Journal: Nucleic acids research

Article Title: A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation.

doi: 10.1093/nar/gkae370

Figure Lengend Snippet: Figure 2. A-MYB regulates Cyclin B2 by indirectly binding to its CHR promoter element through MuvB. ( A ) siRNA knockdown was performed in HCT116 ( n = 5) and U2OS ( n = 4) cells. Tw enty -f our hours af ter knoc kdo wn, cells w ere transfected with luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates. Differences between B-MYB KD and A-MYB + B-MYB dKD are statistically not significant. ( B ) To rescue HCT116 cells from siRNA knockdown of A-MYB and B-MYB , cells were transfected with the mouse A-Myb-overexpressing plasmid mA-Myb-GFP or the empty vector pEGFP as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter in parallel to the siRNA transfections. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 3). ( C ) For luciferase assa y s after mouse A-Myb o v ere xpression, HCT116, RPE-1, Hep3B or HeLa cells were transfected with the mAmyb-GFP overexpression vector as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 12). ( D ) Binding of A-MYB to LIN37 was tested in CoIP assays with HCT116 native protein extracts. As controls, E2F4 representing the DREAM complex and B-MYB as MMB component were precipitated (representative experiment of n = 4). ( E ) Binding of A-MYB to the MuvB core components LIN9, LIN37 and LIN54 was tested in CoIP assays with RPE-1 native protein extracts. As controls, LIN9 and LIN37 representing the MuvB core complex and B-MYB as an MMB component were precipitated (representative experiment of n = 3). ( F ) Binding of the MuvB core to A-MYB was tested in CoIP assa y s with RPE-1 native protein extracts. As negative control, p130 representing the DREAM complex was precipitated (representative experiment of n = 3). ( G ) T98G cells were synchronized in the cell cycle by density arrest and released for 15 h or 24 h into the cell cycle to obtain cell populations enriched in G 0 , S and G 2 / M phase, respectively. From each time point, native protein extracts were isolated and subjected to a LIN37 CoIP. Binding of DREAM and MMB components B-MYB, E2F4, and LIN37 as well as A-MYB was analyzed by Western blot (representative from n = 3). ( H ) Binding of A-MYB to wild-type (wt) and CHR mutant (CHRmut) Cyclin B2 promoter probes in DNA-affinity purification was analyzed by Western blot (representative of n = 3). Mean ± SD are given, and significances were calculated by two-way ANO V A (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Cell culture and cell count analysis Human fibroblast BJ cells (DSMZ, Braunschweig, Germany), human colorectal carcinoma HCT116 cells (provided by Bert Vogelstein), human embryonic kidney HEK293 cells (DSMZ), human papillomavirus-related cervical adenocarcinoma HeLa cells (DSMZ), human pediatric hepatocellular carcinoma Hep3B cells (DSMZ), human retinal pigment epithelial hTERT immortalized RPE-1 cells (DSMZ), human glioblastoma T98G cells (DSMZ), and human bone osteosarcoma U2OS cells (DSMZ) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Lonza).

Techniques: Binding Assay, Knockdown, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation, Over Expression, Negative Control, Isolation, Western Blot, Mutagenesis, Affinity Purification

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: PRR34-AS1 expression is elevated in HCC cells (A) UCSC database presented PRR34-AS1 expression profile in multiple human normal tissues, including liver tissues. (B) NONCODE database indicated the profile of PRR34-AS1 expression in different normal tissue samples. (C) GEPIA2 database exhibited PRR34-AS1 expression pattern in 369 cases of LIHC tissues and 160 cases of normal tissues. (D) Quantitative real-time RT-PCR analyzed PRR34-AS1 expression in HCC cell lines (Hep 3B, SK-HEP-1, Huh7, MHCC97-H, and HCCLM3) and human normal hepatocyte cell line (THLE-3). (E and F) Subcellular fractionation and FISH experiments determined PRR34-AS1 location in HCC cells. (G) Knockdown efficiencies of sh-PRR34-AS1#1&2&3 in MHCC97-H and HCCLM3 cells, as well as overexpression efficiency of pcDNA3.1/PRR34-AS1 in Hep 3B cells, were evaluated via quantitative real-time RT-PCR. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR, Fractionation, Knockdown, Over Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: PRR34-AS1 facilitates HCC cell proliferation in vitro and tumor growth in vivo (A) CCK-8 assays tested the changes in cell proliferation ability induced by PRR34-AS1 silencing in MHCC97-H and HCCLM3 cells and induced by PRR34-AS1 upregulation in Hep 3B cells. (B) Colony formation assays measured the number of colonies formed under PRR34-AS1 silencing in MHCC97-H, HCCLM3 cells, or under PRR34-AS1 upregulation in Hep 3B cells. (C and D) TUNEL assays and flow cytometry analysis assessed the apoptosis rate when PRR34-AS1 was downregulated in HCCLM3 cells (E) Representative pictures of xenografts derived from HCCLM3 cells transfected with sh-NC or sh-PRR34-AS1#1. (F) The grow curves of tumors from two different groups. (G and H) The final tumor volume and weight were measured. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vitro, In Vivo, CCK-8 Assay, TUNEL Assay, Flow Cytometry, Derivative Assay, Transfection

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: PRR34-AS1 contributes to cell migration, invasion, and EMT process in HCC cells and activates the Wnt/β-catenin pathway (A) Wound healing assays detected the migration ability of indicated HCC cells when PRR34-AS1 was knocked down or upregulated. (B and C) Transwell experiments analyzed the migration and invasion properties in PRR34-AS1 silenced MHCC97-H and HCCLM3 cells, as well as in PRR34-AS1 upregulated Hep 3B cells. (D) Western blot assay examined the protein levels of E-cadherin, N-cadherin, and Vimentin in HCC cells under PRR34-AS1 depletion or overexpression. (E) H&E staining evaluated the metastatic ability of HCCLM3 cells after PRR34-AS1 depletion. (F) TOP Flash/FOP Flash reporter assays assessed the activity of Wnt/β-catenin in HCC cells with PRR34-AS1 depletion or overexpression. (G–I) Western blot evaluated the levels of indicated proteins in HCC cells after PRR34-AS1 depletion or overexpression. ∗p < 0.05.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Migration, Western Blot, Over Expression, Staining, Activity Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: PRR34-AS1 sponges miR-296-5p in HCC cells (A) LncBase database was used to predict underlying miRNAs that could combine with PRR34-AS1. (B) RNA pull-down assays assessed the abundance of candidate miRNAs in biotinylated PRR34-AS1 group in Hep 3B cells. (C) ENCORI database predicted the underlying binding sites between PRR34-AS1 and miR-296-5p. (D) Quantitative real-time RT-PCR analyzed miR-296-5p expression pattern in HCC cells (Hep 3B, SK-HEP-1, Huh7, MHCC97-H, and HCCLM3) and human normal hepatocyte cells (THLE-3). (E) RIP assays assessed the abundance of PRR34-AS1 and miR-296-5p in AGO2 complex. (F) Luciferase reporter assays detected the luciferase activity of PRR34-AS1-WT or PRR34-AS1-Mut under miR-296-5p upregulation in HCC cells. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Luciferase, Activity Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: PRR34-AS1 aggravates HCC cell proliferation, migration, and EMT through targeting miR-296-5p (A) Quantitative real-time RT-PCR detected the expression of miR-296-5p in HCCLM3 cells under indicated transfections. (B and C) CCK-8 and colony formation assays examined the proliferation ability of indicated HCCLM3 cells. (D and E) TUNEL and flow cytometry experiments analyzed the apoptosis rate of indicated HCCLM3 cells. (F–H) Wound healing and Transwell assays detected the migratory and invasive capacities of indicated HCCLM3 cells. (I) Western blot examined the protein levels of E-cadherin, N-cadherin, and Vimentin in HCCLM3 cells under different conditions. ∗p < 0.05.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Migration, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, TUNEL Assay, Flow Cytometry, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: E2F2 and SOX12 are identified as the targets of miR-296-5p in HCC cells (A) The mirDIP, microT, PITA, and miRmap databases were used to screen underlying mRNAs that potentially combined with miR-296-5p. (B) Quantitative real-time RT-PCR analyzed the expression of candidate targets (ZNF76, HMGA1, FAM53B, FGFR3, E2F2, HIPK1, SOX12, CDK16, BMF, and SLC16A3) in PRR34-AS1 silenced HCCLM3 cells. (C) ENCORI predicted the binding sites between E2F2/SOX12 and miR-296-5p. (D) Quantitative real-time RT-PCR detected E2F2 and SOX12 mRNA levels in HCC cells (Hep 3B, SK-HEP-1, Huh7, MHCC97-H, and HCCLM3) and THLE-3 cells. (E) Western blot assay analyzed the protein levels of E2F2 and SOX12 in PRR34-AS1 silenced or overexpressed HCC cells. (F) RIP assays assessed the enrichments of PRR34-AS1, miR-296-5p, and E2F2/SOX12 in anti-AGO2 complex. (G) Luciferase reporter experiments assessed the luciferase activity of E2F2-3′ UTR-WT/Mut and SOX12-WT/Mut in HEK293T cells with different transfections. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Expressing, Binding Assay, Western Blot, Luciferase, Activity Assay, Transfection

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12

doi: 10.1016/j.omtn.2021.04.016

Figure Lengend Snippet: E2F2 activates PRR34-AS1 transcription in HCC cells (A) Western blot analyzed the knockdown or overexpression efficiencies of E2F2 in HCC cells. (B and C) The levels of E2F2 and PRR34-AS1 in indicated HCC cells were examined via quantitative real-time RT-PCR. (D) ChIP experiments measured the connection between PRR34-AS1 promoter and E2F2 in HCC cells. (E) The binding motif of E2F2 were predicted by JASPAR. (F) Luciferase reporter experiments assessed the luciferase activities of PRR34-AS1-Pro-WT and PRR34-AS1-Pro-Mut in HCC cells after E2F2 expression was increased or silenced. ∗∗p < 0.01.

Article Snippet: Human HCC cell lines (Hep 3B and SK-HEP-1), human normal hepatocyte cell line (THLE-3), and human embryonic kidney cell line (HEK293T) were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Western Blot, Knockdown, Over Expression, Quantitative RT-PCR, Binding Assay, Luciferase, Expressing

Journal: Journal of Gastrointestinal Oncology

Article Title: The deficiency of FKBP-5 inhibited hepatocellular progression by increasing the infiltration of distinct immune cells and inhibiting obesity-associated gut microbial metabolite

doi: 10.21037/jgo-21-71

Figure Lengend Snippet: FKBP-5 was highly expressed in HCC. (A) The expression of FKBP-5 was examined by the IHC staining of para-carcinoma tissues and HCC tissues. The representative images were displayed. Magnification, 100×; Scale bar =100 µm. (B) The western blotting analysis of FKBP-5 expression in HCC tissues (He) and para-carcinoma tissues (Pa), GAPDH was used as an internal reference. (C) The mRNA expression level of FKBP-5 was confirmed by RT-qPCR assay in HL-7702 and five HCC cells (SMMC-7721, Hep 3B, Huh7, Hep G2, and LO2), *, P<0.05; ***, P<0.001 vs. HL-7702 cells.

Article Snippet: Cell culture Human liver (HL)-7702 and HCC cells (i.e., SMMC-7721, Hep 3B, Huh7, Hep G2, and LO2) were purchased from the American Type Culture Collection.

Techniques: Expressing, Immunohistochemistry, Western Blot, Quantitative RT-PCR

Journal: OncoTargets and therapy

Article Title: circ_0003418 Inhibits Tumorigenesis And Cisplatin Chemoresistance Through Wnt/β-Catenin Pathway In Hepatocellular Carcinoma

doi: 10.2147/OTT.S229507

Figure Lengend Snippet: circ_0003418 downregulation in HCC tissues and cells. Notes: ( A ) The expression profile of circ_0003418 in HCC tissues and adjacent noncancerous tissues were detected by qRT-PCR assays. ( B ) qRT-PCR was used to analyze the level of circ_0003418 in five HCC cell lines and one normal human hepatocyte cell line. ( C and D ) The knockdown efficiency of LV3-circ_0003418 on circ_0003418 in Huh-7 and Hep-3B cells was verified via qRT-PCR. ** P<0.01 and *** P<0.001 compared to control group. Abbreviations: HCC, hepatocellular carcinoma; NC, negative control.

Article Snippet: Human HCC cell lines (Hep-3B, Huh-7, Sk-hep-1, SMMC-7721 and PLC) and normal human hepatocyte line (HL-77O2) were purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Control, Negative Control

Journal: OncoTargets and therapy

Article Title: circ_0003418 Inhibits Tumorigenesis And Cisplatin Chemoresistance Through Wnt/β-Catenin Pathway In Hepatocellular Carcinoma

doi: 10.2147/OTT.S229507

Figure Lengend Snippet: circ_0003418 suppresses proliferation, migration, and invasion and promotes apoptosis in HCC cells. Notes: ( A and B ) CCK-8 assays were performed to measure the effect of silencing circ_0003418 on the proliferation in Huh-7 and Hep-3B cells. ( C–F ) Effect of silencing circ_0003418 on cell migration ( C and D ) and invasion ( E and F ) were analyzed by transwell migration and invasion assays, respectively. * P<0.05, ** P<0.01 and *** P<0.001 compared to control group. Abbreviations: CCK-8, cell counting kit 8; NC, negative control.

Article Snippet: Human HCC cell lines (Hep-3B, Huh-7, Sk-hep-1, SMMC-7721 and PLC) and normal human hepatocyte line (HL-77O2) were purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Migration, CCK-8 Assay, Control, Cell Counting, Negative Control

Journal: OncoTargets and therapy

Article Title: circ_0003418 Inhibits Tumorigenesis And Cisplatin Chemoresistance Through Wnt/β-Catenin Pathway In Hepatocellular Carcinoma

doi: 10.2147/OTT.S229507

Figure Lengend Snippet: circ-0003418 sensitizes HCC cells to cisplatin in vitro. Notes: ( A and B ) Huh-7 and Hep-3B cells infected with LV3-NC or LV3-circ_0003418 were treated with different doses of cisplatin (1, 2, 4, 8, 16, 32, 64 and 128 mg/L) for 24 hrs, and then cell viability was determined by CCK-8 assays. ( C–F ) Huh-7 cells were treated with cisplatin (11.39 mg/L) for 24 hrs as well as Hep-3B cells were treated with cisplatin (20.18 mg/L) for 24 hrs, and then cell migration and invasion were detected by transwell migration ( C and D ) and invasion ( E and F ) assays, respectively. * P<0.05, ** P<0.01 and *** P<0.001 compared to control group. Abbreviation: NC, negative control.

Article Snippet: Human HCC cell lines (Hep-3B, Huh-7, Sk-hep-1, SMMC-7721 and PLC) and normal human hepatocyte line (HL-77O2) were purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: In Vitro, Infection, CCK-8 Assay, Migration, Control, Negative Control

Journal: OncoTargets and therapy

Article Title: circ_0003418 Inhibits Tumorigenesis And Cisplatin Chemoresistance Through Wnt/β-Catenin Pathway In Hepatocellular Carcinoma

doi: 10.2147/OTT.S229507

Figure Lengend Snippet: circ-0003418 enhances sensitivity of HCC cells to cisplatin in vivo. Female BALB/c nude mice were implanted subcutaneously with Huh-7 cells infected with LV3-NC or LV3-circ_0003418. Twelve days later, the LV3-NC tumor-bearing mice were treated with saline or cisplatin (5 mg/kg) by intraperitoneal injection twice a week up to 36 days posttreatment. The mice bearing LV3-circ_0003418 tumors received the same treatment. Notes: ( A ) Image of the tumors in the nude mice. ( B ) The tumors growth curve of xenograft model mice. ( C and D ) The weight and volume of the tumors in the nude mice. * P<0.05, ** P<0.01 and *** P<0.001 compared to control group. Abbreviation: NC, negative control.

Article Snippet: Human HCC cell lines (Hep-3B, Huh-7, Sk-hep-1, SMMC-7721 and PLC) and normal human hepatocyte line (HL-77O2) were purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: In Vivo, Infection, Saline, Injection, Control, Negative Control

Journal: OncoTargets and therapy

Article Title: circ_0003418 Inhibits Tumorigenesis And Cisplatin Chemoresistance Through Wnt/β-Catenin Pathway In Hepatocellular Carcinoma

doi: 10.2147/OTT.S229507

Figure Lengend Snippet: Silencing circ_0003418 induces cisplatin resistance of HCC cells through activating the Wnt/β-catenin pathway. Notes: ( A and B ) Huh-7 cells were treated with cisplatin (11.39 mg/L) for 24 hrs as well as Hep-3B cells were treated with cisplatin (20.18 mg/L) for 24 hrs, and then the protein levels of β-catenin and c-Myc in the Huh-7 and Hep-3B cells were detected by Western blotting. ( C and D ) The inhibition efficiency of ICG-001 was detected by Western blotting. ( E and F ) Cell proliferation assay showed that inhibition of Wnt/β-catenin pathway in cells infected with LV3-circ_0003418 inhibited cell proliferation. ( G and H ) Chemotherapy sensitivity assay showed that inhibition of Wnt/β-catenin pathway in cells infected with LV3-circ_0003418 enhanced sensitivity of HCC cells to cisplatin. * P<0.05, ** P<0.01 and *** P<0.001 compared to control group. Abbreviation: NC, negative control.

Article Snippet: Human HCC cell lines (Hep-3B, Huh-7, Sk-hep-1, SMMC-7721 and PLC) and normal human hepatocyte line (HL-77O2) were purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Western Blot, Inhibition, Proliferation Assay, Infection, Sensitive Assay, Control, Negative Control

Journal: Cell Reports Methods

Article Title: Rapid manipulation of mitochondrial morphology in a living cell with iCMM

doi: 10.1016/j.crmeth.2021.100052

Figure Lengend Snippet:

Article Snippet: Human cervical adenocarcinoma HeLa cells (CCL-2), human hepatocellular carcinoma Hep 3B cells (HB-8064), and human osteosarcoma U-2 OS cells (HTB-96) were purchased from the American Type Culture Collection and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher, 11965118) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher, 10270-106) and 1% Zell Shield (Minerva Biolabs GmbH, 13-0050) at 37°C in 5% CO 2 .

Techniques: Virus, Recombinant, Modification, Saline, Membrane, Staining, Lysis, Mutagenesis, Cloning, Cell Viability Assay, BIA-KA, Western Blot, Sequencing, Control, Plasmid Preparation, Marker, Software, Sterility, Cell Culture, Electroporation, Microscopy