Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. wtEGFR and EGFRvIII bands are marked with * and **, respectively. ( b ) Densitometric quantification of galectin1 protein level normalized to tubulin in different BTSC lines is shown. ( c-d ) EGFR / EGFRvIII KD (si EGFR ) and control BTSCs (siCTL) were analyzed by immunoblotting as described in a. ( e-h ) BTSCs were treated with 1 or 5 µM lapatinib and galectin1 expression was assessed by immunoblotting (e-f) and immunostaining (g-h). Nuclei were stained with DAPI. Scale bar = 10 μm. ( i ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. ( j ) Pearson correlation analysis of pSTAT3-Y705 and galectin1 protein expression in different BTSCs is shown. ( k-l ) STAT3 KD (si STAT3 ) and siCTL BTSCs were analyzed by immunoblotting as described above. ( m-p ) BTSCs were subjected to immunoblotting or immunostaining following treatment with 25 or 50 µM of the STAT3 inhibitor, S3I-201. Scale bar = 10 μm. ( q-s ) EGFRvIII-expressing BTSCs were subjected to ChIP using an antibody to STAT3 or IgG control followed by qPCR using two different pairs of primers ( LGALS1 -a and LGALS1 -b). OSMR , and HPRT loci were used as positive and negative controls, respectively. ( t-u ) Luciferase reporter assay was performed in BTSC73 following KD of STAT3 using siRNA (t) or treatment with STAT3 inhibitors, 5 µM WP1066 or 50 μM S3I-201 (u). Data are presented as the mean□±□SEM, n ≥ 3. Unpaired two-tailed t -test (q, r and s); one-way ANOVA followed by Dunnett’s test (b) or Tukey’s test (t and u),*p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1 and S2.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Western Blot, Expressing, Immunostaining, Staining, Luciferase, Reporter Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) Cell viability was assessed by CellTiter-Glo assay in LGALS1 CRISPR and CTL BTSCs. ( c ) Population growth curves for LGALS1 CRISPR and CTL BTSC73 are shown. ( d-f ) Cell viability assay (d-e) and population growth curves (f) of BTSC73 treated with 1 or 10 µM OTX008 are shown. ( g ) Representative images of EdU staining in LGALS1 CRISPR and CTL BTSC73 are shown. ( h ) The number of EdU positive cells was quantified using Fiji software. ( i ) EdU incorporation was analyzed by flow cytometry in LGALS1 CRISPR and CTL BTSC73. Representative scatter plots of flow cytometry analyses are shown. Data are presented as the mean□±□SEM, n = 3. Unpaired two-tailed t -test (a, b, c and h); one-way ANOVA followed by Dunnett’s test (d, e and f), **p < 0.01, ***p < 0.001. See also Figures S3 and S4.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Glo Assay, CRISPR, Viability Assay, Staining, Software, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) LGALS1 CRISPR or CTL BTSC73 were subcutaneously injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (a). Graph represents tumour mass (b). ( c-f ) BTSC73 or BTSC147 were injected subcutaneously into SCID mice and treated with 10 mg/kg OTX008. Representative bioluminescence real-time images tracing tumour growth are shown (c, e). Graphs represent tumour mass (d, f). ( g-j ) LGALS1 CRISPR or CTL BTSC73 were intracranially injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (g). Intensities of luciferase signal were quantified at different time points using Xenogen IVIS software (h). Graph represents quantification of animal weight (i). KM survival plot was graphed to evaluate mice lifespan in each group (j). Data are presented as the mean□±μSEM, n ≥ 4 mice. Unpaired two-tailed t -test (b, d, f, h and i); log-rank test (j), **p < 0.01, ***p < 0.001.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Injection, Luciferase, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) Volcano plot representing LGALS1 differentially regulated genes is shown. ( b-c ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal (b) and proneural (c) subtypes of glioblastoma. ( d ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal-like meta-module (MES1-like) signature. ( e-f ) GSEA analysis demonstrates enrichment for gene sets corresponding to recruitment of NuMA to mitotic centrosomes (e) and mitotic G2−G2/M phases (f). ( g-h ) RNA-seq data was validated by RT-qPCR in BTSC73 and BTSC147. ( i-j ) Cell cycle distribution was assessed by flow cytometry after PI staining in LGALS1 CRISPR BTSCs. Data are presented as the mean□±□SEM, n = 3. One-way ANOVA followed by Dunnett’s test (g and h); unpaired two- tailed t -test (i and j), *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Flow Cytometry, Staining, CRISPR, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-d ) LGALS1 CRISPR and CTL EGFRvIII-expressing BTSCs were subjected to LDA (a-b) or ELDA (c-d). ( e-f ) EGFRvIII-expressing LGALS1 CRISPR and CTL BTSCs were subjected to clonogenicity assay performed by culturing one single cell per well. ( g-h ) BTSCs that don’t harbour the EGFRvIII mutation were electroporated with siCTL or si LGALS1 and subjected for ELDA analysis. ( i-p ) EGFRvIII-expressing BTSCs were subjected to LDA (i, j, m and n) or ELDA (k, l, o and p) following the treatment with 1 or 10 µM OTX008. ( q-t ) BTSCs that don’t harbour the EGFRvIII mutation were subjected to LDA (q-r) or ELDA (s-t) following the treatment with 1 or 10 µM OTX008. *p < 0.05, **p < 0.01, ***p < 0.001; unpaired two-tailed t -test (a, b, e and f); one-way ANOVA followed by Dunnett’s test (i, j, m and n), n = 3. Data are presented as the mean□±□SEM. See also Figure S6.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Expressing, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) ELDA was performed following 4 Gy of IR in LGALS1 CRISPR or CTL BTSCs. ( b-c ) LGALS1 CRISPR and CTL BTSC73 were subjected to IR (8□Gy). Apoptosis analysis was performed by flow cytometry 48□h following IR using annexin V and PI double staining. Representative scatter plots of flow cytometry analyses are shown (b). The percentage of cell death (annexin V positive cells) is presented in the histogram (c), n□=□3. ( d ) Schematic diagram of the experimental procedure is shown. BTSC73 were intracranially injected into SCID mice and then treated with OTX008, 4□Gy of IR or a combination of OTX008 and IR. ( e ) Representative bioluminescence real-time images tracing tumour growth are shown, n□=□6 mice. ( f ) Coronal sections of mouse brains were stained with hematoxylin and eosin on day 22 after injection. Representative images of 3 different tumour sections are shown. Scale bar = 1□mm, scale bar (inset) = 0.2 mm. ( g ) Intensities of luciferase signal were quantified at different time points, n = 6 mice. ( h ) KM survival plot was graphed to assess animal lifespan, n□=□6 mice. ( i ) Survival extension of mice bearing BTSC-derived tumours treated with OTX008, IR, or OTX008 + IR relative to those treated with the vehicle control. Data are presented as the mean□±□SEM. One-way ANOVA followed by Tukey’s test (c and i); log-rank test (h), *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Flow Cytometry, Double Staining, Injection, Staining, Luciferase, Derivative Assay

