Figure 2.
Treatment of ML-2 and MV-4-11 cells with the RTA combination triggers the UPR and the oxidative stress response. (A) The RNA of ML-2 and MV-4-11 cells, treated as described in Figure 1A for 24 or 72 hours, was analyzed by quantitative real-time polymerase chain reaction for the expression of UPR target genes (the transcription factor CHOP, the ER chaperone BiP, and the spliced, active form of the transcription factor XBP1 [sXBP1]) and for the expression of HMOX, a gene with a main role in the oxidative stress response. sXBP1 induces the expression of a plethora of genes that increase the ER folding capacity, whereas CHOP is involved in the proapoptotic branch of the UPR (ML-2, n = 5 ± SEM; MV-4-11, n = 3 ± SEM; ML-2 and MV-4-11 HMOX, n = 2 ± SEM). Student t test vs C: *P < .05, **P < .005, ***P < .001. Student t test vs RA:  †P < .05, ††P < .005, †††P < .001. (B) Western blot of ML-2 and MV-4-11 protein extracts, from cells treated as in panel A for 24 or 72 hours, to detect CHOP and BiP proteins. The graphs below each western blot report densitometric quantification (n = 2 ± SEM). Student t test *P < .05. (C) Confocal analysis of the expression of the ER chaperone BiP in ML-2 and MV-4-11 cells vehicle or RTA treated. (D) Western blot of protein extracts from ML-2 and MV-4-11 cells vehicle or RTA treated for 72 hours to detect the complexes among the chaperone BiP and its client misfolded proteins. The 72-KDa BiP monomer is indicated by the arrow. Unbalance of ER proteostasis upon treatment with RTA determined accumulation of misfolded proteins bound by BiP, indicated as BiP complexes. (E) ML-2 cells, treated for 72 hours as in Figure 1A in the presence or not of 20 mM NAC or of 2.5 mM sodium-4-phenylbutyrate (PBA), were analyzed for propidium iodide (PI) uptake to evaluate cell death (NAC, n = 4 ± SEM; PBA, n = 3 ± SEM). Student t test *P < .05, ****P < .0001. (F) Western blot of protein extracts from ML-2 cells, treated as in panel A, to detect the BiP misfolded protein complexes. NAC relieved oxidative stress induced by RTA and rescued the functionality of the ER, as indicated by the reduction of BiP protein level and by the loss of BiP complexes. A similar effect, although in minor measure, was achieved by PBA.

Treatment of ML-2 and MV-4-11 cells with the RTA combination triggers the UPR and the oxidative stress response. (A) The RNA of ML-2 and MV-4-11 cells, treated as described in Figure 1A for 24 or 72 hours, was analyzed by quantitative real-time polymerase chain reaction for the expression of UPR target genes (the transcription factor CHOP, the ER chaperone BiP, and the spliced, active form of the transcription factor XBP1 [sXBP1]) and for the expression of HMOX, a gene with a main role in the oxidative stress response. sXBP1 induces the expression of a plethora of genes that increase the ER folding capacity, whereas CHOP is involved in the proapoptotic branch of the UPR (ML-2, n = 5 ± SEM; MV-4-11, n = 3 ± SEM; ML-2 and MV-4-11 HMOX, n = 2 ± SEM). Student t test vs C: *P < .05, **P < .005, ***P < .001. Student t test vs RA:  †P < .05, ††P < .005, †††P < .001. (B) Western blot of ML-2 and MV-4-11 protein extracts, from cells treated as in panel A for 24 or 72 hours, to detect CHOP and BiP proteins. The graphs below each western blot report densitometric quantification (n = 2 ± SEM). Student t test *P < .05. (C) Confocal analysis of the expression of the ER chaperone BiP in ML-2 and MV-4-11 cells vehicle or RTA treated. (D) Western blot of protein extracts from ML-2 and MV-4-11 cells vehicle or RTA treated for 72 hours to detect the complexes among the chaperone BiP and its client misfolded proteins. The 72-KDa BiP monomer is indicated by the arrow. Unbalance of ER proteostasis upon treatment with RTA determined accumulation of misfolded proteins bound by BiP, indicated as BiP complexes. (E) ML-2 cells, treated for 72 hours as in Figure 1A in the presence or not of 20 mM NAC or of 2.5 mM sodium-4-phenylbutyrate (PBA), were analyzed for propidium iodide (PI) uptake to evaluate cell death (NAC, n = 4 ± SEM; PBA, n = 3 ± SEM). Student t test *P < .05, ****P < .0001. (F) Western blot of protein extracts from ML-2 cells, treated as in panel A, to detect the BiP misfolded protein complexes. NAC relieved oxidative stress induced by RTA and rescued the functionality of the ER, as indicated by the reduction of BiP protein level and by the loss of BiP complexes. A similar effect, although in minor measure, was achieved by PBA.

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