Volume 118, Issue 3, Pages 507-514 (March 2000) Overexpression of protein kinase C-β1 isoenzyme suppresses indomethacin-induced apoptosis in gastric epithelial cells  Geng Hui Zhu*, Benjamin Chun Yu Wong*, Eric D. Slosberg‡, Margaret C. Eggo§, Chi Kong Ching*, Siu Tsan Yuen∥, Kam Chuen Lai*, Jae Won Soh‡, I.Bernard Weinstein‡, Shiu Kum Lam*  Gastroenterology  Volume 118, Issue 3, Pages 507-514 (March 2000) DOI: 10.1016/S0016-5085(00)70256-3 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of PKC isoforms in AGS cells with indomethacin treatment or PKC-β1 transfection. AGS cells were treated with 200 μmol/L indomethacin and collected at different time points up to 24 hours. Whole cell lysates (60 μg/lane) were purified on SDS-PAGE and transferred onto nitrocellulose membrane. Western blotting was performed using PKC isoform–specific antibodies, quantified by densitometry, and expressed relative to control levels. (A) Quantitative analysis by densitometric scanning. Bars represent the mean ± SE of 3 different experiments. (B) A representative result. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of PKC isoforms in AGS cells with indomethacin treatment or PKC-β1 transfection. AGS cells were treated with 200 μmol/L indomethacin and collected at different time points up to 24 hours. Whole cell lysates (60 μg/lane) were purified on SDS-PAGE and transferred onto nitrocellulose membrane. Western blotting was performed using PKC isoform–specific antibodies, quantified by densitometry, and expressed relative to control levels. (A) Quantitative analysis by densitometric scanning. Bars represent the mean ± SE of 3 different experiments. (B) A representative result. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Overexpression of PKC-β1 and PKC-β2 by transient transfection in AGS cells. After 48 hours of transfection, whole cell lysates (60 μg/ lane) were prepared, purified on SDS-PAGE, and transferred onto nitrocellulose membrane. The blot was processed for immunoblotting with anti-PKC-β1 and -PKC-β2 polyclonal antibodies. (A) Quantitative analysis by densitometric scanning. Bars represent the mean ± SE of 3 different transfections and scans of the films. (B) A representative result. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Overexpression of PKC-β1 and PKC-β2 by transient transfection in AGS cells. After 48 hours of transfection, whole cell lysates (60 μg/ lane) were prepared, purified on SDS-PAGE, and transferred onto nitrocellulose membrane. The blot was processed for immunoblotting with anti-PKC-β1 and -PKC-β2 polyclonal antibodies. (A) Quantitative analysis by densitometric scanning. Bars represent the mean ± SE of 3 different transfections and scans of the films. (B) A representative result. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Effects of PKC-β1 and PKC-β2 overexpression on the degree of apoptosis induced by indomethacin. AGS cells transfected with empty vectors or plasmids containing PKC-β1 or -β2 cDNA were treated with various concentrations of indomethacin for 24 hours. The percentage of cell apoptosis was determined by acridine orange staining. The results are expressed as mean ± SE from 3 different experiments. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Total PKC activity in PKC-β1-transfected AGS cells. After 48 hours of transfection, cells transfected with empty vector and PKC-β1 were harvested, and cytosolic and particulate fractions were prepared as described in Materials and Methods. The total cellular activity of PKC in cell was measured by ELISA and expressed as OD per milligram of protein. The results are the mean ± SE from 3 different experiments. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of PKC-β1 overexpression on the mRNA and protein of p21, p53, and c-myc. Both the vector- and PKC-β1-transfected AGS cells were treated with 200 μmol/L indomethacin for 24 hours. Expressions of p21, p53, and c-myc were determined by Northern and Western blot. (A) Western blotting quantified by densitometry and expressed as relative to control level; (B) representative result for Western blotting; and (C) representative result for Northern blotting. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of PKC-β1 overexpression on the mRNA and protein of p21, p53, and c-myc. Both the vector- and PKC-β1-transfected AGS cells were treated with 200 μmol/L indomethacin for 24 hours. Expressions of p21, p53, and c-myc were determined by Northern and Western blot. (A) Western blotting quantified by densitometry and expressed as relative to control level; (B) representative result for Western blotting; and (C) representative result for Northern blotting. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of PKC-β1 overexpression on the mRNA and protein of p21, p53, and c-myc. Both the vector- and PKC-β1-transfected AGS cells were treated with 200 μmol/L indomethacin for 24 hours. Expressions of p21, p53, and c-myc were determined by Northern and Western blot. (A) Western blotting quantified by densitometry and expressed as relative to control level; (B) representative result for Western blotting; and (C) representative result for Northern blotting. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of antisense p21waf1/cip1 on the antiapoptotic effect of PKC-β1. Cotransfection of PKC-β1 with antisense cDNA p21waf1/cip1. (A) The protein level of p21waf1/cip1 was quantified and expressed as relative to control level. (B) The representative for Western blot. (C) Apoptotic response to indomethacin was assessed and compared with transfection with PKC-β1 only. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of antisense p21waf1/cip1 on the antiapoptotic effect of PKC-β1. Cotransfection of PKC-β1 with antisense cDNA p21waf1/cip1. (A) The protein level of p21waf1/cip1 was quantified and expressed as relative to control level. (B) The representative for Western blot. (C) Apoptotic response to indomethacin was assessed and compared with transfection with PKC-β1 only. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of antisense p21waf1/cip1 on the antiapoptotic effect of PKC-β1. Cotransfection of PKC-β1 with antisense cDNA p21waf1/cip1. (A) The protein level of p21waf1/cip1 was quantified and expressed as relative to control level. (B) The representative for Western blot. (C) Apoptotic response to indomethacin was assessed and compared with transfection with PKC-β1 only. Gastroenterology 2000 118, 507-514DOI: (10.1016/S0016-5085(00)70256-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions