santa cruz wt11 ⁄ 4wilm tumor gene 1  (Santa Cruz Biotechnology)

Journal: Cell Death & Disease

Article Title: Origin recognition complex 6 overexpression promotes growth of glioma cells

doi: 10.1038/s41419-024-06764-w

Figure Lengend Snippet: Multiple databases predicted the common potential transcription factors of ORC6 ( A ). P1 glioma cells were transfected with the specified siRNAs (at 200 nM) targeting the described transcription factors or a scramble non-sense siRNA (siC, at 200 nM) for 48 h, the expression of ORC6 mRNA was assessed ( B ). P1 glioma cells were genetically engineered to stably express the lentiviral RBPJ shRNA (shRBPJ-Sq1 or shRBPJ-Sq2) ( C and D ), scramble control shRNA (“shC”) ( C , D ), the lentiviral RBPJ-expressing construct (oeRBPJ) ( E , F ), or an empty vector (“Vec”) ( E , F ); The expression of the listed mRNAs and proteins was assessed ( C – F ). Chromatin Immunoprecipitation (ChIP) assay results showed the relative levels of RBPJ-bound ORC6 promoter region in specified glioma tissues (“T”) and matched adjacent normal brain tissues (“N”) ( G ) as well as in listed glioma cells and human astrocytes ( H ). RBPJ protein expression in primary human astrocytes (“Astrocytes1/2”), immortalized (A172) or primary (“P1,” “P2,” “P3”) glioma cells was presented ( I ). Values were mean ± standard deviation (SD). * P < 0.05 versus “siC” ( B ). * P < 0.05 versus “shC”/ “Vec” cells ( C – F ). * P < 0.05 versus “N” tissues or “Astryocytes1” ( G , H ). “n. s.” indicates non-statistical difference ( P > 0.05). The experiments were conducted five times (biological repeats), yielding consistent results.

Article Snippet: Genechem (Shanghai, China) provided validated siRNAs targeting specific transcription factors (STAT5A, RBPJ, NFYA, NFATC1, WT1, SP1 and STAT1).

Techniques: Transfection, Expressing, Stable Transfection, shRNA, Control, Construct, Plasmid Preparation, Chromatin Immunoprecipitation, Standard Deviation

Journal: PLOS Pathogens

Article Title: Wilms’ tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP

doi: 10.1371/journal.ppat.1011881

Figure Lengend Snippet: A. A representative KS case showing KSHV LANA (brown, nuclear) (top panel, with zoom-in on right panels), WT1 (brown, nuclear and cytoplasmic) (middle panels), and by double IHC for WT1 (brown, nuclear and cytoplasmic and KSHV LANA (red, nuclear) (lower panels) (Original magnification, 20X). B. Five KS tissue biopsies were analyzed using HALO analysis software for WT1 in the lesional tissue and adjacent normal subcutaneous tissue, demonstrating significant differential WT1 expression in KS compared to normal skin, p< 0.0001(***) using a two-sided unpaired t-test. C. Higher proportion of WT1-positive cells occurred with advanced histopathologic subtype demonstrated by scatter plot, patch vs. plaque p<0.0001(****), patch vs. nodule, p<0.0001(****), and plaque vs. nodule, p<0.0001(****), using Tukey’s multiple comparison test. WT1 analysis according to histopathologic subtype is also demonstrated in . D. Proportion of WT1 expressing cells correlated significantly with proportion of LANA+ cells, r = 0.5564, p<0.0001(****), as determined by IHC and image analysis using Spearman’s correlation analysis, N = 261. E . The VECTRA panels performed of WT1/LANA demonstrating representative examples in Case 1 and Case 2 of the heterogeneity of KS tumors, with WT1 at times colocalizing (Case 1) and other times in neighboring but separate cells (Case 2). F . WT1 expression examined in 13 tumor biopsies from HIV positive and 13 from HIV negative patients, demonstrating increased WT1 expression in the HIV positive group, p = .023(*) using a two-sided unpaired t-test.

Article Snippet: The WT1 siRNA and control siRNA were obtained from Thermo Fisher.

Techniques: Software, Expressing, Comparison

Journal: PLOS Pathogens

Article Title: Wilms’ tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP

doi: 10.1371/journal.ppat.1011881

Figure Lengend Snippet: A. Representative immunohistochemistry of KS within a lymph node used for RNA extraction to evaluate WT1 isoform expression. B. RT-qPCR of total WT1, p = 0.0010 (***) and major WT1 isoforms: A, p<0.05 (*), B, p = 0.3178 (ns), C, p = 0.0652(ns), and D, p<0.05(*) was performed, and RNA found to be more abundant in a Kaposi Sarcoma compared to tonsil tissue, using two-sided, unpaired student t-tests. Data shown is representative of two independent experiments performed in triplicate. C. RT-qPCR of viral genes, LANA, vFLIP, and K8.1 was performed and is shown. Statistical significance was determined by one way ANOVA, Tukey’s multiple comparisons test in iSLK-BAC16 with and without lytic induction compared to KS tissue, p<0.05(*), p<0.01(**), p <0.001(***), p <0.0001(****).

Article Snippet: The WT1 siRNA and control siRNA were obtained from Thermo Fisher.

Techniques: Immunohistochemistry, RNA Extraction, Expressing, Quantitative RT-PCR

Journal: PLOS Pathogens

Article Title: Wilms’ tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP

doi: 10.1371/journal.ppat.1011881

Figure Lengend Snippet: A. RT-qPCR demonstrating mRNA levels of WT1, p<0.01(**) in mock and KSHV-infected HuARLT-1 cells. B. Western blot of WT1, LANA, vFLIP upon KSHV infection of HuARLT-1 and corresponding densitometry analysis from 3 independent experiments shown of WT1/GAPDH ratio, p<0.05(*), using a one-sided unpaired t-test. C . (Top panel) Cell blocks of Mock vs. KSHV infected HuARLT-1, demonstrating IHC for LANA (nuclear brown), documenting infection. (Lower panel) Representative images of monolayer of mock vs KSHV infected HuARLT-1, obtained under fluoroscopy for GFP, indicative of KSHV BAC-16 infection in HuARLT-1 cells. Right panels show RT-qPCR mRNA levels of untreated and after lytic induction with 1mM Sodium butyrate (NaB) for LANA, p<0.0001 (****), vFLIP, p<0.05(*) and K8.1, p<0.0001(****), using two-sided, unpaired student t-tests. D. Western blots of WT1, LANA and vFLIP in primary HUVEC and HUVEC ORFE4 from 24 hours to 72 hours showing higher levels of WT1 in the setting of KSHV infection. Image densitometry analysis is shown below as the WT1/GAPDH ratio.

Article Snippet: The WT1 siRNA and control siRNA were obtained from Thermo Fisher.

Techniques: Quantitative RT-PCR, Infection, Western Blot

Journal: PLOS Pathogens

Article Title: Wilms’ tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP

doi: 10.1371/journal.ppat.1011881

Figure Lengend Snippet: A. vFLIP expression upregulates WT1, demonstrated by immunofluorescence (green) in HuARLT-1 cells transduced with a doxycycline inducible pLVX vFLIP-FLAG lentivirus for wild type vFLIP vs mutant vFLIP compared to untransduced control cells. A mutant vFLIP that is unable to bind IKKγ and induce NFκB is defective in its ability to upregulate WT1. B . Quantitation of WT1 immunofluorescence (Arbitrary units) upon vFLIP induction, performed by averaging the GFP fluorescence for six distinct areas, showed increased WT1 expression with wild type vFLIP compared to control (HuARLT-1 not transduced with vFLIP), and mutant vFLIP: control vs. vFLIP. Statistical significance was determined using one way ANOVA, Tukey’s multiple comparisons, p<0.01(**), p<0.0001(****) C. Western blots show that WT1 protein is increased upon vFLIP-FLAG induction in HuARLT-1 cells. EMD Millipore WT1-NT antibody (clone 6F-H2) against WT1 was used. This increase was not seen to the same extent when the mutant vFLIP-FLAG is expressed, which is also less stable than the wild type form, as seen with antibodies to FLAG. Quantification of three independent experiments is shown, p = 0.05(*), using one-sided, student’s t test. D. Inhibition of NFκB signaling with BMS-345541 demonstrated reduction of WT1 expression, with quantification for WT1 in three independent experiments shown below. E. RT-qPCR for WT1 showed induction of WT1 mRNA with wild type vFLIP expression, which did not occur to the same extent upon mutant vFLIP expression: control vs. vFLIP, p<0.0001(****) and vFLIP vs. mutant vFLIP,p<0.0001(****) using two-way ANOVA with Tukey’s multiple comparison’s tests. F. Major WT1 isoforms, A, B, C, D are all found to be upregulated by RT-qPCR for WT1 in vitro upon vFLIP induction in HuARLT-1 cells, p<0.05(*), p<0.01(**), p <0.001(***), p<0.0001(****), not significant (ns), using ordinary one way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: The WT1 siRNA and control siRNA were obtained from Thermo Fisher.

Techniques: Expressing, Immunofluorescence, Transduction, Mutagenesis, Quantitation Assay, Fluorescence, Western Blot, Inhibition, Quantitative RT-PCR, In Vitro

Journal: PLOS Pathogens

Article Title: Wilms’ tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP

doi: 10.1371/journal.ppat.1011881

Figure Lengend Snippet: A. Western blotting demonstrates WT1 knockdown in iSLK KSHV BAC-16 cells transfected with WT1 siRNA in comparison to a control siRNA. Corresponding densitometry analysis is noted as protein fold expression compared to GAPDH for WT1, p<0.001(***), vFLIP(ns), LANA p<0.05(*), pAKT p<0.05(*), and BCL2 p <0.05(*) using two-sided, unpaired student t-tests. B. Introduction of WT1 siRNA leads to decrease total cell number using trypan blue to assess cell viability compared to control siRNA, p<0.01 (**) using two-sided, unpaired student’s t-tests. C. RT-qPCR of WT1, p<0.05(*), vFLIP(ns), LANA, p<0.0001(****), K8.1, p<0.0001(****), and BCL2 p<0.05(*) in the setting of WT1 knockdown in ISLK BAC-16 with WT1 siRNA in comparison to a control siRNA using two-sided, unpaired student’s t-tests. D. iSLK-KSHV BAC-16 cells transfected with WT1 siRNA compared to control siRNA, demonstrating flow cytometry for DAPI and Annexin V revealing increased apoptosis, performed in duplicate.

Article Snippet: The WT1 siRNA and control siRNA were obtained from Thermo Fisher.

Techniques: Western Blot, Transfection, Comparison, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: PLOS Pathogens

Article Title: Wilms’ tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP

doi: 10.1371/journal.ppat.1011881

Figure Lengend Snippet: A. Using HALO analysis software of KS tumors from the AMC066/A5263 clinical trial, pseudocolor images were created for IHC of LANA, WT1, CD8, and CD4 cells. B. WT1 expression in all cases was inversely correlated with the number of CD8+T cells, using Spearman’s correlation test. Of note, melanin is noted along the epidermis and was excluded upon analysis of WT1 expression. C. Overall assessment of cellular populations according to histological subtype showed lower CD8 and CD4 T cells with advanced histological subtype, and the reverse for WT1 and LANA, p <0.001(***), p <0.0001(****) using two way ANOVA, Tukey’s multiple comparisons test. D. Clusters of CD8+ cells were seen at the periphery of nodular KS lesions, a representative case. E . Areas with numerous LANA+ cells were selected and quantified for CD8 positivity in a sequential tissue section, and vice versa using ordinary one-way ANOVA, Tukey’s multiple comparisons test. This spatial analysis was performed for WT1, LANA, CD4+T cells and CD8+T cells in areas of high LANA (intranodular areas) or high CD8 +T cells (along the periphery of nodular lesions), and lymphocytic infiltrates in corresponding regions for WT1/LANA/CD8/CD4 in plaques and patches. Ordinary one-way ANOVA multiple comparisons was performed as shown in , p<0.05 (*), p <0.01 (**), p <0.001(***), p <0.0001(****), ns (not significant).

Article Snippet: The WT1 siRNA and control siRNA were obtained from Thermo Fisher.

Techniques: Software, Expressing

Journal: PLOS Pathogens

Article Title: Wilms’ tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP

doi: 10.1371/journal.ppat.1011881

Figure Lengend Snippet: A diagram demonstrating KSHV infection contributing to vFLIP mediated upregulation of WT1 through NFκB activation. WT1 upregulation in combination with KSHV, potentially impact the tumor microenvironment, contributing to an immunosuppressed state, in the suppression of T cells. WT1 directed immunotherapy such as TCRm mAb may aid in reversing the effects of WT1 mediated tumorigenesis through the targeting of KS spindle cells overexpressing WT1 through the induction of antibody-dependent cellular cytotoxicity (ADCC). ( Image created with Biorender . com) .

Article Snippet: The WT1 siRNA and control siRNA were obtained from Thermo Fisher.

Techniques: Infection, Activation Assay

Journal: Non-Coding RNA

Article Title: Therapeutic Effects of WT1 Silencing via Respiratory Administration of Neutral DOPC Liposomal-siRNA in a Lung Metastasis Melanoma Murine Model

doi: 10.3390/ncrna9020021

Figure Lengend Snippet: Effect of WT1 silencing over protein expression and cellular motility on melanoma cells. ( A ) Western blot showing silencing of the WT1 protein and densitometry quantification for each group after 72 h of treatment. ( B ) Cell nuclei stained with DAPI after WT1 silencing showing a reduced migration after 48 h and ( C ) a complete loss of invasion capacity through the Matrigel- transwell chamber after 72 h. Average number of nuclei per field was normalized to untreated group. Scale bars represent 100 µm. Groups U: Untreated, C: Control siRNA, W: WT1 siRNA. One-way ANOVA was used as statistical test for group’s comparison. * p < 0.05, *** p < 0.001.

Article Snippet: siRNA sequences were commercially acquired from Sigma-Aldrich, including the WT1 -silencing siRNA from the reported sequence (5′-AAGCUGUCCCACUUACAGAUGCdTdT-3′) and a universal negative control siRNA (Sigma-Aldrich, Cat. SIC001).

