Journal: PLoS ONE

Article Title: The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation

doi: 10.1371/journal.pone.0136546

Figure Lengend Snippet: (A) RT-PCR analysis of cannabinoid receptor mRNA expression in HOBs. Human astrocytes (HA) were used as a positive control for CB1R, CB2R and TRPV1 expression. Hypoxanthine-guanine PhosphoRibosylTransferase (HPRT) was used as the control house-keeping gene. (B) ALP production, normalised to DNA content, was measured in HOBs grown for 4 days and treated with 10μM anandamide (AEA) only, or 10μM anandamide and the following antagonists and inhibitors. Cells were treated with AM251 (100nM, CB 1 R antagonist), AM630 (100nM, CB 2 R antagonist) or Capsazepine (1μM, Cap, TRPV1 antagonist) for 30 minutes before the addition of vehicle or anandamide. (C) Cells were treated with KT5720 (500nM, PKA inhibitor), PD98059 (1μM, MEK inhibitor) or SP600125 (10μm, JNK inhibitor) for 30 minutes before the addition of vehicle or anandamide. Data given as mean ± S.E.M., n = 9, from 3 experiments. * P <0.05, ** P <0.01, compared to antagonist/inhibitor only; one-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: HOBs were treated with media supplemented with 100nM AM251 (CB 1 R antagonist), 100nM AM630 (CB 2 R antagonist), 1μM capsazepine (TRPV1 antagonist), 500nM KT5720 (PKA inhibitor, PKA is upstream of CREB), 1μM PD98059 (MEK inhibitor) or 10μm SP600125 (JNK inhibitor) for 30 minutes before the addition of vehicle (0.1% ethanol) or 10μM anandamide (all Tocris, UK).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Ccl3 enhances docetaxel chemosensitivity in breast cancer by triggering proinflammatory macrophage polarization

doi: 10.1136/jitc-2021-003793

Figure Lengend Snippet: DTX induces Ccl3 by relieving the inhibitory effect of Creb via ROS accumulation. (A) GSEA based on RNA-Seq results of iBMMs and 4T1 cells treated with Veh and DTX (10 nM for iBMMs and 5 nM for 4T1). The NES and p value are shown. (B) ROS levels in iBMMs and 4T1 cells treated with DTX (10 nM for iBMMs and 5 nM for 4T1) for 2 days were determined via flow cytometry. (C) Western blotting was performed to detect the expression of Ccl3, Creb and p-Creb in iBMMs treated with different concentrations of DTX (0, 5, 10 and 20 nM) for 1 day. (D) Western blotting was used to detect the expression of Creb and p-Creb, whereas qRT-PCR was used to quantify Ccl3 expression in 4T1 treated with different concentrations of DTX (0, 3, 5 and 10 nM) for 1 day. (E) Various concentrations of H 2 O 2 (0, 50, 100 and 250 µM) were used to induce ROS in iBMMs for 16 hours. Ccl3, Creb and p-Creb expression levels were determined via western blotting. (F) Various concentrations of H 2 O 2 (0, 50, 100, 250 and 500 µM) were used to induce ROS in 4T1 for 16 hours. Western blotting was used to detect the expression of Creb and p-Creb, whereas qRT-PCR was used to quantify Ccl3 expression. (G) NAC (10 mM) was used to neutralize ROS in combination with DTX treatment (10 nM) for 1 day in iBMMs. Ccl3 and Creb expression levels were determined via western blotting. (H) NAC (10 mM) was used to neutralize ROS in combination with DTX treatment (5 nM) for 1 day in 4T1. Creb and p-Creb expression levels were determined via western blotting, whereas Ccl3 expression was quantified by qRT-PCR. (I) Creb inhibitor 666–15 (200 nM) was administered to iBMMs for 12 hours. The cells were collected for western blotting and qRT-PCR analysis to detect Ccl3 expression. (J) Creb inhibitor 666–15 (200 nM) was administered to 4T1 for 12 hours. qRT-PCR was performed to quantify Ccl3 expression. (K) Ccl3 expression was determined via western blotting in iBMMs knocking down Creb. (L) Ccl3 expression was quantified by qRT-PCR in 4T1 knocking down Creb. (M) iBMM stable cell line overexpressing Creb was subjected to DTX (10 nM) and doxycycline (DOC, 1 µg/mL) treatment for 2 days. Creb was cloned into an inducible pTRIPZ overexpression vector induced with DOC. The cells were collected and lysed for western blotting to detect Ccl3. (N) 4T1 stable cell line overexpressing Creb was subjected to DTX (5 nM) and DOC (1 µg/mL) treatment for 2 days. Creb was cloned into an inducible pTRIPZ overexpression vector induced with DOC. The cells were collected for qRT-PCR to quantify Ccl3 expression. Gapdh was used as the loading control. Tbp was used as the internal control. Data are presented as mean±SEM. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001; NS, no significance. Ccl3, C–C motif chemokine ligand 3; Creb, cAMP-response element binding protein; DOC, doxycycline; DTX, docetaxel; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; GSEA, gene set enrichment analysis; H 2 O 2 , hydrogen dioxide; iBMMs, immortalized bone marrow-derived macrophages; NAC, N-acetyl-L-cysteine; NES, normalized enrichment score; qRT-PCR, quantitative real-time PCR; RNA-Seq, RNA sequencing; ROS, reactive oxygen species; sh, short hairpin RNA; shNT, short hairpin RNA of non-target; Tbp, TATA-binding protein; Veh, vehicle.

