Inhibition of heat-shock protein 70 induction in intestinal cells overexpressing cyclooxygenase 2 Richard T. Ethridge, Mark R. Hellmich, Raymond N. DuBois, B.Mark Evers Gastroenterology Volume 115, Issue 6, Pages 1454-1463 (December 1998) DOI: 10.1016/S0016-5085(98)70024-1 Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 1 Analysis of COX-2 expression and PGE2 amounts. (A) Total protein (300 μg) was extracted from the RIE cell lines over a time course after heat shock and immunoprecipitated with antibody to COX-2 (Santa Cruz Biotechnology), and Western immunoblot analysis was performed. Results represent 2 separate experiments. (B) Culture medium was taken from RIE-P, RIE-AS, and RIE-S cell lines after heat shock at 43°C for 1 hour (+) and measured for PGE2 production. −, Control (nontreated) cells. (C) The COX-2 selective inhibitor NS-398 (0.6 μmol/L) was added before heat shock, and culture medium was analyzed for PGE2 production. Results are expressed as mean ± SEM and represent 2 separate experiments. *P < 0.05 vs. respective control (nonheated) cells. Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 1 Analysis of COX-2 expression and PGE2 amounts. (A) Total protein (300 μg) was extracted from the RIE cell lines over a time course after heat shock and immunoprecipitated with antibody to COX-2 (Santa Cruz Biotechnology), and Western immunoblot analysis was performed. Results represent 2 separate experiments. (B) Culture medium was taken from RIE-P, RIE-AS, and RIE-S cell lines after heat shock at 43°C for 1 hour (+) and measured for PGE2 production. −, Control (nontreated) cells. (C) The COX-2 selective inhibitor NS-398 (0.6 μmol/L) was added before heat shock, and culture medium was analyzed for PGE2 production. Results are expressed as mean ± SEM and represent 2 separate experiments. *P < 0.05 vs. respective control (nonheated) cells. Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 1 Analysis of COX-2 expression and PGE2 amounts. (A) Total protein (300 μg) was extracted from the RIE cell lines over a time course after heat shock and immunoprecipitated with antibody to COX-2 (Santa Cruz Biotechnology), and Western immunoblot analysis was performed. Results represent 2 separate experiments. (B) Culture medium was taken from RIE-P, RIE-AS, and RIE-S cell lines after heat shock at 43°C for 1 hour (+) and measured for PGE2 production. −, Control (nontreated) cells. (C) The COX-2 selective inhibitor NS-398 (0.6 μmol/L) was added before heat shock, and culture medium was analyzed for PGE2 production. Results are expressed as mean ± SEM and represent 2 separate experiments. *P < 0.05 vs. respective control (nonheated) cells. Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 2 Northern and Western blot analysis. (A) Total RNA (15 μg/lane) was isolated from the RIE-P, RIE-AS, and RIE-S cell lines after heat shock at 43°C for 1 hour and analyzed by Northern blotting. Blots were probed initially for hsp70 mRNA and then stripped and reprobed for the constitutively expressed GAPDH to confirm intact RNA. Results represent 4 different experiments. (B) Western immunoblot analysis of protein lysates (50 μg/lane) extracted from the RIE cell lines over a time course after heat shock using an antibody to HSP70 (StressGen). The filter was stripped and reprobed with the constitutively expressed HSC70 antibody to confirm intact protein and relatively equal loading. Results represent 3 different experiments. Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 2 Northern and Western blot analysis. (A) Total RNA (15 μg/lane) was isolated from the RIE-P, RIE-AS, and RIE-S cell lines after heat shock at 43°C for 1 hour and analyzed by Northern blotting. Blots were probed initially for hsp70 mRNA and then stripped and reprobed for the constitutively expressed GAPDH to confirm intact RNA. Results represent 4 different experiments. (B) Western immunoblot analysis of protein lysates (50 μg/lane) extracted from the RIE cell lines over a time course after heat shock using an antibody to HSP70 (StressGen). The filter was stripped and reprobed with the constitutively expressed HSC70 antibody to confirm intact protein and relatively equal loading. Results represent 3 different experiments. Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 3 Immunofluorescence microscopy. (A) RIE-AS and (B) RIE-S cells were heat-shocked at 43°C for 1 hour followed by 90 minutes' incubation at 37°C (+, right). − (left), control (nontreated) cells. The cells were fixed with 100% methanol, stained with antiserum to the inducible form of HSP70 protein as a primary antibody and FITC-conjugated goat anti-mouse IgG antibody as the secondary antibody; cells were visualized by fluorescence microscopy (bottom panels). Top panels show the same field by phase-contrast microscopy. The heated RIE-AS cells showed a strong cytoplasmic and nuclear staining compared with the heated RIE-S cells (original magnification for all fields 63×). Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 3 Immunofluorescence microscopy. (A) RIE-AS and (B) RIE-S cells were heat-shocked at 43°C for 1 hour followed by 90 minutes' incubation at 37°C (+, right). − (left), control (nontreated) cells. The cells were fixed with 100% methanol, stained with antiserum to the inducible form of HSP70 protein as a primary antibody and FITC-conjugated goat anti-mouse IgG antibody as the secondary antibody; cells were visualized by fluorescence microscopy (bottom panels). Top panels show the same field by phase-contrast microscopy. The heated RIE-AS cells showed a strong cytoplasmic and nuclear staining compared with the heated RIE-S cells (original magnification for all fields 63×). Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 4 Analysis of HSP27, HSP60, and HSP90 protein levels. (A) Western immunoblot analysis of protein lysates (50 μg/lane) extracted from RIE-P, RIE-AS, and RIE-S cell lines over a time course after heat shock using an antibody to HSP27 (StressGen). (B) Western immunoblot analysis of protein lysates (50 μg/lane) extracted from the RIE cell lines over a time course after heat shock using antibodies to HSP60 and HSP90 (both from StressGen). The filters (A and B) were stripped and reprobed with the antibody to HSC70 to confirm intact protein and relatively equal loading. Results represent 3 different experiments. Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 4 Analysis of HSP27, HSP60, and HSP90 protein levels. (A) Western immunoblot analysis of protein lysates (50 μg/lane) extracted from RIE-P, RIE-AS, and RIE-S cell lines over a time course after heat shock using an antibody to HSP27 (StressGen). (B) Western immunoblot analysis of protein lysates (50 μg/lane) extracted from the RIE cell lines over a time course after heat shock using antibodies to HSP60 and HSP90 (both from StressGen). The filters (A and B) were stripped and reprobed with the antibody to HSC70 to confirm intact protein and relatively equal loading. Results represent 3 different experiments. Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 5 Electrophoretic mobility shift assay analysis of HSF binding. (A) Whole-cell extracts (50 μg) were prepared 30 and 90 minutes after heat shock at 43°C for 1 hour. Extracts were assayed for HSF binding activity by incubation of extract with an oligonucleotide corresponding to an idealized HSE sequence.42 Heat shock–treated HeLa cell extract was used as a positive control (lane 12). Competition experiments were performed with a 100-fold molar excess of unlabeled HSE oligonucleotide (lane 5). Results represent 4 different experiments. (B) The same extracts from above are used to analyze Oct-1 DNA binding activity. Competition experiments were performed with a 100-fold molar excess of unlabeled Oct-1 oligonucleotide (lane 5). Results represent 2 different experiments. Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 5 Electrophoretic mobility shift assay analysis of HSF binding. (A) Whole-cell extracts (50 μg) were prepared 30 and 90 minutes after heat shock at 43°C for 1 hour. Extracts were assayed for HSF binding activity by incubation of extract with an oligonucleotide corresponding to an idealized HSE sequence.42 Heat shock–treated HeLa cell extract was used as a positive control (lane 12). Competition experiments were performed with a 100-fold molar excess of unlabeled HSE oligonucleotide (lane 5). Results represent 4 different experiments. (B) The same extracts from above are used to analyze Oct-1 DNA binding activity. Competition experiments were performed with a 100-fold molar excess of unlabeled Oct-1 oligonucleotide (lane 5). Results represent 2 different experiments. Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions
Fig. 6 Northern blot analysis after NS-398 treatment. Total RNA (15 μg/lane) was extracted from RIE cells, either untreated or subjected to heat shock. The RIE cells were heated for 1 hour at 43°C, returned to 37°C for 90 minutes, and then washed with the selective COX-2 inhibitor NS-398 (0.2, 0.6, 1.7, and 5 μmol/L) in PBS for 15 minutes at room temperature; total RNA was extracted. Blots were probed for hsp70 mRNA and then stripped and reprobed for GAPDH as a loading control and for confirmation of intact RNA. Results represent 3 different experiments. Gastroenterology 1998 115, 1454-1463DOI: (10.1016/S0016-5085(98)70024-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions