Journal: Cancer cell

Article Title: Synthetic Lethality of Combined Bcl-2 Inhibition and p53 Activation in AML: Mechanisms and Superior Antileukemic Efficacy

doi: 10.1016/j.ccell.2017.11.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies for immunoblotting were purchased from the following sources: p53 (sc-126), MDM2 (sc-5304), Bcl-2 (sc-7382), Mcl-1 (sc-819), Puma (sc-28226), ERK (sc-1647), N-Ras (sc-31) and GSK3 (sc-7291) from Santa Cruz Biotechnology (Dallas, TX, USA); pERK (T202/Y204, 4370), pMEK (S217/S221, 9121), MEK (4694), AKT (2920), pAKT(S473, 9271), pGSK3 (S21/S9, 8566), PARP-1 (9542), Bim (2819), Bak (12105), caspase-3 (9664), caspase-9 (9508), pMcl-1 (T163, 14765), B-Raf (9433), pB-Raf (S445, 2696), JNK (9252) and pJNK (T183Y185, 9255) from Cell Signaling Technology; Bax (B8554) and β-Actin (A2228) from Sigma-Aldrich; Noxa (ab13654) and pMcl-1 (T163S159, ab111574) from Abcam; p21 (OP64) from EMD Millipore (Darmstadt, Germany).

Techniques: Recombinant, Imaging, Staining, Mutagenesis, Transfection, shRNA, Negative Control, Plasmid Preparation, Software, In Vivo Imaging, Microscopy, Flow Cytometry

Journal: Life Science Alliance

Article Title: MAPK pathway mutations in head and neck cancer affect immune microenvironments and ErbB3 signaling

doi: 10.26508/lsa.201900545

Figure Lengend Snippet: (A) Kaplan–Meier overall survival curves for head and neck squamous cell carcinoma (HNSCC) patients, whose tumors overexpressed versus underexpressed phospho-ErbB3(Y1289) (median cutoff) in TCPA HNSCC cohort (N = 344). (B) Western blot results of phospho-ErbB3(Y1289) protein levels upon ectopic expression of the MAPK1 , ARAF , BRAF , HRAS , and MAP2K1 as well as MAP2K2 wild-type and mutant constructs in FaDu cells by retroviral infection (pool of at least four independent repeats). (C) Western blot results of phospho-ErbB3(Y1289) levels of HNSCC Pt.25 primary tumor cultures (carrying both HRAS p.G12S and MAPK1 p.R135K mutations), HSC-6 cell line (carrying MAPK1 p.E322K mutation), and HSC-4 (MAPK pathway WT, Cancer Cell Line Encyclopedia [CCLE]) upon MAPK inhibitor GDC-0994 treatment for 30 min. 50 µg of protein lysate was used for Pt.25 and HSC-4 samples, 50 μg of protein lysate was used, whereas for HSC-6 (because of the relatively low endogenous p-RSK levels intrinsic to this cell line), 100 μg of protein lysate was loaded for presentation of signal clarity. Bar graphs showing the quantified changes of p-ErbB3(Y1289) levels upon GDC-0994 treatment (N ≥ 4 independent experiments). (D) Negative correlation between p-ErbB3(Y1289) and p-MAPK(T202/Y204) levels in MAPK-mutant HNSCC cell lines based on the published CCLE-proteomic data and in MAPK-mutated HNSCC patient tumors (allele frequencies [AFs] >40%) based on TCPA HNSCC RPPA cohort ( , ). (E) Immunohistochemical staining for p-ErbB3(Y1289) in MAPK-mutated HNSCC patient tumors with high AFs close to 40%: T40 ( MAPK1 p.D321N with AF = 39.1%) and T43 ( HRAS p.G12S with AF = 37.3%) versus T47 and T82 (both are MAPKWT). Source data are available for this figure.

Article Snippet: Primary antibodies include AKT (#9272), pi-AKT (#9271), ARAF(#4432), pi-ARAF(S299) (#4431), BRAF (#2696), pi-BRAF(S445) (#2696), pi-ErbB3 (#2842), ErbB3 (#12708), MAPK (#9102), pi-MAPK (#9101), pi-MEK1/2 (#9154), and RSK1 (#8408), all from Cell Signaling Technology, USA.

Techniques: Western Blot, Expressing, Mutagenesis, Construct, Retroviral, Infection, Immunohistochemical staining, Staining

Journal: Life Science Alliance

Article Title: MAPK pathway mutations in head and neck cancer affect immune microenvironments and ErbB3 signaling

doi: 10.26508/lsa.201900545

Figure Lengend Snippet: Cumulative phospho-ErbB3(Y1289)/total-ErbB3 ratio of MAPK-mutants expressing as well as GDC-0994 inhibited FaDu cel ls. (A) Cumulative quantification plots of phospho-ErbB3(Y1289)/total-ErbB3 ratio upon ectopic expression of mutants and WTs of MAPK1 , ARAF , BRAF , HRAS , MAP2K1 , and MAP2K2 in FaDu cells (N ≥ 4 independent experiments). (B) Cumulative quantification plots of phospho-ErbB3(Y1289) and total-ErbB3 levels in HNSCC Pt.25 primary tumor cultures (carrying both HRAS p.G12S and MAPK1 p.R135K mutations), HSC-6 cell line (carrying MAPK1 p.E322K mutation) and HSC4 (MAPK pathway WT, CCLE) upon MAPK inhibitor GDC-0994 treatment for 30 min. The quantified changes of p-ErbB3(Y1289)/total-ErbB3 ratio upon GDC-0994 treatment were showed (N ≥ 4 independent experiments).

Article Snippet: Primary antibodies include AKT (#9272), pi-AKT (#9271), ARAF(#4432), pi-ARAF(S299) (#4431), BRAF (#2696), pi-BRAF(S445) (#2696), pi-ErbB3 (#2842), ErbB3 (#12708), MAPK (#9102), pi-MAPK (#9101), pi-MEK1/2 (#9154), and RSK1 (#8408), all from Cell Signaling Technology, USA.

Techniques: Expressing, Mutagenesis

Journal: Life Science Alliance

Article Title: MAPK pathway mutations in head and neck cancer affect immune microenvironments and ErbB3 signaling

doi: 10.26508/lsa.201900545

Figure Lengend Snippet: (A) Oncoprints (20% and 50% cutoffs respectively) showing that MAPK pathway-mutated patients with low p-ErbB3 protein expression, and high IFN-γ functionality, T-effector signature and CYT score were not significantly overlapping (P = n.s.). The upper panel shows the oncoprint with deep blue color ( ) denoting those with bottom 20% of pErbB3 level (i.e., p-ErbB3 down-regulation), and levels above that as noncolored white bars ( ), whereas individuals without available RPPA data on p-ErbB3 are denoted by grey bars ( ). Similarly, those with top 20% immune scores, IFN-γ score, CYT score and T-effector are indicated by deep pink ( ), light pink ( ) and orange ( ), respectively, while non-colored white bars ( ) denote patients with immune scores lower than the top 20%. In the lower panel, the same color coding is adopted, but the colored bars refer to patients with a median (i.e., 50% cutoffs), that is, lower 50% for p-ErbB3 level and top 50% for IFN-γ, CYT, and T-effector scores. (B) A schematic summarizing two plausible mechanisms for markedly improved clinical outcomes in HNSCC tumors with MAPK aberrations.

Article Snippet: Primary antibodies include AKT (#9272), pi-AKT (#9271), ARAF(#4432), pi-ARAF(S299) (#4431), BRAF (#2696), pi-BRAF(S445) (#2696), pi-ErbB3 (#2842), ErbB3 (#12708), MAPK (#9102), pi-MAPK (#9101), pi-MEK1/2 (#9154), and RSK1 (#8408), all from Cell Signaling Technology, USA.

Techniques: Expressing

Journal: Life Science Alliance

Article Title: MAPK pathway mutations in head and neck cancer affect immune microenvironments and ErbB3 signaling

doi: 10.26508/lsa.201900545

Figure Lengend Snippet: (A) Kaplan–Meier overall survival curves for head and neck squamous cell carcinoma (HNSCC) patients, whose tumors overexpressed versus underexpressed phospho-ErbB3(Y1289) (median cutoff) in TCPA HNSCC cohort (N = 344). (B) Western blot results of phospho-ErbB3(Y1289) protein levels upon ectopic expression of the MAPK1 , ARAF , BRAF , HRAS , and MAP2K1 as well as MAP2K2 wild-type and mutant constructs in FaDu cells by retroviral infection (pool of at least four independent repeats). (C) Western blot results of phospho-ErbB3(Y1289) levels of HNSCC Pt.25 primary tumor cultures (carrying both HRAS p.G12S and MAPK1 p.R135K mutations), HSC-6 cell line (carrying MAPK1 p.E322K mutation), and HSC-4 (MAPK pathway WT, Cancer Cell Line Encyclopedia [CCLE]) upon MAPK inhibitor GDC-0994 treatment for 30 min. 50 µg of protein lysate was used for Pt.25 and HSC-4 samples, 50 μg of protein lysate was used, whereas for HSC-6 (because of the relatively low endogenous p-RSK levels intrinsic to this cell line), 100 μg of protein lysate was loaded for presentation of signal clarity. Bar graphs showing the quantified changes of p-ErbB3(Y1289) levels upon GDC-0994 treatment (N ≥ 4 independent experiments). (D) Negative correlation between p-ErbB3(Y1289) and p-MAPK(T202/Y204) levels in MAPK-mutant HNSCC cell lines based on the published CCLE-proteomic data and in MAPK-mutated HNSCC patient tumors (allele frequencies [AFs] >40%) based on TCPA HNSCC RPPA cohort ( , ). (E) Immunohistochemical staining for p-ErbB3(Y1289) in MAPK-mutated HNSCC patient tumors with high AFs close to 40%: T40 ( MAPK1 p.D321N with AF = 39.1%) and T43 ( HRAS p.G12S with AF = 37.3%) versus T47 and T82 (both are MAPKWT). Source data are available for this figure.

Article Snippet: Primary antibodies include AKT (#9272), pi-AKT (#9271), ARAF(#4432), pi-ARAF(S299) (#4431), BRAF (#2696), pi-BRAF(S445) (#2696), pi-ErbB3 (#2842), ErbB3 (#12708), MAPK (#9102), pi-MAPK (#9101), pi-MEK1/2 (#9154), and RSK1 (#8408), all from Cell Signaling Technology, USA.

Techniques: Western Blot, Expressing, Mutagenesis, Construct, Retroviral, Infection, Immunohistochemical staining, Staining

Journal: Life Science Alliance

Article Title: MAPK pathway mutations in head and neck cancer affect immune microenvironments and ErbB3 signaling

doi: 10.26508/lsa.201900545

Figure Lengend Snippet: Cumulative phospho-ErbB3(Y1289)/total-ErbB3 ratio of MAPK-mutants expressing as well as GDC-0994 inhibited FaDu cel ls. (A) Cumulative quantification plots of phospho-ErbB3(Y1289)/total-ErbB3 ratio upon ectopic expression of mutants and WTs of MAPK1 , ARAF , BRAF , HRAS , MAP2K1 , and MAP2K2 in FaDu cells (N ≥ 4 independent experiments). (B) Cumulative quantification plots of phospho-ErbB3(Y1289) and total-ErbB3 levels in HNSCC Pt.25 primary tumor cultures (carrying both HRAS p.G12S and MAPK1 p.R135K mutations), HSC-6 cell line (carrying MAPK1 p.E322K mutation) and HSC4 (MAPK pathway WT, CCLE) upon MAPK inhibitor GDC-0994 treatment for 30 min. The quantified changes of p-ErbB3(Y1289)/total-ErbB3 ratio upon GDC-0994 treatment were showed (N ≥ 4 independent experiments).

Article Snippet: Primary antibodies include AKT (#9272), pi-AKT (#9271), ARAF(#4432), pi-ARAF(S299) (#4431), BRAF (#2696), pi-BRAF(S445) (#2696), pi-ErbB3 (#2842), ErbB3 (#12708), MAPK (#9102), pi-MAPK (#9101), pi-MEK1/2 (#9154), and RSK1 (#8408), all from Cell Signaling Technology, USA.

Techniques: Expressing, Mutagenesis

Journal: Life Science Alliance

Article Title: MAPK pathway mutations in head and neck cancer affect immune microenvironments and ErbB3 signaling

doi: 10.26508/lsa.201900545

Figure Lengend Snippet: (A) Oncoprints (20% and 50% cutoffs respectively) showing that MAPK pathway-mutated patients with low p-ErbB3 protein expression, and high IFN-γ functionality, T-effector signature and CYT score were not significantly overlapping (P = n.s.). The upper panel shows the oncoprint with deep blue color ( ) denoting those with bottom 20% of pErbB3 level (i.e., p-ErbB3 down-regulation), and levels above that as noncolored white bars ( ), whereas individuals without available RPPA data on p-ErbB3 are denoted by grey bars ( ). Similarly, those with top 20% immune scores, IFN-γ score, CYT score and T-effector are indicated by deep pink ( ), light pink ( ) and orange ( ), respectively, while non-colored white bars ( ) denote patients with immune scores lower than the top 20%. In the lower panel, the same color coding is adopted, but the colored bars refer to patients with a median (i.e., 50% cutoffs), that is, lower 50% for p-ErbB3 level and top 50% for IFN-γ, CYT, and T-effector scores. (B) A schematic summarizing two plausible mechanisms for markedly improved clinical outcomes in HNSCC tumors with MAPK aberrations.

Article Snippet: Primary antibodies include AKT (#9272), pi-AKT (#9271), ARAF(#4432), pi-ARAF(S299) (#4431), BRAF (#2696), pi-BRAF(S445) (#2696), pi-ErbB3 (#2842), ErbB3 (#12708), MAPK (#9102), pi-MAPK (#9101), pi-MEK1/2 (#9154), and RSK1 (#8408), all from Cell Signaling Technology, USA.

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: Cryptotanshinone suppresses tumorigenesis by inhibiting lipogenesis and promoting reactive oxygen species production in KRAS-activated pancreatic cancer cells

doi: 10.3892/ijo.2022.5398

Figure Lengend Snippet: Effects of CPT on human PDAC (MIAPaCa-2 and BxPC3) and CRC (DLD-1 and DKO4) cell lines. (A) Spheroid viability of PDAC (left) and CRC (right) cell lines treated with CPT. A total of 1×10 3 cells were seeded into a 96-well V-bottom plate and incubated with CPT for 72 h in triplicate. Spheroid viability was determined by measuring the ATP content using the CellTiter-Glo 3D assay. Closed and open circles represent the cell lines with mutant KRAS (MIAPaCa-2 and DLD-1) and wild-type KRAS (BxPC3 and DKO4), respectively. Error bars represent standard deviation. * P<0.05 and *** P<0.001 vs. the wild-type KRAS cells treated with the same CPT concentration. (B) Immunoblot analyses of K-Ras protein in cells treated with CPT. A total of 5×10 5 cells were seeded into poly-HEMA-coated 60 mm dishes and incubated with CPT at 10 and 20 µ M for 48 h. GAPDH was used as the internal control. CPT, cryptotanshinone; PDAC, pancreatic ductal adenocarcinoma; CRC, colorectal cancer; KRAS, Kirsten rat sarcoma viral oncogene homolog.

Article Snippet: Mouse monoclonal antibodies for GAPDH (1:3,000; cat. no. AC002) and rabbit monoclonal antibodies for KRAS (1:3,000; cat. no. A12704) and IDH1 (1:3,000; cat. no. A13245) were purchased from ABclonal Biotech Co., Ltd. Rabbit monoclonal antibodies for FASN (1:1,000; cat. no. #3189S), anti-ACLY rabbit polyclonal (1:1,000; cat. no. 4332S), anti-phosphorylated (p)ACLY rabbit polyclonal (1:1,000; p-S445; cat. no. 4331S) and anti-ACC1 rabbit polyclonal (1:1,000; cat. no. 3662) antibodies and anti-p-ACC1 rabbit polyclonal (1:1,000; p-S79; cat. no. 3661S) were purchased from Cell Signaling Technology, Inc.

Techniques: Incubation, Mutagenesis, Standard Deviation, Concentration Assay, Western Blot, Control

Journal: International Journal of Oncology

Article Title: Cryptotanshinone suppresses tumorigenesis by inhibiting lipogenesis and promoting reactive oxygen species production in KRAS-activated pancreatic cancer cells

doi: 10.3892/ijo.2022.5398

Figure Lengend Snippet: Immunoblot analyses of proteins regulating glutaminolysis and lipogenesis in nonadherent MIAPaCa-2 (mutant KRAS) and BxPC3 (wild-type KRAS) cancer cells treated with CPT. A total of 5×10 5 cells were seeded into poly-HEMA-coated 60 mm dishes and treated with CPT at 10 and 20 µ M for 48 h. GAPDH was used as the internal control. KRAS, Kirsten rat sarcoma viral oncogene homolog; CPT, cryptotanshinone; GOT, glutamic-oxaloacetic transaminase; GLUD, glutamate transport system permease protein; GLS, glutaminase; IDH, isocitrate dehydrogenase; FASN, fatty acid synthase; ACC1, acetyl-CoA carboxylase 1; ACLY, ATP-citrate lyase; p-, phosphorylated.

Article Snippet: Mouse monoclonal antibodies for GAPDH (1:3,000; cat. no. AC002) and rabbit monoclonal antibodies for KRAS (1:3,000; cat. no. A12704) and IDH1 (1:3,000; cat. no. A13245) were purchased from ABclonal Biotech Co., Ltd. Rabbit monoclonal antibodies for FASN (1:1,000; cat. no. #3189S), anti-ACLY rabbit polyclonal (1:1,000; cat. no. 4332S), anti-phosphorylated (p)ACLY rabbit polyclonal (1:1,000; p-S445; cat. no. 4331S) and anti-ACC1 rabbit polyclonal (1:1,000; cat. no. 3662) antibodies and anti-p-ACC1 rabbit polyclonal (1:1,000; p-S79; cat. no. 3661S) were purchased from Cell Signaling Technology, Inc.

Techniques: Western Blot, Mutagenesis, Control

Journal: International Journal of Oncology

Article Title: Cryptotanshinone suppresses tumorigenesis by inhibiting lipogenesis and promoting reactive oxygen species production in KRAS-activated pancreatic cancer cells

doi: 10.3892/ijo.2022.5398

Figure Lengend Snippet: Immunoblot analyses of proteins regulating glutaminolysis and lipogenesis in MIAPaCa-2 cells treated with control (siC) and KRAS siRNA (siKRAS). (A) A total of 1×10 5 cells were treated with siRNA for 24 h, followed by nonadherent culture (48 h). GAPDH was used as the internal control. (B) Quantitative analysis of protein expression levels of K-Ras, FASN and ACC1 after siKRAS treatment in MIAPaCa-2 cells. The bars indicate the relative expression value normalized to those of GAPDH and are presented as mean ± standard deviation of three independent assays. * P<0.05 compared with siC using Welch's t-test. KRAS, Kirsten rat sarcoma viral oncogene homolog; si, short interfering; FASN, fatty acid synthase; ACC1, acetyl-CoA carboxylase 1; ACLY, ATP-citrate lyase; GOT, glutamic-oxaloacetic transaminase; IDH, isocitrate dehydrogenase; GLUD, glutamate transport system permease protein; GLS, glutaminase; p-, phosphorylated.

Article Snippet: Mouse monoclonal antibodies for GAPDH (1:3,000; cat. no. AC002) and rabbit monoclonal antibodies for KRAS (1:3,000; cat. no. A12704) and IDH1 (1:3,000; cat. no. A13245) were purchased from ABclonal Biotech Co., Ltd. Rabbit monoclonal antibodies for FASN (1:1,000; cat. no. #3189S), anti-ACLY rabbit polyclonal (1:1,000; cat. no. 4332S), anti-phosphorylated (p)ACLY rabbit polyclonal (1:1,000; p-S445; cat. no. 4331S) and anti-ACC1 rabbit polyclonal (1:1,000; cat. no. 3662) antibodies and anti-p-ACC1 rabbit polyclonal (1:1,000; p-S79; cat. no. 3661S) were purchased from Cell Signaling Technology, Inc.

Techniques: Western Blot, Control, Expressing, Standard Deviation

Journal: International Journal of Oncology

Article Title: Cryptotanshinone suppresses tumorigenesis by inhibiting lipogenesis and promoting reactive oxygen species production in KRAS-activated pancreatic cancer cells

doi: 10.3892/ijo.2022.5398

Figure Lengend Snippet: Effect of siRNA against KRAS (siKRAS) and FASN (siFASN) on (B) 3D-spheroid formation, (C-F) lipogenesis and (G and H) redox regulation in MIAPaCa-2 cells. (A) Immunoblot analysis of MIAPaCa-2 cells treated with control (siC), siKRAS and siFASN. A total of 1×10 5 cells were treated with the siRNAs for 24 h, followed by nonadherent culture for 48 h. GAPDH was used as the internal control. (B) 3D-spheroid viability of cell lines treated with siC, siKRAS and siFASN. A total of 1×10 5 cells were treated with siRNA for 24 h, followed by spheroid culture in a 96-well V-bottom plate for 72 h in triplicate. 3D-spheroid viability was determined by measuring adenosine triphosphate (ATP) content using the CellTiter-Glo ® 3D assay. Error bars represent standard deviation. *** P<0.001 vs. siC using Tukey's test. (C) Box plot of the areas of lipid droplets in MIAPaCa-2 cells treated with siC, siKRAS and siFASN. The cells were seeded into culture dishes at a density of 1.0×10 5 cells/dish. When the cell density reached ~60%, siKRAS and siFASN were added and incubated for 72 h. The cells were fixed in 4% paraformaldehyde and stained with LipiDyeII and DAPI. The integrated density of LipiDyeII staining (n=20 for each group) was measured using ImageJ software and normalized to the average of the control (siC) group. In the plots, the box indicates the lower and upper quartiles, the horizontal bar represents the median, the whiskers are the highest and lowest data points that fall within 1.5 times the interquartile range from the lower and upper quartiles and the dots are the outliers. *** P<0.001 compared with siC using Tukey's test. Representative images of lipid droplet formation treated with (D) siC, (E) siKRAS and (F) siFASN for 72 h in MIAPaCa-2 cells. The nuclei of the cells were stained with DAPI (blue) and the lipid droplets were stained with LipiDyeII (red). Scale bars=10 µ m. (G) H 2 O 2 content in MIAPaCa-2 cells treated with siC, siKRAS and siFASN. A total of 1×10 5 cells were treated with siKRAS and siFASN for 24 h, followed by nonadherent culture for 48 h. H 2 O 2 concentration in the culture medium was determined using the Amplite fluorimetric H 2 O 2 kit and normalized to cellular protein content. The bars indicate the mean ± standard deviation. *** P<0.001 compared with siC using Tukey's test. n.s., not significant. (H) GSH/GSSG in MIAPaCa-2 cells treated with siC, siKRAS and siFASN. A total of 1×10 5 cells were treated with siKRAS and siFASN for 24 h, followed by nonadherent culture for 48 h. GSH/GSSG was determined by measuring GSH and GSSG using the GSH/GSSG-Glo Assay kit. The bars indicate mean ± standard deviation. * P<0.05 compared with siC using Tukey's test. si, short interfering; KRAS, Kirsten rat sarcoma viral oncogene homolog; FASN, fatty acid synthase; GSH, glutathione-SH; GSSG, glutathione disulfide.

Article Snippet: Mouse monoclonal antibodies for GAPDH (1:3,000; cat. no. AC002) and rabbit monoclonal antibodies for KRAS (1:3,000; cat. no. A12704) and IDH1 (1:3,000; cat. no. A13245) were purchased from ABclonal Biotech Co., Ltd. Rabbit monoclonal antibodies for FASN (1:1,000; cat. no. #3189S), anti-ACLY rabbit polyclonal (1:1,000; cat. no. 4332S), anti-phosphorylated (p)ACLY rabbit polyclonal (1:1,000; p-S445; cat. no. 4331S) and anti-ACC1 rabbit polyclonal (1:1,000; cat. no. 3662) antibodies and anti-p-ACC1 rabbit polyclonal (1:1,000; p-S79; cat. no. 3661S) were purchased from Cell Signaling Technology, Inc.

Techniques: Western Blot, Control, Standard Deviation, Incubation, Staining, Software, Concentration Assay, Glo Assay