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) LGALS1 -differentially regulated genes were subjected to enrichment analysis of TF binding motifs using oPOSSUM-3 software. ( b ) Volcano plot representing the HOXA5 target genes among the LGALS1 -differentially-regulated genes is shown. ( c ) BTSCs were analyzed by immunoblotting using the antibodies indicated on the blots. ( d ) Pearson correlation analysis of HOXA5 and galectin1 protein expression is shown. ( e ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients is shown. ( f ) Relative positions of HOXA5 ChIP-seq peaks to the adjacent TSS of LGALS1 -differentially regulated genes are shown. The x-axis indicates the distance between peak centers and the TSS of adjacent LGALS1 -differentially regulated genes. The y-axis denotes the expression ratios (log2) of the LGALS1 -differentially regulated gene. Circle size indicates HOXA5 peak height, and color denotes the conservation score of HOXA5 peaks. ( g-h ) HOXA5 KD (si HOXA5 ) and siCTL BTSCs were subjected to RT-qPCR analysis. ( i ) ELDA was performed following 4LGy of IR in si HOXA5 vs. siCTL. ( j - m ) Endogenous Co-IP experiments were performed in different BTSC lines using an anti-HOXA5 antibody, followed by immunoblotting with galectin1 and HOXA5 antibodies. ( n ) Co-IP experiment was performed using anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-HOXA5 antibodies. ( o - r ) PLA of galectin1 and HOXA5 were performed in different BTSC lines. Primary antibodies were omitted for the controls. Nuclei were stained with DAPI. Scale bar = 10 μm. ( s ) LGALS1 CRISPR and CTL BTSC73 were subjected to ChIP using an antibody to HOXA5 followed by qPCR for HOXA5 candidate target genes. HBB locus was used as a negative control. ( t-u ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients treated with radiotherapy (microarray G4502A Agilent, level 3, n = 489). Data are presented as the meanL±LSEM, n = 3. Log-rank test (e, t and u); one-way ANOVA followed by Dunnett’s test (g and h); unpaired two-tailed t -test (s). *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Binding Assay, Software, Western Blot, Expressing, ChIP-sequencing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Staining, CRISPR, Negative Control, Microarray, Two Tailed Test

Journal: PLoS ONE

Article Title: Functional Genomic Analyses of Two Morphologically Distinct Classes of Drosophila Sensory Neurons: Post-Mitotic Roles of Transcription Factors in Dendritic Patterning

doi: 10.1371/journal.pone.0072434

Figure Lengend Snippet: To access differences in gene expression, RNA from enriched C-I and C-IV neuronal populations were compared with that of whole larvae in microarray experiments. Live confocal images of neuronal populations labeled by ( A ) GAL4 221 (C-I) and ( B ) GAL4 ppk1.9 (C- IV) visualized by the trans-membrane fusion construct mCD8::GFP. Neurons have been pseudo-colored to distinguish individual subtypes and dendritic territories. ( C ) GAL4 221 strongly labels C-I da neurons along with weakly labelling of C-IV neurons in the background (dotted red trace). ( D ) GAL80 driven by a ppk promoter was combined in the background of GAL4 221 (ppk-GAL80; GAL4 221 ) that results in highly class I specific GAL4 expression. ( E ) A representative whole larval image of GAL4 ppk1.9 driving the expression of UAS-mCD8::GFP (gut is auto-fluorescent). ( F-J ) Strategy of class-specific neuronal isolation. Larvae expressing mCD8::GFP under the control of either ppk-GAL80; GAL4 221 or GAL4 ppk1.9 ( F ) were dissociated ( G ) filtered and incubated with superparamagnetic beads coated with anti-mCD8 antibody ( H ). The C-I/C-IV neurons bound to the magnetic beads were purified using a strong magnet ( I ), washed several time and used to perform microarray gene expression profiling ( J ). An identical region from the C-I and C-IV microarray are represented to show their dramatic qualitative differences ( J ). The microarray replicates were highly correlated, as represented in the correlation map ( K ). Principle component analysis revealed the three microarray samples from C-I, C-IV and whole larval lysate cluster into three distinct and well-defined clusters ( L ).

Article Snippet: The filtrate is then incubated with superparamagnetic beads (Dynabeads MyOne Streptavidin T1, Invitrogen) coupled with biotinylated mouse anti-CD8a antibody (eBioscience) for 60 minutes.

Techniques: Gene Expression, Microarray, Labeling, Membrane, Construct, Expressing, Isolation, Control, Incubation, Magnetic Beads, Purification

Journal: International Journal of Molecular Sciences

Article Title: G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer

doi: 10.3390/ijms23020589

Figure Lengend Snippet: G9a knockdown may lead to increase in BMP5 expression. ( A ) Regulatory factors suppressing tumor aggressiveness were identified by using microarray analysis. ( B ) Expression levels for each gene were compared (ratios between gene expression levels of non-silencing control). In two G9a knockdown cells (shG9a #1 and shG9a #2), expression levels for each gene were compared to those of non-silencing control. Then the most upregulated and downregulated genes were screened. ( C ) G9a knockdown cells showed increase in BMP5 expression. ( D ) Reduced G9a occupancy was found at the BMP5 promoter region. G9a knockdown cells also showed decreased H3K9me2 occupancy at the promoter region of BMP5. p values were calculated using ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Western blot analysis was performed with antibodies specific for the following proteins: G9a (#229455, Abcam, Cambridge, MA, USA), E-cadherin (#3195S, Cell Signaling, Boston, MA, USA), β-catenin (#8480P, Cell Signaling, Boston, MA, USA), ZO-1 (#5406P, Cell signaling, Boston, MA, USA), PARP (#9542S, Cell signaling, Boston, MA, USA), Caspase-7 (#9492S, Cell signaling, Boston, MA, USA), BMP5 (#13253-1-AP, Proteintech, Chicago, IL, USA), p -Smad1/5/9 (#13820T, Cell Signaling, Boston, MA, USA), p -Smad1/5 (#9516T, Cell Signaling, Boston, MA, USA), Smad1 (#6944T, Cell Signaling, Boston, MA, USA) and β-actin.

Techniques: Knockdown, Expressing, Microarray, Gene Expression, Control

Journal: International Journal of Molecular Sciences

Article Title: G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer

doi: 10.3390/ijms23020589

Figure Lengend Snippet: BMP5 contributes to reduce migration and invasion abilities of tumor cells. ( A ) Low BMP5 expression is associated with poor survival outcomes of breast cancer patients. ( B ) Patients with stage 3 disease showed higher BMP5 levels than those of stage 2 patients. ( C , D ) Migration/invasion abilities were decreased by treatment of recombinant BMP5; however, the capabilities were enhanced following BMP5 downregulation. p values were calculated using ANOVA. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Western blot analysis was performed with antibodies specific for the following proteins: G9a (#229455, Abcam, Cambridge, MA, USA), E-cadherin (#3195S, Cell Signaling, Boston, MA, USA), β-catenin (#8480P, Cell Signaling, Boston, MA, USA), ZO-1 (#5406P, Cell signaling, Boston, MA, USA), PARP (#9542S, Cell signaling, Boston, MA, USA), Caspase-7 (#9492S, Cell signaling, Boston, MA, USA), BMP5 (#13253-1-AP, Proteintech, Chicago, IL, USA), p -Smad1/5/9 (#13820T, Cell Signaling, Boston, MA, USA), p -Smad1/5 (#9516T, Cell Signaling, Boston, MA, USA), Smad1 (#6944T, Cell Signaling, Boston, MA, USA) and β-actin.

Techniques: Migration, Expressing, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer

doi: 10.3390/ijms23020589

Figure Lengend Snippet: G9a knockdown facilitates Smad protein phosphorylation via BMP5 activation. ( A , B ) G9a-knockdown-induced increase in BMP5 expression had no effect on the total level of either Smad1 or Smad5. G9a knockdown increased phosphorylation of Smad1/5/9. ( C ) ICC demonstrated that nuclear translocation of pSmad1/5/9 was increased in G9a-depleted MCF7 cells. ( D ) Similar tendency was found after treatment of recombinant BMP5. p values were calculated using ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant.

Article Snippet: Western blot analysis was performed with antibodies specific for the following proteins: G9a (#229455, Abcam, Cambridge, MA, USA), E-cadherin (#3195S, Cell Signaling, Boston, MA, USA), β-catenin (#8480P, Cell Signaling, Boston, MA, USA), ZO-1 (#5406P, Cell signaling, Boston, MA, USA), PARP (#9542S, Cell signaling, Boston, MA, USA), Caspase-7 (#9492S, Cell signaling, Boston, MA, USA), BMP5 (#13253-1-AP, Proteintech, Chicago, IL, USA), p -Smad1/5/9 (#13820T, Cell Signaling, Boston, MA, USA), p -Smad1/5 (#9516T, Cell Signaling, Boston, MA, USA), Smad1 (#6944T, Cell Signaling, Boston, MA, USA) and β-actin.

