Journal: bioRxiv

Article Title: Microsecond pulse electrical stimulation modulates cell migration

doi: 10.1101/2022.10.23.513372

Figure Lengend Snippet: (a) A graphical illustration showed the effects of μsPEF (pulse width:20 μs, frequency: 10 Hz, duration: 5 s) on the skin wound, promoting cell migration and extracellular matrix remodeling. (b) Representative time-lapse images showing the fibroblasts morphology after μsPEF (i.e., 750 and 1500 V/cm) treatment of different intensity at different time points (i.e., 0, 1 and 2 h). Detached cells are marked by yellow arrow. Scale bar, 50 μm. (c) The line graph representing the cell migration average speed per 1 h from 0 to 12 h after different intensity μsPEF (i.e., 750 and 1500 V/cm) treatment (blue circle: control; orange square: 750 V/cm; pink triangle: 1500 V/cm). Results are presented as mean ± standard deviation with 95% CI (n CTRL =42 cells, n 750 v/cm =48 cells, n 1500 v/cm =47 cells); * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 versus control by one-way ANOVA for multiple comparisons. (d) Box plot showing the average cell migration speed with different intensity μsPEF (i.e., 750 and 1500 V/cm) and control treatments in 24 h. Results are presented as mean ± standard deviation with 95% CI (n CTRL =470 cells, n 750 V/cm =979 cells, n 1500 V/cm =535 cells). (e) Effects of μsPEF on the secretion of COLA2. The level of collagen type I α2 in cellular supernatants was measured after 48 h. The concentration of COLA2 was 0.109 ± 0.018 ng/mL in the control group, 0.257 ± 0.058 ng/mL in the 750 V/cm group and 0.363 ± 0.034 ng/mL in the 1500 V/cm group. Results are presented as mean ± standard deviation with 95% CI (n=5); * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 versus control by one-way ANOVA for multiple comparisons. (f) Effects of μsPEF on the expression of FGF2. The level of FGF2 in cellular supernatants was measured after 48 h. The concentration of FGF2 was 44.139 ± 0.360 ng/mL in the control group, 48.012 ± 1.488 ng/mL in the 750 V/cm group and 48.523 ± 1.944 ng/mL in the 1500 V/cm group. Results are presented as mean ± standard deviation with 95% CI (n=3); ns=0.7329, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 versus control by one-way ANOVA for multiple comparisons.

Article Snippet: Then, the cells were cultured in serum-free medium for 48 h. The content of type I α collagen and basic fibroblast growth factor (FGF-2) in the supernatant were measured using commercially available Human COL1A2 (Collagen Type I Alpha 2) ELISA Kit (Elabscience, Wuhan, China) and Human bFGF/FGF2 (Basic Fibroblast Growth Factor) ELISA Kit (Elabscience, Wuhan, China) according to the manufacturer’s protocol respectively.

Techniques: Migration, Control, Standard Deviation, Concentration Assay, Expressing

Journal: Cancer Research

Article Title: Targeting LRRC15 in Cancer-Associated Fibroblasts Modifies the Extracellular Matrix and Enhances Tumor Immune Responses to Suppress Lung Cancer Progression

doi: 10.1158/0008-5472.CAN-25-2871

Figure Lengend Snippet: LRRC15 promotes M2 polarization by modulating the ECM production. A, Bar plot showing the enriched pathways of the upregulated genes in TGFβ-treated human CAFs compared with those of LRRC15 -KO CAFs, based on bulk RNA-seq data. B, Scatter plot showing the correlation between the ECM organization signature score and LRRC15 expression level in lung cancer from TCGA. Pearson correlation coefficient R is indicated. C, Bar plot showing the enriched pathways in human LRRC15 + CAFs from lung cancer, based on scRNA-seq data. D, Bar plot showing the enriched pathways in mouse LRRC15 + CAFs from TC1-GFP tumor co-injected with TGFβ-treated CAFs, based on scRNA-seq data. E and F, Collagen expression detected using Sirius Red staining in TGFβ-treated mouse ( E ) and human ( F ) WT or LRRC15 -KO CAFs ( n = 3 wells per group). G, Western blot plots showing collagen I expression in WT or LRRC15 -KO human CAFs treated with or without TGFβ for 48 hours. H, Sirius Red staining of tumors from Col1a2- CreER; Lrrc15 fl/fl and littermate Lrrc15 fl/fl mice bearing KPS tumors ( n = 3–4 mice per group). I, Cell number and proportion of CD206 + BMDMs in mouse BMDM and CAF ECM coculture assay ( n = 3 wells per group). J, Cell number and proportion of CD206 + mo-MΦs in human mo-MΦs and CAF ECM coculture assay ( n = 3 wells per group). K, Proportion and cell number of CD206 + mo-MΦs of CD206 in human mo-MΦs cultured with or without recombinant human collagen I ( n = 3 wells per group). Data are presented as mean ± SEM, Statistical significance was determined using the hypergeometric test ( A , C , and D ) or unpaired two-tailed Student t test ( E , F , H , I , J , and K ). P values were adjusted using the Benjamini–Hochberg method ( A , C , and D ).

Article Snippet: For macrophage–collagen stimulation assays, 500 μg/mL human collagen I (MedChemExpress, cat. #HY-NP160) was coated in 12-well plates for culturing mo-MΦs.

Techniques: RNA Sequencing, Expressing, Injection, Staining, Western Blot, Co-culture Assay, Cell Culture, Recombinant, Two Tailed Test

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