Journal: Journal of the American Society of Nephrology : JASN

Article Title: The Extracellular Matrix Receptor Discoidin Domain Receptor 1 Regulates Collagen Transcription by Translocating to the Nucleus

doi: 10.1681/ASN.2018111160

Figure Lengend Snippet: DDR1 is upregulated in injured kidney proximal tubules. (A) Paraffin kidney sections from control and biopsy specimens from three different patients with transplant AKI (Tx-AKI) were stained with anti-DDR1 antibody. Upregulated DDR1 expression is evident in the tubules of injured kidneys. (B) Paraffin kidney sections from control or one patient with Tx-AKI were stained with anti-DDR1 antibody and Lotus tetragonolobus agglutinin (LTA, a marker of proximal tubule) and analyzed by confocal microscopy. Expression of DDR1 is evident both in the cytoplasm and in the nuclei of injured proximal tubules (arrow). (C) Orthogonal projection of confocal images of kidney sections from the patient shown in (B) was performed using the imaging program Zen (black edition). Red, DDR1; blue, DAPI. (D) Non-nuclear and nuclear fractions (20 µg/lane) from kidney cortices of wild-type mice uninjured or 3 days after ischemia-reperfusion (3d-I/R) were analyzed by western blot for levels of DDR1. (E and F) Non-nuclear DDR1 and GAPDH (E) or nuclear DDR1 and PARP1 (F) bands were quantified by densitometry. Values represent DDR1/GAPDH or DDR1/PARP1 ratio and are the mean±SD of four animals. (G) Serum-starved HK-2 cells were treated with collagen I (50 µg/ml) for the time indicated. Time 0 represents cells incubated with vehicle (20 mM acetic acid) for 60 minutes. Nuclear fractions (20 µg/lane) were analyzed by western blot for levels of DDR1. (H) Nuclear DDR1 and PARP1 bands were quantified by densitometry. Values represent DDR1/PARP1 ratio and are the mean±SD of two experiments performed in triplicate. PARP1 (nuclear marker), GAPDH, or α-tubulin (non-nuclear markers) was used to evaluate fraction purity. (I) Schematic representation of the biotinylation assay performed on HK-2 cells. See text for details. (J) Nuclear fractions of HK-2 cells biotinylated (+ biotin) and treated at 37°C with collagen I for the time indicated were analyzed for levels of DDR1 or total biotinylated proteins using HRP-avidin. Nonbiotinylated (- biotin) cells treated with collagen I for the times indicated served as control. (K) Nuclear DDR1 and PARP1 of biotinylated cells were quantified and expressed as indicated above. (L) Nuclear fractions (200 µg) of biotinylated HK-2 cells treated at 37°C with collagen I for the times indicated were immunoprecipitated using streptavidin beads. Immunoprecipitated biotinylated proteins were analyzed for levels of DDR1. Cells treated at 37°C with collagen I for the time indicated in the absence of biotinylation (- biotin) or biotinylated by kept at 4°C served as negative (background for streptavidin beads) and positive (total biotinylated DDR1) controls, respectively. (M) Nuclear biotinylated DDR1 was quantified to the Coomassie protein band shown. IP, immunoprecipitation; IB, immunoblot.

Article Snippet: For double immunostaining, paraffin sections were stained with anti-DDR1 antibody, anti-collagen IV antibody (600–401–106–0.5; Rockland), collagen I antibody (ab34710; Abcam), and anti–NMHC-IIA antibody (ab89837; Abcam), together with biotinylated Lotus tetragonolobus agglutinin (cat. B-1325; Vector Laboratories), followed by secondary antibodies conjugated to AlexaFluor 555 and Fluorescein-Streptavidin (cat. SA-5001; Vector Laboratories), and mounted using ProLong Gold Antifade Mountant with DAPI (cat. {"type":"entrez-protein","attrs":{"text":"P36931","term_id":"2506707","term_text":"P36931"}} P36931 ; Thermo Scientific).

Techniques: Staining, Expressing, Marker, Confocal Microscopy, Imaging, Western Blot, Incubation, Cell Surface Biotinylation Assay, Avidin-Biotin Assay, Immunoprecipitation

Journal: Nature Communications

Article Title: Mitochondrial fission induces immunoescape in solid tumors through decreasing MHC-I surface expression

doi: 10.1038/s41467-022-31417-x

Figure Lengend Snippet: A Tumor volume measurements after subcutaneous implantation, arrow indicates the time of Mdivi-1 or DMSO treatment (mean ± s.e.m; n = 6; p = 0.0124; ** p < 0.001 by two-way ANOVA followed by Dunnett’s tests for multiple comparisons). B Weights of harvested tumors ( n = 6; p < 0.001; ** p < 0.001 by two-tailed t -test). C Photograph of harvested tumors. D Membrane expression of H-2K b and SIINFEKL-H-2K b complex of isolated primary cancer cells from harvested tumors by flow cytometry (mean ± s.e.m; n = 5; p < 0.0001 for H-2K b and SIINFEKL-H-2K b complex; ** p < 0.001 by two-tailed t -test). E IFN-γ-producing CTLs as quantified by ELISpot (mean ± s.e.m; n = 6; p < 0.0001; ** p < 0.001 by two-tailed t -test). F The isolated tumor-infiltrated CD8 + T cells were stained with anti-OVA-H-2K b tetramer and anti-CD8a, and the percentages of infiltrated OVA-specific CTLs were quantified by flow cytometry (mean ± s.e.m; n = 3; p = 0.0012; * p < 0.01 by two-tailed t -test). G Biodistribution of cancer cells 12 and 25 days after inoculation ( n = 6). Color scales represent photon intensities. H Kaplan–Meier survival curve was plotted for tail vein tumor inoculation model, and survival difference was analyzed using log-rank test ( n = 6; p = 0.0012).

