human hepatocellular carcinoma hep 3b cells  (ATCC)

Journal: The Febs Journal

Article Title: Label‐free imaging of intracellular structures in living mammalian cells via external apodization phase‐contrast microscopy

doi: 10.1111/febs.70286

Figure Lengend Snippet: Movie S13. FCCP treatment‐induced changes in mitochondrial morphology in Hep3B cells.

Article Snippet: Human lung carcinoma A549 cells (RRID:CVCL_0023, ATCC CCL‐185), human cervical adenocarcinoma HeLa cells (RRID:CVCL_0030, ATCC CCL‐2), human colon carcinoma HCT116 cells (RRID:CVCL_0291, ATCC CCL‐247), human hepatocellular carcinoma Hep 3B cells (RRID:CVCL_0326, ATCC HB‐8064), mouse fibroblast NIH/3T3 cells (RRID:CVCL_0594, ATCC CRL‐1658), human breast adenocarcinoma MDA‐MB‐231 cells (RRID:CVCL_0062, ATCC CRM‐HTB‐26), human osteosarcoma U2‐OS cells (RRID:CVCL_0042, ATCC HTB‐96), and human breast carcinoma ZR75‐1‐1 (RRID:CVCL_0588, ATCC CRL‐1500) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques:

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