Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Inhibitors of actin polymerization improve monolayer integrity and reverse plasticity. (A–C) LatA (an actin polymerization inhibitor) and U0126 (a MAPK inhibitor) are the only drugs which reduced cellular velocity (A), TEER (B) and correlation length (C) on the CS-exposed epithelia [cells treated with pathway modulators such as LatA, the actin-polymerizing agent JaspA, U0126, the TGFβ1 neutralizer (1D11), or an antagonist of Wnt signaling pathway (DKK1) and compared to vehicle control]. Data is representative of two donors (three to six inserts per donor). (D) None of the inhibitors restored the CS-induced decrease in basal mRNA expression of the epithelial marker CDH1 . (E–J) The levels of the mesenchymal markers ( CDH2 , VIM , SNAI1 , SNAI2 , ZEB1 and ZEB2 ) were significantly reduced relative to GAPDH in CS-exposed epithelia treated with several pathway modulators (LatA, U0126, 1D11 and DKK1) as analyzed by qPCR. (K) LatA and U0126 treatment reduced mean square displacement (MSD) in CS exposed epithelia. Data is representative of three to six inserts from two donors. (L–O) LatA and U0126 treatment improved TEER in CHBE cells (L), LatA and U0126 treatment decreased cellular velocity in COPD epithelia (CHBE) (M), LatA and U0126 treatment reduced correlation length in CHBE cells (N), and LatA and U0126 treatment improved the height of pseudostratified epithelia on COPD epithelia (CHBE) (O). (P) Representative DIC image from three inserts from one donor taken with a 40× oil objective of CHBE treated with or without LatA or U0126 showing improved monolayer height. Scale bars: 25 µm. (Q) LatA and U0126 treatment reduced the MSD with time in CHBE cells approaching levels similar to that of NHBE. Of note, there is not a significant increase in MSD with time in pEMT-induced NHBE. Data is representative of nine inserts, except for cellular velocity and correlation length (six inserts) and height of pseudostratified epithelia (three inserts) from one donor of CHBE. Graphs are shown with median bars. Statistics determined by Kruskal–Wallis test followed by Dunn's multiple comparison test, with P <0.05 considered statistically significant.

Article Snippet: The NHBE cells at the ALI were exposed to CS for 10 days and simultaneously basolaterally treated with an antagonist of Wnt signaling pathway (recombinant human DKK1 protein, 50 ng/ml, R&D Systems), TGFβ1 neutralizer [TGFβ-1,2,3 monoclonal antibody (1D11), 1.25 μg/ml, Thermo Fisher Scientific], MAPK inhibitor (U0126, 15 μM, Cell Signaling Technology ® ), an inhibitor of actin polymerization (Latrunculin A, 0.625 μM, Sigma-Aldrich) or the actin filament-polymerizing and stabilizing agent (Jasplakinolide A, 0.25 μM, Sigma-Aldrich).

Techniques: Control, Expressing, Marker, Comparison

Journal: Frontiers in Oncology

Article Title: TIPE2 Suppresses Malignancy of Pancreatic Cancer Through Inhibiting TGFβ1 Mediated Signaling Pathway

doi: 10.3389/fonc.2021.680985

Figure Lengend Snippet: TIPE2 inhibited PI3K/AKT and Raf/MEK/ERK signaling pathways triggered by TGFβ1. (A) The total proteins extracted from cells were analyzed for the expression of AKT, ERK and Bax using western blot. The antibodies used were anti-pAKT, anti-AKT, anti-pERK, anti-ERK, anti-Bax and anti-GAPDH. (B) ELISA analysis of TGFβ1 secretion from AsPC-1/vector and AsPC-1/TIPE2 cells. (C) Immunohistochemistry analysis of the TGFβ1 expression in AsPC-1/vector and AsPC-1/TIPE2 tumor tissues. (D) Western blot analysis of the expression of p-TGFBR1 and total TGFBR1 in AsPC-1/vector and AsPC-1/TIPE2 cells. (E) AsPC-1 cells were seeded in 6-well plate and added with or without anti-TGFβ1 antibody or rhTGFβ1 protein. After 48 h incubation, the total proteins extracted from the cultured AsPC-1 cells were analyzed for the expression of AKT and ERK using western blot. The antibodies used were anti-pAKT, anti-AKT, anti-pERK, anti-ERK and anti-GAPDH. Data shown were representative of three independent experiments. Values are presented as means ± SD. ***p < 0.001.

Article Snippet: In AsPC-1 cells, the neutralizing anti-TGFβ1 monoclonal antibody (1 μg/ml, ProteinTech) or 5 ng/ml human recombinant TGFβ1 (ProteinTech) was added for 48 h incubation, respectively.

