Journal: Biology Open

Article Title: The anti-motility signaling mechanism of TGFβ3 that controls cell traffic during skin wound healing

doi: 10.1242/bio.20122246

Figure Lengend Snippet: Serum-starved HDFs and HKCs in duplicate culture plates were either unstimulated or stimulated with TGFβ3 (3 ng/ml) for 10 min. The stimulation was stopped by removing the medium from the plates and rinsing of the plates three times with ice-cold PBS solution on ice. The cells were then subjected to Protein Kinase Array screening strictly following the manufacturer's procedures (Materials and Methods), including lysing cells with a special lysis buffer provided. ( A ) Images of ECL-developed films, where antibody identities on the spots were based on the information given by the manufacturer. ( B , C ) Phosphoimager quantitation of phospho-ERK, phospho-CREB and phospho-Src in HDFs and HKCs. ( D ) Stronger activation of PKA pathway, i.e. phosophorylation of CREB, in HDFs than in HKCs by TGFβ3 (10 min) was confirmed by Western blots (lane 2 versus lane 4). ( E ) Downregulation of Smad4 blocks TGFβ3-induced CREB phosphorylation (lane 4 versus lane 2). The Array experiment was repeated twice and the Western blotting experiment four times. Similar results were obtained.

Article Snippet: The third or fourth passages of cells were used for experiments. rhPDGF-bb, rhTGFα, rhTGFβ3, anti-phospho-CREB (Ser-133) antibody and anti-Smad4 antibody were purchased from R&D System (Minneapolis, MN).

Techniques: Lysis, Quantitation Assay, Activation Assay, Western Blot, Phospho-proteomics

Journal: Biology Open

Article Title: The anti-motility signaling mechanism of TGFβ3 that controls cell traffic during skin wound healing

doi: 10.1242/bio.20122246

Figure Lengend Snippet: ( A ) HDFs were serum starved overnight and subjected to colloidal gold migration assays in the absence or presence of PDGF-bb (15 ng/ml), TGFβ3 (3 ng/ml) and PKA inhibitor, H89 (1.0 µM). Representative images of migrated cells under the indicated conditions are shown (a to d). Quantitative analyses of the migration tracks are shown underneath as Migration index (MI) (%). Average size migration tracks are marked with dotted circles. This experiment was repeated multiple times and similar results were obtained. ( B ) Serum-starved HDFs were treated without or with TGFβ3 or TGFβ3 plus H89. Total cell lysates were subjected to immunoblotting analyses with indicated antibodies. ( C ) Serum-starved HDFs were subjected to colloidal gold migration assay, as previously described, in the absence or presence of PDGF-bb (bar 1 and 2) or PDGF-bb plus increasing concentrations of forskolin, an activator of PKA (bars 3 to 7). The effectiveness of forskolin (10.0 µM) on HDFs is indicated by causing an increased phosphorylation of CREB (insert image) in the cells. These experiments were repeated three times. *Statistically significant over the control, P <0.05.

Article Snippet: The third or fourth passages of cells were used for experiments. rhPDGF-bb, rhTGFα, rhTGFβ3, anti-phospho-CREB (Ser-133) antibody and anti-Smad4 antibody were purchased from R&D System (Minneapolis, MN).

Techniques: Migration, Western Blot, Phospho-proteomics, Control

Journal: Biology Open

Article Title: The anti-motility signaling mechanism of TGFβ3 that controls cell traffic during skin wound healing

doi: 10.1242/bio.20122246

Figure Lengend Snippet: ( A ) HDFs were infected with FG-12 lentivirus carrying either a GFP control gene or a shRNAs against PKA-Cα. After 48 hours, downregulation of the endogenous PKA-Cα was confirmed by immunoblotting the lysates of the cells with an anti-PKA-Cα antibody. ( B ) Downregulation of PKA-Cα blocks forskolin-stimulated CREB phosphorylation (lane 4 versus lane 2). ( C ) Downregulation of PKA-Cα showed little effect on TGFβ3-stimulated phosphorylation of Smad3 (lanes 4 and 6 versus lane 2). ( D ) Two sets of PKA-Cα-downregulated HDFs were subjected to colloidal gold migration assay in response to PDGF-bb (15 ng/ml) in the absence or presence of TGFβ3 (3 ng/ml). Representative images of the migrated cells under the indicated conditions are shown (a to i). ( E ) The computer-assisted quantitative analyses of the migration tracks are shown as Migration index (MI) (%). The experiment was repeated four times ( n = 3, P ≤0.05). *Statistically significant over the control.

