Journal: iScience

Article Title: An integrative proteomics approach identifies tyrosine kinase KIT as a therapeutic target for SPINK1-positive prostate cancer

doi: 10.1016/j.isci.2024.108794

Figure Lengend Snippet: Global quantitative proteome and phosphoproteome profiling of the SPINK1-knockdown prostate cancer cells (A) Heatmap depicting the Z-scaled abundance of significant proteins in 22RV1-shSCRM and 22RV1-shSPINK1 cells. (B) Same as in (A), except for significantly enriched phosphoproteins in 22RV1-shSCRM and 22RV1-shSPINK1 cells. Each sample was analyzed in biological triplicates. (C) Volcano plot showing the differentially enriched proteins in 22RV1-shSPINK1 versus 22RV1-shSCRM cells; blue dots are downregulated, and red dots are upregulated proteins in shSPINK1, whereas black dots signify no change. (D) Kinase-substrate enrichment analysis of the phosphopeptides enriched in 22RV1-shSPINK1 versus 22RV1-shSCRM cells. Identified kinases are plotted with their respective Z scores; blue kinases have negative Z score, and red kinases have positive Z score in 22RV1-shSPINK1 versus 22RV1-shSCRM cells. A negative Z score corresponds to a collective dephosphorylation of the kinase’s substrates; the inverse is true for a positive score. (E) Pathway enrichment analysis for differential phosphoproteins in 22RV1-shSPINK1 versus 22RV1-shSCRM cells using pathfindR; size of the dot represents number of genes, and the color symbolizes the -log 10 (p value) of the enriched pathway. See also Figure S1 and .

Article Snippet: 1:100 diluted SPINK1 [4D4] (Novus Biologicals, H00006690-M01), 1:20 diluted CD117/KIT (DAKO, A4502), 1:25 diluted CD44 (DAKO, M7082), Ki67 [MIB-1] (DAKO, IR626) in EnVision FLEX Antibody Diluent (Agilent DAKO, K800621-2) was added to each slide and incubated overnight at 4°C.

Techniques: Knockdown, De-Phosphorylation Assay

Journal: iScience

Article Title: An integrative proteomics approach identifies tyrosine kinase KIT as a therapeutic target for SPINK1-positive prostate cancer

doi: 10.1016/j.isci.2024.108794

Figure Lengend Snippet: KIT tyrosine kinase is highly expressed in androgen-independent prostatic tumors (A) KIT mRNA expression in Beltran et al., 2016 dataset. p value was calculated using Unpaired Student’s t test with Welch’s correction. (B) Same as (A), except for Aggarwal et al., 2018 dataset. (C) Scatterplot showing correlation of KIT mRNA expression (FPKM polyA) [log 10 (value + 1)] with AR signaling score in metastatic prostate adenocarcinoma (SU2C/PCF 2019) dataset. Coefficient values for both Spearman and Pearson correlation along with the p value are depicted in the plot. (D) Same as in (C), except for the correlation of KIT mRNA expression with NEPC score. (E) Micrographs representing immunohistochemical (IHC) staining for KIT and SPINK1 expression in tissue microarrays of prostate cancer specimens, SPINK1-positive patient (left) and SPINK1-negative patient (right). Scale bar represents 300 μm. (F) Bar plot depicting the percentage of SPINK1-positive patients' specimens for KIT intensity (high/medium/low/negative). (G) Dot plot showing KIT expression in PCa patients with or without ADT, GSE48403 . Statistical significance was calculated using paired t test. (H) Bar plots showing SPINK1 and KIT expression in 22 weeks ADT-treated PCa patient with Gleason score 8 and TNM stage 3b. See also Figure S3 and .

Article Snippet: 1:100 diluted SPINK1 [4D4] (Novus Biologicals, H00006690-M01), 1:20 diluted CD117/KIT (DAKO, A4502), 1:25 diluted CD44 (DAKO, M7082), Ki67 [MIB-1] (DAKO, IR626) in EnVision FLEX Antibody Diluent (Agilent DAKO, K800621-2) was added to each slide and incubated overnight at 4°C.

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: iScience

Article Title: An integrative proteomics approach identifies tyrosine kinase KIT as a therapeutic target for SPINK1-positive prostate cancer

doi: 10.1016/j.isci.2024.108794

Figure Lengend Snippet: KIT tyrosine kinase inhibition attenuates oncogenic characteristics (A) Immunoblot showing phospho-KIT (Y703), KIT, phospho-EGFR (Y845), and EGFR levels in 22RV1 cells stimulated with recombinant SPINK1 (100 ng/mL) at different time points. β-actin was used as the loading control. (B) Line plot representing relative cell proliferation of 22RV1 cells along with different concentrations of KIT tyrosine kinase inhibitor, KITi, and control (CTL). (C) Same as in (B), except for 42D ENZR cells. (D) Representative images for foci of 22RV1 cells in the respective conditions (top). Bar plot showing percent foci formation efficiency of 22RV1 cells treated with indicated concentrations of KITi and CTL. Scale bar represents 1,000 μm. (E) Same as in (D), except for 42D ENZR cells. (F) Representative images (top) and bar plots showing percent tumorsphere formation efficiency (left) and mean area of 22RV1 tumorspheres (right) treated with indicated concentrations of KITi and CTL. Scale bar represents 100 μm. (G) Immunoblot showing KIT levels in KIT -silenced 22RV1 cells. β-actin was used as the loading control (top). Same as in (B), except for KIT -silenced 22RV1 cells (bottom). (H) Same as in (D), except for KIT -silenced 22RV1 cells. (I) Same as in (F), except for KIT -silenced 22RV1 cells. Each experiment was performed in biological triplicates (N = 3); the bar represents mean ± SEM, and each dot represents individual value. Statistical significance was calculated using two-way or one-way ANOVA followed by Dunnett’s multiple comparison test. p value: ∗<0.05 and ∗∗<0.01. See also Figure S5 .

Article Snippet: 1:100 diluted SPINK1 [4D4] (Novus Biologicals, H00006690-M01), 1:20 diluted CD117/KIT (DAKO, A4502), 1:25 diluted CD44 (DAKO, M7082), Ki67 [MIB-1] (DAKO, IR626) in EnVision FLEX Antibody Diluent (Agilent DAKO, K800621-2) was added to each slide and incubated overnight at 4°C.

Techniques: Inhibition, Western Blot, Recombinant, Control, Comparison

Journal: iScience

Article Title: An integrative proteomics approach identifies tyrosine kinase KIT as a therapeutic target for SPINK1-positive prostate cancer

doi: 10.1016/j.isci.2024.108794

Figure Lengend Snippet: KIT tyrosine kinase inhibition abrogates SPINK1-positive tumor growth and metastases (A) Line plot representing tumor volume of 22RV1 cells subcutaneously implanted in NOD/SCID mice and administered with KITi (50 mg/kg)/CTL via oral gavage (each group, N = 5). (B) Bar plot showing the relative percent reduction in tumor volume. (C) Same as in (A), except for the plot depicting mice’s body weight. (D) Micrographs representing IHC staining for CD44 and Ki67 in tumor sections from xenografted mice as shown in (A). Scale bar represents 200 μm and 50 μm (for insets). (E) Bar plots showing quantification of IHC staining for CD44 and Ki67. Twenty random fields were quantified for each group. (F) Bar plots representing the number of cells metastasized to the bone marrow in xenografted mice, as shown in (A). (G) Same as in (F), except for the number of cells metastasized to the lungs. Bar represents mean ± SEM, and each dot represents individual value. Statistical significance was calculated using unpaired Student’s t test. p-value: ∗<0.05, ∗∗<0.01 and NS = non-significant.

Article Snippet: 1:100 diluted SPINK1 [4D4] (Novus Biologicals, H00006690-M01), 1:20 diluted CD117/KIT (DAKO, A4502), 1:25 diluted CD44 (DAKO, M7082), Ki67 [MIB-1] (DAKO, IR626) in EnVision FLEX Antibody Diluent (Agilent DAKO, K800621-2) was added to each slide and incubated overnight at 4°C.

Techniques: Inhibition, Immunohistochemistry

Journal: iScience

Article Title: An integrative proteomics approach identifies tyrosine kinase KIT as a therapeutic target for SPINK1-positive prostate cancer

doi: 10.1016/j.isci.2024.108794

Figure Lengend Snippet:

Article Snippet: 1:100 diluted SPINK1 [4D4] (Novus Biologicals, H00006690-M01), 1:20 diluted CD117/KIT (DAKO, A4502), 1:25 diluted CD44 (DAKO, M7082), Ki67 [MIB-1] (DAKO, IR626) in EnVision FLEX Antibody Diluent (Agilent DAKO, K800621-2) was added to each slide and incubated overnight at 4°C.

Techniques: Virus, Recombinant, Protease Inhibitor, cDNA Synthesis, Software

Journal: The Journal of nutrition

Article Title: Suppression of High-Fat Diet-Induced Obesity by Platycodon Grandiflorus in Mice Is Linked to Changes in the Gut Microbiota.

doi: 10.1093/jn/nxaa159

Figure Lengend Snippet: FIGURE 2 Fatty acid catabolism and lipid anabolism in the adipose tissues of male C57BL/6J mice fed CON, HFD, or HFPG. (A) Map of genes involved in fat accumulation and fatty-acid metabolism affected by PG supplementation. (B) Western blot analysis for p-AMPK, p-ACC, and FASN in epididymal adipose tissues. (C) Fold change in densitometric concentrations of p-AMPK/AMPK, p-ACC/ACC, and FASN/ACTB relative to the CON group (n = 3). (D) Relative levels of mRNA expression in epididymal adipose tissues tested by qRT-PCR (n = 3). Values represent means ± SEMs, n = 7 if not specified. Labeled means without a common letter differ, P < 0.05. ACC, acetyl-CoA carboxylase; ACTB, actin, β; Adipoq, adiponectin; AMPK, AMP-activated protein kinase; ATGL, adipose triglyceride lipase; CEBPA, CCAAT/enhancer binding protein α; CON, control diet; FASN, fatty acid synthase; HFD, high-fat diet; HFPG, high-fat diet and PG supplementation; Lep, leptin; p-ACC, phosphorylated acetyl-CoA carboxylase; p-AMPK, phosphorylated AMP-activated protein kinase; PG, Platycodon grandiflorus; PPARG, peroxisome proliferator– activated receptor γ ; Srebf1, sterol regulatory element binding transcription factor 1; SREBP-1c, sterol regulatory element binding protein-1c; WAT, white adipose tissue.

Article Snippet: The primary antibodies against AMP-activated protein kinase α (AMPKα) (no. 5831), phosphorylated-AMPKα (p-AMPKα) (Thr172) (no. 2535), acetyl-CoA carboxylase (ACC) (no. 3676), phosphorylated-ACC (p-ACC) (Ser79) (no. 11818), fatty acid synthase (FASN) (no. 3180), and horseradish peroxidase–conjugated anti-rabbit secondary antibody (no. 7074) were obtained from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Labeling, Binding Assay, Control

Journal: Chinese Medical Journal

Article Title: Metformin improved oxidized low-density lipoprotein-impaired mitochondrial function and increased glucose uptake involving Akt-AS160 pathway in raw264.7 macrophages

doi: 10.1097/cm9.0000000000000333

Figure Lengend Snippet: Figure 3: Metformin improved lipid and glucose oxidation in Ox-LDL-loaded macrophages. (A) Effects of metformin on protein expression of enzymes mediating lipid synthesis and oxidation. (B) Metformin increased Ox-LDL-impaired glucose consumption. Glucose consumption was normalized by protein concentrations of cellular lysates. (C) Metformin decreased lactic acid production in Ox-LDL-loaded macrophages. Lactic acid levels were normalized by protein concentrations of cellular lysates. (D) Metformin down-regulated the ratio of lactic acid output by glucose input in the presence of Ox-LDL. ∗P < 0.05, †P < 0.01, xP < 0.001 vs. control; ‡P < 0.01, jjP < 0.001 vs. Ox-LDL. CPT-1b: Carnitine palmitoyltransferase 1b; NDUFS3: Nicotinamide adenine dinucleotide:ubiquinone oxidoreductase core sub-unit S3; Ox-LDL: Oxidized low-density lipoprotein; p-ACC: Acetyl-CoA carboxylase phosphorylation.

Article Snippet: After being blocked with 5% bovine serum albumin (BSA), blots were incubated with various primary antibodies including rabbit anti-Akt 1/2/3 (Thermo Fisher), rabbit anti-dynamin-related protein 1 (DRP-1), optic atrophy 1 (OPA1), mitofusin 2 (MFN2), Akt substrate of 160 kDa Cellular reactive oxygen species detection (AS160), nicotinamide adenine dinucleotide:ubiquinone oxidoreductase core sub-unit S3 (NDUFS3; Abcam, Cambridge, UK), rabbit anti-AMPK, P-AMPK (Thr172), acetyl-CoA carboxylase (ACC), ACC phosphorylation (pACC; Ser79), P-Akt (Ser473), TBC1 family domain member 1 (TBC1D1) phosphorylation (p-TBC1D1; Ser660) (CST, Beverly, MA, USA), anti-P-AS160 (T642) (Biorbyt, Cambridge, UK), anti-carnitine palmitoyltransferase (CPT)-1b (SAB, Nanjing, China), and rabbit anti-b-actin (Bio-world, St. Louis Park, MN, USA) at 4°C overnight.

Techniques: Expressing, Control, Phospho-proteomics

Journal: Nutrients

Article Title: Anti-Obesity Effect of Different Opuntia stricta var. dillenii ’s Prickly Pear Tissues and Industrial By-Product Extracts in 3T3-L1 Mature Adipocytes

doi: 10.3390/nu16040499

Figure Lengend Snippet: Effects of Opuntia stricta var. dillenii ’s extracts on GLUT4, ACC, and HSL protein expressions and the ratios of phosphorylated-ACC/total phosphorylated-HSL/total HSL ratio in 3T3-L1 mature adipocytes treated for 24 h. Values are means ± SEM. Comparisons of extracts and the control for each gene expression were carried out using Student’s t -test. The asterisks (*) represent differences versus the controls ( p < 0.05).

Article Snippet: The protein levels were detected via specific antibodies for phosphorylated ACC (Serenine 79, 1:1000), phosphorylated HSL (Serenine 660, 1:1000) (Cell Signaling Technology, Danvers, MA, USA) and GLUT4 (1:1000) (Santa Cruz Biotech, Dallas, TX, USA).

Techniques: Control, Gene Expression

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Melanocortin-induced PKA activation inhibits AMPK activity via ERK-1/2 and LKB-1 in hypothalamic GT1-7 cells.

doi: 10.1210/me.2011-1218

Figure Lengend Snippet: FIG. 3. -MSH-induced dephosphorylation of AMPK (Thr172) or of ACC (Ser79) in GT1-7 cells monitored by whole-cell ELISA. Approximately 25,000 GT1-7 cells were seeded into white 96-well dishes. After 20 h of serum starvation, cells were stimulated with 1 M -MSH for 10 min and phosphorylation of AMPK at Thr172 (A), total AMPK levels (B), or phosphorylation of ACC at Ser79 (C) monitored by specific antibodies in whole cells. Cell-bound, HRP-conjugated secondary antibody was detected by automatic injection of SuperSignal West Femto HRP substrate in a FLUOstar Omega plate reader at time point 1 sec (as indicated by the arrow) and total luminescence recorded for additional 8 sec. As a control, luminescence signals were measured in samples without any first antibody. A–C, Raw data (eight replicates) of one representative experiment are shown as the mean SEM. D, Data of three independent experiments were compiled, control values subtracted and normalized by setting RLU values of unstimulated cells (basal) at time point 9 sec as 100%. -MSH-induced dephosphorylation was than calculated by subtracting values of stimulated cells from 100%. Hashed signs (##, P 0.01) indicate a significant difference to unstimulated cells (basal).

Article Snippet: Primary antibodies phospho (p)-ERK-1/2 or total (t)-ERK-2 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and t-AMPK- , p-AMPKThr172, p-ACC-Ser79, or LKB-1, were from Cell Signaling (Danvers, MA).

Techniques: De-Phosphorylation Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Injection, Control