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CircRERE(4-5) upregulates <t>ONECUT2</t> by sponging miR-571. (A) qRT–PCR demonstrating the enrichment of circRNAs in a representative anti-AGO2 RIP assay conducted in GC cells. (B) Venn diagram illustrating the overlap of candidate miRNAs predicted to interact with circRERE(4-5) by two open-access databases. (C) qRT–PCR showing the enrichment of miRNAs following circRERE(4-5) pull-down in lysates of GC cells. (D) Venn diagram depicting the intersection of candidate mRNAs targeted by miR-571 as predicted by three open-access databases. (E) qRT–PCR analysis of ONECUT2 mRNA expression in GC cells under control conditions (sh-NC) or with circRERE(4-5) knockdown (sh-circRERE(4-5)). (F) qRT–PCR analysis of ONECUT2 mRNA expression in GC cells transfected with control inhibitors or miR-571 inhibitors. (G) Western blotting revealing ONECUT2 protein levels in GC cells under control conditions (sh-NC) or with circRERE(4-5) knockdown (sh-circRERE(4-5)), with GAPDH serving as a loading control. (H) Western blotting showing ONECUT2 protein levels in GC cells transfected with control inhibitors or miR-571 inhibitors. (I) qRT–PCR analysis of ONECUT2 mRNA expression in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and miR-571 inhibitors. (J) Representative western blot of ONECUT2 in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and miR-571 inhibitors. (K) Sequence alignment analysis highlighting the binding sites of circRERE(4-5) and ONECUT2 3’ UTR on miR-571. Data are presented as the mean ± SD. P -values were calculated using a two-tailed unpaired Student’s t -test (E, F, I) ; ** P < 0.01, *** P < 0.001. See also <xref ref-type=

Supplementary Figure 4 . " width="250" height="auto" />
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1) Product Images from "Silencing of circRERE(4-5) inhibits ONECUT2-mediated tumorigenesis and metastasis in gastric cancer"

Article Title: Silencing of circRERE(4-5) inhibits ONECUT2-mediated tumorigenesis and metastasis in gastric cancer

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2026.1686702

CircRERE(4-5) upregulates ONECUT2 by sponging miR-571. (A) qRT–PCR demonstrating the enrichment of circRNAs in a representative anti-AGO2 RIP assay conducted in GC cells. (B) Venn diagram illustrating the overlap of candidate miRNAs predicted to interact with circRERE(4-5) by two open-access databases. (C) qRT–PCR showing the enrichment of miRNAs following circRERE(4-5) pull-down in lysates of GC cells. (D) Venn diagram depicting the intersection of candidate mRNAs targeted by miR-571 as predicted by three open-access databases. (E) qRT–PCR analysis of ONECUT2 mRNA expression in GC cells under control conditions (sh-NC) or with circRERE(4-5) knockdown (sh-circRERE(4-5)). (F) qRT–PCR analysis of ONECUT2 mRNA expression in GC cells transfected with control inhibitors or miR-571 inhibitors. (G) Western blotting revealing ONECUT2 protein levels in GC cells under control conditions (sh-NC) or with circRERE(4-5) knockdown (sh-circRERE(4-5)), with GAPDH serving as a loading control. (H) Western blotting showing ONECUT2 protein levels in GC cells transfected with control inhibitors or miR-571 inhibitors. (I) qRT–PCR analysis of ONECUT2 mRNA expression in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and miR-571 inhibitors. (J) Representative western blot of ONECUT2 in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and miR-571 inhibitors. (K) Sequence alignment analysis highlighting the binding sites of circRERE(4-5) and ONECUT2 3’ UTR on miR-571. Data are presented as the mean ± SD. P -values were calculated using a two-tailed unpaired Student’s t -test (E, F, I) ; ** P < 0.01, *** P < 0.001. See also <xref ref-type=

Supplementary Figure 4 . " title="CircRERE(4-5) upregulates ONECUT2 by sponging miR-571. (A) qRT–PCR demonstrating the enrichment ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: CircRERE(4-5) upregulates ONECUT2 by sponging miR-571. (A) qRT–PCR demonstrating the enrichment of circRNAs in a representative anti-AGO2 RIP assay conducted in GC cells. (B) Venn diagram illustrating the overlap of candidate miRNAs predicted to interact with circRERE(4-5) by two open-access databases. (C) qRT–PCR showing the enrichment of miRNAs following circRERE(4-5) pull-down in lysates of GC cells. (D) Venn diagram depicting the intersection of candidate mRNAs targeted by miR-571 as predicted by three open-access databases. (E) qRT–PCR analysis of ONECUT2 mRNA expression in GC cells under control conditions (sh-NC) or with circRERE(4-5) knockdown (sh-circRERE(4-5)). (F) qRT–PCR analysis of ONECUT2 mRNA expression in GC cells transfected with control inhibitors or miR-571 inhibitors. (G) Western blotting revealing ONECUT2 protein levels in GC cells under control conditions (sh-NC) or with circRERE(4-5) knockdown (sh-circRERE(4-5)), with GAPDH serving as a loading control. (H) Western blotting showing ONECUT2 protein levels in GC cells transfected with control inhibitors or miR-571 inhibitors. (I) qRT–PCR analysis of ONECUT2 mRNA expression in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and miR-571 inhibitors. (J) Representative western blot of ONECUT2 in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and miR-571 inhibitors. (K) Sequence alignment analysis highlighting the binding sites of circRERE(4-5) and ONECUT2 3’ UTR on miR-571. Data are presented as the mean ± SD. P -values were calculated using a two-tailed unpaired Student’s t -test (E, F, I) ; ** P < 0.01, *** P < 0.001. See also Supplementary Figure 4 .

Techniques Used: Quantitative RT-PCR, Expressing, Control, Knockdown, Transfection, Western Blot, Cotransfection, Sequencing, Binding Assay, Two Tailed Test

CircRERE(4-5) promotes oncogenic activity through ONECUT2 in GC cells. (A) Representative western blot of ONECUT2 protein in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. (B, C) Plate colony formation assay evaluating colony formation in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. (D) CCK-8 assay depicting the proliferation of AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. (E, F) Transwell migration assay illustrating the migration of AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. Scale bar, 100 µm. (G, H) Wound healing assay demonstrating the migration of AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. Scale bar, 200 µm. Data are presented as the mean ± SD. P- values were calculated using a two-tailed unpaired Student’s t -test (C, F, H) or two-way ANOVA (D) ; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also <xref ref-type=

Supplementary Figure 5 . " title="CircRERE(4-5) promotes oncogenic activity through ONECUT2 in GC cells. (A) Representative western blot of ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: CircRERE(4-5) promotes oncogenic activity through ONECUT2 in GC cells. (A) Representative western blot of ONECUT2 protein in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. (B, C) Plate colony formation assay evaluating colony formation in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. (D) CCK-8 assay depicting the proliferation of AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. (E, F) Transwell migration assay illustrating the migration of AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. Scale bar, 100 µm. (G, H) Wound healing assay demonstrating the migration of AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. Scale bar, 200 µm. Data are presented as the mean ± SD. P- values were calculated using a two-tailed unpaired Student’s t -test (C, F, H) or two-way ANOVA (D) ; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also Supplementary Figure 5 .

Techniques Used: Activity Assay, Western Blot, Control, Knockdown, Cotransfection, Plasmid Preparation, Colony Assay, CCK-8 Assay, Transwell Migration Assay, Migration, Wound Healing Assay, Two Tailed Test

