Journal: Cancer gene therapy

Article Title: In vitro and in vivo delivery of novel anticancer fusion protein MULT1E/FasTI via adenoviral vectors.

doi: 10.1038/cgt.2009.69

Figure Lengend Snippet: Figure 1 RT-PCR and FACS analyses. TC-1 cells were infected with Ad-MULT1E/FasTI at different MOIs. (a) RNAs were isolated from the infected cells and RT-PCR was performed. Lanes 1 and 14 are DNA markers; lines 2, 4, 6, 8, 10 and 12 are 1134bp RT-PCR products of total RNA from TC1 cells infected with Ad-MULT1E/FasTI at 500, 250, 100, 50, 25 or 0 MOIs and amplified with MULT1E forward and Fas reverse primers; lines 3, 5, 7, 9, 11 and 13 are b-actin controls for each sample. (b) 5 105 TC1 cells were harvested from each group that were infected with Ad-MULT1E/FasTI at 500, 250, 100, 50, 25 or 0 MOIs for 24 h and stained with purified rat anti-mouse MULT1 antibody followed by goat anti-mouse IgG F(ab0)-FITC. Dashed lines represent uninfected TC-1 cells.

Article Snippet: TC-1 cells infected with different MOIs of Ad-MULT1E/ FasTI (500, 250, 100, 50, 25 and 0) for 24 h were harvested and 5 105 cells from each sample were washed twice with staining buffer and incubated with 1 mg monoclonal rat anti-mouse MULT1 antibody (R&D Systems, Minneapolis, MN) for 30min at 4 1C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Isolation, Amplification, Staining, Purification

Journal: Cancer Biology & Medicine

Article Title: The PI3K/Akt/GSK-3β/ROS/eIF2B pathway promotes breast cancer growth and metastasis via suppression of NK cell cytotoxicity and tumor cell susceptibility

doi: 10.20892/j.issn.2095-3941.2018.0253

Figure Lengend Snippet: eIF2B functions as a downstream molecule of PI3K/AKT/GSK-3β/ROS pathway to upregulate NKG2D ligands. (A) eIF2B, CCND1, and NFAT1 levels were investigated by RT-PCR. (B) mRNA levels of GSK-3β and eIF2B, protein levels of pSer9-GSK-3β, pSer535-eIF2B, H60, and Rae1 were quantified. (C) The expression of p-eIF2B induced by H 2 O 2 , NAC, and SOD (1,000 U/mL) as well as TWS119, LY294002, and Wortmannin (2 μmol/L, another inhibitor of PI3K/AKT) was detected by Western blot. (D) The protein levels of p-GSK-3β, p-eIF2B, H60 and Rae1 expressed in tumor tissue homogenate after intraperitoneal injection of TWS119 and LY294002 (5 mg/kg) was assayed by Western blot. (E) Consecutive sections of above tissues were stained by DAB. The mean densities in the positive areas were quantitatively by the IPP software. *, indicates values of P < 0.05.

Article Snippet: Antibodies were purchased as follows: NKG2D and ULBP1 or Mult-1 (Proteintech, America), GSK-3β (Boster, Wuhan, China), pSer9-GSK-3β (Santa Cruz, CA, USA), Rae1 (Santa Cruz), H60 (R&D system, Minnesota, USA), pSer535-eIF2B (Eno Gene, Nanjing, China), and β-actin (Santa Cruz).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Injection, Staining, Software

Journal: Oncogene

Article Title: CBP/p300 acetyltransferases regulate the expression of NKG2D ligands on tumor cells

doi: 10.1038/onc.2016.259

Figure Lengend Snippet: HDACi-induced NKG2D ligand regulation did not involve the DDR. ( a ) HEK-293 cells were treated with 100 n M LBH589 for 16 h, washed and incubated with primary human NK cells of healthy donors in indicated ratios for 3 h. Mean and s.e. of four independent experiments. ( b ) FACS analysis of HEK-293 cells after 1 h of preincubation with 2 μ M actinomycin D or 10 μ M cycloheximide, and treatment with LBH589 (100 n M ) for 6 h. ( c ) Real-time PCR for HEK-293 cells treated with 10 μ M Ara-C or 100 n M LBH589 for the indicated periods relative to GAPDH. ( d ) HEK-293 cells were treated with the ATM/ATR inhibitor CGK733 (5 μ M ) together with the damage inducer Ara-C (10 μ M ) or the HDACis LBH589 (100 n M ) and trichostatin A (TSA; 250 n M ) for 16 h to analyze the surface expression of MICA/B, ULBP1, ULBP2 and ULBP3. ( e ) MDA-231 and 911 cells were incubated with ATM/ATR inhibitor CGK733 (5 μ M ) or ATM inhibitor KU55933 (10 μ M ) and TSA (250 n M ) for 16 h and subsequently studied for MICA/B expression by FACS. ( f ) Intracellular FACS staining of HEK-293 cells for DNA damage marker phospho-γH2AX (S139 phosphorylated) after incubation with 10 μ M Ara-C or 100 n M LBH589 for 1 h.

Article Snippet: The following specific antibodies were used: ac-Lysine (9441, CST, Leiden, Netherlands); MICA (AMO1, BamOmaB); MICA/B (6D4, BioLegend); MICB (BMO2, BamOmaB, Gräfelfing, Germany); MULT-1 (237104, R&D); p-γH2AX (S139, 9718, CST); Rae-1 (R&D, Wiesbaden-Nordenstadt, Germany); ULBP1 (AUMO2, BamOmaB); ULBP2 (BUMO1, BamOmaB); ULBP2 (BAF1298, R&D); ULBP3 (CUMO3 BamOmaB) and isotype controls purchased from BioLegend.

Techniques: Incubation, Real-time Polymerase Chain Reaction, Expressing, Staining, Marker