![Figure 7. ASCT2 silencing by lentiviral vector impairs MM cell growth in vitro and in vivo. (A) RPMI 8226 cells were incubated in growth medium ([Gln] = 0.6 mM) in the absence (control) or in the presence of MeAIB (20 mM), GPNA (3 mM), or BCH (20 mM). After 72 hours, cell viability was assessed, and results were expressed as % of control. Data represent means ± SD of 3 experiments with 3 determinations each. **P < .01, ***P < .001 vs control as assessed with a 2-tailed Student t test for unpaired data. (B-C) ASCT2 expression in scramble and (B) ΔASCT2 RPMI 8226 and (C) JJN3 cells. Gene expression was evaluated with qRT-PCR and normalized to the expression of RPL-15. ASCT2 protein expression was evaluated with western blot and β-tubulin was used for loading control. (D) 1-Min uptake of Gln (0.6 mM) was performed in scramble and ΔASCT2 RPMI 8226 cells in culture medium in the absence (−) or in the presence of GPNA (3 mM). ***P < .001 vs control. $$$P<.001 vs scramble, as assessed with a 2-tailed Student t test for unpaired data. (E) Scramble and ΔASCT2 RPMI 8226 and JJN3 cells, both at 5 × 105 cells/mL, were grown for 72 hours in medium at 0.6 mM Gln. Cell growth was monitored at the indicated times with the resazurin assay. Data represent means ± SD of 2 experiments with 3 determinations each. SD are shown when greater than the size of the point. (F-H) Two groups of 8 SCID/NOD animals each were injected subcutaneously with 5 × 106 JJN3 cells transfected with a lentiviral vector containing shRNA against ASCT2 (ΔASCT2) or with the control vector (Scramble). Twenty-one days after cell inoculation, mice were killed, and tumors were removed and measured as described in “Patients, materials, and methods.” (F) The box plot graph represents the median volume of the masses (P calculated by Mann-Whitney test). (G) Representative picture of tumors obtained from mice injected with JJN3 Scramble and ΔASCT2 cells stained with hematoxylin-eosin (original magnification, 1×). (H) ASCT2 expression was assessed in plasmacytomas removed after animal death. β-Tubulin was used for loading control.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/128/5/10.1182_blood-2016-01-690743/4/667f7.jpeg?Expires=1735810745&Signature=WUPRN27njTWGiAlFneqBmyZUm38xezKiU6H917IeTmURKgfGC20BlNBu29n1BrxW3YMD82PRNO4924lEjgu7Vj2th26LIMihf6C2~bbtc5-BQ-nzlO2seotw3RXb7vawQzmzET7ibBHYmqMxmObdP7EpugqbehQC9uQh-x~5K09fU~o8h74jTcZg27eK-yMwqHGM4rtzjiwHarES6fJZJygeoEIetSxivhwLf3gtMndAgW4Vg~quIQT1fQJ-OfuOik1wQTy9gp-KJBjEcYNy5Fnn7Nym4Hn-4cBfaQMxkOwCxDRrcZZAK29oOWVlrd4goZckMGQvLQO3ppDCl0VZCQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
ASCT2 silencing by lentiviral vector impairs MM cell growth in vitro and in vivo. (A) RPMI 8226 cells were incubated in growth medium ([Gln] = 0.6 mM) in the absence (control) or in the presence of MeAIB (20 mM), GPNA (3 mM), or BCH (20 mM). After 72 hours, cell viability was assessed, and results were expressed as % of control. Data represent means ± SD of 3 experiments with 3 determinations each. **P < .01, ***P < .001 vs control as assessed with a 2-tailed Student t test for unpaired data. (B-C) ASCT2 expression in scramble and (B) ΔASCT2 RPMI 8226 and (C) JJN3 cells. Gene expression was evaluated with qRT-PCR and normalized to the expression of RPL-15. ASCT2 protein expression was evaluated with western blot and β-tubulin was used for loading control. (D) 1-Min uptake of Gln (0.6 mM) was performed in scramble and ΔASCT2 RPMI 8226 cells in culture medium in the absence (−) or in the presence of GPNA (3 mM). ***P < .001 vs control. $$$P<.001 vs scramble, as assessed with a 2-tailed Student t test for unpaired data. (E) Scramble and ΔASCT2 RPMI 8226 and JJN3 cells, both at 5 × 105 cells/mL, were grown for 72 hours in medium at 0.6 mM Gln. Cell growth was monitored at the indicated times with the resazurin assay. Data represent means ± SD of 2 experiments with 3 determinations each. SD are shown when greater than the size of the point. (F-H) Two groups of 8 SCID/NOD animals each were injected subcutaneously with 5 × 106 JJN3 cells transfected with a lentiviral vector containing shRNA against ASCT2 (ΔASCT2) or with the control vector (Scramble). Twenty-one days after cell inoculation, mice were killed, and tumors were removed and measured as described in “Patients, materials, and methods.” (F) The box plot graph represents the median volume of the masses (P calculated by Mann-Whitney test). (G) Representative picture of tumors obtained from mice injected with JJN3 Scramble and ΔASCT2 cells stained with hematoxylin-eosin (original magnification, 1×). (H) ASCT2 expression was assessed in plasmacytomas removed after animal death. β-Tubulin was used for loading control.
