Figure 8.

PACS2 depletion in HeLa confers resistance to perforin/hGrzB-mediated cell death and reduced Bid processing. (a) PCAF-KD or ADA3-KD HeLa have reduced PACS2 expression. PCAF-KD, ADA3-KD or non-silencing cells were examined for PACS2 expression by RNA extraction and relative mRNA expression of PACS2 was measured by qRT-PCR. (b) HeLa transduced with PACS2 shRNA have reduced PACS2 expression. RNA was extracted from non-silencing, or PACS2-KD HeLa and relative mRNA expression for PACS2 was detected by qRT-PCR. (c) PACS2-depleted HeLa are resistant to perforin/hGrzB-mediated cell death and have reduced Bid processing. i. PACS2-KD or non–silencing HeLa were treated with sublytic perforin in the absence or presence of hGrzB at 30 nM and 60 nM and a 4 h ⁵¹Cr release assay was completed. ii. Non- silencing, or PACS2-KD HeLa were treated for 20 min with sublytic perforin in the absence or presence of hGrzB at the indicated concentrations and whole-cell lysates were subjected to immunoblot for Bid detecting full length (FL) and cleaved (tBid, p11). Total protein was evaluated by detecting Actin levels. (d) A simplified schematic depicting the role that PCAF and ADA3 have in perforin-mediated human perforin/hGrzB-induced cell death and their effect on Bid processing via the transport protein PACS2. The suggested mechanism shows the following: PCAF and ADA3 influence the expression of PACS2; PACS2 binds to full-length Bid and assists in its trafficking and cleavage (tBid) by human GrzB; tBid instigates Bax/Bak oligomerisation which induces mitochondrial outer membrane permeabilisation (MOMP); inducing cytochrome-c release (as well as Smac/Diablo) which activates caspases and leads to cell death. Error bars represent S.E.M., n=3. Statistical analysis performed: one way ANOVA versus non-silencing and a Bonferroni test, **P<0.01