Journal: Oncoimmunology

Article Title: Calpain 2 regulates IL-1α secretion and inhibits tumor development via modulating calpain 1 expression in the tumor microenvironment

doi: 10.1080/2162402X.2025.2451444

Figure Lengend Snippet: Calpain 2 deficiency enhances HCC development in an IL-1α dependent manner. Calpain 2 was knocked out in the transfected IL1α + C2 + cells using CRISPR/Cas9 and the absence of calpain 2 mRNA and protein were confirmed by qPCR (a) and Western blotting (b), respectively. (c) The proliferation of the IL1α + C2 + and IL1α + C2 − cells were compared using the CCK8 assay at 24 h, 48 h and 72 h. (d) Rates of apoptosis in the IL1α + C2 + and IL1α + C2 − cells were compared after UV irradiation for 30 min through annexin V and DAPI staining. Cells stained annexin V + DAPI − were defined as early apoptotic cells and those stained annexin V + DAPI + were late apoptotic cells. (e) IL-1α was measured in the supernatants of IL1α + C2 + and IL1α + C2 − cells by ELISA. For these experiments, cells were cultured at 1.0 × 10 5 /ml for 24 h in triplicates. (f) IL1α + C2 + or IL1α + C2 − cells (1.0 × 10 6 ) were injected orthotopically into the liver of C57BL/6 mice ( n = 6–9 each group). After two weeks, tumor size and liver weight were measured. (g) After two weeks, the level of IL-1α in the mouse sera and liver lysates were determined by ELISA in both the IL1α + C2 + and IL1α + C2 − mice. Calpain 2 was also knocked out in wild type Hepa-1–6 cells and the deletion of calpain 2 in the Hepa-1–6-C2 KO cells was confirmed by qPCR (h) and Western blotting (i). Wild type Hepa-1–6-mock cells were used as a control. (j) Rate of Hepa-1–6-C2 KO cell proliferation was compared with control cells using the CCK8 assay. (k) These cells (1.0 × 10 6 ) were injected orthotopically in the liver of C57BL/6 mice ( n = 4 each group). Tumor size and liver weight were measured two weeks post-tumor transplantation. Values are presented as mean ± SEM.

Article Snippet: Murine IL1α + C2 + cells were transfected with either calpain 2 CRISPR/Cas9 KO plasmid or scrambled control plasmid (Santa Cruz Biotechnology, Dallas, TX) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) transfection reagent.

Techniques: Transfection, CRISPR, Western Blot, CCK-8 Assay, Irradiation, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture, Injection, Control, Transplantation Assay

Journal: Oncoimmunology

Article Title: Calpain 2 regulates IL-1α secretion and inhibits tumor development via modulating calpain 1 expression in the tumor microenvironment

doi: 10.1080/2162402X.2025.2451444

Figure Lengend Snippet: Calpain 2 deletion in the Hepa 1–6 tumor cells suppressed mouse anti-tumor T cell response. IL1α + C2 + and IL1α + C2 − cells (1.0 × 10 6 ) were injected orthotopically into the liver of C57BL/6 mice ( n = 5 each group). After one week, splenocytes and liver lymphocytes were isolated from these mice for flow cytometry analysis. The percentage of activated, naive, effector and memory CD4 + and CD8 + T cells were determined in the spleen (a) and liver (b). The functionality of the isolated splenic (c) and liver (d) CD4 + and CD8 + T cells were examined by staining intracellular IFN-γ and TNF-α. Results are presented as mean ± SEM.

Article Snippet: Murine IL1α + C2 + cells were transfected with either calpain 2 CRISPR/Cas9 KO plasmid or scrambled control plasmid (Santa Cruz Biotechnology, Dallas, TX) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) transfection reagent.

Techniques: Injection, Isolation, Flow Cytometry, Staining

Journal: Oncoimmunology

Article Title: Calpain 2 regulates IL-1α secretion and inhibits tumor development via modulating calpain 1 expression in the tumor microenvironment

doi: 10.1080/2162402X.2025.2451444

Figure Lengend Snippet: The pro-tumoral effect observed upon calpain 2 deletion was mediated by MDSCs. IL1α + C2 + and IL1α + C2 − cells (1.0 × 10 6 ) were injected orthotopically into the liver of C57BL/6 mice ( n = 4–5 each group). After one week, splenocytes (a) and liver-infiltrating leukocytes (b) were isolated. Cells were stained for CD11b and CD45 to measure the percentage of total MDSCs. PMN-MDSC were identified by staining CD11b + Ly6G + Ly6C low , and M-MDSC were identified as CD11b + Ly6G – Ly6C high cells. (c) After orthotopical tumor cell (1.0 × 10 6 ) injection into the mouse livers, mice were also injected with GEM (100 mg/kg) every 3 d to deplete MDSC. As a control, PBS was injected every 3 d. After two weeks, tumor size and liver weight were measured. Results were presented as mean ± SEM.

Article Snippet: Murine IL1α + C2 + cells were transfected with either calpain 2 CRISPR/Cas9 KO plasmid or scrambled control plasmid (Santa Cruz Biotechnology, Dallas, TX) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) transfection reagent.

Techniques: Injection, Isolation, Staining, Control

Journal: Oncoimmunology

Article Title: Calpain 2 regulates IL-1α secretion and inhibits tumor development via modulating calpain 1 expression in the tumor microenvironment

doi: 10.1080/2162402X.2025.2451444

Figure Lengend Snippet: Calpain 1 expression was upregulated in Hepa-1–6 tumor cells after calpain 2 deletion. (a) Total calpain activity was measured in the supernatant and cell lysates using the Calpain-Glo TM protease assay. The levels of calpain 1 mRNA and protein in these cells were determined by qPCR (b) and Western blotting (c), respectively. (d) RNA sequencing was performed with IL1α + C2 + and IL1α + C2 − cells and the data were analyzed by volcano plot and heatmap to show differentially expressed genes. Data were also subjected to signaling pathway analysis using the GO (e) and KEGG (f) methods. Data are presented as mean ± SEM.

Article Snippet: Murine IL1α + C2 + cells were transfected with either calpain 2 CRISPR/Cas9 KO plasmid or scrambled control plasmid (Santa Cruz Biotechnology, Dallas, TX) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) transfection reagent.

Techniques: Expressing, Activity Assay, Protease Assay, Western Blot, RNA Sequencing

Journal: Oncoimmunology

Article Title: Calpain 2 regulates IL-1α secretion and inhibits tumor development via modulating calpain 1 expression in the tumor microenvironment

doi: 10.1080/2162402X.2025.2451444

Figure Lengend Snippet: FoxO3 and FoxP2 are significantly upregulated in HCC tumor cells upon calpain 2 deletion. (a) Expression of nine fox family transcription factors in IL1α + C2 − cells by qPCR. (b) FoxO3 and FoxP2 expression was also determined by Western blotting using β-actin as loading controls. The band signal intensities were estimated by densitometry to estimate the folds of change. The FoxO3 gene was knockdown using three specific siRNAs and the expression levels of FoxO3 (c) and calpain 1 (d) mRNA in these cells was determined by qPCR. (e) Calpain activity in these cells were measured using the calpain-GloTM protease assay. (f) IL-1α in the supernatants was measured by ELISA. FoxP2 expression was also knocked down using three specific siRNAs in the IL1α + C2 − cells. The expression levels of FoxP2 (g) and calpain 1 (h) in these cells was determined by qPCR. (i) Calpain 1 activity was measured in the cell lysates using the Calpain-Glo TM protease assay. (j) IL-1α in the supernatant was measured by ELISA. (k) Ectopic overexpression of FoxO3 and FoxP2 in IL1α + C2 − cells. The elevated FoxO3 and FoxP2 expression was examined by Western blotting and qPCR, respectively. (l) Measurement of calpain 1 protein expression in FoxO3 or FoxP2 over-expressed IL1α + C2 − by Western blotting and densitometry. (m) pcDNA3.1 plasmid vectors containing the FoxO3 or FoxP2, or calpain 1 promoter with luciferase reporter gene were transfected into HeLa cells using lipofectamine 3000. As a control, empty pcDNA3.1 vector or luciferase reporter vector was used to transfect HeLa cells. After 24 h, luciferase activity was detected in the cell lysate using the Dual-Luciferase® Reporter assay kit. Data are presented as mean ± SEM.

Article Snippet: Murine IL1α + C2 + cells were transfected with either calpain 2 CRISPR/Cas9 KO plasmid or scrambled control plasmid (Santa Cruz Biotechnology, Dallas, TX) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) transfection reagent.

Techniques: Expressing, Western Blot, Knockdown, Activity Assay, Protease Assay, Enzyme-linked Immunosorbent Assay, Over Expression, Plasmid Preparation, Luciferase, Transfection, Control, Reporter Assay