Journal: Theranostics

Article Title: Tamoxifen activates Nrf2-dependent SQSTM1 transcription to promote endometrial hyperplasia

doi: 10.7150/thno.19135

Figure Lengend Snippet: TAM activated SQSTM1 transcription in a Nrf2-dependent manner. A, SQSTM1 mRNA levels in RL95-2 (left), AN3CA (middle) or MCF7 (right) cells incubated with different concentrations of 4-OH-TAM for 24 hours were measured by qRT-PCR. B, ChIP-PCR experiment was performed to detect the binding of Nrf2 to SQSTM1 promoter in AN3CA cells before and after 4 hours of 4-OH-TAM (20μM) treatment. IgG was used as the negative control. The experiments were performed in triplicate and repeated three times. Representative results from one triplicated experiment were shown as Mean±SD. Student's T test was used for the statistical analysis. The Asterisk indicates the statistical significance. C, p62-pro/pGL2 or pGL2 vector were co-transfected with Nrf2 plasmid or vector in AN3CA cells. Forty-eight hours later, SQSTM1 promoter activity was measured by dual luciferase reporter assay. The experiments were repeated three times. Representative results from one triplicated experiment were shown as Mean±SD. Student's T test was used for the statistical analysis. The Asterisk indicates the statistical significance. D, Forty-eight hours after transfection of Nrf2 siRNAs, 4-OH-TAM (15 μM) was added for another 24 hours, and the effect of 4-OH-TAM on SQSTM1 and Nrf2 protein expression in AN3CA cells transfected with or without Nrf2 siRNAs were analyzed by Western blotting. E, Effects of 4-OH-TAM (as in D) on SQSTM1 mRNA expression in AN3CA cells transfected with or without Nrf2 siRNAs were analyzed by qRT-PCR. The experiments were repeated three times. Representative results from one triplicated experiment were shown as Mean±SD. Student's T test was used for the statistical analysis. The Asterisk indicates the statistical significance. F, Effects of 4-OH-TAM on growth of AN3CA cells transfected with or without Nrf2 knockdown were analyzed by MTS assay. Forty-eight hours after transfection of Nrf2 siRNAs, various concentrations of 4-OH-TAM were added for another 72 hours. Two way ANOVA test was used to compare the statistical difference (NC vs siNrf1 #1, p<0.01; NC vs siNrf1 #1, p<0.01). The experiments were repeated three time. Representative results from one triplicated experiment were shown as Mean±SD. G, Nrf2 inhibitor brusatol (Bru) (40 or 80 nM) was added together with 4-OH-TAM (15 μM) for 24 hours in AN3CA cells. SQSTM1 expression was determined by western blotting. H, SQSTM1 mRNA expression in AN3CA cells treated with brusatol (Bru) and 4-OH-TAM incubation (as in G) were analyzed by qRT-PCR. The results were shown as in E. I, The growth of AN3CA cells treated with various concentrations of brusatol (Bru) and 4-OH-TAM (15 μM) incubation were explored by MTS assay. The experiments were repeated three times. Representative results from one triplicated experiment were shown as Mean±SD.

Article Snippet: Anti-Nrf2 antibody was purchased from Proteintech (Wuhan, China), anti-Nrf2 (phospho S40) antibody and anti-Histone H3 were purchased from Abcam (Shanghai, China), anti-GLS antibody was purchased from Abgent (Suzhou, China), anti-PKC delta antibody was purchased from BD biosciences (Shanghai, China), anti-phospho PKC delta (Thr505) and anti-Actin antibody were purchased from Cell Signaling Technology (Shanghai, China).

Techniques: Incubation, Quantitative RT-PCR, Binding Assay, Negative Control, Plasmid Preparation, Transfection, Activity Assay, Luciferase, Reporter Assay, Expressing, Western Blot, MTS Assay

Journal: Theranostics

Article Title: Tamoxifen activates Nrf2-dependent SQSTM1 transcription to promote endometrial hyperplasia

