Journal: Research

Article Title: Mapping Immune-Inflammatory Niches on Zirconia Bone Implants: Single-Cell Transcriptomic Profiling

doi: 10.34133/research.1162

Figure Lengend Snippet: Intercellular cross talk and functional enrichment in the bone-marrow microenvironment following titanium (Ti) implantation. (A and B) Cells extracted from the Ti implant (A) and their proportions compared with those in the sham group (B). (C) Fuzzy C -means clustering of the differentially expressed genes (DEGs) and their functional annotation via Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses. BP, Biological Process; CC, Cellular Component; MF, Molecular Function. (D and E) Outgoing–incoming cross talk analysis among the bone-marrow cells and their subpopulations. (F) Outgoing–incoming signaling patterns and associated interaction strengths. (G) Analysis of collagen-related signaling networks and intercellular interaction strengths. (H) Interaction-strength analysis of candidate ligand–receptor pairs in bone-marrow fibroblasts. (I) KEGG and GO (BP, CC, and MF) enrichment analyses of the DEGs in bone-marrow fibroblasts. (J and K) Multiplex fluorescence colocalization and costaining analysis of candidate ligand–receptor pairs. Scale bars: 100 and 20 μm. COL1A1, collagen type I alpha 1 chain; SDC1, syndecan 1; ATP, adenosine triphosphate; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The following antibodies were applied sequentially: CD44 (Bioss, bsm-54767R, 1:300, China), CD68 (Proteintech, 66231-2-Ig, 1:500, China), CD206 (Bioss, bsm-55604R, 1:300, China), NOS2 (Bioss, bs-0162R, 1:300, China), COL6A2 (Bioss, bs-13963R, 1:300, China), S100A4 (Bioss, bs-3759R, 1:300, China), COL1A1 (Bioss, bs-10423R, 1:300, China), and SDC1 (Bioss, bs-1309R, 1:300, China), followed by nuclear counterstaining with 4′,6-diamidino-2-phenylindole.

Techniques: Functional Assay, Multiplex Assay, Fluorescence

Journal: Research

Article Title: Mapping Immune-Inflammatory Niches on Zirconia Bone Implants: Single-Cell Transcriptomic Profiling

doi: 10.34133/research.1162

Figure Lengend Snippet: Validation of the specific ligand–receptor pairs between the implants and bone-marrow cells. (A) Schematic diagram of the rat model of intramedullary femoral implantation and bulk RNA sequencing workflow. (B) Violin plots showing the expression levels of Col6a2 , Cd44 , Col1a1 , and Sdc1 . (C and D) Hierarchical clustering heatmaps of pairwise DEGs among the sham, Ti, and ZrO 2 groups. (E to G) Identification of the gene modules associated with Ti and ZrO 2 via weighted gene coexpression network analysis. (H) Overlapping genes between module-related genes and DEGs in each group. (I) Contributions of Ti and ZrO 2 -associated feature genes to predictive outcomes, assessed using least absolute shrinkage and selection operator (LASSO) regression analysis. (J and K) Temporal expression patterns and correlations of Col6a2 , Cd44 , Col1a1 , and Sdc1 . Statistical significance was assessed using the t test.

Article Snippet: The following antibodies were applied sequentially: CD44 (Bioss, bsm-54767R, 1:300, China), CD68 (Proteintech, 66231-2-Ig, 1:500, China), CD206 (Bioss, bsm-55604R, 1:300, China), NOS2 (Bioss, bs-0162R, 1:300, China), COL6A2 (Bioss, bs-13963R, 1:300, China), S100A4 (Bioss, bs-3759R, 1:300, China), COL1A1 (Bioss, bs-10423R, 1:300, China), and SDC1 (Bioss, bs-1309R, 1:300, China), followed by nuclear counterstaining with 4′,6-diamidino-2-phenylindole.

Techniques: Biomarker Discovery, RNA Sequencing, Expressing, Selection

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-D) qPCR analysis for the lipogenic marker genes PLIN2 and PPARg , as well as COL1A1 in human lung fibroblasts treated with metformin or vehicle. (E, F) Staining of lipid droplets in fibroblasts using LipidTOX (red). Nuclei were counterstained with DAPI (blue). (G-H) Gating strategy for detecting LipidTOX + cells by flow cytometry. (I) Quantification of LipidTOX + cells in response to metformin treatment. (J) Heatmap representation of the top 100 differentially expressed genes in fibroblasts following metformin treatment. Scale bars: (E, F) 25 µm. (B-D) Each data point within a given group corresponds to one patient. Vehicle-treated group: n=12, Metformin-treated group: n=11. (I) n=3 per group. ** P<0.01, *** P<0.001, **** P<0.0001.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Marker, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-D) qPCR analysis for PLIN2, PPARg and COL1A1 in human lung fibroblasts treated with TGFβ1 or vehicle for 72h. (E-H) Staining of TGFβ1- and vehicle-treated cells with LipidTOX (red), anti-ACTA2 antibodies (green) and DAPI (blue). (I-K) qPCR analysis for PLIN2, PPARg and COL1A1 in human lung fibroblasts treated with TGFβ1 or vehicle for 72 h, followed by treatment with metformin or vehicle for 72 h. (L-M) Staining of TGFβ1-and vehicle-treated cells with LipidTOX (red) and DAPI (blue) at the end of treatment (t=144 h). Scale bars: (E-H) and (L-M) 25 µm. (B-D, I-K) Each data point within a given group corresponds to one patient. (B-D) n=4 per group. (I-K) n=9-10 per group. * P<0.05, **P<0.01, ****P<0.0001.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Staining

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-E) Bright-field imaging of PCLS treated with metformin or vehicle for five days. (F, G) Hematoxylin and eosin staining and COL1A1 immunostaining of PCLS prepared from a non-IPF donor lung. (H-M) Hematoxylin and eosin staining, Masson’s trichrome staining and COL1A1 immunostaining of PCLS prepared from an IPF lung and treated with metformin or vehicle for five days. (N, O) 3D-reconstruction of z-stacks of metformin- and vehicle-treated PCLS stained for COL1A1 (green) and lipid droplets (red). (P) Gating strategy for flow cytomety-based quantification of LipidTOX + cells that are negative for hematopoeitic (CD45), endothelial (CD31) and epithelial (EpCAM) cell markers. (Q) Quantification of flow cytometry measurements on metformin- and vehicle-treated cells. (R) Total collagen assay for metformin- and vehicle-treated cells. Scale bars: (B-E) 2 mm, (F) 500 µm, (G, L, M) 50 µm, (H-K) 200 µm. (Q, R) Each data point within a given group corresponds to one patient. (Q) n=4 per group. ® n=3 per group. * P<0.05, **P<0.01.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Imaging, Staining, Immunostaining, Flow Cytometry, Collagen Assay

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the Acta2-Cre-ERT2 and tdTomato flox construct. (B) Schematic representation of the timeline of the experiment. Bleomycin was administered intratracheally at day 0. Between days 5 and 14, mice were fed tamoxifen-containing pellets and starting at day 14, metformin (1.5 mg/mL) or vehicle was administered through drinking water. Mice were sacrificed at day 28. (C-F) Hematoxylin and eosin and Masson’s trichrome staining of metformin- and vehicle-treated lungs. (G) Quantification of fibrosis in metformin- and vehicle-treated lungs. (H, I) Immunofluorescence for COL1A1 (green). Endogenous tdTomato signal (red) and DAPI (blue) are also shown. (J) LipidTOX staining (green) and tdTomato + cells (red) are shown. The box in (J) is magnified in (K). Arrowheads indicate LipidTOX + tdTomato + cells. (L-S) Gating strategy (to detect CD45 - CD31 - EpCAM - tdTomato + and/or LipidTOX + cells) and quantification of various cell populations based on tdTomato and LipidTOX detection. Scale bars: (C-F) 1 mm, (H, I) 50 µm, (J) 25 µm. (G, Q-S) Each data point within a given group corresponds to one animal. n=5 per group. * P<0.05, **P<0.01. IF: Immunofluorescence, ns: Not significant.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Construct, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the gain-of-function experimental setup for AMPK signaling. (B-E) qPCR analysis of PLIN2, PPARg , COL1A1 and BMP2 in IPF fibroblasts treated with AMPK agonist GSK621 or vehicle. (F) Schematic representation of the loss-of-function experimental setup for AMPK signaling. (G-I) qPCR analysis of PLIN2, PPARg and COL1A1 in IPF fibroblasts treated with AMPK siRNA or scramble siRNA. The decrease of AMPK protein levels at the time of analysis is shown in (J, K). (L-N) Staining of GSK621- and vehicle-treated cells with LipidTOX (red) and DAPI (blue). Metformin-treated cells were used as a positive control for lipid-droplet accumulation (M). Scale bars: (L-N) 25 µm. (B-E, G-I, K) Each data point corresponds to one patient. (B-E) Vehicle-treated group: n=7-8, GSK621-treated group: n=6-8. (G-I) n=4 per group. (K) n=3 per group. * P<0.05. ns: Not significant.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Staining, Positive Control