Techniques: Expressing, Western Blot, Staining, Migration

Journal: Non-Coding RNA

Article Title: Therapeutic Effects of WT1 Silencing via Respiratory Administration of Neutral DOPC Liposomal-siRNA in a Lung Metastasis Melanoma Murine Model

doi: 10.3390/ncrna9020021

Figure Lengend Snippet: Induction of cell cycle arrest decreased viability and apoptosis activation after WT1 silencing on melanoma cells. ( A ) WT1 silencing induces G1 cell cycle arrest and decreased G2/M progression measured via PI cytometry. ( B ) AlamarBlue assay shows a reduced viability in the group treated with WT1 siRNA. ( C ) Apoptosis detection via a TUNEL assay after WT1 silencing using fluorescent microscopy. The graph shows the percentage of TUNEL-positive nuclei per field compared to the untreated group. All experiments were performed after 72 h of treatment. Groups U: Untreated, C: Control siRNA, W: WT1 siRNA. Scale bars represent 100 µm. One-way ANOVA was used as statistical test for group’s comparison. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: siRNA sequences were commercially acquired from Sigma-Aldrich, including the WT1 -silencing siRNA from the reported sequence (5′-AAGCUGUCCCACUUACAGAUGCdTdT-3′) and a universal negative control siRNA (Sigma-Aldrich, Cat. SIC001).

Techniques: Activation Assay, Cytometry, Alamar Blue Assay, TUNEL Assay, Microscopy

Journal: Non-Coding RNA

Article Title: Therapeutic Effects of WT1 Silencing via Respiratory Administration of Neutral DOPC Liposomal-siRNA in a Lung Metastasis Melanoma Murine Model

doi: 10.3390/ncrna9020021

Figure Lengend Snippet: Respiratory administration of liposomal WT1 siRNA reduces the tumor size by decreasing WT1 protein expression in a lung metastatic melanoma model in mice. ( A ) L-WT1 siRNA administration reduces the size of the tumor and number of metastases in the lungs after 2 weeks of treatment. ( B ) There is a decrease in the weight of the lungs treated with L-WT1 siRNA when compared with control groups. Dot line represents average weight of a healthy tumor as a reference. ( C ) WT-1 protein expression is reduced in the lung’s melanoma metastases of the L-WT1 siRNA-treated group observed via a WB and ( D ) WT-1 quantification via densitometry of the bands. Groups L-E: L-Empty, L-C: L-Control siRNA, L-W: L-WT1 siRNA. One-way ANOVA was used as statistical test for group’s comparison. *** p < 0.001.

Article Snippet: siRNA sequences were commercially acquired from Sigma-Aldrich, including the WT1 -silencing siRNA from the reported sequence (5′-AAGCUGUCCCACUUACAGAUGCdTdT-3′) and a universal negative control siRNA (Sigma-Aldrich, Cat. SIC001).

Techniques: Expressing

Journal: Non-Coding RNA

Article Title: Therapeutic Effects of WT1 Silencing via Respiratory Administration of Neutral DOPC Liposomal-siRNA in a Lung Metastasis Melanoma Murine Model

doi: 10.3390/ncrna9020021

Figure Lengend Snippet: Survival assay for melanoma lung cancer metastasis model in mice treated with respiratory L-WT1 siRNA. Respiratory therapy of L-WT1 siRNA twice per week improved the survival rate of the treated animals. Log-rank test was performed as a statistical analysis. *** p < 0.001.

Article Snippet: siRNA sequences were commercially acquired from Sigma-Aldrich, including the WT1 -silencing siRNA from the reported sequence (5′-AAGCUGUCCCACUUACAGAUGCdTdT-3′) and a universal negative control siRNA (Sigma-Aldrich, Cat. SIC001).

Techniques: Clonogenic Cell Survival Assay

Journal: Open Medicine

Article Title: Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis

doi: 10.1515/med-2022-0595

Figure Lengend Snippet: Primer sequences for PCR

Article Snippet: HTR-8/Svneo cells (5 × 10 4 cells per well) were cultured in six-well plates overnight and subsequently transfected with control plasmid, control-small interfering (si) RNA (Guangzhou Ribobio Co., Ltd., China), 100 nM inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Shanghai GenePharma Co., Ltd., China), 100 nM mimic control (5′-UUCUCCGAACGUGUCACGUTT-3′; Shanghai GenePharma Co., Ltd.), WT1-AS plasmid, WT1-AS-siRNA (cat no. siG180524011008-1-5; Guangzhou Ribobio Co., Ltd.; https://www.ribobio.com/product_detail/?sku=siG180524011008-1-5 ), 100 nM miR-186-5p inhibitor (5′-AGCCCAAAAGGAGAAUUCUUUG-3′; Shanghai GenePharma Co., Ltd.), 100 nM miR-186-5p mimic (5′-CAAAGAAUUCUCCUUUUGGGCU-3′; Shanghai GenePharma Co., Ltd.), or CADM2 plasmid for 48 h, using Lipofectamine ® 2000 reagent (Invitrogen, USA), according to the manufacturer’s protocol.

Techniques:

Journal: Open Medicine

Article Title: Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis

doi: 10.1515/med-2022-0595

Figure Lengend Snippet: Effects of WT1-AS-plasmid on HTR-8/SVneo cell viability, migration, and invasion. (a) qRT-PCR analysis was used to determine the transfection efficiency of control plasmid and WT1-AS plasmid in HTR-8/SVneo cells. (b) The viability of HTR-8/SVneo cells was determined using the MTT assay. HTR-8/SVneo cell migration (c) and invasion (e) (magnification: ×100; bar = 100 μm) were assessed by Transwell assay. The number of migratory cells (d) and invasive cells (f) were determined. ** P < 0.01 vs control plasmid. WT1-AS, WT1 antisense RNA.

Article Snippet: HTR-8/Svneo cells (5 × 10 4 cells per well) were cultured in six-well plates overnight and subsequently transfected with control plasmid, control-small interfering (si) RNA (Guangzhou Ribobio Co., Ltd., China), 100 nM inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Shanghai GenePharma Co., Ltd., China), 100 nM mimic control (5′-UUCUCCGAACGUGUCACGUTT-3′; Shanghai GenePharma Co., Ltd.), WT1-AS plasmid, WT1-AS-siRNA (cat no. siG180524011008-1-5; Guangzhou Ribobio Co., Ltd.; https://www.ribobio.com/product_detail/?sku=siG180524011008-1-5 ), 100 nM miR-186-5p inhibitor (5′-AGCCCAAAAGGAGAAUUCUUUG-3′; Shanghai GenePharma Co., Ltd.), 100 nM miR-186-5p mimic (5′-CAAAGAAUUCUCCUUUUGGGCU-3′; Shanghai GenePharma Co., Ltd.), or CADM2 plasmid for 48 h, using Lipofectamine ® 2000 reagent (Invitrogen, USA), according to the manufacturer’s protocol.

Techniques: Plasmid Preparation, Migration, Quantitative RT-PCR, Transfection, MTT Assay, Transwell Assay

Journal: Open Medicine

Article Title: Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis

doi: 10.1515/med-2022-0595

Figure Lengend Snippet: Effects of WT1-AS plasmid on apoptosis of HTR-8/SVneo cells. (a and b) Flow cytometry analysis of apoptotic HTR-8/SVneo cells. ** P < 0.01 vs control plasmid. WT1-AS, WT1 antisense RNA.

Article Snippet: HTR-8/Svneo cells (5 × 10 4 cells per well) were cultured in six-well plates overnight and subsequently transfected with control plasmid, control-small interfering (si) RNA (Guangzhou Ribobio Co., Ltd., China), 100 nM inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Shanghai GenePharma Co., Ltd., China), 100 nM mimic control (5′-UUCUCCGAACGUGUCACGUTT-3′; Shanghai GenePharma Co., Ltd.), WT1-AS plasmid, WT1-AS-siRNA (cat no. siG180524011008-1-5; Guangzhou Ribobio Co., Ltd.; https://www.ribobio.com/product_detail/?sku=siG180524011008-1-5 ), 100 nM miR-186-5p inhibitor (5′-AGCCCAAAAGGAGAAUUCUUUG-3′; Shanghai GenePharma Co., Ltd.), 100 nM miR-186-5p mimic (5′-CAAAGAAUUCUCCUUUUGGGCU-3′; Shanghai GenePharma Co., Ltd.), or CADM2 plasmid for 48 h, using Lipofectamine ® 2000 reagent (Invitrogen, USA), according to the manufacturer’s protocol.

Techniques: Plasmid Preparation, Flow Cytometry

Journal: Open Medicine

Article Title: Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis

doi: 10.1515/med-2022-0595

Figure Lengend Snippet: miR-186-5p directly targets lncRNA WT1-AS. (a) A schematic of miR-186a-5p binding site in lncRNA WT1-AS 3′-UTR. (b) The interaction between miR-186a-5p and lncRNA WT1-AS was confirmed via dual-luciferase reporter assay. ** P < 0.01 vs mimic control. miR, microRNA; lncRNA, long noncoding RNA; WT1-AS, WT1 antisense RNA; WT, wild-type; MUT, mutant; UTR, untranslated region.

Article Snippet: HTR-8/Svneo cells (5 × 10 4 cells per well) were cultured in six-well plates overnight and subsequently transfected with control plasmid, control-small interfering (si) RNA (Guangzhou Ribobio Co., Ltd., China), 100 nM inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Shanghai GenePharma Co., Ltd., China), 100 nM mimic control (5′-UUCUCCGAACGUGUCACGUTT-3′; Shanghai GenePharma Co., Ltd.), WT1-AS plasmid, WT1-AS-siRNA (cat no. siG180524011008-1-5; Guangzhou Ribobio Co., Ltd.; https://www.ribobio.com/product_detail/?sku=siG180524011008-1-5 ), 100 nM miR-186-5p inhibitor (5′-AGCCCAAAAGGAGAAUUCUUUG-3′; Shanghai GenePharma Co., Ltd.), 100 nM miR-186-5p mimic (5′-CAAAGAAUUCUCCUUUUGGGCU-3′; Shanghai GenePharma Co., Ltd.), or CADM2 plasmid for 48 h, using Lipofectamine ® 2000 reagent (Invitrogen, USA), according to the manufacturer’s protocol.

Techniques: Binding Assay, Luciferase, Reporter Assay, Mutagenesis

Journal: Open Medicine

Article Title: Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis

doi: 10.1515/med-2022-0595

Figure Lengend Snippet: WT1-AS knockdown upregulates miR-186a-5p expression in HTR-8/SVneo cells. (a) qRT-PCR analysis of WT1-AS in HTR-8/SVneo cells transfected with control siRNA or WT1-AS siRNA. (b) Determination of miR-186-5p levels in HTR-8/SVneo cells transfected with inhibitor control or miR-186-5p inhibitor. (c) mRNA expression of miR-186-5p in HTR-8/SVneo cells transfected with control siRNA, WT1-AS-siRNA, WT1-AS-siRNA + inhibitor control, or WT1-AS-siRNA + miR-186-5p inhibitor was determined using qRT-PCR. ** P < 0.01 vs control siRNA; ## P < 0.01 vs inhibitor control; && P < 0.01 vs WT1-AS-siRNA + inhibitor control. WT1-AS, WT1 antisense RNA; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering.

Article Snippet: HTR-8/Svneo cells (5 × 10 4 cells per well) were cultured in six-well plates overnight and subsequently transfected with control plasmid, control-small interfering (si) RNA (Guangzhou Ribobio Co., Ltd., China), 100 nM inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Shanghai GenePharma Co., Ltd., China), 100 nM mimic control (5′-UUCUCCGAACGUGUCACGUTT-3′; Shanghai GenePharma Co., Ltd.), WT1-AS plasmid, WT1-AS-siRNA (cat no. siG180524011008-1-5; Guangzhou Ribobio Co., Ltd.; https://www.ribobio.com/product_detail/?sku=siG180524011008-1-5 ), 100 nM miR-186-5p inhibitor (5′-AGCCCAAAAGGAGAAUUCUUUG-3′; Shanghai GenePharma Co., Ltd.), 100 nM miR-186-5p mimic (5′-CAAAGAAUUCUCCUUUUGGGCU-3′; Shanghai GenePharma Co., Ltd.), or CADM2 plasmid for 48 h, using Lipofectamine ® 2000 reagent (Invitrogen, USA), according to the manufacturer’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Real-time Polymerase Chain Reaction

Journal: Open Medicine

Article Title: Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis

doi: 10.1515/med-2022-0595

Figure Lengend Snippet: Effects of WT1-AS-siRNA on the function of HTR-8/SVneo cells. (a) Viability of HTR-8/SVneo cells was checked by the MTT assay. HTR-8/SVneo cell migration (b) and invasion (d) (magnification: ×100; bar = 100 μm) were evaluated using Transwell assay. The number of migratory cells (c) and invasive cells (e) were determined. (f and g) Flow cytometry was applied to assess apoptosis of HTR-8/SVneo cells. One-way ANOVA followed by Tukey’s post hoc test was used for data analysis. ** P < 0.01 vs control-siRNA; ## P < 0.01 vs WT1-AS-siRNA + inhibitor control. WT1-AS, WT1 antisense RNA; si, small interfering.

Article Snippet: HTR-8/Svneo cells (5 × 10 4 cells per well) were cultured in six-well plates overnight and subsequently transfected with control plasmid, control-small interfering (si) RNA (Guangzhou Ribobio Co., Ltd., China), 100 nM inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Shanghai GenePharma Co., Ltd., China), 100 nM mimic control (5′-UUCUCCGAACGUGUCACGUTT-3′; Shanghai GenePharma Co., Ltd.), WT1-AS plasmid, WT1-AS-siRNA (cat no. siG180524011008-1-5; Guangzhou Ribobio Co., Ltd.; https://www.ribobio.com/product_detail/?sku=siG180524011008-1-5 ), 100 nM miR-186-5p inhibitor (5′-AGCCCAAAAGGAGAAUUCUUUG-3′; Shanghai GenePharma Co., Ltd.), 100 nM miR-186-5p mimic (5′-CAAAGAAUUCUCCUUUUGGGCU-3′; Shanghai GenePharma Co., Ltd.), or CADM2 plasmid for 48 h, using Lipofectamine ® 2000 reagent (Invitrogen, USA), according to the manufacturer’s protocol.

Techniques: MTT Assay, Migration, Transwell Assay, Flow Cytometry

Journal: Open Medicine

Article Title: Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis

doi: 10.1515/med-2022-0595

Figure Lengend Snippet: WT1-AS-siRNA-reduced CADM2 expression in HTR-8/SVneo cells. HTR-8/SVneo cells were transfected with control siRNA, WT1-AS siRNA, WT1-AS siRNA + inhibitor control, or WT1-AS siRNA + miR-186-5p inhibitor. (a) Protein expression of CADM2 in HTR-8/SVneo cells was determined using western blotting. (b) mRNA expression of CADM2 in HTR-8/SVneo cells was determined using RT-qPCR. ** P < 0.01 vs control siRNA; ## P < 0.01 vs WT1-AS siRNA + inhibitor control. WT1-AS, WT1 antisense RNA; si, small interfering; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HTR-8/Svneo cells (5 × 10 4 cells per well) were cultured in six-well plates overnight and subsequently transfected with control plasmid, control-small interfering (si) RNA (Guangzhou Ribobio Co., Ltd., China), 100 nM inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Shanghai GenePharma Co., Ltd., China), 100 nM mimic control (5′-UUCUCCGAACGUGUCACGUTT-3′; Shanghai GenePharma Co., Ltd.), WT1-AS plasmid, WT1-AS-siRNA (cat no. siG180524011008-1-5; Guangzhou Ribobio Co., Ltd.; https://www.ribobio.com/product_detail/?sku=siG180524011008-1-5 ), 100 nM miR-186-5p inhibitor (5′-AGCCCAAAAGGAGAAUUCUUUG-3′; Shanghai GenePharma Co., Ltd.), 100 nM miR-186-5p mimic (5′-CAAAGAAUUCUCCUUUUGGGCU-3′; Shanghai GenePharma Co., Ltd.), or CADM2 plasmid for 48 h, using Lipofectamine ® 2000 reagent (Invitrogen, USA), according to the manufacturer’s protocol.

Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Autophagy

Article Title: Autophagy regulates differentiation of ovarian granulosa cells through degradation of WT1

doi: 10.1080/15548627.2021.2005415

Figure Lengend Snippet: Knockdown of ATG5 and BECN1 inhibited autophagy in GCs. The negative control siRNA (NC), ATG5 siRNA (si ATG5 ), and BECN1 siRNA (si BECN1 ) were transfected into KGN cells, and 48 h later the knockdown efficiencies were determined in both mRNA (A) and protein (B) levels. GAPDH and ACTB/β-actin served as the internal control for mRNA and protein, respectively. Data are presented as mean ± SD, n = 3, * P < 0.05, ** P < 0.01 vs. NC. (C) The expression of LC3 and SQSTM1 proteins in these three groups was detected by Western blot. ACTB was used as the loading control. Quantitative analysis showed lower LC3-II:LC3-1 ratios (D) and higher levels of SQSTM1 (E) in the si ATG5 and si BECN1 groups, compared to the NC group. Data are presented as mean ± SD, n = 3, * P < 0.05, ** P < 0.01. (F) KGN cells were transfected with NC, si ATG5 , and si BECN1 for 24 h, followed by infection with LC3 reporter adenovirus for 48 h. The distribution of green (GFP) LC3 puncta was observed under a fluorescence microscope after starvation for 6 h. Cell nuclei were counterstained with Hoechst 33342. Scale bar: 5 μm. (G) Quantification of green LC3 puncta in KGN cells of each group. Data are presented as mean ± SD, n = 3, *** P < 0.001.

Article Snippet: The siRNAs for silencing the expression of ATG5, BECN1 , and WT1 , as well as negative control siRNA, were designed and produced by Genepharma Inc. (Shanghai, China) and were transfected using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, L3000015) into KGN cells at 80 nM or 100 nM when the cell confluence reached 80%.

Techniques: Knockdown, Negative Control, Transfection, Control, Expressing, Western Blot, Infection, Fluorescence, Microscopy

