Journal: Molecular Human Reproduction

Article Title: ADAM8 localizes to extravillous trophoblasts within the maternal–fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism

doi: 10.1093/molehr/gay034

Figure Lengend Snippet: ADAM8 preferentially localizes to anchoring columns of placental villi. (A) Representative immunofluorescence, immunohistochemistry and RNAScope microscopy images of serially sectioned first trimester placental villi (6–11 weeks gestation; n = 5) and decidua (10–12 weeks gestation; n = 4) showing ADAM8 mRNA transcript localization (pink punctate signal) within trophoblast subtypes. Proximal and distal column, as well as interstitial EVT subtypes of trophoblasts are identified by immunostaining of cytokeratin (KRT7) and HLA-G. ‘PCT’ indicates proximal column trophoblast; ‘DCT’ indicates distal column trophoblast; ‘VT’ indicates villous trophoblast; ‘SynT’ indicates syncytiotrophoblast; ‘MC’ indicates placenta mesenchymal core; ‘iEVT’ indicates interstitial extravillous trophoblast; ‘GC’ indicates trophoblast giant cell. The perforated white box indicates enlarged inset image. Black arrowheads denote ADAM8 signal. Bars = 100 μm. (B) Fluorescence activated cell-sorting (FACS) plots demonstrate the trophoblast isolation strategy used to purify mesencymal core cells (MC), distal column trophoblasts (DCT) and villous/proximal column trophoblasts (VT). Live cells, depleted of CD31+ (endothelial) and CD45+ (immune) cells using immuno-magnetic beads, were positively gated by 7AAD exclusion. Cells were further segregated by excluding CD45+ immune cells and by cell surface labeling of HLA-G and CD49f. Cell subtype proportions are indicated within each gated population (percent of cells within FACS plot). (C) ADAM8 mRNA levels in FACS-purified MCs, VTs and DCTs. Trophoblast subtype purity was assessed by qPCR analysis targeting the pan-trophoblast marker KRT7, the EVT-marker HLA-G and the mesenchymal lineage marker VIM. GAPDH was used for normalization. Results are presented as mean ± SD in bar graphs from four distinct placental villi specimens (n = 4); results were analyzed by one-way ANOVA and Dunn’s multiple comparisons test. (D) Immunoblot showing protein levels of pro- and active-ADAM8, HLA-G, and EGFR in MC, VT and DCT isolated from placental villi using immuno-magnetic beads. Molecular weights (kDa) are shown to the left and β-actin indicates loading control.

Article Snippet: Contaminating immune cells were removed from the cell admixture by EasySep CD45 immuno-magnetic bead purification (StemCell Technologies) where remaining cells were plated onto plastic culture plates for 30 min to remove stromal cells.

Techniques: Immunofluorescence, Immunohistochemistry, RNAscope, Microscopy, Immunostaining, Fluorescence, FACS, Isolation, Magnetic Beads, Labeling, Purification, Marker, Western Blot, Control

Journal: RSC Advances

Article Title: Effects of a natural PTP1B inhibitor from Rhodomela confervoides on the amelioration of fatty acid-induced insulin resistance in hepatocytes and hyperglycaemia in STZ-induced diabetic rats

doi: 10.1039/c9ra10660j

Figure Lengend Snippet: BPN is a competitive PTP1B inhibitor. (A) Chemical structure of BPN. (B) The Lineweaver–Burk plot for inhibition of PTP1B enzyme reaction by BPN. The experiment was performed at 37 °C and pH 7.5. BPN concentrations were 4 (◆), 8 (■) and 16 μM (▲), respectively. (C) K i determination for BPN. The plot of apparent K m versus the concentrations of BPN. BPN concentrations were 4 (◆), 8 (■) and 16 μM (▲), respectively. (D) SPR dose–response curves of BPN. The K a , K d and K D values of BPN were 4.238 × 10 3 Ms −1 , 1.406 × 10 −2 s −1 , 3.318 × 10 −6 M, respectively. K D values of BPN was obtained by fitting the data sets to 1 : 1 binding model using Biacore T200 Evaluation Software.

