Figure 2
Malat1 upregulation during UPR is in response to activation of PERK.
(A) Schematic representation of the activation of PERK and IRE1α sensors during the UPR. The biochemical steps that are regulated by the UPR-influencing drugs PERKi, STF083010 and CCT020312 used in subsequent experiments are indicated. (B) Equal quantity of total protein isolated from Neuro2a cells which were untreated (lane 1) or treated with either TG alone (lane 2) or TG supplemented with increasing concentration of PERKi (lanes 3–6), was resolved in SDS-PAGE and immunoblotted using either anti-PERK or anti-phospho-PERK antibody. The numbers on the left indicate the migration of relative protein molecular weight marker bands. (C) Total RNA was isolated from Neuro2a cells treated with either DMSO (white bar) or TG (grey bar) or TG supplemented with PERKi (hashed bar) or TG supplemented with STF083010 (black bar), at 6 h after addition of drugs. The total RNA was reverse-transcribed and used for qPCR estimation of Malat1 transcript level, normalized to that of Gapdh mRNA. The relative transcript level in DMSO-treated cells was taken arbitrarily as 1 and that in others represented as fold-change over it. (D) Total RNA was isolated from Neuro2a cells treated with either DMSO (white bar) or TG (grey bar) or TG supplemented with PERKi (black bar), at 6 h after addition of drugs. The total RNA was reverse-transcribed and used for qPCR estimation of Ppp1r15a transcript level, normalized to that of Gapdh mRNA. The relative transcript level in DMSO-treated cells was taken arbitrarily as 1 and that in others represented as fold-change over it. (E) Total RNA was isolated from Neuro2a cells treated with either DMSO or CCT020312 at 6 h after addition of drugs. The total RNA was reverse-transcribed and used for qPCR of the respective gene transcripts, normalized to that of Gapdh mRNA. The transcript level in DMSO-treated cells was arbitrarily taken as 1 (white bar) and that from CCT020312-treated cells (grey bar) represented as fold-change over it. All experiments were done in triplicates and the error bars represent the standard deviation (** signifies p < 0.01).