Journal: Autophagy

Article Title: Autophagy regulates differentiation of ovarian granulosa cells through degradation of WT1

doi: 10.1080/15548627.2021.2005415

Figure Lengend Snippet: Autophagy inhibition leads to insufficient differentiation of GCs. (A) After siRNA transfection of KGN cells for 48 h, the mRNA expression of the genes related to GC differentiation and steroidogenesis, including CYP19A1, FSHR, GATA4, GATA6, INHBA, SF1, SP1, HSD3B1 , and STAR , showed significant downregulation in both the si ATG5 and si BECN1 groups. GAPDH served as the internal control. Data are presented as mean ± SD, n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001. vs. NC. (B) After treatment of KGN cells with CQ, the mRNA expression of CYP19A1 and FSHR was downregulated compared to controls. GAPDH served as the internal control. Data are presented as mean ± SD, n = 3. ** P < 0.01 vs. CON. (C and D) Protein levels of CYP19A1 and FSHR were decreased in KGNs transfected with si ATG5 and si BECN1 or pretreated with CQ as measured by Western blot. ACTB was used as the loading control. (E) E 2 production in KGN cell supernatant was decreased in both the si ATG5 (6.32 ± 1.41 ng/mg) and si BECN1 (8.81 ± 0.56 ng/mg) groups compared to the NC group (15.33 ± 0.36 ng/mg). Data are presented as mean ± SD, n = 3, ** P < 0.01 vs. NC. (F) E 2 production in CQ-treated KGN cells (7.96 ± 0.47 ng/mg) was lower than that in the control group (18.11 ± 1.10 ng/mg). Data are presented as mean ± SD, n = 3, ** P < 0.01 vs. CON. (G) Mouse GCs were harvested and cultured in vitro . After 48 h treatment with 50 μM CQ, the protein levels of CYP19A1 and FSHR were lower than those in the control group. GAPDH was used as the loading control. (H) The E 2 production was decreased in CQ-treated mouse GCs (0.12 ± 0.03 ng/mg) compared to the control group (0.82 ± 0.18 ng/mg). Data are presented as mean ± SD, n = 3, ** P < 0.01 vs. CON.

Article Snippet: The siRNAs for silencing the expression of ATG5, BECN1 , and WT1 , as well as negative control siRNA, were designed and produced by Genepharma Inc. (Shanghai, China) and were transfected using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, L3000015) into KGN cells at 80 nM or 100 nM when the cell confluence reached 80%.

Techniques: Inhibition, Transfection, Expressing, Control, Western Blot, Cell Culture, In Vitro

Journal: Autophagy

Article Title: Autophagy regulates differentiation of ovarian granulosa cells through degradation of WT1

doi: 10.1080/15548627.2021.2005415

Figure Lengend Snippet: Accumulation of WT1 in GCs results from autophagy inhibition. (A) Western blot showed that, compared to NC, there was an increase in WT1 protein in both the si ATG5 and si BECN1 groups, but there were no significant changes in FOXO1 protein. GAPDH was used as the loading control. (B) The qRT-PCR results showed that, compared to NC, the WT1 mRNA did not change significantly and FOXO1 mRNA decreased in the si ATG5 group, while both WT1 and FOXO1 mRNA decreased significantly in the si BECN1 group. Data are presented as mean ± SD, n = 3, * P < 0.05, ** P < 0.01 vs. NC. (C) After 0 h, 2 h, 4 h, and 6 h of CHX treatment to inhibit protein synthesis, Western blot was performed to detect the remaining amount of WT1 protein in KGN cells transfected with NC, si ATG5 , and si BECN1 . GAPDH was used as the loading control. (D) The results of the quantitative analysis showed that the degradation rate of the WT1 protein in the si ATG5 and si BECN1 groups was significantly slower compared to the NC. Data are presented as mean ± SD, n = 3. (E) Compared to control, there was a significant increase in WT1 protein level in KGN cells treated with CQ, but no significant change in FOXO1 protein. GAPDH was used as the loading control. (F and G) The degradation rate of WT1 protein in KGN cells pretreated with CQ was also delayed significantly. GAPDH was used as the loading control. Data are presented as mean ± SD, n = 3. It was noted that because the CHX chase assay was carried out after 48 h of siRNA infection or CQ treatment, the WT1 and SQSTM1 proteins had already accumulated in the siRNA or CQ group at 0 h (C and F). (H) Compared to control, the WT1 protein in the nucleus was elevated in KGN cells pretreated with CQ. LMNB1 and GAPDH were used as the loading controls for the nuclear and cytoplasmic proteins, respectively.

Article Snippet: The siRNAs for silencing the expression of ATG5, BECN1 , and WT1 , as well as negative control siRNA, were designed and produced by Genepharma Inc. (Shanghai, China) and were transfected using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, L3000015) into KGN cells at 80 nM or 100 nM when the cell confluence reached 80%.

Techniques: Inhibition, Western Blot, Control, Quantitative RT-PCR, Transfection, Infection

Journal: Autophagy

Article Title: Autophagy regulates differentiation of ovarian granulosa cells through degradation of WT1

doi: 10.1080/15548627.2021.2005415

Figure Lengend Snippet: Antibody information.

Article Snippet: The siRNAs for silencing the expression of ATG5, BECN1 , and WT1 , as well as negative control siRNA, were designed and produced by Genepharma Inc. (Shanghai, China) and were transfected using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, L3000015) into KGN cells at 80 nM or 100 nM when the cell confluence reached 80%.

Techniques: