Journal: PLoS ONE

Article Title: Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors

doi: 10.1371/journal.pone.0176057

Figure Lengend Snippet: (A), (B) and (C): Inhibition of the CXCL12/CXCR4 interaction by (A) the peptide analogs T22, T140, TC14012 and CTCE-9908 and the small molecules (B) AMD3100, AMD3465, AMD11070, IT1t and (C) WZ811, Me6TREN and gambogic acid. Jurkat cells were incubated with 2.9 nM CXCL12 AF647 in the presence of different concentrations of compound. Data are represented as % inhibition of maximal CXCL12 binding to CXCR4. Mean ± SEM of three or four independent experiments is shown.

Article Snippet: The compounds TC14012 (MW: 2,066.4 g/mol) [ ], CTCE-9908 (MW: 1,927.3 g/mol) [ ], IT1t (MW: 479.6 g/mol) [ ] and AMD3465 Hexahydrobromide (MW: 896.1 g/mol) [ ] were obtained from Tocris (Bristol, UK).

Techniques: Inhibition, Incubation, Binding Assay

Journal: PLoS ONE

Article Title: Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors

doi: 10.1371/journal.pone.0176057

Figure Lengend Snippet: Fluo-2 AM loaded cells were treated with serial dilutions of compound and subsequently stimulated with 6.25 nM CXCL12. Fluorescence changes were measured over time in all wells simultaneously. (A): The inhibitory effect of different concentrations of AMD11070 on CXCL12-induced calcium release is shown. The red and blue line represent the negative (untreated, without CXCL12 stimulation) and positive (untreated, CXCL12 stimulation) control, respectively. Calcium fluxes are represented as % of baseline ( i . e . mean relative light units in each well during a fixed time interval before CXCL12 addition). One representative experiment out of three is shown with each data point corresponding to the mean fluorescence of three to six replicates. (B), (C) and (D): Inhibitory effect of (B) T22, T140, TC14012, CTCE-9908, (C) AMD3100, AMD3465, AMD11070, IT1t, (D) WZ811, Me6TREN and gambogic acid on CXCL12-induced calcium flux. Data are represented as % inhibition of CXCL12-induced calcium mobilization relative to the positive and negative control. Mean ± SEM of three independent experiments is shown.

Article Snippet: The compounds TC14012 (MW: 2,066.4 g/mol) [ ], CTCE-9908 (MW: 1,927.3 g/mol) [ ], IT1t (MW: 479.6 g/mol) [ ] and AMD3465 Hexahydrobromide (MW: 896.1 g/mol) [ ] were obtained from Tocris (Bristol, UK).

Techniques: Fluorescence, Control, Inhibition, Negative Control

Journal: PLoS ONE

Article Title: Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors

doi: 10.1371/journal.pone.0176057

Figure Lengend Snippet: SUP-T1 cells were stained with a mAb targeting the N-terminus of CXCR4 (clone 1D9) and then treated with different concentrations of compound. CXCR4 internalization was induced by stimulating samples with CXCL12 at 37°C and quantified by flow cytometry. (A): Flow cytometric analysis of the negative (untreated, no CXCL12 stimulation; red histogram) and positive (untreated, CXCL12 stimulation; dark blue histogram) control and TC14012-treated samples (50 nM to 0.5 nM; light blue, green and orange histogram, respectively). (B), (C) and (D): Inhibitory effect of (B) T22, T140, TC14012, CTCE-9908, (C) AMD3100, AMD3465, AMD11070, IT1t, (D) WZ811, Me6TREN and gambogic acid on CXCR4 endocytosis. Data are represented as % of CXCR4 internalization relative to the positive and negative control. Mean ± SEM of at least two independent experiments is shown.

Article Snippet: The compounds TC14012 (MW: 2,066.4 g/mol) [ ], CTCE-9908 (MW: 1,927.3 g/mol) [ ], IT1t (MW: 479.6 g/mol) [ ] and AMD3465 Hexahydrobromide (MW: 896.1 g/mol) [ ] were obtained from Tocris (Bristol, UK).

Techniques: Staining, Flow Cytometry, Control, Negative Control

Journal: PLoS ONE

Article Title: Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors

doi: 10.1371/journal.pone.0176057

Figure Lengend Snippet: (A), (B) and (C): Effect of (A) T22, T140, TC14012, CTCE-9908, (B) AMD3100, AMD3465, AMD11070, IT1t, (C) WZ811, Me6TREN and gambogic acid on chemotaxis of CXCR4-positive Jurkat cells. After compound treatment, cells were allowed to migrate towards 6.25 nM CXCL12, present in the lower compartment of the insert, for two hours. The migrated cells were counted by flow cytometry. Data are represented as % inhibition of migration relative to the negative and positive control (spontaneously migrated cells and cells migrated towards CXCL12, respectively). ± SEM of two to three independent experiments, each performed in triplicate.

Article Snippet: The compounds TC14012 (MW: 2,066.4 g/mol) [ ], CTCE-9908 (MW: 1,927.3 g/mol) [ ], IT1t (MW: 479.6 g/mol) [ ] and AMD3465 Hexahydrobromide (MW: 896.1 g/mol) [ ] were obtained from Tocris (Bristol, UK).

Techniques: Chemotaxis Assay, Flow Cytometry, Inhibition, Migration, Positive Control

Journal: PLoS ONE

Article Title: Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors

doi: 10.1371/journal.pone.0176057

Figure Lengend Snippet: Anti-HIV-1 (X4) activity of CXCR4 compounds evaluated in CD4-positive MT-4 cells and PHA-stimulated PBMCs.

Article Snippet: The compounds TC14012 (MW: 2,066.4 g/mol) [ ], CTCE-9908 (MW: 1,927.3 g/mol) [ ], IT1t (MW: 479.6 g/mol) [ ] and AMD3465 Hexahydrobromide (MW: 896.1 g/mol) [ ] were obtained from Tocris (Bristol, UK).

Techniques: Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: DAPK1 Promotes Extrasynaptic GluN2B Phosphorylation and Striatal Spine Instability in the YAC128 Mouse Model of Huntington Disease

doi: 10.3389/fncel.2020.590569

Figure Lengend Snippet: Increased DAPK1 protein expression and activation occur in affected regions of the YAC128 brain and require mHTT cleavage at D586. (A) Cortical (CTX), (B) striatal (STR), or (C) cerebellar (CB) tissues from 1-month-old WT, YAC128 (Y128; line 53), and C6R (line W13) mice were lysed in a stringent buffer and total lysate was assessed by Western blotting for DAPK1, pS308, and β-actin protein levels. Data are normalized to WT ( n = 6–12 biological replicates, two technical replicates each; Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001). (D) Dapk1 mRNA expression in WT and YAC128 brains at 1 month of age was quantified by qPCR. Data are normalized to WT ( n = 6 biological replicates; Student’s t -test). (E) Cortical, striatal, and cerebellar tissues from 1-month-old WT FVB mice were evaluated by Western blot for DAPK1, pS308, and β-actin protein levels. Data are normalized to CTX values ( n = 8 biological replicates, two technical replicates each; Student’s t -test, ### p < 0.001; one-way ANOVA with Bonferroni post hoc analysis, *** p < 0.001).

Article Snippet: The DAPK1 inhibitor TC-DAPK6 (DKI; Tocris Bioscience; Cat# 4301) was dissolved in DMSO and used at a final concentration of 1 μM (0.01% DMSO).

Techniques: Expressing, Activation Assay, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: DAPK1 Promotes Extrasynaptic GluN2B Phosphorylation and Striatal Spine Instability in the YAC128 Mouse Model of Huntington Disease

doi: 10.3389/fncel.2020.590569

Figure Lengend Snippet: Extrasynaptic GluN2B S1303 phosphorylation and interaction with DAPK1 are elevated in the YAC128 brain. Cortical or striatal tissues were subjected to subcellular fractionation to yield (A,C) synaptic (PSD), (B) cortical extrasynaptic (non-PSD), or (D) striatal “non-synaptic” membrane fractions, which were run by SDS–PAGE and Western blotting for GluN2B expression and S1303 phosphorylation ( n = 6–14 biological replicates, two technical replicates each; Student’s t -test, * p < 0.05, ** p < 0.01). (E) DAPK1 was immunoprecipitated from WT and YAC128 cortical lysates, followed by the detection of co-immunoprecipitated GluN2B by Western blot. All sample blot images in panel (E) are cropped from the same Western blot membrane which contained multiple biological replicates run side-by-side. Data are normalized to WT values ( n = 14 biological replicates, Student’s t -test, * p < 0.05, ** p < 0.01).

