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Cancers 2018, 10, 144 8 of 15 in only 10% survival (Figure 5H). These data also suggest that the combination of OAdmCherry and TMZ has a potent inhibitory effect upon colony formation in murine TNBC cells. Cancers 2018, 10, x FOR PEER REVIEW 7 of 15 Figure 5. Combined therapy increases autophagosome formation and light chain 3 (LC3)-II Figure 5. Combined therapy increases autophagosome formation and light chain 3 (LC3)-II accumulation: (A) Human triple-negative breast cancer (TNBC) cells were transfected with pEGFP- accumulation: (A) Human triple-negative breast cancer (TNBC) cells were transfected with pEGFP-LC3 LC3 followed by treatment with temozolomide (TMZ) at 0.4 mM or oncolytic adenovirus mCherry followed by treatment with temozolomide (TMZ) at 0.4 mM or oncolytic adenovirus mCherry (OAdmCherry) at a multiplicity of infection concentration of 10 alone or in combination. Integration (OAdmCherry) at a multiplicity of infection concentration of 10 alone or in combination. Integration of GFP-LC3 into the autophagosome is depicted by punctate structures (arrows) and was analyzed of GFbyP-fLluCo3reisncetonctehme aicurotoscpohpaygaots4o8mhepiossdt terpeaictmteednbt.yImpaugnecstwateresttraukcetnuartes40(×armroawgnsi)fiacantdionwwasitahnthaleyzed by flEuVorOeSscFeLnIcme amgincrgoSsycsotpemy a(At 4d8vahnpceodstMtriceraotsmcoepnyt.GIrmouagpe) sunwdere3t5a7k/4e4naantd4404×7/6m0anganiofimcaeteiorsn(wnmit)h the EVOeSxcFiLtaItimonagainndgeSmyistseiomn (vAisduvalaiznacteiodnM, reicsrpoescctiovpeylyG. (Bro)uCpo)mupnadriesron35o7f/n4u4mabnedr o4f4G7/FP60dnotasnpoemr ceetlelrisn(nm) untreated cells or cells treated with TMZ, OAdmCherry, or a combination of both. A representative excitation and emission visualization, respectively. (B) Comparison of number of GFP dots per cell in experiment is shown from three performed (* p < 0.05). (C) Whole cell protein lysates were collected untreated cells or cells treated with TMZ, OAdmCherry, or a combination of both. A representative 24 h post treatment. Expression of LC3-I and LC3-II was detected by Western blot analysis; actin was experiment is shown from three performed (* p < 0.05). (C) Whole cell protein lysates were collected used as a loading control. A representative experiment is shown from three performed. 24 h post treatment. Expression of LC3-I and LC3-II was detected by Western blot analysis; actin was used as a loading control. A representative experiment is shown from three performed. 2.5. Combination of OAdmCherry with TMZ Alters Autophagy in Human TNBC CellsPDF Image | Temozolomide Enhances Triple-Negative Breast Cancer Virotherapy
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