Journal: BMC Veterinary Research

Article Title: The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention

doi: 10.1186/1746-6148-8-73

Figure Lengend Snippet: Schematic diagram of the class I PI3K/Akt/mTOR axis pathway. The targets of the inhibitors (ZSTK474, Wortmannin, KP372-1 and Rapamycin) used in this study are indicated.

Article Snippet: ZSTK474 (pan-PI3K inhibitor, Z-1066, LC Laboratories, USA), Wortmannin (pan-PI3K inhibitor, 1232, Tocris bioscience, USA), KP372-1 (Akt inhibitor, B-0102, Echelon, USA) and Rapamycin (mTOR inhibitor, R0395, Sigma-Aldrich, USA) were dissolved in dimethyl sulfoxide (DMSO) as concentrated stocks that were stored at -70 °C and diluted freshly in cell medium before use.

Techniques:

Journal: BMC Veterinary Research

Article Title: The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention

doi: 10.1186/1746-6148-8-73

Figure Lengend Snippet: Sensitivity of canine and human cancer cells to inhibitors targeting class I PI3K/Akt/mTOR pathway. Cells were treated with a range of doses of the pan-class I PI3K inhibitor ZSTK474 for 3 days ( A ), Akt inhibitor KP372-1 for 2 days ( B ), or mTOR inhibitor Rapamycin for 3 days ( C ). After drug treatment, the number of viable cells was determined by using CellTiter-Glo® Luminescent Cell Viability Assay. Results were expressed as mean (±SD) counts of quadruplicate wells. The values of the viability rates of the drug-treated cells were compared with the vehicle (DMSO)-treated cells on the same culture plates.

Article Snippet: ZSTK474 (pan-PI3K inhibitor, Z-1066, LC Laboratories, USA), Wortmannin (pan-PI3K inhibitor, 1232, Tocris bioscience, USA), KP372-1 (Akt inhibitor, B-0102, Echelon, USA) and Rapamycin (mTOR inhibitor, R0395, Sigma-Aldrich, USA) were dissolved in dimethyl sulfoxide (DMSO) as concentrated stocks that were stored at -70 °C and diluted freshly in cell medium before use.

Techniques: Cell Viability Assay

Journal: BMC Veterinary Research

Article Title: The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention

doi: 10.1186/1746-6148-8-73

Figure Lengend Snippet: Effects of the inhibitors on class I PI3K/Akt/mTOR axis signaling in canine and human cancer cells. Cells were seeded at a density of 20,000 cells per ml overnight, followed by treatment with 1 μM ZSTK474 ( A ), 400 nM KP372-1 ( B ), or 100 nM Rapamycin ( C ) for 5 hrs. Whole cell lysates (comprising 50 μg total protein) were subjected to western blot with the indicated antibodies. β-actin was used as a loading control.

Article Snippet: ZSTK474 (pan-PI3K inhibitor, Z-1066, LC Laboratories, USA), Wortmannin (pan-PI3K inhibitor, 1232, Tocris bioscience, USA), KP372-1 (Akt inhibitor, B-0102, Echelon, USA) and Rapamycin (mTOR inhibitor, R0395, Sigma-Aldrich, USA) were dissolved in dimethyl sulfoxide (DMSO) as concentrated stocks that were stored at -70 °C and diluted freshly in cell medium before use.

Techniques: Western Blot

Journal: BMC Veterinary Research

Article Title: The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention

doi: 10.1186/1746-6148-8-73

Figure Lengend Snippet: Effects of the inhibitors on class I PI3K/Akt/mTOR axis signaling in canine C2 cells. Cells were treated with pan-class I PI3K inhibitor Wortmannin (W) at 1 μM and ZSTK474 (Z) at 1 μM, mTOR inhibitor Rapamycin (R) at 100 nM ( A ) and Akt inhibitor KP372-1 at 0, 150, 200 and 400 nM ( B ) for the indicated period of time. Whole cell lysates (comprising 50 μg total protein) were subjected to western blot with the indicated antibodies. β-actin was used as a loading control. N/A indicates data unavailable due to induction of apoptosis in all cells.

Article Snippet: ZSTK474 (pan-PI3K inhibitor, Z-1066, LC Laboratories, USA), Wortmannin (pan-PI3K inhibitor, 1232, Tocris bioscience, USA), KP372-1 (Akt inhibitor, B-0102, Echelon, USA) and Rapamycin (mTOR inhibitor, R0395, Sigma-Aldrich, USA) were dissolved in dimethyl sulfoxide (DMSO) as concentrated stocks that were stored at -70 °C and diluted freshly in cell medium before use.

Techniques: Western Blot

Journal: BMC Veterinary Research

Article Title: The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention

doi: 10.1186/1746-6148-8-73

Figure Lengend Snippet: Effects of ZSTK474, KP372-1 and Rapamycin on induction of apoptosis. Cells were treated with 20 μM ZSTK474 for 2 days, 400 nM KP372-1 for 1 day, 20 μM Rapamycin for 2 days, or vehicle control as described in Materials and Methods. Induction of apoptosis was determined by annexin V/propidium iodide (PI) staining and flow cytometry analysis. Scatter pots with percentages of live, early apoptosis and late apoptosis were indicated at lower left, lower right and upper right quadrants, respectively in A while B demonstrates this data in a bar chart format. This data is representative of two independent experiments.

Article Snippet: ZSTK474 (pan-PI3K inhibitor, Z-1066, LC Laboratories, USA), Wortmannin (pan-PI3K inhibitor, 1232, Tocris bioscience, USA), KP372-1 (Akt inhibitor, B-0102, Echelon, USA) and Rapamycin (mTOR inhibitor, R0395, Sigma-Aldrich, USA) were dissolved in dimethyl sulfoxide (DMSO) as concentrated stocks that were stored at -70 °C and diluted freshly in cell medium before use.

Techniques: Staining, Flow Cytometry

Journal: BMC Veterinary Research

Article Title: The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention

doi: 10.1186/1746-6148-8-73

Figure Lengend Snippet: Effects of the combination of the class I PI3K/Akt/mTOR pathway inhibitors and Doxorubicin on cell viability. SB ( A ) and REM ( B ) cells were treated with the indicated does of the class I PI3K/Akt/mTOR pathway inhibitors, Doxorubicin, the combination of the former two drugs or vehicle control for 3 days (2 days for KP372-1). Additive, synergistic or antagonistic inhibitory effects of the drug combination on cell viability were determined when the experiment values (cell viability percentages) of the drug combination were overlapped with, lower than, or higher than Bliss theoretical values respectively.

Article Snippet: ZSTK474 (pan-PI3K inhibitor, Z-1066, LC Laboratories, USA), Wortmannin (pan-PI3K inhibitor, 1232, Tocris bioscience, USA), KP372-1 (Akt inhibitor, B-0102, Echelon, USA) and Rapamycin (mTOR inhibitor, R0395, Sigma-Aldrich, USA) were dissolved in dimethyl sulfoxide (DMSO) as concentrated stocks that were stored at -70 °C and diluted freshly in cell medium before use.

Techniques:

Journal: Scientific Reports

Article Title: DNA damage induced by KP372-1 hyperactivates PARP1 and enhances lethality of pancreatic cancer cells with PARP inhibition

doi: 10.1038/s41598-020-76850-4

Figure Lengend Snippet: Elevated NQO1 expression sensitizes pancreatic cancer cells to KP372-1. ( A , B ) Relative survival measured by DNA content assay in the presence of indicated concentrations (µM) of KP372-1 ± dicoumarol (DIC, NQO1 inhibitor) for 2 h. ( C ) Relative survival of siSCR or siNQO1 knockdown cells in the presence of indicated concentrations (µM) of KP372-1 ± DIC for 2 h. ( D ) Clonogenic survival in the presence of indicated concentrations (µM) of KP372-1 ± DIC for 2 h. ( E – H ) Relative survival in the presence of indicated concentrations (µM) of KP372-1 ± DIC for 2 h. ( I , J ) Relative survival in the presence of 0.2 µM concentration of KP372-1 ± 50 µM DIC for indicated time points. ( K , L ) Relative survival in the presence of KP372-1 ± 50 µM DIC and β-lap ± 50 µM DIC. Graphs represent means ± SEM for drug treatment over control (i.e., DMSO) treated (T/C) samples for MIA PaCa-2 n = 5 ( A ), Capan-2 n = 3 ( B ), MIA PaCa-2 ± siSCR/siNQO1 n = 3 ( C ), MIA PaCa-2: clonogenic survival n = 4 ( D ), hTERT-HPNE n = 3 ( E ), PANC-1 n = 3 ( F ), AsPC-1 n = 4 ( G ), and BxPC-3 n = 3 ( H ), MIA PaCa-2: time course ( I ), Capan-2: time course n = 3 ( J ), MIA PaCa-2: β-lap treatment from n = 4 ( K ), and Capan-2: β-lap treatment n = 4 ( L ). Each biological replicate was performed in triplicate. p values were obtained via two-tailed student’s t-tests. * p < 0.05; ** p < 0.01; *** p < 0.001, comparing KP372-1 alone with KP372-1 + DIC or KP372-1 + β-lap. For the clonogenic survival assay, p values were obtained via an ordinary one-way ANOVA using the Dunnett’s multiple comparisons test. **** p < 0.0001; ns, not significant, comparing indicated drug treatments to the DMSO control.

Article Snippet: KP372-1 was also purchased from Echelon Biosciences Inc. (Salt Lake City, UT, USA) for comparison.

Techniques: Expressing, Concentration Assay, Two Tailed Test, Clonogenic Cell Survival Assay

Journal: Scientific Reports

Article Title: DNA damage induced by KP372-1 hyperactivates PARP1 and enhances lethality of pancreatic cancer cells with PARP inhibition

doi: 10.1038/s41598-020-76850-4

Figure Lengend Snippet: KP372-1 treatment enhances ROS production in pancreatic cancer cells. ( A – D ) Relative levels of H 2 O 2 production in control (DMSO), KP 372-1 or KP372-1 ± 50 µM DIC treated cells were measured using the Promega ROS-Glo H 2 O 2 assay kit. MIA PaCa-2 cells treated, with indicated dose of KP372-1 (µM) for 30 min ( A ), and with 0.2 µM KP372-1 for indicated time (min) points ( B ). Capan-2 cells treated, with indicated dose of KP372-1 (µM) for 30 min ( C ), and with 0.2 µM KP372-1 for indicated time (min) points ( D ). ( E , F ) Relative levels of H 2 O 2 production in control (DMSO), KP372-1 and KP372-1 ± N-acetylcysteine amide [NAC, 1 mM or 5 mM for total of 5 h (pre-treatment for 3 h and co-treatment for 2 h)] treated MIA PaCa-2 cells ( E ) or Capan-2 cells ( F ) with indicated concentrations . Bar graphs represent means ± SEM for treated/control samples from n = 4, each performed in duplicate. p values were obtained via two-tailed student’s t-tests. * p < 0.05; ** p < 0.01; *** p < 0.001, comparing treated with control samples.

Article Snippet: KP372-1 was also purchased from Echelon Biosciences Inc. (Salt Lake City, UT, USA) for comparison.

Techniques: Two Tailed Test

Journal: Scientific Reports

Article Title: DNA damage induced by KP372-1 hyperactivates PARP1 and enhances lethality of pancreatic cancer cells with PARP inhibition

doi: 10.1038/s41598-020-76850-4

Figure Lengend Snippet: KP372-1 treatment instigates oxidative DNA damage and DNA breaks in pancreatic cancer cells. ( A – D ) Relative levels of nuclear 8-oxoG signal in control (DMSO), 0.15 µM KP372-1, 0.15 µM KP372-1 ± 50 µM DIC or KP372-1 ± N-acetylcysteine amide [NAC, 5 mM for total of 4 h (pre-treatment for 3 h and co-treatment for 1 h)] treated cells were measured by immunofluorescence confocal microscopy. Representative images of MIA PaCa-2 cells ( A ), and quantification of fluorescence signal ( B ). Representative images of Capan-2 cells ( C ), and quantification of fluorescence signal ( D ). Cells treated with H 2 O 2 (1 mM, 15 min in 1× PBS) served as positive control. The scale bar represents 10 µm. Graphs represent the means (red bar) for treated/control samples from n = 3, each performed in duplicate for total of 150 cells. Relative fluorescence intensities were determined by ImageJ software (version 1.53c, https://imagej.net ). ( E , F ) Relative levels of comet tail moment of control (DMSO), 0.15 µM KP372-1 ± 50 µM DIC or KP372-1 ± N-acetylcysteine amide [NAC, 5 mM for total of 4 h (pre-treatment for 3 h and co-treatment for 1 h)] treated cells measured by confocal microscopy. Representative images of MIA PaCa-2 cells ( E ), and quantification of fluorescence signal ( F ). Cells treated with H 2 O 2 (1 mM, 15 min in 1× PBS) served as positive control. The scale bar represents 10 µm. Tail moments were obtained using the ImageJ (version 1.53c, https://imagej.net ) plug-in OpenComet v1.3 ( www.biocomet.org ). Graphs represent the means (red bar) for treated/control samples from n = 3, each performed in duplicate for total of 150 cells. p values were obtained via an ordinary one-way ANOVA using the Dunnett’s multiple comparisons test. * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant, comparing indicated drug treatments to the DMSO control.

Article Snippet: KP372-1 was also purchased from Echelon Biosciences Inc. (Salt Lake City, UT, USA) for comparison.

Techniques: Immunofluorescence, Confocal Microscopy, Fluorescence, Positive Control, Software

Journal: Scientific Reports

Article Title: DNA damage induced by KP372-1 hyperactivates PARP1 and enhances lethality of pancreatic cancer cells with PARP inhibition

doi: 10.1038/s41598-020-76850-4

Figure Lengend Snippet: KP372-1 elicits robust DNA damage signaling in pancreatic cancer cells. ( A – H ) Assessment of phosphorylated H2AX (yH2AX) via Western blotting as a marker of DNA damage response induced by KP372-1. MIA PaCa-2 cells treated with 0.15 µM KP372-1 ± 50 µM DIC for indicated time (min) points ( A , B ), blot image and quantification, respectively, and with indicated dose of KP372-1 (µM) ± 50 µM DIC for 120 min ( C , D ), blot image and quantification, respectively. Capan-2 cells treated with 0.15 µM KP372-1 ± 50 µM DIC for indicated time (min) points (E–F), blot image and quantification, respectively, and with indicated dose of KP372-1 (µM) ± 50 µM DIC for 120 min ( G , H ), blot image and quantification, respectively. Representative Western blot images are presented. Bar graphs represent quantification of band intensities (means ± SEM) of yH2AX normalized to α-tubulin of respective sample from n = 5. Band intensities were detected by ImageJ software (version 1.53c, https://imagej.net ) and α-tubulin was used as loading control. Cell lysates prepared from H 2 O 2 -treated cells (1 mM, 15 min in 1× PBS) served as positive control. p values were obtained via two-tailed student’s t-tests. * p < 0.05; ** p < 0.01; *** p < 0.001, comparing treated with control (DMSO) samples. WB; Western blot. Raw data for all the Western blot images are provided in the supplementary information.

Article Snippet: KP372-1 was also purchased from Echelon Biosciences Inc. (Salt Lake City, UT, USA) for comparison.

Techniques: Western Blot, Marker, Software, Positive Control, Two Tailed Test

Journal: Scientific Reports

Article Title: DNA damage induced by KP372-1 hyperactivates PARP1 and enhances lethality of pancreatic cancer cells with PARP inhibition

doi: 10.1038/s41598-020-76850-4

Figure Lengend Snippet: KP372-1 hyperactivates PARP1 in pancreatic cancer cells. ( A – H ) Assessment of PAR (poly-(ADP-ribose)) via Western blotting as a marker of PARP1 hyperactivation by KP372-1-induced DNA damage. MIA PaCa-2 cells treated with 0.15 µM KP372-1 ± 50 µM DIC for indicated time (min) points ( A , B ), blot image and quantification, respectively, and with indicated dose of KP372-1 (µM) ± 50 µM DIC for 10 min ( C , D ), blot image and quantification, respectively. Capan-2 cells treated with 0.15 µM KP372-1 ± 50 µM DIC for indicated time (min) points ( E , F ), blot image and quantification, respectively, and with indicated dose of KP372-1 (µM) ± 50 µM DIC for 10 min ( G , H ), blot image and quantification, respectively. Representative Western blot images are presented. Bar graphs represent quantification of band intensities (means ± SEM) of PAR normalized to α-tubulin of respective sample from n = 5. Band intensities were detected by ImageJ software (version 1.53c, https://imagej.net ) and α-tubulin was used as loading control. Cell lysates prepared from H 2 O 2 -treated cells (1 mM, 15 min in 1× PBS) served as positive control. p values were obtained via two-tailed student’s t-tests. * p < 0.05; ** p < 0.01; *** p < 0.001, comparing treated with control (DMSO) samples. WB; Western blot. Raw data for all the Western blot images are provided in the supplementary information.

Article Snippet: KP372-1 was also purchased from Echelon Biosciences Inc. (Salt Lake City, UT, USA) for comparison.

Techniques: Western Blot, Marker, Software, Positive Control, Two Tailed Test

Journal: Scientific Reports

Article Title: DNA damage induced by KP372-1 hyperactivates PARP1 and enhances lethality of pancreatic cancer cells with PARP inhibition

doi: 10.1038/s41598-020-76850-4

Figure Lengend Snippet: KP372-1 treatment activates caspase-3 in pancreatic cancer cells. ( A ) Evaluation of caspase activation via confocal immunofluorescence microscopy using cleaved caspase-3 antibody (i.e., activated caspase-3). Capan-2 cells were treated with 0.15 µM KP372-1 ± 50 µM DIC for 2 h and released for indicated time points (h) prior to processing for immunofluorescence. Note strong staining of capan-2 cells with activated caspase-3 from 24 to 96 h, whereas, DMSO treated cells do not show appreciable signal. Representative image from n = 4 biological repeats has shown. ( B , C ) Model providing mechanistic insight into cellular consequences of KP372-1 exposure to NQO1 overexpressing cancer cells.

Article Snippet: KP372-1 was also purchased from Echelon Biosciences Inc. (Salt Lake City, UT, USA) for comparison.

Techniques: Activation Assay, Immunofluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: DNA damage induced by KP372-1 hyperactivates PARP1 and enhances lethality of pancreatic cancer cells with PARP inhibition

doi: 10.1038/s41598-020-76850-4

Figure Lengend Snippet: KP372-1 + PARP inhibition combination enhances cytotoxicity of pancreatic cancer cells. Relative survival measured by DNA content assay in the presence of indicated µM concentrations of KP372-1 ± BMN 673 (PARP1/2 inhibitor). ( A ) MIA PaCa-2 cells were treated with BMN 673 and KP372-1 in the order indicated by the outline above the graph. Bar graph represent means ± SEM for individual or combination treatment over control (i.e., DMSO) treated (T/C) samples from n = 3, each in triplicate. p values were obtained via two-tailed student’s t-tests. * p < 0.05; ** p < 0.01; *** p < 0.001, comparing combination with individual treatments. ( B ) Dose reduction index (DRI) values were calculated using the survival data from panel A as input via CompuSyn 1.0 software ( www.combosyn.com ) where any value above 1 indicates a reduction in dose for individual agents to achieve the given fraction affected (Fa) upon combination. p values were obtained via two-tailed student’s t-tests. * p < 0.05; ** p < 0.01; *** p < 0.001, comparing the dose reduction value to the additive value of 1.

Article Snippet: KP372-1 was also purchased from Echelon Biosciences Inc. (Salt Lake City, UT, USA) for comparison.

Techniques: Inhibition, Two Tailed Test, Software

Journal: International Journal of Molecular Sciences

Article Title: Testosterone Promotes the Proliferation of Chicken Embryonic Myoblasts Via Androgen Receptor Mediated PI3K/Akt Signaling Pathway

doi: 10.3390/ijms21031152

Figure Lengend Snippet: Effect of T and inhibitors treatment on cell cycle of myoblasts cultured in vitro. ( A–B ): Cell cycle of myoblasts treated with T and/or inhibitors (Bicalutamide, B; LY294002, LY; KP372-1, KP) was detected by flow cytometry. The percentage of S or G2/M stage cells was measured in different treatment compared with control (Con). A minimum of 15,000 myoblast cells were analysed for each sample. Values are means + SE of four independent experiments with triplicate determinations. Asterisk (*) represents statistically different ( p < 0.05), ** indicates that the difference is extremely significant ( p < 0.01).

Article Snippet: Specific inhibitors including 50 μM AR inhibitor (Bicalutamide, Shanghai Civi Chemical Technology, Shanghai, China), 10 μM PI3K inhibitor (LY294002, Sigma Aldrich, St. Louis, MO, USA), and 1 μM Akt inhibitor (KP372-1, Echelon, Logan, UT, USA) were added into culture medium for 30 min before T addition.

Techniques: Cell Culture, In Vitro, Flow Cytometry