Article Snippet: The DAPK1 inhibitor TC-DAPK6 (DKI; Tocris Bioscience; Cat# 4301) was dissolved in DMSO and used at a final concentration of 1 μM (0.01% DMSO).

Techniques: Phospho-proteomics, Fractionation, Membrane, SDS Page, Western Blot, Expressing, Immunoprecipitation

Journal: Frontiers in Cellular Neuroscience

Article Title: DAPK1 Promotes Extrasynaptic GluN2B Phosphorylation and Striatal Spine Instability in the YAC128 Mouse Model of Huntington Disease

doi: 10.3389/fncel.2020.590569

Figure Lengend Snippet: Low-dose memantine normalizes cortical DAPK1 activation and extrasynaptic pS1303 levels in YAC128 mice. Cortical tissues from 1-month-old WT and YAC128 mice treated with low-dose memantine from conception were processed to obtain (A) total or (B) extrasynaptic (non-PSD) membrane protein fractions. Samples were processed for DAPK1 or GluN2B protein expression and phosphorylation levels by Western blot. Data are normalized to WT H 2 O values ( n = 8 biological replicates, two technical replicates each; two-way ANOVA with Bonferroni post hoc analysis, * p < 0.05, ** p < 0.01; Student’s t -test, # p < 0.05; two-way ANOVA interaction * p < 0.05 for DAPK1 protein level and pS308, two-way ANOVA interaction ** p < 0.01 for pS1303).

Article Snippet: The DAPK1 inhibitor TC-DAPK6 (DKI; Tocris Bioscience; Cat# 4301) was dissolved in DMSO and used at a final concentration of 1 μM (0.01% DMSO).

Techniques: Activation Assay, Membrane, Expressing, Phospho-proteomics, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: DAPK1 Promotes Extrasynaptic GluN2B Phosphorylation and Striatal Spine Instability in the YAC128 Mouse Model of Huntington Disease

doi: 10.3389/fncel.2020.590569

Figure Lengend Snippet: DAPK1 inhibition normalizes extrasynaptic GluN2B phosphorylation and DAPK1 protein levels in the YAC128 model. (A) Corticostriatal primary cultures from WT and YAC128 embryos were treated at DIV21 with DMSO (0.1%) or a DAPK1 inhibitor (DKI, 10 μM) for 1 h followed by lysis in a gentle buffer to solubilize non-synaptic protein. Lysates were processed by Western blot to detect GluN2B, pS1303, and DAPK1 levels. Data are normalized to WT DMSO values ( n = 8 biological replicates; two-way ANOVA with Bonferroni post hoc analysis, * p < 0.05; Student’s t -test, # p < 0.05). (B) Four-week-old YAC128 mice were treated intranasally with 50 nmol of DAPK1 inhibitor (DKI; TC-DAPK6) or vehicle control (VEH). After 6 h, cortical extrasynaptic fractions were isolated to measure GluN2B, pS1303, and DAPK1 levels ( n = 9 biological replicates, two technical replicates each; Student’s t -test, * p < 0.05). (C) Quantitative measurement of DAPK1-FLAG protein turnover using bio-orthogonal labeling, FLAG immunoprecipitation, CLICK chemistry, and detection of AHA/Streptavidin (Strep)-labeled DAPK1 in transiently transfected COS-7 cells treated with DMSO or 10 μM DKI (TC-DAPK6) for 24 h ( n = 3 for 8 h and 10 h chase time points, n = 7 for all other chase time points; two-way ANOVA ** p < 0.01 for DKI treatment effect; Sidak’s multiple comparisons test, ** p < 0.01).

Article Snippet: The DAPK1 inhibitor TC-DAPK6 (DKI; Tocris Bioscience; Cat# 4301) was dissolved in DMSO and used at a final concentration of 1 μM (0.01% DMSO).

