Journal: BMC Cancer

Article Title: Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells

doi: 10.1186/s12885-015-1568-3

Figure Lengend Snippet: siRNA mediated Knock Down of 14-3-3 zeta/delta reduces soft agar colony formation. a HCT116 cells were transfected with siRNA to 14-3-3 zeta/delta, or equivalent amount of control siRNA solution. After 48 h incubation with siRNA to 14-3-3 zeta/delta, or equivalent amount of control, HCT116 cells were harvested for soft agar cloning and saved for Western Blot analysis. EVs were harvested from the HCT116 cells transfected with siRNA to 14-3-3 zeta/delta. EVs were resuspended in PBS and co-cultured with 1459 cells. After 7 days of co-culture, soft agar coloning was performed. There were 5 dishes/condition. The data represents the mean +/− s.d. of 3 independent experiments performed in quadruplet. A paired t-test was performed to analyze the decrease in soft agar colony formation of HCT116 transfected with 14-3-3 zeta/delta siRNA, compared to HCT116 transfected with scrambled siRNA as control, *p < 0.00001. Decrease in colony formation of 1459 + HCT116 14-3-3siRNA EV when compared to 1459 + HCT116 scramble siRNA EV was assessed, ** p = 0.0039. b After co-culture, cells were harvested and a portion of the cells were prepared as whole cell lysates for Western blot analysis. Western blot analysis confirmed the reduced expression of 14-3-3 zeta/delta in HCT116 + 14-3-3siRNA compared to HCT116 and HCT116 + scramble siRNA controls. Reduced expression of 14-3-3 zeta/delta was also seen in the co-culture of 1459 + HCT116 14-3-3 siRNA EV as compared to 1459 + HCT116 EV and 1459 + HCT116 scramble siRNA EV controls. c Western blot analysis of 14-3-3 sigma and epsilon protein levels of 1459 cells after co-culture with HCT116 EVs. d Western blot of 14-3-3 zeta/delta protein from vesicles isolated from 3 differential centrifugation preparations. MV = microvesicle fraction; EX = exosome fraction; T = MV and EX fraction

Article Snippet: The primary antibodies to Raf-1 (sc-133), actin (sc-1616), prohibitin (sc-6705), STAT1 (sc-346), pRKIP (sc-32623) and 14-3-3 zeta (sc-1019) and 14-3-3 zeta siRNA (sc-29583) were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Knockdown, Transfection, Control, Incubation, Cloning, Western Blot, Cell Culture, Co-Culture Assay, Expressing, Isolation, Centrifugation

Journal: Aging (Albany NY)

Article Title: Targeting regulation of ATP synthase 5 alpha/beta dimerization alleviates senescence

doi: 10.18632/aging.203858

Figure Lengend Snippet: Maintenance of 14–3–3ζ expression is a prerequisite for KB1541-mediated senescence alleviation. ( A ) Expression levels of 14–3–3ζ and ATP synthase 5 alpha/beta proteins in young and senescent fibroblasts. ( B ) shRNA-mediated 14–3–3ζ knockdown significantly reduced the endogenous expression level of 14–3–3ζ mRNA. ** P < 0.01, student t -test. Mean ± S.D., n = 3. ( C ) Immunoblot of total lysates from senescent cells transduced with lenti-virus expressing control shRNA or 14–3–3ζ shRNA. ( D ) Quantification of SA-β gal positive cells. * P < 0.05, student t -test. Mean ± S.D., n = 3.

Article Snippet: HEK 293T cells were transfected with 10 μg shRNA plasmid (control shRNA or 14–3–3ζ shRNA (sc-156019-SH; Santa Cruz Biotechnology), 5 μg PAX2 plasmid, and 5 μg VSV-G plasmid using Lipofectamine 2000 (11668019; Invitrogen).

Techniques: Expressing, shRNA, Knockdown, Western Blot, Transduction, Virus, Control

Journal: Current protocols

Article Title: O-GlcNAc engineering on a target protein in cells with nanobody-OGT and -splitOGA

doi: 10.1002/cpz1.117

Figure Lengend Snippet: Number of wells and DNA amounts can be scaled up accordingly for additional replicates or material.

Article Snippet: Materials 1 pcDNA3.1-GFP-Flag-JunB-EPEA (Addgene, cat. No. 160743).

Techniques: Plasmid Preparation

Journal: Current protocols

Article Title: O-GlcNAc engineering on a target protein in cells with nanobody-OGT and -splitOGA

doi: 10.1002/cpz1.117

Figure Lengend Snippet: The mixture is incubated in a thermocycler with the conditions described below

Article Snippet: Materials 1 pcDNA3.1-GFP-Flag-JunB-EPEA (Addgene, cat. No. 160743).

Techniques: Incubation