Journal: Gut Microbes

Article Title: Midbiotics: conjugative plasmids for genetic engineering of natural gut flora

doi: 10.1080/19490976.2019.1591136

Figure Lengend Snippet: Bacterial strains and plasmids used in the experiments and the spacer sequences of pCRISPR plasmid. Only the resistance genes relevant to the experiments are mentioned here.

Article Snippet: In this study, the so-called midbiotic system consists of the conjugative RP4 blaTEM−2∆172−714 plasmid (delivery plasmid) and mobilizable pCas9 plasmid (pCRISPR plasmid, a gift from Luciano Marraffini, Addgene plasmid # 42876) encoding the S. pyogenes CRISPR/Cas9 system with crRNA(s) targeting conservative sites of different beta-lactamase resistance genes in ESBL plasmids ( ). pCas9 was made mobilizable by cloning RP4 oriT site , (50980–51793 bps, amplified with primers RP4oriT-F and RP4oriT-R, Supplementary Table 1) into pCas9 digested with SalI (ThermoScientific; Waltham, Massachusetts, United States) into region spanning 7377–7486 bps.

Techniques: Plasmid Preparation, Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: Inhibiting PLA2G7 reverses the immunosuppressive function of intratumoral macrophages and augments immunotherapy response in hepatocellular carcinoma

doi: 10.1136/jitc-2023-008094

Figure Lengend Snippet: PLA2G7 is upregulated in HCC tissues and mainly expressed in intratumoral macrophages. (A) Bubble plot of the expression levels of the seven genes in each single cell subcluster in Data set 1. (B) t-SNE plot of all single cells colored by the expression level of PLA2G7 in Data set 1. (C) Bubble plot of the expression levels of the seven genes in each single cell subcluster in Data set 2. (D) t-SNE plot of all single cells colored by the expression level of PLA2G7 in Data set 2. (E) The violin plot displaying the expression levels of PLA2G7 in macrophage from tumor and adjacent normal tissues in Data set 1. (F) The violin plot displaying the expression levels of PLA2G7 in macrophage from tumor, adjacent normal tissues, blood, and ascites in Data set 2. (G) Representative IHC images of PLA2G7 and CD68 in HCC tissues and peritumor tissues. Scale bar: 200 µm (left) and 20 µm (right). (H) IHC expression pattern of CD68 (left panel) and PLA2G7 (right panel) in HCC tissues versus peritumor tissues (n=130). χ 2 . ns, no significance; *p<0.05. HCC, hepatocellular carcinoma; IHC, immunohistochemistry; PLA2G7, phospholipase A2 Group VII; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: Non-specific binding was blocked by incubating the slides with 5% bovine serum albumin (BSA), followed by overnight staining with primary antibodies against the following antigens at 4°C: PLA2G7 (Proteintech, #15 526–1-AP), CD68 (Abcam, #ab283654), and CD8 (Abcam, #ab237709).

Techniques: Expressing, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Inhibiting PLA2G7 reverses the immunosuppressive function of intratumoral macrophages and augments immunotherapy response in hepatocellular carcinoma

doi: 10.1136/jitc-2023-008094

Figure Lengend Snippet: The prognostic value of macrophage-specific PLA2G7 in HCC. (A) Representative IHC images of low-expressions and high-expressions of PLA2G7 and CD68 in HCC tissues. Scale bar: 200 µm (left) and 20 µm (right). (B–C) OS curves for patients with HCC with low-expression and high-expression of PLA2G7 or CD68 in the Zhongshan cohort (n=115). (D) OS curves for patients with HCC with low-co-expressions and high-co-expressions of PLA2G7/CD68 in the Zhongshan cohort (n=115). (E) Forest plots for univariate and multivariate Cox proportional hazards regression models of OS in the Zhongshan cohort. (F–I) OS and DFS curves for patients with HCC with low-mRNA and high-mRNA levels of PLA2G7 or CD68 in TCGA-LIHC cohort (n=369). (J–K) OS and DFS curves for patients with HCC with low-co-expressions and high-co-expressions of PLA2G7/CD68 in TCGA-LIHC cohort (n=369). Log-rank test. * indicates statistical significance. AFP, α-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; DFS, disease-free survival; HBsAg, hepatitis B surface antigen; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; mRNA, messenger RNA; OS, overall survival; PLA2G7, phospholipase A2 Group VII; TCGA-LIHC, The Cancer Genome Atlas liver hepatocellular carcinoma; TNM, tumor-nodes-metastasis.

Article Snippet: Non-specific binding was blocked by incubating the slides with 5% bovine serum albumin (BSA), followed by overnight staining with primary antibodies against the following antigens at 4°C: PLA2G7 (Proteintech, #15 526–1-AP), CD68 (Abcam, #ab283654), and CD8 (Abcam, #ab237709).

