Journal: Experimental & Molecular Medicine

Article Title: IL-30 † (IL-27A): a familiar stranger in immunity, inflammation, and cancer

doi: 10.1038/s12276-021-00630-x

Figure Lengend Snippet: a The biogenesis of human (h) IL-27 is shown. Human IL-30 depends on assembly with Ebi3 to fold, be released from the endoplasmic reticulum (ER) chaperone Bip and function once outside the cell. If it remains unpaired, it is degraded by ER-associated degradation (ERAD). b In contrast, murine (m) IL-30 has two structurally adjacent cysteines c that can form a disulfide bond, so murine IL-30 can fold and be secreted in isolation or as a pair with Ebi3.

Article Snippet: Liu et al. reported that Il30 ( Il27p28 ) mRNA expression and IL-30 (IL-27p28) protein secretion (IL-27p28/IL-30 Quantikine ELISA kit from R&D Systems) in macrophages are induced through a MyD88-mediated activation pathway and that combined stimulation with IFNγ dramatically enhances the production.

Techniques: Isolation

Journal: Journal of Hematology & Oncology

Article Title: CRISPR/Cas9-mediated deletion of Interleukin-30 suppresses IGF1 and CXCL5 and boosts SOCS3 reducing prostate cancer growth and mortality

doi: 10.1186/s13045-022-01357-6

Figure Lengend Snippet: IL30-dependent regulation of IGF1 production in human PC cells and IGF1-mediated autocrine growth loop. A , B Elisa assay of IGF1 release by wild-type, EV and IL30 gene-transfected DU145 ( A ) and PC3 ( B ) cells . ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with WT and EV-transfected cells. Results are expressed as mean ± SD. C Cytofluorimetric analyses of IGF1R expression in PC3 and DU145 cells. Red lines: isotype control. Experiments were performed in triplicate. D , E Elisa assay of IGF1 release by wild-type DU145 ( D ) and PC3 ( E ) cells, after the treatment with anti-IL30 Abs. (D) ANOVA: p < 0.05. * p < 0.05, Tukey HSD test compared with DU145 cells untreated or treated with 5 μg/mL. E ANOVA: p < 0.001. * p < 0.01, Tukey HSD test compared with untreated PC3 cells. Results are expressed as mean ± SD. F , G MTT assay of DU145 ( F ) and PC3 ( G ) cells, untreated (0.0 ng/mL) or treated with rhIGF1 (5.0, 10, 30, 50 ng/mL). ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with 0 ng/mL. ** p < 0.01, Tukey HSD test compared with 0 and 5 ng/mL. *** p < 0.01, Tukey HSD test compared with 0, 5 and 10 ng/mL. Results are expressed as mean ± SD. H , I MTT assay of DU145 (H) and PC3 (I) cells, untreated (0.0 μg/mL) or treated with anti-IGF1 Abs (0.1, 0.4, 0.8 μg/mL in DU145; 0.25, 0.50, 0.70 μg/mL in PC3). (H) ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with 0.0 μg/mL. ** p < 0.01, Tukey HSD test compared with 0.0 and 0.1 μg/mL. *** p < 0.05, Tukey HSD test compared with 0.0, 0.1 and 0.4 μg/mL. (I) ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with 0.00 µg/mL. ** p < 0.01, Tukey HSD test compared with 0.00 and 0.25 μg/mL. Results are expressed as mean ± SD. J , K MTT assay of wild-type and IL30 gene-transfected DU145 ( J ) and PC3 ( K ) cells, untreated (0.0 μg/mL), or treated with anti-IGF1 Abs (30 μg/mL). ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with wild-type cells. ** p < 0.01, Tukey HSD test compared with wild-type and IL30-transfected cells. Results are expressed as mean ± SD

Article Snippet: Quantitation of IL30, CXCL5 and IGF1, in the supernatant derived from murine or human PC cells, was carried out using the following ELISA kits, according to manufacturer’s protocols: Human CXCL5/ENA-78 Quantikine ELISA Kit (#DX000, R&D Systems, Minneapolis, MN, USA); human IGF1 ELISA Kit (#ab211651, Abcam, Cambridge, UK); and mouse Interleukin-27 subunit alpha ELISA Kit (#CSB-E08466m, Cusabio, Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Expressing, MTT Assay

Journal: Journal of Hematology & Oncology

Article Title: CRISPR/Cas9-mediated deletion of Interleukin-30 suppresses IGF1 and CXCL5 and boosts SOCS3 reducing prostate cancer growth and mortality