Techniques: Knockdown, Phospho-proteomics, Activation Assay, Expressing, Translocation Assay, Recombinant

Journal: Frontiers in Medicine

Article Title: Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis

doi: 10.3389/fmed.2022.1013660

Figure Lengend Snippet: TaqMan gene expression assays used in the study (Thermo-Fisher Scientific Cat Number 4331182).

Article Snippet: The membranes were probed overnight at 4°C with 2 μg/mL of CNA.42 or 1:200 FBXO2 rabbit polyclonal antibody (Proteintech).

Techniques: Gene Expression

Journal: Frontiers in Medicine

Article Title: Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis

doi: 10.3389/fmed.2022.1013660

Figure Lengend Snippet: RNA-Seq analysis of the PEAC cohort illustrates the differential correlation of the FDC genes associated with early perivascular and late mature developmental stages in the RA synovium. Strong positive correlations of the pericyte/fibroblast markers NG2, THY1 and αSMA with the PDGFR-β/PDGF-BB axis (A-I) , NG2 and FDC-CNA.42/FBXO2 (A-II) , and NG2, THY1 and αSMA (A-III) in RA. (B) Correlations of PDGF-BB and TNFα/LTβ with the expression of each other's receptors and early FDC developmental genes. PDGF-BB positively correlates with its receptor and the TNFα/LTβ receptors (B-I) , TNFα and LTβ negatively correlate with PDGFR-β expression and the early FDC markers NG2 and αSMA (B-II) . (C) Converse correlations of the PDGF-BB/PDGFR-β and the TNF-α/LT-β axes with the expression of mature FDC markers. PDGF-BB/PDGFR-β and TNF-α/LT-β differently correlate with the mature FDC related genes CXCL13 (B cell chemoattractant), BAFF (B cell survival factor), and antigen display and presentation to B cells namely complement receptors (CR1/CD35, CR2/CD21), and Fcg receptors (FcγRIIA/CD32A, FcγRIIB/CD32A). (D) Correlation of the RA synovial pathotypes with the expression of PDGF-BB, PDGFR-β, TNF-α, and LT-β. Person correlation coefficient (r) and adjusted p -values are shown with the corresponding plots and tables.

Article Snippet: The membranes were probed overnight at 4°C with 2 μg/mL of CNA.42 or 1:200 FBXO2 rabbit polyclonal antibody (Proteintech).

Techniques: RNA Sequencing, Expressing

Journal: Frontiers in Medicine

Article Title: Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis

doi: 10.3389/fmed.2022.1013660

Figure Lengend Snippet: Activation of sorted tonsillar stromal cell subsets with PDGF-BB and TNF-α/LT-β induces early and mature FDC markers in vitro . (A) The CD45 − tonsillar stromal subsets were sorted using combinations of NG2/αSMA, NG2/CNA.42, CNA.42/αSMA and CNA.42/CR2 Abs. (B) Type-1 Pericytes [NG2 + /αSMA + ; indicated by red * in (A,B) ], early FDCs; (CNA.42 + /NG2 + , CNA.42 + /αSMA + , CNA.42 + /CR2 − ; indicated in A and B by blue, magenta, and green *, respectively) and mature FDCs [CNA.42 + /CR2 + , indicated by brown * in (A,B) ] were treated with 300 ng/ml PDGF-BB or 100 ng/ml TNF-α+ 100 ng/ml LT-αβ and the fold change in FBXO2 (CNA.42), αSMA, Collagen 1, CR2, and FcγRIIB gene expression compared to untreated cells was calculated by Livak's DD equation and shown in bar graphs. (C) Treatment of the NG2 + /αSMA + type-1 pericyte subset with PDGF-BB in slide cultures for 6 days induced the expression of the FDC marker CNA.42 compared to untreated cells as demonstrated by Immunocytochemistry. Image J quantification of the mean fluorescence intensity (MFI) of CNA-42 of the different conditions is shown in the histogram. Data is representative of three different experiments and is expressed as the mean ± SEM.

Article Snippet: The membranes were probed overnight at 4°C with 2 μg/mL of CNA.42 or 1:200 FBXO2 rabbit polyclonal antibody (Proteintech).

Techniques: Activation Assay, In Vitro, Gene Expression, Expressing, Marker, Immunocytochemistry, Fluorescence

Journal: Frontiers in Medicine

Article Title: Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis

doi: 10.3389/fmed.2022.1013660

Figure Lengend Snippet: Correlation of IL-6 expression with synovial pathotypes and FDC markers. (A) Boxplots displaying the correlation of synovial IL-6, JAK2, STAT1, and blood IL-6 expression with synovial pathotypes. (B,C) Correlation blots of synovial IL-6, IL-6R, JAK1, and JAK2 expression with the FDC markers complement receptor 1 (CR1/CD35) and CXCL13, respectively. Correlation of STAT3 and STAT1 with CR1 and CXCL13 respectively are also shown. (D) Correlation of synovial IL-6 expression with the markers associated with early FDC differentiation including NG2 (pericytes), αSMA (myofibroblasts), and FBXO2 (CNA.42). (E) IL-6 release from synovial organ and fibroblast cultures stimulated with 300 ng/ml PDGF-BB for 24 hrs and 6 days respectively. (E-I) Synovial organ culture showing a piece of synovial tissue placed in cell culture inserts mounted in 24-well plates (Upper). Diagrammatic representation of the synovial organ culture setup (S = Synovial Tissue, M = Culture Medium, F = Filter Device). (E-II) Rheumatoid arthritis synovial fibroblasts (RASFs) at base line (Day 1 = D1) and after 6-day (D6) stimulation with PDGF-BB. IL-6 levels at baseline and after stimulation are shown in (E-III) . Cultures were run in triplicates and data is expressed as the mean ± SEM. Significance was calculated using 2-tailed unpaired student T test and the p -value between baseline and PDGF-BB stimulation is shown.

Article Snippet: The membranes were probed overnight at 4°C with 2 μg/mL of CNA.42 or 1:200 FBXO2 rabbit polyclonal antibody (Proteintech).

Techniques: Expressing, Organ Culture, Cell Culture

Journal: Frontiers in Medicine

Article Title: Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis

doi: 10.3389/fmed.2022.1013660

Figure Lengend Snippet: The FDC mAb CNA.42 recognizes FBXO2. (A) Immunoprecipitation (IP) and characterization of the CNA-42 binding protein. (A-I) Western blotting of total cell lysate (a), negative control (agarose beads only) and the CNA.42-immunoprecipitated proteins (c and d, 1.5 and 6 uls/lane respectively) from tonsillar single cell suspension probed with CNA-42. A single band is detectable at 120 Kd. (A-II) the reactivity of the CNA.42 mAb on the HuProt™ human proteome microarray showing subarray 9-1 of array 1300017931 (used for the CNA.42) with fluorescence detection at 633 nm excitation (a) and 543 nm excitation (b). (a) Staining with biotinylated anti-GST and Streptavidin-647. Rows 1-28 show generic staining of the GST-tagged immobilized human proteins, among them FBXO2 in row 11. (b) Probing with CNA.42 and Cy3 labeled anti-mouse IgM shows one hit, the human protein FBXO2 in the subarray. (A-III) Western blotting of tonsillar lysates with FBXO2 and CNA-42-specific antibodies recognize 120 Kd bands in the lysates [CNA.42 BP = CNA.42 binding protein]. (B) In situ hybridization of FBXO2 mRNA (green) showing intracellular signal in tonsillar CD21 + FDC reticula (red). (C) Western blotting of lysates from the CAN.42 expressing CEM cell line using mAb CAN.42 and anti FBXO2. CEM were untreated or treated either with Accell human FBXO2 siRNA (1 uM), or non-targeting control (NTC). GAPDH is used as a loading control. Compared to untreated cells, densitometric analysis with Image J indicates that FBXO2 siRNA-treated cells expressed 50% (*) and 35% (**) less FBXO2 and CNA.42, respectively.

