Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: Lacidipine attenuates the enzymatic activity and expression of IDO1. A) Chemical structure of lacidipine. B) MDA‐MB‐231 and MCF‐7 cells were treated with lacidipine (5 × 10 −6 , 10 × 10 −6 , 20 × 10 −6 , 40 × 10 −6 , and 80 × 10 −6 m ) for 2 h, and then treated with IFN γ (100 ng mL −1 ) for 24 h. The concentration of kynurenine in the cell supernatants was determined. Bars, ± standard error of mean (SEM). The curves were plotted using a variable slope (four‐parameter) non‐linear fit. C) Effect of lacidipine on the IDO1 expression. MDA‐MB‐231 cells were pre‐treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 m ) and JAK inhibitor I (5 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of IDO1, IDO2, and TDO2 was analyzed by western blotting. GAPDH was used as the loading control. D) MDA‐MB‐231 and MCF‐7 cells were pre‐treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 m ) and JAK inhibitor I (5 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The mRNA expressions of IDO1 examined by qRT‐PCR. The mRNA levels of these genes were normalized against GAPDH expression levels. Bars, ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus IFN γ treatment group (unpaired two‐tailed Student's t ‐test). E) HEK293A cells are transfected with pCMV3‐IDO1 plasmids for 48 h, and then treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 Μ) and epacadostat (25 × 10 −9 m ) for 6 h. Cell supernatants were used to detect the activity of IDO1. Bars, ± SEM. * p < 0.05, ** p < 0.01 versus pCMV3‐IDO1 group (unpaired two‐tailed Student's t ‐test). The cell lysates were immunoblotted with IDO1 antibody. GAPDH was used as the loading control. All experiments were conducted with three independent replicates. F) The catalytic activity of the recombinant expressed IDO1 protein was measured. G) The binding location of lacidipine in the IDO1 protein. H) Detailed interactions between lacidipine and the active binding site of the IDO1 protein.

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: Activity Assay, Expressing, Concentration Assay, Western Blot, Control, Quantitative RT-PCR, Two Tailed Test, Transfection, Recombinant, Binding Assay

Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: Lacidipine exerts potential anti‐tumor effects in vivo. A) Tumor volumes were recorded in the DOX only, and DOX + lacidipine (0.5 or 1 mg kg −1 ) groups ( n = 6, each group) and are presented as the mean ± SEM. B) Image of the tumors in the DOX only, and DOX + lacidipine (0.5 or 1 mg kg −1 ) groups ( n = 6, each group). C) Tumor mass was recorded in the DOX only, and DOX + lacidipine (0.5 or 1 mg kg −1 ) groups ( n = 6, each group) and the results are presented as the mean ± SEM; * p < 0.05 versus control group and # p < 0.05, ### p < 0.001 versus DOX‐treated group (unpaired two‐tailed Student's t ‐test). D) Tumor volumes were recorded in the DOX only, lacidipine (10 or 20 mg kg −1 ) only, and DOX + lacidipine (20 mg kg −1 )/1‐MT groups ( n = 12, each group) and are presented as the mean ± SEM. E) Image of the tumors in the DOX only, lacidipine (10 or 20 mg kg −1 ) only, and DOX + lacidipine (20 mg kg −1 )/1‐MT groups ( n = 6, each group). F) Tumor mass was recorded in the DOX only, lacidipine (10 or 20 mg kg −1 ) only, and DOX + lacidipine (20 mg kg −1 )/1‐MT groups ( n = 6, each group) and the results are presented as the mean ± SEM; ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control group and #### p < 0.0001 versus DOX‐treated group (unpaired two‐tailed Student's t ‐test). G) The ratio of Kyn/Try in mouse serum was determined by HPLC at the end of the treatment. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus control group and # p < 0.05, ## p < 0.01 versus DOX‐treated group (unpaired two‐tailed Student's t ‐test). H,I) The expression of IDO1, IDO2 and TDO2 were analyzed by western blotting in mouse tumor tissue homogenate of at the end of treatment. GAPDH was used as the loading control. The experiments were conducted with three independent replicates.

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: In Vivo, Control, Two Tailed Test, Expressing, Western Blot

Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: Lacidipine inhibits the IDO1 expression through suppression of JAK/STAT and NF‐κB signaling pathways. A) Gene set enrichment analysis (GSEA) for JAK/STAT and NF‐κB signaling pathways. Rank statistics (bottom; see y axis) and normalized enrichment scores (top; see y axis) indicate downregulation and upregulation, respectively. JAK/STAT signaling pathways, normalized enrichment score (NES) = −1.37, p < 0.001, FDR q = 0.11; NF‐κB signaling pathways, NES = ‐1.64, p = 0.01, FDR q = 0.17. B) Heat map hierarchical clustering displays the differentially expressed genes in the JAK/STAT and NF‐κB signaling pathways with a p < 0.05 ( n = 3, each group). La, Lacidipine. C) MDA‐MB‐231 cells were pre‐treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 m ) and JAK inhibitor I (5 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 12 h. The expression of key proteins of the JAK/STAT signaling pathway was analyzed by western blotting. GAPDH was used as the loading control. D) MDA‐MB‐231 cells were pre‐treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 m ) and BAY11‐7082 (10 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 12 h. The expression of key proteins of the NF‐κB signaling pathway was analyzed by western blotting. GAPDH was used as the loading control. E,F) The the key proteins expression of the JAK/STAT and NF‐κB signaling pathways were analyzed by western blotting in mouse tumor tissue homogenate of at the end of treatment. GAPDH was used as the loading control. All western blot experiments were conducted with three independent replicates.

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: Expressing, Western Blot, Control

Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: Ca 2+ participates in lacidipine regulation of IDO1 expression. A) Effect of IFN γ only and lacidipine + IFN γ on intracellular Ca 2+ concentration. MDA‐MB‐231 cells were pre‐treated with lacidipine (20 × 10 −6 m ) for 2 h, and then stimulated with IFN γ (100 ng mL −1 ) for different durations (0, 0.5, 1, 2, and 4 h). The cells were loaded with 5 × 10 −6 m fluo‐3 AM, and intracellular Ca 2+ levels were analyzed using flow cytometry. B–E) MDA‐MB‐231 cells were pre‐treated with lacidipine (20 × 10 −6 m ) and diltiazem (1 × 10 −6 , 10 × 10 −6 , 25 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of B,C) IDO1 proteins and D) the key proteins of the JAK/STAT and E) NF‐κB signaling pathways were analyzed by western blotting. GAPDH was used as the loading control. F,G) MDA‐MB‐231 cells were pre‐treated with lacidipine (20 × 10 −6 m ) and BAY K 8644 (0.5 × 10 −6 , 1 × 10 −6 , and 5 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of IDO1 proteins were analyzed by western blotting. GAPDH was used as the loading control. H) MDA‐MB‐231 cells were pre‐treated with lacidipine (20 × 10 −6 m ) and BAPTA‐AM (10 × 10 −6 , 25 × 10 −6 , and 50 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of IDO1 proteins was analyzed using western blotting. GAPDH was used as the loading control. All experiments were conducted with three independent replicates.

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: Expressing, Concentration Assay, Flow Cytometry, Western Blot, Control

Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: Ca V 1.2/1.3 regulates IDO1 expression via Pyk2 and calmodulin. A,B) MDA‐MB‐231 cells were pre‐treated with lacidipine (10 × 10 −6 , 20 × 10 −6 , and 40 × 10 −6 m ) and tyrphostin A9 (5 × 10 −6 m ) or W‐7 (35 × 10 −6 m ) for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of Pyk2 phosphorylated at A) Tyr402, Pyk2 and B) calmodulin (B) proteins was analyzed by western blotting. GAPDH was used as the loading control. MDA‐MB‐231 cells were pre‐treated with lacidipine (20 × 10 −6 m ) and C) tyrphostin A9 (5 × 10 −6 m ) or D) W‐7 for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of IDO1 proteins was analyzed by western blotting. GAPDH was used as the loading control. MDA‐MB‐231 cells were pre‐treated with E) lacidipine (20 × 10 −6 m ) and tyrphostin A9 (5 × 10 −6 m ) or F) W‐7 for 2 h, then stimulated with IFN γ (100 ng mL −1 ) for 24 h. The expression of phosphorylated JAK at Tyr1022, phosphorylated STAT1 at Tyr701, phosphorylated IKK α/β, phosphorylated IκBα, and IκBα proteins was analyzed by western blotting. GAPDH was used as the loading control. G) MDA‐MB‐231 cells were treated with lacidipine (20 × 10 −6 m ) for 3 h and then co‐immunoprecipitated with Pyk2 antibodies or control immunoglobulin G (IgG) and analyzed for antibody‐specific JAK1, calmodulin, and Pyk2. The MDA‐MB‐231 cells were transfected with the indicated H) Ca V 1.2 and I) Ca V 1.3 siRNA. After transfection for 48 h, cells were treated with IFN γ (100 ng mL −1 ) for 24 h and cell lysates were collected for western blot analysis using the indicated antibodies to assess the effect of Ca V 1.2 and Ca V 1.3. GAPDH was used as the loading control. All experiments were conducted with three independent replicates.

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: Expressing, Western Blot, Control, Immunoprecipitation, Transfection

Journal: Advanced Science

Article Title: Calcium Channel Blocker Lacidipine Promotes Antitumor Immunity by Reprogramming Tryptophan Metabolism

doi: 10.1002/advs.202409310

Figure Lengend Snippet: The expression levels of IDO1 and Pyk2 phosphorylation correlate with human breast cancer development. A) Statistical analysis of IDO1 expression in the breast tumor tissues ( n = 136). B,C) Expression of IDO1 in different subtypes of breast cancer ( n = 136). D) Statistical analysis of Pyk2 phosphorylation expression in the breast tumor tissues ( n = 136). E,F) Expression levels of Pyk2 phosphorylation in different subtypes of breast cancer ( n = 136). G) The correlation of IDO1 and Pyk2 phosphorylation, R = 0.3, p = 0.00042. H) Expression levels of IDO1 in the breast tumor tissues, adjacent normal breast tissues, and normal breast tissue based on TCGA data ( n = 1034). I) Expression levels of IDO1 in different subtypes of breast cancer based on TCGA data, p < 0.001 ( n = 1034).

Article Snippet: [ ] In brief, the IDO activity was measured with the standard reaction mixture (250 L) containing potassium phosphate buffer (50 × 10 −3 m , pH = 6.5), L‐tryptophan (200 × 10 −6 m ), methylene blue (10 × 10 −6 m ) (#7220‐79‐3, Macklin), ascorbic acid (20 × 10 −3 m ) (#A8100, Solarbio), catalase (100 µg mL −1 ) (#C8070, Solarbio), and recombinant expressed IDO1 enzyme (10 µg mL −1 ) (#11650‐H70E, SinoBiological).

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: Counter-regulation of T cell effector function by differentially activated p38

doi: 10.1084/jem.20131917

Figure Lengend Snippet: p38 Alternative activation is required for TCR-induced expression of NFATc1 and IRF4 in CD4 + T cells. (A) Purified CD4 + T cells were stimulated with anti-CD3/CD28 in the presence or absence of SB203580 and immunoblotted for the indicated transcription factors 48 h later. (B and C) Purified CD4 + T cells were stimulated with anti-CD3/CD28 in the presence of 11R-VIVIT or 11R-VEET. Irf4 mRNA (B) and protein (C) were determined 24 h later. (D and E) Purified CD4 + T cells from WT and DKI mice were stimulated with anti-CD3/CD28 or PMA plus ionomycin, as indicated for the indicated times and analyzed for expression of Nfatc1 and Irf4 mRNA (D) and protein (E). Results shown in A–E are representative of three independent experiments each. (F) Naive CD4 + T cells were stimulated with either anti-CD3/CD28 or PMA and ionomycin under neutral or Th17-skewing conditions for 3 d. IL-17A and IFN-γ expression were determined by intracellular staining and flow cytometry after restimulation with PMA and ionomycin. Results shown in the left panel are representative of three independent experiments and bar graphs in the right panel show the mean ± SEM of all three. *, P < 0.05 (unpaired two-tailed Student’s t test).

Article Snippet: Recombinant full-length NFATc1 was purchased from SignalChem.

Techniques: Activation Assay, Expressing, Purification, Staining, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Counter-regulation of T cell effector function by differentially activated p38

doi: 10.1084/jem.20131917

Figure Lengend Snippet: MAPK-cascade activated p38 inhibits functions distal to alternatively activated p38. (A) Purified CD4 + T cells were cultured overnight without stimulation, and then treated with either UV (50 J/m 2 or 100 J/m 2 or 200 J/m 2 ), 0.6 M sorbitol for 30 min, or PMA (10 ng/ml) and ionomycin (1 µg/ml) for 1 h. Cell lysates were immunoblotted with anti-phospho-p38. The results are representative of three independent experiments. (B) Purified CD4 + T cells were treated with either 0.6 M sorbitol for 30 min, 50 J/m 2 UV, or PMA and ionomycin were stimulated with anti-CD3/CD28 for 24 h. The expression of Nfatc1 and Irf4 was determined by quantitative real-time PCR. Results are the mean ± SEM of three independent experiments. *, P < 0.05 (unpaired two-tailed Student’s t test). (C) Cells were treated as in B, and IRF4 expression was determined by Western blot. The results are representative of two independent experiments. (D) Freshly purified T cells were stimulated or not with anti-CD3/CD28 for 48 h. Cytosolic and nuclear fractions were separated in a low percentage SDS-PAGE (8%) gel and immunoblotted for NFATc1. The results are representative of three independent experiments. (E) CD4 + T cells were stimulated for 48 h with anti-CD3/CD28, and then treated with sorbitol in the presence or absence of SB203580 and/or SP600125 for 30 min, rested for 1 h, then immunoblotted for NFATc1 in cytosolic and nuclear fractions. Results are representative of three independent experiments. (F) Purified CD4 + T cells were treated or not with sorbitol for 30 min and stimulated with anti-CD3/CD28 for 48 h, and then immunoblotted for NFATc1 in cytosolic and nuclear fractions. The results are representative of three independent experiments. (G) Jurkat T cells were stimulated for 48 h with an agonistic anti-TCR antibody (C305), and then treated with sorbitol in the presence or absence of SB203580 and/or SP600125 for 30 min. After 1 h without further stimulation, cytosolic fractions were immunoblotted for phospho-NFATc1 (p-NFATc1). The results are representative of three independent experiments. (H) Recombinant p38 was activated with Zap70, MKK6, or buffer alone in in vitro kinase buffer. After 1 h, recombinant ATF2 and 10 µCi [ 32 P]ATP were added for 30 min before separation on SDS-PAGE and PhosphorImager analysis. The phosphorylation state of p38 Y323 was determined by immunoblotting. The results are representative of three independent experiments. (I) Recombinant p38 was activated with Zap70, MKK6, or buffer alone and an in vitro kinase assay using recombinant NFATc1 as substrate was performed as in (H). The results are representative of two independent experiments. (J) Recombinant p38 was activated with MKK6 or buffer alone in in vitro kinase buffer. Active JNK1 was kept in in vitro kinase buffer. After 1 h, recombinant NFATc1 was added and incubated for an additional hour before separation on SDS-PAGE and immunoblotting with an antibody specific for NFATc1 phosphorylated on residue S172. The results are representative of two independent experiments. (K) Recombinant p38 was activated with Zap70, MKK6, or buffer alone in an in vitro kinase assay with recombinant NFATc1 as the p38 substrate, as in I. Samples were separated on a low percentage SDS-PAGE (8%) and immunoblotted for NFATc1 phosphorylated on residue S172. The results are representative of three independent experiments.

Article Snippet: Recombinant full-length NFATc1 was purchased from SignalChem.

Techniques: Purification, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, SDS Page, Recombinant, In Vitro, Phospho-proteomics, Kinase Assay, Incubation, Residue

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Representative HDAC6 PET tracers with IC 50 values for HDAC6.

Article Snippet: The binding assay using AS-MS was performed at rt in a final volume of 10 μL using the recombinant HDAC6 (HDAC6, Active, H88-30G-10, Lot:B2010-9; SignalChem Biotech Inc., Richmond, BC, Canada).

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Affinity selection (AS)–MS binding study of HDAC6 with 16a and bavarostat. Data points are the mean ± standard error (SE) (four technical replicates). (A) Saturation binding curve. (B) Time course for the dissociation. (C) Binding parameters calculated from AS–MS data. Functional IC 50 values are shown for comparison.

Article Snippet: The binding assay using AS-MS was performed at rt in a final volume of 10 μL using the recombinant HDAC6 (HDAC6, Active, H88-30G-10, Lot:B2010-9; SignalChem Biotech Inc., Richmond, BC, Canada).

Techniques: Selection, Binding Assay, Functional Assay, Comparison

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Brain and plasma concentration of 16a in wild-type (WT) and HDAC6 knockout (KO) mice. Compound 16a (1 mg/kg) was intravenously administered to WT and HDAC6 KO mice. Two hours after administration, the hippocampus and plasma were collected and the concentration of 16a in the brain (A) and plasma (B) was measured using liquid chromatography with tandem mass spectrometry (LC–MS/MS). The data are expressed in percentage of injected dose per gram of brain tissue (%ID/g) or per milliliter of plasma (%ID/mL), respectively, as mean ± standard error (SE), n = 4. ** P < 0.01, n.s.: not significant.

Article Snippet: The binding assay using AS-MS was performed at rt in a final volume of 10 μL using the recombinant HDAC6 (HDAC6, Active, H88-30G-10, Lot:B2010-9; SignalChem Biotech Inc., Richmond, BC, Canada).

Techniques: Clinical Proteomics, Concentration Assay, Knock-Out, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Injection

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Comparison of HDAC6 expression levels in a mouse and monkey using Western blotting. Hippocampus (Hip) was obtained from two mice, and HDAC6 expression was examined. Recombinant HDAC6 conjugated with GST (0.03, 0.1, 0.3, and 1 ng) was also used for the positive controls (A). Lysates of the hippocampus and corpus callosum (CC) obtained from a monkey were applied to Western blotting in duplicate. The same controls were used for comparison (B).

Article Snippet: The binding assay using AS-MS was performed at rt in a final volume of 10 μL using the recombinant HDAC6 (HDAC6, Active, H88-30G-10, Lot:B2010-9; SignalChem Biotech Inc., Richmond, BC, Canada).

Techniques: Comparison, Expressing, Western Blot, Recombinant

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Immunohistochemical (IHC) staining of HDAC6 protein in a monkey brain. Coronal brain sections, including the areas below were prepared from a monkey (a, putamen; b, caudate nucleus; c, corpus callosum; d, white matter; e, anterior cingulate cortex; f, thalamus; g, hippocampus; h, cerebellum; i, brain stem [pons]). They were immunostained with HDAC6 antibody.

Article Snippet: The binding assay using AS-MS was performed at rt in a final volume of 10 μL using the recombinant HDAC6 (HDAC6, Active, H88-30G-10, Lot:B2010-9; SignalChem Biotech Inc., Richmond, BC, Canada).

