Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Diabetic uterus environment may play a key role in alterations of DNA methylation of several imprinted genes at mid-gestation in mice

doi: 10.1186/1477-7827-11-119

Figure Lengend Snippet: Oligonucleotides and annealing temperature utilized for qAMP and qRT-PCR of imprinted genes

Article Snippet: The embedded placentas were sectioned at 8 μm and incubated with rabbit anti-Peg3 (1:500, Bioss, China, code: bs-1870R) overnight at 4°C.

Techniques: Sequencing

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Diabetic uterus environment may play a key role in alterations of DNA methylation of several imprinted genes at mid-gestation in mice

doi: 10.1186/1477-7827-11-119

Figure Lengend Snippet: Quantitative DNA methylation in DMRs of imprinted genes in 10.5dpc placentas as revealed by qAMP. Diabetic (n = 26 from 5 litters) and nondiabetic (n = 25 from 5 litters) placentas at mid-gestation were recovered. (A) The paternally methylated H19 ; (B, C) Percentage methylation values at single and groups of restriction sites in the maternally methylated DMRs of Snrpn and Peg3. The lines represent the DMRs which were analyzed and the letter indicates the recognition sites of the enzymes. M, McrBc; Hh, HhaI; Hp, HpaII. (D) Confirmation of gene methylation level was determined by qAMP to expected values. HhaI and McrBc sites in chr9:106724005–106724149 are unmethylated in the genome. A primer pair flanking these sites was utilized to amplify DNA (NIH 3 T3 mouse genomic DNA and CpG methylated NIH 3 T3 mouse genomic DNA, NEB) mixed at the ratios: 100:0, 75:25, 55:45, 50:50, 45:55, 0:100. The qAMP values were close to expected values. Data are presented as mean ± SE for each enzyme employed. White bar, non-diabetic group; black bar, diabetic group; *P < 0.05, **P < 0.01.

Article Snippet: The embedded placentas were sectioned at 8 μm and incubated with rabbit anti-Peg3 (1:500, Bioss, China, code: bs-1870R) overnight at 4°C.

Techniques: DNA Methylation Assay, Methylation

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Diabetic uterus environment may play a key role in alterations of DNA methylation of several imprinted genes at mid-gestation in mice

doi: 10.1186/1477-7827-11-119

Figure Lengend Snippet: Average DNA methylation levels in DMRs of imprinted genes in 10.5dpc fetus as revealed by qAMP. Diabetic (n = 12 from 4 litters) and control (n = 12 from 4 litters) fetus were collected at 10.5dpc of gestation. DNA was digested with Hha1 (Hh), HpaII (Hp) or McrBC (M) and amplified using real-time PCR. The locations of flanked restriction sites were displayed for each DMR. (A) Shown are the average methylation levels of paternally methylated gene H19 DMR; (B, C) represented the DNA methylation status in DMRs of Snrpn and Peg3 . Data were presented as mean ± SE for each enzyme employed. White bar, non-diabetic group; black bar, diabetic group.

Article Snippet: The embedded placentas were sectioned at 8 μm and incubated with rabbit anti-Peg3 (1:500, Bioss, China, code: bs-1870R) overnight at 4°C.

Techniques: DNA Methylation Assay, Amplification, Real-time Polymerase Chain Reaction, Methylation

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Diabetic uterus environment may play a key role in alterations of DNA methylation of several imprinted genes at mid-gestation in mice

doi: 10.1186/1477-7827-11-119

Figure Lengend Snippet: Analysis of the mRNA and protein expression levels of imprinted genes in 10.5dpc placentas and fetus. (A, B) Placentas (n = 30 from 6 litters for each group) and fetus (n = 12 from 4 litters for each group) were collected at 10.5dpc of gestation. Total RNA was purified and reverse transferred into cDNA and then amplified using qRT-PCR. (A) Relative expression levels of H19, Snrpn and Peg3 in placentas. (B) Relative expression levels of H19, Snrpn and Peg3 in fetus. (C) The protein expression of Peg3 in placentas (n = 6) was investigated by western blot analysis and (D) the relative intensity of Peg3/beta-actin was evaluated by gel level analysis. (E) Placentas (n = 6) from diabetic and non-diabetic groups were stained by anti-Peg3 at 1:500 for histological analysis. Scale bar, 100 μm. Data are presented as mean ± SD. White bar, non-diabetic group; black bar, diabetic group; *P < 0.05, **P < 0.01.

Article Snippet: The embedded placentas were sectioned at 8 μm and incubated with rabbit anti-Peg3 (1:500, Bioss, China, code: bs-1870R) overnight at 4°C.

