Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: Exposure of esophageal cells to bile acids at low pH leads to accumulation of the ΔNp73 protein and its translocation to the nucleus. A. Representative immunohistochemical staining for ΔNp73 in the human Barrett’s esophagus (top left panel), esophageal tumor (top right panel), normal stomach and esophagus (bottom panels). Patients with Barrett’s metaplasia express high levels of ΔNp73 in epithelial cells whereas expression of this protein is low in the normal stomach and esophagus. Esophageal tumor specimen was used as a positive control. B. Endogenous ΔNp73 protein was immunoprecipitated from cellular extracts of CP-A cells 7 hours after treatment with 100 µM BA cocktail, pH 4.0 for 30 min. Non-specific rabbit antibody was used as a negative control (N/S). Input protein levels were normalized to β-actin. C. Western blot analysis of total cell extracts from SK-GT-4 cells stably transfected with ΔNp73α plasmid. Cells were collected at the indicated time after treatment with 100 µM BA cocktail, pH 4.0 for 30 min. D. Representative immunofluorescent staining for the ΔNp73 protein using an anti-FLAG tag antibody in stably transfected SK-GT-4 cells before and after BA/A treatment. Cells were fixed 7 hours after treatment with 100 µM BA cocktail, pH4.0 for 30 min. Graph shows percentage of cells with nuclear localization of ΔNp73α protein (***p<0.001). E. Levels of ΔNp73 mRNA were not increased following BA/A treatment. Real-time PCR analysis was performed in CP-A cells at the indicated time after treatment with BA/A (100 µM, 30 min).

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Translocation Assay, Immunohistochemical staining, Staining, Expressing, Positive Control, Immunoprecipitation, Negative Control, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, FLAG-tag, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: A. Dynamics of c-Abl phosphorylation in BA/A-treated cells. SK-GT-4 cells were treated with 100 µM BA cocktail, pH 4.0. Extracts were collected at the indicated time and analyzed by Western blotting. Early (5–30 minutes) and late (4–24 hours) time points are shown. B. Western blot analysis for phospho-ΔNp73(Y50) in SK-GT-4 cells stably transfected with ΔNp73α. Cells were collected at indicated time after treatment with BA/A (100 µM, 30 min). C. c-Abl chemical inhibitor Imatinib downregulates the ΔNp73 protein. SK-GT-4 cells were pretreated with Imatinib (30 µM) for 1 hour, treated with BA/A (100 µM) for 30 minutes and then incubated for an additional 7 hours in the presence of the indicated inhibitor. Total cell extracts were analyzed by Western blotting. D. Inhibition of c-Abl by siRNA leads to downregulation of the ΔNp73 protein in BA/A-treated cells. SK-GT-4 cells were transfected with c-Abl-specific siRNA for 48 hours, treated with BA/A (100 µM) for 30 minutes, and then collected 7 hours after treatment. Levels of ΔNp73 were analyzed by Western blotting. E. Transfection of constitutively active c-Abl leads to upregulation of the ΔNp73 protein. SK-GT-4 cells were transfected with the c-Abl (P242E/P249E)-pcDNA3 vector for 24 hours and analyzed by Western blotting.

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Western Blot, Stable Transfection, Transfection, Incubation, Inhibition, Plasmid Preparation

Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: A. Dynamics of IKKα/β phosphorylation in BA/A-treated cells. SK-GT-4 cells were treated with 100 µM BA cocktail, pH 4.0. Extracts were collected at the indicated time points and analyzed by Western blotting. Early (5–30 minutes) and late (4–24 hours) time points are shown. B. IKK kinase inhibitor Bay11–7085 downregulates the ΔNp73 protein. SK-GT-4 cells were pretreated with Bay11–7085 (10 µM) for 1 hour, treated with BA/A (100 µM) for 30 minutes and then incubated for an additional 7 hours in the presence of the indicated inhibitor. Total cell extracts were analyzed by Western blotting. C. Inhibition of IKK kinases by siRNA leads to downregulation of the ΔNp73 protein in BA/A-treated cells. SK-GT-4 cells were transfected with IKKα- or IKKβ- specific siRNA for 48 hours, treated with BA/A (100 µM) for 30 minutes, and then collected 7 hours after treatment. Levels of ΔNp73 were analyzed by Western blotting. D. Transfection of constitutively active IKKα and IKKβ mutants leads to upregulation of the ΔNp73 protein. SK-GT-4 cells were transfected with the indicated mutants for 24 hours and analyzed by Western blotting.

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Western Blot, Incubation, Inhibition, Transfection

Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: A. Dynamics of p38 protein phosphorylation in BA/A-treated cells. SK-GT-4 cells were treated with 100 µM BA cocktail, pH 4.0. Extracts were collected at the indicated time and analyzed by Western blotting. Early (5–30 minutes) and late (4–24 hours) time points are shown. B. p38 MAPK kinase inhibitor SB203580 downregulates the ΔNp73 protein. SK-GT-4 cells were pretreated with SB203580 inhibitor (10 µM) for 1 hour, treated with BA/A (100 µM) for 30 minutes and then incubated for an additional 7 hours in the presence of the inhibitor. Total cell extracts were analyzed by Western blotting. C. Downregulation of p38 MAPK by siRNA leads to inhibition of the ΔNp73 protein in BA/A-treated cells. SK-GT-4 cells were transfected with p38-specific siRNA for 48 hours, treated with BA/A (100 µM) for 30 minutes, and collected 7 hours after treatment. Protein levels of ΔNp73 were analyzed by Western blotting. D. Co-transfection of p38 MAPK with MKK6 (S207E/T211E) mutant, which activates p38 kinase, leads to upregulation of the ΔNp73 protein. SK-GT-4 cells were transfected with the indicated constructs for 24 hours and then analyzed by Western blotting.

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Western Blot, Incubation, Inhibition, Transfection, Cotransfection, Mutagenesis, Construct

Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: A. Dynamics of Aurora A phosphorylation in BA/A-treated cells. SK-GT-4 cells were treated with 100 µM BA cockail, pH 4.0. Extracts were collected at the indicated time points and analyzed by Western blotting. Early (5–30 minutes) and late (4–24 hours) time points are shown. B. Chemical inhibitor of Aurora A MLN8237 does not affect the ΔNp73 protein. SK-GT-4 cells were pretreated with MLN8237 (0.5 µM) for 1 hour, treated with BA/A (100 µM) for 30 minutes and then incubated for an additional 7 hours in the presence of the indicated inhibitor. Total cell extracts were analyzed by Western blotting. C. Aurora A kinase inhibitor MLN8237 efficiently blocked autophoshorylation of Aurora A kinase.

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Western Blot, Incubation

Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: A. SK-GT-4 cells were treated with either BA/A (100 µM, 30 min) or cytokines (IL-1β, TNFα) or their combinations and then analyzed at the indicated time points. A combined treatment with BA/A and cytokines further enhances induction of ΔNp73 protein. B. Western blot analysis of ΔNp73 in SK-GT-4 cells treated as in A in the presence of siRNAs against p38 or IKKβ kinases. Bottom panel: downregulation of p38 and IKKβ kinases by siRNA is shown. C. Real-time PCR analysis of ΔNp73 mRNA was performed 8 hours after treatment with either IL-1β (20 ng/ml) or TNFα (20 ng/ml) in CP-A cells. D. BAR-T1 cells were treated with BA/A (100 µM) and TNFα (20 ng/ml). Cellular extracts were collected from treated and untreated cells. The endogenous ΔNp73 protein was immunoprecipitated using the ΔNp73 (N-16) antibody. The ΔNp73-p73 binding was analyzed by Western blotting with p73 antibody (Bethyl), which specifically recognizes p73 protein and does not cross-react with ΔNp73. Non-specific goat antibody was used as a negative control (N/S). Input protein levels were normalized to β-actin. Bottom panel: Relative binding of ΔNp73 to p73 was measured by densitometry; p73/IgG ratio is shown (**p<0.01). ΔNp73-p73 binding in untreated cells was arbitrarily set at 1. E. Overexpression of ΔNp73α significantly increases (**p<0.01) the survival of SK-GT-4 cells exposed to BA/A. SK-GT-4 cells, stably transfected with either ΔNp73α (ΔNp73) or empty (Vector) plasmid, were treated with BA/A (100 µM) for 20 min and cell survival was analyzed using MTT assay 24 hours after BA/A treatment.

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, Binding Assay, Negative Control, Over Expression, Stable Transfection, Transfection, Plasmid Preparation, MTT Assay

Journal: Nature Communications

Article Title: A Klotho-derived peptide protects against kidney fibrosis by targeting TGF-β signaling

doi: 10.1038/s41467-022-28096-z

Figure Lengend Snippet: a Amino acid sequence alignment of KP1 in various species. A light green background indicates the regions with identical amino acids in different species. Differences in amino acids among various species are highlighted by red color. b KP1 inhibited the induction of α-SMA mRNA by TGF-β1 in NRK-49F cells. NRK-49F cells were incubated with TGF-β1 (2 ng/ml) in the absence or presence of KP1 (10 µg/ml) or sKlotho (10 µg/ml). The expression of α-SMA mRNA was assessed by qRT-PCR. P values (from left to right): <0.001, 0.001, <0.001. n = 4 biologically independent cells. c – e KP1 inhibited the protein expression of fibronectin and α-SMA. NRK-49F cells were preincubated with KP1 (10 µg/ml; 3 µM) for 1 h and then treated with TGF-β1 (2 ng/ml) for 24 h. Western blot ( c ) and quantitative data ( d , e ) are presented. P values (from left to right): 0.001 and 0.004 (fibronectin); 0.017 and 0.026 (α-SMA). n = 3 biologically independent cells. f KP1 inhibited TGF-β1-mediated fibronectin expression and deposition by NRK-49F cells. Representative micrographs with immunofluorescence staining of fibronectin are shown. Scale bar, 50 µm. Arrows indicated positive staining. g – i KP1 inhibited the expression of fibronectin and α-SMA in primary tubular cells. Western blot analyses ( g ) and quantitative data of fibronectin ( h ) and α-SMA ( i ) are shown. P values (from left to right): 0.001 and 0.001 (fibronectin); 0.001 and 0.003 (α-SMA). n = 3 biologically independent cells. j – l The protein level of fibronectin and collagen I expressed by primary cardiomyocytes. Western blot analyses ( j ) and quantitative data of fibronectin ( k ) and collagen I ( l ) are shown. P values (from left to right):0.001 and 0.001 (fibronectin); <0.001 and <0.001 (collagen I). n = 3 biologically independent cells. Ctrl controls. Data are presented as mean values ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher’s Least-significant Difference (LSD) post hoc test. Source data are provided as a Source Data file.

Article Snippet: Recombinant TGF-β receptor 2 protein (43.4 kDa, >95% as determined by SDS-PAGE) (10358-H03H-B; Sino Biological Inc., Beijing, China) diluted at 50 µg/ml in 10 mM acetate pH 4.0, was covalently immobilized on a CM5 sensor chip (GE Healthcare) using amine-coupling chemistry.

Techniques: Sequencing, Incubation, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: A Klotho-derived peptide protects against kidney fibrosis by targeting TGF-β signaling

doi: 10.1038/s41467-022-28096-z

Figure Lengend Snippet: a , b Co-immunoprecipitation (Co-IP) demonstrated that KP1 bound to TβR2. NRK-49F cell lysates (500 µg) and FITC-KP1 (10 µg) were immunoprecipitated (IP) with the anti-TβR2 antibody at 4 °C overnight, followed by immunoblotted (IB) for FITC and TβR2, respectively ( a ). In the reciprocal experiment, mixtures of cell lysates and FITC-KP1 were immunoprecipitated with anti-FITC, followed by immunoblotted for TβR2 and FITC ( b ). c The affinity of TβR2 to KP1 was shown. SPR analyses showed concentration-dependent (0.78–25 μM) binding of KP1 to TβR2 immobilized on a sensor chip. A representative sensorgram of two independent experiments is presented, in which the curves (color) with an overlay of the fitting (black) are shown. The fitted constants are k a = 407.7 M −1 S −1 ; k d = 5.8 × 10 −4 S −1 ; K D = 1.4 × 10 −6 M. d The affinity of TβR2 to KP12 was shown. SPR analyses showed concentration-dependent (1.56–100 μM) binding of KP12 to TβR2 immobilized on a sensor chip. A representative sensorgram of two independent experiments is presented, in which the curves (color) with an overlay of the fitting (black) are shown. The fitted constants are k a = 159.5 M −1 S −1 ; k d = 2.3 × 10 −3 S −1 ; K D = 14.6 × 10 −6 M. e The specific binding of FITC-KP1 to TβR2 coated on a microplate. The fluorescence intensity of FITC-KP1 (arbitrary units) was assessed after incubating on a microplate coated with TβR2 protein. P values (from left to right): <0.001, <0.001, and <0.001 (one-way ANOVA with Fisher’s LSD post hoc test). ( n = 4 biologically independent cells). Data are presented as mean values ± SEM. f KP1 inhibited the interaction between TβR2 and TGF-β1 dose-dependently. NRK-49F cells were pre-incubated with different amounts of KP1 for 1 h, and then treated with TGF-β1 (2 ng/ml) for 5 min. Cells were collected and immunoprecipitated with anti-TβR2 or anti-IgG. g Negative peptide KP12 did not block the binding of TβR2 and TGF-β1. h KP1 inhibited the binding of TβR1 and TβR2. i KP1 inhibited the interaction of TβR2 and sKlotho. Cells were transfected with sKlotho plasmid for 24 h and incubated with TGF-β1 in the absence or presence of KP1. Cells were collected and immunoprecipitated with anti-TβR2. Source data are provided as a Source Data file.

Article Snippet: Recombinant TGF-β receptor 2 protein (43.4 kDa, >95% as determined by SDS-PAGE) (10358-H03H-B; Sino Biological Inc., Beijing, China) diluted at 50 µg/ml in 10 mM acetate pH 4.0, was covalently immobilized on a CM5 sensor chip (GE Healthcare) using amine-coupling chemistry.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Concentration Assay, Binding Assay, Fluorescence, Incubation, Blocking Assay, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: A Klotho-derived peptide protects against kidney fibrosis by targeting TGF-β signaling

doi: 10.1038/s41467-022-28096-z

Figure Lengend Snippet: a KP1 inhibited the phosphorylation of Smad2 and Smad3 induced by TGF-β1. Numbers (1–3) indicate each individual wells of cells. Serum-starved NRK-49F cells were pre-treated with KP1 or vehicle for 1 h and then stimulated by TGF-β1 for 45 min. b , c Quantitative data of p-Smad3 ( b ) and p-Smad2 ( c ) in different groups are shown. P values (from left to right): <0.001 and 0.001 (p-Smad3); <0.001 and 0.001 (p-Smad2). n = 3 biologically independent cells. d Immunofluorescence staining of p-Smad3 showed that KP1 blocked TGF-β1-mediated Smad3 phosphorylation and nuclear translocation. DAPI denotes the nuclei. Arrows indicate p-Smad3 positive cells. Scale bar, 20 µm. e – h KP1 inhibited TGF-β1-induced phosphorylation of ERK1/2, JNK, and p38. Western blot analyses ( e ) and quantitative data of p-ERK1/2 ( f ), p-JNK ( g ), and p-p38 ( h ) are shown. P values (from left to right): <0.001 and 0.001 (p-ERK1/2); 0.001 and 0.002 (p-JNK); 0.005 and 0.010 (p-p38). n = 3 biologically independent cells. i – k KP1 inhibited TGF-β/Smad signaling in primary tubular cells. Western blot analyses ( i ) and quantitative data of p-Smad2 ( j ) and p-Smad3 ( k ) in different groups are shown. P values (from left to right): 0.032 and 0.034 (p-Smad2); 0.041 and 0.049 (p-Smad3). n = 3 biologically independent cells ( l – n ) KP1 was more effective in inhibiting TGF-β signaling compared to TβR2 inhibitor ITD-1. NRK-49F cells were pre-incubated with KP1 (10 µg/ml, 3 μM) or ITD-1 (3 μM) for 1 h, and then treated with TGF-β1 (2 ng/ml) for 45 min. Western blot analyses ( l ) and quantitative data of p-Smad3 ( m ) and p-p38 ( n ) are shown. P values (from left to right): < 0.001, 0.008 and <0.001 (p-Smad3); <0.001, <0.001 and <0.001 (p-p38). n = 3 biologically independent cells. Ctrl controls. Data are presented as mean values ± SEM. P values were calculated by one-way ANOVA with Fisher’s LSD post hoc test ( b , c , f , g , h , m , n ) or Dunnett’s T3 test ( j , k ). Source data are provided as a Source Data file.

Article Snippet: Recombinant TGF-β receptor 2 protein (43.4 kDa, >95% as determined by SDS-PAGE) (10358-H03H-B; Sino Biological Inc., Beijing, China) diluted at 50 µg/ml in 10 mM acetate pH 4.0, was covalently immobilized on a CM5 sensor chip (GE Healthcare) using amine-coupling chemistry.

Techniques: Immunofluorescence, Staining, Translocation Assay, Western Blot, Incubation

Journal: Nature Communications

Article Title: A Klotho-derived peptide protects against kidney fibrosis by targeting TGF-β signaling

doi: 10.1038/s41467-022-28096-z

Figure Lengend Snippet: a Co-immunoprecipitation showed that KP1 blocked the interaction between TGF-β1 and TβR2 in vivo. b , c KP1 inhibited Smad2/3 activation in vivo. Representative Western blot analyses ( b ) and quantitative data ( c ) of p-Smad3 and p-Smad2 are shown. P values: <0.001, <0.001 (p-Smad3); 0.002, 0.002 (p-Smad2). n = 6 biologically independent animals. d Representative micrographs of immunohistochemical staining of p-Smad3 and p-ERK1/2. Boxed areas are enlarged. Arrows indicate positive staining. Scale bar, 50 µm. e , f Quantitative data of p-Smad3 positive cells number ( e ) and p-ERK positive staining area ( f ) in each high-power field. P values: < 0.001, < 0.001 (p-Smad3); < 0.001, < 0.001 (p-ERK); n = 6 biologically independent animals. g KP1 blocked the activation of MAPK and TβR2 expression in vivo. h – k Quantitative data of p-ERK1/2 ( h ), p-JNK ( i ), p-p38 ( j ), and TβR2 ( k ) are shown. P values (from left to right): 0.006, 0.018 (p-ERK1/2); < 0.001, 0.046 (p-JNK); < 0.001, 0.035 (p-p38); 0.002, 0.004 (TβR2). n = 6 biologically independent animals. Data are presented as mean values ± SEM. Statistical significance was determined by one-way ANOVA followed by Fisher’s LSD post hoc test ( c , i , k ) or Dunnett’s T3 test ( e , f , h , j ). Source data are provided as a Source Data file.

Article Snippet: Recombinant TGF-β receptor 2 protein (43.4 kDa, >95% as determined by SDS-PAGE) (10358-H03H-B; Sino Biological Inc., Beijing, China) diluted at 50 µg/ml in 10 mM acetate pH 4.0, was covalently immobilized on a CM5 sensor chip (GE Healthcare) using amine-coupling chemistry.

Techniques: Immunoprecipitation, In Vivo, Activation Assay, Western Blot, Immunohistochemical staining, Staining, Expressing

Journal: Molecular Cancer Research

Article Title: Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells

doi: 10.1158/1541-7786.mcr-21-0366

Figure Lengend Snippet: Figure 1. Chk1 inhibitors block tyrosine phosphorylation of STAT3 (p-Y705) in multiple myeloma cells. A, U266 cells were treated with 100 nmol/L PF-477736 (PF) for 4 and 16 hours. Gene expression profiling was performed using Human Signal Transduction PathwayFinder R2 PCR Array (84 genes). The heatmap shows absolute mRNA copy numbers, which were calculated from PCR cycle thresholds (Ct) and generated from Qiagen RT2 Profiler PCR Data Analysis Web page. B, Fold change of STAT3 pathway-related genes upon PF stimulation in U266 cells as compared with untreated controls. C–E, Western blot analysis of p-Y705 STAT3, p-S727 STAT3, and total STAT3 in U266 cells treated with indicated doses of PF, LY, or other STAT3 inhibitors. CEP ¼ CEP-3891; AQ ¼ Atovaquone; Cry ¼ Cryptotanshinone; BBI ¼ BBI-608; TTI ¼ TTI-101. b-actin controls were assayed to ensure equivalent loading and transfer. Images were quantified and analyzed by using ImageJ software. Data were normalized by the ratio of indicated protein and b-actin versus control. F, Untreated and treated (PF 100 nmol/L, 3 hours) cells were stained with p-Y705 STAT3 and DAPI, and then visualized by fluorescence microscope and by ImageStream and representative cells are shown (BF ¼ brightfield; Scale bar ¼ 20 mmol/L). Histogram of p-Y705 STAT3 intensity and fold change were shown. Representative data of at least three replicates. , P < 0.05.

Article Snippet: Under a flow rate of 25 mL/minute, STAT3 protein (MW 95,000, SignalChem, catalog no. S54-54BH) was injected over both channels at concentrations of 0.1, 0.2, 0.45, 0.9, and 2 mmol/L, respectively, for 6 minutes followed by a 6-minute dissociation in running buffer.