Techniques: Expressing, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Inhibiting PLA2G7 reverses the immunosuppressive function of intratumoral macrophages and augments immunotherapy response in hepatocellular carcinoma

doi: 10.1136/jitc-2023-008094

Figure Lengend Snippet: The association between macrophage-specific PLA2G7 and immunotherapy response in patients with HCC receiving anti-PD-1 treatment. (A) Representative MRIs of patients with HCC receiving anti-PD-1 treatment with PD and PR. (B–D) Representative IF images depicting the expressions of PLA2G7, CD68, and CD8 in tumor tissues from patients with HCC with PD and PR. Scale bar: 100 µm (left) and 20 µm (right). (E) Expression pattern of CD8 among patients with anti-PD-1 treated HCC with PLA2G7 high &CD68 high versus others (n=29). (F) The distributions of treatment responses among patients with anti-PD-1 treated HCC with PLA2G7 high &CD68 high versus others (n=29). (G) Kaplan-Meier analysis comparing the overall survival of patients with anti-PD-1 treated HCC with PLA2G7 high &CD68 high versus others (n=21). Statistical analysis was performed using the χ 2 test in (E) the Fisher’s exact test in (F) and the log-rank test in (G). *p<0.05. HCC, hepatocellular carcinoma; IF, immunofluorescence; PD, progressive disease; PD-1, programmed cell death protein 1; PLA2G7, phospholipase A2 Group VII; PR, partial response.

Article Snippet: Non-specific binding was blocked by incubating the slides with 5% bovine serum albumin (BSA), followed by overnight staining with primary antibodies against the following antigens at 4°C: PLA2G7 (Proteintech, #15 526–1-AP), CD68 (Abcam, #ab283654), and CD8 (Abcam, #ab237709).

Techniques: Expressing, Immunofluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Inhibiting PLA2G7 reverses the immunosuppressive function of intratumoral macrophages and augments immunotherapy response in hepatocellular carcinoma

doi: 10.1136/jitc-2023-008094

Figure Lengend Snippet: PLA2G7 preserves the immunosuppressive phenotype in macrophages. (A) t-SNE plots showing the PLA2G7 low and PLA2G7 high macrophages in Data set 1. (B) The expression-based scores for pro-inflammation (left panel) and T-cell activation (right panel) were calculated in PLA2G7 low and PLA2G7 high macrophages at the single-cell level. (C) Western blot analysis of PLA2G7 in untreated or TCM-educated THP-1-differentiated macrophages. β-actin was used as loading control. (D) Flow cytometric analysis of CD86 and CD206 on TCM-education THP-1-differentiated macrophages treated with darapladib (0.5 µM, 1 µM, and 2 µM) or vehicle. (E) Schematic representation of in vitro induction of TAM-like BMDMs. (F) Western blot analysis of PLA2G7 in untreated or TCM-educated BMDMs. β-actin was used as loading control. (G) Flow cytometric analysis of CD86 and CD206 on TCM-educated BMDMs treated with either darapladib (0.5 µM, 1 µM, and 2 µM) or vehicle. (H) Analysis of IFN-γ+ GZMB+ CD8 T cells after co-culturing with TCM-educated BMDMs treated with either darapladib (0.5 µM, 1 µM, and 2 µM) or vehicle. Statistical analysis was performed using the Mann-Whitney U test in (B) and the Student’s t-test in (D) and (G–H). Data was presented as median with IQR in (B) and mean with SD in (D) and (G–H). *p<0.05, **p<0.01, ***p<0.001. BMDM, bone marrow-derived macrophages; GZMB, granzyme; IFN-γ, interferon-γ; PLA2G7, phospholipase A2 Group VII; TCM, tumor-conditioned medium; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: Non-specific binding was blocked by incubating the slides with 5% bovine serum albumin (BSA), followed by overnight staining with primary antibodies against the following antigens at 4°C: PLA2G7 (Proteintech, #15 526–1-AP), CD68 (Abcam, #ab283654), and CD8 (Abcam, #ab237709).

Techniques: Expressing, Activation Assay, Western Blot, Control, In Vitro, MANN-WHITNEY, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Inhibiting PLA2G7 reverses the immunosuppressive function of intratumoral macrophages and augments immunotherapy response in hepatocellular carcinoma