Techniques: Immunohistochemical staining, Immunohistochemistry

Journal: International Journal of Nanomedicine

Article Title: Nanosized functional miRNA liposomes and application in the treatment of TNBC by silencing Slug gene

doi: 10.2147/IJN.S207837

Figure Lengend Snippet: Functional miRNA liposomes enabling to silence Slug gene and suppressing TGF-β1-Smad signaling pathway in TNBC cells. (A) The relative mRNA levels of transcription factor Slug, Snail, Blimp-1, and BMP5 in MDA-MB-231 cells after transfection of functional miRNA-NC liposomes and functional miRNA liposomes. The results indicate that functional miRNA liposomes are able to silence Slug and Blimp-1 and to up-regulate BMP5. (B) The protein expressions of Slug, Snail, Blimp-1, and BMP5 after transfections. a , functional miR-NC liposomes; b , functional miRNA liposomes; c , functional miRNA liposomes with TGF-β1/Smad inhibitor; d , functional miRNA liposomes with NF-κB inhibitor. The results show that the functional miRNA liposomes significantly increase BMP5 protein level while reducing Slug and Blimp-1 proteins. Besides, functional miRNA liposomes do not affect the Snail protein level. In contrast, the silencing effect of functional miRNA liposomes on Slug protein is evidently inhibited by TGF-β inhibitor, and the changed BMP5 and Blimp-1 protein levels are recovered to those of negative control. (C) Smad2/3/4 protein complex in MDA-MB-231 cells. 1 , input: positive control, lysates of MDA-MB-231 cells; 2 , mouse IgG: negative control; 3 , protein samples after immunomagnetic bead sorting by Smad2/3 ChIP grade antibodies. a , functional miR-NC liposomes; b , functional miRNA liposomes; c , functional miRNA liposomes with TGF-β1/Smad inhibitor; d , functional miRNA liposomes with NF-κB inhibitor. The results show that Smad2/3/4 complexes exist in un-treated MDA-MB-231 cells while functional miRNA liposomes inhibit the formation of Smad2/3/4 complex. TGF-β inhibitor and NF-κB inhibitor relieve the inhibition caused by functional miRNA liposomes through a phosphorylation of Smad protein. The results demonstrate that the functional miRNA liposomes are able to silence Slug gene and to inhibit TGF-β1/Smad pathway. (D) Illustration for action mechanism of miRNA.

Article Snippet: To evaluate the Smad2/3/4 complex, MDA-MB-231 cells were transfected with the same reagents as those listed in the section titled “protein expression by Western blotting.” The collected protein samples (crude) were incubated with Smad2/3 antibody-bound immunomagnetic beads for immunoprecipitation (Sino Biological, Beijing, China).

Techniques: Functional Assay, Transfection, Negative Control, Positive Control, Inhibition

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular HMGB1 negatively regulates efferocytosis

doi: 10.4049/jimmunol.1101500

Figure Lengend Snippet: (A–B) Cell lysates (500 µg) of NIH3T3 mouse fibroblasts were incubated with anti-Src antibody (2 µg) (A) or anti-HMGB1 antibody (2 µg) (B) overnight. Immunocomplexes were precipitated by incubation with Protein G agarose beads for 2 hours. Cell lysates incubated without antibodies, but with Protein G agarose beads were used as a negative control. Levels of HMGB1 and Src in the immunocomplexes were determined by Western blotting. (C) Cytoplasmic fractions (500 µg) of the control and HMGB1 knockdown NIH3T3 fibroblasts were incubated with anti-Src antibody overnight followed by immunoprecipitation. Cell lysates not exposed to anti-Src antibody, but incubated with Protein G agarose beads, were used as negative controls. The levels of Src and FAK in the immunocomplexes were determined by Western blotting. (D) Recombinant Src-GST protein was incubated with FAK-1-Myc in the presence of BSA or of HMGB1-Flag for 1 hour. Immunoprecipitation followed by Western blotting was performed to determine the association of the various proteins. Representative gels are shown. Similar results were obtained from a second independent experiment.

Article Snippet: Recombinant FAK-1 Myc protein (OriGene Technologies) and Src-GST (Sino Biological Inc) were obtained commercially.

Techniques: Incubation, Negative Control, Western Blot, Immunoprecipitation, Recombinant