doi: 10.1186/s13045-022-01357-6

Figure Lengend Snippet: Tumor growth and survival of mice-bearing IL30-deficient or IL30-overexpressing PC of murine or human origin. A Mean volume of tumors developed in NSG mice, after s.c. implantation of wild type, EV- or IL30-DU145 cells. ANOVA, p < 0.0001; Tukey HSD test, p < 0.01 versus wild type or EV-transfected DU145 cells. Results are expressed as mean ± SD. B Mean volume of tumors developed in NSG mice, after s.c. implantation of wild type, NTgRNA-treated or IL30KO-DU145 cells. ANOVA, p < 0.0001; Tukey HSD test, p < 0.01 versus wild type or NTgRNA-treated DU145 cells. Results are expressed as mean ± SD. C Average number of lung metastasis spontaneously developed in NSG mice, which developed tumors after s.c. implantation of wild type, NTgRNA-treated or IL30KO-DU145 cells. ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test versus DU145 or NTgRNA-treated DU145 cells. Results are expressed as mean ± SD. D Kaplan–Meier survival curves of mice-bearing tumors developed after s.c. implantation of wild type, NTgRNA-treated or IL30KO-DU145 cells. Log-rank test: p = 0.000009. E Immunopathological features of tumors developed in NSG mice, after s.c. implantation of IL30KO-DU145, wild type DU145 and IL30-DU145 cells. Expression of IGF1, proliferation (Ki67 Abs) and vascularization (CD31 Abs) were prominent in IL30-overexpressing tumors, and scanty in IL30KO tumors, compared with wild type tumors. Cytoplasmic and nuclear expression of NFKB1 was strong in IL30-overexpressing tumors and faint in IL30KO tumors. The immunopathological features of tumors developed after implantation of control, EV-transfected or NTgRNA-treated, cells were comparable to those of wild type tumors. Magnification: × 400; CD31, × 200. F Immunopathological features of tumors developed in NSG mice, after s.c. implantation of IL30KO-DU145 and wild type DU145 cells. Expression of tumor suppressor genes CDH1/E-Cadh, DKK3, PTEN, RARb and SOCS3 was stronger in IL30KO-DU145 tumors, when compared to wild type tumors, whereas the expression of PTGS2 was weaker. The immunopathological features of control tumors, developed in NSG after implantation of NTgRNA-treated cells, were comparable to those of wild type tumors. Magnification: × 400. G Automated immune cell count and microvessel density in tumors developed in NSG mice, after implantation of wild type, EV- or IL30 gene-transfected, NTgRNA-treated and IL30KO-DU145 cells, assessed by immunohistochemistry, as described in Methods. ANOVA, p < 0.0001. * p < 0.01, Tukey HSD test compared with DU145, NTgRNA-treated DU145 and EV-transfected DU145. ** p < 0.01, Tukey HSD test compared with DU145, NTgRNA-treated DU145, IL30KO-DU145 and EV-transfected DU145. Results are expressed as mean ± SD. H The immune cell contexture of tumors developed in NSG mice, after s.c. implantation of IL30KO and IL30-DU145 cells, revealed a higher content of macrophages (anti-F4/80 Abs) and granulocytes (anti-Ly-6G Abs) in IL30-overexpressing tumors, compared to wild type tumors. By contrast, these immune cell populations were scarce to absent in IL30-deficient tumors. Magnification: × 400. Scale bars: 30 μm. I Western blot analysis of IL30 protein expression in wild type, EV- and IL30 gene-transfected TRAMP-C1 cells. J Mean volume of tumors developed in C57BL/6J mice, after s.c. implantation of wild type, EV- or IL30-TRAMP-C1 cells. ANOVA, p < 0.0001. Tukey HSD test, p < 0.01 versus wild type or EV-TRAMP-C1 cells. Results are expressed as mean ± SD. K Immunopathological features of tumors developed in C57BL/6J mice, after s.c. implantation of wild type or IL30-TRAMP-C1 cells revealed that proliferation (PCNA Abs), microvascular density (CD31 Abs) and granulocyte content (Ly-6G Abs) were higher in IL30-overexpressing tumors, than in control tumors. Cancer cells that formed IL30-TRAMP-C1 tumors showed reduced cytoplasmic expression of PTEN and an increased nuclear and cytoplasmic expression of NFKB1. Magnification: × 400; CD31, × 200. L Automated immune cell count in tumors developed in C57BL/6J mice, after s.c. implantation of wild type, EV or IL30 gene-transfected TRAMP-C1 cells, assessed by immunohistochemistry, as described in Methods. ANOVA, p < 0.001. * p < 0.01, Tukey HSD test compared with wild type or EV-TRAMP-C1 cells. Results are expressed as mean ± SD. M Percentage of lung metastasis spontaneously developed in C57BL/6J mice, which developed tumors after s.c. implantation of wild type or IL30 gene-transfected TRAMP-C1 cells. *Fisher’s exact test, p = 0.03 versus EV-TRAMP-C1 or TRAMP-C1. Results from mice implanted with EV-TRAMP-C1 cells were comparable to those obtained from mice implanted with wild type cells. N Kaplan–Meier survival curves of mice-bearing tumors developed after s.c. implantation of wild type, EV- or IL30 gene-transfected TRAMP-C1 cells. Log-rank test: p = 0.000124

Article Snippet: Quantitation of IL30, CXCL5 and IGF1, in the supernatant derived from murine or human PC cells, was carried out using the following ELISA kits, according to manufacturer’s protocols: Human CXCL5/ENA-78 Quantikine ELISA Kit (#DX000, R&D Systems, Minneapolis, MN, USA); human IGF1 ELISA Kit (#ab211651, Abcam, Cambridge, UK); and mouse Interleukin-27 subunit alpha ELISA Kit (#CSB-E08466m, Cusabio, Wuhan, China).

Techniques: Transfection, Expressing, Cell Counting, Immunohistochemistry, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: IL-30 † (IL-27A): a familiar stranger in immunity, inflammation, and cancer

doi: 10.1038/s12276-021-00630-x

Figure Lengend Snippet: a The biogenesis of human (h) IL-27 is shown. Human IL-30 depends on assembly with Ebi3 to fold, be released from the endoplasmic reticulum (ER) chaperone Bip and function once outside the cell. If it remains unpaired, it is degraded by ER-associated degradation (ERAD). b In contrast, murine (m) IL-30 has two structurally adjacent cysteines c that can form a disulfide bond, so murine IL-30 can fold and be secreted in isolation or as a pair with Ebi3.

Article Snippet: ELISA experiments measuring IL-27 production in this study utilized the IL-27p28/IL-30 Quantikine kit from the R&D Systems, which primarily measures IL-30 with modest cross-reactivity with recombinant IL-27 (Ebi3/p28 fusion protein) ( https://resources.rndsystems.com/pdfs/datasheets/m2728.pdf ).

Techniques: Isolation