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OriGene dnmt1 shrna vector
Dnmt1 Shrna Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/dnmt1+shrna+vector/DNMT1+(NM_001130823)+Human+Tagged+ORF+Clone/pm38446659-799-13-23
Average 92 stars, based on 3 article reviews

dnmt1 shrna vector - by Bioz Stars, 2026-09

92/100 stars

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shRNA:

Article Title: Cellular senescence is associated with the spatial evolution toward a higher metastatic phenotype in colorectal cancer.
Article Snippet: To introduce vectors and small interfering RNAs (siRNAs) into cells, cells were transfected with vectors or siRNA duplexes using FuGENEHD (E2311; Promega, Madison, WI) or Oligofectamine Reagent (12252011; Invitrogen, Waltham, MA) for 2 days, respectively. .. The LAMC2 vector [pDNA(VB900003-3865dfu)], theMMP7 vector (RC201225), the DNMT1 vector (RG226414), and the DNMT1 shRNA vector were purchased from VectorBuilder, Inc. (Chicago, IL), OriGene Technologies, Inc. (Rockville, MD), OriGene Technologies, Inc., and Sigma-Aldrich, respectively. .. To generate DNMT1-knocked down stable cell line, DNMT1 shRNA vector (TRCN0000232751 and TRCM0000021893, Sigma-Aldrich)-transfected cells were selected with 7.5 mg/mL puromycin (Sigma-aldrich) for 2 weeks.

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High expression of DNMT1 promotes DNA methylation of the SOX21 promoter. A Prediction of CpG islands in the SOX21 promoter region using the MethPrimer database. B The methylation of the SOX21 promoter region was predicted on the Mexpress database. C The correlation between DNMT3A, DNMT1, DNMT3B, DNMT3L, and SOX21 methylation levels in GC from the Meth450 platform in the LinkOmics database using Spearman analysis. D DNMT1 expression was predicted in the STAD-UALCAN database. E The protein expression of DNMT1 in adjacent and tumor tissues in GC patients was examined using western blot analysis ( n = 13). F The protein expression of DNMT1 in GC cells and GES-1 cells was examined using western blot analysis. G The mRNA expression of DNMT1 in GC cells infected with shRNAs targeting DNMT1 was examined using RT-qPCR. H The protein expression of DNMT1 in GC cells infected with shRNAs targeting DNMT1 was examined using western blot analysis. I The methylation level of the SOX21 promoter in GC cells infected with sh-NC or sh-DNMT1 #2 was examined using the MSP assay. J The binding relation between DNMT1 and the SOX21 promoter was verified using a ChIP assay. K The binding relation between DNMT1 and the SOX21 promoter was examined using luciferase assays. L The mRNA expression of SOX21 in GC cells infected with sh-DNMT1 #2 was examined using RT-qPCR. M The protein expression of SOX21 in GC cells infected with sh-DNMT1 #2 was examined using western blot analysis. Paired or unpaired t-tests were used to compare the data between two groups, and ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means of three independent experiments

Journal: BMC Cancer

Article Title: DNMT1 blocks SOX21-repressed CKS2 transcription to promote gastric cancer progression

doi: 10.1186/s12885-025-14577-z

Figure Lengend Snippet: High expression of DNMT1 promotes DNA methylation of the SOX21 promoter. A Prediction of CpG islands in the SOX21 promoter region using the MethPrimer database. B The methylation of the SOX21 promoter region was predicted on the Mexpress database. C The correlation between DNMT3A, DNMT1, DNMT3B, DNMT3L, and SOX21 methylation levels in GC from the Meth450 platform in the LinkOmics database using Spearman analysis. D DNMT1 expression was predicted in the STAD-UALCAN database. E The protein expression of DNMT1 in adjacent and tumor tissues in GC patients was examined using western blot analysis ( n = 13). F The protein expression of DNMT1 in GC cells and GES-1 cells was examined using western blot analysis. G The mRNA expression of DNMT1 in GC cells infected with shRNAs targeting DNMT1 was examined using RT-qPCR. H The protein expression of DNMT1 in GC cells infected with shRNAs targeting DNMT1 was examined using western blot analysis. I The methylation level of the SOX21 promoter in GC cells infected with sh-NC or sh-DNMT1 #2 was examined using the MSP assay. J The binding relation between DNMT1 and the SOX21 promoter was verified using a ChIP assay. K The binding relation between DNMT1 and the SOX21 promoter was examined using luciferase assays. L The mRNA expression of SOX21 in GC cells infected with sh-DNMT1 #2 was examined using RT-qPCR. M The protein expression of SOX21 in GC cells infected with sh-DNMT1 #2 was examined using western blot analysis. Paired or unpaired t-tests were used to compare the data between two groups, and ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means of three independent experiments

Article Snippet: Lentiviral vectors containing shRNAs targeting DNMT1 or SOX21 (Table ), overexpression of SOX21, and overexpression of CKS2 (all from VectorBuilder, Guangzhou, Guangdong, China) were used to infect AGS and NCI-N87 cells, and Polybrene was added (1 μL/mL, C0351-1 ml, Beyotime, Shanghai, China).