CircRERE(4-5) knockdown suppresses GC tumorigenesis and metastasis. (A) Tumour images of AGS cell derived xenograft (CDX) from mice receiving intratumoral injections of control ASOs or in vivo -optimized circRERE(4-5) ASOs. (B) Tumour growth trajectories in each group of mice. (C) Tumour weights in each group of mice. (D) qRT–PCR demonstrating the expression levels of circRERE(4-5) and ONECUT2 mRNA in tumours from control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups. (E, F) IHC staining illustrating the expression levels of ONECUT2 protein in tumours from control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups. Scale bar, 50 µm (left), 20 µm (right). (G, H) The dissemination of AGS cells in control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups was visualized by bioluminescence imaging using the IVIS ® Spectrum imaging system. Bioluminescent signals were captured after intraperitoneal injection of 150 mg/kg D-luciferin 10 minutes prior to imaging. (I) Lung metastasis nodules in mice receiving intravenous injections of control ASOs or in vivo -optimized circRERE(4-5) ASOs. Scale bar, 100 µm. (J) The count of lung metastasis nodules in control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups. Data are presented the as mean ± SD. P -values were calculated using a two-tailed unpaired Student’s t -test (C, D, F, H, J) or two-way ANOVA (B) ; ** P < 0.01, *** P < 0.001. See also <xref ref-type=

Supplementary Figure 6 . " title="... qRT–PCR demonstrating the expression levels of circRERE(4-5) and ONECUT2 mRNA in tumours from control (ASO-NC) or circRERE(4-5) ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: CircRERE(4-5) knockdown suppresses GC tumorigenesis and metastasis. (A) Tumour images of AGS cell derived xenograft (CDX) from mice receiving intratumoral injections of control ASOs or in vivo -optimized circRERE(4-5) ASOs. (B) Tumour growth trajectories in each group of mice. (C) Tumour weights in each group of mice. (D) qRT–PCR demonstrating the expression levels of circRERE(4-5) and ONECUT2 mRNA in tumours from control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups. (E, F) IHC staining illustrating the expression levels of ONECUT2 protein in tumours from control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups. Scale bar, 50 µm (left), 20 µm (right). (G, H) The dissemination of AGS cells in control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups was visualized by bioluminescence imaging using the IVIS ® Spectrum imaging system. Bioluminescent signals were captured after intraperitoneal injection of 150 mg/kg D-luciferin 10 minutes prior to imaging. (I) Lung metastasis nodules in mice receiving intravenous injections of control ASOs or in vivo -optimized circRERE(4-5) ASOs. Scale bar, 100 µm. (J) The count of lung metastasis nodules in control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups. Data are presented the as mean ± SD. P -values were calculated using a two-tailed unpaired Student’s t -test (C, D, F, H, J) or two-way ANOVA (B) ; ** P < 0.01, *** P < 0.001. See also Supplementary Figure 6 .

Techniques Used: Knockdown, Derivative Assay, Control, In Vivo, Quantitative RT-PCR, Expressing, Immunohistochemistry, Imaging, Injection, Two Tailed Test

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Supplementary Figure 4 . " width="100%" height="100%">

Journal: Frontiers in Immunology

Article Title: Silencing of circRERE(4-5) inhibits ONECUT2-mediated tumorigenesis and metastasis in gastric cancer

doi: 10.3389/fimmu.2026.1686702

Figure Lengend Snippet: CircRERE(4-5) upregulates ONECUT2 by sponging miR-571. (A) qRT–PCR demonstrating the enrichment of circRNAs in a representative anti-AGO2 RIP assay conducted in GC cells. (B) Venn diagram illustrating the overlap of candidate miRNAs predicted to interact with circRERE(4-5) by two open-access databases. (C) qRT–PCR showing the enrichment of miRNAs following circRERE(4-5) pull-down in lysates of GC cells. (D) Venn diagram depicting the intersection of candidate mRNAs targeted by miR-571 as predicted by three open-access databases. (E) qRT–PCR analysis of ONECUT2 mRNA expression in GC cells under control conditions (sh-NC) or with circRERE(4-5) knockdown (sh-circRERE(4-5)). (F) qRT–PCR analysis of ONECUT2 mRNA expression in GC cells transfected with control inhibitors or miR-571 inhibitors. (G) Western blotting revealing ONECUT2 protein levels in GC cells under control conditions (sh-NC) or with circRERE(4-5) knockdown (sh-circRERE(4-5)), with GAPDH serving as a loading control. (H) Western blotting showing ONECUT2 protein levels in GC cells transfected with control inhibitors or miR-571 inhibitors. (I) qRT–PCR analysis of ONECUT2 mRNA expression in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and miR-571 inhibitors. (J) Representative western blot of ONECUT2 in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and miR-571 inhibitors. (K) Sequence alignment analysis highlighting the binding sites of circRERE(4-5) and ONECUT2 3’ UTR on miR-571. Data are presented as the mean ± SD. P -values were calculated using a two-tailed unpaired Student’s t -test (E, F, I) ; ** P < 0.01, *** P < 0.001. See also Supplementary Figure 4 .

