Journal: Cancers

Article Title: miR-1227 Targets SEC23A to Regulate the Shedding of Large Extracellular Vesicles

doi: 10.3390/cancers13225850

Figure Lengend Snippet: MiR-1227 induces shedding of L-EV and inhibits the shedding of S-EV in PC cells. ( A ) MiR-1227 (teal) and miR-21 (grey) expression in different biological fluids from Weber et al. 2010 . ( B ) Taqman qPCR quantification of miR-1227 in EV isolated from pooled normal or PC patient plasma ( n = 1). ( C ) Transwell migration of RWPE-2 PC and U87 glioma cells stably expressing miR-1227 (teal) or empty vector control (grey) ( n = 3) normalized to corresponding vector control; ( D ) qPCR for miR-1227 from S-EV (blue) and L-EV (red) isolated from PC3 cells stably expressing miR-1227( n = 1). ( E , F ) Digital droplet qPCR quantification of miR-1227 per S-EV ( E ) and L-EV ( F ) ( n = 3). ( G , H ) Atomic force microscopy quantifications of S-EV ( G ) and L-EV ( H ) shed from PC3 cells stably expressing miR-1227 ( n = 3). ( I – L ) Quantification of S-EV ( I ) and L-EV ( J ) shed from PC3 cells stably expressing miR-1227 by TRPS with size distribution plots in ( K , L ), respectively ( n = 3); * p < 0.05.

Article Snippet: PC3 (prostate cancer, human), RWPE-2 (prostate cancer, human), and U87 (glioma, human) cells were purchased from ATCC.

Techniques: Expressing, Isolation, Clinical Proteomics, Migration, Stable Transfection, Plasmid Preparation, Control, Microscopy

Journal: Cancers

Article Title: miR-1227 Targets SEC23A to Regulate the Shedding of Large Extracellular Vesicles

doi: 10.3390/cancers13225850

Figure Lengend Snippet: SEC23A is a direct target of miR-1227; ( A ) qPCR for high confidence miR-1227 target genes involved in vesicle formation from PC3 cells stably expressing miR-1227 ( n = 3). ( B ) Western blot validation of SEC23A reduced expression in PC3, RWPE-2, and U87 cells stably expressing miR-1227. ( C ) Prostate cancer transcriptome atlas analysis of SEC23A in prostate cancer, showing reduced expression levels of SEC23A in cancer versus benign prostate tissue and in metastatic castration resistant versus primary prostate cancer. ( D – F ) Luciferase assays from RWPE-2 cells transfected with miR-1227 or control miRNA, plus the SEC23A 3′UTR luciferase construct ( D ) or the SEC23A 3′UTR luciferase construct with the deletion of 2 different predicted miR-1227 binding sites ( E , F ) ( n = 3); * p < 0.05; ** p < 0.005.

Article Snippet: PC3 (prostate cancer, human), RWPE-2 (prostate cancer, human), and U87 (glioma, human) cells were purchased from ATCC.

Techniques: Stable Transfection, Expressing, Western Blot, Biomarker Discovery, Luciferase, Transfection, Control, Construct, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Correlation between CPN1 and RAI14. (A) Co-immunoprecipitation (Co-IP) in 293T cells with CPN1 overexpression. (B) A total of 29 differential proteins with more than identified five peptides in immunoprecipitation. (C) Validation of CPN1 interacting with RAI14.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Over Expression, Biomarker Discovery

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: High expression of RAI14 in breast cancer. (A) Expression levels of human RAI14 in different tumor types from TCGA database. (B) The mRNA expression of RAI14 in breast cancer and normal tissues from GEPIA. (C) The protein expression of RAI14 in breast cancer and normal tissues from UALCAN database. (D) High expression of RAI14 in human breast cancer from Human Protein Atlas (HPA) database. * p < 0.05, *** p < 0.001.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Correlation analysis of RAI14 expression levels with pathological type parameters of breast cancer. (A) The mRNA levels of RAI14 in mammary carcinoma with different molecular types. (B) Protein levels of RAI14 in mammary carcinoma with different molecular types. (C) Expression of RAI14 in different mammary carcinoma stages. (D) RAI14 levels in different menopausal states. * p < 0.05, *** p < 0.001.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Relationship between mRNA expression of RAI14 and clinicopathological parameters of breast cancer.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing, Immunohistochemistry, Sequencing

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Differential expression of RAI14 in mammary carcinoma specimens with different clinicopathological parameters from BC-GenExMiner v4.4 data analysis. (A–C) Receptor status (ER + vs. ER-, PR + vs. PR-, HER2+ vs. HER2-). (D) The differential expression of RAI14 in the TNBC and non-TNBC. (E–I) The differential expression of (E) wild-type and mutant, (F) age (≤51 and >51 years), (G) molecular types, (H) clinical tumor stage (I, II, III, IV), and (I) clinicopathological type (IDC, ILC).

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Quantitative Proteomics, Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Kaplan–Meier survival curves of RAI14 in different breast cancer molecular subtypes. (A, B) Survival curves for overall survival (OS) and recurrence-free survival (RFS) in triple-negative breast cancer (n = 1494). (C, D) Survival curves for OS and RFS in luminal B breast cancer (n = 2015). (E, F) Survival curves for OS and RFS in luminal A breast cancer (n = 3511). (G, H) Survival curves for OS and RFS in HER2+ breast cancer for OS and RFS (n = 515).

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Correlation analysis between RAI14 and related genes and markers of immune cells in breast cancer.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Correlation of RAI14 levels with immune infiltration in TNBC. (A) The expression level of RAI14 was positively correlated with programmed cell death-ligand 1 (CD274) and programmed cell death protein 1(PDCD1). X-axis represents the expression level of immune cell gene markers, and Y-axis represents RAI14. Log2TPM that indicates gene expression level. (B) The expression of immunohistochemical (IHC)staining of CPN1, RAI14, and PDL1 in patients with TNBC tissues. (C) Correlation among IHC scores of CPN1, RAI14, and PDL1.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: In TNBC cell lines, CPN1 positively correlated with RAI14 and PDL1 expression. (A) Expression levels of CPN1, RAI14, and PDL1 in breast cancer cell lines. (B) Increased expression of RAI14 and PDL1 in MDA-MB-231 cell lines with overexpression of CPN1. (C) Our protocol summarized in a schematic diagram.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing, Over Expression

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Detailed view of blocking loop 2 (BL2), which occludes the active site of USP14 (PDB access code 2AYN). The BL2 loop, which contains Ser432, is shown in stick model, in the apo form. ( B ) Combined ribbon representation and stick model showing a comparison of the conformations of the BL2 loop contained in the apo form (blue, PDB access code 2AYN) and in the USP14- Ub-aldehyde (Ubal) adduct (orange, PDB access code 2AYO). In this drawing, the Ser432 and Cys114 residues are shown in stick model, and the bound Ubal (a ubiquitin derivative in which the C-terminal carboxylate is replaced by an aldehyde) in the complex is drawn in green. ( C ) A surface charge potential representation (contoured at ± 7 kT/eV; blue/red) of USP14 (PDB accession 2AYN) showing that the S432 residue is very close to a highly negatively charged patch mainly formed by the acidic E188, D199, and E202 residues. When S432 is phosphorylated, the negatively charged phosphate group may induce a repulsive force, thereby relieving inhibition of the catalytic activity of USP14. ( D ) USP14 domain organization and sequence alignment of the Akt phosphorylation site within USP14 orthologs from different species. Two BLs (BL1 and BL2) covering the USP14 active site are shown. The Akt phosphorylation site in USP14 from different species as predicted by Scansite. ( E ) S432 is the major phosphorylation site in USP14. HEK293T cells were treated as in , followed by electrospray ionization mass spectrometry (ESI-MS) analysis. Spectral counts were determined by ESI-MS. ( F ) Akt phosphorylates USP14 in vitro. Bacterially expressed and purified USP14 was incubated with active Akt in the presence of ATP. Reaction products were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and phosphorylated species were detected by a phospho-Ser antibody. DOI: http://dx.doi.org/10.7554/eLife.10510.003

