi: 10.1038/s41556-019-0321-6

Figure Lengend Snippet: ( a ) Predicted transmembrane domains of GPR137B. ( b ) Confocal images of expressed GPR137B-YFP, lysosome marker Lamp2 and mTOR in Hs68 cells. ( c ) siRNA knockdown of GPR137B reduces amino acid-induced mTOR translocation. ( d ) Analysis of mTOR and Lamp2 co-localization and rpS6 phosphorylation for siRNAs targeting GPR137B, Rheb and RagC. Error bars, ± SD of mean; n=3 independent experiments. ( e ) Confocal images of Hela cells showing increased amino acid-induced mTOR translocation in cells expressing GPR137B-YFP or Lamp1-GFP as control. ( f ) Binary mask analysis showing that amino acid-triggered increase in mTOR localization at lysosomes is regulated by GPR137B-YFP overexpression. Error bars, ± SD of mean, 8 independent experiments, p=1.6×10 −13 (two-tailed Student’s t-test). ( g ) Lysosome localization of GPR137-YFP and GPR137C-YFP (HeLa cells). ( h ) siRNA knockdown of GPR137B enhances autophagy as measured by LC3-GFP. Error bars, ± SD of mean; n=3 independent experiments (two-tailed Student’s t-test). ( i, j ) siRNA knockdown of GPR137B causes autophagy flux defects as measured by p62 staining. Scale bar, 20 μm. Error bars, ± SD of mean (P value, ratio paired, two-tailed Student’s t-test; n=3 independent experiments). ( k ) GPR137B-YFP expression causes phosphorylation of 4E-BP1 during amino acid starvation. Error bars, ± SEM of mean (P values, comparison of transfected, n= 2329 cells, no aa; 2211 cells, plus aa; 2308 cells, Torin; to untransfected cells, n=3256 cells, no aa; 3099 cells, plus aa; 3048 cells, Torin; two-tailed Student’s t-test). ( l ) Knockdown of GPR137B inhibits amino acid-induced S6K and 4E-EP1 phosphorylation. HEK293T cells transfected with siControl, siGPR137B, or siRagA/C were amino acid-starved for 1 hour and stimulated w/wo amino acid for 30 min. Western blot analysis of phosphorylation of S6K and 4E-BP1. ( m ) Same as ( l ) but with transiently transfected GPR137B-3xFLAG or Lamp1–3xFLAG. Amino acid starvation for 3 hours and amino acid re-addition for 30 min w/wo rapamycin. Results in b, c , g were confirmed by 3 independent experiments, in l and m by 2 independent experiments. In all figures, *, p=0.01 to 0.05; **, p=0.001 to 0.1, ***, p<0.001. All scale bars are 10 μm except when noted.

Article Snippet: Amino acid starvation medium (DMEM without amino acids) was purchased from Athena Enzyme Systems as a custom media.

Techniques: Marker, Knockdown, Translocation Assay, Phospho-proteomics, Expressing, Control, Over Expression, Two Tailed Test, Staining, Comparison, Transfection, Western Blot

Journal: Nature cell biology

Article Title: The lysosomal GPCR-like protein GPR137B regulates Rag and mTORC1 localization and activity