Article Snippet: The commercial Creb inhibitor 666–15 (HY-101120, MedChemExpress) was used at 200 ng/mL in vitro and 10 mg/kg in vivo.

Techniques: RNA Sequencing, Flow Cytometry, Western Blot, Expressing, Quantitative RT-PCR, Stable Transfection, Clone Assay, Over Expression, Plasmid Preparation, Control, Binding Assay, Derivative Assay, Real-time Polymerase Chain Reaction, shRNA

Journal: Journal for Immunotherapy of Cancer

Article Title: Ccl3 enhances docetaxel chemosensitivity in breast cancer by triggering proinflammatory macrophage polarization

doi: 10.1136/jitc-2021-003793

Figure Lengend Snippet: High CCL3 expression predicts better prognosis, and Creb inhibitor or rmCcl3 increases DTX chemosensitivity in breast cancer. (A) Overall survival analysis of CCL3 in breast cancer using the Kaplan-Meier tool ( https://kmplot.com/analysis/ ). HR and log-rank p value are as shown. (B) The serum CCL3 levels of TNC-receiving patients before chemotherapy (before, n=41), at earlier chemotherapy cycles (earlier, the second or third cycle, n=52) and at later chemotherapy cycles (later, the fifth or sixth cycle, n=51) were determined using ELISA. (C–D) IHC staining was performed using paired tumor sections of patients before or after TNC, and the patients were separated into response and non-response cohorts according to the MP score. (C) CCL3 expression levels of cancer cells and TME cells were separately calculated by H-score (non-response: n=47, response: n=53). (D) IHC staining of CD68 and COX2 were performed. The changes of CD68 + COX2 + macrophages after TNC were defined as upregulated, no change and downregulated and presented as percentages of these three groups (non-response: n=17, response: n=24). (E) Indicated concentrations of 666–15 and DTX were combined to treat iBMMs for 20 hours. Western blotting was performed to determine the expression levels of Ccl3 and proinflammatory macrophage markers. (F) Indicated concentrations of 666–15 and DTX were combined to treat 4T1 for 20 hours, and qRT-PCR was performed to quantify Ccl3 expression. (G) 4T1 cells were orthotopically injected into BALB/c mice for the treatment of different dosed DTX in the presence or absence of 666–15 (n=5). The tumor volume was measured, and the tumor growth curves are shown. The arrows indicate the time of DTX and 666–15 administration. (H) Flow cytometry was used to analyze the percentage of F4/80 + MHCII + proinflammatory macrophages in cells isolated from tumors in . (I) 4T1 cells were orthotopically injected into BALB/c mice for the treatment of different dosed DTX in the presence or absence of rmCcl3 (n=5). The tumor volume was measured, and the tumor growth curves are shown. The arrows indicate the time of DTX and rmCcl3 administration. (J) Flow cytometry was used to analyze the percentage of F4/80 + MHCII + proinflammatory macrophages in cells isolated from tumors in . (K) Schematic diagram for the findings of the present work. DTX administration led to the intracellular ROS accumulation in both macrophages and cancer cells, which further upregulated Ccl3 by relieving the inhibitory effect of Creb on Ccl3. Then, the binding of Ccl3 to Ccr5 activated p38 and Irf5, resulting in the proinflammatory polarization of macrophages. These DTX educated macrophages suppressed tumor progression and augmented DTX chemosensitivity in breast cancer by enhancing phagocytosis of both bulk cancer cells and ALDH + CSCs. Gapdh was used as the loading control. Tbp was used as the internal control. Data are presented as mean±SEM. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001; NS, no significance. ALDH, aldehyde dehydrogenase; Ccl3, C–C motif chemokine ligand 3; Ccr, C–C motif chemokine receptor; Cox2, cyclooxygenase 2; Creb, cAMP-response element binding protein; CSCs, cancer stem cells; TNC, Taxane-containing neoadjuvant chemotherapy; DTX, docetaxel; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; H-score, histo-score; iBMMs, immortalized bone marrow-derived macrophages; IHC, immunohistochemistry; iNos, inducible nitric oxide synthase; MHCII, major histocompatibility complex class II; MP, Miller-Payne; qRT-PCR, quantitative real-time PCR; rmCcl3, recombinant Ccl3; ROS, reactive oxygen species; Tbp, TATA-binding protein; TME, tumor microenvironment.

Article Snippet: The commercial Creb inhibitor 666–15 (HY-101120, MedChemExpress) was used at 200 ng/mL in vitro and 10 mg/kg in vivo.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Western Blot, Quantitative RT-PCR, Injection, Flow Cytometry, Isolation, Binding Assay, Control, Derivative Assay, Immunopeptidomics, Real-time Polymerase Chain Reaction, Recombinant

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: Figure 5C

Article Snippet: Mouse Monoclonal Anti-CREB (86B10) , Cell Signaling Technology , Cat. # 9104S; RRID:AB_490881.

Techniques: Control, Virus, Recombinant, Expressing, Protease Inhibitor, Lysis, Transfection, Electron Microscopy, Immunodepletion, Cloning, Protein Purification, Endotoxin Assay, Bradford Assay, Staining, Mass Spectrometry, Mutagenesis, Software, Isolation

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-Phospho-CREB (Ser133) (87G3) , Cell Signaling Technology , Cat# 9198; RRID AB_2561044.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Modification, Saline, Concentration Assay, Over Expression, Protein Extraction, Membrane, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Protease Inhibitor, CCK-8 Assay, Reverse Transcription, Selection, Plasmid Preparation, Knock-Out, Software, Real-time Polymerase Chain Reaction, Flow Cytometry