Techniques: Blocking Assay, Phospho-proteomics, Gene Expression, Transduction, Generated, Western Blot, Software, Control, Staining, Microscopy

Journal: Molecular Cancer Research

Article Title: Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells

doi: 10.1158/1541-7786.mcr-21-0366

Figure Lengend Snippet: Figure 2. Chk1 inhibitors block tyrosine phosphorylation of STAT3 (p-Y705) in multiple myeloma cell lines. A–C, right, -6/1, or PSR cells were treated with the indicated concentrations of PF, CEP, Cry 2.5 mmol/L, or AQ 20 mmol/L for 6 hours. Western blot analysis of p-Y705 STAT3, p-S727 STAT3, and total STAT3 were performed. b-actin controls were assayed to ensure equivalent loading and transfer. C, left, bortezomib-resistant PS-R cells were exposed (24 hours) to 10 nmol/L bortezomib (BTZ), followed by flow cytometry to monitor the percentage of apoptotic (7-AADþ) cells. Values represent the means SD for three experiments performed in triplicate. , P < 0.001. D, OPM2 cells were pretreated with HS-5 CM for 16 hours, and then treated with 500 nmol/L PF for 6 and 24 hours. Western blot analysis was carried out to asmentioned in A–C.E, OPM2 cellswere pretreated with IL6 (1 or 5 ng/mL) for 15 minutes,and then treated with 500 nmol/L PF for 8 hours. Western blot analysis was carried out as in A–C. Images were quantified and analyzed using ImageJ software. Data were normalized by the ratio of indicated protein/b-actin versus control. F, OMP2 cells were pretreated with CM or IL6 as in D and E, Immunofluorescence staining was performed to monitor p-Y705 STAT3.

Article Snippet: Under a flow rate of 25 mL/minute, STAT3 protein (MW 95,000, SignalChem, catalog no. S54-54BH) was injected over both channels at concentrations of 0.1, 0.2, 0.45, 0.9, and 2 mmol/L, respectively, for 6 minutes followed by a 6-minute dissociation in running buffer.

Techniques: Blocking Assay, Phospho-proteomics, Western Blot, Cytometry, Software, Control, Staining

Journal: Molecular Cancer Research

Article Title: Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells

doi: 10.1158/1541-7786.mcr-21-0366

Figure Lengend Snippet: Figure 3. Chk1 inhibitors disrupt STAT3 activation in multiple myeloma cells. A, U266 cells were treated with indicated concentrations of PF for 3 and 6 hours. Native nuclear protein extracts were prepared and were separated on a 6% native PAGE gel. Electrophoresis was performed in the absence of SDS. Proteins were immunoblotted with p-Y705 STAT3 and total STAT3. Total STAT3 served as a loading control. B, Nuclear extracts from U266 cells treated with the indicated concentrations of PF for 6 and 16 hours were subjected to EMSA analysis. C, U266 cells were treated with indicated concentrations of PF, CEP, or LY for 6 and 16 hours. A STAT3 DNA-binding ELISA was employed to evaluate the ability of STAT3 in cellular nuclear extracts to bind to its corresponding consensus sequence that had been immobilized on a 96- well plate. D, U266 cells were treated with the indicated concentrations of PF for 16 hours. ChIP experiments were performed to detect the presence of STAT3 at the promotors of the BCL-XL, MCL-1, and c-Myc genes after treatment with PF. Data were normalized with non-immune IgG (negative control) and STAT3 antibody, and the relative fold change (vs. untreated controls; UT) is presented. E, Relative mRNA expressions of c-Myc, MCL-1, and BCL-XL were determined by real-time RT-PCR analysis. GAPDH served as an internal control. , P < 0.01; , P < 0.001; , P < 0.0001.

Article Snippet: Under a flow rate of 25 mL/minute, STAT3 protein (MW 95,000, SignalChem, catalog no. S54-54BH) was injected over both channels at concentrations of 0.1, 0.2, 0.45, 0.9, and 2 mmol/L, respectively, for 6 minutes followed by a 6-minute dissociation in running buffer.

Techniques: Activation Assay, Clear Native PAGE, Nucleic Acid Electrophoresis, Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Negative Control, Quantitative RT-PCR

Journal: Molecular Cancer Research

Article Title: Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells

doi: 10.1158/1541-7786.mcr-21-0366

Figure Lengend Snippet: Figure 4. Mechanisms of STAT3 inhibition by Chk1 inhibitors. A, SPR analyses are shown to monitor Chk1 and STAT3 interactions. SPR characterizes the interaction between Chk1 and STAT3 by detecting the soluble STAT3 (analyte) binding to immobilized Chk1 on a carboxymethyldextran sensor chip. The STAT3 was injected over the Chk1 surface in a series of concentrations (0.1, 0.2, 0.45, 0.45, 0.9, and 2 mmol/L that are labeled with different colors). Left, The data were fit to a 1:2 binding model in a kinetic analysis evaluation graph; the black lines show the global 1:2 model fit). The binding constants are reported in the insets. Right, Equilibrium fit plot displays the STAT3 concentration–response curve between Chk1 and STAT3 obtained from affinity SPR analysis. B, U266 cells were treated with 200 nmol/L PF for 16 hours. Nuclear and cytoplasmic extracts were collected for performance of co-immunoprecipitation assays. Chk1 pulldown proteins were immunoblotted with p-Y705 STAT3, total STAT3, and Chk1. Total lysates were immunoblotted for p-Y705 STAT3, total STAT3, and Chk1. P84 and b-actin were used as controls for nuclear and cytoplasmic protein levels, respectively. C, U266 cells were treated with 100 mmol/L PV and 50 nmol/L PF for 6 and 24 hours. Western blot analysis of p-Y705 STAT3, p-S727 STAT3, and total STAT3 were then performed. b-actin controls were assayed to ensure equivalent loading and transfer. D and E, Purified His-Chk1 was used in an in vitro kinase assay using purified GST-STAT3 as substrate. Kinase assay products were separated by SDS-PAGE (6%–12%) and visualized by autoradiography (P32) and CBB. F, A kinase assay was performed as in E in the presence or absence of cold ATP, and kinase assay products were transferred to a nitrocellulose membrane and immunoblotted with p-Y705 STAT3, p-S727 STAT3, and p-S296 Chk1. Ponceau S stained blots served as loading controls. G, left, U266 cells were treated with 200 PF or 500 nmol/L Rux (ruxolitinib) for 6 hours, followed by lysis and immunoprecipitation with JAK2. The blots were probed for p-Y570 JAK2 and total JAK2. Total lysates were immunoblotted for p-Y705 STAT3, p-S727 STAT3, total STAT3, and JAK2. b-actin was used as a loading control. Right, A non- radioactive JAK2 kinase assay was performed. The graph was generated by expressing fold-phosphorylation changes (normalized to control). Images were quantified and analyzed using ImageJ software. Data were normalized by the ratio of the indicated protein/b-actin or Ponceau S staining versus control.

