Fig. 4.

RNAi-mediated knockdown of αB-crystallin in Bcl2L12-expressing astrocytes enhances apoptotic and decreases necrotic cell death. (A) Western blot analysis for αB-crystallin in pBabe- and Bcl2L12-expressing astrocytes transfected with RNAi oligonucleotides (50 nM; siNT, nontargeting oligonucleotide; siCRYAB-1 to siCRYAB-4, αB-crystallin-specific oligonucleotides). Hsp70 is shown as a loading control. (B) pBabe- and Bcl2L12-expressing astrocytes transfected with siNT and αB-crystallin-targeting RNAi oligonucleotides were treated with STS (0.5 μM) for the indicated periods of time and subsequently subjected to Western blot analysis using antibodies specific for cleaved caspase-3 and caspase-7 species. The migration positions of the large subunits (LS) are indicated. Hsp70 is shown as a loading control. (C) pBabe- and Bcl2L12-expressing astrocytes transfected with siRNA oligonucleotides were treated with the indicated doses of STS for 24 h, and DNA fragmentation was assessed by FACS-based quantification of subdiploid DNA content. The means of three independent experiments are shown. Error bars represent SDs, and P values were calculated by using Student's t test. (D) TEM of Bcl2L12-expressing astrocytes transfected with siNT or siCRYAB-1, siCRYAB-2 oligonucleotides. Shown are representative micrographs of untreated and STS-treated cells (1 μM). (Bar: 1 μm). Filled arrowheads point to condensed chromatin as a hallmark characteristic of apoptotic cells. Note the absence of chromatin condensation in siNT-transfected Bcl2L12-expressing astrocytes, and the occurrence of such structures within the nucleus of siCRYAB-treated cells. (E) Quantification of apoptotic and necrotic cells as determined by the absence or presence of plasma membrane integrity and chromatin condensation of TEM micrographs. Error bars depict SDs, and P values were calculated by using Student's t test.