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. Author manuscript; available in PMC: 2015 Sep 26.
Published in final edited form as: Oncogene. 2014 Apr 28;34(13):1718–1728. doi: 10.1038/onc.2014.93

Figure 3. ORCTL3 induces apoptosis via the ARCosome and elicits an incomplete ER stress.

Figure 3

(a) Expression constructs for Bap31-EYFP and ORCTL3 or β-gal were co-transfected into HeLa cells in a 1:3 ratio, cellular extracts were probed for cleavage of the Bap31-GFP fusion protein with an anti-GFP antibody at the indicated time points. (b) the percentage of cleaved product to the total signal of the Bap31-EYFP fusion constructs was quantified after scanning in the Western blots. Each bar represents the mean ± SD (n=2). (c,d) HeLa cells harbouring shRNA constructs against the indicated genes (inserts) were transfected with an expression construct for ORCTL3 and apoptosis was scored after 24 hours. Raw data were normalised to transfection efficiency estimated by GFP for each cell type. Data represents the means ± SD (n=6). (e) Protein levels of the ER stress factors Grp78/Bip chaperone protein, ATF4/CREB2, and CHOP were determined in Hela cells at different times post-transfection of either ORTCL3 or β-galactosidase using 5 ng/μl tunicamycin for eight hours as a positive control and ß-actin and GAPDH as loading controls. (f) After transfection of an expression plasmid for ORCTL3 the conversion of XBP-1 from its unspliced form (uXBP-1) into its spliced form (sXBP-1) was monitored by RT-PCR with β-actin as a control. (g) HeLa cells were stably transfected with an shRNA construct against ATF4 (insert), followed by introducing an expression construct for ORCTL3, Bax or β-gal. After 24 hours apoptosis was quantified evaluated by flow cytometry analysis. Data represent means ± SD (n=3).