Journal: Molecular cell

Article Title: Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1

doi: 10.1016/j.molcel.2018.07.030

Figure Lengend Snippet: (A) BT-549 cells were treated with metformin (5 mM) for the indicated time. Detection of endogenous AMPKα and PD-L1 binding (red dots) by Duolink II assay. Three different positions were randomly selected at each point, and the number of red dots were divided by the number of nuclei. Data represent mean ± SD. n = 3. *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. Scale bar, 20 μm. Right, MDA-MB-231 WT, PD-L1 KO and AMPKα KO cells were used as negative controls. Scale bar, 25 μm. (B) MDA-MB-231 cells were cultured for 6 hr with or without metformin (5 mM) and MG132 (10 μM). Endogenous PD-L1 and AMPKα were immunoprecipitated and their binding was analyzed with immunoblotting. (C) In vitro kinase activity of AMPK toward PD-L1 with 32P-labeled ATP. (D) Kinetics of PD-L1 phosphorylation by AMPK. Acetyl-CoA carboxylase (ACC) was used as positive control. Km and Vmax were calculated using the Michaelis-Menten equation. (E) In vitro phosphorylation assay and phospho-tag gel shifting assay. W, PD-L1/WT. A, PD-L1/S195A. (F) PD-L1/S195 phosphorylation was examined using anti-PDL1/S195-p antibody at different time points after metformin (5 mM) treatment. (G) Western blot analysis of MDA-MB-231 WT and AMPKα KO cells after metformin treatment (5 mM) for 8 hr. Endogenous PD-L1 purified by IP was subjected to immunoblotting with PDL1 S195-p antibody after PNGase F reaction. (H) PD-L1 and AMPK subcellular localization of MDA-MB-231 WT and AMPKα KO cells. (I) Trypsin digestion of ER fractions with or without permeabilization. (J, K) BT-549 cells were treated with metformin (5 mM) for 3 hr. (J) BT-549 cells were subjected to Duolink II assay combined with immunofluoresence staining using markers for ER (HSP90B1), Golgi (TNG46), and nuclei (Hoechst). Scale bar, 20 μm (inset, 10 μm). (K) Duolink assay with antibodies specific for ECD (Ab205921 and 86744S) and ICD (13684S and GTX104763) of PD-L1. Scale bar, 50 μm (inset, 25 μm). Red dots: AMPK-PD-L1 binding in (J) and (K).

Article Snippet: In antibody reaction buffer (PBS plus 1% BSA, 0.3% Triton X-100, pH 7.4), samples were stained with primary antibodies against active caspase 3 (9661L, 1:300; Cell Signaling Technology, Danvers, MA, USA ), CD8 (MCA609G, 1:100; BioRad, Hercules, CA, USA), GranzymeB (AF1865, 1:500; R&D Systems, Minneapolis, MN, USA), PD-L1 (13684, 1:200; Cell Signaling Technology), HSP90B1 (NBP2–42379, 1:300; Novus Biologicals, Littleton, CO, USA), GM130 (610823, 1:200; BD Biosciences, San Jose, CA, USA), Alexa fluor 488 Giantin (908701, 1:200; BioLegend, San Diego, CA, USA), TNG46 (NB110–62093, 1:400; Novus Biologicals) overnight at 4 °C, followed by Alexa 350, 488, 546, and 647 (1:3,000, Life Technologies) secondary antibodies at RT for 1 hr.

Techniques: Binding Assay, Ii Assay, Cell Culture, Immunoprecipitation, Western Blot, In Vitro, Activity Assay, Labeling, Positive Control, Phosphorylation Assay, Purification, Staining

Journal: Molecular cell

Article Title: Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1

doi: 10.1016/j.molcel.2018.07.030

Figure Lengend Snippet: (A) WT, S195A S195D, S195E, and 4NQ PD-L1 stable cells were treated with or without tunicamycin (5 μg/ml) for 24 hr. (B) Schematic diagram of PD-L1 showing the position of S195 and the 4 N-glycosylation sites. (C) Comparison of the glycan structure between WT and S195E PD-L1 by IP/Mass analysis. (D) BT-549 and MDA-MB-231 stable cells expressing WT, S195E, or S195A PD-L1 were treated with metformin (5 mM) for 24 hr. (E) Expression pattern of PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells by IF staining. (F) MDA-MB-231 stable cells co-stained with antibodies against PD-L1 and Golgi markers (GM130: cis, Giantin: medial, TNG46: trans). (G) IF staining with antibodies against PD-L1 and ER marker (HSP90B1) (H) Flow cytometric analysis of membrane PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells. Data represent mean ± SD. n = 3. (I) Binding of green fluorescent-labeled PD-1/Fc to MDA-MB-231 WT, S195A and S195E PD-L1 stable cells was quantified. Data represent mean ± SD. n = 3. (J) PD-L1 localization in MDA-MB-231 expressing WT, S195E or NXT motif mutant (glycosylation site mutant) PD-L1 by IF staining. For experiments shown in (E), (F), (G) and (J), MG132 (10 μM) was added 6 hr prior to fixation to prevent degradation of PD-L1. Hoechst: nuclear counter staining. Scale bar, 20 μm (inset, 10 μm). *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. NS, not significant.

