Fig 1: Smad3 promoted ATF4 transcription in brown adipocytes(A) Relative mRNA levels of ATF4, Smad3 and Smad5 of brown adipocytes pre-infected with pAd-Sirt1 or si-Sirt1 for 24 h (n = 3); (B) Predicate translation factors of ATF4 by using Genomatix software; (C) Dual luciferase reporter assay of ATF4 and Smad3. HEK293 cells were transfected with PGL3-basic or PGL3-ATF4 plasmids, and pc-Smad3 plasmid (n = 3); (D) ChIP analysis between ATF4 and Smad3 (n = 3); (E) Relative mRNA levels of Smad3 and ATF4 with pAd-Smad3 infection of brown adipocytes (n = 3); (F) Protein levels of Smad3, ATF4, CHOP, GRP78, Cleaved-Caspase3, Bax and Bcl-2 of brown adipocytes pre-incubated with TM and treated with pAd-Smad3 or not (n = 3); (G) Protein levels of ATF4, Smad3, IRE1 and ERdj4 of brown adipocytes pre-incubated with TM, and infected with pAd-Sirt1 or si-Sirt1 (n = 3). pAd-Smad3: recombinant adenovirus over-expression vector of Smad3, pc-Smad3: Overexpression plasmid of Smad3; pAd-Sirt1: recombinant adenovirus over-expression vector of Sirt1, si-Sirt1: recombinant lentiviral interference vector of Sirt1. Values are means ± SEM. *p < 0.05, #p < 0.05 compared with the control group.
Fig 2: Sirt1 decreased brown adipocytes apoptosis and reduced ATF4 levelBrown adipocytes were pre-infected with recombinant vectors of Sirt1 (pAd-Sirt1 or si-Sirt1) for 48 h. n = 3 for each treatment. (A) Protein level of Sirt1 and Sirt2 in brown adipocytes; (B) ATP level of brown adipocytes; (C) Cytosolic Ca2+ concentration and Flow cytometry (FCM) analysis of Cytosolic Ca2+ in brown adipocytes; (D) Annexin V-FITC/PI double staining and flow cytometry analysis of brown adipocyte apoptosis stages; (E) Protein levels of GRP78, Smad3, Chop, Bcl-2, Bax, Apaf-1, Cleaved-Caspase9 and Cleaved-Caspasw3 in brown adipocytes. pAd-Sirt1: recombinant adenovirus over-expression vector of Sirt1, si-Sirt1: recombinant lentiviral interference vector of Sirt1. Values are means ± SEM. *p < 0.05 compared with the control group.
Fig 3: Thapsigargin (Tg) induced ER stress triggered brown adipocytes apoptosis and reduced UCP1(A) Protein of Sirt1 of brown adipocytes treated with thapsigargin (Tg, 1 µM) for 12 h (n = 3); (B) Protein level of UCP1 and PRDM16 of brown adipocytes treated with 1 µM Tg for 12 h (n = 3); (C) ATP level of brown adipocytes treated with 1 µM Tg for 12 h (n = 3); (D) Protein level of GRP78, Chop, ATF4, Bcl-2 and Bax of brown adipocytes treated with 1 µM Tg for 12 h (n = 3). Values are means ± SEM, *p < 0.05 compared with the control group.
Fig 4: Induction of ferroptosis reversed the effects of ATF4 suppression on Sev-induced ferroptosis in glioma cells. Ferroptosis inducer Erastin was used to incubate in Sev-treated and ATF4-suppressed U87 and U251 cells. Cell viability was detected using CCK-8 assay in U87 (A) and U251 cells (B). Fe2+ concentrations were determined by colorimetric assay in U87 and U251 cells (C). ROS assay combined with flow cytometry was used to observe the content of ROS generation in U87 and U251 cells (D); the ratio of ROS generation was calculated (E). The expression of ATF4 and CHAC1 in U87 and U251 cells was detected using Western blotting; GAPDH was used as the internal control (F, G). Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, compared with control.
Fig 5: PERK downregulation compromises the inhibition of HC growth induced by 125I and LBP in SMMC7721 xenograft tumors in nude mice.To further verify the effect of the PERK-eIF2a-ATF4-CHOP pathway on 125I-mediated apoptosis and anti-proliferation of HCC cells, SMMC7721 cells transfected with PERK-RNAi or Control-RNAi xenograft tumors were treated with 125I and LBP. When the volume of the tumors reached 500 mm3, the mice were randomly divided to three groups with four mice in each group. After 30 days of treatment, the mice were killed and the tumors were exfoliated. The tumor weight (b) and diameters (c) were measured every other day for 30 days. One-way ANOVA with Tukey’s multiple comparison test was utilized to analyze the subcutaneous tumor growth. All the experiments were performed in triplicate and the data are presented as the mean ± SD. The t-test was used for data analysis. *P < 0.05, **P < 0.01
Supplier Page from Abcam for Anti-ATF-4 antibody [EPR18111]