Figure 8

SDIM1 attenuates cell death induced by DNAJB4 overexpression. Changes in DNAJB4 protein levels in NT2 neurons (A) and mouse primary neurons (B) treated with OGD and OGD plus 16 h recovery were determined by Western blotting with anti-DNAJB4 antibody. C. Percentage of cell death after DNAJB4 overexpression alone or co-overexpression with SDIM1 in N2a cells. Cell death percentage was assessed 21 h after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (p-value = 0.0016 for pCMV-Tag1 and DNAJB4; p-value = 0.0022 for DNAJB4 and DNAJB4/SDIM1). D. Percentage of cell death of the transfected cells after OGD treatment. The single or double transfected cells shown in C were treated with OGD for 6 h and allowed to recover for 16 h. The cell death assay was performed as described for C. (p-value = 0.0033 for pCMV-Tag1 and DNAJB4; p-value = 0.0027 for pEGFP-N1 and SDIM1; p-value = 0.057 for DNAJB4 and DNAJB4/SDIM1). E. Assessment of gene overexpression efficiency. Protein samples were collected 21 h after transfection. Overexpression of SDIM1 and DNAJB4 proteins was analyzed by Western blotting. F. RT-PCR analysis of RNA levels of DNAJB4 and SDIM1 in N2a cells transfected with pCMV-DNAJB4 or control vector pCMV-tag1. G. Western analysis of protein levels of the same samples shown in F. H. Assessment of siRNA silencing efficiency. NT2 cells were transfected or double transfected with synthetic siRNAs targeted for DNAJB4 and/or SDIM1. Cells were collected for protein extractions 24 h after transfection. I. Percentage of cell death 24 h after siRNA transfection followed by OGD treatment. (p-value = 0.032 for DNAJB4 and control; p-value = 0.029 for DNAJB4 and DNAJB4/SDIM1; p-value = 0.023 for control and SDIM1).