Journal: JCI Insight

Article Title: Collagen-binding C-type natriuretic peptide enhances chondrogenesis and osteogenesis

doi: 10.1172/jci.insight.198959

Figure Lengend Snippet: ( A ) Collagen-binding ability and thermal stability of CBD-CNP. Unbound CBD-CNP was assessed by SDS-PAGE. M, molecular weight marker; B, buffer control; No heat, no preheating; Heat, preheating at 56°C 30 minutes; (–), no collagen powder was added; (+), collagen powder was added. ( B ) Surface plasmon resonance analysis of CBD-CNP binding to recombinant NPR-B immobilized on a CM5 chip. The Kd value was calculated from sensorgrams as described under the experimental procedures. ( C ) Ligand-receptor binding assay with CBD-CNP and NPR-B, the receptor for CNP. ( D ) Inhibitory effect of FGF2-FGFR3 downstream signaling pathway of RCS cells owing to the binding of CNP to NPR-B. Phosphorylation of ERK1/2 in RCS cells was assessed by FGF2 alone and combined treatment with CNP-22 or CBD-CNP. ( E ) Cell proliferation assessment of RCS cells after treatment with FGF2 alone or in combination with CNP-22 or CBD-CNP ( n = 5). The cell proliferation rate was calculated based on the cell count with no drug treatment as 100%. ( F ) Alcian blue staining of RCS cells in each group. Scale bars: 200 μm in F . Statistical analysis was performed by using 1-way ANOVA followed by Tukey post hoc tests. SDS-PAGE, sodium dodecyl sulfate polyacrylamide electrophoresis; RU, response unit; IP, immunoprecipitation; IB, immunoblot; NPR-B, natriuretic peptide receptor B; CNP, C-type natriuretic peptide; FGF2, fibroblast growth factor 2; FGFR3, fibroblast growth factor receptor 3; RCS, rat chondrosarcoma; ERK, extracellular signal-regulated kinase; P-ERK, phospho-extracellular signal-regulated kinase.

Article Snippet: After 72 hours, cell numbers were evaluated using the Cell Counting Kit-8 (Dojindo), and the cell proliferation rate was calculated by setting the number of cells with untreated control as 100%.

Techniques: Binding Assay, SDS Page, Molecular Weight, Marker, Control, SPR Assay, Recombinant, Reporter Assay, Phospho-proteomics, Cell Characterization, Staining, Electrophoresis, Immunoprecipitation, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Targeting lysosomal protease CTSL promotes anti-tumor immunity and sensitizes HNSCC to PD-1 blockade by stabilizing PDK1 and activating Akt–PD-L1 axis

doi: 10.1016/j.neo.2025.101228

Figure Lengend Snippet: CTSL regulates PD-L1 expression and malignant phenotypes through the AKT pathway. (A, B) Bubble plots illustrating pathway enrichment of DEGs derived from an integrated analysis of the TCGA-HNSCC, GSE178537 , GSE127165 , and GSE227919 cohorts, stratified by CTSL expression: (A) top Gene Ontology biological processes and (B) top KEGG pathways. (C, D) Western blots of CTSL, total and phosphorylated AKT, and PD-L1 in Cal27 cells with CTSL knockdown alone (C) or knockdown combined with the AKT activator SC-79 (D). (E, F) Western blots of the same proteins in HN8 cells with CTSL overexpression (E) or overexpression combined with the AKT inhibitor MK-2206 (F). (G) Transwell invasion assay images and quantification for Cal27 cells comparing SH#NC, SH2#CTSL, and SH2#CTSL + SC-79 groups. (H) Transwell invasion assay images and quantification for HN8 cells comparing Vector, OE#CTSL, and OE#CTSL + MK-2206 groups. (I) Quantification of wound-healing assays in Cal27 cells after CTSL knockdown and SC-79 rescue. (J) CCK-8 proliferation curves for Cal27 cells in the same groups. (K) Quantification of wound-healing assays in HN8 cells after CTSL overexpression and MK-2206 treatment. (L) CCK-8 proliferation curves for HN8 cells in the same groups.

Article Snippet: Cell Proliferation (CCK-8) Assay: Cell proliferation rates were measured using the Cell Counting Kit-8 (CCK-8, Dojindo), which assesses metabolic activity as a proxy for viable cell number.

Techniques: Expressing, Derivative Assay, Western Blot, Knockdown, Over Expression, Transwell Invasion Assay, Plasmid Preparation, CCK-8 Assay