Article Snippet: The third or fourth passages of cells were used for experiments. rhPDGF-bb, rhTGFα, rhTGFβ3, anti-phospho-CREB (Ser-133) antibody and anti-Smad4 antibody were purchased from R&D System (Minneapolis, MN).

Techniques: Infection, Control, Western Blot, Phospho-proteomics, Migration

Journal: Frontiers in Immunology

Article Title: Granulocytic Myeloid-Derived Suppressor Cell Exosomal Prostaglandin E2 Ameliorates Collagen-Induced Arthritis by Enhancing IL-10 + B Cells

doi: 10.3389/fimmu.2020.588500

Figure Lengend Snippet: G-MDSC exosomal PGE2 promotes IL-10 + B cells (A) COX-2 expression in G-MDSCs was detected after treatment with the COX-2 inhibitor celecoxib. (B) The ratio of COX2 and GAPDH in each group was statistically analyzed. (C) The level of PGE2 in the G-MDSC culture supernatant treated with celecoxib was detected by ELISA. (D) The level of PGE2 in the G-exo treated with celecoxib was detected by ELISA. (E) IL-10–producing B cells after treatment with celecoxib-treated G-exo in the present of LPS were analyzed by flow cytometry. (F) The level of IL-10 in culture supernatant after treatment with celecoxib-treated G-exo was detected by ELISA. (G) The p-GSK-3β, T-GSK-3β, p-CREB, and T-CREB were detected by Western blot analysis. (H) The ratio of p-CREB and T-CREB in each group was statistically analyzed. (I) The ratio of p-GSK-3β and T-GSK-3β in each group was statistically analyzed. Bar graphs show the means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ns: indicates no significance.

Article Snippet: The membranes were then incubated with specific rabbit antibodies against phosphorylated (p)-GSK-3β (Santa Cruz Biotechnology, Texas, USA ) , total (t)- GSK-3β (Wanleibio, Shenyang, China), phosphorylated (p)-cAMP response element binding protein (CREB) (Wanleibio, Shenyang, China) and total (t)-CREB (Wanleibio, Shenyang, China) followed by an incubation with the secondary HRP-conjugated goat anti-rabbit IgG (CST, Danvers, MA, USA) according to manufacturer’s protocol.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Western Blot

Journal: Frontiers in Immunology

Article Title: Granulocytic Myeloid-Derived Suppressor Cell Exosomal Prostaglandin E2 Ameliorates Collagen-Induced Arthritis by Enhancing IL-10 + B Cells

doi: 10.3389/fimmu.2020.588500

Figure Lengend Snippet: Schematic image demonstrating that Granulocytic myeloid-derived suppressor cell exosomal prostaglandin E2 ameliorates collagen-induced arthritis by inducing IL-10 + B cells production via affecting GSK-3β and CREB phosphorylation.

Article Snippet: The membranes were then incubated with specific rabbit antibodies against phosphorylated (p)-GSK-3β (Santa Cruz Biotechnology, Texas, USA ) , total (t)- GSK-3β (Wanleibio, Shenyang, China), phosphorylated (p)-cAMP response element binding protein (CREB) (Wanleibio, Shenyang, China) and total (t)-CREB (Wanleibio, Shenyang, China) followed by an incubation with the secondary HRP-conjugated goat anti-rabbit IgG (CST, Danvers, MA, USA) according to manufacturer’s protocol.

Techniques: Derivative Assay, Phospho-proteomics