Journal: mBio

Article Title: Dengue virus NS1 secretion is regulated via importin-subunit β1 controlling expression of the chaperone GRp78 and targeted by the clinical drug ivermectin

doi: 10.1128/mbio.01441-23

Figure Lengend Snippet: Nuclear transport blockage prevents the secretion and activity of secreted alkaline phosphatase (SEAP). ( A ) A549 cells expressing SEAP or control (mock) cells were treated for 48 h with DMSO or IVM (1 µM). Lysates and supernatants were analyzed by western blot using a SEAP-specific antibody. iSEAP and eSEAP, intra- and extracellular SEAP, respectively. ( B ) Quantification of the SEAP signal in lysates and culture supernatant of cells treated like in panel A . Values are displayed as the ratio of supernatant/lysate and normalized to those of DMSO-treated cells. N = 5 biological replicates. ( C ) Extracellular phosphatase activity released from cells treated like in panel A . Values were normalized to those of DMSO-treated cells. N = 4 biological replicates. ( D ) Intracellular phosphatase activity normalized to SEAP abundance determined by western blot of cell lysates obtained like in panel A . The ratio was normalized to values obtained for DMSO-treated cells. N = 4 biological replicates. ( E ) A549 cells expressing SEAP were transfected with KPNB1-targeting or NT siRNAs. Forty-eight hours post-transfection, the medium was changed for 48 h, and cell lysate and culture supernatant were harvested 48 h later. Samples were analyzed by western blot using a SEAP-specific antibody. ( F ) Quantification of SEAP signals in western blots as shown in panel E . N = 7 biological replicates. ( G ) Quantification of SEAP activities as in panel C . N = 8 biological replicates. ( H ) SEAP activity normalized to total intracellular SEAP amounts. Values in panels F – G were normalized to those obtained with siNT-transfected cells. N = 7 biological replicates. ( I ) Lysates of mock vs DENV-infected A549 cells were analyzed by western blot after sample loading under reducing or non-reducing conditions and detection of NS1 using conformation-specific DN3 antibody. ( J ) Lysates of A549 cells transfected with given siRNAs for 48 h and infected with DENV for 48 h (left panel) or infected with DENV and treated with IVM (1 µM) or DMSO for 48 h (right panel) were analyzed by NS1-specific western blot after sample loading under reducing or non-reducing conditions. For quantification, NS1 signal was normalized to GAPDH and the ratio of non-reducing over reducing sample signal was calculated. Values were normalized to those obtained with siNT-transfected or IVM-treated cells. Data are represented as mean ± SEM of three or six independent experiments. ( K ) NS1 in lysate (intra) and culture supernatant (extra) of mock and DENV-infected A549 cells. ( L ) Lysate (intra) and supernatant (extra) of DENV-infected A549 cells were treated with endoH or PNGase prior to NS1-specific western blot analysis. ( M ) Lysates of A549 cells transfected with given siRNAs for 48 h and infected with DENV for 48 h (top panel) or infected with DENV and treated with IVM (1 µM) or DMSO for 48 h (bottom panel) were analyzed by NS1-specific western blot. Empty and filled arrowheads point to mature and immature forms of NS1, respectively. Quantification of seven or eight independent biological replicates is shown on the right. Data are represented as mean ± SEM. Each dot in the graphs corresponds to the value of an individual experiment. In all graphs, statistical significance was determined by one-sample t -test or ratio-paired t -test for panel M. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For activity measurement, cell lysates and supernatants were analyzed using the colorimetric Secreted Alkaline Phosphatase Reporter Assay Kit (Novus Biologicals) according to the manufacturer’s instructions.

Techniques: Activity Assay, Expressing, Control, Western Blot, Transfection, Infection

Journal: mBio

Article Title: Dengue virus NS1 secretion is regulated via importin-subunit β1 controlling expression of the chaperone GRp78 and targeted by the clinical drug ivermectin

doi: 10.1128/mbio.01441-23

Figure Lengend Snippet: Key resource table ^a

Article Snippet: For activity measurement, cell lysates and supernatants were analyzed using the colorimetric Secreted Alkaline Phosphatase Reporter Assay Kit (Novus Biologicals) according to the manufacturer’s instructions.

Techniques: Virus, Recombinant, Cell Viability Assay, Extraction, Reverse Transcription, SYBR Green Assay, Reporter Assay, Negative Control, Software

Journal: Cancer Science

Article Title: Hippo pathway monomerizes STAT3 to regulate prostate cancer growth

doi: 10.1111/cas.15463

Figure Lengend Snippet: MST2 impairs IL6‐induced STAT3 activation at high cell density. A‐F, Immunoblotting analyses were performed using indicated antibodies. Immunoprecipitation with anti‐STAT3 or anti‐YAP1 antibody was performed. STAT3 transcription activity was measured. Data represent the mean and SD from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. A, DU145 and LNCaP cells at high (~80%) or low (~10%) density were treated with 50 ng/ml IL6 for 30 min. B, DU145 and LNCaP cells at high (~80%) density were treated with 5 μM XMU‐MP‐1 for 2 h, and then treated with 50 ng/ml IL6 for 30 min. C, DU145 cells with expression of MST2 shRNAs at high (~80%) density were treated with 50 ng/ml IL6 for 30 min. D, DU145 cells with expression of LATS1/2 shRNAs at high (~80%) density were treated with 50 ng/ml IL6 for 30 min. E, DU145 cells with expression of MST1 shRNA at high (~80%) density were treated with 50 ng/ml IL6 for 30 min. F, DU145 cells with expression of YAP1 and TAZ at high (~80%) density were treated with 50 ng/ml IL6 for 30 min

Article Snippet: STAT3 transcription activity assay was performed by using a STAT3 reporter kit obtained from BPS Bioscience (#79730), following the manufacturer's instructions.

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Activity Assay, Expressing, shRNA

Journal: Cancer Science

Article Title: Hippo pathway monomerizes STAT3 to regulate prostate cancer growth

doi: 10.1111/cas.15463

Figure Lengend Snippet: MST2 phosphorylates STAT3 at T622. A, B, D‐G, Immunoblotting analyses were performed using indicated antibodies. A, Purified GST‐STAT3 protein was incubated with purified WT His‐MST2 or His‐MST2 K56R proteins for an in vitro kinase assay. A GST pulldown assay was performed. B, Purified WT GST‐STAT3 or indicated mutant protein was incubated with purified WT His‐MST2 for an in vitro kinase assay. A GST pulldown assay was performed. C, Alignment analyses of STAT3 T622 among indicated species were performed. D, DU145 cells with expression of Flag‐STAT3 at high (H, ~80%) or low (L, ~10%) density were harvested and lysed. Immunoprecipitation was performed using an anti‐Flag antibody. Immunoblotting analysis was performed in the presence or absence of the blocking peptide. E, DU145 cells with expression of Flag‐STAT3, MST2 shRNA, or SAV1 shRNA at high (H, ~80%) or low (L, ~10%) density were harvested and lysed. Immunoprecipitation was performed using an anti‐Flag antibody. F, DU145 cells with expression of WT Flag‐STAT3 or Flag‐STAT3 T622A at high (H, ~80%) or low (L, ~10%) density were harvested and lysed. Immunoprecipitation was performed using an anti‐Flag antibody. G, Purified WT GST‐STAT3 or GST‐STAT3 T622A protein was incubated with purified WT His‐MST2 proteins for an in vitro kinase assay

Article Snippet: STAT3 transcription activity assay was performed by using a STAT3 reporter kit obtained from BPS Bioscience (#79730), following the manufacturer's instructions.

Techniques: Western Blot, Purification, Incubation, In Vitro, Kinase Assay, GST Pulldown Assay, Mutagenesis, Expressing, Immunoprecipitation, Blocking Assay, shRNA

Journal: Cancer Science

Article Title: Hippo pathway monomerizes STAT3 to regulate prostate cancer growth

doi: 10.1111/cas.15463

Figure Lengend Snippet: MST2‐dependent STAT3 T622 phosphorylation impairs STAT3 dimerization and activation. C‐H, Immunoblotting analyses were performed using indicated antibodies. A, A schematic of the SH2 domain in STAT3 protein sequence. B, Human STAT3 protein structure (PDB code: 6TLC) shows the spatial location of the T622 site. The SH2 domain is boxed and enlarged. T622 site is shown in yellow. C, DU145 cells with expression of HA‐STAT3 and Flag‐STAT3 at high (~80%) or low (~10%) density were treated with 50 ng/ml IL6 for 30 min. Immunoprecipitation was performed using an anti‐Flag antibody. D, DU145 cells with expression of HA‐STAT3, Flag‐STAT3 or MST2 shRNA at high (~80%) density were treated with 50 ng/ml IL6 for 30 min. Immunoprecipitation was performed using an anti‐Flag antibody. E, DU145 cells with expression of WT HA‐STAT3, WT Flag‐STAT3, or indicated mutants at high (~80%) density were treated with 50 ng/ml IL6 for 30 min. Immunoprecipitation was performed using an anti‐Flag antibody. F, Purified GST‐STAT3, Flag‐STAT3, or indicated mutant protein was incubated with purified His‐MST2 protein for an in vitro kinase assay and then incubated with His‐JAK2 protein for another in vitro kinase assay. Immunoprecipitation was performed using an anti‐Flag antibody. G, Purified Flag‐STAT3 or indicated mutant protein was incubated with purified His‐MST2 protein for an in vitro kinase assay and then incubated with His‐JAK2 protein for another in vitro kinase assay. Size exclusion chromatography assay was performed. H, Endogenous STAT3‐depleted DU145 cells were stably expressed with WT Flag‐STAT3 and Flag‐STAT3 T622A. STAT3 shRNA targets noncoding region. I‐K, Endogenous STAT3‐depleted DU145 cells with expression of WT Flag‐STAT3 or indicated mutant at high (~80%) density were treated with 50 ng/ml IL6 for 30 min (I‐J) or 2 h (K). DNA‐binding affinity (I) and transcription activity (J) of STAT3 were measured. The level of IL6 mRNA was measured by RT‐PCR (K). L, M, DU145 cells with expression of MST2 shRNA at high (~80%) density were treated with 50 ng/ml IL6 for 30 min (L) or 2 h (M). DNA‐binding affinity of STAT3 was measured (L). The level of IL6 mRNA was measured by RT‐PCR (M)