Techniques: Protein-Protein interactions, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Immunohistochemistry, Incubation, Cell Culture

Journal: Frontiers in Oncology

Article Title: TIPE2 Suppresses Malignancy of Pancreatic Cancer Through Inhibiting TGFβ1 Mediated Signaling Pathway

doi: 10.3389/fonc.2021.680985

Figure Lengend Snippet: TIPE2 suppressed the growth of pancreatic cancer through inhibiting TGFβ1 expression in subcutaneous tumor model. (A) The tumor volume was measured weekly one week after tumor inoculation and the tumor growth curve was calculated. (B, C) The tumors were isolated, weighted and photographed five weeks after tumor cells inoculation. (D) ELISA analysis of TGFβ1 secretion from Panc02/vector and Panc02/TIPE2 cells. (E) Immunohistochemistry analysis of the TGFβ1 expression in Panc02/vector and Panc02/TIPE2 tumor tissues. (F) Western blot analysis of the expression of p-TGFBR1 and total TGFBR1 in Panc02/vector and Panc02/TIPE2 cells. The data shown are the representative of three experiments. Values are presented as means ± SD. *p < 0.05, ***p < 0.001.

Article Snippet: In AsPC-1 cells, the neutralizing anti-TGFβ1 monoclonal antibody (1 μg/ml, ProteinTech) or 5 ng/ml human recombinant TGFβ1 (ProteinTech) was added for 48 h incubation, respectively.

Techniques: Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Immunohistochemistry, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Fibroblast-like cells Promote Wound Healing via PD-L1-mediated Inflammation Resolution

doi: 10.7150/ijbs.69890

Figure Lengend Snippet: TGF and FGF up-regulate PD-L1 and skew macrophage polarization . (a, b) Expression of PD-L1 on the surface of fibroblast-like cells detected by FCM. (c, d, f, g) Expression of CD86 and CD206 in WT-MEF or PD-L1 KO MEF detected by FCM. Macrophages were co-cultured with untreated, or FGF-2 and TGF-β1 pretreated fibroblasts were washed with PBS, blocked in 5% FBS and two color-stained to identify M1 (CD86) and M2 (CD206) macrophage subpopulations.(e, h) Expression of CD16, CD86 and CD206 in macrophages co-cultured with pretreated fibroblast-like cells detected by immunofluorescence staining.(i) Pretreating WT or PD-L1-/- fibroblasts (fixed with Paraformaldehyde or not) affect activated macrophages TNF-α, IL-6 and IL-10 production in a contact-dependent manner. *p < 0.05, **p < 0.01.

Article Snippet: 20 mg/mL anti-FGF-2 neutralizing antibody (Sigma Aldrich, St. Louis, MO, USA) and 50 mg/mL anti-TGFβ1 neutralizing antibody (Thermo Fischer Scientific; former Savant, MA, USA) were incubated along with Wound exudate in FACS, where indicated.

Techniques: Expressing, Cell Culture, Staining, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: Fibroblast-like cells Promote Wound Healing via PD-L1-mediated Inflammation Resolution

doi: 10.7150/ijbs.69890

Figure Lengend Snippet: TGF-β1 together with FGF-2 significantly upregulated fibroblast-like cells PD-L1 at translational levels. (a, b) Polysome profiles of MEF cells treated 24 h with FGF-2 and TGF-β1. One representative profile from three independent experiments is shown. Percentage of transcripts in each polysomal fraction obtained by sucrose-gradient ultracentrifugation was quantified by qRT-PCR (n = 3). (c) Western blot analysis of the indicated proteins in MEF when stimulated by FGF-2 and TGF-β1. Representative blots from two independent experiments are shown. (d, e) PD-L1 is visualized by flow cytometry in MEF cells stimulated by FGF-2 and TGF-β1 and eIF4E inhibitor. One representative experiment of two is shown. *p < 0.05,**p < 0.01.

Article Snippet: 20 mg/mL anti-FGF-2 neutralizing antibody (Sigma Aldrich, St. Louis, MO, USA) and 50 mg/mL anti-TGFβ1 neutralizing antibody (Thermo Fischer Scientific; former Savant, MA, USA) were incubated along with Wound exudate in FACS, where indicated.

Techniques: Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: Fibroblast-like cells Promote Wound Healing via PD-L1-mediated Inflammation Resolution

doi: 10.7150/ijbs.69890

Figure Lengend Snippet: PD-L1 positive fibroblast-like cells pretreated by TGF-β1 and FGF-2 promote chronic refractory wound healing in vivo. (a) Representative photographs showing macroscopic excisional wound closure treated with PD-L1 positive and negative MEF in PD-L1-/- C57 BL6 mice. The wild-type and PD-L1-/- mice fibroblast-like cells were isolated and cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin and 50 units/mL streptomycin (Sigma). Followed by pretreated with TGF-β1 and FGF-2, approximately 1x 10 5 fibroblast-like cells were further suspended in saline and sprayed over the wound area of the mice. (b) Planimetric analysis of wound photographs reveals significantly delayed at 3-5 days after wounding when PD-L1-/- MEF were used. (c) The schematic diagram of the mechanism of PD-L1 regulating wound healing.

Article Snippet: 20 mg/mL anti-FGF-2 neutralizing antibody (Sigma Aldrich, St. Louis, MO, USA) and 50 mg/mL anti-TGFβ1 neutralizing antibody (Thermo Fischer Scientific; former Savant, MA, USA) were incubated along with Wound exudate in FACS, where indicated.

Techniques: In Vivo, Isolation, Cell Culture, Modification, Saline