Article Snippet: The binding affinity of BPN towards PTP1B was assayed using the SPR-based Biacore T200 instrument. hPTP1B 1-321 in 10 mM sodium acetate buffer (pH 5.0) was immobilized onto CM5 sensor chips using standard amine coupling method to 10 000 response units.

Techniques: Inhibition, Binding Assay, Software

Journal: Science Advances

Article Title: Targeting conserved TIM3 + VISTA + tumor-associated macrophages overcomes resistance to cancer immunotherapy

doi: 10.1126/sciadv.adm8660

Figure Lengend Snippet: ( A ) Basal immunophenotyping of LLC and MC38 tumors, showing percentages of CD8 + T cells, CD4 + T cells, and TAMs ( n = 5). See fig. S3B for the violin plots and statistics. ( B and C ) Tumor volume curve of (B) LLC and (C) MC38 tumor–bearing mice treated with anti-PD1/PD-L1/CTLA4 antibody or phosphate-buffered saline (PBS) [ n = 4; Kruskal-Wallis test corrected for false discovery rate (FDR)]. ( D to G ) KDE plots showing TAM populations in LLC and MC38 tumors from flow cytometry data of CD45 + cell fraction obtained from LLC and MC38 tumors. KDE plots were made on the basis of the Uniform Manifold Approximation and Projections (UMAPs) shown in fig. S3 (E and F). ( H ) Percentage of TIM3 + VISTA + TAMs in LLC and MC38 tumors ( n = 9; Mann-Whitney test). ( I ) Different markers used in for LLC and MC38 tumor samples. Size of dots represents fraction of cells and color intensity represents mean expression levels in each group. ( J and K ) CD45 + cell fraction from LLC and MC38 tumors. (J) Ratio of M1-like (MHCII HIGH CSF1R LOW ) TAMs to M2-like (CSF1R HIGH MHCII LOW ) TAMs and (K) ratio of M1-like (MHCII HIGH CD206 LOW ) TAMs to M2-like (CD206 HIGH MHCII LOW ) TAMs of TIM3 + VISTA + TAMs ( n = 6; Mann-Whitney test). ( L ) UMAP of scRNA-seq data from LLC tumors. ( M ) scRNA-seq LLC tumor tissue dataset, showing Vsir and Havcr2 in different immune cell subsets. ( N ) Percentage of TIM3 + VISTA + on CD8 + and CD4 + T cells, TAMs, and DCs—conventional DC 1 (cDC1) and cDC2, monocytic DC (moDC), and plasmacytoid DC (pDC)—in LLC tumors ( n = 4; Kruskal-Wallis test corrected for FDR, comparison to TAMs). ( O ) pHrodo Green + TIM3 + VISTA + CD11b + F4/80 + or TIM3 − VISTA − CD11b + F4/80 + in cocultures of BMDMs or TAMs from LLC or MC38 tumors, cocultured with pHrodo labeled E. coli bioparticles ( n = 4; Kruskal-Wallis test corrected for FDR). N number represents number of biological repeats or number of independent animals used.

Article Snippet: Tumor-infiltrating leukocytes (TILs) were isolated through magnetic bead separation via CD45 (Miltenyi, #130-110-618).

Techniques: Saline, Flow Cytometry, MANN-WHITNEY, Expressing, Comparison, Labeling

Journal: Science Advances

Article Title: Targeting conserved TIM3 + VISTA + tumor-associated macrophages overcomes resistance to cancer immunotherapy

doi: 10.1126/sciadv.adm8660

Figure Lengend Snippet: ( A and B ) Tumor volume curve of (A) LLC tumors and (B) MC38 tumors treated with CDDP or PBS with anti-TIM3/VISTA antibody (LLC, n = 8; MC38, n = 4; Kruskal-Wallis test corrected for FDR). ( C and D ) Tumor volume curve of (C) LLC tumors and (D) MC38 tumors treated with PTX or PBS with anti-TIM3/VISTA antibody (LLC, n = 10; MC38, n = 4; Kruskal-Wallis test corrected for FDR). ( E to G ) CD45 + cell fraction obtained from (C). (E) Percentage of TIM3 + VISTA + TAMs ( n = 8; Kruskal-Wallis test corrected for FDR). (F) Ratio of M1-like (MHCII HIGH CSF1R LOW ) TAMs to M2-like (CSF1R HIGH MHCII LOW ) TAMs ( n = 8; Kruskal-Wallis test corrected for FDR). (G) Percentage of CD8 + T cells ( n = 8; Kruskal-Wallis test corrected for FDR). ( H and I ) Tumor volume curve of LLC tumors treated with PTX or PBS and/or anti-TIM3/VISTA antibody (H) combined with clodronate liposomes ( n = 9; Kruskal-Wallis test corrected for FDR, comparison to PBS) and (I) combined with anti-CD8 antibody ( n = 8; Kruskal-Wallis test corrected for FDR, comparison to PBS). ( J and K ) Lysate from CD45 − cell fraction obtained from (C). (J) Protein levels of different analytes. Incalculable values are depicted as gray ( n = 4). (K) Pro–caspase 9, cleaved caspase 9, and actin Western blot. Ratio of quantification of pixel intensity is represented in a violin plot ( n = 4; Kruskal-Wallis test corrected for FDR.) ( L ) Percentages of cancer cell death after coincubation with TAMs with different inhibitors [ n = 4; two-way analysis of variance (ANOVA) corrected for FDR]. ( M ) Overview of a CRISPR-Cas9–mediated Vsir and Havcr2 knockout in CSF1R + cells (MΦs) (created with BioRender.com ). WT, wild-type. ( N to P ) LLC tumor–bearing TIM3 − VISTA − MΦ or control knockout MΦ mice, treated with PTX or PBS. (N) Percentages of CSF1R + and CSF1R − TIM3 + VISTA + TAMs ( n = 5; Kruskal-Wallis test corrected for FDR). (O) Percentages of TIM3 + VISTA + TAMs ( n = 5; Kruskal-Wallis test corrected for FDR). (P) Tumor volume curve (control knockout PTX, n = 8; others, n = 6; Kruskal-Wallis test corrected for FDR).

Article Snippet: Tumor-infiltrating leukocytes (TILs) were isolated through magnetic bead separation via CD45 (Miltenyi, #130-110-618).

Techniques: Liposomes, Comparison, Western Blot, CRISPR, Knock-Out, Control

Journal: Science Advances

Article Title: Targeting conserved TIM3 + VISTA + tumor-associated macrophages overcomes resistance to cancer immunotherapy

doi: 10.1126/sciadv.adm8660

Figure Lengend Snippet: ( A to C ) Basal immunophenotyping of (A) YUMM1.7, (B) YUMM1.7-OVA, and (C) YUMMER1.7 tumors showing percentages of CD8 + T cells, CD4 + T cells, and TAMs ( n = 6). See fig. S14 (A to C) for the violin plots and statistics corresponding to these pie charts. ( D ) Flow cytometry analysis of CD45 + cell fraction obtained from subcutaneous YUMM1.7, YUMM1.7-OVA, and YUMMER1.7 tumors. Percentage of TIM3 + VISTA + CSF1R + TAMs (YUMM1.7, n = 5; others, n = 6; Mann-Whitney test). ( E to G ) Tumor volume curve of (E) YUMM1.7, (F) YUMM1.7-OVA, and (G) YUMMER1.7 tumor–bearing mice treated with anti-PD1 antibody ( n = 4; Kruskal-Wallis test corrected for FDR). ( H to J ) Tumor volume curve of (H) YUMM1.7, (I) YUMM1.7-OVA, and (J) YUMMER1.7 tumor–bearing mice treated with PTX or PBS and/or combination with anti-TIM3 or anti-VISTA antibody (YUMM1.7, n = 8; others, n = 4; Kruskal-Wallis test corrected for FDR). ( K to P ) Flow cytometry analysis of CD45 + cell fraction obtained from subcutaneous YUMM1.7, YUMM1.7-OVA, and YUMMER1.7 tumors treated with PTX or PBS and/or combination with anti-TIM3 or anti-VISTA antibody. (K to M) Percentage of TIM3 + VISTA + CSF1R + TAMs, YUMM1.7 (K), YUMM1.7-OVA (L), and YUMMER1.7 (M) subcutaneous tumors (YUMM1.7, n = 8; others, n = 4; Kruskal-Wallis test corrected for FDR). (N to P) Percentage of CD3 + CD8 + T cells of live cells in YUMM1.7 (N), YUMM1.7-OVA (O), and YUMMER1.7 (P) subcutaneous tumors (YUMM1.7, n = 8; others, n = 4; Kruskal-Wallis test corrected for FDR). N number represents the number of biological repeats or number of independent animals used.

Article Snippet: Tumor-infiltrating leukocytes (TILs) were isolated through magnetic bead separation via CD45 (Miltenyi, #130-110-618).

Techniques: Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Lung Epithelial Cells Can Produce Antibodies Participating In Adaptive Humoral Immune Responses

doi: 10.1101/2021.05.13.443498

Figure Lengend Snippet: (A) The left panel: the local expression of IgA in lung tissue of μ MT mice were detected by immunohistochemical staining. The selected representative image was repeated 3 times independently. Bar=50μm in the figure. The right panel: lung epithelial cells in bronchi or alveoli of μMT mice were detected with anti-mouse CD326 by immunofluorescence (top) and FACS (bottom). (B) The analysis of the various class (for heavy chain) or types (for light chain) of Ig expression in different lung epithelial cells from a set of mouse lung tissue single-cell sequencing data (GSE124872) in the GEO database. (C) The expression of different Igs in the culture supernatant of primary cultured BALB/c mouse lung epithelial cells were detected by the Quantibody ® Mouse Ig Isotype Array. (D) The secretion level of anti-NP-IgG and anti-NP-IgA of lung epithelial cells from BALB/c mice and μMT mice after immunized with NP-KLH were detected by ELISPOT. (E) The level of anti-NP-IgG in the culture supernatant of lung epithelial cells (CD45 − EpCAM + ) at 0h, 12h, 24, 36h, 48h from μMT mice immunized with NP-KLH were detected by ELISA. n=3, and individual data was represented by each line. (F) We knocked down Igκ in lung epithelial cells by infecting Igc flox/flox mice with AAV-Cre intranasally, then confirmed that AAV-Cre significantly reduced the Igk expression level in lung tissue by Western blot. The levels of anti-NP-IgM, anti-NP-IgA and anti-NP-IgG in BALF, lung tissue and serum of the mice were detected by ELISA.GFP-PBS: n=3, GFP-NP-KLH: n=3, Cre-NP-KLH: n=5 (G) We knocked down Igκ in Clara cells by infecting Igκ flox/flox mice with AAV-CC10-Cre intranasally, the levels of anti-NP-IgG in BALF serum of the mice were detected by ELISA.AAV-PBS: n=4, AAV-NP-KLH: n=4, AAV-Cre-KLH: n=3, and statistical difference between AAV-NP-KLH and AAV-Cre-KLH was analyzed. ****P□<□0.0001, ***P□<□0.001, **P□<□0.01, *P□<□0.05, ns P□>□0.05.

Article Snippet: Lung epithelial cells separated by magnetic bead: the anti-mouse CD45 magnetic bead (Miltenyi Biotec) was used to enrich the epithelial cells in single-cell suspension, and then the anti-mouse EpCAM magnetic bead (Miltenyi Biotec) was used to separate the epithelial cells.

Techniques: Expressing, Immunohistochemical staining, Staining, Immunofluorescence, Sequencing, Cell Culture, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Western Blot