Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: ( A ) The pathway analysis of CXCL13 associated genes. The data from the microarray data sets of the eight highly polluted region (HPR) lung cancers are shown. See also . ( B ) Gene ranking according to the correlation with CXCL13 expression. The genes were filtered based on extracellular localization to identify paracrine mediators. The list on the right shows the genes that correlate most significantly with CXCL13 . ( C ) The mRNA expression of the candidate gene was detected in THP-1 cells by qPCR. ( D ) The concentration of SPP1 in supernatants of THP-1 cells in the absence or presence of CXCL13. ( E ) Trans-well migration assays were performed by plating THP-1 cells (transfected with siSPP1 or siNC) in the lower chambers, and the indicated cells (CXCL13-treated or untreated) in the upper chambers. ( F ) Spp1 serum concentrations of mice treated with benzo(a)pyrene (BaP) and/or dexamethasone (DEX) were detected by ELISA. ( G ) Spp1 expression in lung section of mice treated with BaP and/or DEX was determined by IHC. ( H ) IHC assays using antibodies against Cd68, Ttf1, and Spp1. ( I ) Immunofluorescence assay using antibodies against Spp1 (green), Cd68 (red), and Ttf1 (white). DAPI was used to stain the nucleus (blue). DOI: http://dx.doi.org/10.7554/eLife.09419.016 10.7554/eLife.09419.017 Figure 6—source data 1. CXCL13 -associated genes in lung cancer. DOI: http://dx.doi.org/10.7554/eLife.09419.017

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques: Microarray, Expressing, Concentration Assay, Migration, Transfection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: ( A ) The expression of indicated genes in lung tissues from A/J mice treated with BaP and/or dexamethasone (DEX) was detected by real-time PCR. ( B ) Western blot analyses of lysates of lung tissues from A/J mice treated with BaP and/or DEX. ( C ) Western blot analyses of cytoplasmic and nucleic protein fractions of lung tissues from A/J mice treated with BaP and/or DEX, using indicated antibodies. ( D ) Western blot analysis of lysates of lung tissues from NOD/SCID mice injected with indicated cells. ( E ) Western blot analyses of cytoplasmic and nucleic protein fractions in lung tissues from NOD/SCID mice injected with indicated cells. ( F ) Western blot analyses of lysates of lung tissues from Cxcr5 mutant mice treated with BaP. ( G ) IHC assays for the expression of β–catenin in Cxcr5 mutant mice upon BaP. ( H ) Immunofluorescence analyses of β-catenin in A549 cells transfected with control vector (V) or SPP1, or treated with THP-1 supernatant (Mϕs.) or supernatant of THP-1 cells co-incubated with CXCL13 (M13s.). ( I ) Western blot analyses of cytoplasmic and nucleic protein fractions of A549 cells transfected with control vector (V) or SPP1, or treated with Mϕs. or M13s. ( J ) Real-time PCR assays of indicated genes in A549 cells transfected with control vector or SPP1, or treated with Mϕs. or M13s. ( K ) Western blot analyses of lysates of A549 cells transfected with control vector (V) or SPP1, or treated with Mϕs. or M13s, using indicated antibodies. DOI: http://dx.doi.org/10.7554/eLife.09419.018

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Mutagenesis, Immunofluorescence, Transfection, Control, Plasmid Preparation, Incubation

Journal: Cell Death & Disease

Article Title: Global transcriptomic changes in glomerular endothelial cells in mice with podocyte depletion and glomerulosclerosis

doi: 10.1038/s41419-021-03951-x

Figure Lengend Snippet: A Representative immunofluorescence images of SPP1 (red) with endothelial marker isolectin GS-IB4 (IB4, green, top panels) or podocyte marker, podocin (green, bottom panels). DNA is counterstained in blue. Scale bar, 50 μm. B Average fluorescence intensity of SPP1 in glomeruli of control vs. diseased mice are shown in arbitrary units ( n = 6 control, 7 diseased mice, 20–30 glomeruli quantified per mouse). C Western blot analysis of cultured GECs expressing short harpin RNA (shRNA) against scrambled control (shScr) or against four independent clones of shRNA against Spp1 (ShSpp1-1, -2, -3, and -4). The densitometric analysis of SPP1 normalized to GAPDH is shown for shScr vs. shSpp1-2 ( n = 3 independent experiments). D In vitro angiogenesis assay of GECs cultured in normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) and plated on Matrigel bed. Images were taken 6 h post plating. Scale bar, 250 μM. ** P < 0.01 between two groups by unpaired two-tailed t -test.

Article Snippet: Equal amounts of protein samples were separated on SDS polyacrylamide gel, transferred to polyvinylidene fluoride membranes (Millipore), and probed with primary antibodies: mouse anti-SPP1 (MPIIIB10, Developmental Studies Hybridoma Bank), mouse anti-CARP/ANKRD1 (sc365056, Santa Cruz Biotechnology), rabbit polyclonal Cleaved Caspase 3 (9664, Cell Signaling), and rabbit anti-GAPDH (2118; Cell Signaling Technologies).

Techniques: Immunofluorescence, Marker, Fluorescence, Western Blot, Cell Culture, Expressing, shRNA, Clone Assay, In Vitro, Angiogenesis Assay, Two Tailed Test