Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: OGD/R model induced THBS1 expression in RAW264.7 cells and apoptosis in AML12 cells. (A) Schematic of OGD/R treatment. AML12 cells (lower chamber) and RAW264.7 cells (upper chamber) were co-cultured in serum-free, glucose-free DMEM under hypoxic conditions (1% O2) for 6 hours, followed by re-oxygenation in complete medium for another 3 hours. Control cells were cultured in complete medium under normoxic conditions. (B) THBS1 protein expression in RAW264.7 cells between Control and OGD/R groups. (C) The contents of IL-1β, IL-6, and TNF-α in RAW264.7 cell culture medium between Control and OGD/R groups. (D) The expressions of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between Control and OGD/R groups. (E) Cell death in the Control and OGD/R groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (F) The mitochondrial membrane potential (ΔψM) of the Control and OGD/R groups was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Cell Culture, Control, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: RAW264.7-specific THBS1 siRNA treatment alleviated OGD/R-induced apoptosis in AML12 cells. (A) Schematic of OGD/R treatment. RAW264.7 cells, separately transfected with siRNA-NC and siRNA-THBS1, were seeded in the upper chamber and co-cultured with AML12 cells (lower chamber) under hypoxic conditions (1% O2) in serum-free, glucose-free DMEM for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (B) THBS1 protein expression in RAW264.7 cells between siRNA-NC and siRNA-THBS1 groups. (C) The contents of IL-1β, IL-6, and TNF-α in RAW264.7 cell culture medium between siRNA-NC and siRNA-THBS1 groups. (D) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between siRNA-NC and siRNA-THBS1 groups. (E) Cell death in the siRNA-NC and siRNA-THBS1 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (F) The mitochondrial membrane potential (ΔψM) of the siRNA-NC and siRNA-THBS1 groups was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Transfection, Cell Culture, Expressing, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: THBS1 promoted hepatocyte apoptosis by inhibiting PI3K-AKT- NF-κB signaling. (A) The expression of p-AKT, AKT, p-NF-κB p65 and NF-κB p65 proteins in AML12 cells between siRNA-NC and siRNA-THBS1 groups. (B) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells among the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 groups. (C) Cell death in the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (D) The mitochondrial membrane potential (ΔψM) of the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: THBS1-mediated hepatocyte apoptosis depended on the CD47 receptor. (A) CD47 protein expression in AML12 cells between Control and OGD/R groups. (B) Schematic of OGD/R treatment. AML12 cells, separately transfected with siRNA-NC and siRNA-CD47, were seeded in the lower chamber and co-cultured with RAW264.7 cells (upper chamber) under hypoxic conditions (1% O2) in serum-free, glucose-free DMEM for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (C) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between siRNA-NC and siRNA-CD47 groups. (D) Cell death in the siRNA-NC and siRNA-CD47 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (E) Schematic of OGD/R treatment. AML12 cells were separately transfected with siRNA-NC and siRNA-CD47, respectively, then cultured in serum-free, glucose-free DMEM supplemented with 1 ng/mL rmTHBS1 under hypoxic conditions (1% O2) for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (F) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells among the siRNA-NC, siRNA-NC+ rmTHBS1 and siRNA-CD47+ rmTHBS1 groups. (G) Cell death in the siRNA-NC, siRNA-NC+ rmTHBS1 and siRNA-CD47+ rmTHBS1 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (H) Schematic diagram of the THBS1-CD47 axis’s role in macrophage-hepatocyte crosstalk during HIRI.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Control, Transfection, Cell Culture, Double Staining

