Journal: Nature cell biology

Article Title: Nucleated transcriptional condensates amplify gene expression.

doi: 10.1038/s41556-020-00578-6

Figure Lengend Snippet: Fig. 2 | Transcriptional condensates colocalize with and recruit additional CTD. a, THZ1 covalently binds to and inhibits the activity of CDK7, preventing phosphorylation of the CTD of Pol II and inhibiting transcription initiation. The net charge per residue (in a 5 amino-acid window) of CTD with 52 repeats of the heptad consensus amino acid sequence YSPTSPS. b, Fluorescence images of live cells expressing optoTAF15 and eGFP–CTD after blue-light illumination. Time since the start of illumination is shown with 0 s being just before illumination. c, Fluorescence intensities of cells in b, showing that optoTAF15 condensates and CTD within the condensates increase with time after blue-light illumination. Data are mean ± s.d. (n = 10 cells). AU, arbitrary units. d, Fluorescence images of cells expressing optoIDRs and eGFP–CTD after blue-light illumination. Cell nucleus is outlined by dotted line. Arrowheads point to optoIDR condensates colocalized with recruited CTD. e, CTD partition coefficients in various optoIDR condensates. OptoTAF15 condensates exhibit a nearly fourfold preferential partition of CTD, which is higher than other optoIDRs. The lines indicate the mean and error bars show s.d. derived from the number of cells as indicated in the panel. The dashed line represents a CTD partition coefficient of 1 (that is, no preferential partitioning). P values by t-test. f, OptoTAF15(10R>Q/10D>S) has a higher saturation concentration (C*) than optoTAF15 in nucleoplasm (Nuc). Inhibition of CTD phosphorylation with THZ1 leads to a decrease in the nucleoplasmic saturation concentration of optoTAF15 and optoTAF15(10R>Q/10D>S), but no significant (P > 0.05) change in the cytoplasm (Cyto). Data are mean ± s.d. derived from the number of cells as indicated in the panel. P values from pairwise one-sided Fisher’s exact tests in e,f. g, The ratio of cytoplasmic to nucleoplasmic saturation concentration correlates with nuclear CTD partitioning into optoIDR condensates. Data in e–g, are mean ± s.d. (sample sizes as shown in e and Extended Data Fig. 2a). The dashed line represents the correlation curve from a second-order polynomial fit, with R2 value shown. The number of cells (n) analysed in each panel was pooled from three independent experiments. Statistical source data are provided in Source Data Fig. 2.

Article Snippet: For CDK7 inhibition experiments, THZ1 (Med Chem Express) was dissolved in DMSO at 1 mM and further diluted with growth medium to a final concentration of 1 μM before being applied to NIH 3T3 cells followed by a 1.5 h incubation before imaging.

Techniques: Activity Assay, Phospho-proteomics, Residue, Sequencing, Fluorescence, Expressing, Derivative Assay, Concentration Assay, Inhibition

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 3. Ability of NAR to promote osteogenesis in human periodontal ligament stem cells is mitigated after L‑NAME (eNOS inhibitor) treatment. (A) Alkaline phosphatase staining in different treatment groups. (B) Alizarin red staining in different treatment groups. (C) NO levels in different treatment groups. (D) Gene expression levels of RUNX2, OPN and sGC in different treatment groups. (E‑G) Protein expression of RUNX2, OPN, sGC and p‑eNOS in various treatment groups. *P<0.05, **P<0.01, ***P<0.001. NAR, naringenin; eNOS, endothelial nitric oxide synthase; RUNX2, Runt‑related transcription factor; OPN, osteopontin; sGC, soluble guanylate cyclase; NO, nitric oxide; p‑, phosphorylated; t‑, total; L‑NAME, NG‑nitro‑L‑arginine methyl ester.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques: Staining, Gene Expression, Expressing

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 6. Impact of AKT inhibitor VIII on NAR‑mediated osteogenic promotion in human periodontal ligament stem cells. (A) Alkaline phosphatase staining in different treatment groups. (B) Alizarin red staining in different treatment groups. (C) Gene expression of RUNX2 and OPN in different treatment groups. (D‑F) Protein levels of RUNX2, OPN, p‑AKT and p‑eNOS in different treatment groups. *P<0.05, **P<0.01, ***P<0.001. NAR, naringenin; RUNX2, Runt‑related transcription factor; OPN, osteopontin; eNOS, endothelial nitric oxide synthase; p‑, phosphorylated; t‑, total.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques: Staining, Gene Expression

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 8. Schematic diagram of the mechanism by which naringenin promotes the osteogenic differentiation of human periodontal ligament stem cells. eNOS, endothelial nitric oxide synthase; p, phosphorylated; NO, nitric oxide; sGC, soluble guanylate cyclase; cGMP, cyclic guanosine monophosphate; TRPC6, transient receptor potential cation channel, subfamily C, member 6; PKG, protein kinase G.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques:

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