doi: 10.1038/s41556-019-0321-6

Figure Lengend Snippet: ( a ) mTORC1 interaction with HA-RagA was markedly reduced in cells treated with siGPR137B. GPR137B was knocked down in HEK293T cells stably expressing HA - RagA. HA-RagA was immunoprecipitated and the interaction with endogenous mTORC1 subunits was detected by Westernblot. ( b ) mTORC1 interaction with HA-RagA was increased in GPR137B overexpressing cells. GPR137B was transiently overexpressed in HEK293T cells stably expressing HA - RagA. In a and b, the cells were amino acid-starved for 2 hours then treated with starvation or amino acid-containing media for 10 min. Cells were then treated with a cell-permeable chemical cross-linker (DSP) and subjected to HA immunoprecipitation. n = 2 independent experiments in a and b . ( c ) Analysis of synergism between GPR137B and Rags for mTORC1 activation. GPR137B co-expression with RagC promotes Rag expression-dependent recruitment of mTORC1 during amino acid starvation (10–12hrs). Control heatmap experiments show co-expression of Lamp1 with RagC does not induce mTORC1 translocation at any expression level of Lamp1 or RagC ( left ), but co-expression of GPR137B with RagC causes synergistic mTORC1 translocation at even low levels of RagC expression in the absence of amino acids ( right ). Heatmaps are plotted from translocation scores pooled from 3 independent experiments. ( d ) Synergy analysis as in c comparing GPR137B expressing and control Lamp1 expressing cells. There is a sharp expression dependence of mTORC1 translocation on RagC expression only when co-expressed with GPR137B. Error bars represent mean ± SD and n=3 independent experiments. ( e ) GPR137B co-expression with wildtype RagA/C in RagA/B−/− MEFs promotes RagA/C expression-dependent mTORC1 translocation in the absence of amino acids, and this effect is significantly lower in RagA/B−/− MEFs co-expressing GPR137B and dominant negative RagA/C (RagA T21L/RagC Q120L). RagA/B−/− MEFs transiently expressing either Lamp1 as control or GPR137B, along with WT or DN Rag A/C proteins, starved for 2hrs, fixed and stained for mTOR and Lamp2. In c, d and e , RagC expression is used as a readout of RagA/C dimer expression.

Article Snippet: Amino acid starvation medium (DMEM without amino acids) was purchased from Athena Enzyme Systems as a custom media.

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Activation Assay, Control, Translocation Assay, Dominant Negative Mutation, Staining

Journal: Nature cell biology

Article Title: The lysosomal GPCR-like protein GPR137B regulates Rag and mTORC1 localization and activity

doi: 10.1038/s41556-019-0321-6

Figure Lengend Snippet: ( a, b ) Confocal images of HAP-1 parental, GPR137B KO and GPR137 KO cells stained with Hoechst and Lysotracker Green. Quantification in b . ( c ) HAP-1 GPR137 KO and GPR137B KO cells have increased number of autophagosomes and/or autolysosomes as measured by endogenous LC3B puncta in response to amino acid starvation. KO clones have statistically significant upregulation from parental. In b and c , mean ± SD, n=4 independent experiments, ratio paired t-test. ( d ) Amino acid-induced mTOR translocation is not statistically significantly different in HAP-1 GPR137B KO and GPR137 KO cells. ( e ) Lysosomal localization of RagA is reduced in HAP-1 GPR137B KO and GPR137 KO cells. Every KO has statistically significant downregulation from parental. n=3 independent experiments in d , e. Error bars representing mean ± SD (one-way ANOVA analysis followed by Tukey’s test). ( f ) Wildtype and gpr137ba −/− zebrafish have comparable numbers of neutral red positive microglia. (g) Quantitative analysis of ( f ) using unpaired, parametric, two-tailed t-test with Welch’s correction (p=0.75), wildtype n=9 animals, gpr137ba −/− n=5 animals. Center values indicate mean and error bars represent SEMs. ( h ) LysoTracker Red staining in wildtype ( left ), and gpr137ba −/− zebrafish larvae ( right ), show enlarged lysosomal compartments in the microglia of gpr137ba −/− fish. Scale bar, 50 μm. LysoTracker Red positive compartments are segmented and analyzed for area in wildtype and gpr137ba −/− ( bottom ). (i) Quantification of area of LysoTracker Red punctae in zebrafish microglia shows that gpr137ba −/− larvae (n=11) have significantly (p=0.008) larger LysoTracker Red positive compartments relative to wildtype larvae (n=14). Data points represent the average area of the punctae in a single animal. Mean values and SEMs shown (unpaired, parametric, two-tailed t-test with Welch’s correction). ( j ) Quantitative PCR analysis of Transcription Factor EB targets in pooled gpr137ba −/− zebrafish relative to a pool of heterozygous ( gpr137ba +/− ) controls (n=15–16 animals in each pool); mutations in gpr137 and gpr137c are randomly assorted in the pools (see text and ). Targets of TFEB significantly upregulated by comparing ΔCt values: arghgdia p=0.01, mapk1 p=0.001, sqstm p=0.001, ctsc p=0.05. Mean values and SEMs shown (unpaired, parametric two-tailed t-test with Welch’s correction).

Article Snippet: Amino acid starvation medium (DMEM without amino acids) was purchased from Athena Enzyme Systems as a custom media.

Techniques: Staining, Clone Assay, Translocation Assay, Two Tailed Test, Real-time Polymerase Chain Reaction