Article Snippet: In antibody reaction buffer (PBS plus 1% BSA, 0.3% Triton X-100, pH 7.4), samples were stained with primary antibodies against active caspase 3 (9661L, 1:300; Cell Signaling Technology, Danvers, MA, USA ), CD8 (MCA609G, 1:100; BioRad, Hercules, CA, USA), GranzymeB (AF1865, 1:500; R&D Systems, Minneapolis, MN, USA), PD-L1 (13684, 1:200; Cell Signaling Technology), HSP90B1 (NBP2–42379, 1:300; Novus Biologicals, Littleton, CO, USA), GM130 (610823, 1:200; BD Biosciences, San Jose, CA, USA), Alexa fluor 488 Giantin (908701, 1:200; BioLegend, San Diego, CA, USA), TNG46 (NB110–62093, 1:400; Novus Biologicals) overnight at 4 °C, followed by Alexa 350, 488, 546, and 647 (1:3,000, Life Technologies) secondary antibodies at RT for 1 hr.

Techniques: Comparison, Expressing, Staining, Marker, Membrane, Binding Assay, Labeling, Mutagenesis

Journal: Medical mycology

Article Title: Paradoxical antifungal activity and structural observations in biofilms formed by echinocandin-resistant Candida albicans clinical isolates

doi: 10.1093/mmy/myt007

Figure Lengend Snippet: . Comparison of echinocandin activity on planktonic (pMIC 80 ) and sessile (sMIC 80 ) cells of Candida albicans fks1 mutants.

Article Snippet: Two fks1 mutant strains (32746 and 5415) were categorized as poor biofilm producers, four (2762, 4254, 4715, and 42286) and the three ATCC reference strains were moderate biofilm producers, and six fks1 mutant strains (41509, 42379, 42996, 43001, 53264, and 41301) were categorized as strong biofilm producers.

Techniques: Activity Assay, Mutagenesis

Journal: Medical mycology

Article Title: Paradoxical antifungal activity and structural observations in biofilms formed by echinocandin-resistant Candida albicans clinical isolates

doi: 10.1093/mmy/myt007

Figure Lengend Snippet: . Comparison of paradoxical effect concentrations of planktonic and sessile cells of Candida albicans fks1 mutants.

Article Snippet: Two fks1 mutant strains (32746 and 5415) were categorized as poor biofilm producers, four (2762, 4254, 4715, and 42286) and the three ATCC reference strains were moderate biofilm producers, and six fks1 mutant strains (41509, 42379, 42996, 43001, 53264, and 41301) were categorized as strong biofilm producers.

Techniques: Mutagenesis

Journal: Medical mycology

Article Title: Paradoxical antifungal activity and structural observations in biofilms formed by echinocandin-resistant Candida albicans clinical isolates

doi: 10.1093/mmy/myt007

Figure Lengend Snippet: Assessment of biofilm mass of Candida albicans reference strains and fks1 clinical isolates. Biofilms were grown in triplicate at a concentration of 1 × 106 cells/ml in buffered RPMI-1640 at 37°C for 24 h. Biofilm mass was quantified using the crystal violet assay. Light absorbance was measured in a plate reader at OD 630 nm. Each experiment was performed independently three times.

Article Snippet: Two fks1 mutant strains (32746 and 5415) were categorized as poor biofilm producers, four (2762, 4254, 4715, and 42286) and the three ATCC reference strains were moderate biofilm producers, and six fks1 mutant strains (41509, 42379, 42996, 43001, 53264, and 41301) were categorized as strong biofilm producers.

Techniques: Concentration Assay, Crystal Violet Assay

Journal: Medical mycology

Article Title: Paradoxical antifungal activity and structural observations in biofilms formed by echinocandin-resistant Candida albicans clinical isolates

doi: 10.1093/mmy/myt007

Figure Lengend Snippet: Assessment of biofilm metabolic activity of Candida albicans reference strains and fks1 clinical isolates. Biofilms were grown in quadruplicate at a concentration of 1 × 106 cells/ml in buffered RPMI-1640 at 37° C for 24 h. The XTT assay was used to assay sessile metabolic activity. Formation of the colored formazan was subsequently measured at OD 490 nm. Each experiment was performed independently three times.

Article Snippet: Two fks1 mutant strains (32746 and 5415) were categorized as poor biofilm producers, four (2762, 4254, 4715, and 42286) and the three ATCC reference strains were moderate biofilm producers, and six fks1 mutant strains (41509, 42379, 42996, 43001, 53264, and 41301) were categorized as strong biofilm producers.

Techniques: Activity Assay, Concentration Assay, XTT Assay

Journal: Medical mycology

Article Title: Paradoxical antifungal activity and structural observations in biofilms formed by echinocandin-resistant Candida albicans clinical isolates

doi: 10.1093/mmy/myt007

Figure Lengend Snippet: Ultrastructural assessment of biofilm morphology using scanning electron microscopy. (A) Scanning electron microscopy of representative Candida albicans fks1 mutants that form poor (4254), moderate (42286), and strong (53264) biofilms compared with reference strain SC5314. (B) Scanning electron microscopy view of pit-like cell surface structures identified on select C. albicans fks1 mutants.

Article Snippet: Two fks1 mutant strains (32746 and 5415) were categorized as poor biofilm producers, four (2762, 4254, 4715, and 42286) and the three ATCC reference strains were moderate biofilm producers, and six fks1 mutant strains (41509, 42379, 42996, 43001, 53264, and 41301) were categorized as strong biofilm producers.

Techniques: Electron Microscopy

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Knockout of ClC-2 reveals critical functions of adherens junctions in colonic homeostasis and tumorigenicity

doi: 10.1152/ajpgi.00087.2018

Figure Lengend Snippet: Absence of chloride channel protein-2 (ClC-2) resulted in increased in vitro tumorigenicity and disrupted adherens junctions (AJ) proteins in HT-29 colon cancer cells. A: equal numbers of control (CT) and ClC-2 short-hairpin RNA (shRNA) cells were plated on a 96-well plate. Cell proliferation was monitored with the cell counting kit-8 (CCK-8) assay at different time points. Downregulated ClC-2 significantly increased HT-29 cell proliferation. B: in vitro tumorigenicity of the CT and ClC-2 shRNA cells was determined by a colony formation assay assessing anchorage-dependent and -independent cell populations. The tumorigenicity of ClC-2 shRNA cells was significantly increased compared with CT cells. C: ClC-2 knockdown induced disruption of AJ proteins E-cadherin and β-catenin. Arrowhead, nuclear distribution of β-catenin. D: proximity ligation assay (PLA) showing the interaction (red dots) between E-cadherin and β-catenin in ClC-2 shRNA cells was significantly reduced compared with ClC-2 wild type (WT). Data are presented as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.01 vs. CT shRNA, Student’s t-test.

Article Snippet: Persistent knockdown of clcn-2 gene expression using short-hairpin RNA (shRNA) was achieved by transfecting HT-29 cells with lentiviral particles encoding ClC-2 shRNA or control shRNA (catalog nos. sc-42379-V and sc-108080; Santa Cruz Biotechnology).

Techniques: In Vitro, shRNA, Cell Counting, CCK-8 Assay, Colony Assay, Proximity Ligation Assay

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Knockout of ClC-2 reveals critical functions of adherens junctions in colonic homeostasis and tumorigenicity

doi: 10.1152/ajpgi.00087.2018

Figure Lengend Snippet: Absence of chloride channel protein-2 (ClC-2) resulted in increased in vitro tumorigenicity and disrupted adherens junctions (AJ) proteins in HT-29 colon cancer cells. A: equal numbers of control (CT) and ClC-2 short-hairpin RNA (shRNA) cells were plated on a 96-well plate. Cell proliferation was monitored with the cell counting kit-8 (CCK-8) assay at different time points. Downregulated ClC-2 significantly increased HT-29 cell proliferation. B: in vitro tumorigenicity of the CT and ClC-2 shRNA cells was determined by a colony formation assay assessing anchorage-dependent and -independent cell populations. The tumorigenicity of ClC-2 shRNA cells was significantly increased compared with CT cells. C: ClC-2 knockdown induced disruption of AJ proteins E-cadherin and β-catenin. Arrowhead, nuclear distribution of β-catenin. D: proximity ligation assay (PLA) showing the interaction (red dots) between E-cadherin and β-catenin in ClC-2 shRNA cells was significantly reduced compared with ClC-2 wild type (WT). Data are presented as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.01 vs. CT shRNA, Student’s t-test.

Article Snippet: Persistent knockdown of clcn-2 gene expression using short-hairpin RNA (shRNA) was achieved by transfecting HT-29 cells with lentiviral particles encoding ClC-2 shRNA or control shRNA (catalog nos. sc-42379-V and sc-108080; Santa Cruz Biotechnology).

Techniques: In Vitro, shRNA, Cell Counting, CCK-8 Assay, Colony Assay, Proximity Ligation Assay