Techniques: Expressing, DNA Methylation Assay, Methylation, Western Blot, Infection, Quantitative RT-PCR, MSP Assay, Binding Assay, Luciferase

Silencing of SOX21 abates the anti-proliferative and pro-apoptotic properties of sh-DNMT1 on GC cells. A The mRNA expression of SOX21 in GC cells infected with sh-DNMT1 + shRNAs targeting SOX21 was examined using RT-qPCR. B The protein expression of SOX21 in GC cells infected with sh-DNMT1 + shRNAs targeting SOX21 was examined using western blot analysis. C The EdU-positive GC cells after infection. D The OD value of GC cells was read at 0, 24, 48, and 72 h using the CCK-8 assay. E The migration and invasion of GC cells were examined using the Transwell assay. F Detection of apoptosis in GC cells by TUNEL assay. ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means of three independent experiments

Journal: BMC Cancer

Article Title: DNMT1 blocks SOX21-repressed CKS2 transcription to promote gastric cancer progression

doi: 10.1186/s12885-025-14577-z

Figure Lengend Snippet: Silencing of SOX21 abates the anti-proliferative and pro-apoptotic properties of sh-DNMT1 on GC cells. A The mRNA expression of SOX21 in GC cells infected with sh-DNMT1 + shRNAs targeting SOX21 was examined using RT-qPCR. B The protein expression of SOX21 in GC cells infected with sh-DNMT1 + shRNAs targeting SOX21 was examined using western blot analysis. C The EdU-positive GC cells after infection. D The OD value of GC cells was read at 0, 24, 48, and 72 h using the CCK-8 assay. E The migration and invasion of GC cells were examined using the Transwell assay. F Detection of apoptosis in GC cells by TUNEL assay. ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means of three independent experiments

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Migration, Transwell Assay, TUNEL Assay

DNMT1/SOX21/CKS2 axis is involved in GC progression in vivo. A The representative images of subcutaneous xenograft tumors in nude mice and the tumor growth curve within 25 d. B The weight of tumors formed by AGS cells with sh-NC, sh-DNMT1 #2 + sh-NC, sh-DNMT1 #2 + sh-SOX21 #3, oe-SOX21 + oe-NC, or oe-SOX21 + oe-CKS2 at day 25. C DNMT1, SOX21, and CKS2 expression in the tumor tissues was examined using western blot analysis. D The representative immunohistochemical images and positive cells of Ki67 and cleaved-caspase-3 in the tumor tissues formed by AGS cells. ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means ( n = 6)

Journal: BMC Cancer

Article Title: DNMT1 blocks SOX21-repressed CKS2 transcription to promote gastric cancer progression

doi: 10.1186/s12885-025-14577-z

Figure Lengend Snippet: DNMT1/SOX21/CKS2 axis is involved in GC progression in vivo. A The representative images of subcutaneous xenograft tumors in nude mice and the tumor growth curve within 25 d. B The weight of tumors formed by AGS cells with sh-NC, sh-DNMT1 #2 + sh-NC, sh-DNMT1 #2 + sh-SOX21 #3, oe-SOX21 + oe-NC, or oe-SOX21 + oe-CKS2 at day 25. C DNMT1, SOX21, and CKS2 expression in the tumor tissues was examined using western blot analysis. D The representative immunohistochemical images and positive cells of Ki67 and cleaved-caspase-3 in the tumor tissues formed by AGS cells. ANOVA and Tukey’s post hoc test were used to compare the data between multiple groups. Data are expressed as means ± standard errors of the means ( n = 6)

Techniques: In Vivo, Expressing, Western Blot, Immunohistochemical staining

Epigenetic modifications of the 5’-flanking region of the cysteine synthetic genes in the hepatic tumors. A and B , The mRNA levels of the enzymes involved in cysteine synthesis ( A ) and DNA methyltransferases ( B ) in the liver and BNL 1ME A.7 R.1-formed tumors in mice. The expression levels were normalized to those of 18s . The values in the liver were set at 1.0. Each value represents the mean with S.D. ( n = 6). ** P < 0.01; significant difference between the two groups ( t 10 = − 15.397, P < 0.001 for Cbs ; t 10 = − 7.424, P < 0.001 for Cth ; t 10 = − 3.258, P = 0.009 for Dnmt1 ; t 10 = 5.041, P = 0.001 for Dnmt3a ; t 10 = − 3.438, P = 0.006 for Dnmt3b ; unpaired t -test, two sided). C and D , Methylation status of the 5’-flanking region in mice Cbs (C) and Cth (D) genes in the BNL 1ME A.7 R.1-formed tumors. Left panels show representative electropherograms of direct-bisulfite sequencing. Triangles indicate the methylation sites. Right panels show the quantification of methylation levels. Each value represents the mean with S.D. ( n = 4). ** P < 0.01, * P < 0.05; significant difference between the two groups (unpaired t -test, two sided)

Journal: Cancer & Metabolism

Article Title: Epigenetic repression of de novo cysteine synthetases induces intra-cellular accumulation of cysteine in hepatocarcinoma by up-regulating the cystine uptake transporter xCT

doi: 10.1186/s40170-024-00352-4

Figure Lengend Snippet: Epigenetic modifications of the 5’-flanking region of the cysteine synthetic genes in the hepatic tumors. A and B , The mRNA levels of the enzymes involved in cysteine synthesis ( A ) and DNA methyltransferases ( B ) in the liver and BNL 1ME A.7 R.1-formed tumors in mice. The expression levels were normalized to those of 18s . The values in the liver were set at 1.0. Each value represents the mean with S.D. ( n = 6). ** P < 0.01; significant difference between the two groups ( t 10 = − 15.397, P < 0.001 for Cbs ; t 10 = − 7.424, P < 0.001 for Cth ; t 10 = − 3.258, P = 0.009 for Dnmt1 ; t 10 = 5.041, P = 0.001 for Dnmt3a ; t 10 = − 3.438, P = 0.006 for Dnmt3b ; unpaired t -test, two sided). C and D , Methylation status of the 5’-flanking region in mice Cbs (C) and Cth (D) genes in the BNL 1ME A.7 R.1-formed tumors. Left panels show representative electropherograms of direct-bisulfite sequencing. Triangles indicate the methylation sites. Right panels show the quantification of methylation levels. Each value represents the mean with S.D. ( n = 4). ** P < 0.01, * P < 0.05; significant difference between the two groups (unpaired t -test, two sided)