Article Snippet: Under a flow rate of 25 mL/minute, STAT3 protein (MW 95,000, SignalChem, catalog no. S54-54BH) was injected over both channels at concentrations of 0.1, 0.2, 0.45, 0.9, and 2 mmol/L, respectively, for 6 minutes followed by a 6-minute dissociation in running buffer.

Techniques: Inhibition, Binding Assay, Injection, Labeling, Concentration Assay, Immunoprecipitation, Western Blot, In Vitro, Kinase Assay, SDS Page, Autoradiography, Membrane, Staining, Lysis, Control, Generated, Expressing, Phospho-proteomics, Software

Journal: Molecular Cancer Research

Article Title: Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells

doi: 10.1158/1541-7786.mcr-21-0366

Figure Lengend Snippet: Figure 5. Chk1 plays a critical role in regulating STAT3 signaling. A, U266 cells were transfected with a Tet-on inducible Chk1 shRNA. Doxycycline (0.5 mg/mL) induced cells were collected at 24, 48, and 72 hours. Total lysateswere immunoblotted for p-Y705 STAT3, total STAT3,and Chk1. b-actin was used as a loading control. Imageswere quantified and analyzed using ImageJ software. Data were normalized by the ratio of indicated protein/b-actin versus control. B–I, U266 cells were transfected with a lentivirus harboring Chk1 shRNA. B, Western blot analyses of Chk1 and p-Y705 STAT3 were performed. b-actin controls were assayed to ensure equivalent loading and transfer. C, The STAT3 DNA-binding ELISA was used to evaluate the ability of STAT3 in cellular nuclear extracts to bind to its corresponding consensus sequence immobilized on a 96-well plate. D, EMSA assay of DNA-binding activity of STAT3 in Chk1 knockdown cells. E–G, ChIP experiments were performed to detect the presence of STAT3 at the promotor regions of BCL-XL, MCL-1, and c-Myc in Chk1 knockdown cells. Data were normalized with non-immune IgG (negative control) and STAT3 antibody, and relative fold change (vs. UT) is presented. H, Relative mRNA expression of c-Myc was determined by real-time RT-PCR analysis. GAPDH served as an internal control. Relative fold change (vs. EV) is presented. I, Cells were stained with p-Y705 STAT3, Chk1 and DAPI, and then visualized by fluorescence microscope and by ImageStream and representative cells are shown (BF ¼ brightfield). Histograms of p-Y705 STAT3 intensity and fold change are shown. Representative data for at least three replicates. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001.

Article Snippet: Under a flow rate of 25 mL/minute, STAT3 protein (MW 95,000, SignalChem, catalog no. S54-54BH) was injected over both channels at concentrations of 0.1, 0.2, 0.45, 0.9, and 2 mmol/L, respectively, for 6 minutes followed by a 6-minute dissociation in running buffer.

Techniques: Transfection, shRNA, Control, Software, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Activity Assay, Knockdown, Negative Control, Expressing, Quantitative RT-PCR, Staining, Microscopy

Journal: Molecular Cancer Research

Article Title: Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells

doi: 10.1158/1541-7786.mcr-21-0366

Figure Lengend Snippet: Figure 6. STAT3 inhibition by Chk1 inhibitor through a STAT3-dependent manner. A–C, U266 were transfected with pcDNA3.1 (empty vector) or pcDNA-STAT3 CA (FLAG fusion), and STAT3-CA01 was selected. STAT3-CA02 was selected in U266 cells infected with a lentivirus harboring STAT3 CA (FLAG fusion). A, The STAT3 DNA- binding ELISA was used to evaluate STAT3 activity in STAT3-CA cell. B, cells were exposed (24 hours) to indicated concentrations of PF, followed by flow cytometry to monitor the percentage of apoptotic (7-AADþ) cells. Values represent the means SD for three experiments performed in triplicate. , P < 0.05; , P < 0.01. C, Western blot analysis of FLAG, cleaved-PARP, cleaved-Caspase-3, and gH2A.X was performed. a-tubulin controls were assayed to ensure equivalent loading and transfer. D–F, U266 cells infected with a lentivirus harboring STAT3 DN (FLAG fusion). STAT3-CA01/02 were selected. Assays were performed as in A–C, , P < 0.01.

Article Snippet: Under a flow rate of 25 mL/minute, STAT3 protein (MW 95,000, SignalChem, catalog no. S54-54BH) was injected over both channels at concentrations of 0.1, 0.2, 0.45, 0.9, and 2 mmol/L, respectively, for 6 minutes followed by a 6-minute dissociation in running buffer.

Techniques: Inhibition, Transfection, Plasmid Preparation, Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Cytometry, Western Blot

Journal: Molecular Cancer Research

Article Title: Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells

doi: 10.1158/1541-7786.mcr-21-0366

Figure Lengend Snippet: Figure 7. Chk1 inhibitors decrease p-Y705 STAT3 expression in primary human multiple myeloma cells ex vivo and in mice invivo. A, top, CD138þ and CD138 mononuclear cells (MNC) were isolated from multiple myeloma primary samples. Bottom, Isolated CD138þ MNCs were treated with 500 nmol/L PF for 16 hours. Western blot analysis of p-Y705 STAT3 and p-S727 STAT3 were then performed. a-tubulin and b-actin controls were assayed to ensure equivalent loading and transfer. A black border indicates that the blotswere cut and spliced from the same membrane and after the same exposure interval. B, Isolated multiple myeloma primary MNCswere stained with p-Y705 STAT3, CD138, and DAPI, and then visualized by fluorescence microscopy and by ImageStream; representative cells are shown (BF ¼ brightfield). Histograms of p-Y705 STAT3 intensity and fold change are shown. C, Isolated multiple myeloma primary MNCs were treated with indicated doses of PF and CEP for 16 hours. Western blot analysis of p-Y705 STAT3, p-S727 STAT3, STAT3, c-Myc, MCL-1, BCL-XL, and gH2A.X were performed. b-actin controls were assayed to ensure equivalent loading and transfer. D, NOD-SCID IL2Rgammanull mice were subcutaneously injected with 5 106 U266 cells into the flank. When tumors grew to 10 mm (length), PF (15 mg/kg) was administrated (intraperitoneally) for 3 days. Control animals received equal volumes of vehicle. Western blot analysis was performed to monitor the indicated candidate pharmacodynamic markers, identified from in vitro experiments, in tumors excised from representative mice. Images were quantified and analyzed using ImageJ software. Data were normalized by determining the ratio of the indicated protein/b-actin versus control. E, Tumor sections from vehicle and PF-treated mice were stained with p-Y705 STAT3 antibody. Sections were visualized and images captured usingVectra Polaris Imaging System (Akoya Biosciences) research microscope and quantified using the Polaris software program. Scale bar ¼ 50 mm. , P < 0.001.

Article Snippet: Under a flow rate of 25 mL/minute, STAT3 protein (MW 95,000, SignalChem, catalog no. S54-54BH) was injected over both channels at concentrations of 0.1, 0.2, 0.45, 0.9, and 2 mmol/L, respectively, for 6 minutes followed by a 6-minute dissociation in running buffer.

Techniques: Expressing, Ex Vivo, Isolation, Western Blot, Membrane, Staining, Microscopy, Injection, Control, In Vitro, Software, Imaging

Journal: eLife

Article Title: Endogenous Syngap1 alpha splice forms promote cognitive function and seizure protection

doi: 10.7554/elife.75707

Figure Lengend Snippet: Figure 5. Validation of SynGAP PDZ binding motif (PBM) mutations and construction of the Syngap1PBM mouse line. (A) Schematic diagram for exon map and alternative use of Exon21 in Syngap1 gene. Exon21b encodes for α1 isoform. Exon 21 a encodes for α2 isoform. Point mutations indicated in red alter exon 21b coding sequence without influencing exon21a open reading frame. (B) Schematics of SynGAPα1 and PSD95 domain structure and the location of point mutations. (C) Illustrations of constructs expressed in HeLa cells to study PDZ-dependent interaction between SynGAP and PSD95.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Biological sample (Mus musculus) Mouse primary forebrain neurons This study 18–21 days in vitro Biological sample (Mus musculus) Cortical and hippocampal tissue Multiple strains (this study) Male and female Strain, strain background (Mus musculus) IRES- TD Spicer et al., 2018 C57BL/6 J background Strain, strain background (Mus musculus) Beta KO (β*) This study C57BL/6 J background Strain, strain background (Mus musculus) PBM This study C57BL/6 J background Cell line (Homo sapiens) HeLa Farzan Lab (Scripps Research) Cell line (Homo sapiens) H293T Kissil Lab (Scripps Research) Genetic reagent (Rattus norvegicus) PSD95- tRFP Addgene #52,671 Plasmid Genetic reagent (Mus musculus) EGFP- CCα1 This study Plasmid Genetic reagent (Mus musculus) EGFP- CCPBM This study Plasmid Genetic reagent (Mus musculus) EGFP- SynGAPα1 This study Plasmid Genetic reagent (Mus musculus) EGFP- SynGAPα1PBM This study Plasmid Antibody Rabbit polyclonal antibody Thermo PA1- 046 Anti- total SynGAP (1:1000) Antibody Rabbit polyclonal antibody Millipore 06–900 Anti- SynGAPα1 (1:1000) Antibody Rabbit monoclonal antibody Abcam ab77235 Anti- SynGAPα2 (1:1000) Antibody Rabbit polyclonal antibody Araki et al., 2020 Huganir Lab (JHU) Anti- SynGAPβ (1:1000) Antibody Mouse monoclonal antibody Thermo MA1- 045 Anti- PSD95 (1:2000) Antibody Rabbit polyclonal antibody Novus NB300- 653 Anti- Synaptophysin (1:1000) Antibody Mouse monoclonal antibody CST 9,106 Anti- phosphoERK (1:1000) Antibody Mouse monoclonal antibody CST 4,696 Anti- ERK (1:1000) Antibody Mouse monoclonal antibody Millipore MAB2263 Anti- GluA1 N- term. (1:500) Antibody Rabbit polyclonal antibody Millipore Ab9876 Anti- TARP (1:500) Antibody Rabbit polyclonal antibody Thermo Pierce PA521097 Anti- LRRTM2 (1:1000) Sequence- based reagent IRES- TD Genotyping Primer Fw IDT AGAT CCAC CAGG CCCT GAA Sequence- based reagent IRES- TD Genotyping Primer Rev IDT GTCT TGAA CTCC ACCA GGTA GTG Sequence- based reagent PBM Genotyping Primer Fw IDT CTGG TTCA AAGG CTCC TGGT A Sequence- based reagent PBM Genotyping Primer Rev IDT CTGT TTGT TTCT CACC TCCA GGAA Kilinc et al. eLife 2022;11:e75707.

