Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: AICAr Inhibition of cardiomyocyte autophagy promotes p62-dependent NRF2 expression and protection against doxorubicin toxicity

doi: 10.1007/s00018-025-05905-4

Figure Lengend Snippet: AICAr effects on autophagy, p62, NRF2 and doxorubicin toxicity in MCF7 cells. a Western analysis of ADK in 20µg protein lysates from mouse hearts, NRVMs, and MCF7 cells. ADK-L and S are indicated. b MCF7 cells were treated with 500µM AICAr for 3 hours or c 24 hours. 50nM bafilomycin was added for the last 2 hours. LC3-II, LC3-II Flux, AMPK Thr172 phosphorylation, p62 and NRF2 levels were analyzed by Western blot and normalized to ß-Actin. d MCF7 cells were incubated with 500µM AICAr or control conditions for 24 hours, then exposed to 2µM doxorubicin for 6 hours. Cell viability was measured via XTT assay 24, 72, or 120 hours after doxorubicin treatment. Box and Whiskers plots represent data from 3 independent experiments. 4-5 wells per condition were examined in each experiment and normalized to intra-experiment controls. * Indicates p <0.05 relative to control, # indicates p <0.05 for doxorubicin treatment compared to doxorubicin + AICAr treatment

Article Snippet: Antibodies for p62 (cat#18420-1-AP), NRF2 (cat#16396-1-AP), GAPDH (cat#60004-1-Ig), Sarcomeric actin (cat#66125-1-Ig), and ßactin (cat#66009-1-Ig) were from Proteintech.

Techniques: Western Blot, Phospho-proteomics, Incubation, Control, XTT Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: AICAr Inhibition of cardiomyocyte autophagy promotes p62-dependent NRF2 expression and protection against doxorubicin toxicity

doi: 10.1007/s00018-025-05905-4

Figure Lengend Snippet: AICAr dose response effects on phosphorylation of AMPK, ULK1 and autophagic flux. NRVMs were treated with 0, 10, 50, 250, or 500µM AICAr for 3 hours with or without 50nM bafilomycin for the last two hours. AMPK Thr172 and Ulk Ser555 phosphorylation, LC3-II, autophagic flux, and p62 levels were analyzed by western blot and normalized to sarcomeric actin. * indicates p <0.05 vs control.# indicates p <0.05 comparing LC3-II levels in control and treatment groups under bafilomycin treatment

Article Snippet: Antibodies for p62 (cat#18420-1-AP), NRF2 (cat#16396-1-AP), GAPDH (cat#60004-1-Ig), Sarcomeric actin (cat#66125-1-Ig), and ßactin (cat#66009-1-Ig) were from Proteintech.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: AICAr Inhibition of cardiomyocyte autophagy promotes p62-dependent NRF2 expression and protection against doxorubicin toxicity

doi: 10.1007/s00018-025-05905-4

Figure Lengend Snippet: ADK inhibition with ABT-702 blocks AICAr suppression of autophagy. a NRVMs were treated with 500µM AICAr with or without ABT-702 for 3 hours in the presence or absence of 50nM bafilomycin for the last 2 hours. AMPK Thr172 , Ulk Ser555 phosphorylation, LC3-II, autophagic flux, and p62 levels were analyzed by western blot and normalized to sarcomeric actin. b In parallel experiments , LC3-positive puncta were examined by immunofluorescence. Box and Whisker plots represent the number of autophagosomes/cell from 3 independent experiments, counting 20-30 cells per condition in each experiment. The + sign indicates the mean. * indicates p <0.05 as compared to controls. # indicates p <0.05 comparing LC3-II levels in control and treatment groups under bafilomycin treatment

Article Snippet: Antibodies for p62 (cat#18420-1-AP), NRF2 (cat#16396-1-AP), GAPDH (cat#60004-1-Ig), Sarcomeric actin (cat#66125-1-Ig), and ßactin (cat#66009-1-Ig) were from Proteintech.

Techniques: Inhibition, Phospho-proteomics, Western Blot, Immunofluorescence, Whisker Assay, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: AICAr Inhibition of cardiomyocyte autophagy promotes p62-dependent NRF2 expression and protection against doxorubicin toxicity

doi: 10.1007/s00018-025-05905-4

Figure Lengend Snippet: Time course of AICAr effects on AMPK activation, autophagy, NRF2 expression and NRF2 transcriptional activity a NRVMs were treated with 500µM AICAr for 3, 6, and 24 hours. To examine autophagic flux, 50nM bafilomycin was added for the last 2 hours. pAMPKThr172, pUlkSer555, p62, LC3-II, autophagic flux, and NRF2 levels were analyzed by western blot and normalized to sarcomeric actin. b immunofluorescent staining of NRF2 after 24 hours of AICAr treatment and c quantification of nuclear NRF2 from two separate experiments, each measuring 30-40 cells per condition. d ARE/NRF2 reporter luciferase activity was measured after 24 hours of AICAr treatment and normalized to co-transfected constitutive renilla luciferase activity. * indicates p <0.05 as compared to control. # indicates p <0.05 comparing LC3-II control values and treatment groups under bafilomycin treatment

Article Snippet: Antibodies for p62 (cat#18420-1-AP), NRF2 (cat#16396-1-AP), GAPDH (cat#60004-1-Ig), Sarcomeric actin (cat#66125-1-Ig), and ßactin (cat#66009-1-Ig) were from Proteintech.

Techniques: Activation Assay, Expressing, Activity Assay, Western Blot, Staining, Luciferase, Transfection, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: AICAr Inhibition of cardiomyocyte autophagy promotes p62-dependent NRF2 expression and protection against doxorubicin toxicity

doi: 10.1007/s00018-025-05905-4

Figure Lengend Snippet: AICAr-induced increases of NRF2, NRF2 target genes, and protection against doxorubicin-induced cell death require p62. a NRVMs were treated with control RNAi or p62 RNAi and exposed to 24 hours AICAr or control conditions.pAMPK Thr172 , p62 and NRF2 protein levels were measured by western blot and normalized to β-actin. b mRNA levels of p62, NRF2, HO-1, NQO1, SOD1 and ATG7 were measured by RT-QPCR and normalized to 18 s RNA. c NRF2 localization was examined by immunofluorescence. d Following 24 hours of AICAr treatment, control RNAi or p62 RNAi-treated NRVMs were exposed to 2µM doxorubicin for 6 hours. 24 hours later, % cell death was determined by XTT assay. Box and Whisker plots represent data from 3 independent experiments with 3-6 wells measured per condition and normalized to intra-experiment controls. * Indicates p <0.05 relative to ctr RNAi. # indicates p <0.05 comparing p62 RNAi to ctr RNAi exposed to the same treatments

Article Snippet: Antibodies for p62 (cat#18420-1-AP), NRF2 (cat#16396-1-AP), GAPDH (cat#60004-1-Ig), Sarcomeric actin (cat#66125-1-Ig), and ßactin (cat#66009-1-Ig) were from Proteintech.

Techniques: Control, Western Blot, Quantitative RT-PCR, Immunofluorescence, XTT Assay, Whisker Assay