Article Snippet: Small hairpin RNA (shRNA) expressing vectors against the mouse Slc7a11 , Dnmt1 , Dnmt3a , or Dnmt3b gene were purchased from VectorBuilder (Chicago, IL).

Techniques: Expressing, Methylation, Methylation Sequencing

DNA methyltransferase suppresses the expression of cysteine synthetic enzymes in BNL 1ME A.7 R.1 cells. A and B , Induction of CBS and CTH expression by pharmacological inhibition of DNA methyltransferase activity. BNL 1ME A.7 R.1 cells were treated with 500 nM decitabine for 24 h. The mRNA and protein levels were normalized to those of 18s and β-ACTIN, respectively. The values in vehicle-treated cells were set at 1.0. Each value represents the mean with S.D. ( n = 4). * P < 0.05; significant difference between the two groups ( t 6 = 2.719, P = 0.035 for Cth mRNA; t 6 = 3.498, P = 0.013 for CBS protein; t 6 = 3.270, P = 0.017 for CTH protein; unpaired t -test, two sided). C and D , Induction of CBS and CTH expressions by down-regulation of DNA methyltransferase. BNL 1ME A.7 R.1 cells were transduced with lentivirus expressing shRNA against Dnmt1 , Dnmt3a , or Dnmt3b . The mRNA and protein levels in mock-transduced and Dnmt -knockdown (KD) cells were normalized to those of 18s and β-ACTIN, respectively. The values in mock-transduced cells were set at 1.0. Each value represents the mean with S.D. ( n = 3). ** P < 0.01, * P < 0.05; significant difference between the indicated groups ( F 3,8 = 160.673, P < 0.001 for Cbs mRNA; F 3,8 = 13.969, P = 0.002 for Cth mRNA; F 3,8 = 13.491, P = 0.002 for CBS protein; F 3,8 = 24.930, P < 0.001 for CTH protein; ANOVA with Tukey–Kramer’s post hoc test)

Journal: Cancer & Metabolism

Article Title: Epigenetic repression of de novo cysteine synthetases induces intra-cellular accumulation of cysteine in hepatocarcinoma by up-regulating the cystine uptake transporter xCT

doi: 10.1186/s40170-024-00352-4

Figure Lengend Snippet: DNA methyltransferase suppresses the expression of cysteine synthetic enzymes in BNL 1ME A.7 R.1 cells. A and B , Induction of CBS and CTH expression by pharmacological inhibition of DNA methyltransferase activity. BNL 1ME A.7 R.1 cells were treated with 500 nM decitabine for 24 h. The mRNA and protein levels were normalized to those of 18s and β-ACTIN, respectively. The values in vehicle-treated cells were set at 1.0. Each value represents the mean with S.D. ( n = 4). * P < 0.05; significant difference between the two groups ( t 6 = 2.719, P = 0.035 for Cth mRNA; t 6 = 3.498, P = 0.013 for CBS protein; t 6 = 3.270, P = 0.017 for CTH protein; unpaired t -test, two sided). C and D , Induction of CBS and CTH expressions by down-regulation of DNA methyltransferase. BNL 1ME A.7 R.1 cells were transduced with lentivirus expressing shRNA against Dnmt1 , Dnmt3a , or Dnmt3b . The mRNA and protein levels in mock-transduced and Dnmt -knockdown (KD) cells were normalized to those of 18s and β-ACTIN, respectively. The values in mock-transduced cells were set at 1.0. Each value represents the mean with S.D. ( n = 3). ** P < 0.01, * P < 0.05; significant difference between the indicated groups ( F 3,8 = 160.673, P < 0.001 for Cbs mRNA; F 3,8 = 13.969, P = 0.002 for Cth mRNA; F 3,8 = 13.491, P = 0.002 for CBS protein; F 3,8 = 24.930, P < 0.001 for CTH protein; ANOVA with Tukey–Kramer’s post hoc test)

Article Snippet: Small hairpin RNA (shRNA) expressing vectors against the mouse Slc7a11 , Dnmt1 , Dnmt3a , or Dnmt3b gene were purchased from VectorBuilder (Chicago, IL).

