FIGURE 2.

PiT1 involvement in TNF-induced apoptosis is (i) not shared with PiT2, (ii) independent of the presence of extracellular P_i and (iii) unrelated to its transport activity. A, HeLa cells were transiently transfected with a siRNA control (Ct), two different siRNAs against PiT1 (P1a and P1b), or an siRNA against PiT2 (P2). 48 h post-transfection, cells were either mock-treated (0) or treated with 50 ng/ml TNF and 10 μg/ml cycloheximide for 4 h. Cells were lysed and analyzed by Western blot with antibodies directed against PARP and actin (as a loading control). B, values are the mean ± S.E. of three independent experiments performed as in A. **, p < 0.005. C, shCt or shPiT1 HeLa cells were incubated with or without a combination of 50 ng/ml TNF and 10 μg/ml cycloheximide (TNF+C) in medium containing either 0 or 0.9 mm P_i for 4 h. Apoptosis was detected by Western blot with antibodies against PARP and actin as loading control. D, three independent experiments were performed as in C and quantified. Values are the mean ± S.E. (error bars). *, p < 0.05. E, PiT1^+/+ and PiT1^−/− MEFs were incubated with or without 50 ng/ml TNF plus 100 μg/ml cycloheximide (TNF+C) for 7 h in medium containing either 0 or 0.9 mm P_i. Apoptosis was detected by the appearance of cleaved caspase-3, and actin was used as a loading control. This representative blot was quantified, and the corresponding values are given below the Western blot panel. F, PiT1^+/+ and PiT1^−/− MEFs were either left untreated (−) or treated (+) with 2 mm AICAR for 7 h. Cell lysates were analyzed by Western blot for the phosphorylation of AMPK (pAMPK) and the total amount of AMPK. The detection of tubulin was used as a loading control. G–K, PiT1^−/− MEFs were transduced with lentiviral vectors expressing either the human WT PiT1 protein or the S621A mutant and the GFP protein from an internal ribosome entry site. Two independent infections (with two different virus stocks) were performed to obtain two independent populations in each case (T1 and T2). GFP-positive cells were enriched by fluorescent cell sorting. G, PiT1 proteins were detected with the anti-PiT1 antibody. H, sodium-dependent P_i uptake was measured in the different transduced populations. *, p < 0.05; **, p < 0.005; ns, nonsignificant. I and J, the different populations were treated (+) or not (−) with 50 ng/ml TNF and 100 μg/ml cycloheximide for 3.5 h. Cell lysates were analyzed for the appearance of cleaved caspase-3, and the detection of actin was used as a loading control. K, quantification of the blots shown in I and J. Three independent experiments (with the two different cell populations for each constructs) were analyzed. The ratio of the intensities of the cleaved caspase-3 band/actin band was quantified and pooled for each construct and is presented as the relative percentage of the intensity of caspase-3 cleavage quantified in GFP expressing control PiT1^−/− MEFs. ***, p < 0.0005.