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. Author manuscript; available in PMC: 2009 Oct 6.
Published in final edited form as: Cell Death Differ. 2008 Jun 13;15(9):1460–1471. doi: 10.1038/cdd.2008.81

Figure 6.

Figure 6

GRP78 knockdown disrupts ER integrity but not PI3KC3-Beclin1 complex formation. (a) Cell lysates from siRNA transfected HEK293 cells were immunoprecipitated with anti-Beclin1 antibody. The immunocomplex was analyzed for PI3KC3 and Beclin1 levels by Western blot using antibodies sequentially. Part of the cell lysates were also analyzed for GRP78, PI3KC3 and Beclin1 expression by Western blot using antibodies sequentially. (b) HEK293 cells were transfected with pcDNA3 (vector control, vec) or indicated amount of pcDNA3-His-GRP78 (His-GRP78). Immunoprecipitation, analysis of immunocomplex and His-GRP78, PI3KC3 and Beclin1 expression were done the same as in (a). (c) Representative electron micrograph of cells transfected with siCtrl (left panel) showing sparse ER structures (white arrow heads) or siGrp78 (right panel) showing increased ER structures with expanded lumens (white arrow heads). N, nucleus; Bar=1 μm. (d) Quantitation of the ER area in the cytoplasm in cells transfected with either siCtrl or siGrp78. The standard deviation is shown, *, p<0.0001. (e) Knockdown of XBP-1 recovered normal levels of stress-induced autophagosome formation inhibited by knockdown of GRP78. The indicated siRNA(s) was transiently co-transfected with GFP-LC3 into HeLa cells. The cells were either non-treated (NT), treated with Tun (3 μM) for 4 h or NS for 2 h. The formation of autophagosome under each condition was assayed by fluorescence microscopy and quantitated. The experiments were repeated 2 times. The results were summarized and expressed as the mean with the indicated standard deviation. The insert shows Western blot analysis of GRP78, XBP-1 and β-actin levels in cells transfected with siCtrl (lane 1), siGrp78 (lane 2), siXbp-1 (lane 3) and siGrp78 plus siXbp-1 (lane 4) respectively.