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Blocking Assay, Comparison, Ubiquitin Proteomics, Residue, Inhibition, Activity Assay, Sequencing, Phospho-proteomics, Mass Spectrometry, In Vitro, Purification, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Akt interacts with USP14. HEK293T cells were transfected with indicated plasmids for 24 hr. The cell lysates were collected for co-immunoprecipitation and western blotting analysis. ( B ) Schematic representation of mass spectrometry assay to determine USP14 phosphorylation sites by Akt. ( C ) Four phosphorylation sites of USP14 were determined by mass spectrometry. ( D ) The representative MS/MS spectrum of phosphorylated tryptic peptide ‘SSSphosSGHYVSWVK’ of human USP14 protein. The peptide sequence ‘SSSphosSGHYVSWVK’ containing phosphorylated S432 was identified by shotgun analysis using mass spectrometry when USP14 was coexpressed with Myr-Akt in HEK293T cells. Fragmentation ion of the amide bond of the peptide result in formation of “b” ion and “y” ion series corresponding to the N- and C-terminal fragments respectively. Representative ions with phosphorylation and H 2 O loss were manually labeled in red on the spectrum. DOI: http://dx.doi.org/10.7554/eLife.10510.004

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Transfection, Immunoprecipitation, Western Blot, Mass Spectrometry, Phospho-proteomics, Tandem Mass Spectroscopy, Sequencing, Labeling

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) In vitro phosphorylation of USP14 at S432 by Akt. Bacterially expressed and purified wild type USP14 or AA mutant incubated with active Akt in the presence of ATP. Reaction products were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and phosphorylation was detected by the phospho-Ser antibody. ( B ) Akt phosphorylates USP14 at S432 in vivo. Western blot analysis of whole cell lysate and immunoprecipitates derived from HEK293T cells transfected with wild type USP14, USP14 S143A, USP14 S432A, and USP14 S143A/S432A (AA) constructs using the phospho-Ser antibody. L.E., long exposure. ( C ) Immunoprecipitation (IP) and IB analysis of HEK293T cells transfected with HA-USP14 and Myr-Akt and preincubated with or without λ-phosphatase as indicated. ( D ) Inhibition of Akt decreased exogenous USP14 phosphorylation. HEK293T cells were transfected with Myc-USP14 for 20 hr then treated with 1 μM MK2206 or deprived of serum for another 4 hr before harvest. ( E ) In vitro kinase assay to detect Akt phosphorylation of USP14 by phospho-Ser432-specific antibody and phos-tag-containing gels. Bacterially expressed and purified wild type USP14 or S432A mutant was incubated with active Akt in the presence of ATP. The reaction products were resolved by SDS-PAGE, and USP14 phosphorylation was detected using an antibody that specifically recognizes Ser432 phosphorylation of USP14 or determined by differential migration on phos-tag gels. ( F ) In vivo detection of endogenous USP14 Ser432 phosphorylation by anti-p-Ser432-specific antibody. Western blot analysis of immunoprecipitates derived from H4 cells transfected with or without Myr-Akt plasmids using the anti-p-Ser432-specific antibody. ( G, H ) Phosphorylation of endogenous USP14 S432 upon stimulation with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). HEK293T cells were serum-starved and pretreated with Akt inhibitor MK2206 (1 μM) for 30 min before stimulation with IGF-1 (100 ng/mL) for 30 min ( G ) or EGF (100 ng/mL) for 1 hr ( H ). The cell lysates were immunoprecipitated with USP14 antibody and western-blotted with anti-p-S432 antibody. ( I ) Phosphorylation of endogenous USP14 S432 in Pten knockout cells with high activity of Akt. Lysates from mouse embryonic fibroblasts (MEFs) with indicated genotypes were immunoprecipitated with USP14 antibody and then Western blotted with p-S432 antibody. The differential migration of phospho-USP14 on phos-tag-containing gels was determined as shown in the bottom panel. DOI: http://dx.doi.org/10.7554/eLife.10510.005

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: In Vitro, Phospho-proteomics, Purification, Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, In Vivo, Western Blot, Derivative Assay, Transfection, Construct, Immunoprecipitation, Inhibition, Kinase Assay, Migration, Knock-Out, Activity Assay

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Akt phosphorylates USP14 at S432 in vivo. Western blotting analysis of whole cell lysate and immunoprecipitates derived from HEK293T cells transfected with wild type USP14, USP14 S143A, USP14 S432A, and USP14 S143A/S432A (AA) constructs using an Akt phosphorylation-consensus motif (R××S/T) antibody. (B, C) Inhibition of Akt decreases USP14 S432 phosphorylation levels. H4 cells were treated with different concentration of Akt inhibitors MK2206 ( B ) or AZD5363 ( C ) as indicated for 4 hr. The cell lysates were collected for immunoprecipitation and western blotting analysis. ( D ) Inhibition of phosphoinositide 3-kinases (PI3K) decreases USP14 S432 phosphorylation levels. H4 cells were treated with different concentration of PI3K inhibitors GDC0941 or Wortmannin as indicated for 4 hr. The cell lysates were collected for immunoprecipitation and western blotting analysis. ( E ) ERK1/2 inhibition has no effect on USP14 S432 phosphorylation. H4 cells were treated with different concentration of ERK1/2 inhibitor U0126 as indicated for 4 hr. The cell lysates were collected for immunoprecipitation and western blotting analysis. ( F ) Pten –/– mouse embryonic fibroblast (MEF) cells were treated with 1 μM Akt inhibitors for 4 hr, then cell lysates were collected for immunoprecipitation and western blotting analysis. DOI: http://dx.doi.org/10.7554/eLife.10510.006

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: In Vivo, Western Blot, Derivative Assay, Transfection, Construct, Phospho-proteomics, Inhibition, Concentration Assay, Immunoprecipitation

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A, B ) A representative curve of USP14 recombinant protein purification before ( A ) and after ( B ) cleavage by 3C protease on size-exclusion chromatography. Bacterially expressed USP14 forms oligomer and dimer, only monomer was used for further experiments. Trx-tag was removed to get tag-free USP14 protein for in vitro kinase assay or Ub-AMC and ubiquitin cleavage assay. ( C ) One dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining of purified USP14 protein before and after cleavage by 3C protease. DOI: http://dx.doi.org/10.7554/eLife.10510.008

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Recombinant, Protein Purification, Size-exclusion Chromatography, In Vitro, Kinase Assay, Ubiquitin Proteomics, Cleavage Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Purification

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Akt activates USP14 DUB activity in vitro. USP14 protein (1 μg) was incubated with or without active Akt (1 μg) in kinase assay buffer in a total volume of 50 μL for 1 hr at 30 ° C, then the reaction mixtures were subjected to Ub-AMC assay. RFU, relative fluorescence units. (B, C) Akt activates USP14 in cells. USP14 was immunoprecipitated from HEK293T cells coexpressed with activated Akt ( B ) or treated with 10 μM MK2206 for 4 hr ( C ) and then eluted with HA-peptide following Ub-AMC hydrolysis assay. ( D ) Activation of USP14 by stimulating cells with IGF-1. HEK293T cells were serum-starved and pretreated with or without Akt inhibitor MK2206 (1 μM) for 30 min before stimulation with IGF-1 (100 ng/mL) for 30 min. USP14 was then immunoprecipitated and eluted with HA-peptide. The activity of USP14 was determined using Ub-AMC hydrolysis assay. ( E ) USP14 activation by Akt is blocked by S432A mutation. Ub-AMC hydrolysis assay of wildde type USP14 or S432A mutant in the presence or absence of active Akt. ( F ) Ub-AMC hydrolysis assay of bacterially expressed and purified wild type USP14 or S432E mutant. ( G ) Lineweaver–Burk analysis of USP14 S432E, obtained by measuring the initial rates at varying Ub-AMC concentrations (see for reference). ( H ) The activity of phospho-mimetic USP14 mutant can be further stimulated by the presence of proteasome. Ub-AMC hydrolysis assay of wild type USP14 or S432E mutant in the presence or absence of Ub-VS-treated human proteasome (VS-proteasome (see ); 1 nM). Ptsm, 26S proteasome. DOI: http://dx.doi.org/10.7554/eLife.10510.007

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Activity Assay, In Vitro, Incubation, Kinase Assay, Ub-AMC Assay, Fluorescence, Immunoprecipitation, Hydrolysis Assay, Activation Assay, Mutagenesis, Purification

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Validation of Ub-AMC assay on immunoprecipitated USP14 from HEK293T cells. Wild type or inactive mutant USP14 was immunoprecipitated from transfected HEK293T cells and then eluted with HA-peptide following Ub-AMC hydrolysis assay. ( B–D ) Ser143 phosphorylation has limited effect on USP14 activity. S143A mutant had similar levels of hydrolyzing activity as that of WT USP14 in the presence of active Akt ( B ). Double E mutant (S143E/S432E) showed similar levels of hydrolyzing activity as that of S432E single mutant ( C ). S143E mutant has minor effect on USP14 activity compared with S432E mutant ( D ). ( E ) Linear kinetics (R 2 > 0.99) of Ub-AMC hydrolysis. ( F ) Distribution of total and phosphorylated USP14 specific in glycerol gradient centrifugation. Soluble lysates of Pten –/– mouse embryonic fibroblast (MEF) cells, prepared as described in Materials and methods, were subjected to glycerol density gradient centrifugation. Gradient fractions were collected and subjected to western blotting with the indicated antibodies. Anti-RPN11 was used as a control for proteasome. DOI: http://dx.doi.org/10.7554/eLife.10510.009

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Biomarker Discovery, Ub-AMC Assay, Immunoprecipitation, Mutagenesis, Transfection, Hydrolysis Assay, Phospho-proteomics, Activity Assay, Gradient Centrifugation, Western Blot, Control

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Dimeric Ub cleavage assay. USP14 S432E cleavage of Lys 48 and Lys 63 Ub chain linkages was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). ( B ) Cleavage of Lys 48, Lys 63 and linear dimeric Ub chain types in the presence of USP14 S432E was measured over time and analyzed using SDS-PAGE. Quantification of the amount of dimer remaining from the data was shown below. ( C ) Dimeric Ub cleavage analysis of immunoprecipitates derived from HEK293T cells transfected with wild type HA-USP14 or S432A mutant plasmids. DOI: http://dx.doi.org/10.7554/eLife.10510.010

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Cleavage Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Derivative Assay, Transfection, Mutagenesis

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) USP14 S432E does not cleave linear di-Ub. USP14 S432E cleavage of linear dimeric Ub chain types and analyzed using SDS-PAGE. Cleavage of Lys48 dimeric Ub chain types was used as a positive control. ( B, C ) USP14 immunoprecipitated from transfected HEK293T cells and eluted with HA-peptide has high deubiquitination activity towards both K48 and K63 di-Ub but not linear di-Ub. DOI: http://dx.doi.org/10.7554/eLife.10510.011

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: SDS Page, Positive Control, Immunoprecipitation, Transfection, Activity Assay

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Inhibition of Akt promotes UPS function. H4-GFP-CL1 cells were treated with different concentrations of MK2206 as indicated for 4 hr. The cells were then harvested and subjected to western blotting analysis using indicated antibodies. ( B ) H4-GFP-CL1 cells were treated as in ( A ) and . Images of the cells were collected using an ArrayScan HCS 4.0 Reader. The average GFP intensity in 2,000 cells from each indicated sample was determined. Data are displayed as mean ± SD of the GFP intensity per cell. ** p <0.01, *** p <0.001 ( C ) Inhibition of Akt or phosphoinositide 3-kinase (PI3K) promotes UPS function. H4-GFP-CL1 cells were treated with Akt inhibitor AZD5363 (1 μM) or PI3K inhibitor GDC0941 (1 μM) as indicated for 4 hr. The cells were then harvested and subjected to western blotting analysis using indicated antibodies. p-GSK3β(S9) was blotted to indicate the inhibition of Akt. ( D ) Activation of Akt inhibits UPS. H4-GFP-CL1 cells were transfected with Myr-Akt for 24 hr. The cells were then harvested and subjected to western blotting analysis using indicated antibodies. ( E ) IGF-1 stimulation inhibits UPS function. H4-GFP-CL1 cells were serum-starved and pretreated with Akt inhibitor MK2206 (1 μM) for 30 min before stimulating with IGF-1 (100 ng/mL) for 30 min. The cells were then imaged and quantified as in (B). ( F ) H4-GFP-CL1 cells were treated as in (D) and . Then cells were imaged and quantified as in (B). ( G ) EGF stimulation inhibits UPS function. H4-GFP-CL1 cells were serum-starved and pretreated with Akt inhibitor MK2206 (1 μM) for 30 min before stimulation with EGF (100 ng/mL) for 1 h. The cells were then harvested and subjected to western blotting analysis using indicated antibodies. ( H ) Akt regulates UPS function through USP14. Myr-Akt was transfected into either wild type or Usp14 –/– H4 cells stably expressing GFP-CL1 for 24 hr. The cells were then harvested and subjected to western blotting analysis using indicated antibodies. ( I ) Akt regulates UPS function through phosphorylation of USP14. Myr-Akt was transfected into either wild type USP14 or USP14 AA reconstitution cell lines stably expressing GFP-CL1 for 24 hr. The cells were then imaged and quantified as in (B). DOI: http://dx.doi.org/10.7554/eLife.10510.012

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Inhibition, Western Blot, Activation Assay, Transfection, Stable Transfection, Expressing, Phospho-proteomics

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: (A, B, C, D) Generation of USP14 knockout H4 cell line using CRISPR/Cas9 system. A schematic of the Cas9/sgRNA/oligo targeting site in the exon2 of Usp14 . The sgRNA coding sequence is underlined and labeled in red. The protospacer-adjacent motif (PAM) sequence is underlined ( A ). The deleted sequences in the Usp14 –/– cell lines are presented. The number of sequences analyzed is indicated right ( B ). Sequencing analysis of Usp14 –/– cell lines. The arrow indicates the missing sequences (TCAAG) ( C ). Western blotting analysis of USP14 expression in WT cells and Usp14 –/– cells ( D ). ( E ) Akt regulates UPS function through phosphorylation of USP14. An expression vector for Myr-Akt was transfected into either wild type USP14 or USP14 AA reconstitution cell lines stably expressing GFP-CL1 and incubated for 24 hr. Then the cell lysates were harvested and subjected to western blotting analysis using indicated antibodies. ( F ) GFP-cODC assay was verified. H4-GFP-cODC cells were treated with 10 μM MG132 for 4 hr, and then the cell lysates were harvested and subjected to western blotting analysis using indicated antibodies. ( G ) H4-GFP-cODC cells were treated with 10 μM MK2206 for 4 hr or transfected with an expression vector for Myr-Akt as indicated for 24 hr. Then the cell lysates were harvested and subjected to western blotting analysis using indicated antibodies. DOI: http://dx.doi.org/10.7554/eLife.10510.014

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Knock-Out, CRISPR, Sequencing, Labeling, Western Blot, Expressing, Phospho-proteomics, Plasmid Preparation, Transfection, Stable Transfection, Incubation