Techniques: Expressing, Inhibition, Activity Assay, Transduction, shRNA, Knockdown

Hit validation for 5-aza-dC. (A and B) Confirmation of Screen-seq results for 5-aza-dC-treated cells using an orthogonal method to measure AI. cDNA samples from day 7 of screening were assessed using ddPCR with allele-specific fluorescent probes. (A) Scatterplots for 20,000 droplets targeting the readout gene, Col6a5 . 5-aza-dC concentration is shown in the plots. Black : empty droplets; blue : droplets with the Cast paternal allele amplified (labeled by FAM fluorophore); red : droplets with the 129 maternal allele amplified (labeled by HEX fluorophore). Ratio of red: blue droplets are shown. AI value written in red is the maternal AI. Note that the double-positive droplets (orange) contained both maternal and paternal templates; a small number of such double-positives is expected with higher concentrations of biallelic template. These droplets are ignored in the quantitative analysis. (B) left— summary of AI measurements shown in (A) for Col6a5 on day 7; right— summary of AI measurements for Dnajc12 on day 7. (C and D) Biological replicate of Abl.1 cells were treated with 5-aza-dC and AI was measured using ddPCR. (C) Scatterplots representation as shown in (A) after 2 days of exposure. (D) Summary of AI measurement for Col6a5 (left) and Dnajc12 (right) after 2, 5, and 7 days of exposure (denoted by color). Gray vertical dashed lines for Col6a5 dose-response were used to determine “low,” “medium,” and “high” 5-aza-dC concentrations for the future experiments. Results for readout gene Adnp2 are in . (E–G) Analysis of Dnmt1 knock-down (KD) in Abl.1 cells. (E) Real-time quantitative PCR (RT-qPCR) analysis of Dnmt1 relative expression (expression in the empty vector control, normalized to Nono , taken as 1.0). Abl.1 cells were transduced with an empty plKO vector (control) or with two separate Dnmt1 shRNA knockdown constructs ( Dnmt1 KD construct 1 or 2) and grown for 2 days. Transduced cells were then selected by growing in the presence of a selection antibiotic for an additional 17 days. RT-qPCR quantification was performed on cells collected 19 days after transduction. Mean and SEM for three technical replicates are shown. (F) Representative scatterplots show AI measurement for Col6a5 in the transduced Abl.1 cells. AI was measured using ddPCR. (G) Summary of the AI measurement for Col6a5 (left) and Dnajc12 (right) after Dnmt1 KD.

Journal: G3: Genes|Genomes|Genetics

Article Title: RNA sequencing-based screen for reactivation of silenced alleles of autosomal genes

doi: 10.1093/g3journal/jkab428

Figure Lengend Snippet: Hit validation for 5-aza-dC. (A and B) Confirmation of Screen-seq results for 5-aza-dC-treated cells using an orthogonal method to measure AI. cDNA samples from day 7 of screening were assessed using ddPCR with allele-specific fluorescent probes. (A) Scatterplots for 20,000 droplets targeting the readout gene, Col6a5 . 5-aza-dC concentration is shown in the plots. Black : empty droplets; blue : droplets with the Cast paternal allele amplified (labeled by FAM fluorophore); red : droplets with the 129 maternal allele amplified (labeled by HEX fluorophore). Ratio of red: blue droplets are shown. AI value written in red is the maternal AI. Note that the double-positive droplets (orange) contained both maternal and paternal templates; a small number of such double-positives is expected with higher concentrations of biallelic template. These droplets are ignored in the quantitative analysis. (B) left— summary of AI measurements shown in (A) for Col6a5 on day 7; right— summary of AI measurements for Dnajc12 on day 7. (C and D) Biological replicate of Abl.1 cells were treated with 5-aza-dC and AI was measured using ddPCR. (C) Scatterplots representation as shown in (A) after 2 days of exposure. (D) Summary of AI measurement for Col6a5 (left) and Dnajc12 (right) after 2, 5, and 7 days of exposure (denoted by color). Gray vertical dashed lines for Col6a5 dose-response were used to determine “low,” “medium,” and “high” 5-aza-dC concentrations for the future experiments. Results for readout gene Adnp2 are in . (E–G) Analysis of Dnmt1 knock-down (KD) in Abl.1 cells. (E) Real-time quantitative PCR (RT-qPCR) analysis of Dnmt1 relative expression (expression in the empty vector control, normalized to Nono , taken as 1.0). Abl.1 cells were transduced with an empty plKO vector (control) or with two separate Dnmt1 shRNA knockdown constructs ( Dnmt1 KD construct 1 or 2) and grown for 2 days. Transduced cells were then selected by growing in the presence of a selection antibiotic for an additional 17 days. RT-qPCR quantification was performed on cells collected 19 days after transduction. Mean and SEM for three technical replicates are shown. (F) Representative scatterplots show AI measurement for Col6a5 in the transduced Abl.1 cells. AI was measured using ddPCR. (G) Summary of the AI measurement for Col6a5 (left) and Dnajc12 (right) after Dnmt1 KD.

Article Snippet: Two shRNA vectors targeting Dnmt1 (SHR000038801.1_TRC001.1 and SHR000373188.1_TRC005.1) and a control empty vector (NUL003.3_TRC021.1) packaged in lentiviral vectors obtained from the Genetic Perturbation Platform at the Broad Institute were tested.

Techniques: Biomarker Discovery, Concentration Assay, Amplification, Labeling, Knockdown, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Plasmid Preparation, Control, Transduction, shRNA, Construct, Selection

Comparison of stemness between U2OS cells and OSLCs. (a) The activity of DNMTs was determined by ELISA. (b) The level of DNMT1 protein was examined by immunoblot (left) and its densitometric analysis (right), with β -actin as a loading control. (c, d) DNMT1 mRNA and miR-34a-5p were assessed by qRT-PCR. (e) Comparison of miR-34a-5p promoter methylation levels in U2OS cells and OSLCs (M, methylated miR-34a-5p promoter; U, unmethylated miR-34a-5p promoter). (f) Representative images of sphere formation (left) (scale bar, 200 μ m), and sphere-forming rate was assessed by the sphere formation assay (right). (g) Representative images of colony formation (left) (scale bar, 200 μ m), and colony-forming rate was determined by the colony formation assay (right). (h) The levels of CD133, CD44, and ABCG2 proteins were examined by immunoblot (left) and their densitometric analysis (right). (i) Bmi1, Sox2, and Oct4 mRNA were assessed, with β -actin serving as a loading control. ∗ p < 0.05 ( n = 3) vs. U2OS cells.

Journal: Stem Cells International

Article Title: The DNMT1/miR-34a Axis Is Involved in the Stemness of Human Osteosarcoma Cells and Derived Stem-Like Cells

doi: 10.1155/2019/7028901

Figure Lengend Snippet: Comparison of stemness between U2OS cells and OSLCs. (a) The activity of DNMTs was determined by ELISA. (b) The level of DNMT1 protein was examined by immunoblot (left) and its densitometric analysis (right), with β -actin as a loading control. (c, d) DNMT1 mRNA and miR-34a-5p were assessed by qRT-PCR. (e) Comparison of miR-34a-5p promoter methylation levels in U2OS cells and OSLCs (M, methylated miR-34a-5p promoter; U, unmethylated miR-34a-5p promoter). (f) Representative images of sphere formation (left) (scale bar, 200 μ m), and sphere-forming rate was assessed by the sphere formation assay (right). (g) Representative images of colony formation (left) (scale bar, 200 μ m), and colony-forming rate was determined by the colony formation assay (right). (h) The levels of CD133, CD44, and ABCG2 proteins were examined by immunoblot (left) and their densitometric analysis (right). (i) Bmi1, Sox2, and Oct4 mRNA were assessed, with β -actin serving as a loading control. ∗ p < 0.05 ( n = 3) vs. U2OS cells.

Article Snippet: Lentiviruses of LV-15 (pGLVH1/RFP/Puro) vectors carrying shRNA targeting human DNMT1 and lentiviruses of LV8N (EF-1 α F/mCherry/Puro) vectors carrying human DNMT1 cDNA were purchased from GenePharma (Shanghai).

Techniques: Comparison, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Quantitative RT-PCR, Methylation, Tube Formation Assay, Colony Assay

Effects of Aza-dC on the stemness feature in OSLCs. (a) The activity of DNMT1 in OSLCs treated with Aza-dC was assessed by ELISA. (b) The level of DNMT1 mRNA of OSLCs treated with Aza-dC was determined by qRT-PCR. (c) Immunoblotting was used to analyze the expression of DNMT1 of OSLCs treated with Aza-dC (left), as well as its densitometric analysis (right), with β -actin serving as a loading control. (d) The miR-34a-5p level of OSLCs treated with Aza-dC was detected by qRT-PCR. (e) The effect of Aza-dC on OSLC miR-34a-5p promoter methylation (M, methylated miR-34a-5p promoter; U, unmethylated miR-34a-5p promoter). (f) Representative images of the sphere formation of OSLCs treated with Aza-dC (left) (scale bar, 200 μ m), and the sphere formation assay was used to assess the sphere-forming rate (right). (g) Representative images of the colony formation of OSLCs treated with Aza-dC (left) (scale bar, 200 μ m), and the colony-forming rate of OSLCs was determined by the colony formation assay (right). (h) CD133, CD44, and ABCG2 proteins of OSLCs treated with Aza-dC (left) and their densitometric analysis (right). (i) Bmi1, Sox2, and Oct4 of OSLCs treated with Aza-dC. ∗ p < 0.05 ( n = 3) vs. the DMSO control; # p < 0.05 ( n = 3) vs. OSLCs treated with Aza-dC (1.0 μ M).

Journal: Stem Cells International

Article Title: The DNMT1/miR-34a Axis Is Involved in the Stemness of Human Osteosarcoma Cells and Derived Stem-Like Cells

doi: 10.1155/2019/7028901

Figure Lengend Snippet: Effects of Aza-dC on the stemness feature in OSLCs. (a) The activity of DNMT1 in OSLCs treated with Aza-dC was assessed by ELISA. (b) The level of DNMT1 mRNA of OSLCs treated with Aza-dC was determined by qRT-PCR. (c) Immunoblotting was used to analyze the expression of DNMT1 of OSLCs treated with Aza-dC (left), as well as its densitometric analysis (right), with β -actin serving as a loading control. (d) The miR-34a-5p level of OSLCs treated with Aza-dC was detected by qRT-PCR. (e) The effect of Aza-dC on OSLC miR-34a-5p promoter methylation (M, methylated miR-34a-5p promoter; U, unmethylated miR-34a-5p promoter). (f) Representative images of the sphere formation of OSLCs treated with Aza-dC (left) (scale bar, 200 μ m), and the sphere formation assay was used to assess the sphere-forming rate (right). (g) Representative images of the colony formation of OSLCs treated with Aza-dC (left) (scale bar, 200 μ m), and the colony-forming rate of OSLCs was determined by the colony formation assay (right). (h) CD133, CD44, and ABCG2 proteins of OSLCs treated with Aza-dC (left) and their densitometric analysis (right). (i) Bmi1, Sox2, and Oct4 of OSLCs treated with Aza-dC. ∗ p < 0.05 ( n = 3) vs. the DMSO control; # p < 0.05 ( n = 3) vs. OSLCs treated with Aza-dC (1.0 μ M).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Expressing, Control, Methylation, Tube Formation Assay, Colony Assay

Effects of DNMT1 knockdown on the stemness feature in OSLCs. (a, b) qRT-PCR and immunoblot were performed to detect DNMT1 mRNA and protein in OSLCs transfected with shRNA DNMT1, respectively, with β -actin serving as a loading control. (c) qRT-PCR analyzed miR-34a-5p expression in OSLCs knocking down DNMT1. (d) Representative images of the spheres of OSLCs transfected with shNC or shDNMT1 (left) (scale bar, 200 μ m) and sphere-forming rate (right). (e) Representative images of the colonies of OSLCs transfected with NC or DNMT1 shRNA (left) (scale bar, 200 μ m) and colony-forming rate (right). (f) CD133, CD44, and ABCG2 proteins in OSLCs transfected with shNC or shDNMT1 (left) and their densitometric analysis (right). (g) Bmi1, Sox2, and Oct4 in OSLCs transfected with shNC or shDNMT1. ∗ p < 0.05 ( n = 3) vs. U2OS cell; # p < 0.05 ( n = 3) vs. shNC. (h) The images of subcutaneous xenografts of OSLC (2 × 10 5 ) expressing red fluorescent protein (RFP) and shNC (left) and comparison of tumor weight of OSLCs expressing RFP and DNMT1 shRNA (right). The data were obtained from xenograft weighing results from 6 inoculation sites ( n = 6). ∗ p < 0.05 vs. OSLCs expressing RFP. The images of immunohistochemistry of DNMT1 protein expression under an optical microscope (right) (scale bar, 50 μ m).

Journal: Stem Cells International

Article Title: The DNMT1/miR-34a Axis Is Involved in the Stemness of Human Osteosarcoma Cells and Derived Stem-Like Cells

doi: 10.1155/2019/7028901

Figure Lengend Snippet: Effects of DNMT1 knockdown on the stemness feature in OSLCs. (a, b) qRT-PCR and immunoblot were performed to detect DNMT1 mRNA and protein in OSLCs transfected with shRNA DNMT1, respectively, with β -actin serving as a loading control. (c) qRT-PCR analyzed miR-34a-5p expression in OSLCs knocking down DNMT1. (d) Representative images of the spheres of OSLCs transfected with shNC or shDNMT1 (left) (scale bar, 200 μ m) and sphere-forming rate (right). (e) Representative images of the colonies of OSLCs transfected with NC or DNMT1 shRNA (left) (scale bar, 200 μ m) and colony-forming rate (right). (f) CD133, CD44, and ABCG2 proteins in OSLCs transfected with shNC or shDNMT1 (left) and their densitometric analysis (right). (g) Bmi1, Sox2, and Oct4 in OSLCs transfected with shNC or shDNMT1. ∗ p < 0.05 ( n = 3) vs. U2OS cell; # p < 0.05 ( n = 3) vs. shNC. (h) The images of subcutaneous xenografts of OSLC (2 × 10 5 ) expressing red fluorescent protein (RFP) and shNC (left) and comparison of tumor weight of OSLCs expressing RFP and DNMT1 shRNA (right). The data were obtained from xenograft weighing results from 6 inoculation sites ( n = 6). ∗ p < 0.05 vs. OSLCs expressing RFP. The images of immunohistochemistry of DNMT1 protein expression under an optical microscope (right) (scale bar, 50 μ m).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Transfection, shRNA, Control, Expressing, Comparison, Immunohistochemistry, Microscopy

Effects of overexpressing DNMT1 on the stemness feature in U2OS cells. (a, b) DNMT1 mRNA and protein were detected by qRT-PCR and immunoblot in U2OS cells transfected with DNMT1 cDNA, with β -actin serving as a loading control. (c) qRT-PCR analyzed the miR-34a-5p level in U2OS cells transfected with DNMT1 cDNA. (d) Representative images of the spheres in U2OS cells transfected with DNMT1 cDNA (left) (scale bar, 200 μ m) and the sphere-forming rates (right). (e) Representative images of the colonies in U2OS cells transfected with DNMT1 cDNA (left) (scale bar, 200 μ m) and the colony-forming rates (right). (f) CD133, CD44, and ABCG2 proteins in U2OS cells transfected with DNMT1 cDNA (left) and their densitometric analysis (right), with β -actin serving as a loading control. (g) Bmi1, Sox2, and Oct4 were assessed in U2OS cells transfected with DNMT1 cDNA. ∗ p < 0.05 ( n = 3) vs. U2OS cell; # p < 0.05 ( n = 3) vs. vector control.

Journal: Stem Cells International

Article Title: The DNMT1/miR-34a Axis Is Involved in the Stemness of Human Osteosarcoma Cells and Derived Stem-Like Cells

doi: 10.1155/2019/7028901

Figure Lengend Snippet: Effects of overexpressing DNMT1 on the stemness feature in U2OS cells. (a, b) DNMT1 mRNA and protein were detected by qRT-PCR and immunoblot in U2OS cells transfected with DNMT1 cDNA, with β -actin serving as a loading control. (c) qRT-PCR analyzed the miR-34a-5p level in U2OS cells transfected with DNMT1 cDNA. (d) Representative images of the spheres in U2OS cells transfected with DNMT1 cDNA (left) (scale bar, 200 μ m) and the sphere-forming rates (right). (e) Representative images of the colonies in U2OS cells transfected with DNMT1 cDNA (left) (scale bar, 200 μ m) and the colony-forming rates (right). (f) CD133, CD44, and ABCG2 proteins in U2OS cells transfected with DNMT1 cDNA (left) and their densitometric analysis (right), with β -actin serving as a loading control. (g) Bmi1, Sox2, and Oct4 were assessed in U2OS cells transfected with DNMT1 cDNA. ∗ p < 0.05 ( n = 3) vs. U2OS cell; # p < 0.05 ( n = 3) vs. vector control.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Control, Plasmid Preparation

Effects of the miR-34a-5p mimic on the stemness feature in OSLCs. (a) qRT-PCR analyzed the level of miR-34a-5p in OSLCs transfected with miR-NC or miR-34a-5p mimic (miR-34a). (b, c) ELISA and qRT-PCR detected the activities and mRNA levels of DNMT1 in OSLCs transfected with miR-NC or miR-34a. (d) Immunoblot analyzed the expression of DNMT1 in OSLCs transfected with miR-NC or miR-34a, with β -actin serving as a loading control. (e) Representative images of the spheres in OSLCs transfected with miR-34a or with miR-NC and untreated cells serving as the controls (left) (scale bar, 200 μ m) and the sphere-forming rate (right). (f) Representative images of the colonies in OSLCs transfected with miR-34a or with miR-NC and untreated cells serving as the controls (left) (scale bar, 200 μ m) and the colony-forming rate (right). (g) CD133, CD44, and ABCG2 proteins in OSLCs transfected with miR-34a or miR-NC (left) and their densitometric analysis (right). (h) Bmi1, Sox2, and Oct4 in OSLCs transfected with miR-34a or miR-NC were assessed by qRT-PCR. ∗ p < 0.05 ( n = 3) vs. OSLCs; # p < 0.05 ( n = 3) vs. OSLCs transfected with miR-NC. (i) Images of subcutaneous xenografts of OSLCs (2 × 10 5 ) expressing red fluorescent protein (RFP) intratumorally injected with miR-NC or miR-34a (left) and comparison of tumor weight from OSLCs expressing RFP intratumorally injected with miR-NC or miR-34a (middle); and data were obtained from xenograft weighing results from 6 inoculation sites ( n = 6). ∗ p < 0.05 vs. miR-NC. The expression level of miR-34a-5p of xenograft tumors intratumorally injected with NC or miR-34a (right). ∗ p < 0.05 vs. cells injected with miR-NC.

Journal: Stem Cells International

Article Title: The DNMT1/miR-34a Axis Is Involved in the Stemness of Human Osteosarcoma Cells and Derived Stem-Like Cells

doi: 10.1155/2019/7028901

Figure Lengend Snippet: Effects of the miR-34a-5p mimic on the stemness feature in OSLCs. (a) qRT-PCR analyzed the level of miR-34a-5p in OSLCs transfected with miR-NC or miR-34a-5p mimic (miR-34a). (b, c) ELISA and qRT-PCR detected the activities and mRNA levels of DNMT1 in OSLCs transfected with miR-NC or miR-34a. (d) Immunoblot analyzed the expression of DNMT1 in OSLCs transfected with miR-NC or miR-34a, with β -actin serving as a loading control. (e) Representative images of the spheres in OSLCs transfected with miR-34a or with miR-NC and untreated cells serving as the controls (left) (scale bar, 200 μ m) and the sphere-forming rate (right). (f) Representative images of the colonies in OSLCs transfected with miR-34a or with miR-NC and untreated cells serving as the controls (left) (scale bar, 200 μ m) and the colony-forming rate (right). (g) CD133, CD44, and ABCG2 proteins in OSLCs transfected with miR-34a or miR-NC (left) and their densitometric analysis (right). (h) Bmi1, Sox2, and Oct4 in OSLCs transfected with miR-34a or miR-NC were assessed by qRT-PCR. ∗ p < 0.05 ( n = 3) vs. OSLCs; # p < 0.05 ( n = 3) vs. OSLCs transfected with miR-NC. (i) Images of subcutaneous xenografts of OSLCs (2 × 10 5 ) expressing red fluorescent protein (RFP) intratumorally injected with miR-NC or miR-34a (left) and comparison of tumor weight from OSLCs expressing RFP intratumorally injected with miR-NC or miR-34a (middle); and data were obtained from xenograft weighing results from 6 inoculation sites ( n = 6). ∗ p < 0.05 vs. miR-NC. The expression level of miR-34a-5p of xenograft tumors intratumorally injected with NC or miR-34a (right). ∗ p < 0.05 vs. cells injected with miR-NC.

Techniques: Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control, Injection, Comparison

Effects of the miR-34a-5p inhibitor on the stemness feature in U2OS cells. (a) qRT-PCR analyzed the levels of miR-34a-5p in U2OS cells transfected with anti-NC or miR-34a inhibitor (anti-34a). ∗ p < 0.05 ( n = 3) vs. U2OS cells; # p < 0.05 ( n = 3) vs. anti-NC. (b, c) ELISA and qRT-PCR detected DNMT1 activities and mRNA in U2OS cells transfected with anti-NC or anti-34a. (d) Immunoblot analyzed the DNMT1 expression in U2OS cells transfected with anti-NC or anti-34a, with β -actin serving as a loading control. (e) Representative images of the spheres of U2OS cells transfected with anti-34a or with anti-NC and untreated U2OS cells serving as the controls (left) (scale bar, 200 μ m) and the sphere-forming rates (right). (f) Representative images of the colony formation of U2OS cells transfected with anti-34a or with anti-NC and untreated U2OS cells serving as the controls (left) (scale bar, 200 μ m) and the colony-forming rates (right). (g) CD133, CD44, and ABCG2 proteins in U2OS cells transfected with anti-34a or with anti-NC and untreated U2OS cells serving as the controls (left) and their densitometric analysis (right). (h) Bmi1, Sox2, and Oct4 of U2OS cells transfected with anti-34a or with anti-NC and untreated U2OS cells serving as the controls. ∗ p < 0.05 ( n = 3) vs. U2OS cells; # p < 0.05 ( n = 3) vs. U2OS cells transfected with anti-NC.

Journal: Stem Cells International

Article Title: The DNMT1/miR-34a Axis Is Involved in the Stemness of Human Osteosarcoma Cells and Derived Stem-Like Cells

doi: 10.1155/2019/7028901

Figure Lengend Snippet: Effects of the miR-34a-5p inhibitor on the stemness feature in U2OS cells. (a) qRT-PCR analyzed the levels of miR-34a-5p in U2OS cells transfected with anti-NC or miR-34a inhibitor (anti-34a). ∗ p < 0.05 ( n = 3) vs. U2OS cells; # p < 0.05 ( n = 3) vs. anti-NC. (b, c) ELISA and qRT-PCR detected DNMT1 activities and mRNA in U2OS cells transfected with anti-NC or anti-34a. (d) Immunoblot analyzed the DNMT1 expression in U2OS cells transfected with anti-NC or anti-34a, with β -actin serving as a loading control. (e) Representative images of the spheres of U2OS cells transfected with anti-34a or with anti-NC and untreated U2OS cells serving as the controls (left) (scale bar, 200 μ m) and the sphere-forming rates (right). (f) Representative images of the colony formation of U2OS cells transfected with anti-34a or with anti-NC and untreated U2OS cells serving as the controls (left) (scale bar, 200 μ m) and the colony-forming rates (right). (g) CD133, CD44, and ABCG2 proteins in U2OS cells transfected with anti-34a or with anti-NC and untreated U2OS cells serving as the controls (left) and their densitometric analysis (right). (h) Bmi1, Sox2, and Oct4 of U2OS cells transfected with anti-34a or with anti-NC and untreated U2OS cells serving as the controls. ∗ p < 0.05 ( n = 3) vs. U2OS cells; # p < 0.05 ( n = 3) vs. U2OS cells transfected with anti-NC.

Techniques: Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control

Effects of overexpressing DNMT1 combined with the miR-34a-5p mimic on stemness in U2OS cells. (a) qRT-PCR analyzed the effect of the miR-34a mimic on the miR-34a expression in U2OS cells overexpressing DNMT1. (b, c) Immunoblotting and qRT-PCR detected the DNMT1 protein and mRNA in U2OS cells overexpressing DNMT1 (DNMT1) transfected with the miR-34a mimic (miR-34a) and their densitometric analysis (right), with β -actin serving as a loading control. ∗ p < 0.05 ( n = 3) vs. U2OS cells; # p < 0.05 ( n = 3) vs. U2OS cells transfected with miR-34a. (d) Representative images of the spheres of U2OS cells overexpressing DNMT1 transfected with miR-34a (left) (scale bar, 200 μ m) and the sphere-forming rates (right). (e) Representative images of the colonies of U2OS cells overexpressing DNMT1 transfected with miR-34a (left) (scale bar, 200 μ m) and the colony-forming rates (right). (f) CD133, CD44, and ABCG2 proteins of U2OS cells overexpressing DNMT1 transfected with miR-34a (left) and their densitometric analysis (right). (g) Bmi1, Sox2, and Oct4 of U2OS cells overexpressing DNMT1 transfected with miR-34a. ∗ p < 0.05 ( n = 3) vs. U2OS cells; # p < 0.05 ( n = 3) vs. U2OS cells transfected with miR-34a.

Journal: Stem Cells International

Article Title: The DNMT1/miR-34a Axis Is Involved in the Stemness of Human Osteosarcoma Cells and Derived Stem-Like Cells

doi: 10.1155/2019/7028901

Figure Lengend Snippet: Effects of overexpressing DNMT1 combined with the miR-34a-5p mimic on stemness in U2OS cells. (a) qRT-PCR analyzed the effect of the miR-34a mimic on the miR-34a expression in U2OS cells overexpressing DNMT1. (b, c) Immunoblotting and qRT-PCR detected the DNMT1 protein and mRNA in U2OS cells overexpressing DNMT1 (DNMT1) transfected with the miR-34a mimic (miR-34a) and their densitometric analysis (right), with β -actin serving as a loading control. ∗ p < 0.05 ( n = 3) vs. U2OS cells; # p < 0.05 ( n = 3) vs. U2OS cells transfected with miR-34a. (d) Representative images of the spheres of U2OS cells overexpressing DNMT1 transfected with miR-34a (left) (scale bar, 200 μ m) and the sphere-forming rates (right). (e) Representative images of the colonies of U2OS cells overexpressing DNMT1 transfected with miR-34a (left) (scale bar, 200 μ m) and the colony-forming rates (right). (f) CD133, CD44, and ABCG2 proteins of U2OS cells overexpressing DNMT1 transfected with miR-34a (left) and their densitometric analysis (right). (g) Bmi1, Sox2, and Oct4 of U2OS cells overexpressing DNMT1 transfected with miR-34a. ∗ p < 0.05 ( n = 3) vs. U2OS cells; # p < 0.05 ( n = 3) vs. U2OS cells transfected with miR-34a.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Control

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