Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) and presence of each drug (10 µM). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Blots of results with fluvastatin (Fluv) and simvastatin (Simv) are shown. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) After testing 1,165 drugs, levels of CD9 and CD81 relative to actin were quantified by densitometry. Fold changes of the expression levels compared with vehicle alone were calculated and plotted. Drugs that increased the level of either CD9 or CD81 more than 1.5-fold compared with vehicle alone were regarded as positive. Correlation between fold changes in CD9 and CD81 levels was analyzed using Pearson’s correlation coefficient. ( C ) RAW264.7 cells were cultured in the absence (V) or presence of multiple statins (10 µM) and levels of CD9 and CD81 were examined by immunoblotting. The statins are arranged in order of decreasing lipophilicity. Ceri, cerivastatin; Simv, simvastatin; Fluv, fluvastatin; Ator, atorvastatin; Rosu, rosuvastatin; Prav, pravastatin. ( D ) RAW264.7 cells were cultured in the absence (shaded histograms) or presence (10 µM) of fluvastatin (open red histograms) and simvastatin (open blue histograms). Surface levels of CD9, CD63, CD81, and the integrin β1 subunit were analyzed by flow cytometry.

Article Snippet: The primary Abs were rat anti-mouse CD9 mAb (KMC8; BD Biosciences), hamster anti-mouse CD81 mAb (Eat2; UK-Serotec), rabbit anti-LAMP3 (CD63) polyclonal Ab (12632-1-AP; Proteintech), mouse anti-human CD9 mAb (MM2/57; Biosource), mouse anti-human CD81 mAb (JS64; Immunotech), rabbit anti-IκBα polyclonal Ab (9242; Cell Signaling Technology), rat anti-mouse integrin β2 subunit mAb (M18/2; BD Biosciences), rat anti-CD14 mAb (rmC5-3; BD Biosciences), rabbit anti-TLR4 polyclonal Ab (IMG577; Imgenex), mouse anti-flotillin-1 mAb (clone 18; BD Biosciences), and goat anti-CD45 polyclonal Ab (R&D Systems).

Techniques: Cell Culture, Western Blot, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence or presence of increasing concentrations of fluvastatin (Fluv) or simvastatin (Simv). The cells were lysed, and levels of CD9, CD63, and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) RAW264.7 cells were untreated (-) or cultured in the absence or presence of increasing concentrations of fluvastatin or simvastatin and stimulated for 24 h with 0.1 µg/ml LPS (+). Levels of CD9, CD63, and CD81 were examined by immunoblotting. Note that LPS downregulates CD9 and CD81 in the absence of statins (arrowheads). ( C ) RAW264.7 cells were cultured in the absence (-) or presence of 3 µM fluvastatin (+), and unstimulated (-) or stimulated for 24 h with 1 µg/ml LPS (+). mRNA levels of CD9 and CD81 were examined by reverse transcription PCR. GAPDH is an internal loading control. ( D ) RAW264.7 cells were cultured in the absence or presence of fluvastatin, and unstimulated or stimulated with LPS. Control (Cont) was an untreated culture. mRNA levels of CD9 and CD81 were examined by real-time PCR. Data shown are from one representative of three similar experiments. ( E ) Human monocytic THP-1 cells were treated for 4 h with 1 µg/ml phorbol 12-myristate 13-acetate, allowed to attach to a plate, and then cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting. ( F ) Mouse 3T3 fibroblasts were cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting.

Article Snippet: The primary Abs were rat anti-mouse CD9 mAb (KMC8; BD Biosciences), hamster anti-mouse CD81 mAb (Eat2; UK-Serotec), rabbit anti-LAMP3 (CD63) polyclonal Ab (12632-1-AP; Proteintech), mouse anti-human CD9 mAb (MM2/57; Biosource), mouse anti-human CD81 mAb (JS64; Immunotech), rabbit anti-IκBα polyclonal Ab (9242; Cell Signaling Technology), rat anti-mouse integrin β2 subunit mAb (M18/2; BD Biosciences), rat anti-CD14 mAb (rmC5-3; BD Biosciences), rabbit anti-TLR4 polyclonal Ab (IMG577; Imgenex), mouse anti-flotillin-1 mAb (clone 18; BD Biosciences), and goat anti-CD45 polyclonal Ab (R&D Systems).

Techniques: Cell Culture, Western Blot, Reverse Transcription, Control, Real-time Polymerase Chain Reaction

Journal: BMC cancer

Article Title: Galectin-9 expression clinically associated with mature dendritic cells infiltration and T cell immune response in colorectal cancer.

doi: 10.1186/s12885-022-10435-4

Figure Lengend Snippet: Fig. 3 DCs infiltrate the tumor microenvironment of CRC patients. A IHC was performed to assess the frequency of CD208 + DCs (red arrow) and CD1a + DCs (blue arrow). B Violin plot showing the frequency of CD208 + DCs in CRC patients with different MMR statuses, * p < 0.05. C Column bar graph showing the frequency of CD1a + DCs in CRC patients with different MMR statuses, *** p < 0.001

Article Snippet: An immunohistochemical (IHC) study on galectin-9 (54330 s, Cell Signaling Technology (CST); 1:500), mouse anti-human CD208 (LAMP3) antibody (to define mDCs; DDX0191P-100; Novusbio USA; 1:10), mouse antihuman CD1a antibody (to define iDCs [17]; 1:100 dilution; Zhongshan Golden Bridge Biotech Co., Ltd, Beijing, China), CD3 (Zhongshan Golden Bridge Biotech Co., Ltd, ready to use, Beijing, China) and CD8 (Zhongshan Golden Bridge Biotech Co., Ltd, ready to use, Beijing, China) was carried out on formalin-fixed paraffin, with a 4-μm-thick serial section of tissue, according to the manufacturer’s recommended protocol.