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-D) qPCR analysis of PLIN2, PPARg and COL1A1 in IPF fibroblasts treated with rhBMP2 or vehicle. (E, F) Staining of rhBMP2- and vehicle-treated cells with LipidTOX (red) and DAPI (blue). (G) Western blot showing the induction of PPARγ phosphorylation in response to rhBMP2 treatment. Lanes 1-4 and lanes 5-8 were run in parallel on different gels under the same conditions. Quantification of the immunoblot is shown in the lower panel. (H) Western blot showing the opposing effects of metformin and TGFβ1 on PPARγ phosphorylation, and the ability of metformin to partially restore PPARγ phosphorylation in TGFβ1-treated cells. Lanes 1-12 and lanes 13-18 were run in parallel on different gels under the same conditions. Quantification of the immunoblot is shown in the lower panel. (I) Model for the antifibrotic mechanism of action of metformin in human lung fibrosis. Metformin activates AMPK signaling in myofibroblasts, leading to suppression of collagen production, and induces lipogenic differentiation via an AMPK-independent mechanism involving BMP2 release and PPARγ activation. Arising lipofibroblasts are known to support type 2 alveolar epithelial stem cells in the lung. Scale bars: (E-F) 50 µm. (B-D, G, H) Each data point corresponds to one patient. (B-D) n=10-11 per group. (G) n=4 per group. (H) n=3 per group. * P<0.05, ns: Not significant.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Staining, Western Blot, Activation Assay

Journal: Aging cell

Article Title: Unraveling Alveolar Fibroblast and Activated Myofibroblast Heterogeneity and Differentiation Trajectories During Lung Fibrosis Development and Resolution in Young and Old Mice.

doi: 10.1111/acel.14503

Figure Lengend Snippet: FIGURE 5 | Characterization of Ct1 and Ct2 cells in saline and Bleo-Tam d14 lungs. (a) RNA-scope of Cthrc1+ aMYF marker Cthrc1 and Ct1- cluster marker Inmt showing colocalization with tdTom+ cells. (b) RNA-scope of Cthrc1+ aMYF marker Cthrc1 combined with immunofluorescence (IF) for Pro-Col1a1 (Ct2-cluster marker), showing colocalization with tdTom+ cells. Scale bars: (a, b): 150 μm.

Article Snippet: Subsequently, the slides were then incubated overnight with anti- Pro- Col1a1 (Phosphosolutions, 321- COLP) and anti- tdTomato (Sicgen, AB8181- 200) at 4°C.

Techniques: Saline, RNAscope, Marker, Immunofluorescence

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Interleukin 1 beta-induced chloride currents are important in osteoarthritis onset: an in vitro study

doi: 10.1093/abbs/gmab010

Figure Lengend Snippet: Expression levels of caspase-1 and caspase-3 detected by immunofluorescence and immunohistochemical staining in normal and OA chondrocytes (A) The detection of caspase-1 and caspase-3 expressions by immunofluorescence. The digested chondrocytes are directly centrifuged on the slide for staining. The nuclei were stained with DAPI (blue), caspase-1 was labeled by Alexa Fluor 488 (green), and caspase-3 was labeled by Cy3 (red). Fluorescent images were obtained by confocal microscopy. (B) The detection of caspase-1 and caspase-3 expression levels by immunohistochemistry. Typical images were obtained with a pathological microscope. (C,D) The rate of positive cells for caspase-1 and caspase-3 ( n =339–411 cells in five views, ** P <0.01 vs the normal group; n =223–269 cells in five views, ** P <0.01 vs the normal group).

Article Snippet: The primary antibodies used included Cy3-conjugated rabbit anti-collagen I antibody (bs-10423R-Cy3; Bioss, Beijing, China), FITC-conjugated rabbit anti-collagen II antibody (bs-10589R-FITC; Bioss), AF488-conjugated rabbit anti-caspase-1 P10 antibody (bs-0169R-AF488; Bioss), Cy3-conjugated mouse anti-caspase-3 antibody (bsm-33284M-Cy3; Bioss), and anti-NLRP3 monoclonal immunoglobulin G (MAB7578; R&D Systems, Minneapolis, USA).

Techniques: Expressing, Immunofluorescence, Immunohistochemical staining, Staining, Labeling, Confocal Microscopy, Immunohistochemistry, Microscopy