Techniques: Inhibition, Phospho-proteomics, Lysis, Gentle, Western Blot, Control, Isolation, Labeling, Immunoprecipitation, Transfection

Journal: Frontiers in Cellular Neuroscience

Article Title: DAPK1 Promotes Extrasynaptic GluN2B Phosphorylation and Striatal Spine Instability in the YAC128 Mouse Model of Huntington Disease

doi: 10.3389/fncel.2020.590569

Figure Lengend Snippet: DAPK1 kinase activity promotes GluN2B surface expression via pS1303. (A) Surface expression (surface/internal YFP fluorescence ratio) and cluster analysis of transfected YFP-GluN2B was performed in 1:1 cortico-striatal co-cultured YAC128 medium spiny neurons (MSNs) treated with DMSO or the DAPK1 inhibitor TC-DAPK6 (DKI; 1 μM for 3 days, 0.01% final DMSO concentration). Absolute values were used for punctae analysis ( n = 24–38 cells from three independent cultures; two-way ANOVA with Bonferroni post hoc analysis, * p < 0.05, *** p < 0.001). Scale bar = 5 μm. (B) Surface expression of GFP-GluN2B [WT or an S1303A (SA) mutant] in transfected COS-7 cells co-expressing GluN1 and either WT-DAPK1 or a kinase-dead DAPK1 mutant (K42A; KA; n = 23–30 cells from three independent culture passages; two-way ANOVA with Bonferroni post hoc analysis, * p < 0.05, *** p < 0.001). Scale bar = 15 μm.

Article Snippet: The DAPK1 inhibitor TC-DAPK6 (DKI; Tocris Bioscience; Cat# 4301) was dissolved in DMSO and used at a final concentration of 1 μM (0.01% DMSO).

Techniques: Activity Assay, Expressing, Fluorescence, Transfection, Cell Culture, Concentration Assay, Mutagenesis

Journal: Frontiers in Cellular Neuroscience

Article Title: DAPK1 Promotes Extrasynaptic GluN2B Phosphorylation and Striatal Spine Instability in the YAC128 Mouse Model of Huntington Disease

doi: 10.3389/fncel.2020.590569

Figure Lengend Snippet: DAPK1 inhibition or extrasynaptic NMDAR blockade prevents spine instability in YAC128 MSNs. (A) WT and YAC128 1:3 CS co-cultures were treated from DIV14 to 21 with DMSO (0.01%) or the DAPK1 inhibitor TC-DAPK6 (DKI, 1 μM), and spine analysis was performed on DARPP32+ MSNs. (B) 1:3 co-cultures were treated with either water, memantine (MEM, 3 μM) or (C) ifenprodil (IFEN, 3 μM) from DIV14 to 21 and processed in the same way as for DKI ( n = 24 cells from three independent cultures; Two-way ANOVA with Bonferroni post hoc analysis, * p < 0.05, ** p < 0.01, *** p < 0.001). Scale bar = 5 μm.

Article Snippet: The DAPK1 inhibitor TC-DAPK6 (DKI; Tocris Bioscience; Cat# 4301) was dissolved in DMSO and used at a final concentration of 1 μM (0.01% DMSO).

Techniques: Inhibition

Journal: Frontiers in Cellular Neuroscience

Article Title: DAPK1 Promotes Extrasynaptic GluN2B Phosphorylation and Striatal Spine Instability in the YAC128 Mouse Model of Huntington Disease

doi: 10.3389/fncel.2020.590569

Figure Lengend Snippet:

Article Snippet: The DAPK1 inhibitor TC-DAPK6 (DKI; Tocris Bioscience; Cat# 4301) was dissolved in DMSO and used at a final concentration of 1 μM (0.01% DMSO).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Targeting Adaptive IRE1α Signaling and PLK2 in Multiple Myeloma: Possible Anti-Tumor Mechanisms of KIRA8 and Nilotinib

doi: 10.3390/ijms21176314

Figure Lengend Snippet: The gene expression profile by KIRA8 in human myeloma cells. ( A ) IM-9 cells were treated with vehicle (DMSO), 10 μM of KIRA8, and its combination with 5 nM of bortezomib (Bor) or 500 nM of Tg in duplicate. The heatmap shows the cluster analysis from each group. Cluster 16 (dotted frame) represents the differentially expressed genes (DEGs) suppressed by KIRA8-based treatments. ( B ) The gene list shows the top 30 downregulated protein-coding DEGs treated with KIRA8 alone; the most downregulated gene is at the bottom. Pink arrow, the Polo-like kinase 2 ( PLK2 ) was focused on in this study. ( C ) The list of Gene Ontology-enriched terms for molecular function in cluster 16. Terms related to PLK2 are highlighted in purple. ( D ) Quantitative RT-PCR of the relative PLK1 and PLK2 mRNA levels from IM-9 cells treated with vehicle (DMSO) or 10 μM of KIRA8 for 24 h. For these experiments, three independent biological samples were used. ( E ) Western blotting of protein extracts from IM-9 cells treated with vehicle (DMSO) or 10 μM of KIRA8 for 24 h using anti-PLK2 antibodies. Numbers above the representative western blotting picture denote the signal intensity ratios of PLK2 and β-actin measured by Image J software. The β-actin level was used as an internal control. For statistical analysis, three independent biological samples data were used. The data shown are the mean ± SEM. *, p < 0.05; **, p < 0.01.

Article Snippet: Two PERK inhibitors, GSK2606414 (CAS#1337531-89-1) and AMG PERK 44 (AMG; CAS# 1883548-84-2), and dominant Polo-like kinase 2 (PLK2) inhibitor TC-S 7005 (CAS#1082739-92-1) were purchased from TOCRIS Bioscience (Bristol, UK).

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot, Software, Control

Journal: International Journal of Molecular Sciences

Article Title: Targeting Adaptive IRE1α Signaling and PLK2 in Multiple Myeloma: Possible Anti-Tumor Mechanisms of KIRA8 and Nilotinib

doi: 10.3390/ijms21176314

Figure Lengend Snippet: PLK2 expression was IRE1α-dependent and its inhibitor exhibited anti-myeloma effects in IM-9 cells. PLK2 protein and mRNA were expressed strongly in patients with NDMM. ( A , B ) Quantitative RT-PCR of relative rat Plk1 and Plk2 mRNA levels in duplicate ( A ) and western blotting of PLK2 protein ( B ) from doxycycline (DOX)-inducible IRE1α-overexpressing INS-1 insulinoma cells treated with vehicle (DMSO) or 0.5 μM of KIRA8 with or without 1 μg/mL of DOX for 24 h. ( C ) Quantitative RT-PCR of the relative PLK1 and PLK2 mRNA levels from IM-9 cells treated with 25 μM of AMG for 24 h ( n = 3). ( D ) The cell viability in IM-9 cells treated with vehicle (DMSO) or the indicated concentrations of TC-S 7005 for 72 h ( n = 3). ( E ) Annexin V-positive/PI-negative IM-9 cells treated with vehicle (DMSO) or 10 μM of TC-S 7005 for 72 h ( n = 3). ( F , G ) The % components of cell-cycle phases and % cells in the G 2 /M phase in IM-9 cells treated with vehicle (DMSO) and 10 μM of TC-S 7005 for 72 h ( F ) or 10 μM of KIRA8 ( G ) for 24 h. For these experiments, four independent biological samples were used. ( H ) Quantitative RT-PCR of relative PLK2 mRNA levels in BM samples of control subjects ( n = 6) and patients with NDMM who attained a complete response receiving bortezomib-based treatment and high-dose melphalan ( n = 6). ( I ) The expression of PLK2 protein in the BM clot specimen of a patient with NDDM was confirmed by immunohistochemical analysis with anti-PLK2 antibodies (magnification, ×200). The rabbit immunoglobulin G control was negative (data not shown). Immunofluorescence at higher magnification illustrates the expression of the PLK2 protein (green) in CD138 + myeloma cells (red). DAPI (4′,6-diamidino-2-phenylindole; blue) was used as a nuclear counterstain. The data shown are the mean ± SEM. *, p < 0.05; **, p < 0.01.

Article Snippet: Two PERK inhibitors, GSK2606414 (CAS#1337531-89-1) and AMG PERK 44 (AMG; CAS# 1883548-84-2), and dominant Polo-like kinase 2 (PLK2) inhibitor TC-S 7005 (CAS#1082739-92-1) were purchased from TOCRIS Bioscience (Bristol, UK).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Immunohistochemical staining, Immunofluorescence

Journal: Molecules

Article Title: Pyridazinones and Structurally Related Derivatives with Anti-Inflammatory Activity

doi: 10.3390/molecules27123749

Figure Lengend Snippet: The effects of the test compounds and FPR peptide agonists on the LPS-induced NF-κB activity in human THP1-Blue cells, cytotoxicity, and Ca 2+ mobilization in FPR1/FPR2-transfected HL-60 cells.

Article Snippet: Cmpd43 (TC-FPR 43) and Boc-MLF were purchased from Tocris Biosciences (Bristol, UK).

Techniques: Activity Assay, Inhibition

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Formoterol (FORM) suppressed EPO‐induced VSMC senescence in mouse aorta. A) GO enrichment for the intersection genes. B) KEGG enrichment for the intersection genes. C) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of γH2AX (red particles) and αSMA (specific for SMCs, green particles) in ApoE −/− mice receiving vehicle, EPO and EPO+ medium‐dose formoterol treatment, respectively (scale bars = 10 µm). D) Quantitative analysis of γH2AX/αSMC colocalization in (C) ( n = 6 per group). E) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of SIRT1 (green particles) and αSMA (specific for SMCs, red particles) in ApoE −/− mice receiving vehicle, EPO and EPO+medium‐dose formoterol treatment, respectively. (scale bars = 10 µm). F) Quantitative analysis of γH2AX/αSMC colocalization in (E) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, One‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Formoterol ameliorated aortic senescence via β2AR. A) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of β2AR (green particles) and αSMA (specific for SMCs, red particles) in the aorta of ApoE −/− mice (scale bars = 10 µm). B) Representative western blot analysis of protein expression of SIRT1 and P21 in VSMCs treated with vehicle, EPO, EPO+0.01 nmol mL −1 formoterol, EPO+0.1 nmol mL −1 formoterol, EPO+1 nmol mL −1 formoterol and EPO+10 nmol mL −1 formoterol, respectively. C,D) Quantitative analysis of western blot in (B) ( n = 6 per group). E) Representative sections of SA‐β‐gal staining of VSMCs transfected with β2AR siRNA (siβ2AR) or negative control siRNA (siNC) and treated with vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. (scale bars = 10 µm). F) Quantitative analysis of percentage of VSMCs with positive SA‐β‐gal staining in (E) ( n = 6 per group). G) Representative western blot analysis of protein expression of SIRT1, P21 in VSMCs transfected with β2AR siβ2AR or siNC and treated with vehicle, EPO and EPO + 0.1 nmol mL −1 formoterol, respectively. H‐I) Quantitative analysis of western blot in (G) ( n = 6 per group). J) Representative images of immunostaining of SIRT1 in VSMCs transfected with β2AR siβ2AR or siNC and treated with vehicle, EPO, and EPO+ 0.1 nmol mL −1 formoterol, respectively. K) Quantitative analysis of colocalization of dapi/SIRT1 in VSMCs in(G) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, Two‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Western Blot, Expressing, Staining, Transfection, Negative Control, Immunostaining, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: cAMP‐regulated the effect of formoterol on VSMC senescence. A) Quantification of intracellular cAMP levels in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. B) Representative images of SA‐β‐gal staining of VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. (scale bars = 10 µm). C) Quantitative analysis of SA‐β‐gal staining in (B) ( n = 6 per group). D) Representative western blot assay of SIRT1, P21 in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. E‐F) Quantitative analysis of protein expression of SIRT1, P21 protein levels by western blot in (D), ( n = 6 per group). G) Representative images of immunostaining of SIRT1 in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. H) Quantitative analysis of colocalization of dapi/SIRT1 in (G) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, Two‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Control, Staining, Western Blot, Expressing, Immunostaining, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Casitas B‐lineage lymphoma (CBL) was essential for EPO‐induced VSMC senescence. A) Representative immunofluorescent analysis of the colocalization (yellow particles) of CBL (red particles) and SIRT1 (green particles) in VSMC receiving vehicle and EPO treatment respectively (scale bars = 10 µm). B) Quantitative analysis of CBL/SIRT1 colocalization in (A) ( n = 6 per group). C) Representative western blot analysis of protein expression of SIRT1 and P21 in VSMC transfected with siCBL or negative control siRNA (siNC) and treated with vehicle and EPO respectively. D‐E) Quantitative analysis of western blot in (C) ( n = 6 per group). F) Representative sections of SA‐β‐gal staining of VSMC transfected with CBL siRNA (siCBL) or negative control siRNA (siNC) and treated with vehicle and EPO respectively (scale bars = 10 µm). G) Quantitative analysis of the percentage of VSMC with positive SA‐β‐gal staining in (F) ( n = 6 per group). ** p < 0.01, *** p < 0.001, unpaired two‐tailed Student's t‐tests with Welch's correction were applied in (B). The other data were analyzed via Two‐way ANOVA followed by the Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Western Blot, Expressing, Transfection, Negative Control, Staining, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Schematic diagram showing the mechanism of therapeutic effects of medium‐dose formoterol on EPO‐induced AAA. Formoterol binds to β2AR and activates cAMP, which increases SIRT1 protein expression, leading to suppressed VSMC senescence induced by EPO. In contrast, SIRT1 is downregulated by EPO via activation of CBL, resulting in aggravated VSMC senescence. Thus, medium‐dose formoterol attenuated EPO‐induced AAA via β2AR/cAMP/SIRT1 pathways, which provides a promising medication for the treatment of AAA.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Expressing, Activation Assay