doi: 10.7150/thno.19135

Figure Lengend Snippet: TAM activated Nrf2 specifically in endometrial cells. A, Nrf2 expression in RL95-2, AN3CA or MCF7 cells incubated with or without 4-OH-TAM (15 μM) for 24 hours were analyzed by western blotting. B, RL95-2, AN3CA or MCF7 cells were incubated with or without 4-OH-TAM (15 μM) for 24 hours. Nrf2 distributions in nuclear and cytoplasmic extracts were explored by western blotting. GLS1 was used as the cytoplasmic protein marker while H3 (Histone H3) was used as the nuclear protein marker. C, NQO1 mRNA level in RL95-2, AN3CA or MCF7 cells incubated with or without 4-OH-TAM (15 μM) for 24 hours were analyzed with qRT-PCR. The experiments were repeated three times. Representative results from one triplicated experiment were shown as Mean±SD. Student's T test was used for the statistical analysis. The asterisk indicates the statistical significance and the octothorpe stands for no significant difference. D, Forty-eight hours after transfection of SQSTM1 siRNAs, 4-OH-TAM (15 μM) was added for another 24 hours, the effect of 4-OH-TAM on Nrf2 expression in RL95-2 and AN3CA cells with or without SQSTM1 knockdown were explored by western blotting. E, Forty-eight hours after transfection of Flag-SQSTM1/Vector, Nrf2 expression in RL95-2 (left) or AN3CA (right) cells were detected by western blotting. F, Nrf2 distribution in nuclear and cytoplasmic extracts of RL95-2 (left) or AN3CA (right) cells with or without Flag-SQSTM1 over-expression were detected by western blotting.

Article Snippet: Anti-Nrf2 antibody was purchased from Proteintech (Wuhan, China), anti-Nrf2 (phospho S40) antibody and anti-Histone H3 were purchased from Abcam (Shanghai, China), anti-GLS antibody was purchased from Abgent (Suzhou, China), anti-PKC delta antibody was purchased from BD biosciences (Shanghai, China), anti-phospho PKC delta (Thr505) and anti-Actin antibody were purchased from Cell Signaling Technology (Shanghai, China).

Techniques: Expressing, Incubation, Western Blot, Marker, Quantitative RT-PCR, Transfection, Plasmid Preparation, Over Expression

Journal: Theranostics

Article Title: Tamoxifen activates Nrf2-dependent SQSTM1 transcription to promote endometrial hyperplasia

doi: 10.7150/thno.19135

Figure Lengend Snippet: Expression of Nrf2 and SQSTM1 in clinical endometrial tissues. A, Represented images of Nrf2 or SQSTM1 expression in primary endometrial tissues. B, Summary of Nrf2 and SQSTM1 expression in normal and hyperplasia endometrial tissues. C, Summary of Nrf2 and SQSTM1 expression in endometrial tissues from cases received TAM treatment or not. D, Correlation of SQSTM1 and Nrf2 expression. Chi-Square test was used for the statistical analysis of staining results.

Article Snippet: Anti-Nrf2 antibody was purchased from Proteintech (Wuhan, China), anti-Nrf2 (phospho S40) antibody and anti-Histone H3 were purchased from Abcam (Shanghai, China), anti-GLS antibody was purchased from Abgent (Suzhou, China), anti-PKC delta antibody was purchased from BD biosciences (Shanghai, China), anti-phospho PKC delta (Thr505) and anti-Actin antibody were purchased from Cell Signaling Technology (Shanghai, China).

Techniques: Expressing, Staining

Journal: Theranostics

Article Title: Tamoxifen activates Nrf2-dependent SQSTM1 transcription to promote endometrial hyperplasia

doi: 10.7150/thno.19135

Figure Lengend Snippet: TAM activated Nrf2-SQSTM1 axis to promote endometrial hyperplasia in rats. A, TAM (10 mg/kg) was given daily to rats 3 days after ovariectomy to induce endometrial hyperplasia. Nrf2 inhibitor brusatol (Bru) (1 mg/kg) was given biweekly 5 days after ovariectomy. B, Representative HE staining of endometrium from rats treated with DMSO or TAM or TAM+Bru. C, The thicknesses of endometrium from 3 groups of rats treated with DMSO or TAM or TAM+Bru were summarized. The results of 5 mice in each group were shown as Mean±SD. Asterisks indicate statistical difference (Student's t test, p<0.05). D, Expressions of SQSTM1 mRNA in endometrium from rats were analyzed by qRT-PCR. Representative results from one triplicated experiment were shown as Mean±SD. Student's T test was used for the statistical analysis. The Asterisk indicates the statistical significance. E, Expressions of Nrf2 and SQSTM1 in endometrium from various rats treated as indicated were detected by Western blotting.

Article Snippet: Anti-Nrf2 antibody was purchased from Proteintech (Wuhan, China), anti-Nrf2 (phospho S40) antibody and anti-Histone H3 were purchased from Abcam (Shanghai, China), anti-GLS antibody was purchased from Abgent (Suzhou, China), anti-PKC delta antibody was purchased from BD biosciences (Shanghai, China), anti-phospho PKC delta (Thr505) and anti-Actin antibody were purchased from Cell Signaling Technology (Shanghai, China).

