Journal: PLoS ONE

Article Title: Hsp70 and DNAJA2 limit CFTR levels through degradation

doi: 10.1371/journal.pone.0220984

Figure Lengend Snippet: (A) Immunoblot (IB) of CFTR-3HA stably expressed in HeLa cells treated with 10 μM MKT077, 50 μM VER155008 or vehicle control for 24 h. Quantitations of CFTR bands B and C are shown, relative to amounts of each in vehicle treated samples, n = 5. (B) Pulse-chase autoradiograph of CFTR as in , in the above cells, chased in the presence of 10 μM MKT077, 50 μM VER155008 or vehicle control. Quantitations of CFTR bands B and C are shown relative to initial amounts of band B, n = 3. (C) Immunoblot of cells treated as in (A) except with 10 μM MKT077, 100 μM CQ or both, or vehicle control. Quantitation of CFTR band C is shown relative to amounts in vehicle treated samples, n = 8 for MKT077, n = 4 for CQ with and without MKT077. (D) Immunoblot of CFTR-3HA in the above cells chased for 5 h in the presence of CHX and 10 μM MKT077 or vehicle control. Quantitations of CFTR bands B and C are shown relative to initial amounts of each, n = 5. (E) Cells were treated as in (A) with 10 μM MKT077 or vehicle control, cell surface biotinylated and pulled down with streptavidin beads. CFTR-3HA and tubulin control were detected by immunoblot in input lysate and biotinylated samples. CFTR band C in biotinylated samples was quantified relative to amounts in vehicle controls, n = 4. (F) The above cells were cell surface biotinylated without any treatment, then chased for 7.5 h in the presence of 10 μM MKT077 or vehicle control. Biotinylated CFTR-3HA was pulled down and detected by immunoblot as in (E). Quantitation of biotinylated CFTR band C is shown relative to the initial amount, with data fit to delayed one phase decay curves, n = 4. (G) Iodide efflux time courses of CFTR in the above cells treated with 10 μM MKT077 or vehicle control for 24 h. Cell surface CFTR was stimulated with cAMP cocktail and iodide efflux from cells was measured, relative to the total iodide released by detergent, n = 6. Error bars show standard deviation from the mean, * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Compounds MKT077 (4621) and VER155008 (3803) were purchased from Tocris Bioscience.

Techniques: Western Blot, Stable Transfection, Control, Pulse Chase, Autoradiography, Quantitation Assay, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: Histone Acetyltransferase (HAT) P300/CBP Inhibitors Induce Synthetic Lethality in PTEN-Deficient Colorectal Cancer Cells through Destabilizing AKT

doi: 10.7150/ijbs.42197

Figure Lengend Snippet: Anacardic acid reduces the level of AKT at both transcription and post-translational levels. (A) Western blots of AKT1 in PTEN -/- cells treated with 10 μM cycloheximide (CHX) combined with or without 100 μM AA for indicated time points. (B) Quantification curve of AKT1 protein level based on the western blot of (A). (C) RT-qPCR analysis of AKT1 mRNA in PTEN -/- cells treated with 100 μM anacardic acid for 9 h. (D) Western blots of AKT1 and GAPDH in PTEN -/- cells treated with anacardic acid, MG132 and the combination of both for 24 h. ** P -values ≤ 0.01 in Student's t-test.

Article Snippet: The kinase inhibitor drug library (#L1200, Selleck), Anacardic acid, C646, cycloheximide (CHX), MG132 and MKT077 were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Western Blot, Quantitative RT-PCR

Journal: International Journal of Biological Sciences

Article Title: Histone Acetyltransferase (HAT) P300/CBP Inhibitors Induce Synthetic Lethality in PTEN-Deficient Colorectal Cancer Cells through Destabilizing AKT

doi: 10.7150/ijbs.42197

Figure Lengend Snippet: Anacardic acid decreased the transcription of Hsp70 family by inhibiting P300/CBP HAT activity on promoters of Hsp70 family and induced destabilization of Hsp70/AKT complex. (A) Western blots of heat shock proteins in PTEN isogenic HCT116 pairs after treated with 0, 20, 40, 80, 100 μM anacardic acid for 24 h. (B) Western blots of co-immunoprecipitation experiment of heat shock protein members with AKT1 in PTEN -/- cells treated with 100 μM AA for 3, 6 and 9 h. (C) Bar charts of cell viability after incubating PTEN +/+ and PTEN -/- HCT116 cell lines with the Hsp70 inhibitor MKT077. (D) Western blots of p-AKT Ser473, AKT1, cleaved-caspase3, PARP1, Hsc70, Hsp70, Hsp90 and beta-actin in PTEN isogenic HCT116 pairs after treating with 0, 2.5, 5, 10, 20 μM MKT077 for 24 h. (E) Western blots of AKT1, Hsp70, Hsp90, Ac-H4 and GAPDH in PTEN -/- cells treated with anacardic acid, MG132 and the combination of both for 24 h. (F) Real-time qPCR showing the changes in the expression level of Hsp70 and Hsp90 family at 0, 6, 9 h with 100 μM anacardic acid in PTEN -/- cells. (G) Bar charts of immunoprecipitated promoter regions of heat shock protein members and AKT1 with acetylation H4 antibody in ChIP experiments after treating with anacardic acid for 9 h in PTEN -/- cells. (H) The effect of the overexpression of Hsp70/Hsc70 on anti-proliferative effect on AA in PTEN -/- cancer cells. PTEN -/- HCT116 cells were transfected with Hsp70 or Hsc70 plasmid and treated with 100 μM AA for 24 h. The cell viability was measured with AlamarBlue assay. * P -values ≤ 0.05; ** P -values ≤ 0.01 in Student's t-test.

Article Snippet: The kinase inhibitor drug library (#L1200, Selleck), Anacardic acid, C646, cycloheximide (CHX), MG132 and MKT077 were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Expressing, Over Expression, Transfection, Plasmid Preparation, Alamar Blue Assay