Journal: The Journal of Cell Biology

Article Title: KIF14 controls ciliogenesis via regulation of Aurora A and is important for Hedgehog signaling

doi: 10.1083/jcb.201904107

Figure Lengend Snippet: AURA activity mediates effects of KIF14 depletion on primary cilia formation. (A) Experimental design of KIF14 siRNA effects rescue using 100 nM TCS7010 (AURA inhibitor). (B–J) hTERT RPE-1 cells were transfected with indicated siRNA 48 h before fixation and last 24 h serum starved and AURA inhibited by TCS7010. (B and C) Examination of FBF1 (green) localization and intensity. Representative images (CAP350 in red; scale bar, 1 µm) are shown in B and intensity quantification (normalized to CAP350) in C. (D and E) Examination of SCLT1 (green) localization and intensity. Representative images (CAP350 in red; scale bar, 1 µm) are shown in D and intensity quantification (normalized to CAP350) in E. (F and G) Examination of IFT57 (green) localization and intensity. Representative images (CAP350 in yellow, Ac-tub in red; scale bar, 2 µm) are shown in F and intensity quantification histograms (normalized to CAP350; N = 5) in G ("norm." means normalized to CAP350). (H) Representative images of AURA inhibition rescue experiment of ciliogenesis defect caused by KIF14 knockdown. Detection of ARL13B + primary cilia (green; γ-tubulin, red; DNA, blue); scale bar, 10 µm. (I) Quantification of ARL13B + primary cilia formation. (J) Effects on ARL13B + primary cilia length. Asterisks or "ns" indicates statistical significance determined using Tukey's multiple comparisons test (C, E, and J), an unpaired t test (I; ciliated cells = short + long), or the Holm–Sidak method (I; categories).

Article Snippet: Where indicated, cells were treated for indicated time period with either vehicle (DMSO) or the following compounds at the indicated dose: 100 nM TCS7010 (AURA inhibitor; Tocris Bioscience; ; and ), 10–20 μM (R)-roscovitine ( ; ), or 10 μM monastrol ( ).

Techniques: Activity Assay, Transfection, Inhibition, Knockdown

Journal: The Journal of Cell Biology

Article Title: KIF14 controls ciliogenesis via regulation of Aurora A and is important for Hedgehog signaling

doi: 10.1083/jcb.201904107

Figure Lengend Snippet: KIF14 regulates HH signaling independently of AURA activity. (A–C) Functional analyses of HH pathway activation following control or KIF14 silencing and mock or 2 µM SAG treatment in nHDFs. (A) Quantitative RT-PCR quantification of the effect of GLI1 on mRNA levels . (B and C) Analysis of the effect of KIF14 depletion on SMO translocation to the ciliary axoneme and its intensity. (B) Representative images of SMO (green) and ARL13B (red) IF detection; scale bar, 2 µm. (C) Quantification of changes in relative SMO intensity. (D–F) Examination of effect of TCS7010 AURA inhibition on response of nHDF cells transfected with the indicated siRNA to HH pathway activation. Experimental setup was the same as in , but with an additional 0.5 µm SAG treatment for the last 24 h. (D) Representative images of SMO (green) and ARL13B (red) IF detection in nHDF cells transfected with ctrl or KIF14 siRNA and treated with mock or AURA inhibitor; scale bar = 2 µm. Quantification of changes in SMO intensity (normalized to ARL13B) is shown in E and quantitative RT-PCR quantification of effect on mRNA level of GLI1 in F. Asterisks or “ns” indicate statistical significance determined using Tukey's multiple comparisons test.

Article Snippet: Where indicated, cells were treated for indicated time period with either vehicle (DMSO) or the following compounds at the indicated dose: 100 nM TCS7010 (AURA inhibitor; Tocris Bioscience; ; and ), 10–20 μM (R)-roscovitine ( ; ), or 10 μM monastrol ( ).

Techniques: Activity Assay, Functional Assay, Activation Assay, Control, Quantitative RT-PCR, Translocation Assay, Inhibition, Transfection