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D inhibits EC migration but not proliferation. ( A ) Representative images from a wound healing scratch assay using control, wild-type TSP1, and phosphomutant TSP1-transfected BPAECs. Scratches were made in a confluent EC monolayer 24 h post-transfection. Images were captured at 0, 8, and 24 h post-scratch. Scale bar = 100 µm. ( B ) Wound closure was evaluated by measuring the open area at each time point, normalized to the 0 h image (**** p < 0.0001). Statistical analysis was completed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SD ( n = 5). ( C ) Representative measurement of an in vitro wound healing assay performed using ECIS. Wounding was applied at 1 h. Each line represents the mean of three replicates ± SD. ( D ) Statistical analysis of EC migration rate was performed using one-way ANOVA with Tukey’s test (**** p < 0.0001). Data are represented as mean ± S.D ( n = 8). ( E ) BPAEC were either untransfected (ctr) or transfected with TSP1 WT -, TSP1 S93A -, or TSP1 S93D -expressing constructs. Cell proliferation was measured by an MTT assay. Absorbance values were normalized to 0 h. No significant differences in proliferation were observed between groups. Data represent SD ( n = 6).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Migration, Wound Healing Assay, Control, Transfection, In Vitro, Expressing, Construct, MTT Assay

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D inhibits FAK signaling and downstream targets during cell migration. ( A ) Control or TSP1-transfected confluent monolayers of BPAECs were scratched 24 h post-transfection. Samples were collected at the indicated time points (0, 4, and 8 h). Overexpression of TSP1 and the levels of signaling protein were analyzed by Western blot. ( B ) Quantitative analysis was performed by densitometry of the Western blot bands. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test ( n = 3–5) (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Migration, Control, Transfection, Over Expression, Western Blot

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D shows enhanced binding to ITGB1. ( A ) Bacterially expressed GST and GST-TSP1 1–221 WT, GST-TSP1 1–221 S93A, and GST-TSP1 1–221 S93D recombinant proteins immobilized on glutathione Sepharose beads were incubated with BPAEC lysate for pull-down assays. EC lysates and the eluted proteins were analyzed by Western blot using ITGB1- and TSP1-specific antibodies. ( B ) Quantitative analysis of pull-down samples. Statistical analysis was performed using one-way ANOVA ( n = 4) (** p < 0.01). ( C ) Control and TSP1-transfected BPAECs were subjected to immunoprecipitation using c-myc antibody to purify recombinant TSP1 proteins. Total cell lysates and immunocomplexes were tested for c-myc and ITGB1 by Western blot. ( D ) Quantitative analysis of IP. Statistical analysis was performed using one-way ANOVA ( n = 4) (*** p < 0.001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Binding Assay, Recombinant, Incubation, Western Blot, Control, Transfection, Immunoprecipitation

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D alters ITGB1 clusterization. ( A ) Representative images of control cells and cells expressing TSP1 recombinant variants, immunostained for ITGB1 (green) and nuclei (DAPI, blue). Images were acquired using the Opera Phenix HCS (PerkinElmer, Inc., Shelton, CT, USA. Scale bar: 100 μm. ( B ) Quantitative analysis of ITGB1 clusters was performed using the built-in Harmony software (version 4.8, Perkin Elmer). Statistical analysis was performed using one-way ANOVA. Data are presented as means ± S.D, and >10,000 cells were analyzed per condition. (**** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Control, Expressing, Recombinant, Software

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: The S93A mutation of TSP1 increases its secretion via N-glycosylation. ( A ) Cell culture supernatants were analyzed for TSP1 levels using ELISA. Statistical analysis of TSP1 concentration was performed using one-way ANOVA followed by Tukey’s post hoc test ( n = 7). Data are reported as means ± S.D. ( B ) TSP1 glycosylation was assessed by immunoprecipitating c-myc–tagged TSP1 proteins from the supernatant of transfected cells. Total cell lysates, supernatants, and IP complexes were tested for c-myc. IP complexes were further analyzed for glycosylation using lectin and O-GlcNac antibody by Western blot. ( C ) Quantification of glycosylation of immunoprecipitated recombinant TSP1 proteins. O-GlcNAc levels were not significantly different among groups. Data are shown as normalized signal intensity. (** p < 0.01 and **** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Mutagenesis, Glycoproteomics, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection, Western Blot, Immunoprecipitation, Recombinant

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D enhances SMC migration and proliferation and induces morphological changes. ( A ) MOVAS cells were seeded at 10% confluence and treated with conditioned media from non-transfected (ctr) or TSP1-transfected BPAECs. Proliferation was assessed using MTT, with absorbance measured at 540 nm at 24, 48, and 72 h. Data are shown as means ± SEM ( n = 12). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (* p < 0.01). ( B ) Migration of treated MOVAS cells was measured using an ECIS-based wound healing assay. Data presented mean ± S.D. from three chambers per condition. ( C ) Statistical analysis of migration rates was performed using one-way ANOVA with Tukey’s post hoc test ( n = 5; means ± S.D.; **** p < 0.0001). ( D ) Representative images of control and TSP1-treated MOVAS cells analyzed by HCS. Actin filaments were stained with Texas Red phalloidin (red) and nuclei with DAPI. White arrows indicate filopodia of cells. Scale bars: 50 μm. ( E ) Morphological parameters of MOVAS cells were analyzed using Harmony software on the Opera Phenix HCS system. Data are presented as mean ± SD (1000–1600 cells per well, n = 4). Statistical analysis was performed using ANOVA (**** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Migration, Transfection, Wound Healing Assay, Control, Staining, Software

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D induces a shift toward a synthetic-like state in SMCs and increases IL-6 secretion. ( A ) MOVAS were treated with conditioned media from TSP1 overexpressing ECs. Expression levels of indicated proteins were tested by Western blot. Actin was used as a loading control. ( B ) Densitometric analysis of Western blot signals. Statistical analysis was performed using one-way ANOVA with Tukey’s test ( n = 4). ( C ) Representative images of control and TSP1-treated MOVAS cells analyzed by HCS. Actin filaments were stained with Texas Red phalloidin (red) and nuclei with DAPI. Scale bars: 50 μm. ( D ) Conditioned media treated and untreated MOVAS cell supernatants were analyzed by ELISA for IL-6. Statistical analysis was performed using one-way ANOVA ( n = 9). Data are reported as means ± S.D. (** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: TSP1 or IL-6 levels in the cell culture supernatant were quantified using a Human TSP1 ELISA Kit from Elabscience Biotechnology (E-EL-H1589) or a Mouse IL-6 ELISA Kit from Fine Test (EM0121), respectively.

Techniques: Expressing, Western Blot, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Cathepsin L as a dual-target to mitigate muscle wasting while enhancing anti-tumor efficacy of anti-PD-L1

doi: 10.1038/s41467-025-64500-0

Figure Lengend Snippet: A Tumor volume and tumor-free body weight (BW) changes at 13 weeks in CT26 mice. αPD-L1 responders (green, n = 5; per original study ; Supplementary Fig. ) versus IgG controls (black, n = 8); BW normalized to initial BW (IBW). B Male C57BL/6 mice were intratracheally (i.t.) injected with Kras IKKαL cells, treated intraperitoneally (i.p.) with the indicated antibodies or IgG three times weekly, and euthanized when any group reached ≥ 10% BW loss versus IBW. Lung size ( C ), tumor burden ( D ), BW changes ( E ), and gastrocnemius (GA) and tibialis anterior (TA) muscle weights ( F ) in TB versus CON mice ( n = 7 mice; BW and muscle weights normalized to IBW). Representative H&E of TA ( G ) and averaged myofiber CSA per mouse ( H ; n = 7; ≥100 myotubes per field; three fields per mouse). I Percentage of CD3 + CD8 + cells within CD45 + infiltrates in tumor and GA by flow cytometry ( n = 5). J Principal component analysis (PCA) of TA transcriptomes (CON, n = 5; IgG, n = 4; αPD-L1, n = 3; αCD8a, n = 3). Top five enriched Gene Ontology (GO) terms for differentially expressed genes (DEGs) in muscles: IgG vs. CON ( K ); αPD-L1 vs. IgG ( L ); αCD8a vs. IgG ( M ). Enrichment score represents −log 10 -transformed adjusted p -values; FDR false discovery rate. N WB analysis of indicated proteins in GA. Vinculin muscle-specific loading control. Samples derive from the same experiment; different gels for MuRF-1, LC3B-I/II, and another for vinculin were processed in parallel. O Final grip strength normalized to IBW ( n = 7 mice). Co-culturing of C2C12 myotubes ( P ) with splenic CD8 + T cells from CON or TB mice in Kras IKKαL cell-conditioned medium (CM); myotube diameter ( Q ) from 12 myotubes per well, biological triplicate wells, each dot means averaged diameter per well. R WB analysis of C2C12 myotube proteins after co-culture. For A , data are mean ± SEM, one-sided t -test; for D – F , H , I , O , and Q , mean ± SEM, two-sided unpaired t -test; for K – M , hypergeometric test with Benjamini–Hochberg correction. Source data are provided as a Source Data file.

Article Snippet: Following differentiation, C2C12 myotubes were treated with 10 ng/ml of mouse recombinant granzyme A (Gzma, HY- P76377 , MedChemExpress, Monmouth Junction, NJ, USA) with or without CTSLi (1 μg/ml) in the presence of either DM or CM for 96 h.

Techniques: Injection, Flow Cytometry, Muscles, Transformation Assay, Control, Co-Culture Assay

Journal: Nature Communications

Article Title: Cathepsin L as a dual-target to mitigate muscle wasting while enhancing anti-tumor efficacy of anti-PD-L1

doi: 10.1038/s41467-025-64500-0

Figure Lengend Snippet: A UMAP of scRNA-seq profiles from CD45 + cells isolated from lung tumors or GA muscles of TB mice (pooled from three animals), colored by tissue origin (left) and by immune cell types (right). Relative proportions of each CD45 + immune cluster in lung tumors ( B ) and GA muscle ( C ). D UMAP expression overlays for selected T cell-related and cytotoxicity genes in CD45 + cells isolated from tumors or muscle tissue (purple indicates expression; scale bar at right). Top five enriched GO/Kyoto Encyclopedia of Genes and Genomes (KEGG) terms for genes upregulated in classical T cells versus CD49a + CD8 + T cells ( E ) and vice versa ( F ). Enrichment scores represent −log 10 of the adjusted p-values from the hypergeometric test with Benjamini–Hochberg correction. G and H Percentage of CD49a + CD8 + T cells ( G , CD45 + CD3 + CD8 + CD49a + ) and CD8 + T cells ( H , CD45 + CD3 + CD8 + ) within CD45 + infiltrating tumor and GA muscle tissues by flow cytometry ( n = 6). I qRT-PCR of Itga1 , Prf1 , Gzma , and Gzmb in CD8 + T cells co-cultured with C2C12 myotubes in the presence or absence of CM as described in Fig. . Vinculin , a muscle-specific housekeeping gene for normalization ( n = 4 independent biological replicates per group). For E and F , hypergeometric test with Benjamini–Hochberg correction; for G – I , data are mean ± SEM, two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: Following differentiation, C2C12 myotubes were treated with 10 ng/ml of mouse recombinant granzyme A (Gzma, HY- P76377 , MedChemExpress, Monmouth Junction, NJ, USA) with or without CTSLi (1 μg/ml) in the presence of either DM or CM for 96 h.

Techniques: Isolation, Muscles, Expressing, Flow Cytometry, Quantitative RT-PCR, Cell Culture

Journal: Nature Communications

Article Title: Cathepsin L as a dual-target to mitigate muscle wasting while enhancing anti-tumor efficacy of anti-PD-L1

doi: 10.1038/s41467-025-64500-0

Figure Lengend Snippet: A Schematic of the animal study. Lung size ( B ), tumor burden ( C ), BW changes ( D ), and GA and TA muscle weights ( E ) in mice ( n = 8; weights normalized to IBW). F Representative H&E and Masson’s Trichrome images of TA muscle. G Muscle CSA quantification ( n = 8 mice per group; ≥ 100 myotubes per field, three fields per section from each mouse). H Final grip strength normalized to IBW ( n = 8 mice per group). I CTSL concentration in tumor and TA muscle ( n = 3 mice per group; normalized to tissue mass). J and K Percentage of indicated immune subsets within CD45 + infiltrating tumor and GA muscle tissues by flow cytometry ( n = 8). L Correlation of CTSL and HLA-B expression in human skeletal muscle: NSCLC (E-MTAB-12781; CON, n = 4; CAC, n = 5) and PDAC ( GSE133523 ; CON, n = 3; CAC, n = 3), log 2 (TPM + 1). M Correlation of Ctsl and H2-D1 expression in lung tumor and muscle (tumor: CON, n = 3; TB, n = 5; muscle: CON, n = 5; IgG, n = 4; αPD-L1, n = 3; αCD8a, n = 3), log 2 (TPM + 1). N WB of MHC-I expression in GA muscle. Two mice per group are shown ( n = 4 per group); quantification of all four mice is presented in Supplementary Fig. . Samples derive from the same experiment; different gels for MHC-I and another for vinculin were processed in parallel. O WB analysis of MHC-I expression in C2C12 myotubes transfected with si-RNA against Ctsl (si-Ctsl) or a non-targeting si-control (si-Cont). Samples derive from the same experiment; different gels for Ctsl, and another for MHC-I, vinculin, were processed in parallel. P C2C12 myotube diameter after transfection with siRNA, followed by co-culturing with splenic CD8 + T cells from TB mice in CM (12 myotubes per well, biological triplicates; total 36 myotubes per group). For C – E , G – K , and P , data are mean ± SEM, two-sided unpaired t -tests; for L and M , two-sided t -test for Pearson correlation. Source data are provided as a Source Data file.

Article Snippet: Following differentiation, C2C12 myotubes were treated with 10 ng/ml of mouse recombinant granzyme A (Gzma, HY- P76377 , MedChemExpress, Monmouth Junction, NJ, USA) with or without CTSLi (1 μg/ml) in the presence of either DM or CM for 96 h.

Techniques: Concentration Assay, Flow Cytometry, Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Cathepsin L as a dual-target to mitigate muscle wasting while enhancing anti-tumor efficacy of anti-PD-L1

doi: 10.1038/s41467-025-64500-0

Figure Lengend Snippet: A Human PPI network of CTSL-BNIP3 neighbors colored by log 2 FC in skeletal muscle from CTSL-BNIP3 -high versus -low individuals (GTEx). B GSEA of DEGs in GTEx muscle: top five positively and negatively enriched pathways (NES, normalized enrichment scores). C Representative GSEA plots for WP_PROTEASOME_DEGRADATION (top) and WP_ELECTRON_TRANSPORT_CHAIN_OXPHOS_SYSTEM_IN_MITOCHONDRIA (bottom) in CTSL-BNIP3 -high versus -low muscles. D Representative WB of indicated proteins in TB muscle. Two mice per group are shown ( n = 4 per group); quantification of all four mice in Supplementary Fig. . Samples derive from the same experiment; different gels for Ctsl, another for Bnip3, another for vinculin, were processed in parallel. E WB and quantification of Bnip3 in C2C12 myotubes 24 h after si-Ctsl or si-Cont transfection; biological triplicates for each group. Vinculin as a loading control. F C2C12 myotube diameter after 24 h transfection with si-Bnip3 or si-Cont and 72 h co-culture with CD8 + T cells in CM; 12 myotubes per well, biological triplicates for each group. Relative mtDNA copy number in GA muscle from TB mice ( G , n = 8); in C2C12 myotubes co-cultured with CD8 + T cells in CM plus or minus CTSLi ( H , biological triplicates for each group); and transfected with si-Ctsl, si-Bnip3, or si-Cont then co-cultured with CD8 + T cells in CM ( I , biological triplicates for each group). C2C12 myotube diameter ( J ) and mtDNA copy number ( K ) after co-culture with CD8 + T cells in CM plus or minus mouse recombinant Gzma (10 ng/ml); biological triplicates for each group. L Working model illustrating how the CTSL-BNIP3 network promotes muscle wasting in cooperation with cytotoxic T cells. For B and C , GSEA for functional enrichment analysis; for E – K , data are mean ± SEM, two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: Following differentiation, C2C12 myotubes were treated with 10 ng/ml of mouse recombinant granzyme A (Gzma, HY- P76377 , MedChemExpress, Monmouth Junction, NJ, USA) with or without CTSLi (1 μg/ml) in the presence of either DM or CM for 96 h.

Techniques: Muscles, Transfection, Control, Co-Culture Assay, Cell Culture, Recombinant, Functional Assay