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. 2010 Aug 27;18(2):350–361. doi: 10.1038/cdd.2010.104

Figure 3.

Figure 3

Downregulation of SPP1 induces ER stress that leads to autophagy. (a, b) Equal amounts of lysates from MCF7 cells transfected with siControl or siSPP1 were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Blots were re-probed for total eIF2α or tubulin to insure equal loading and transfer. (c) Duplicate cultures were lysed and caspase 2 and caspase 4 activities were determined by fluorescence and luminescence, respectively. Data are means±S.D. of three independent experiments each carried out in triplicate. (d) MCF7 cells were transfected with siControl or siRNAs targeted to IRE1α or ATF6, and downregulation efficiency was examined by immunoblotting. (e) MCF7 cells were also co-transfected with vector or dominant-negative PERK (dnPERK) and inhibition of PERK activity confirmed by immunoblotting with p-eIF2α antibody. (f) MCF7 cells were co-transfected with siControl, siSPP1, siIRE1α, siATF6, or dnPERK and EGFP-LC3, as indicated. Autophagy was quantified by confocal microscopy. Values are the means±S.D. of three independent experiments. (g–i) Downregulation of SPP1 does not trigger apoptosis or induce cell death. (g) MCF7 cells were transfected with siControl or siSPP1, cultured for the indicated times, and nuclei were stained with Hoechst 33342. Apoptosis was determined by scoring the percentage of cells displaying fragmented, condensed nuclei indicative of apoptosis. At least three fields were analyzed, scoring a minimum of 300 cells. Data are the means±S.D. of three independent experiments. Insert: Equal amounts of lysates from duplicate 1-day cultures were resolved by SDS-PAGE and immunoblotted with anti-PARP antibody. Blots were re-probed with anti-tubulin to insure equal loading and transfer. (h, i) MCF7 cells were transfected with siControl siRNA, siSPP1, or individual siRNAs targeted to Atg5, ATF6, or IRE1α, or transfected with dnPERK. Apoptosis was quantified by Hoechst 33342 staining (h) and viability was quantified by Trypan blue exclusion (i). Data are means±S.D. of three independent experiments. *P<0.01 compared with siControl