Techniques:

Journal: BMC cancer

Article Title: Galectin-9 expression clinically associated with mature dendritic cells infiltration and T cell immune response in colorectal cancer.

doi: 10.1186/s12885-022-10435-4

Figure Lengend Snippet: Fig. 4 Correlation between galectin-9 expression and DC infiltration. A Representative IHC images of CD208 (red arrow) and CD1a (blue arrow) obtained from a patient with high galectin-9 expression and a patient with low galectin-9 expression. B Linear analysis of galectin-9 expression and CD208 + DC infiltration (N = 139), ***p < 0.001; C Linear analysis of galectin-9 expression and CD1a + DC infiltration (N = 75), * p < 0. (D) Linear analysis of galectin-9 expression and DC maturity rate (N = 75), * p < 0.05

Article Snippet: An immunohistochemical (IHC) study on galectin-9 (54330 s, Cell Signaling Technology (CST); 1:500), mouse anti-human CD208 (LAMP3) antibody (to define mDCs; DDX0191P-100; Novusbio USA; 1:10), mouse antihuman CD1a antibody (to define iDCs [17]; 1:100 dilution; Zhongshan Golden Bridge Biotech Co., Ltd, Beijing, China), CD3 (Zhongshan Golden Bridge Biotech Co., Ltd, ready to use, Beijing, China) and CD8 (Zhongshan Golden Bridge Biotech Co., Ltd, ready to use, Beijing, China) was carried out on formalin-fixed paraffin, with a 4-μm-thick serial section of tissue, according to the manufacturer’s recommended protocol.

Techniques: Expressing

Journal: bioRxiv

Article Title: Cell division tracing combined with single-cell transcriptomics reveals new cell types and differentiation paths in the regenerating mouse lung

doi: 10.1101/2023.01.18.524384

Figure Lengend Snippet: A. Schematic of Sftpc-CreER, Scgb1a1-CreER, Rosa26R-DTA, and CycB1-GFP transgenes in SfSRC mice. B. IF staining of SfSRC mouse lungs with Cyp2f2 (white; club cells), Lamp3 (red; AT2 cells), CycB1-GFP (green; dividing cells), and DAPI (blue; nuclei), on days 0, 2, 3, 14, 28, and 56 after tamoxifen injections. Aw: airway. Scale bar: 100 μm. Images are representative of at least three animals analyzed per group. C. Outline of cells used for scRNA-seq. Created with BioRender.com. D. UMAP embedding showing cell type assignment of epithelial cells before (day 0, n=5 mice) and two (n=9 mice) and three days (n=6 mice) after tamoxifen injection. At day 2, basal_Krt13 + and club_Krt15 + cells are highlighted. E. Dot plot showing normalized enrichment score (NES) and significance for published signatures of epithelial cell types in all SfSRC mouse epithelial populations (all timepoints merged). DATP: damage-associated transient progenitors; PATS: pre-alveolar type-1 transitional cell state; RAS: respiratory airway secretory; BC: basal cell; TRB-SC: terminal and respiratory bronchioles secretory cell; pre-TB-SC: pre-terminal bronchiole secretory cell; AEP: alveolar epithelial progenitor; BASC: bronchioalveolar stem cell. Cell types described in the human lung are depicted in red. F. Heatmap of the top 20 marker genes for basal_Krt13 + and club_Krt15 + cells, and expression of Krt15, Krt13, and Krt4, in basal and club cells of SfSRC mice (all timepoints merged).

Article Snippet: The following antibodies were used: acetylated tubulin (1:1000, #T7451, Sigma), Agr2 (1:100, #12275-1-AP, Proteintech), CC10 (1:500, #07-623, Millipore), CD34 (1:200, #553731, BD), Cyp2f2-Alexa Fluor 647 (1:1000, #sc-374540, Santa Cruz), GFP-FITC (1:500, #600-102-215, Rockland), Krt5 (1:500, # ab53121, Abcam), Lamp3 (1:500, #DDX0192A647-100, Origene), Npnt (1:50, PA547610, Thermo Fischer Scientific), Pdpn (1:2000, #ab11936, Abcam), SPC (1:1000, # AB3786, Millipore), donkey anti-rabbit Alexa Fluor 568 (1:500, Thermo Fischer Scientific), goat anti-rat Alexa Fluor 568 (1:500, Thermo Fischer Scientific), and goat anti-hamster Alexa Fluor 647 (1:500, Thermo Fischer Scientific).

Techniques: Staining, Injection, Marker, Expressing