Techniques: Expressing, Purification, Amplification, Quantitative RT-PCR, Western Blot, Staining

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Diabetic uterus environment may play a key role in alterations of DNA methylation of several imprinted genes at mid-gestation in mice

doi: 10.1186/1477-7827-11-119

Figure Lengend Snippet: The methylation level and expression of Peg3 in different litters. (A, B) the methylation level of Peg3 in different litters (n = 6 litters) at McrBc and HpaII was analyzed by qAMP; (C) the expression of Peg3 in s2, s3, s5 and s6 was evaluated by qRT-PCR. White bar, nondiabetic; black bar, diabetic; number and letter under X-axis, litter; *P < 0.05; **P < 0.01.

Article Snippet: The embedded placentas were sectioned at 8 μm and incubated with rabbit anti-Peg3 (1:500, Bioss, China, code: bs-1870R) overnight at 4°C.

Techniques: Methylation, Expressing, Quantitative RT-PCR

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Diabetic uterus environment may play a key role in alterations of DNA methylation of several imprinted genes at mid-gestation in mice

doi: 10.1186/1477-7827-11-119

Figure Lengend Snippet: DNA methylation and expression levels of Peg3 in placentas at mid-gestation. (A, B) Non-diabetic pronuclear embryos were transferred to normal/diabetic (NN/ND) pseudopregnant female and the DNA methylation and expression levels of Peg3 (n, NN:ND = 12:15) in 10.5d placentas were analyzed by qAMP and qRT-PCR, respectively. (A) methylation level of Peg3 in dpc10.5 placenta; (B) expression of Peg3 in placenta. (C, D) DN’s (n = 15 from 4 litters) and NN’s (n = 15 from 4 litters) placentas were collected at mid-gestation. DNA was digested with HpaII or McrBC and amplified using real-time PCR. (C) the methylation in DMRs of Peg3 in placentas; (D) Relative expression levels of Peg3 in placentas. Data were presented as mean ± SD. *P < 0.05, **P < 0.01.

Article Snippet: The embedded placentas were sectioned at 8 μm and incubated with rabbit anti-Peg3 (1:500, Bioss, China, code: bs-1870R) overnight at 4°C.

Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Methylation, Amplification, Real-time Polymerase Chain Reaction

Journal: Oncology Reports

Article Title: HMGB1 promotes the development of castration-resistant prostate cancer by regulating androgen receptor activation

doi: 10.3892/or.2022.8412

Figure Lengend Snippet: Roles of HMGB1 mRNA in the development of PCa and the correlation with the expression of AR mRNA, analysed using bioinformatics. (A) The levels of HMGB1 mRNA in normal PG and PCa were analysed using the Oncomine and GEPIA databases. (B) The levels of AR mRNA in normal PG and PCa were analysed using the Oncomine and GEPIA databases. (C) The correlation between the expression of HMGB1 mRNA and AR mRNA was detected by the GEPIA database. (D) The levels of AR mRNA and HMGB1 mRNA in VCaP, 22RV1, LNCaP, and MDAPCA2B cells were analysed using the DepMap Portal database. HMGB1, high-mobility group protein B1; PCa, prostate cancer; AR, androgen receptor; PG, prostate gland; PRAD, prostate adenocarcinoma.

Article Snippet: Subsequently, the histological sections were stained with rabbit anti-AR antibody (product code ab74272; 1:200; Abcam) and anti-HMGB1 antibody (product code A00066-1; 1:200; Boster Biological Technology, Inc.) at 4°C overnight.

Techniques: Expressing

Journal: Oncology Reports

Article Title: HMGB1 promotes the development of castration-resistant prostate cancer by regulating androgen receptor activation

doi: 10.3892/or.2022.8412

Figure Lengend Snippet: Expression levels of HMGB1 and AR proteins in the specimens of patients with PCa detected by IHC staining. (A) The expression levels of HMGB1 protein in PCa specimens with different Gleason scores. (B) The expression levels of AR protein in PCa specimens with different Gleason scores. (C) The association between HMGB1 protein expression in the specimens with Gleason scores and PSA levels. (D) The association between AR protein expression in the specimens and Gleason scores and PSA levels. (E) The correlation between HMGB1 protein expression and AR protein expression in the specimens of the patients. *P<0.05. HMGB1, high-mobility group protein B1; AR, androgen receptor; PCa, prostate cancer; IHC, immunohistochemical; MOD, mean optical density.

Article Snippet: Subsequently, the histological sections were stained with rabbit anti-AR antibody (product code ab74272; 1:200; Abcam) and anti-HMGB1 antibody (product code A00066-1; 1:200; Boster Biological Technology, Inc.) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, Immunohistochemical staining

Journal: Oncology Reports

Article Title: HMGB1 promotes the development of castration-resistant prostate cancer by regulating androgen receptor activation

doi: 10.3892/or.2022.8412

Figure Lengend Snippet: Correlation of AR and HMGB1 expression with Gleason score, PSA and age.

Article Snippet: Subsequently, the histological sections were stained with rabbit anti-AR antibody (product code ab74272; 1:200; Abcam) and anti-HMGB1 antibody (product code A00066-1; 1:200; Boster Biological Technology, Inc.) at 4°C overnight.

