Journal: The Journal of Biological Chemistry

Article Title: The ER stress sensor inositol-requiring enzyme 1α in Kupffer cells promotes hepatic ischemia-reperfusion injury

doi: 10.1016/j.jbc.2021.101532

Figure Lengend Snippet: IRE1α deficiency diminishes iNos expression in Kupffer cells. A–C, control or MφKO mice were subjected to sham or hepatic warm ischemia/reperfusion (n=3–4 per group). A, immunoblot analysis of iNos protein in liver lysates. β-Actin was used as the loading control. B, qRT-PCR analysis of the mRNA abundance of Nos2 in isolated Kupffer cells. C, immunoblot analysis of iNos protein in freshly isolated Kupffer cells. HSP90 was used as the loading control. The relative levels of iNos protein were quantified. D–G, Kupffer cells were isolated from control and MφKO mice. Cells were then treated with 100 ng/ml LPS for the indicated time intervals ( D and E ), or with 0.1 or 1 μg/ml LPS for 12 h ( F and G ). D and F, immunoblot analysis of IRE1α and iNos protein. E and G, qRT-PCR analysis of the mRNA abundance of Xbp1s and Nos2 . H, primary Kupffer cells were treated with PBS (veh) or 100 ng/ml LPS with or without 10 μM 4μ8C. Immunoblot analysis of the indicated proteins. The relative levels of iNos protein were quantified. All data represent the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by one way or two-way ANOVA.

Article Snippet: For iNOS inhibition, mice were injected with 1400W (35 mg per kg body weight; Targetmol, #T3491) or 10% DMSO, 40% PEG300, 5% Tween 80 in saline as vehicle control at 1 h prior to hepatic I/R surgery.

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, Isolation

Journal: The Journal of Biological Chemistry

Article Title: The ER stress sensor inositol-requiring enzyme 1α in Kupffer cells promotes hepatic ischemia-reperfusion injury

doi: 10.1016/j.jbc.2021.101532

Figure Lengend Snippet: Blocking IRE1α RNase attenuates Kupffer-cell-mediated inflammation during hepatic I/R injury. A, immunohistochemical staining of Ly6G + neutrophils in liver sections of the indicated group. Quantification of Ly6G + cells is shown per field. Scale bar, 100 μm. B, ELISA analysis of serum levels of the indicated inflammatory cytokines. C and D, qRT-PCR analysis of the mRNA abundance of the indicated UPR ( C ) or inflammatory genes ( D ) in freshly isolated Kupffer cells. E, immunoblot analysis of the indicated proteins in lysates of freshly isolated Kupffer cells. HSP90 was used as the loading control. The relative levels of NLRP3, c-Casp1 and iNos proteins were quantified. All data represent the mean ± SD. ∗ p < 0.05 and ∗∗ p < 0.01 by one-way ANOVA. NLRP3, nucleotide-binding domain, leucine-rich repeat containing protein 3; UPR, unfolded protein response.

Article Snippet: For iNOS inhibition, mice were injected with 1400W (35 mg per kg body weight; Targetmol, #T3491) or 10% DMSO, 40% PEG300, 5% Tween 80 in saline as vehicle control at 1 h prior to hepatic I/R surgery.

Techniques: Blocking Assay, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Isolation, Western Blot, Control, Binding Assay

Journal: Journal for immunotherapy of cancer

Article Title: Inhibiting CMTM4 reverses the immunosuppressive function of myeloid-derived suppressor cells and augments immunotherapy response in cervical cancer.

doi: 10.1136/jitc-2025-011776

Figure Lengend Snippet: Figure 2 CMTM4 increases MDSCs infiltration and inhibits CD8+ T cell accumulation in CC. (A–B) Flow cytometric anaysis of MDSCs (A), G-MDSC or M-MDSC (B) in TC1-C4KO and TC1-C4WT tumor-bearing mice. (C–D) Composition of immune cells in the TC1 spleen (C) and tumor (D) was shown in the heatmap. (E–F) Flow cytometric analysis of the ability of MDSC, G- MDSC, and M-MDSC to secrete INOS (E) or Arg-1 (F). INOS and Arg-1 expressions were described by percentage and mean fluorescence intensity (MFI). (G) Flow cytometric analysis of the infiltration of IFN-γ and GzmB CD8+ T cells. (H) Correlation between tumor weight with tumorous MDSCs abundance, tumor-infiltrating CD8+ T cells IFN-γ level in TC1-C4KO and TC1- C4WT tumor-bearing mice model (All n=6). (I) Flow cytometric analysis of PMN-MDSC and M-MDSC in PBMCs isolated from patients with benign tumors (n=21) and cervical cancer (n=18). (J) Correlation analysis of S100A8 or INOS expression and CMTM4 expression in clinical samples. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Arg-1, arginase-1; CC, cervical cancer; CKLF, chemokine-like factor; CMTM, CKLF-like MARVEL transmembrane domain-containing family; DC, dendritic cell; G- MDSC, granulocyte-like MDSC; GzmB, granzyme B; IFN, interferon; iNOS, inducible nitric oxide synthase; KO, knockout; MDSC, myeloid-derived suppressor cell; M-MDSC, monocyte-like MDSC; mRNA, messenger RNA; NK, natural killer; PMN- MDSC, polymorphonuclear myeloid-derived suppressor cell; Th1, T helper type 1 cell; Treg, regulatory T cell; WT, wild type.