Article Snippet: Additionally, the overexpression vectors (GV367) for ONECUT2 were obtained from GeneChem.

Techniques: Quantitative RT-PCR, Expressing, Control, Knockdown, Transfection, Western Blot, Cotransfection, Sequencing, Binding Assay, Two Tailed Test

Supplementary Figure 5 . " width="100%" height="100%">

Journal: Frontiers in Immunology

Article Title: Silencing of circRERE(4-5) inhibits ONECUT2-mediated tumorigenesis and metastasis in gastric cancer

doi: 10.3389/fimmu.2026.1686702

Figure Lengend Snippet: CircRERE(4-5) promotes oncogenic activity through ONECUT2 in GC cells. (A) Representative western blot of ONECUT2 protein in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. (B, C) Plate colony formation assay evaluating colony formation in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. (D) CCK-8 assay depicting the proliferation of AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. (E, F) Transwell migration assay illustrating the migration of AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. Scale bar, 100 µm. (G, H) Wound healing assay demonstrating the migration of AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5), or following cotransfection with sh-circRERE(4-5) and ONECUT2 vector. Scale bar, 200 µm. Data are presented as the mean ± SD. P- values were calculated using a two-tailed unpaired Student’s t -test (C, F, H) or two-way ANOVA (D) ; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also Supplementary Figure 5 .

Article Snippet: Additionally, the overexpression vectors (GV367) for ONECUT2 were obtained from GeneChem.

Techniques: Activity Assay, Western Blot, Control, Knockdown, Cotransfection, Plasmid Preparation, Colony Assay, CCK-8 Assay, Transwell Migration Assay, Migration, Wound Healing Assay, Two Tailed Test

Supplementary Figure 6 . " width="100%" height="100%">

Journal: Frontiers in Immunology

Article Title: Silencing of circRERE(4-5) inhibits ONECUT2-mediated tumorigenesis and metastasis in gastric cancer

doi: 10.3389/fimmu.2026.1686702

Figure Lengend Snippet: CircRERE(4-5) knockdown suppresses GC tumorigenesis and metastasis. (A) Tumour images of AGS cell derived xenograft (CDX) from mice receiving intratumoral injections of control ASOs or in vivo -optimized circRERE(4-5) ASOs. (B) Tumour growth trajectories in each group of mice. (C) Tumour weights in each group of mice. (D) qRT–PCR demonstrating the expression levels of circRERE(4-5) and ONECUT2 mRNA in tumours from control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups. (E, F) IHC staining illustrating the expression levels of ONECUT2 protein in tumours from control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups. Scale bar, 50 µm (left), 20 µm (right). (G, H) The dissemination of AGS cells in control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups was visualized by bioluminescence imaging using the IVIS ® Spectrum imaging system. Bioluminescent signals were captured after intraperitoneal injection of 150 mg/kg D-luciferin 10 minutes prior to imaging. (I) Lung metastasis nodules in mice receiving intravenous injections of control ASOs or in vivo -optimized circRERE(4-5) ASOs. Scale bar, 100 µm. (J) The count of lung metastasis nodules in control (ASO-NC) or circRERE(4-5) knockdown (ASO-circRERE(4-5)) groups. Data are presented the as mean ± SD. P -values were calculated using a two-tailed unpaired Student’s t -test (C, D, F, H, J) or two-way ANOVA (B) ; ** P < 0.01, *** P < 0.001. See also Supplementary Figure 6 .

Article Snippet: Additionally, the overexpression vectors (GV367) for ONECUT2 were obtained from GeneChem.

Techniques: Knockdown, Derivative Assay, Control, In Vivo, Quantitative RT-PCR, Expressing, Immunohistochemistry, Imaging, Injection, Two Tailed Test

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