Techniques: Expressing

Journal: Oncology Reports

Article Title: HMGB1 promotes the development of castration-resistant prostate cancer by regulating androgen receptor activation

doi: 10.3892/or.2022.8412

Figure Lengend Snippet: HMGB1 protein activates the AR signalling pathway by directly interacting with AR protein in PCa in vitro. (A) Following transfection with exogenous HMGB1, the expression levels of AR and HMGB1 in LNCaP were detected by western blotting. *P<0.05 compared with pLVX or sh-Scramble. (B) Following transfection with sh-HMGB1, the expression levels of AR and HMGB1 in 22RV1 cells were detected by western blotting. *P<0.05 compared with pLVX or sh-Scramble. (C) The growth abilities of LNCaP-neo, LNCaP-HMGB1, 22RV1-shScramble, and 22RV1-shHMGB1 cells were detected by CCK-8 assay. *P<0.05 compared with the 0-h group. (D) The effects of HMGB1 on the transcription levels of PSA and TMPRSS2 in LNCaP-neo, LNCaP-HMGB1, 22RV1-shScramble, and 22RV1-shHMGB1 cells were examined by RT-qPCR. *P<0.05 compared with pLVX or sh-Scramble. (E) After treatment with pLVX-shAR, AR, HMGB1, PSA, and TMPRSS2 protein expression levels in LNCaP-neo, LNCaP-HMGB1 and LNCaP-HMGB1/shAR cells were detected by western blotting. *P<0.05 compared with the control group; #P<0.05 compared with the pLVX-HMGB1 group. (F) The effects of HMGB1 and AR on the transcription levels of PSA and TMPRSS2 in LNCaP-neo, LNCaP-HMGB1 and LNCaP-HMGB1/shAR cells were examined by RT-qPCR. *P<0.05 compared with the pLVX group; #P<0.05 compared with the pLVX-HMGB1 group. (G) Following transfection with exogenous HMGB1 or sh-HMGB1, the transactivating activity of AR in LNCaP-neo, LNCaP-HMGB1, 22RV1-shScramble, and 22RV1-shHMGB1 cells was determined by a reporter gene assay. *P<0.05 compared with the pLVX group. (H and I) ChIP assay followed by RT-qPCR was used to detect the effect of HMGB1 on the ability of AR to bind to the promoters of PSA and TMPRSS2 in LNCaP and 22RV1 cells. *P<0.05 compared with the pLVX group or sh-Scramble group. (J) AR-Turbo and HMGB1-Rluc, the BRET fusion constructs, were cotransfected into PC3 cells, and the BRET signal was measured after the addition of the coelenterazine substrate. Western blotting revealed the fold changes of the expression levels of the fusion proteins. *P<0.05 compared with the AR-Turbo/HMGB1-Rluc ratio=0 group. (K) A schematic of the principle of the BRET assay. HMGB1, high-mobility group protein B1; AR, androgen receptor; PCa, prostate cancer; PSA, prostate specific antigen; TMPRSS2, transmembrane protease, serine 2; RT-qPCR, reverse transcription-quantitative PCR; ChIP, chromatin immunoprecipitation; BRET, bioluminescence resonance energy transfer.

Article Snippet: Subsequently, the histological sections were stained with rabbit anti-AR antibody (product code ab74272; 1:200; Abcam) and anti-HMGB1 antibody (product code A00066-1; 1:200; Boster Biological Technology, Inc.) at 4°C overnight.

Techniques: In Vitro, Transfection, Expressing, Western Blot, CCK-8 Assay, Quantitative RT-PCR, Control, Activity Assay, Reporter Gene Assay, Construct, Bioluminescence Resonance Energy Transfer, Reverse Transcription, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: Oncology Reports

Article Title: HMGB1 promotes the development of castration-resistant prostate cancer by regulating androgen receptor activation

doi: 10.3892/or.2022.8412

Figure Lengend Snippet: HMGB1 promotes the development of prostate cancer in vivo. (A-C) HMGB1 significantly increased the volume and weight of tumours in a LNCaP xenograft model. (D) The effect of HMGB1 on the level of PSA in mouse serum was examined by ELISA. (E) HMGB1 protein expression in mouse tumour tissues detected by IHC. (F) AR protein expression in mouse tumour tissues detected by IHC. *P<0.05. HMGB1, high-mobility group protein B1; PSA, prostate specific antigen; IHC, immunohistochemical; AR, androgen receptor; MOD, mean optical density.

Article Snippet: Subsequently, the histological sections were stained with rabbit anti-AR antibody (product code ab74272; 1:200; Abcam) and anti-HMGB1 antibody (product code A00066-1; 1:200; Boster Biological Technology, Inc.) at 4°C overnight.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Expressing, Paraffin-embedded Immunohistochemistry, Immunohistochemical staining