Article Snippet: STAT3 transcription activity assay was performed by using a STAT3 reporter kit obtained from BPS Bioscience (#79730), following the manufacturer's instructions.

Techniques: Activation Assay, Western Blot, Sequencing, Expressing, Immunoprecipitation, shRNA, Purification, Mutagenesis, Incubation, In Vitro, Kinase Assay, Size-exclusion Chromatography, Stable Transfection, Binding Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Science

Article Title: Hippo pathway monomerizes STAT3 to regulate prostate cancer growth

doi: 10.1111/cas.15463

Figure Lengend Snippet: STAT3 T622 phosphorylation promotes prostate cancer growth and predicts patient outcome. A, B, Endogenous STAT3‐depleted DU145 cells with stable expression of WT Flag‐STAT3 or indicated mutant were subcutaneously injected in mice. The images of two representative xenografts from each group are shown (A). The volume of mice tumor xenografts ( n = 7) was measured at indicated time points after injection (B). Data represent the mean and SD. *** p < 0.001. C, The specificity of the anti‐STAT3 pT622 antibody was measured by immunohistochemistry (IHC) staining using phosphorylated or nonphosphorylated blocking peptide. Scale bar, 80 μm. D, E, IHC staining using indicated antibodies was performed using mouse tumor samples (D, scale bar, 80 μm) or human prostate cancer samples (E, scale bar, 50 μm). F, The IHC staining scores were analyzed by linear regression. G, Kaplan‐Meier plots of the overall survival time of prostate cancer patients ( n = 55) with high or low STAT3 T622 phosphorylation or MST2 expression were generated. P value was calculated using the log‐rank test

Article Snippet: STAT3 transcription activity assay was performed by using a STAT3 reporter kit obtained from BPS Bioscience (#79730), following the manufacturer's instructions.

Techniques: Expressing, Mutagenesis, Injection, Immunohistochemistry, Blocking Assay, Generated

Journal: Oncogene

Article Title: Dichotomous roles of ACBD3 in NSCLC growth and metastasis

doi: 10.1038/s41388-025-03360-w

Figure Lengend Snippet: A Boyden chamber transwell migration and invasion assays in H1299 cells transfected with EGFP-ACBD3 or EGFP-ACBD3 lacking the NUMB-binding domain (ΔNUMB). B Correlation between ACBD3, NUMB, NOTCHs, or NOTCH target genes mRNA levels and EMT scores. C Schema: The NOTCH luciferase reporter. CBF1/RBPJκ/Suppressor of Hairless/Lag-1 (CSL) response element (RE) is fused upstream of the firefly luciferase gene. NICD Notch intracellular domain, Graph luciferase reporter assay in H441 cells co-transfected with luciferase reporter and shACBD3 or NOTCH1 NICD. n = 4. D WB analysis of ACBD3 and EGFP-ACBD3 proteins in H1299 cells transfectants. E NOTCH luciferase reporter assay. F , G , I qPCR analysis of mRNA levels of ACBD3 and NOTCH target genes in shACBD3-tranfected H441 ( F ) and A549 ( G ), and ACBD3-transfected H1299 ( I ) cells. n = 4. H , J , K WB analysis of SNAI1 and ACBD3 protein levels in lung cancer cells transfected with shACBD3 ( H ), ACBD3 expression vector ( J ), or shACBD3 and SNAI1 siRNA ( K ). L , M Transwell migration ( L ) and apoptosis ( M ) assays of cell transfectants in ( K ). N Working model: ACDB3 inhibits the NOTCH-SNAI1 axis to suppress cancer cell motility and metastasis. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. P -values were determined using two-tailed Student’s t-test (for F , G , I ), one-way ANOVA test (for A , E , L , M ).

Article Snippet: NOTCH activity was assessed using the Notch1 Pathway Reporter Kit (#79503, BPS Bioscience) following the manufacturer’s instructions.

Techniques: Migration, Transfection, Binding Assay, Luciferase, Reporter Assay, Expressing, Plasmid Preparation, Two Tailed Test