Techniques: Staining, Quantitative RT-PCR, Western Blot

Journal: Theranostics

Article Title: Tamoxifen activates Nrf2-dependent SQSTM1 transcription to promote endometrial hyperplasia

doi: 10.7150/thno.19135

Figure Lengend Snippet: TAM activated Nrf2 through phosphorylation at ser40. A, Two or four hours after 4-OH-TAM (20 μM) incubation, nuclear and cytoplasmic distributions of Nrf2 in RL95-2 and AN3CA cells were explored by western blotting. B, Ser40 phosphorylation of Nrf2 (p-Nrf2) and total Nrf2 levels in RL95-2 and AN3CA cells after 1 hour 4-OH-TAM (20 μM) incubation were determined by western blotting. C, Nuclear and cytoplasmic distributions of p-Nrf2 in RL95-2 and AN3CA cells treated with or without 1-hour treatment of 4-OH-TAM (20 μM) were analyzed by western blotting. D, P62-pro/pGL2 or pGL2 vector were co-transfected with Nrf2 wild type (Nrf2-WT) or S40 phosphorylation deficient mutant (Nrf2-S40A) or S40 phosphorylation mimic mutant (Nrf2-S40E) or vector in AN3CA cells. Forty-eight hours later, the effects of wild type or mutated Nrf2 on SQSTM1 promoter activity were determined by luciferase activity assay. The experiments were performed in triplicate and repeated three times. Representative results were shown as Mean±SD. Asterisks indicate statistical significance (Student's T test, p<0.05). E, Forty-eight hours after Nrf2-WT or Nrf2-S40A or Nrf2-S40E or vector transfection in AN3CA cells, the expression and nuclear distribution of wild type or mutated Nrf2 and SQSTM1 protein expression were analyzed by western blotting. F, Nrf2-WT or Nrf2-S40A was transfected into AN3CA cells for 48 hours. Nuclear distributions of Nrf2-WT or Nrf2-S40A in AN3CA cells before and after 2h incubation of 4-OH-TAM (20 μM) were explored by western blotting.

Article Snippet: Anti-Nrf2 antibody was purchased from Proteintech (Wuhan, China), anti-Nrf2 (phospho S40) antibody and anti-Histone H3 were purchased from Abcam (Shanghai, China), anti-GLS antibody was purchased from Abgent (Suzhou, China), anti-PKC delta antibody was purchased from BD biosciences (Shanghai, China), anti-phospho PKC delta (Thr505) and anti-Actin antibody were purchased from Cell Signaling Technology (Shanghai, China).

Techniques: Incubation, Western Blot, Plasmid Preparation, Transfection, Mutagenesis, Activity Assay, Luciferase, Expressing

Journal: Theranostics

Article Title: Tamoxifen activates Nrf2-dependent SQSTM1 transcription to promote endometrial hyperplasia

doi: 10.7150/thno.19135

Figure Lengend Snippet: TAM activated PRKCD to activate Nrf2-SQSTM1 axis. A, Phosphorylation of PRKCD (p-PRKCD) and total PRKCD expression in RL95-2 and AN3CA cell upon 4-OH-TAM treatment at different times were analyzed by western blotting. B, Forty-eight hours after PRKCD siRNAs transfection, 4-OH-TAM (20 μM) was added for another 1 hour. The effect of PRKCD depletion on 4-OH-TAM-induced phosphorylation and expression of Nrf2 were explored by western blotting. The effects of PRKCD depletion on 4-OH-TAM induced SQSTM1 mRNA ( C ) and protein ( D ) expression were explored by qRT-PCR or western blotting, respectively. RT-PCR results were shown as Mean±SD. Asterisks indicate statistical significance (Student's T test, p<0.05, n=3). E, Working model: Tamoxifen activated PRKCD to promote Nrf2 S40 phosphorylation. Phosphorylated Nrf2 translocated to the nucleus and stimulate SQSTM1 transcription to promote endometrial hyperplasia, eventually leading to endometrial tumorigenesis.

Article Snippet: Anti-Nrf2 antibody was purchased from Proteintech (Wuhan, China), anti-Nrf2 (phospho S40) antibody and anti-Histone H3 were purchased from Abcam (Shanghai, China), anti-GLS antibody was purchased from Abgent (Suzhou, China), anti-PKC delta antibody was purchased from BD biosciences (Shanghai, China), anti-phospho PKC delta (Thr505) and anti-Actin antibody were purchased from Cell Signaling Technology (Shanghai, China).

Techniques: Expressing, Western Blot, Transfection, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction