Journal: Cell Death & Disease

Article Title: Transient receptor potential ion channel TRPM2 promotes AML proliferation and survival through modulation of mitochondrial function, ROS, and autophagy

doi: 10.1038/s41419-020-2454-8

Figure Lengend Snippet: a Western blotting was performed to determine expression of HIF-1α, HIF-2α, FOXO3a, IQGAP1, Nrf2, CREB, and CREB phosphorylation in TRPM2-depleted U937 cells. In these experiments, two knockout (KO-1-2) and two scrambled clones (Scr-2-3) were selected randomly for western blots with or without treatment with 0.3 µM doxorubicin. b Expression of autophagy proteins ULK1, Atg7, Atg5, Atg13, FIP200, Atg101, and transcription factor ATF4 was examined by western blotting. p62, Tom20, and LC3B-I and II, which are modified in autophagy, were also studied. Actin was probed to confirm equivalent loading. In a and b , western blots performed for each protein are shown on the left. One blot for each protein is shown here and the others in Supplemental Information Figs. , . Densitometry measurements from two to three experiments for each protein were standardized to results for each experiment’s average untreated scrambled control and means ± s.e.m. calculated and shown on the right ( n = 4-6, Tom20 n = 8). * p ≤ 0.001, ** p < 0.03, group effect, Scr vs KO, two-way ANOVA. c RT-PCR was used to measure TRPM2, HIF-1α, HIF-2α, CREB, ULK1, and Atg7 mRNA in TRPM2-depleted leukemia cells. Results summarizing two (TRPM2, ULK1), three (Atg7) or four (HIF-1/2α, CREB) experiments are shown (mean ± s.e.m., n = 8-16). * p ≤ 0.0001, ** p < 0.015, one-way ANOVA. d U937 cell were incubated with or without bafilomycin A1, and conversion of LCB-I to II was examined with western blotting. Four experiments were performed. One blot is shown here and the others in Supplemental Information Fig. . Densitometry measurements were obtained. Relative autophagic flux was calculated as: (O.D. LC3B-II + Bafilomycin/O.D. Actin) - (O.D. LC3B-II-Bafilomycin/O.D. Actin) for each band. Means ± s.e.m. for the four experiments for each cell line are shown below the figure ( n = 8). * p < 0.05, unpaired, two-tailed t -test. e Western blotting was performed to measure TRPM2, CREB, ATF4, ULK1, Atg7, Atg5, and LC3B-I and II expression in four experiments after TRPM2-L reconstitution. One blot is shown here and the others in Supplemental Information Fig. . Densitometry measurements were normalized to each blots’ untreated scrambled control, and mean densitometry measurements ± s.e.m. for experiments with each protein are shown on the right. * p < 0.04 ( n = 4) one-way ANOVA.

Article Snippet: Blots were probed with anti-TRPM2-C (1:300; Bethyl Laboratories, Montgomery, TX, USA); with antibodies to ATF4 (1:400), Atg7 (1:2000), Atg13 (1:500), Atg101 (1:500), pCREB1 and CREB1 (1:250), FIP200 (1:300), forkhead box transcription factor 3a (FOXO3a; 1:400), HIF-1α (1:250), Nrf2 (1:400), and ULK1 (1:1000) from Cell Signaling Technology (Boston, MA, USA); Atg5 (1:1000) from Medical Biological Lab (Japan); COX6B1 (1:500), MT-CO2 (1:500), MT-ND2 (1:500), and NDUFA13 (1:300) from LSBio (Seattle, WA, USA); HIF-2α (1:500) and LC3B (1:2000) from Novus (Littleton, CO, USA); IQGAP1 (1:5000) from Abcam (Cambridge, MA, USA); p62 (1:5000) from American Research Products (Waltham, MA, USA); Tom20 (1:5000) from Santa Cruz Biotech (Dallas, TX, USA); and actin (1:5000) from Sigma (St. Louis, MO, USA).

Techniques: Western Blot, Expressing, Knock-Out, Clone Assay, Modification, Reverse Transcription Polymerase Chain Reaction, Incubation, Two Tailed Test

Journal: Genes & Nutrition

Article Title: Anthocyanins protect human endothelial cells from mild hyperoxia damage through modulation of Nrf2 pathway

doi: 10.1007/s12263-012-0324-4

Figure Lengend Snippet: Effects of 24-h pretreatment with AS or FS on Nrf2 nuclear localization in HUVECs exposed for 24 h to 32 % O2 (mild hyperoxic conditions) or to 21 % O2 (normoxic conditions). The figure shows a representative image from three independent experiments. Nrf2 bands intensity values were normalized to the corresponding Lamin B values. Results by densitometry are reported as fold change against FS in normoxia and expressed as mean ± SD of at least three independent experiments. *P < 0.05 versus FS-treated cells exposed to normoxic conditions. °P < 0.05 versus FS-treated cells exposed to hyperoxic conditions. §P < 0.05 versus AS-treated cells exposed to normoxic conditions. AS, human serum obtained after Medox® capsules consumption; FS, human serum obtained immediately before Medox® capsules consumption

Article Snippet: Membranes were then probed with specific primary antibodies: rabbit anti-Nrf2 polyclonal antibody (Santa Cruz Biotechnology) (1:200); rabbit anti-p-ERK1/2 polyclonal antibody (Santa Cruz Biotechnology) (1:150); mouse anti-Lamin B monoclonal antibody (Santa Cruz Biotechnology) (1:200); rabbit anti-cytoskeletal actin (Bethyl Laboratories) (1:5,000), followed by peroxidase-conjugated secondary antibody HRP-labeled goat anti-rabbit Ig (BD Pharmigen) (1:5,000) and visualized with an ECL plus detection system (Amersham Biosciences).

Techniques: Capsules

Journal: Genes & Nutrition

Article Title: Anthocyanins protect human endothelial cells from mild hyperoxia damage through modulation of Nrf2 pathway

doi: 10.1007/s12263-012-0324-4

Figure Lengend Snippet: Effect of MAPKK inhibition on the Nrf2 activation pathway in HUVECs pretreated for 24 h with AS or FS and then exposed for 24 h to 32 % O2 (mild hyperoxic conditions) or to 21 % O2 (normoxic conditions), as evaluated by Western blot analysis. Cells were incubated with the pharmacological MAPKK inhibitor PD98059 (25 μM) for 1 h before AS or FS addition. Cultures treated with FS and exposed to normoxic conditions were used as negative controls. Each point represents mean ± SD of three experiments. The figure shows a representative image from three independent experiments. Results by densitometry are reported as fold change against controls and expressed as mean ± SD of three experiments. Nrf2 bands intensity values were normalized to the corresponding Lamin B values. *P < 0.05 versus FS-treated cells exposed to normoxic conditions. °P < 0.05 versus FS-treated cells exposed to hyperoxic conditions. @P < 0.05 versus FS-treated cells exposed to hyperoxic conditions and treated with PD98059. §P < 0.05 versus AS-treated cells exposed to normoxic conditions. #P < 0.05 versus AS-treated cells exposed to hyperoxic conditions. ^P < 0.05 versus AS-treated cells exposed to hyperoxic conditions and treated with PD98059. AS, human serum obtained after Medox® capsules consumption; FS, human serum obtained immediately before Medox® capsules consumption

Article Snippet: Membranes were then probed with specific primary antibodies: rabbit anti-Nrf2 polyclonal antibody (Santa Cruz Biotechnology) (1:200); rabbit anti-p-ERK1/2 polyclonal antibody (Santa Cruz Biotechnology) (1:150); mouse anti-Lamin B monoclonal antibody (Santa Cruz Biotechnology) (1:200); rabbit anti-cytoskeletal actin (Bethyl Laboratories) (1:5,000), followed by peroxidase-conjugated secondary antibody HRP-labeled goat anti-rabbit Ig (BD Pharmigen) (1:5,000) and visualized with an ECL plus detection system (Amersham Biosciences).

Techniques: Inhibition, Activation Assay, Western Blot, Incubation, Capsules

Journal: FEBS letters

Article Title: Sulforaphane induces autophagy through ERK activation in neuronal cells.

doi: 10.1016/j.febslet.2014.06.036

Figure Lengend Snippet: Fig. 7. Nrf2 activity is not involved in autophagy. (A) Mouse cortical cells (CN1.4) transiently transfected with mock or Myc-Nrf2 expression plasmid using the Lipofectamine 2000 (Invitrogen) were treated with 50 lM CQ for 12 h. Cell lysates were analyzed by immunoblotting using anti-Myc (9B11) and LC3 antibodies, respectively. (B) Bar graph indicates the relative ratio of LC3-II to actin of triplicate experiments. (C) CN1.4 cells were transfected with siRNA specific Nrf2 or scrambled siRNA as a control using the Oligofectamine (Invitrogen) and kept for 36 h. Cell lysates were analyzed by immunoblotting using anti-Nrf2 and LC3 antibodies, respectively. (D) Bar graph indicates the relative ratio of LC3-II to actin of triplicate experiments. Data shown are mean ± S.E.M. of three independent experiments.

Article Snippet: Anti-phospho-specific mTOR, mTOR, phospho-specific stressactivated protein kinase/c-jun N-terminal kinase (SAPK/JNK), SAPK/JNK, phospho-specific p38, p38, Myc (9B11) and Nrf2 antibodies were obtained from Cell Signaling Technology.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Control

Journal: Nature

Article Title: Collagenolysis-dependent DDR1 signalling dictates pancreatic cancer outcome.

doi: 10.1038/s41586-022-05169-z

Figure Lengend Snippet: Fig. 2 | Col I cleavage controls PDAC metabolism. a–c, Genes differentially expressed between KPC cells grown on wild-type or R/R ECM in LG (0.5 mM) medium for 24 h. Blue, replicates with low expression (z-score = −2); red, replicates with high expression (z-score = 2). Mitochondrial ETC genes (a), mitochondrial ribosome subunit genes (b) and macropinocytosis-related and NRF2-target genes (c). d,e, Fractional labelling (mole per cent enrichment) of TCA cycle intermediates (d) and intracellular amino acids (e) in KPC cells incubated for 24 h in LG medium after plating on [U-13C]-glutamine-labelled wild-type or R/R ECM. α-KG-, α-ketoglutarate. f, KPC cells plated on wild-type or R/R ECM or plastic were incubated in CM or LG medium with or without EIPA, MBQ-167 (MBQ), MRT68921 (MRT), EIPA + MRT or MBQ + MRT for 24 h. Total cellular ATP is presented relative to untreated plastic-plated cells. CM, complete medium. Data in d,e (n = 3 per condition) and f (n = 3 independent experiments) are mean ± s.e.m. Statistical significance determined by two-tailed t-test. Exact P values are shown in the Source Data. ***P < 0.001; ****P < 0.0001.

Article Snippet: Immunoprecipitation was performed using antibodies that specifically recognize NRF2 (CST, 12721).

Techniques: Expressing, Incubation, Two Tailed Test

Journal: Nature

Article Title: Collagenolysis-dependent DDR1 signalling dictates pancreatic cancer outcome.

doi: 10.1038/s41586-022-05169-z

Figure Lengend Snippet: Fig. 4 | The Col I–DDR1–NRF2 axis controls macropinocytosis and mitochondrial biogenesis. a, Representative images and quantification of mitochondria and macropinocytosis in TMR-DEX-incubated parental and variant KPC cells grown on wild-type ECM. b, Immunoblot analysis of the indicated proteins in KPC cells grown on plastic or wild-type or R/R ECM and incubated in LG or LQ medium for 24 h. The effects of wild-type and R/R ECM on DDR1 signalling are summarized on the right. mito., mitochondria; pDDR1, phosphorylated DDR1. c, Representative images and quantification of mitochondria and macropinocytosis in TMR-DEX-incubated parental and NRF2E79Q (E79Q) KPC cells plated on wild-type or R/R ECM in LG medium with or without 7rh or ML120B for 24 h. d, Immunoblot analysis of the indicated proteins in parental, E79Q, DDR1Δ and E79Q/DDR1Δ KPC cells plated with or

Article Snippet: Immunoprecipitation was performed using antibodies that specifically recognize NRF2 (CST, 12721).

Techniques: Incubation, Variant Assay, Western Blot

Journal: Nature

Article Title: Collagenolysis-dependent DDR1 signalling dictates pancreatic cancer outcome.

doi: 10.1038/s41586-022-05169-z

Figure Lengend Snippet: Fig. 6 | Therapeutic targeting of the DDR1–NF-κB–NRF2 axis inhibits PDAC growth and metabolism. a, Parental and E79Q KPC cells plated on wild-type or R/R ECM were incubated in LG medium with or without 7rh, ML120B or ML385. Total viable cells are presented relative to parental cells that were treated with vehicle and plated on wild-type ECM. b, Oxygen consumption rate (OCR) of parental and E79Q KPC cells plated on wild-type or R/R ECM and incubated in LG medium for 24 h before and after treatment with oligomycin (Omy), FCCP or rotenone/antimycin A. c, Representative images and sizes of parental and NHE1KD MIA tumours grown with or without wild-type or R/R fibroblasts in nude mice. Right, immunoblot analysis of NHE1 in MIA cells. d,e, Liver and pancreas morphology (d) and weight (e) four weeks after orthotopic transplantation of KPC E79Q cells into CAE-pretreated Col IWT and Col Ir/r mice.

Article Snippet: Immunoprecipitation was performed using antibodies that specifically recognize NRF2 (CST, 12721).

Techniques: Incubation, Western Blot, Transplantation Assay

Journal: Nature

Article Title: Collagenolysis-dependent DDR1 signalling dictates pancreatic cancer outcome.

doi: 10.1038/s41586-022-05169-z

Figure Lengend Snippet: Fig. 8b–f). NRF2(E79Q)-expressing cells grew faster than parental cells and were resistant to R/R ECM, DDR1 inhibition or IKKβ inhibition but not NRF2 inhibition. IKKαKD cells with high rates of macropinocytosis and high levels of nuclear NRF2 also grew faster than parental cells on wild-type ECM but were more sensitive to R/R ECM and macropinocyto- sis inhibitors (Extended Data Fig. 8b,c). Inhibition of macropinocytosis, DDR1, IKKβ or NRF2 did not decrease the low growth of parental cells on R/R ECM (Fig. 6a and Extended Data Fig. 8b–f). Moreover, paren- tal KPC or 1305 cells that were plated on wild-type ECM were more sensitive to the mitochondrial protein synthesis inhibitor tigecycline than cells plated on R/R ECM or DDR1KD cells grown on wild-type ECM (Extended Data Fig. 8g). NRF2E79Q cells showed higher rates of oxygen consumption and mitochondrial ATP production than did parental cells; these rates were diminished by R/R ECM but only in the parental cells (Fig. 6b and Extended Data Fig. 8h). Thus, the fibrolytic stroma may support PDAC cell growth through Col I-stimulated macropinocy- tosis and mitochondrial biogenesis. R/R fibroblasts inhibited human PDAC (MIA PaCa-2) tumour growth, but wild-type fibroblasts were stimulatory. NHE1 ablation or EIPA inhibited tumour growth with or without co-transplanted wild-type fibroblasts or in wild-type livers, but had little effect on tumours growing with R/R fibroblasts or in Col Ir/r livers (Fig. 6c and Extended Data Fig. 8i). Tumours growing with wild-type fibroblasts were more fibrotic than tumours without added fibroblasts, and small tumours growing with R/R fibroblasts had the highest collagen content (Extended Data Fig. 8j), indicating that deposi- tion of Col I enhances the growth of PDAC only when Col I is cleaved by MMPs. NRF2E79Q cells in Col Ir/r hosts exhibited similar growth, NRF2,

Article Snippet: Immunoprecipitation was performed using antibodies that specifically recognize NRF2 (CST, 12721).

Techniques: Expressing, Inhibition

Journal: Genes & Diseases

Article Title: X-box binding protein 1 caused an imbalance in pyroptosis and mitophagy in immature rats with di-(2-ethylhexyl) phthalate-induced testis toxicity

doi: 10.1016/j.gendis.2023.02.030

Figure Lengend Snippet: DEHP exposure caused testicular injury in immature rats. (A) Histological changes in the testes of the corn oil- and DEHP-treated groups. The vehicle group (a, d) showed an organized seminiferous epithelium, and spermatogenesis progressed normally. Exfoliated germ cells were observed in the 250 mg/kg group (b, e) . In the 500 mg/kg group (c, f) , atrophy of Sertoli cells and a disorganized seminiferous epithelium were noted. Black arrows indicate germ cells. Red arrows indicate Sertoli cells (a–c) Scale bar = 50 μm (d–f) Scale bar = 25 μm. (B, C) The beginning (B) and end (C) weights of the control, DEHP 250 mg/kg, and DEHP 500 mg/kg groups. No significant difference was observed among the three groups. (D) The testis organ coefficient of the three groups. Compared with the control group, the organ coefficient of the 500 mg/kg group was noticeably reduced. The organ coefficient of the 250 mg/kg group was similar to that of the control group. (E) The levels of proteins in the antioxidant pathway in immature testes. The expression levels of NRF2, HO-1 and SOD were elevated after DEHP treatment. (F) The ER stress and mitophagy levels of the control, 250 mg/kg, and 500 mg/kg groups. Following DEHP exposure, the expression levels of ATF6 and XBP1S were reduced, along with the p-PERK/PERK and p-IRE1α/IRE1α ratios. PINK1 and PARKIN expression levels were obviously increased. (G) The level of pyroptosis in pubertal testes. The expression levels of NLRP3, CASPASE1 and IL-1β were significantly different in the DEHP-treated group. Compared with the control group, IL-10 expression was decreased in the other groups. This finding indicates that the anti-inflammatory response was reduced. (H) Immunofluorescence staining showing NRF2 expression in the three groups. Upon DEHP exposure, the intensity of NRF2 staining was increased. (I) Immunofluorescence staining for XBP1S in the three groups. Upon DEHP exposure, XBP1 expression in somatic cells was reduced, similar to its total intensity, while it was up-regulated in germ cells. The data were compared with the control group. One-way ANOVA was employed to analyze all the data. Each bar shows the mean ± SD of three or more independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Primary antibodies (1:200) against NRF2 (Proteintech, 16396-1-AP) and XBP1S (Proteintech, 24868-1-AP) were used.

Techniques: Control, Expressing, Immunofluorescence, Staining

Journal: Genes & Diseases

Article Title: X-box binding protein 1 caused an imbalance in pyroptosis and mitophagy in immature rats with di-(2-ethylhexyl) phthalate-induced testis toxicity

doi: 10.1016/j.gendis.2023.02.030

Figure Lengend Snippet: ER stress, mitophagy, and pyroptosis were involved in MEHP-induced germ cell damage. (A, B) The expression levels of NRF2, HO-1 and SOD were increased in GC-1 (A) and GC-2 cells (B) from the MEHP-treated groups. (C, D) The expression levels of ATF6 and XBP1S were increased, and the ratios of p-PERK/PERK and p-IRE1α/IRE1α in GC-1 (C) and GC-2 (D) cells were also increased, indicating an elevated ER stress level. PINK1 and PARKIN expression levels were elevated, indicating that mitophagy was activated. (E, F) Following MEHP exposure, the expression levels of NLRP3, CASPASE1, IL-1β, and IL-10 were increased in GC-1 (E) and GC-2 cells (F). Based on these data, pyroptosis was enhanced and the levels of anti-inflammatory molecules were increased. The data were compared with the control group. One-way ANOVA was employed to analyze all the data. Each bar shows the mean ± SD of three or more independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Primary antibodies (1:200) against NRF2 (Proteintech, 16396-1-AP) and XBP1S (Proteintech, 24868-1-AP) were used.

Techniques: Expressing, Control

Journal: Genes & Diseases

Article Title: X-box binding protein 1 caused an imbalance in pyroptosis and mitophagy in immature rats with di-(2-ethylhexyl) phthalate-induced testis toxicity

doi: 10.1016/j.gendis.2023.02.030

Figure Lengend Snippet: NAC rescued MEHP-induced germ cell injury by inhibiting ROS generation. (A, E) ROS generation in the control (a) , NAC (b) , MEHP (c) , and MEHP + NAC (d) groups was detected. Compared with the MEHP group, ROS generation in the MEHP + NAC group was reduced by NAC in both GC-1 (A) and GC-2 (E) cells. (B, C) After the NAC intervention, the expression of NRF2, HO-1, and SOD in the MEHP + NAC group was down-regulated in GC-1 (B) and GC-2 (C) cells compared with the MEHP group. (D, F) NAC reduced the levels of ER stress-related proteins (p-PERK, PERK, p-IRE1α, IRE1α, ATF6, and XBP1S) and down-regulated PINK1 and PARKIN expression in both GC-1 (D) and GC-2 (F) cell lines. (H, I) After excess ROS generation was eliminated by NAC, the pyroptosis level in the MEHP + NAC group was reduced in GC-1 (H) and GC-2 (I) cells. The data were compared with the control group. One-way ANOVA was employed to analyze all the data. Each bar shows the mean ± SD of three or more independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Primary antibodies (1:200) against NRF2 (Proteintech, 16396-1-AP) and XBP1S (Proteintech, 24868-1-AP) were used.

Techniques: Control, Expressing