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Figure S6. Axonal Tctp interacts with pro-survival Mcl1. (A) PLA signal (green) obtained using anti-Tctp and anti-Mcl1 sera in control- or tctp-shRNA-infected HCT116 cells, counterstained with DAPI (blue) and phalloidin (red). (B) PLA signal obtained using anti-Tctp and anti-Mcl1 sera in cultured rat cortical neurons (E18.5 + 14 DIV), counterstained with DAPI and phalloidin. (C) PLA signal obtained using anti-Tctp and anti-Mcl1 sera in cultured rat cortical neurons (E18.5 + 3 DIV), counterstained with MitoTracker. Arrowheads denote PLA positive puncta co-localizing with mitochondria. The bottom three panels are centred on a distal region of the neurite. The data indicates that approximately 5-10% of Tctp-Mcl1 PLA puncta co-localize with mitochondria in neurites. This is in agreement with observations by Yang and colleagues who found by immunocytochemistry means that Tctp and Bcl-XL partially co-localize not only in mitochondria but also in the cytosol (Yang et al., 2005). It is noteworthy that while anti-apoptotic Bcl-2 family proteins are generally integrated within the outer mitochondrial membrane, they can also be found in the cytosol or in the endoplasmic reticulum membrane (Chipuk et al., 2010). Furthermore, pro-apoptotic Bax, whose homodimerization in the outer mitochondrial membrane is prevented by Tctp (Susini et al., 2008), is usually cytosolic (Chipuk et al., 2010). Likewise, the cellular localization of Tctp is mainly cytoplasmic in healthy cells (Bommer and Thiele, 2004; Gachet et al., 1999; Yang et al., 2005). One possibility is that Tctp shuttles between the cytosol and the outer mitochondrial membrane in non-apoptotic conditions. (D) Stage 43 Mcl1-depleted retina probed with anti-Opsin antibody, counterstained with phalloidin and DAPI. Signs of morphological disruption were not detected on examination of the retina in Mcl1 morphants, suggesting that the mcl1-MO does not elicit widespread toxicity. Scale bars: 5 μm in (A), 10 μm in (B) and (C), 50 μm in (D).
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