Journal: Pharmaceutical Biology

Article Title: Berberine regulates the Notch1/PTEN/PI3K/AKT/mTOR pathway and acts synergistically with 17-AAG and SAHA in SW480 colon cancer cells

doi: 10.1080/13880209.2020.1865407

Figure Lengend Snippet: BBR did not inhibit Hsp90 and HDACs in SW480 cells. (A) The binding effect between BBR (100 μM) and Hsp90 was detected by the thermal stabilization-based CESTA method at 48–76 °C after 30 min of incubation at room temperature. (B) Hsp90 protein expression was quantified and the data are presented as means ± S.D. of three independent experiments. (C) Western blot analysis of Hsp70, IKKβ and EGFR in BBR treated SW480 cells after 24 h incubation. The Hsp90 inhibitor 17-AAG was included as positive control. (D) Western blot analysis of HDAC proteins in BBR treated SW480 cells after 24 h incubation.

Article Snippet: Tanespimycin (17-AAG) and Vorinostat (SAHA) were from Medchem Express (Monmouth Junction, NJ).

Techniques: Binding Assay, Incubation, Expressing, Western Blot, Positive Control

Journal: Pharmaceutical Biology

Article Title: Berberine regulates the Notch1/PTEN/PI3K/AKT/mTOR pathway and acts synergistically with 17-AAG and SAHA in SW480 colon cancer cells

doi: 10.1080/13880209.2020.1865407

Figure Lengend Snippet: BBR enhanced the anti-proliferative activity of 17-AAG and SAHA, and the combination of the three has more potent synergistic effect on SW480 cells. The incubation time of SW480 cells and the test drugs was 24 h. (A) SW480 cells were exposed to specified concentrations of 17-AAG for 24 h in the presence of 0–2 µM of BBR. (B) The CI values of BBR and 17-AAG combinations. (C) SW480 cells were exposed to specified concentrations of SAHA for 24 h in the of 0–2 µM of BBR. (D) The CI values of BBR and SAHA combinations. (E) SW480 cells were treated with specified concentrations of 17-AAG and SAHA in the presence of 0–2 µM of BBR. (F) The CI values for BBR, 17-AAG and SAHA combinations. The above data are all presented as the means ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01 when comparing with 17-AAG, SAHA and both 17-AAG and SAHA treatment in the absence of BBR.

Article Snippet: Tanespimycin (17-AAG) and Vorinostat (SAHA) were from Medchem Express (Monmouth Junction, NJ).

Techniques: Activity Assay, Incubation

Journal: Oncotarget

Article Title: TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death

doi: 10.18632/oncotarget.12411

Figure Lengend Snippet: a. Apoptosis was measured after 20 h by quantifying nuclear fragmentation by ImageStream analysis: U937 cells were left untreated (~2%) or TNF treated (~30%) and in combination with Pepstatin A (PepA) (~20%) or 17AAG (~70%). PepA and 17AAG showed no cytotoxicity alone. b. HSP90-V5 was either mutated at F437 to L or at Y465 to W or then overexpressed in U937 (left) and Jurkat (right) cells. The expression was analysed by Western blot via a V5-tag, tubulin served as loading control. c. Overexpression of mutated HSP90 resulted in reduced apoptosis compared to mock transfected cells (ctrl). The Y465W mutant showed higher protective efficiency than the F437L mutant. d. Scheme of the molecular mass matched (compared to the Western blots from Figure ) V5-tagged N- and C-terminal cleavage products. e. The expression of the two V5-tagged fragments in U937 (left panels) and Jurkat cells (right panels) is shown and indicated by asterisks. The lower panels show the endogenous HSP90 and actin as loading control. f. The normalised apoptotic response towards TNF after transfecting the cells with the V5 tagged constructs or mock plasmid (ctrl) is shown. For quantification, the nuclear fragmentation measured by ImageStream was analysed after 20h incubation with the apoptosis inducing agent.

Article Snippet: 17AAG was purchased from Tocris.

Techniques: Expressing, Western Blot, Control, Over Expression, Transfection, Mutagenesis, Construct, Plasmid Preparation, Incubation

Journal: Oncotarget

Article Title: TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death

doi: 10.18632/oncotarget.12411

Figure Lengend Snippet: a. The impact of TNF treatment in combination with HSP90 inhibition on the activation of caspase 9 and Bid was analysed by Western blot. The blots show the decrease in pro-caspase 9 levels and the increase in cleaved caspase 9 levels after 120 min. After the same time, more Bid-cleavage can be observed. Densitometric quantification of the Western blots is shown below (representative result of three experiments). b. Increases in enzymatic activity of caspase 9 after TNF and combined TNF/17AAG treatment compared to untreated cell lysates was analysed by luminescence measurement (n=3).

Article Snippet: 17AAG was purchased from Tocris.

Techniques: Inhibition, Activation Assay, Western Blot, Activity Assay

Journal: Oncotarget

Article Title: TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death

doi: 10.18632/oncotarget.12411

Figure Lengend Snippet: Binding of TNF to TNF-R1 initially triggers survival signalling via TRADD/RIP1/TRAF2 and NF-kB activation. K63 ubiquitination of TNF-R1 mediates internalization of the receptor and formation of TNF-receptosomes, concomitantly switching off NF-kB signalling and recruiting the DISC proteins FADD and caspase-8. The TNF-receptosomes maturate by fusing with trans-golgi vesicles to form multivesicular body compartments. Cathepsin D is activated and released into the cytosol, where it can cleave Bid and also HSP90. Cleavage of HSP90 or inhibition of HSP90 by 17AAG finally results in enhanced Bid cleavage, Caspase 9 activation and apoptosis.

Article Snippet: 17AAG was purchased from Tocris.

Techniques: Binding Assay, Activation Assay, Ubiquitin Proteomics, Inhibition

Journal: Neurobiology of disease

Article Title: DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.

doi: 10.1016/j.nbd.2018.02.013

Figure Lengend Snippet: Fig. 1. Enhanced cell size in human DEPDC5- or NPRL3- associated brain tissue specimens. There is an increase in maximal cell soma width standard error of the mean ( ± SEM) in PS6 (Ser 235/236) im- munoreactive neurons in resected specimens from in- dividuals with DEPDC5 (A; n = 2) and NPRL3 (B; n = 2) mutations compared with age-matched post-mortem control layer V neurons (measured by MAP2 staining; C and D; n = 2). Cells from the DEPDC5 specimen were 1.6 times larger than control (E; n = 1500 cells per group; p < 0.001) and cells from the NPRL3 specimen were 1.4 times larger than control (E; 1500 cells per group, p < 0.001). Scale bar in (B) applies to all micrographs. *** = significantly different from control with a p < 0.001.

Article Snippet: In separate experiments, mouse DEPDC5 expression plasmid (MR21587, Myc-DDK tag), NPRL3 expression plasmid (MR208964; Myc-DDK tag), mouse DEPDC5 shRNA-plasmid (TG508165, tGFP) or NPRL3 shRNA containing plasmid (TG501301, tGFP) were transfected (all plasmids from Origene, Rockville, MD) into each cell type.

Techniques: Control, Staining

Journal: Neurobiology of disease

Article Title: DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.

doi: 10.1016/j.nbd.2018.02.013

Figure Lengend Snippet: Fig. 3. DEPDC5 or NPRL3 KD lead to enhanced PS6 levels. Increased phospho-S6 levels were observed in N2aC (A) and HEK293FT cells (B) after shRNA KD of DEPDC5 or NPRL3 compared to scramble control and wildtype cells. Application of rapamycin to cultured cells resulted in a decrease in PS6 levels (A and B). C) Overexpression or co- transfection of DEPDC5 or NPRL3 overexpression plasmid with shRNA or scramble control did not produce changes in total S6 levels. OE = overexpression; KD = knockdown, Scram = scramble control, DEP = DEPDC5, WT = Wildtype, rapa = rapamycin. Western blots performed in duplicate. Densitometry data are in Supplemental Fig. 4.

Article Snippet: In separate experiments, mouse DEPDC5 expression plasmid (MR21587, Myc-DDK tag), NPRL3 expression plasmid (MR208964; Myc-DDK tag), mouse DEPDC5 shRNA-plasmid (TG508165, tGFP) or NPRL3 shRNA containing plasmid (TG501301, tGFP) were transfected (all plasmids from Origene, Rockville, MD) into each cell type.

Techniques: shRNA, Control, Cell Culture, Over Expression, Cotransfection, Plasmid Preparation, Knockdown, Western Blot

Journal: Neurobiology of disease

Article Title: DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.

doi: 10.1016/j.nbd.2018.02.013

Figure Lengend Snippet: Fig. 4. DEPDC5 or NPRL3 KD result in morphological changes in N2aC and mNPCs. Representative images (A) of DEPDC5, NPRL3, and scramble control shRNA (white line in (A) depicts representative area of measurement) in mNPCs. (B) The soma diameter was significantly larger in DEPDC5 or NPRL3 shRNA transfected mNPCs than scramble control and wildtype cells (n = 50 per group- 25 cells from each of 2 replicates). (C) Nuclear diameter measurements taken from digital images of transfected mNPCs revealed no increase in nuclear size (inset above (C) represents an area of measurement; n = 50 cells per group- 25 cells from each 2 replicates). (D) An increase in cell size was observed in N2aC and HEK 293FT after DEPDC5 or NPRL3 shRNA KD, compared to scramble control transfected cells, by FACS forward scatter analysis (n = 100,000 cells per group; red = scramble construct size, blue = size after KD). In (B) and (C), each box represents SEM with a mean line shown, whiskers reflect 5–95% confidence intervals. Scramble control transfected cells were not significantly larger than wildtype cells suggesting no effect of lipofectamine alone on cell size. DEP = DEPDC5, Scram = scramble control, GFP = green fluorescent protein, *** = significant difference between scramble and WT control with a p < 0.001.

Article Snippet: In separate experiments, mouse DEPDC5 expression plasmid (MR21587, Myc-DDK tag), NPRL3 expression plasmid (MR208964; Myc-DDK tag), mouse DEPDC5 shRNA-plasmid (TG508165, tGFP) or NPRL3 shRNA containing plasmid (TG501301, tGFP) were transfected (all plasmids from Origene, Rockville, MD) into each cell type.

Techniques: Control, shRNA, Transfection, Construct

Journal: Neurobiology of disease

Article Title: DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.

doi: 10.1016/j.nbd.2018.02.013

Figure Lengend Snippet: Fig. 5. DEPDC5 or NPRL3 KD lead to enhanced filopodial number. DEPDC5 or NPRL3 shRNA transfected mNPCs had higher numbers of filopodia (defined by actin labeling) than scramble control or wildtype mNPCs (n = 50 per group- 25 cells from each of 2 replicates; A and B). Filopodia outgrowth in scramble control and wildtype mNPCs was not significantly different. In (B), each box represents SEM with a mean line shown, whiskers reflect 5–95% confidence intervals. Scram = scramble control, GFP = green fluorescent proteins, WT = wildtype; ** = significant difference between scramble and WT control with a p < 0.01.

Article Snippet: In separate experiments, mouse DEPDC5 expression plasmid (MR21587, Myc-DDK tag), NPRL3 expression plasmid (MR208964; Myc-DDK tag), mouse DEPDC5 shRNA-plasmid (TG508165, tGFP) or NPRL3 shRNA containing plasmid (TG501301, tGFP) were transfected (all plasmids from Origene, Rockville, MD) into each cell type.

Techniques: shRNA, Transfection, Labeling, Control

Journal: Neurobiology of disease

Article Title: DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.

doi: 10.1016/j.nbd.2018.02.013

Figure Lengend Snippet: Fig. 6. Soma size and process outgrowth are mTOR-dependent. After rapamycin treatment (50 nM, 48 h), a decrease in soma diameter (B) and number of filopodia (C) was observed following DEPDC5/NPRL3 KD in mNPCs compared to scramble and wildtype mNPCs treated with vehicle (DMSO; representative images in A). Statistical significance of morphological changes using ANOVA can be found in Table 1 (n = 50 cells per group– 25 cells from each of 2 replicates). In (B) and (C), each box represents SEM with a mean line shown, whiskers reflect 5–95% confidence intervals. WT = wildtype cells, GFP = green fluorescent protein, veh = vehicle, rapa = rapamycin.

Article Snippet: In separate experiments, mouse DEPDC5 expression plasmid (MR21587, Myc-DDK tag), NPRL3 expression plasmid (MR208964; Myc-DDK tag), mouse DEPDC5 shRNA-plasmid (TG508165, tGFP) or NPRL3 shRNA containing plasmid (TG501301, tGFP) were transfected (all plasmids from Origene, Rockville, MD) into each cell type.

Techniques:

Journal: Neurobiology of disease

Article Title: DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.

doi: 10.1016/j.nbd.2018.02.013

Figure Lengend Snippet: Fig. 7. Soma size is mTOR-dependent. FACS forward-scatter analysis of N2aC (A) and HEK293FT cells (B) demonstrate a decrease in soma size after rapamycin treatment (50 nM; 48 h; blue) following DEPDC5 or NPRL3 KD, and in scramble and wildtype cells versus vehicle control (DMSO; red).

Article Snippet: In separate experiments, mouse DEPDC5 expression plasmid (MR21587, Myc-DDK tag), NPRL3 expression plasmid (MR208964; Myc-DDK tag), mouse DEPDC5 shRNA-plasmid (TG508165, tGFP) or NPRL3 shRNA containing plasmid (TG501301, tGFP) were transfected (all plasmids from Origene, Rockville, MD) into each cell type.

Techniques: Control

Journal: Neurobiology of disease

Article Title: DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.

doi: 10.1016/j.nbd.2018.02.013

Figure Lengend Snippet: Fig. 8. DEPDC5 or NPRL3 KD promote constitutive changes in subcellular mTOR localization and enhanced PS6 levels during amino acid deprivation. Colocalization analysis was performed on DEPDC5 KD, NPRL3 KD, scramble control, and wildtype N2aC in amino acid free (AAF) or amino acid replete (complete) media (A). A fluorescence intensity surface plot was generated (see 3D reconstruction) for each region of interest (dashed boxes). Areas of pink represent colocalization of mTOR (blue) and the lysosomal membrane protein 2 (LAMP2, red). To quantify fluorescence, a Pearson's correlation coefficient (R) was calculated for each cell and then averaged across all cells in a group (n = 10 per group). Statistical significance was determined using ANOVA. mTOR and LAMP2 were colocalized after DEPDC5 or NPRL3 KD and incubation in AAF media (B). Reduced colocalization was observed in scramble control and wildtype N2aC after incubation in AAF media (B). High degrees of colocalization between mTOR and LAMP2 were observed in wildtype N2aC incubated in complete media (B). In AAF media, DEPDC5 and NPRL3 KD in both N2aC (C) and HEK293FT cells (D) resulted in elevated levels of PS6 while scramble control and wildtype cells display little or no S6 phosphorylation. Rapamycin application after KD and amino acid starvation dramatically reduced PS6 levels. Western blots performed in duplicate. Densitometry data are in Supplemental Fig. 5. LAMP2 = lysosome associated membrane protein 2, mTOR = mechanistic target of rapamycin, GFP = green fluorescent protein, AAF = amino acid free. Area of 3D reconstruction (ROI) is noted by the dashed boxes in the “merge” column. ** = significant difference between scramble and WT control with a p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: In separate experiments, mouse DEPDC5 expression plasmid (MR21587, Myc-DDK tag), NPRL3 expression plasmid (MR208964; Myc-DDK tag), mouse DEPDC5 shRNA-plasmid (TG508165, tGFP) or NPRL3 shRNA containing plasmid (TG501301, tGFP) were transfected (all plasmids from Origene, Rockville, MD) into each cell type.

Techniques: Control, Generated, Membrane, Incubation, Phospho-proteomics, Western Blot