Article Snippet: Human Arg- 1 or INOS protein levels in culture supernatants, and serum samples were measured using the Human Arg- 1 ELISA Kit (catalog no. RK07687, Abclonal) or Human INOS ELISA Kit (catalog no. RK01945, Abclonal).

Techniques: Fluorescence, Isolation, Expressing, Knock-Out, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Inhibiting CMTM4 reverses the immunosuppressive function of myeloid-derived suppressor cells and augments immunotherapy response in cervical cancer.

doi: 10.1136/jitc-2025-011776

Figure Lengend Snippet: Figure 3 Knockdown of CMTM4 results in the dysregulation of immune-associated cytokines and chemokines. (A–B) CM collected from CMTM4 KO-HeLa and CMTM4 KO-SiHa or from control were placed in the lower chambers. Freshly isolated PBMCs MDSCs were seeded in the upper chambers and allowed to transwell for 24 hours. Total numbers of transwelled MDSCs found in the lower chambers were counted. Data are presented as the mean±SEM (n=3). (C–D) CM collected from CMTM4 KO-HeLa and CMTM4 KO-SiHa or control was cocultured with mononuclear cells from cord blood for 24 hours. The proportion of MDSCs was detected by flow cytometry (n=3). (E) After treating umbilical cord blood mononuclear cells with CM from CMTM4 KO HeLa and CMTM4 KO SiHa or the control group for 24 hours, the coculture supernatant was collected and the secretion of Arg-1 or INOS was detected by ELISA. (F) Heatmap of 43 different human cytokine expression levels with a concentration greater than 100 pg/mL in CMTM4 KO-HeLa and CMTM4 NC-HeLa using Luminex-MultiDTX-43-Human. (G–H) RT-qPCR and ELISA assays analyze expression of IL-6 and CCL2 in CMTM4 KO-HeLa and CMTM4 KO-SiHa (n=3). (I–J) ELISA analysis of CCL2 and IL-6 expression in the serum of patients with benign tumors (CCL2 n=17; IL-6 n=10) and cervical cancer (CCL2 n=11; IL-6 n=18). (K–L) Correlation analysis of PMN-MDSC or M-MDSC expression and CCL2 or IL-6 expression in clinical samples. (M) GEPIA overall survival curves analysis of patients with CC with high or low expression of CCL2 or IL-6. N. Tumor mRNA level and serum protein concentration of CCL2 and IL-6 in TC1 tumor-bearing mice models (n=6). (O) Correlation analysis of CCL2 or IL-6 mRNA expression and CMTM4 mRNA expression in clinical cervical tissue. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Arg-1, arginase-1; CCL, C-C motif chemokine ligand; CM, conditioned medium; CKLF, chemokine-like factor; CMTM, CKLF-like MARVEL transmembrane domain-containing family; CXCL, C-X-C motif chemokine ligand; GEPIA, Gene Expression Profiling Interactive Analysis; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; KO, knockout; MDSC, myeloid-derived suppressor cell; mRNA, messenger RNA; PB, peripheral blood; PBMCs, peripheral blood mononuclear cells; PDGF-AA, platelet-derived growth factor-AA; PD-L1, programmed death-ligand 1; RT-qPCR, real-time quantitative PCR; Th1, T helper type 1 cell; TPM, transcripts per million; WT, wild type.

Article Snippet: Human Arg- 1 or INOS protein levels in culture supernatants, and serum samples were measured using the Human Arg- 1 ELISA Kit (catalog no. RK07687, Abclonal) or Human INOS ELISA Kit (catalog no. RK01945, Abclonal).

Techniques: Knockdown, Control, Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay, Luminex, Quantitative RT-PCR, Protein Concentration, Gene Expression, Knock-Out, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs

doi: 10.1371/journal.pone.0258427

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: NANOG , REA314 , PE , Recombinant human IgG1 , anti-human , 130-117-526 , Miltenyi Biotec.

Techniques: Cytometry, Conjugation Assay, Recombinant, Control