Journal: EBioMedicine

Article Title: Inhibition of oncogenic Src induces FABP4-mediated lipolysis via PPARγ activation exerting cancer growth suppression

doi: 10.1016/j.ebiom.2019.02.015

Figure Lengend Snippet: Src negatively regulates lipolysis. (a) Correlation of endogenous Src expression to lipid droplets in a panel of lung cancer (left) and renal cell carcinoma cell lines (right). Together with basal protein expression of pSrc and Src, lipid contents were assayed using Oil-red O staining in the panel. (b) Expression of lipolytic genes in lung cancer cells. The mRNA expression of FABP4 and LPL was shown using the RT-PCR assay. (c) Reverse correlation between Src and FABP or LPL. The analysis showed negative Pearson's correlation coefficient for the expression pattern between Src and FABP4 as well as Src and LPL. For biostatistics analysis, data were obtained for lung adenocarcinoma or RCC patients from the cbioportal database. (d-e) Pharmacological inhibition of Src induces FABP4 expression. (d) Expression of FABP4 mRNA (left) and protein (right) was assayed upon treatment of Src inhibitor SU6656 compound in the lung cancer cell lines. Cells were treated with 5 μM of SU6656 for 24, 48, and 72 h and assayed for FABP4 protein expression or 24 h for mRNA expression. (e) Oncogenic Src was exogenously overexpressed in Calu6 cells and followed by FABP4 expression upon SU6656 treatment. Cells were transfected with pEGFP control or wtSrc-GFP followed by SU6656 treatment for 24 h. Data represent mean ± SEM of triplicates. Similar results were observed in at least two independent experiments. Asterisks refer to ** P ≤ 0.01; *** P ≤ 0∙001; **** P ≤ 0∙001 (Student's t -test (d) and one-way ANOVA, Tukey's post test (e)). In all RT-PCR data, y-axis represents fold change in gene expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies include β-actin (Abcam Cat# ab6276, RRID: AB_2223210 ), 4HNE (Abcam Cat# ab46545, RRID: AB_722490 ), GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID: AB_10167668 ), FYN (BD Cat# 610164, RRID: AB_397565 ), phospho-Stat3 (Y705) (Cell Signaling Technology Cat# 9131, RRID: AB_331586 ), Stat3 (Cell Signaling Technology Cat# 9139, RRID: AB_331757 ), pSrc (Y416) (Cell Signaling Technology Cat# 6943, RRID: AB_10013641 ), Src (Cell Signaling Technology Cat# 2108, RRID: AB_331137 ), PPARγ (Cell Signaling Technology Cat# 2435, RRID: AB_2166051 ), FABP4 (Cell Signaling Technology Cat# 3544, RRID: AB_2278257 ), GFP (Cell Signaling Technology Cat# 2955, RRID: AB_1196614 ), Cyclin A2 (Cell Signaling Technology Cat# 4656, RRID: AB_2071958 ), PARP (Cell Signaling Technology Cat# 9542, RRID: AB_2160739 ), pAMPK (Cell Signaling Technology Cat# 2535, RRID: AB_331250 ), AMPK (Cell Signaling Technology Cat# 5832, RRID: AB_10624867 ), Cleaved Caspase 9 (Cell Signaling Technology Cat# 9501, RRID: AB_331424 ), and Cleaved Caspase 3 (Cell Signaling Technology Cat# 9664, RRID: AB_2070042 ).

Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Inhibition, Transfection, Control, Gene Expression

Journal: EBioMedicine

Article Title: Inhibition of oncogenic Src induces FABP4-mediated lipolysis via PPARγ activation exerting cancer growth suppression

doi: 10.1016/j.ebiom.2019.02.015

Figure Lengend Snippet: High FABP4 or LPL expression shows the better prognosis. (a) Expression of pSrc, Src, PPARγ and FABP4 in pair-matched patient tissues. Five pair-matched tissue samples were obtained from lung cancer patients upon IRB approval, and followed by measuring proteins of interest expression. (b–c) Kaplan-Meier plots for lung and renal cancer patient survival. Data were obtained from TCGA database for lung cancer and RCC or from GEO database (Fig. S6) for lung cancer. Disease free or overall survival was analyzed for groups with high (high, >75 th percentile), in-between (middle, from 25 th percentile to 75 th percentile) or low expression (low, <25 th percentile) of FABP4 or LPL in lung (c) and renal (c) cancer patients. Note that log-rank test was used for the statistical analysis. (d) Proposed model for Src regulation on lipid droplets.

Article Snippet: Primary antibodies include β-actin (Abcam Cat# ab6276, RRID: AB_2223210 ), 4HNE (Abcam Cat# ab46545, RRID: AB_722490 ), GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID: AB_10167668 ), FYN (BD Cat# 610164, RRID: AB_397565 ), phospho-Stat3 (Y705) (Cell Signaling Technology Cat# 9131, RRID: AB_331586 ), Stat3 (Cell Signaling Technology Cat# 9139, RRID: AB_331757 ), pSrc (Y416) (Cell Signaling Technology Cat# 6943, RRID: AB_10013641 ), Src (Cell Signaling Technology Cat# 2108, RRID: AB_331137 ), PPARγ (Cell Signaling Technology Cat# 2435, RRID: AB_2166051 ), FABP4 (Cell Signaling Technology Cat# 3544, RRID: AB_2278257 ), GFP (Cell Signaling Technology Cat# 2955, RRID: AB_1196614 ), Cyclin A2 (Cell Signaling Technology Cat# 4656, RRID: AB_2071958 ), PARP (Cell Signaling Technology Cat# 9542, RRID: AB_2160739 ), pAMPK (Cell Signaling Technology Cat# 2535, RRID: AB_331250 ), AMPK (Cell Signaling Technology Cat# 5832, RRID: AB_10624867 ), Cleaved Caspase 9 (Cell Signaling Technology Cat# 9501, RRID: AB_331424 ), and Cleaved Caspase 3 (Cell Signaling Technology Cat# 9664, RRID: AB_2070042 ).

Techniques: Expressing

Journal: British journal of pharmacology

Article Title: Exploring the pathophysiology underlying clozapine-induced enhancement of glutamatergic transmission through L-glutamate and D-serine release associated with pannexin1 hemichannels.

doi: 10.1111/bph.70112

Figure Lengend Snippet: FIGURE 2 Dose-dependent effects of chronic administration of clozapine on expression of pannexin1 and phosphorylated Src in the mPFC. Rats were chronically administered with clozapine (0, 5 and 10 mg kg1 day1 for 28 days). In the upper histograms, ordinate: mean ± SD (n = 6) of the relative levels of pannexin1 (PANX) per GAPDH (a) and phosphorylated-Src (pSrc) per Src (b). *P < 0.05: relative to control (clozapine free) by one-way ANOVA with Scheffe's post hoc test. F-values of the chronic clozapine administration on the expression of pannexin1 and pSrc in the rat frontal cortex using one-way ANOVA were [F(3,15) = 17.3 (P < 0.01)] and [F(3,15) = 18.0 (P < 0.01)], respectively. The lower panels indicate pseudo-gel images of capillary immunoblotting.

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), pannexin1 (12595-1-AP, RRID:AB_ 2159186, 1:100; Proteintech, Rosemont, IL, USA), Src (2109, RRID: AB_2106059,1:50; Cell Signalling Technology, Danvers, MA, USA) and phosphorylated Src (pSrc; MAB2685, RRID:AB_3657975, 1 μg ml 1, R&D Systems, Minneapolis, MN, USA) were diluted in an antibody diluent (Immuno Shot Platinum; CosmoBio, Tokyo, Japan).

Techniques: Expressing, Control, Western Blot

Journal: British journal of pharmacology

Article Title: Exploring the pathophysiology underlying clozapine-induced enhancement of glutamatergic transmission through L-glutamate and D-serine release associated with pannexin1 hemichannels.

doi: 10.1111/bph.70112

Figure Lengend Snippet: FIGURE 6 Concentration-dependent effects of chronic administration of clozapine on expression of pannexin1 and phosphorylated Src in cultured astrocytes. (a and b) Concentration-dependent effects of chronic exposure to clozapine (3 and 10 μM for 28 days) on expression of pannexin1 and phosphorylated Src in the astroglial plasma membrane fraction, respectively. In the upper histograms, ordinate: mean ± SD (n = 6) of the relative levels of pannexin1 (PANX) per GAPDH or phosphorylated Src (pSrc) per Src. *P < 0.05: relative to control (clozapine free) by one- way ANOVA with Scheffe's post hoc test. F-values of the concentration-dependent effects of chronic clozapine administration on the expression of pannexin1 and pSrc using one-way ANOVA were [F(2,15) = 21.3 (P < 0.01)] and [F(2,15) = 14.9 (P < 0.01)], respectively. The lower panels indicate pseudo-gel images of capillary immunoblotting.

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), pannexin1 (12595-1-AP, RRID:AB_ 2159186, 1:100; Proteintech, Rosemont, IL, USA), Src (2109, RRID: AB_2106059,1:50; Cell Signalling Technology, Danvers, MA, USA) and phosphorylated Src (pSrc; MAB2685, RRID:AB_3657975, 1 μg ml 1, R&D Systems, Minneapolis, MN, USA) were diluted in an antibody diluent (Immuno Shot Platinum; CosmoBio, Tokyo, Japan).

Techniques: Concentration Assay, Expressing, Cell Culture, Clinical Proteomics, Membrane, Control, Western Blot

Journal: British journal of pharmacology

Article Title: Exploring the pathophysiology underlying clozapine-induced enhancement of glutamatergic transmission through L-glutamate and D-serine release associated with pannexin1 hemichannels.

doi: 10.1111/bph.70112

Figure Lengend Snippet: FIGURE 7 Interaction among clozapine and inhibitors of Src and III-mGluR on the expression of pannexin1 and phosphorylated Src in the cultured astrocytes. (a) Interaction between 10 μM PP2 (Src inhibitor) and 10 μM clozapine on pannexin1 expression in the astroglial plasma membrane. (b) Interaction between 100 μM CPPG (III-mGluR inhibitor) and 10 μM clozapine on phosphorylated-Src expression in the astroglial plasma membrane. In the upper histograms, ordinate: mean ± SD (n = 6) of the relative levels of pannexin1 (PANX) per GAPDH or phosphorylated Src (pSrc) per Src. *P < 0.05: relative to control (clozapine free) by two-way ANOVA with Scheffe's post hoc test. @: P < 0.05: relative to 10 μM clozapine exposure by two-way ANOVA with Scheffe's post hoc test. F-values of the interaction between 10 μM PP2 and chronic exposure to 10 μM clozapine on the expression of pannexin1 using two-way ANOVA were [Fclozapine(1,20) = 55.5 (P < 0.01), FPP2(1,20) = 0.2 (P > 0.05), Fclozapine*PP2(1,20) = 1.1 (P > 0.05)]. F- values of the interaction between 100 μM CPPG and chronic exposure to 10 μM clozapine on the expression of pSrc using two-way ANOVA were [Fclozapine(1,20) = 56.0 (P < 0.01), FCPPG(1,20) = 27.7 (P < 0.01), Fclozapine*CPPG(1,20) = 0.4 (P > 0.05)]. The lower panels indicate pseudo-gel images of capillary immunoblotting.

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), pannexin1 (12595-1-AP, RRID:AB_ 2159186, 1:100; Proteintech, Rosemont, IL, USA), Src (2109, RRID: AB_2106059,1:50; Cell Signalling Technology, Danvers, MA, USA) and phosphorylated Src (pSrc; MAB2685, RRID:AB_3657975, 1 μg ml 1, R&D Systems, Minneapolis, MN, USA) were diluted in an antibody diluent (Immuno Shot Platinum; CosmoBio, Tokyo, Japan).

Techniques: Expressing, Cell Culture, Clinical Proteomics, Membrane, Control, Western Blot

Journal: British journal of pharmacology

Article Title: Exploring the pathophysiology underlying clozapine-induced enhancement of glutamatergic transmission through L-glutamate and D-serine release associated with pannexin1 hemichannels.

doi: 10.1111/bph.70112

Figure Lengend Snippet: FIGURE 9 Effects of chronic administration of L-BAIBA on Src signalling in the cultured astrocytes. (a and b) Interaction among chronic exposure to 30 μM L-BAIBA for 28 days and inhibitors of III-mGluR (100 μM CPPG) and NMDAR (1 μM MK801), respectively. In the upper side histogram, ordinate: mean ± SD (n = 6) of the relative levels of phosphorylated-Src (pSrc) per Src. *P < 0.05: relative to control (L-BAIBA free) by two-way ANOVA with Scheffe's post hoc test. @: P < 0.05: relative to 30 μM L-BAIBA exposure alone. F-values of the interaction between 100 μM CPPG and chronic exposure to 30 μM L-BAIBA on the expression of pSrc using two-way ANOVA were [FL-BAIBA(1,20) = 27.6 (P < 0.01), FCPPG(1,20) = 59.3 (P < 0.01), FL-BAIBA*CPPG(1,20) = 2.7 (P > 0.05)]. F-values of the interaction between 1 μM MK801 and chronic exposure to 30 μM L-BAIBA on the expression of pSrc using two-way ANOVA were [FL-BAIBA(1,20) = 46.3 (P < 0.01), FMK801(1,20) = 1.0 (P > 0.05), FL-

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), pannexin1 (12595-1-AP, RRID:AB_ 2159186, 1:100; Proteintech, Rosemont, IL, USA), Src (2109, RRID: AB_2106059,1:50; Cell Signalling Technology, Danvers, MA, USA) and phosphorylated Src (pSrc; MAB2685, RRID:AB_3657975, 1 μg ml 1, R&D Systems, Minneapolis, MN, USA) were diluted in an antibody diluent (Immuno Shot Platinum; CosmoBio, Tokyo, Japan).

Techniques: Cell Culture, Control, Expressing