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: The quantitative analysis of proteome change in USP14 knockout or USP14 mutant cells were performed by tandem mass tag (TMT)-isobaric labeling followed by shotgun analysis. The heat map was plotted based on the set of 87 proteins that are down-regulated greater than or equal to 1.2-fold in H4 KO cells compared to H4 WT cells or to H4 KO cells complemented with WT USP14 (KO-WT). The log base 2 of average ratios was plotted as indicated. DOI: http://dx.doi.org/10.7554/eLife.10510.015

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Knock-Out, Mutagenesis, Labeling

Journal: bioRxiv

Article Title: S100A8/A9 predicts triple-negative breast cancer response to PIM kinase and PD-1/PD-L1 inhibition

doi: 10.1101/2023.09.21.558870

Figure Lengend Snippet: (a) Schematic representation of the bioinformatics methods used in this study. FDR: false discovery rate, LOOCV: leave one out cross-validation. ( b, c ) Kaplan-Meier graphs of patients with TNBC (b) or all BC clinical subtypes (c), dichotomized by S100A8 and - A9 mRNA expression at an optimal threshold, from the indicated cohorts. Samples with elevated S100A8 and - A9 expression levels are represented by red lines. The log-rank test was used to calculate p- values. The shaded areas represent the 95% confidence interval. ( d ) S100A8 and - A9 mRNA expression in primary breast tumor samples from the indicated cohorts, stratified by receptor status. The four datasets used include the three datasets described in and Supplementary Fig. 1a, plus the TCGA Breast dataset. Each box representing a patient group has a number that indicates the sample size. Box plots show the 25 th , 50 th (median), and 75 th percentiles. A pairwise two-tailed t -test between the respective groups was used to calculate p- values. NS: not significant.

Article Snippet: Real-time PCR reactions were run on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) paired with the CFX Manager software (Bio-Rad) using TaqMan probes (Thermo Fisher) specific for the following genes: human S100A8 (Hs00374264_g1), human S100A9 (Hs00610058_m1), mouse S100A8 (Mm00496696_g1), and mouse S100A9 (Mm00656925_m1).

Techniques: Biomarker Discovery, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: S100A8/A9 predicts triple-negative breast cancer response to PIM kinase and PD-1/PD-L1 inhibition

doi: 10.1101/2023.09.21.558870

Figure Lengend Snippet: (a) Representative western blots showing protein expression levels of S100A8 and -A9 in the indicated human TNBC cell lines and non-tumorigenic human mammary epithelial cells (HMEC). (b) Schematic representation of the ELISA screen performed in this study. MDA-MB-468 cells were treated with 30 targeted anticancer agents at their respective IC50 values or 10 µM, whichever was lower. (c) Results from the ELISA screen described in (n = 3). (d) Representative western blots showing the effects of the indicated pan-PIM kinase inhibitors on S100A8 expression in the indicated TNBC cell lines (72 h). (e) Representative western blots showing expression of PIM1, -2, -3, S100A8, and -A9 in the cells transiently transfected with Cas9 proteins and the indicated PIM -specific, synthetic multi-guide sgRNA or a nonspecific control sgRNA. The resulting cells represent heterogenous KO pools and not single-cell clones. (f) Relative S100A8/A9 secretion (72 h) in Cas9/ PIM -specific, sgRNA-transfected MDA-MB-468 cells shown in (n = 3). (g) Relative mRNA expression of S100A8 (left) and - A9 (right), as determined by qPCR, in Cas9/ PIM -specific, sgRNA-transfected MDA-MB-468 cells shown in (n = 3). (h) Representative western blots showing the effects of exogenously introduced PIM1 on S100A8 and -A9 expression in the cells transiently transfected with Cas9/ PIM1,2,3 -specific sgRNAs. (i) The effects of exogenously introduced PIM1 on relative S100A8/A9 secretion in MDA-MB-468 cells transiently transfected with Cas9/ PIM1,2,3 -specific sgRNAs (n = 3). (j) The effects of exogenously introduced PIM1 on relative mRNA expression of S100A8 (left) and - A9 (right), as determined by qPCR, in MDA-MB-468 cells transiently transfected with Cas9/ PIM1,2,3 -specific sgRNAs (n = 3). Throughout this figure, actin serves as a loading control. Error bars represent means +/- SEM. A two-tailed t -test was used to calculate p- values; ** p < 0.01, *** p < 0.001, **** p < 0.0001. In (c), only the drugs that achieved p < 0.001 are indicated.

Article Snippet: Real-time PCR reactions were run on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) paired with the CFX Manager software (Bio-Rad) using TaqMan probes (Thermo Fisher) specific for the following genes: human S100A8 (Hs00374264_g1), human S100A9 (Hs00610058_m1), mouse S100A8 (Mm00496696_g1), and mouse S100A9 (Mm00656925_m1).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Control, Clone Assay, Two Tailed Test

Journal: bioRxiv

Article Title: S100A8/A9 predicts triple-negative breast cancer response to PIM kinase and PD-1/PD-L1 inhibition

doi: 10.1101/2023.09.21.558870

Figure Lengend Snippet: (a) Representative western blots showing S100A8 and -A9 expression in the cells treated with the indicated siRNAs. Two siRNA sequences per gene were used as indicated. Actin serves as a loading control. (b) Representative western blots showing the effects of cycloheximide on the protein abundance of S100A8 and MYC. MYC serves as a positive control for protein degradation. Actin serves as a loading control. The numbers in red indicate relative protein expression. (c) Gene co-expression analysis of S100A8 or - A9 and the indicated genes in the TCGA breast (n = 817) and breast cancer METABRIC (n = 2,509) datasets.

Article Snippet: Real-time PCR reactions were run on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) paired with the CFX Manager software (Bio-Rad) using TaqMan probes (Thermo Fisher) specific for the following genes: human S100A8 (Hs00374264_g1), human S100A9 (Hs00610058_m1), mouse S100A8 (Mm00496696_g1), and mouse S100A9 (Mm00656925_m1).

Techniques: Western Blot, Expressing, Control, Quantitative Proteomics, Positive Control

Journal: bioRxiv

Article Title: S100A8/A9 predicts triple-negative breast cancer response to PIM kinase and PD-1/PD-L1 inhibition

doi: 10.1101/2023.09.21.558870

Figure Lengend Snippet: (a) Venn diagram showing the TFs most reproducibly identified as responsible for S100A8 and - A9 expression across four public datasets (ENCODE 2015, ChEA 2016, TF Perturbations/Expression, and TRUST TF 2019) in the Enrichr database (Supplementary Data 3). (b) Gene co-expression analysis of S100A8 or - A9 and their candidate TFs in the TCGA breast and breast cancer METABRIC datasets. (c) Representative western blots showing protein expression levels of the candidate TFs identified in and PIM1, -2, and -3 in the indicated TNBC cell lines and HMEC cells. (d) Representative western blots showing the effects of knocking down the candidate TFs identified in on S100A8 and -A9 expression in the indicated TNBC cell lines. Two siRNA sequences per gene were used as indicated. (e) Representative western blots showing the effects of GDC-0339 alone or in the presence of MG-132 (1 µM) on the levels of total or T235-phosphorylated C/EBPβ. (f) Representative western blots showing the effects of GDC-0339 on the abundance of ubiquitinated C/EBPβ. IP: immunoprecipitation; WB: western blotting. (g) Representative western blots showing the effects of GDC-0339 on the levels of total or S727-phosphorylated STAT3. (h) Representative western blots showing the levels of total and phosphorylated C/EBPβ and STAT3 in the cells transiently transfected with Cas9/ PIM1,2,3 -specific sgRNAs, in the presence or absence of exogenously introduced PIM1. (i) Schematic representation of the proposed signaling mechanisms by which PIM controls S100A8 and -A9 expression. Throughout this figure, GDC-0339 was used at 5 µM. Actin serves as a loading control. The numbers in red indicate relative protein expression.

Article Snippet: Real-time PCR reactions were run on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) paired with the CFX Manager software (Bio-Rad) using TaqMan probes (Thermo Fisher) specific for the following genes: human S100A8 (Hs00374264_g1), human S100A9 (Hs00610058_m1), mouse S100A8 (Mm00496696_g1), and mouse S100A9 (Mm00656925_m1).

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

Journal: bioRxiv

Article Title: S100A8/A9 predicts triple-negative breast cancer response to PIM kinase and PD-1/PD-L1 inhibition

doi: 10.1101/2023.09.21.558870

Figure Lengend Snippet: (a) Schematic representation of the flow of the experiments and downstream analyses conducted. (b) Representative western blots showing protein abundance of S100A8, -A9, PIM1, -2, and -3 in the indicated tumors (two samples per line) or non-tumor tissues. #This panel is a composite of two sides of identical western blot. (c) Flow analysis showing PD-1/PD-L1-positivity in the indicated cell populations in size-controlled tumors (500 mm 3 ; n = 4-5 per line). (d) Representative western blots showing the effects of the indicated pan-PIM inhibitors (5 µM) on the total abundance or phosphorylation levels of the indicated proteins in CD11b+Gr1+ myeloid cells isolated from D2A1-M1 tumor-bearing mice. ( e, f ) Growth of indicated tumors in mice treated with the indicated drugs or drug combinations for 2 weeks (n = 5 per treatment group). Linear regression analysis was used to calculate p- values. (g) Growth of Py8119 tumors in mice treated with GDC-0339 plus an anti-PD-1 antibody (n = 5 per treatment group). (h) The effects of the indicated drugs or drug combinations on the indicated cytotoxic immune response-related parameters (scoring system) adopted by the NanoString IO360 TM platform (n = 3-5 per treatment group as indicated). Box plots show the 25 th , 50 th (median), and 75 th percentiles. These parameters have not been designed to generate data indicative of statistical significance. TILs, tumor-infiltrating lymphocytes. (i) Heat map representation of the effects of the indicated drug treatment on the relative expression of the indicated cytotoxic immune response-related genes. The individual tumor samples represented in this heat map correspond to those in . Throughout this figure, GDC-0339 was used at 100 mg/kg/dose, and anti-PD-1/PD-L1 antibodies were used at 12 mg/kg/dose in vivo. Error bars represent means +/- SEM. Unless otherwise indicated, a two-tailed t -test was used to calculate p- values; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. NS: not significant. Actin serves as a loading control.

Article Snippet: Real-time PCR reactions were run on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) paired with the CFX Manager software (Bio-Rad) using TaqMan probes (Thermo Fisher) specific for the following genes: human S100A8 (Hs00374264_g1), human S100A9 (Hs00610058_m1), mouse S100A8 (Mm00496696_g1), and mouse S100A9 (Mm00656925_m1).

Techniques: Western Blot, Quantitative Proteomics, Phospho-proteomics, Isolation, Expressing, In Vivo, Two Tailed Test, Control

Journal: bioRxiv

Article Title: S100A8/A9 predicts triple-negative breast cancer response to PIM kinase and PD-1/PD-L1 inhibition

doi: 10.1101/2023.09.21.558870

Figure Lengend Snippet: ( a, b ) Flow analysis showing the abundance of the indicated cell types in size-controlled tumors (n = 4-5 per line). (c) Gating strategy for the identification of MDSCs in tumor samples. (d) Representative western blots showing expression of the indicated proteins in the CD45- or CD45+ cell populations isolated from developed D2A1-M1 tumors (approximately 500 mm 3 ). Each lane represents two pooled CD45-sorted samples. MYC serves as a marker for TNBC cells. Actin serves as a loading control. (e) Volcano plot showing the distribution of 770 IO-related genes, included in the NanoString IO360 TM panel (mouse; NanoString Technologies, Inc.), in the indicated comparison group (n = 5 for the vehicle group, n = 3 for the GDC-0339 group). (f) Relative mRNA levels of S100A8 and - A9 in the D2A1-M1 tumors treated with GDC-0339 or vehicle for 2 weeks (n = 8-9 per treatment group). Box plots show the 25 th , 50 th (median), and 75 th percentiles. (g) Top: Schematic representation of the flow of the experiments. Bottom: Growth of the indicated tumors in mice treated with the indicated drugs or drug combinations (n = 5-6 per treatment group). Linear regression analysis was used to calculate p- values. *Tumors at the time of initiating drug treatment, compared to those used in , were significantly larger (approx. 120-160 mm 3 , as opposed to 75-100 mm 3 ). Throughout this figure, error bars represent means +/- SEM. Unless otherwise indicated, a two-tailed t -test was used to calculate p- values; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. NS: not significant.

Article Snippet: Real-time PCR reactions were run on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) paired with the CFX Manager software (Bio-Rad) using TaqMan probes (Thermo Fisher) specific for the following genes: human S100A8 (Hs00374264_g1), human S100A9 (Hs00610058_m1), mouse S100A8 (Mm00496696_g1), and mouse S100A9 (Mm00656925_m1).

Techniques: Western Blot, Expressing, Isolation, Marker, Control, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: S100A8/A9 predicts triple-negative breast cancer response to PIM kinase and PD-1/PD-L1 inhibition

doi: 10.1101/2023.09.21.558870

Figure Lengend Snippet: (a) Schematic representation of the flow of the experiments and downstream analyses conducted. (b) Relative mRNA levels of S100A8 and - A9 , as determined by qPCR, in the D2A1-M1 tumors treated with GDC-0339 or vehicle for five days (n = 33-34 per treatment group). (c) Changes in serum S100A8/A9 levels, as determined by ELISA, in D2A1-M1 tumor-bearing mice treated with GDC-0339 or vehicle for 5 days (n = 9 for Day 0 samples, n = 21-22 for Day 5 samples). (d) Flow analysis showing the abundance of the indicated T cell types in tumors isolated from D2A1-M1 tumor-bearing mice treated with GDC-0339 or vehicle for 5 days (n = 23-24 per treatment group). (e) Flow analysis showing the total abundance of MDSCs (PMN- and M-MDSCs combined) in tumors isolated from D2A1-M1 tumor-bearing mice treated with GDC-0339 or vehicle for 5 days (n = 23-24 per treatment group). (f) Serum S100A8/A9 concentrations, as determined by ELISA, in samples from healthy donors and patients with TNBC at the indicated breast cancer stages. Each sample was subjected to the assay three times independently, and the concentrations shown represent the average values with SEM. All donors were females. ND: not detectable (concentrations < approximately 0.002 µg/mL). (g) Schematic representation of the in vitro chemotaxis assay performed. PBMCs: peripheral blood mononuclear cells. OE: overexpression. (h) Box plots showing the effects of conditioned media from the indicated MDA-MB-468 CRISPR lines on CD11b+ cell migration (8 h) (n = 9 per experimental group). (i) Relative abundance of total MDSCs (left) or neutrophils (right) in the migrated populations of CD11b+ cells as shown in (n = 3 per experimental group). Throughout this figure, GDC-0339 was used at 100 mg/kg/dose. Error bars represent means +/- SEM. Thick black lines indicate the median in violin plots. A two-tailed t -test was used to calculate p- values; ** p < 0.01, *** p < 0.001, **** p < 0.0001. NS: not significant.

