Journal: Biochemical and biophysical research communications

Article Title: Downregulation of exocyst Sec10 accelerates kidney tubule cell recovery through enhanced cell migration.

doi: 10.1016/j.bbrc.2018.01.013

Figure Lengend Snippet: Fig. 1. Exocyst Sec10 expression correlated with kidney injury and repair following ischemia/reperfusion (I/R). (a) Plasma creatinine (PCr) was measured at the indicated time points after 30 min of bilateral renal I/R or sham operation. (b) Representative Western blots for Sec10 in the kidney at the indicated time points after I/R. (c) The graph summarizes Sec10 expression in the kidney at the indicated time points after I/R. Data are presented as means ± SE (n ¼ 4e6). S ¼ sham, I/R ¼ ischemia and reperfusion. (d) Representative Western blots for Sec10 in cultured mouse proximal tubule cells with (A) or without (C) ATP depletion. (e) The graph summarizes Sec10 expression in cultured mouse proximal tubule cells. Data are presented as means ± SE (n ¼ 3). C ¼ control, A ¼ ATP depletion.

Article Snippet: Protein samples were electrophoresed on 10e12% polyacrylamide gel with 0.1% SDS and transferred to PVDF membranes, and then subjected to an immunoblotting with antibodies against Sec10 (Proteintech, Rosemont, IL, USA), DGKg (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and GAPDH (Novus Biologicals, Littleton, CO, USA) as a loading control.

Techniques: Expressing, Clinical Proteomics, Western Blot, Cell Culture, Control

Journal: Biochemical and biophysical research communications

Article Title: Downregulation of exocyst Sec10 accelerates kidney tubule cell recovery through enhanced cell migration.

doi: 10.1016/j.bbrc.2018.01.013

Figure Lengend Snippet: Fig. 2. Kidney tubule cell recovery after scratch injury inversely correlated with expression levels of Sec10. Sec10 overexpressing (OE) and knockdown (KD), along with control, cells were scratched and cultured for 6 h. Wound healing was determined by measuring the newly grown area. (a) Representative Western blots of Sec10 levels in Sec10 OE and knockdown KD cells. (b, d) Representative pictures of Sec10 OE, KD, and control cells at the indicated times following the scratch. (c, e) The graphs summarize the wound healing rates in Sec10 OE (c) and Sec10 KD (e) cells. (f, h) Representative pictures of H&E-stained migrated cells of Sec10 OE, KD, and control cells. Cells are pinkish stained. Small circles are pores of the membrane filters. (g, i) Graphs summarize the number of migrated cells of Sec10 OE and control (g) and Sec10 KD and control (i) cells. Data are presented as means ± SE. Data were obtained from 3 independent experiments. C ¼ control.

Article Snippet: Protein samples were electrophoresed on 10e12% polyacrylamide gel with 0.1% SDS and transferred to PVDF membranes, and then subjected to an immunoblotting with antibodies against Sec10 (Proteintech, Rosemont, IL, USA), DGKg (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and GAPDH (Novus Biologicals, Littleton, CO, USA) as a loading control.

Techniques: Cell Recovery, Expressing, Knockdown, Control, Cell Culture, Western Blot, Staining, Membrane

Journal: Biochemical and biophysical research communications

Article Title: Downregulation of exocyst Sec10 accelerates kidney tubule cell recovery through enhanced cell migration.

doi: 10.1016/j.bbrc.2018.01.013

Figure Lengend Snippet: Fig. 3. Ruffle formation in kidney tubule cells during wound healing was dependent on the level of Sec10 expression. (a, c) Representative immunofluorescence (IF) pictures of actin staining using Phalloidin (red) in Sec10 OE (a) and KD cells (c). White lines range ruffles at the leading edge during wound healing following the scratch injury. (b, d) Graphs summarize the area of ruffles of Sec10 OE and control (b), and Sec10 KD and control (d) cells. Data are presented as means ± SE. Data were obtained with 18e21 cells from 3 independent experiments. Sec10 OE¼Sec10 overexpressing cells; Sec10 KD¼Sec10 knockdown cells.

Article Snippet: Protein samples were electrophoresed on 10e12% polyacrylamide gel with 0.1% SDS and transferred to PVDF membranes, and then subjected to an immunoblotting with antibodies against Sec10 (Proteintech, Rosemont, IL, USA), DGKg (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and GAPDH (Novus Biologicals, Littleton, CO, USA) as a loading control.

Techniques: Expressing, Staining, Control, Knockdown

Journal: Biochemical and biophysical research communications

Article Title: Downregulation of exocyst Sec10 accelerates kidney tubule cell recovery through enhanced cell migration.

doi: 10.1016/j.bbrc.2018.01.013

Figure Lengend Snippet: Fig. 4. Pharmacological inhibition of DGKg reversed Sec10-mediated inhibition of ruffle formation and wound healing of MDCK cells. Control and Sec10 OE cells were scratched and treated with either vehicle or 1 mM of R59949. Cells were further incubated for 13 h. Wound healing was determined by measuring newly grown area. (a) Representative IF of DGKg at the leading edge during wound healing. (b) The graph summarizes expression of DGKg at the leading edge. Data are presented as means ± SE. Data were obtained with 68e72 cells from 3 independent experiments. (c) Representative IF of F-actin and DGKg at the leading edge during wound healing in control and Sec10 OE cells. (d) Representative bright field images of control and Sec10 OE cells before and after scratch with/without treatment of DGK inhibitor. (e) The graph summarizes wound healing rates in control and Sec10 OE cells with/without treatment of DGK inhibitor after scratch injury. (f) Representative Western blots for Sec10 and DGKg in control and Sec10 OE cells. (g, h) The graphs summarize Sec10 (g) and DGKg (h) expression in control and Sec10 OE cells. (i, k) IF of actin staining using Phalloidin (red) in control (i) and Sec10 OE cells (k) with/without treatment of DGK inhibitor R59949 after scratch injury. (j, l) Graphs summarize the ruffle area at the leading edge in control (j) and Sec10 OE (l) cells. White lines indicate ruffles. Data are presented as means ± SE. Data were obtained with 75e90 cells from 3 independent experiments. Sec10 OE¼Sec10 overexpressing cells.

Article Snippet: Protein samples were electrophoresed on 10e12% polyacrylamide gel with 0.1% SDS and transferred to PVDF membranes, and then subjected to an immunoblotting with antibodies against Sec10 (Proteintech, Rosemont, IL, USA), DGKg (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and GAPDH (Novus Biologicals, Littleton, CO, USA) as a loading control.

Techniques: Inhibition, Control, Incubation, Expressing, Western Blot, Staining