Clusterin Gene Expression Mediates Resistance to Apoptotic Cell Death Induced by Heat Shock and Oxidative Stress  Isabelle Viard, Philippe Wehrli, Lan Jornot, Roberto Bullani, Jean-Luc Vechietti, Lars E. French  Journal of Investigative Dermatology  Volume 112, Issue 3, Pages 290-296 (March 1999) DOI: 10.1046/j.1523-1747.1999.00531.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Heat shock increases clusterin gene expression in A431 and HepG2 cells. Northern blot analysis of total cellular RNA isolated from: (A) A431 cells at 1, 4, or 8 h after a 20 min heat shock at 42°C; (B) HepG2 cells at 4 h after a 1 h heat shock at 43°C. Northern blots were processed using clusterin (CLU), hsp70, and GAPDH cRNA probes as described in the Materials and Methods. Control (Co) represents RNA extracted from A431 cells treated in the same manner at 37°C. (C) Nuclear run-on analysis of A431 nuclei isolated under control conditions at 37°C (Co) or 2 h after a 20 min heat shock at 42°C. Journal of Investigative Dermatology 1999 112, 290-296DOI: (10.1046/j.1523-1747.1999.00531.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Heat shock increases the synthesis and secretion of clusterin protein in A431 cells. A431 cells exposed for 20 min at 42°C (Heat shock) or 37°C (Control) in a water bath and returned to 37°C for 4 h, were pulsed for 20 min with [35S] methionine, and then chased with medium containing a 10-fold excess of unlabeled methionine. Thereafter, clusterin was immunoprecipitated from the cell lysate (top panels) or cell media (bottom panels) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Clusterin migrates as a unique band of ≈80 kDa in these conditions. NI: immunoprecipitation of cell lysates (T = 0) or media (T = 240 min) with preimmune serum. Journal of Investigative Dermatology 1999 112, 290-296DOI: (10.1046/j.1523-1747.1999.00531.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 No protein coimmunoprecipitates with clusterin under nondenaturing conditions in heat shocked A431 cells. A431 cells exposed for 20 min at 42°C (Heat shock) or 37°C (Control) and returned to 37°C for 4 h, were labeled with [35S] methionine as described in the Materials and Methods. Thereafter, clusterin was immunoprecipitated from nondenatured cell lysates under nondenaturing conditions (see Materials and Methods) with an anti-human clusterin (SP40,40) antibody preincubated (+) or not (–) with an excess (1 μg) of affinity-purified cold clusterin. Journal of Investigative Dermatology 1999 112, 290-296DOI: (10.1046/j.1523-1747.1999.00531.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Oxidative stress increases clusterin gene expression in A431 cells. Northern blot analysis of total cellular RNA isolated from A431 cells: (A) at 4, 8, or 24 h after exposure for 30 min to 0.5 mM H2O2; (B) at 4, 8, or 24 h after exposure for 1 h to a flux of 6.8 nmol O2– per ml per min generated by the HX-XO enzymatic system; (C) just after exposure for 4, 8, 24, or 48 h to 95% O2; (D) at 6, 16, 24, or 48 h after exposure to 20 or 40 J UVA radiation per cm2. Control A431 cells were kept in normal medium (Co). Journal of Investigative Dermatology 1999 112, 290-296DOI: (10.1046/j.1523-1747.1999.00531.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Clusterin expression in A431 sense and anti-sense clusterin transfected clones. (A) Northern blot analysis of total cellular RNA isolated from wild-type A431 cells (WT), a pool of control vector transfected A431 cells (PV), sense (S21 and S81) and anti-sense (AS2 and AS10) transfected clusterin clones, under control conditions, after heat shock or UVA exposure. Cells were recovered 4 h after a 20 min heat shock at 42°C or 16 h after exposure to 40 J UVA radiation per cm2. Control conditions correspond to cells kept in normal medium at 37°C. Exposure times and hybridization conditions were identical for all three blots. e-CLU, endogenous clusterin mRNA (1656 bp); tg-CLU, transgenic clusterin mRNA containing 220 bp SV40 polyA (1906 bp); 18S, identical blot hybridized to human 18S cRNA probe. (B) Western blot analysis of clusterin protein in supernatants of: control cells (WT and PV); sense (S21 and S81) and anti-sense (AS2 and AS10) clusterin transfected clones. After 24 h of culture in the absence of serum, cell-free supernatants were collected and analyzed as described in the Materials and Methods. Journal of Investigative Dermatology 1999 112, 290-296DOI: (10.1046/j.1523-1747.1999.00531.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Anti-sense transfected clusterin A431 clones are more sensitive to heat shock and oxidative stress-induced apoptotic cell death. A431 cells (WT), a pool of control vector transfected A431 cells (PV), sense (S21 and S81) and anti-sense (AS2 and AS10) clusterin transfected clones were exposed at 45°C for 6 h (A), or to 120 J UVA radiation per cm2 (B). Apoptosis was quantified 24 h after exposure by FACS analysis of Annexin V positive cells. Journal of Investigative Dermatology 1999 112, 290-296DOI: (10.1046/j.1523-1747.1999.00531.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions