Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 2. Keap1 protein decreases in differentiated adipocytes, while steady-state mRNA expression for Nrf2 and Keap1 increases. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 0, 4, 8, 11, and 14. (A) Nrf2 and (C) Keap1 (70 kDa) protein expression was examined by immunoblotting. Black bars, full-length (FL) Nrf2 (56 kDa); gray bars, degraded (Deg) Nrf2 (40 kDa); white bars, total (Tot) Nrf2; the sums of full-length Nrf2 and degraded Nrf2 were determined to obtain relative total Nrf2 protein expression. (B) Relative steady-state Nrf2 and (D) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 0, bars indicate Pr0.05 between samples. Data are expressed as mean7SD; n¼3–4.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Expressing, Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 3. NQO1 protein increases during limited clonal expansion and postmitotic growth arrest of differentiation. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 0, 1, 2, 3, and 4. (A) NQO1 and (D) Keap1 protein expressions were examined by immunoblotting. (B) Relative NQO1, (C) Nrf2, and (E) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 0, bars indicate Pr0.05 between samples. Data are expressed as mean7SD; n¼3–4.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 4. NQO1 and Keap1 proteins, but not mRNA, decreases as adipocyte differentiation progresses. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 4, 5, 6, 7, and 8. (A) NQO1 and (D) Keap1 protein expressions were examined by immunoblotting. (B) Relative NQO1, (C) Nrf2, and (E) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 4. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 6. LiCl treatment increases NQO1 protein, but not NQO1 mRNA. 3T3-L1 cells were differentiated as described under Materials and methods, and treated with varying concentrations of LiCl at Day 8 and harvested on Day 11. A 40 mM NaCl was used as a vehicle control (VC). (A) NQO1 was examined by immunoblotting. (B) Relative NQO1 and (C) Nrf2 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s; * Pr0.05 as compared to VC for protein and mRNA. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Control, Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 7. Sulforaphane increases NQO1 protein and mRNA levels. 3T3-L1 cells were differentiated as described under Materials and methods, and treated with varying concentrations of sulforaphane (SFN). A 0.3% DMSO was used as a VC. (A) NQO1 was examined by immunoblotting. (B) Relative NQO1 and (C) Nrf2 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s; * Pr0.05 as compared to VC for protein and mRNA. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot

Journal: American Journal of Translational Research

Article Title: PIAS3/SOCS1-STAT3 axis responses to oxidative stress in hepatocellular cancer cells

doi:

Figure Lengend Snippet: Effect of H2O2 treatment on oxidative stress induction and malignant processes in HCC cells. A. H2O2 (50 μM for 24 h) and/or antioxidant TA treatment. Total antioxidant capacity (TAC) in HepG2 cells was determined. VCEAC: vitamin C equivalent antioxidant capacity (n=3). B. Protein carbonyl content in HepG2 cells was detected (n=3). C. Western blotting detected the expression of Nrf2 protein in cells (n=3). The band density for Nrf2 protein are shown (n=3). D. HepG2 cell proliferation 48 h after different treatments (n=3). E. Cell growth of HepG2 cells 24-72 h post treatment (n=3). F. Cell apoptosis in HepG2 cells. G. Invasion ability of HepG2 cells (n=3). Scale bar, 50 μm. H. Migration capacity of HepG2 cells (n=3). Scale bar, 50 μm. *P<0.05, **P<0.01.

Article Snippet: Immunohistochemical staining Immunohistochemical staining was performed according to the manufacturer’s instructions with the following reagents and instruments: horse serum (RTU Vectastain Kit, PK-7200), 1:200 mouse anti-Nrf2 antibody (AF3925, R&D Systems), ABC reagent (LS-J1026-1, Vector labs), and IHC slide staining system (NanoMtrx 100, BioGenex).

Techniques: Western Blot, Expressing, Migration

Journal: American Journal of Translational Research

Article Title: PIAS3/SOCS1-STAT3 axis responses to oxidative stress in hepatocellular cancer cells

doi:

Figure Lengend Snippet: Effects of PIAS3 and SOCS1 overexpression and colivelin treatment on oxidative stress induction and malignant processes of HCC cells. HepG2 cells were transfected with PIAS3 or SOCS1 overexpression vector for 36 h and then treated with H2O2 (50 μM) and/or 0.5 μM CO for 24 h. A. Total antioxidant capacity (TAC) in HepG2 cells was determined. VCEAC: vitamin C equivalent antioxidant capacity (n=3). B. Protein carbonyl content in HepG2 cells was detected (n=3). C. Western blotting detected the expression of Nrf2 protein in cells (n=3). Quantification data from three independent repeats were showed below the blot. D. HepG2 cell proliferation 48 h after different treatments (n=3). E. MTT assays detected cell growth of HepG2 cells 24-72 h post treatment (n=3). F. Cell apoptosis of HepG2 cells. G. Invasion ability of HepG2 cells (n=3). Scale bar, 50 μm. H. Migration capacity of HepG2 cells (n=3). Scale bar, 50 μm. *P<0.05, **P<0.01.

Article Snippet: Immunohistochemical staining Immunohistochemical staining was performed according to the manufacturer’s instructions with the following reagents and instruments: horse serum (RTU Vectastain Kit, PK-7200), 1:200 mouse anti-Nrf2 antibody (AF3925, R&D Systems), ABC reagent (LS-J1026-1, Vector labs), and IHC slide staining system (NanoMtrx 100, BioGenex).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Expressing, Migration

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Kaplan-Meier survival analysis of mice hydrodynamically injected with the MYC transposon system and the indicated NRF2 activating guide RNAs (sgRNA) and Cas9; B) Representative diseased livers from mice injected with MYC and sgKeap1 or sgNrf2 (targeting the Keap1 interacting ETGE domain); C) In vivo growth of murine MYC/sgKeap1 tumors transduced with Nrf2-targeted or control shRNAs; Data from HCC lines 1 & 2 were combined and plotted (n=5 each); D) Kaplan-Meier survival analysis of mice that were hydrodynamically injected with the MYC transposon system and an sgRNA/Cas9 targeting Keap1 and subsequently treated with glutathione synthesis inhibitor BSO. (* indicates p-value < 0.05 by two-tailed student t test). See also Figure S1 and Table S1.

Article Snippet: Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Injection, In Vivo, Transduction, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Measuring NRF2 stability using an NRF2-nanoluciferase fusion protein in lysates of KEAP1 proficient HepG2 and KEAP1 mutant Huh1 cells transduced with control or shRNA against FN3K; mean of n=6 for HepG2 and n=3 for Huh1 cells ± SD; B) Relative expression of ~80 antioxidative genes in KEAP1N414Y mutant Huh1 cells transduced with control or FN3K shRNAs; average of four replicates (n=2 for each FN3K-specific shRNAs) relative to control shRNA; C) Unsupervised clustering of total proteomics data from indicated Huh1 cell lysates; GSEA analysis of over- and underrepresented proteins in FN3K-deficient cells shows reduction of NRF2 target proteins (top) and proteins involved in xenobiotic metabolism (bottom); D) Luminescence-based quantification of oxidized and reduced glutathione in KEAP1N414Y Huh1 cells expressing control vector or shRNA against FN3K; error bar represents SD from n ≥ 5 replicates; E) Nuclear extracts from Huh1 cells with control vector or FN3K knockdown immunoprecipitated with NRF2 or IgG antibodies and probed for MAFG and β-actin; nuclear lysates (bottom) were loaded on a separate gel and probed with αNRF2 and αLamin B as indicated; F) Schematic of KEAP1-dependent and independent mechanisms of NRF2 inhibition by glycation. (*denotes two-tailed t test calculated p-value of <0.05). See also Figure S4 and Table S4.

Article Snippet: Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Mutagenesis, Transduction, shRNA, Expressing, Plasmid Preparation, Immunoprecipitation, Inhibition, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Mass spectrometric identification of tryptic peptides from unglycated (upper) and in vitro glycated recombinant NRF2 (lower); peptides that decreased in abundance upon in vitro glycation are shown in black while those that are unaffected in red; B) AUC analysis of indicated tryptic peptides generated from non-glycated and in vitro glycated NRF2; data are represented relative to R25-R34 peptide and error bar represents SD from 3 replicates; C) Spectral plot of K487-R499 peptide from unmodified (left) and in vitro glycated NRF2 (right, 5 g/L glucose for 14 days); D) Spectral intensity graphs of indicated peptides obtained from tryptic digest of immunoprecipitated nuclear NRF2 from control and FN3K deficient Huh1 cells; E) Normalized AUC analysis of indicated tryptic peptides generated by digesting immunoprecipitated NRF2 from parental (n=2) or FN3K-silenced Huh1 cells (n=3) as indicated; error bar represents SD. (* indicates p-value < 0.05). See also Figures S5, S6, and Table S5.

Article Snippet: Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: In Vitro, Recombinant, Generated, Immunoprecipitation

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Diagram of dual gene-targeting strategy in murine HCCs; B) Ultrasound of murine livers 4 weeks after injection with indicated plasmids; tumors are marked in red; C) Ex vivo images of livers from animals injected with indicated sgRNA combinations; D) Subcutaneous Huh1 xenografts with and without FN3K knockdown and NAC treatment as indicated measured ~30 days after implantation; E) Decreased NRF2 target protein expression by immunoblot on the FN3K deficient Huh1 tumors from panel 5D; F) Phenyl borate enrichment and immunoblot shows NRF2 glycation in the FN3K deficient Huh1 xenografts. See also Figure S7 and Table S6.

Article Snippet: Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Injection, Ex Vivo, Expressing, Western Blot

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Diagram of our strategy for a genome-wide screen against NRF2 driven expression of the HSV-TK suicide gene; B) Map of the lentiviral vector directing ARE-controlled HSV-TK and luciferase expression; C) Change in sgRNA library representation comparing untreated cells and cells treated with the NRF2 inducer tBHQ and ganciclovir; D) Predicted sites of NRF2 protein glycation using indicated algorithms; TAD: Transactivation domain; E) Phenyl borate affinity purification and immunoblotting reveals NRF2 glycation upon FN3K knockdown in KEAP1 mutant Huh1 cells; values on top refers to % of glycated NRF2 represented by the ratio of NRF2 signal intensity in PB-bound (PB) to the sum PB-bound and flow through (FT); F) Immunoblot for nuclear (upper panel) and cytoplasmic (lower panel) levels of the indicated proteins in KEAP1 wild type HepG2 cells transduced and treated as indicated; G) Chromatin immunoprecipitation (ChIP) on indicated HepG2 nuclear lysates with anti-NRF2 antibody followed by amplification of indicated promoters; shown as % of input DNA and error bar is SD of 4 replicates; H) Viability of HepG2 cells untreated or treated with H2O2 (400 μM, 24 hours) with and with without pre-incubation with NAC (10 mM, 3 hours); mean of 9 replicates ± SD. (* indicates p-value < 0.05 by two-tailed student t test). See also Figure S3 and Table S3.

Article Snippet: Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Genome Wide, Expressing, Plasmid Preparation, Luciferase, Affinity Purification, Western Blot, Mutagenesis, Chromatin Immunoprecipitation, Amplification, Incubation, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: A) Unbiased, pan-cancer analysis of mutations that are significantly related (co-occurring or mutual exclusive) with KEAP1 and NRF2 using the SELECT algorithm (see text and methods for details); B) Oncoprint showing mutual exclusive relation between NRF2/ KEAP1 and EGFR mutations in human cancers; C) Nuclear extracts from HepG2 cells treated with NRF2 inducer DLS (1 μM, 24 hours), EGF (10 ng/mL, 24 hours), TGFα (1 nM, 24 hours) or DMSO and probed with antibodies against NRF2 and lamin B; D) Nuclear (upper panel) and cytoplasmic extracts (lower panel) from H3255 (EGFRL858R) cells transduced with EGFR-specific shRNA or control and immunoblotted with indicated antibodies; E) Viability of isogenic H3255 cells transduced with sgRNAs targeting KEAP1 or LacZ (control) and treated with erlotinib; error bars represent SD from 3 replicates; F) Lysates from paired PC9 cells transduced with indicated sgRNA-Cas9 constructs and treated with DMSO or erlotinib (10 nM, 6 hours) probed with the indicated antibodies. (* indicates p-value < 0.05 by two-tailed student t test). See also Figure S2 and Table S2.

Article Snippet: Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Transduction, shRNA, Construct, Two Tailed Test

Journal: Cell

Article Title: The oncogenic action of NRF2 depends on de-glycation by Fructosamine-3-kinase

doi: 10.1016/j.cell.2019.07.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant human NRF2 (Origene) was glycated in vitro in PBS supplemented with 1, 5 or 10 g/L glucose at 37°C for 3 hours, 3 days, or 14 days.

Techniques: Western Blot, Recombinant, Affinity Chromatography, SYBR Green Assay, Software, RNA Sequencing Assay

Journal: Antioxidants

Article Title: Targeting the NRF2/HO-1 Antioxidant Pathway in FLT3-ITD-Positive AML Enhances Therapy Efficacy

doi: 10.3390/antiox11040717

Figure Lengend Snippet: NRF2 inhibition decreases HO-1 expression and inhibits viability in AML. ( A ) Western blot analysis was performed on a panel of cell lines expressing FLT3-ITD (ITD, MOLM13) or FLT3-ITD mutants (D835V, -F691V, and MOLM13) to determine NRF2 and actin protein expression ( B , C ). MOLM13 and MV4.11 human ITD-expressing cell lines were treated with brusatol and cells were harvested at different time points for lysate preparation and Western blot analysis of NRF2 ( B ), HO-1 ( C ) and actin (both) protein expression was determined at indicated timepoints. ( D ) TKI-sensitive Ba/F3-Flt3-ITD cells and -resistant ITD cells (-ITDR, -D835V, -F691V) were treated with brusatol at indicated concentrations for 72 h, and then cell growth was analyzed. The brusatol concentration that inhibited 50% of cell viability (IC 50 ) was determined from the dose–response curves. ( E ) MOLM13-sensitive and MOLM13-TKIR (tyrosine kinase inhibitor resistant) cells and ( F ) three patients’ primary AML samples were treated with NRF2 inhibitor brusatol (10 nM for 4-days) and vehicle, samples were stained with Alamar blue, and cell proliferation was measured quantitatively. ( G ) Cell viability assay was performed in a panel of AML cell lines after NRF2-si transfection in vitro. Transient knockdown of NRF2 by siRNA or control siRNA was allowed for 48 h, and cell viability was measured using ViCell counter. ( H ) HO-1 gene expression was measured from RNA lysates harvested from CD34+ve normal PBMC, and patients’ primary AML samples treated with 50 nM of brusatol for 24 h were harvested. HO-1 transcripts levels were measured by qRT-PCR. ( I ) HO-1 gene expression was measured from RNA lysates harvested from a panel of AML cell lines treated with 50 nM of brusatol for 24 h were harvested. HO-1 transcripts levels were measured by qRT-PCR. Statistical significance is shown as p -values less than 0.05 and 0.01 (* p < 0.05; ** p < 0.01). Experiments were repeated at least three times.

Article Snippet: HO-1 siRNA (purchased from Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-35555) was transfected into all cell lines using lipofectamine RNAiMAX (Life Technologies, Waltham, MA, USA), and Nucleofector Kit (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. shRNA Nrf2 (Cat#: TG311194) and OriGene retroviral particles were transduced using protocol published earlier [ ].

Techniques: Inhibition, Expressing, Western Blot, Concentration Assay, Staining, Viability Assay, Transfection, In Vitro, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Targeting the NRF2/HO-1 Antioxidant Pathway in FLT3-ITD-Positive AML Enhances Therapy Efficacy

doi: 10.3390/antiox11040717

Figure Lengend Snippet: Targeted NRF2 inhibition promotes anti-leukemic effects in AML in vitro and ex vivo. ( A ). Flow cytometry detection of ROS in MOLM13 and MOLM-TKIR cells treated with brusatol and H 2 O 2 for 24 h using DCF. ( B ). ROS levels were measured after brusatol treatment at 10 nM in patients’ primary AML samples (after 24 h) using DCF. ( C ). qRT-PCR based GPx gene expression levels were detected in samples from panel B. ( D ). Cell viability measured after 36 h in brusatol cells from panel B. ( E – G ). Same parameters as in ( B – D ), respectively, after NRF2 knock-down cells. ( H ). Ex vivo analysis of CD34 + cells from normal PBMC and patients’ primary AML cells, treated with brusatol for 24 h, and reduced/oxidized glutathione was measured as an indicator of reductive stress in AML. Biologically independent samples were plated in triplicates (as technical replicates), but statistics were performed only on biologically independent samples ( n = 5), except for panel A and B. All experiments were repeated three times. Statistical significance is shown by p -values less than 0.05 and 0.01 (* p < 0.05; ** p < 0.01).

Article Snippet: HO-1 siRNA (purchased from Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-35555) was transfected into all cell lines using lipofectamine RNAiMAX (Life Technologies, Waltham, MA, USA), and Nucleofector Kit (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. shRNA Nrf2 (Cat#: TG311194) and OriGene retroviral particles were transduced using protocol published earlier [ ].

Techniques: Inhibition, In Vitro, Ex Vivo, Flow Cytometry, Quantitative RT-PCR, Expressing

Journal: Antioxidants

Article Title: Targeting the NRF2/HO-1 Antioxidant Pathway in FLT3-ITD-Positive AML Enhances Therapy Efficacy

doi: 10.3390/antiox11040717

Figure Lengend Snippet: Brusatol potentiates quizartinib’s anti-leukemic effects in AML. ( A ) Bar diagram shows percentage of sub-diploid cells from the ratio of the mean DNA content of MOLM13 and MV4.11 human ITD-expressing cell lines and, ( B ) Ba/F3-Flt3-ITDR cell lines were treated with brusatol at the indicated concentrations and combined with quizartinib at the specified concentrations for 48 h. ( C ) Combination index values for combinations of the NRF2 inhibitor, brusatol, and FLT3 inhibitor, quizartinib, were measured in FLT3-ITD AML cell lines, MOLM13 and MV4.11. Synergy was determined based on DNA fragmentation assays using CalcuSyn software (Biosoft, Cambridge, United Kingdom), with combination index (CI) values < 1 considered synergistic, equal to 1 additive, and greater than 1 antagonistic. ( D ) Combination index values were obtained for the Ba/F3 Flt3-ITD-resitant cells treated with an inhibitor of NRF2 and an FLT3 inhibitor, quizartinib. ( E ) Prolonged survival upon combination of brusatol and quaizartinib in AML: overall survival of AML cell line xenograft-0derived AML-CDX. Briefly, 0.5 × 10 6 MOLM13 cells in 200 µL of PBS were injected via the lateral tail vein in 250 Gy irradiated 5–6-week-old NSG-SGM3 mice ( n = 7 per group). Engraftment of cells were monitored on a weekly basis through tail vein bleeding and staining of MOLM13 cells using human CD45 (hCD45) vs. mouse CD45 (mCD45) markers. When peripheral leukemic burden started showing, i.e., 0.5 to 2% treatment was started, Brusatol 2 mg/kg, 3 times per week, i.v.; quizartinib 10 mg/kg, 3 times per week, p.o. treatment was started. Each treatment was ended when individual mice reach moribund and survival data were recorded for interpretation. ( F ) In vivo efficacy of quizartinib (AC220), a uniquely potent and selective inhibitor of FLT3 and brusatol, an NRF2 inhibitor for the treatment of AML-PDX measured by bioluminescence imaging (BLI). Treatment schedules and doses of drug administered are mentioned in ( E ). Violin plots illustrate the individual whole mouse per each group responding to treatments over time. BLI represents bioluminescence intensity for each treatment group (p/s, 10 5 ). ( G ) Human HO-1 gene expression was measured in mRNA extracts from mouse spleen treated with quizartinib or brusatol or combination of quizartinib and brusatol. Statistical significance is shown as p -values < 0.05 and 0.01 (* p < 0.05; ** p < 0.01).

Article Snippet: HO-1 siRNA (purchased from Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-35555) was transfected into all cell lines using lipofectamine RNAiMAX (Life Technologies, Waltham, MA, USA), and Nucleofector Kit (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. shRNA Nrf2 (Cat#: TG311194) and OriGene retroviral particles were transduced using protocol published earlier [ ].

Techniques: Expressing, Software, Injection, Irradiation, Staining, In Vivo, Imaging

Journal: Antioxidants

Article Title: Targeting the NRF2/HO-1 Antioxidant Pathway in FLT3-ITD-Positive AML Enhances Therapy Efficacy

doi: 10.3390/antiox11040717

Figure Lengend Snippet: Pharmacologic inhibition of NRF2 potentiates chemotherapy mediated anti-leukemia activity in AML. ( A ) MOLM13 and MOLM13-TKIR cells were used for shNRF2 knockdown, treated with standard chemotherapy combination for 48 h in vitro, annexin-V FITC with PI staining was used for flow cytometry analysis. ( B ) Normal PBMC and primary AML samples treated with brusatol, BSO, DOX and their combinations for 48 h, annexin-V FITC with PI staining was used for flow cytometry analysis ex vivo. ( C ) ROS (total-cellular) levels in BM from AML-1-PDX was measured after targeted antioxidant inhibition using BSO, and brusatol, and chemotherapy treatment using DCF based flow cytometry ex vivo. ( D ) In vitro analysis showing mean fluorescence intensity (MFI) of mitochondrial-ROS levels in BM from AML-PDX, and MOLM13, and MOLM13-TKIR xenografted models are shown. Study cohorts treated with targeted antioxidant inhibition (brusatol) and chemotherapy (daunorubicin) were used for BM-Mitosox (5 µM/30 min incubation at 37 °C) staining, and fluorescence measured via flow cytometry. ( E ) Kaplan–Meier survival analysis from AML-PDX cohorts. ( F ) NSG-SGM3 mice engrafted with primary AML patients cells (AML-1, AML-13; 2 × 10 6 cells were injected and mice with 2–5% of leukemic cells in peripheral blood (reflecting 20–30% tumor burden in bone marrow, observed in the third from initial injection) were treated, beginning 2 weeks after initial tail vein injection, with vehicle (control), brusatol (2 mg/kg), daunorubicin (2 mg/kg) by gavage thrice per week on alternative days for 2 weeks ( n = 10 mice per group) and continuously monitored for 2 additional weeks. Mice were euthanized between weeks 4 and 5 due to features of moribundity such as hunched posture or hind leg paralysis after beginning treatment. ( F - G ) Leukemia burden was measured in the peripheral blood ( F ) or bone marrow ( G ) by hCD45 staining. Note: Biologically independent samples were plated in triplicates (as a technical replicates) but statistics were performed only on biologically independent samples, except panel A, B, and C). Statistical significance is shown by p -values less than 0.05 and 0.01 (* p < 0.05; ** p < 0.01) for the study arms or groups compared.

Article Snippet: HO-1 siRNA (purchased from Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-35555) was transfected into all cell lines using lipofectamine RNAiMAX (Life Technologies, Waltham, MA, USA), and Nucleofector Kit (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. shRNA Nrf2 (Cat#: TG311194) and OriGene retroviral particles were transduced using protocol published earlier [ ].

Techniques: Inhibition, Activity Assay, In Vitro, Staining, Flow Cytometry, Ex Vivo, Fluorescence, Incubation, Injection