Article Snippet: Cells were stained with H-2Kb-FITC (Cat. No. MHC2163, JPT), SIINFEKL-H-2Kb-FITC (Cat. No. MA5-17999, Invitrogen), anti-Mouse CD8a-APC (Cat. No. 553035, BD Pharmingen), anti-OVA-H-2K b tetramer-BV421 (Cat. No. TB-5001-4, MBL), HLA-ABC-PE (Cat. No. 560168, BD Pharmingen), Perforin-BV421 (Cat. No. 563393, BD Pharmingen), Granzyme B-FITC (Cat. No. 560211, BD Pharmingen), CD8-PE-CF594 (Cat. No. 562282, BD Pharmingen), EpCAM-BV510 (Cat. No. 563181, BD Pharmingen), ITGB1-PE (Cat. No. 555443, BD Pharmingen), TfR1-APC (Cat. No. 561940, BD Pharmingen), E-Cadherin-BV421 (Cat. No. 743712, BD Pharmingen), CD80-PE (Cat. No. 560925, BD Pharmingen), CD83-BV421 (Cat. No. 562630, BD Pharmingen), and CD86-FITC (Cat. No. 560958, BD Pharmingen).

Techniques: Two Tailed Test, Expressing, Isolation, Flow Cytometry, Enzyme-linked Immunospot, Staining

Journal: Nature Communications

Article Title: Mitochondrial fission induces immunoescape in solid tumors through decreasing MHC-I surface expression

doi: 10.1038/s41467-022-31417-x

Figure Lengend Snippet: A Membrane expression of MHC-I in TSCCs and B16F10 was assessed by flow cytometry with indicated treatments (mean ± s.e.m; n = 6; p = 0.0029, 0.0063 for SCC-9, 0.0037, 0.0002 for CAL-27, 0.001, 0.002 for B16F10; * p < 0.01, ** p < 0.001, siTPP2 compared with NC and siTPP2+XBP-1s, XBP-1s compared with vector and siTPP2+XBP-1s by two-way ANOVA followed by Dunnett’s tests for multiple comparisons). M indicates the mean fluorescence intensity. B SIINFEKL-H-2K b complex expression was evaluated by flow cytometry of B16F10 (mean ± s.e.m; n = 4; p = 0.0113 and p < 0.0001; * p < 0.01, ** p < 0.001 compared with shCtrl by two-tailed t -test). C Scheme of in vitro antigen presentation assays. D Quantification of mouse IFN-γ production by ELISA after 24 h (mean ± s.e.m; n = 5; p = 0.001, p < 0.0001, p < 0.001 for OVA and p < 0.0001 for SIINFEKEL; * p < 0.01, ** p < 0.001 compared with shCtrl by two-tailed t -test). E Twelve hours after coculture, GFP + cancer cells were harvested and cell death was examined by flow cytometry based on the uptake of PI and Annexin V (mean ± s.e.m; n = 4; p = 0.0003, p < 0.0001, p < 0.0001; * p < 0.01, ** p < 0.001 compared with shCtrl by two-tailed t -test). E indicates effector cells namely T cells. T indicates targeted cells namely cancer cells. F Additional transmembrane molecules expression after DRP-1 knockdown was assessed by flow cytometry ( n = 3). Both shCtrl and NC indicate negative control. G Graphic abstract of this study.

Article Snippet: Cells were stained with H-2Kb-FITC (Cat. No. MHC2163, JPT), SIINFEKL-H-2Kb-FITC (Cat. No. MA5-17999, Invitrogen), anti-Mouse CD8a-APC (Cat. No. 553035, BD Pharmingen), anti-OVA-H-2K b tetramer-BV421 (Cat. No. TB-5001-4, MBL), HLA-ABC-PE (Cat. No. 560168, BD Pharmingen), Perforin-BV421 (Cat. No. 563393, BD Pharmingen), Granzyme B-FITC (Cat. No. 560211, BD Pharmingen), CD8-PE-CF594 (Cat. No. 562282, BD Pharmingen), EpCAM-BV510 (Cat. No. 563181, BD Pharmingen), ITGB1-PE (Cat. No. 555443, BD Pharmingen), TfR1-APC (Cat. No. 561940, BD Pharmingen), E-Cadherin-BV421 (Cat. No. 743712, BD Pharmingen), CD80-PE (Cat. No. 560925, BD Pharmingen), CD83-BV421 (Cat. No. 562630, BD Pharmingen), and CD86-FITC (Cat. No. 560958, BD Pharmingen).

Techniques: Expressing, Flow Cytometry, Plasmid Preparation, Fluorescence, Two Tailed Test, In Vitro, Enzyme-linked Immunosorbent Assay, Negative Control