doi: 10.1136/jitc-2023-008094

Figure Lengend Snippet: PLA2G7 promotes macrophage phenotypic transition via modulating the NF-κB pathway. (A–B) GSEA analysis demonstrates the downregulation of signature genes of NF-κB and MAPK pathways in PLA2G7 high macrophages versus PLA2G7 low macrophages in both Data set 1 (A) and Data set 2 (B). (C–D) Western blot analysis of NF-κB and MAPK pathways in tumor-associated macrophages treated with either darapladib (2 µM) or vehicle. (E–G) Flow cytometric analysis of CD86 and CD206 on TCM-educated BMDMs treated with either darapladib (2 µM), SB203580 (10 µM), SB203580 (10 µM) + darapladib (2 µM), JSH-23 (10 µM), JSH-23 (10 µM) + darapladib (2 µM), or vehicle. (H–I) Analysis of IFN-γ+ GZMB+ CD8 T cells after co-culturing with TCM-educated BMDMs treated with either darapladib (2 µM), SB203580 (10 µM), SB203580 (10 µM) + darapladib (2 µM), JSH-23 (10 µM), JSH-23 (10 µM) + darapladib (2 µM), or vehicle. Student’s t-test. Data was presented as mean with SD. ns, no significance; *p<0.05, **p<0.01, ***p<0.001. BMDM, bone marrow-derived macrophages; GZMB, granzyme B; IFN-γ, interferon-γ; NES,normalized enrichment score; PLA2G7, phospholipase A2 Group VII; TCM, tumor-conditioned medium; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: Non-specific binding was blocked by incubating the slides with 5% bovine serum albumin (BSA), followed by overnight staining with primary antibodies against the following antigens at 4°C: PLA2G7 (Proteintech, #15 526–1-AP), CD68 (Abcam, #ab283654), and CD8 (Abcam, #ab237709).

Techniques: Western Blot, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Inhibiting PLA2G7 reverses the immunosuppressive function of intratumoral macrophages and augments immunotherapy response in hepatocellular carcinoma

doi: 10.1136/jitc-2023-008094

Figure Lengend Snippet: Phospholipase A2 Group VII inhibition by darapladib enhances the efficacy of anti-PD-1 treatment in HCC in vivo. (A) Schematic representation of the treatment schedule for clodronate liposomes, anti-PD-1 and darapladib in C57BL/6 mice bearing HCC tumors. (B–C) Murine orthotopic HCC tumors treated with either darapladib, anti-PD-1, darapladib+anti-PD-1, or isotype control. Representative bioluminescence images and statistical diagram illustrating tumor weights are presented (n=5, each). (D) Representative pictures of mIF analysis for F4/80, MHC-II, and CD8 markers in the orthotopic HCC tumors treated with either darapladib, anti-PD-1, darapladib+anti-PD-1, or isotype control. Scale bar: 50 µm. (E–G) Quantification of corresponding immune cells by mIF analysis. (H–J) Tumor growth curves and weights of Hepa 1–6 subcutaneous tumors in C57BL/6 mice treated with either darapladib, anti-PD-1, darapladib+anti-PD-1, or isotype control (n=5 each). Student’s t-test (or one-way analysis of variance with a post hoc LSD test in (E)). Data was presented as mean with SD. ns, no significance; *p<0.05, **p<0.01, ***p<0.001. HCC, hepatocellular carcinoma; mIF, multiplex immunofluorescence; MHC, major histocompatibility complex; PD-1, programmed cell death protein 1; ROI, region of interest.

Article Snippet: Non-specific binding was blocked by incubating the slides with 5% bovine serum albumin (BSA), followed by overnight staining with primary antibodies against the following antigens at 4°C: PLA2G7 (Proteintech, #15 526–1-AP), CD68 (Abcam, #ab283654), and CD8 (Abcam, #ab237709).

Techniques: Inhibition, In Vivo, Liposomes, Control, Multiplex Assay, Immunofluorescence, Immunopeptidomics

Journal: bioRxiv

Article Title: RalB uncoupled exocyst mediates endothelial Weibel-Palade body exocytosis

doi: 10.1101/2024.09.16.613344

Figure Lengend Snippet: (A) HUVECs depleted of RalA or RalB using specific siRNAs and compared with control siRNA. Western blot of total cell lysates showing RalA and RalB knockdown after 48 hours of transfection of siRNA, with β -actin as loading control. (B) Bar graphs showing total vWF antigen in media as estimated by ELISA 30 minutes after 1 U/ml thrombin stimulation 48 hours after siRNA transfection (** P <0.01; from 3 individual experiments each with duplicates). (C) HUVECs treated with increasing concentrations of dihydroartemisinin for 2 hours or DMSO. Western blot showing degradation of RalB but not RalA by dihydroartemisinin, with β-actin as loading control. (D) Bar graphs showing total vWF antigen in media as estimated by ELISA 30 minutes after 1 U/ml thrombin stimulation 2 hours after dihydroartemisinin treatment (** P <0.01, *** P <0.001; **** P <0.0001 from 3 individual experiments each with duplicates). (E) Total cell lysates of resting and thrombin-treated (1 U/ml for 2 minutes) HUVECs were incubated with RalBP1-RBD conjugated agarose slurry and eluates immunoblotted with anti-RalB antibody to determine total GTP-loaded RalB in each condition. Coomassie stain showing RalBP1-RBD as loading control (left). Bar graphs showing densitometric analysis (right). (** P <0.01; from 3 individual experiments).

Article Snippet: Experiments were performed on RalB and RalA knockdown cells 48 h post-transfection. pLA-CMV-N-Flag-RalBWT (plasmid#50990), pLX301 (plasmid#25895), pEGFP-C2-CD63 (plasmid#62964) were obtained from Addgene.

Techniques: Control, Western Blot, Knockdown, Transfection, Enzyme-linked Immunosorbent Assay, Incubation, Staining