Article Snippet: Real-time PCR reactions were run on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) paired with the CFX Manager software (Bio-Rad) using TaqMan probes (Thermo Fisher) specific for the following genes: human S100A8 (Hs00374264_g1), human S100A9 (Hs00610058_m1), mouse S100A8 (Mm00496696_g1), and mouse S100A9 (Mm00656925_m1).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, In Vitro, Chemotaxis Assay, Over Expression, CRISPR, Migration, Two Tailed Test

Journal: bioRxiv

Article Title: S100A8/A9 predicts triple-negative breast cancer response to PIM kinase and PD-1/PD-L1 inhibition

doi: 10.1101/2023.09.21.558870

Figure Lengend Snippet: (a) Left: Representative western blots showing expression of S100A8 and -A9 in the MDA-MB-468 cells that underwent the indicated genetic manipulations. Actin serves as a loading control. Right: S100A8/A9 secretion (48 h), as determined by ELISA, in the genetically modified MDA-MB-468 cells shown in (a) (n = 3). OE: overexpression. (b) Schematic representation of the in vitro immune cell proliferation/activation assay. (c) The effects of conditioned media from (a) on relative cell proliferation and activation levels of the indicated immune cell types (n = 6 per experimental group). Throughout this figure, error bars represent means +/- SEM. A two-tailed t -test was used to calculate p- values; ** p < 0.01, **** p < 0.0001. NS: not significant.

Article Snippet: Real-time PCR reactions were run on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) paired with the CFX Manager software (Bio-Rad) using TaqMan probes (Thermo Fisher) specific for the following genes: human S100A8 (Hs00374264_g1), human S100A9 (Hs00610058_m1), mouse S100A8 (Mm00496696_g1), and mouse S100A9 (Mm00656925_m1).

Techniques: Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Genetically Modified, Over Expression, In Vitro, Activation Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: A and B , images of GPR30 staining intensity level 3 without plasma-membrane staining (GPR30 PM -) ( A ) and with plasma-membrane staining (GPR30 PM +) ( B ). C - G , images of GPR30 total staining (GPR30 TOT ) as negative ( C ), very weak (level 1) ( D ), weak (level 2) ( E ), moderate (level 3) ( F ) and very strong (level 4) ( G ). Bar , 50 μm.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques: Staining, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: Association between total GPR30 intensity score (GPR30 TOT ), plasma membrane GPR30 (GPR30 PM ) status, and various clinicopathological variables.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques: Clinical Proteomics, Membrane, Biomarker Discovery, Staining

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: The change in GPR30 intensity (level 0–4) between paired BC and LGL. In relation to the tumor assumed to have developed earlier, the intensity shift in the second tumor is characterized as decreasing, stable or increasing for each tumor pair respectively. The intensity shift was assessed statistically using Wilcoxon matched pairs signed rank test.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques:

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: Prognostic effect of total GPR30 staining (GPR30 TOT ) and PM-specific GPR30 (GPR30 PM ) staining of BC2 and LGL2 calculated by Cox proportional hazards model with Wald test.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques: Staining

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: A , cumulative incidence of BCD in relation to GPR30 PM staining of BC2. B , cumulative incidence of BCD in relation to GPR30 PM staining of LGL2. In A and B , cumulative incidences of competing event (death from other cause than BC) are shown for comparison.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques: Staining, Comparison

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: A , cumulative incidence of BCD in relation to GPR30 TOT of BC2 in the whole cohort. B , cumulative incidence of BCD in relation to the five GPR30 staining intensity levels of BC2. C , cumulative incidence of BCD in relation to GPR30 TOT of LGL2. In A - C , cumulative incidences of competing event (death from other cause than BC) are shown for comparison.

Article Snippet: HFF11 cells stably expressing the T-Rex system (HeLa TET-On/Off cells), in which the expression of human GPR30 is under the strict control by tetracycline, were constructed according to vendor instructions (ThermoFisher Scientific).

Techniques: Staining, Comparison

Journal: Journal of Neuroinflammation

Article Title: Esketamine alleviates COPD-depression comorbidity in rats via MAPK/NF-κB inhibition and gut-lung-brain axis modulation

doi: 10.1186/s12974-026-03699-1

Figure Lengend Snippet: Activation of MAPK/NF-κB signaling accompanied by systemic inflammation and oxidative stress in the COPD-depression comorbid rat model. a , Serum cytokines and oxidative stress markers, including IL-6, IL-8, TNF-α, IL-10, MPO, SOD, and MDA ( n = 7). b , Representative western blots of lung tissue showing p-p38, p38, p-JNK, JNK, p-p65, p65, and GAPDH, with quantification of phosphorylation ratios ( n = 3). c , Representative western blots of hippocampal tissue showing p-p38, p38, p-JNK, JNK, p-p65, p65, and GAPDH, with quantification of phosphorylation ratios ( n = 3). d , Representative immunohistochemical staining of lung sections for p-p38, p-JNK, p-p65, with quantitative analysis of mean optical density ( n = 3). e , Representative immunohistochemical staining of hippocampal sections for p-p38, p-JNK, and p-p65, with quantitative analysis of mean optical density ( n = 3). Con, control; COPD, COPD model; YY, depression model; CY, comorbid model. Data are shown as mean ± SD. a : one-way ANOVA with Bonferroni post-hoc correction was used (* P < 0.05, ** P < 0.01 vs. Con); Welch test with Dunnett’s T3 correction was used where appropriate ( △ P < 0.05 vs. Con). b - e : one-way ANOVA with LSD post-hoc correction (* P < 0.05 vs. Con)

Article Snippet: After blocking with 5% non-fat milk in TBS-T, membranes were incubated overnight at 4 °C with the following primary antibodies at validated dilutions: phospho-p65 (Affinity, AF2006, 1:1,000), total p65 (Proteintech, 10745-1-AP, 1:2,000), phospho-p38 (Bioss, bs-0636R, 1:1,000), total p38 (Proteintech, 14064-1-AP, 1:2,000), phospho-JNK (Proteintech, 80024-1-RR, 1:1,000), total JNK (Proteintech, 24164-1-AP, 1:2,000), and GAPDH (Proteintech, 60004-1-Ig, 1:5,000).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Immunohistochemical staining, Staining, Control

Journal: Journal of Neuroinflammation

Article Title: Esketamine alleviates COPD-depression comorbidity in rats via MAPK/NF-κB inhibition and gut-lung-brain axis modulation

doi: 10.1186/s12974-026-03699-1

Figure Lengend Snippet: Esketamine suppresses MAPK/NF-κB signaling and restores inflammatory and oxidative balance in comorbid COPD-depression rats. a , Serum cytokines and oxidative stress markers, including IL - 6, IL - 8, TNF - α, IL - 10, MDA, MPO, and SOD ( n = 7). b , Representative western blots of lung tissue showing p-p38, p38, p - JNK, JNK, p - p65, p65, and GAPDH, with quantification of phosphorylation ratios ( n = 3). c , Representative western blots of hippocampal tissue showing p-p38, p38, p-JNK, JNK, p-p65, p65, and GAPDH, with quantification of phosphorylation ratios ( n = 3). d , Representative lung sections stained for p-p38, p - JNK, and p-p65, with quantification of mean optical density (AOD) ( n = 3). e , Representative hippocampal sections stained for p - p38, p - JNK, and p-p65, with quantification of mean optical density (AOD) ( n = 3).Con, control; CY, comorbid model; CYK, CY rats treated with esketamine; CYKD, CY rats treated with esketamine and Diprovocim. Data are expressed as mean ± SD. a : one-way ANOVA with Bonferroni post-hoc correction was used (** P < 0.01 vs. Con; ## P < 0.01 vs. CY; ▲ P < 0.05, ▲ ▲ P < 0.01 vs. CYK). b - e : one-way ANOVA with LSD post-hoc correction was used (* P < 0.05 vs. Con; # P < 0.05 vs. CY; ▲ P < 0.05 vs. CYK)

Article Snippet: After blocking with 5% non-fat milk in TBS-T, membranes were incubated overnight at 4 °C with the following primary antibodies at validated dilutions: phospho-p65 (Affinity, AF2006, 1:1,000), total p65 (Proteintech, 10745-1-AP, 1:2,000), phospho-p38 (Bioss, bs-0636R, 1:1,000), total p38 (Proteintech, 14064-1-AP, 1:2,000), phospho-JNK (Proteintech, 80024-1-RR, 1:1,000), total JNK (Proteintech, 24164-1-AP, 1:2,000), and GAPDH (Proteintech, 60004-1-Ig, 1:5,000).

Techniques: Western Blot, Phospho-proteomics, Staining, Control

Journal: Journal of Neuroinflammation

Article Title: Esketamine alleviates COPD-depression comorbidity in rats via MAPK/NF-κB inhibition and gut-lung-brain axis modulation

doi: 10.1186/s12974-026-03699-1

Figure Lengend Snippet: Cellular validation of MAPK/NF-κB signaling in alveolar macrophages and microglia. a - b , Dose-dependent effects of CSE and esketamine on NR8383 cell viability (CCK-8 assay). c , Western blot analysis of p-p38, p38, p-JNK, JNK, p-p65, p65, and GAPDH in NR8383 cells, with quantification of phosphorylation ratios ( n = 3). d - f , Grouped NR8383 assays showing cell viability (CCK-8) and release of inflammatory cytokines (IL-6, IL-8, TNF-α, IL-10) and oxidative stress markers (MDA, MPO, SOD) ( n = 3). g - h , Dose-dependent effects of LPS + CSE and esketamine on HAPI cell viability (CCK-8 assay). i , Western blot analysis of p-p38, p38, p-JNK, JNK, p-p65, p65, and GAPDH in HAPI cells, with quantification of phosphorylation ratios ( n = 3). j - l , Grouped HAPI assays showing cell viability (CCK-8) and release of inflammatory cytokines and oxidative stress markers ( n = 3). Con, control; CSE, cigarette smoke extract; LPS, lipopolysaccharide. Data are expressed as mean ± SD; * P < 0.05 vs. Con; # P < 0.05 vs. CSE or LPS + CSE; ▲ P < 0.05 vs. CSE + Esketamine or LPS + CSE + Esketamine (LSD correction)

Article Snippet: After blocking with 5% non-fat milk in TBS-T, membranes were incubated overnight at 4 °C with the following primary antibodies at validated dilutions: phospho-p65 (Affinity, AF2006, 1:1,000), total p65 (Proteintech, 10745-1-AP, 1:2,000), phospho-p38 (Bioss, bs-0636R, 1:1,000), total p38 (Proteintech, 14064-1-AP, 1:2,000), phospho-JNK (Proteintech, 80024-1-RR, 1:1,000), total JNK (Proteintech, 24164-1-AP, 1:2,000), and GAPDH (Proteintech, 60004-1-Ig, 1:5,000).

Techniques: Biomarker Discovery, CCK-8 Assay, Western Blot, Phospho-proteomics, Control

Journal: medRxiv

Article Title: Integration of cell-type resolved spatial proteomics and transcriptomics reveals novel mechanisms in early ovarian cancer

doi: 10.1101/2025.08.25.25333715

Figure Lengend Snippet: Spatial proteomics reveals progressive molecular changes during fallopian tube transformation (A) Principal component analysis of epithelial cell proteomes of normal FT epithelium, FT precursors, HGSC, and corresponding metastasis (met). FOXJ1pos, FOXJ1-positive. FOXJ1neg, FOXJ1-negative. P53, p53 signature. (B) K-means clustering analysis of epithelial proteins in seven clusters, excluding FOXJ1pos and metastasis (Table S2C). Heatmap of normalized protein abundance (z-score) across different cell states. Scale indicates relative protein expression levels. (C) Gene set enrichment analysis (GSEA) of pathway alterations during disease progression (Table S2E). Circle size indicates enrichment significance (logarithm of the FDR values); color intensity represents normalized enrichment score (fold-change). (D) Single protein abundance plots for core SUMO machinery and E3 ligases along the disease trajectory. Data shown as mean ± SEM. (E) Heatmap. Protein levels of the specific REACTOME pathway “SUMOlyation of DNA damage response and repair proteins” across the indicated sample types. Columns represent individual samples; rows represent proteins. The scale indicates normalized protein abundance. Protein names are displayed alternating between the left and right sides of the heatmap. (F-G) Protein abundance of TRIP13 and CBY1 by proteomics (F) and immunohistochemistry (G). In G: black dashed line: STIC. Red dashed line: invasive HGSC. Arrow, apical expression of CBY1 in normal FT epithelium. Arrowhead, lack of expression of CBY1 in STIC. (H) Volcano plot comparing protein expression of epithelial cells in STIC lesions from women without (STIC_only) and with (HG_STIC) concurrent HGSC (Table S2B). FDR < 0.05, fold change > 1.5.

Article Snippet: Membranes were incubated in primary antibodies for SULF1 (ThermoFisher, PA5-115984, 1:1000), TRIP13 (Proteintech, 19602-1-AP, 1:1000), CBY1 (Proteintech, 12239-1-AP, 1:1500), BGN (Proteintech, 16409-1-AP, 1:1000), and GAPDH (Cell Signaling, 5174, 1:2000) in 5% bovine serum albumin in TBST at 4 °C overnight.

Techniques: Spatial Proteomics, Transformation Assay, Quantitative Proteomics, Expressing, Biomarker Discovery, Immunohistochemistry

Journal: Immunity

Article Title: Longitudinal Analysis Reveals Early Development of Three MPER-Directed Neutralizing Antibody Lineages from an HIV-1-Infected Individual

doi: 10.1016/j.immuni.2019.02.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 1,2-Dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DMPG) , Avanti Polar Lipids , Cat# 840445P.

Techniques: Purification, Recombinant, Staining, Plasmid Preparation, Luciferase, Cell Culture, Lysis, Reporter Gene Assay, Protease Inhibitor, Modification, Amplification, Binding Assay, Transfection, Crystallization Assay, Sequencing, Expressing, Software

Journal: Experimental Dermatology

Article Title: Targeting the Hippo/ YAP / TAZ signalling pathway: Novel opportunities for therapeutic interventions into skin cancers

doi: 10.1111/exd.14655

Figure Lengend Snippet: YAP/TAZ nuclear shuttling inhibitors

Article Snippet: Verteporfin , Visudyne , Inhibitor of YAP nuclear localization , NR , Co‐IP, TEAD luciferase reporter, PLA, YAP‐TEAD target gene qPCR , NR , 8 nM–11.16 μM (Cell line‐dependent) , , , , Selleckchem (#S1786) Tocris (#5305).

Techniques: Binding Assay, Biomarker Discovery, Activity Assay, Luciferase, Phospho-proteomics, Expressing

Journal: Clinical and Experimental Pediatrics

Article Title: Pediatric patients with cancer exhibit increased neutrophil extracellular traps and reduced active deoxyribonuclease I: diagnostic, prognostic, and therapeutic opportunities

doi: 10.3345/cep.2026.01004

Figure Lengend Snippet: Plasma levels of NETosis biomarkers and deoxyribonuclease I (DNaseI) according to tumor risk in patients with treatment-naive plasma. (A) Calprotectin. (B) Cell-free DNA (cfDNA). (C) Neutrophil elastase. (D) Citrullinated histone 3 (CitH3)-DNA complexes. (E) DNaseI. * P <0.05. ** P <0.01. **** P <0.0001.

Article Snippet: Different markers of NETosis were quantified in plasma as previously described: cell-free DNA (cfDNA, Quant-iT PicoGreen dsDNA kit, Life Technologies, USA), calprotectin (Human Calprotectin ELISA kit, Hycult Biotech, Netherlands), neutrophil elastase (PMN Elastase Human ELISA Kit, Abcam, UK) [ - ] and citrullinated histone 3 (citH3)-DNA complexes [ ].

Techniques: Clinical Proteomics

Journal: Clinical and Experimental Pediatrics

Article Title: Pediatric patients with cancer exhibit increased neutrophil extracellular traps and reduced active deoxyribonuclease I: diagnostic, prognostic, and therapeutic opportunities

doi: 10.3345/cep.2026.01004

Figure Lengend Snippet: Receiver operating characteristic (ROC) curves of neutrophil extracellular traps (NETs) as biomarkers of pediatric cancer for diagnosis (A), stratification in all tumors (B), stratification in neuroblastoma (C), and their levels according to death-related disease (D), survival in all tumors (E), and survival in children with high-risk (HR) tumors. (A) ROC curves of calprotectin and cell-free DNA (cfDNA) in children with cancer versus controls. (B) ROC curves of calprotectin and cfDNA in children with HR tumors versus lowrisk (LR) tumors in all patients. (C) ROC curves of calprotectin and cfDNA in children with HR tumors versus low-risk (LR) tumors in neuroblastoma. (D) Calprotectin and deoxyribonuclease I (DNaseI) levels according to the death-related disease. (E) Diagnostic metrics for each biomarker derived from the ROC curve analysis of NET markers and DNaseI as biomarkers of pediatric cancer. The optimal cutoff values were determined using Youden’s index. Sensitivity and specificity are reported with 95% confidence intervals. AUC, area under the curve; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value.

Article Snippet: Different markers of NETosis were quantified in plasma as previously described: cell-free DNA (cfDNA, Quant-iT PicoGreen dsDNA kit, Life Technologies, USA), calprotectin (Human Calprotectin ELISA kit, Hycult Biotech, Netherlands), neutrophil elastase (PMN Elastase Human ELISA Kit, Abcam, UK) [ - ] and citrullinated histone 3 (citH3)-DNA complexes [ ].

Techniques: Biomarker Discovery, Diagnostic Assay, Derivative Assay

Journal: Clinical and Experimental Pediatrics

Article Title: Pediatric patients with cancer exhibit increased neutrophil extracellular traps and reduced active deoxyribonuclease I: diagnostic, prognostic, and therapeutic opportunities

doi: 10.3345/cep.2026.01004

Figure Lengend Snippet: Association of plasma calprotectin and deoxyribonuclease I (DNaseI) levels with diseaserelated mortality and survival outcomes in pediatric cancer patients. (A) Plasma levels of calprotectin and DNaseI stratified by death-related disease status. (B) Kaplan-Meier analysis of survival according to calprotectin levels in all patients. (C) Kaplan-Meier analysis of survival according to calprotectin levels in HR group. * P <0.05. ** P <0.01. AUC, area under the receiver operating characteristic curve; CI, confidence interval; HR, high-risk patient samples; LR, low-risk patient samples; PPV, positive predictive value; NPV, negative predictive value.

Article Snippet: Different markers of NETosis were quantified in plasma as previously described: cell-free DNA (cfDNA, Quant-iT PicoGreen dsDNA kit, Life Technologies, USA), calprotectin (Human Calprotectin ELISA kit, Hycult Biotech, Netherlands), neutrophil elastase (PMN Elastase Human ELISA Kit, Abcam, UK) [ - ] and citrullinated histone 3 (citH3)-DNA complexes [ ].

Techniques: Clinical Proteomics

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: (a) TGFβ stimulates p38α activity, inhibited by SB2036580 or β-LGND. Bar graph is the mean±SD from 3 exps combined. *p < 0.05 vs. control, +p < 0.05 for TGFβ vs same + SB2036580 or β-LGND. (b) TGFβ inhibits KLF15 mRNA and protein in cardiomyocytes, blocked by the p38 antagonist SB2036580 (0.1μM) (c) TAK1 activating phosphorylation is stimulated by AngII or TGFβ, inhibited by β-LGND. *p<0.05 vs. control, + p<0.05 for TGFβ or AngII vs same plus β-LGND, n=3 exps. (d) TAK1 siRNA diminishes TGFβ or AngII-stimulated p38α activity. The latter was seen as phosphorylation at tyrosine182. *p<0.05 vs control, +p<0.05 for TGFβ or AngII vs same + β-LGND, n=3 exps. TAK1 siRNA validation is also shown. (e) Flow cytometry analysis of β-LGND inhibition of phospho-kinases due to cAMP/PKA. *p<0.05 for control vs. AngII-stimulated phospho-TAK1, phospho-p38α, or KLF15 proteins. +p<0.05 for AngII vs AngII + β-LGND, ++p<0.05 for AngII + β-LGND vs same + either H-89 (PKA inhibitor) or RP-8-Br-cAMP (cAMP inhibitor), n=3 exps.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Activity Assay, Control, Phospho-proteomics, Biomarker Discovery, Flow Cytometry, Inhibition

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: AngII acting through TGFβ stimulates a TAK1-p38α kinase axis that inhibits KLF15 expression and nuclear localization of the protein. This contributes to increased gene expression and cardiomyocyte hypertrophy. ERβ acting through protein kinase A opposes TAK1-p38α activation. This restores KLF15 abundance and nuclear localization, contributing in part to inhibition of AngII-induced gene expression and cardiomyocyte hypertrophy.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Expressing, Gene Expression, Activation Assay, Inhibition

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Retinoids repress human cardiovascular cell calcification with evidence for distinct selective retinoid modulator effects

doi: 10.1161/ATVBAHA.119.313366

Figure Lengend Snippet: Peretinoin inhibits human cardiovascular cell calcification without inducing liver cell APOC3 secretion. (A) APOC3 and (B) APOA1 mRNA levels and secreted protein in HepG2 treated for 24 hours with 0.1% DMSO vehicle (control), 1 μmol/L ATRA, 1 μmol/L 9-cis retinoic acid (9-cis RA), 1 μmol/L peretinoin, 13-cis retinoic acid (13-cis RA), or 1 μmol/L AGN 193109 (AGN). (C) CYP7A1 and (D) HNF4α, PPARGC1α mRNA levels in HepG2 cells treated for 24 hours. Alizarin red stain and relative quantification for (E) primary human coronary artery SMC and (F) VIC treated for three weeks, and (G) primary human femur osteoblasts treated for one three weeks in CM, OM, or PM with DMSO vehicle (0.01%), 1 μmol/L peretinoin, or 1 μmol/L ATRA. (H) RAR siRNA knockdown validation by mRNA level quantification, and Alizarin red stain and relative quantification for primary human femur osteoblasts treated for two weeks in OM with negative control, RARα, RARβ, or RARγ siRNA. Error bars indicate STDEV of mean. aP < 0.0001 vs control, bP < 0.0001 vs peretinoin, cP < 0.0001 vs AGN, dP < 0.001 vs control, eP < 0.001 vs peretinoin, fP < 0.01 vs peretinoin, gP < 0.01 vs control, hP < 0.01 vs AGN, iP < 0.001 vs AGN, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 analyzed by ANOVA or Student’s t-test; n = 6 independent experiments for HepG2 and n = 3 donors for primary human VIC, SMC, and osteoblasts.

Article Snippet: Cells were treated with combinations of the following reagents: 0.1% DMSO vehicle; ATRA (Sigma, St. Louis, MO); AGN 193109 (Sigma), 9-cis retinoic acid (Sigma), or peretinoin (MedChem Express LLC, Monmouth Junction, NJ).

Techniques: Control, Staining, Quantitative Proteomics, Knockdown, Biomarker Discovery, Negative Control