Techniques: Biomarker Discovery, Binding Assay, Sequencing, Construct

Journal: eLife

Article Title: Endogenous Syngap1 alpha splice forms promote cognitive function and seizure protection

doi: 10.7554/elife.75707

Figure Lengend Snippet: Figure 7. Characterization of native PSD95 complexes from Syngap1PBM animals. (A) Volcano plot demonstrating the label-free quantitative mass- spectrometry profile of the logarithmic difference in protein levels in the immunoprecipitated PSD95 complexes derived from DIV21 +/+ and PBM/ PBM cultures in inactivated state. Only Gpm6a (shown in black) was significantly altered beyond p > 0.001 cutoff. Blue dots represent proteins with type 1 PDZ-ligands. Green dots represent DLG family proteins. p Values were calculated via t-test for each protein. Samples were derived from individual cultures (4 per genotype) which are immunoprecipitated separately. Log2FC was calculated as ratio of PBM/PBM over +/+. (B) List of proteins that are differentially expressed beyond p > 0.05 cutoff. Note that Iqseq2 and Dlgap3 are PDZ-binding proteins. (C) Mass-spectrometry profile of type-1 PDZ binding motif containing proteins in immunoprecipitated PSD95 complex in +/+ vs PBM/PBM inactivated cultures. (D) Western blots showing relative expression of TARPs and Lrrtm2 in PSD fractions from adult hippocampi in +/+ vs PBM/PBM. (E–G) Quantifications of (D). (E) TARPg8 t(6)=0.6961, P = 0.5124. (F) TARPg2-4 t(6)=0.7924, p = 0.4583 (G) Lrrtm2 t(6)=0.5542, p = 0.5995. Each sample represents hippocampi pooled from 2 mice. (H) Comparison of PSD95 and SynGAP IP complexes as reported by Li et al., 2017 and Wilkinson et al., 2017. Note that PSD95 and SynGAP complexes share diverse range of components involving cytoskeletal and scaffolding proteins.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Biological sample (Mus musculus) Mouse primary forebrain neurons This study 18–21 days in vitro Biological sample (Mus musculus) Cortical and hippocampal tissue Multiple strains (this study) Male and female Strain, strain background (Mus musculus) IRES- TD Spicer et al., 2018 C57BL/6 J background Strain, strain background (Mus musculus) Beta KO (β*) This study C57BL/6 J background Strain, strain background (Mus musculus) PBM This study C57BL/6 J background Cell line (Homo sapiens) HeLa Farzan Lab (Scripps Research) Cell line (Homo sapiens) H293T Kissil Lab (Scripps Research) Genetic reagent (Rattus norvegicus) PSD95- tRFP Addgene #52,671 Plasmid Genetic reagent (Mus musculus) EGFP- CCα1 This study Plasmid Genetic reagent (Mus musculus) EGFP- CCPBM This study Plasmid Genetic reagent (Mus musculus) EGFP- SynGAPα1 This study Plasmid Genetic reagent (Mus musculus) EGFP- SynGAPα1PBM This study Plasmid Antibody Rabbit polyclonal antibody Thermo PA1- 046 Anti- total SynGAP (1:1000) Antibody Rabbit polyclonal antibody Millipore 06–900 Anti- SynGAPα1 (1:1000) Antibody Rabbit monoclonal antibody Abcam ab77235 Anti- SynGAPα2 (1:1000) Antibody Rabbit polyclonal antibody Araki et al., 2020 Huganir Lab (JHU) Anti- SynGAPβ (1:1000) Antibody Mouse monoclonal antibody Thermo MA1- 045 Anti- PSD95 (1:2000) Antibody Rabbit polyclonal antibody Novus NB300- 653 Anti- Synaptophysin (1:1000) Antibody Mouse monoclonal antibody CST 9,106 Anti- phosphoERK (1:1000) Antibody Mouse monoclonal antibody CST 4,696 Anti- ERK (1:1000) Antibody Mouse monoclonal antibody Millipore MAB2263 Anti- GluA1 N- term. (1:500) Antibody Rabbit polyclonal antibody Millipore Ab9876 Anti- TARP (1:500) Antibody Rabbit polyclonal antibody Thermo Pierce PA521097 Anti- LRRTM2 (1:1000) Sequence- based reagent IRES- TD Genotyping Primer Fw IDT AGAT CCAC CAGG CCCT GAA Sequence- based reagent IRES- TD Genotyping Primer Rev IDT GTCT TGAA CTCC ACCA GGTA GTG Sequence- based reagent PBM Genotyping Primer Fw IDT CTGG TTCA AAGG CTCC TGGT A Sequence- based reagent PBM Genotyping Primer Rev IDT CTGT TTGT TTCT CACC TCCA GGAA Kilinc et al. eLife 2022;11:e75707.

Techniques: Mass Spectrometry, Immunoprecipitation, Derivative Assay, Binding Assay, Western Blot, Expressing, Comparison, Scaffolding