Article Snippet: The membranes were probed overnight at 4°C with 2 μg/mL of CNA.42 or 1:200 FBXO2 rabbit polyclonal antibody (Proteintech).

Techniques: Immunoprecipitation, Binding Assay, Western Blot, Negative Control, Suspension, Microarray, Fluorescence, Staining, Labeling, In Situ Hybridization, Expressing, Control

Journal: Frontiers in Oncology

Article Title: FDXR drives primary and endocrine-resistant tumor cell growth in ER+ breast cancer via CPT1A-mediated fatty acid oxidation

doi: 10.3389/fonc.2023.1105117

Figure Lengend Snippet: The effect of FDXR on metabolic pathways in ER+ breast cancer cells. (A) Metabolite sets enrichment analysis of targeted metabolomics assays in T47D cells infected with lentivirus encoding Ctrl shRNA and FDXR sh434. (B) Heatmaps showing the metabolites involved in the indicated metabolic pathways positively regulated by FDXR. (C) Gene set enrichment analysis (GSEA) of gene expression profile in T47D cells infected with lentivirus encoding Ctrl shRNA and FDXR sh434. (D) Heatmaps showing genes related to fatty acid metabolism positively regulated by FDXR. (E) The diagram for FDXR regulation on fatty acid metabolic pathway. (F) The relative mRNA levels of fatty acid associated genes under FDXR depletion from gene expression microarray. (G) Q-PCR and immunoblots assay to detect FDXR and CPT1A level from T47D cells infected with lentivirus encoding Ctrl shRNA and FDXR sh434. (H–J) . Seahorse assays (H) and their quantifications of basal OCR (I) and indicated 1 and 2 (J) for measurement of FAO-dependent OCR under the treatment of an FAO inhibitor etomoxir (40 μM) in T47D cells infected with lentivirus encoding Ctrl shRNA and FDXR sh434. *, **, and *** denote P-value of < 0.05, 0.01, and 0.005, respectively. NS denotes not significant.

Article Snippet: Rabbit FDXR (15584-1-AP) and CPT1A antibodies (15184-1-AP) were purchased from Proteintech.

Techniques: Infection, shRNA, Gene Expression, Microarray, Western Blot

Journal: Frontiers in Oncology

Article Title: FDXR drives primary and endocrine-resistant tumor cell growth in ER+ breast cancer via CPT1A-mediated fatty acid oxidation

doi: 10.3389/fonc.2023.1105117

Figure Lengend Snippet: FDXR regulates fatty acid oxidation and tumor cell growth through CPT1A in ER+ breast cancer cells. (A, B) Seahorse assays (A) and their quantifications of basal, maximal respiration, or ATP production (B) from T47D cells transfected with Ctrl siRNA and FDXR si434. (C–G) . Immunoblots (C) , Seahorse assays (D) and their quantifications of basal, maximal respiration, or ATP production (E) , soft agar assays (F) and their quantifications (G) from T47D cells infected with vector (control) or CPT1A followed by transfection with Ctrl siRNA and FDXR si434. (H) Immunoblots of the breast cell lines as indicated. (I–L) . The Kaplan–Meier curves of overall survival (OS) (I, L) , disease free survival (DFS) (J) , and disease special survival (DSS) (K) based on CPT1A expression in ERα-positive patients from METABRIC (I–K) and TCGA (L) cohorts. Patients were rank-ordered and divided into two equal groups (low in blue and high in red), using the CPT1A gene expression levels. *, **, and *** denote P-value of < 0.05, 0.01, and 0.005, respectively. ns denotes not significant.

Article Snippet: Rabbit FDXR (15584-1-AP) and CPT1A antibodies (15184-1-AP) were purchased from Proteintech.

Techniques: Transfection, Western Blot, Infection, Plasmid Preparation, Control, Expressing, Gene Expression

Journal: Frontiers in Oncology

Article Title: FDXR drives primary and endocrine-resistant tumor cell growth in ER+ breast cancer via CPT1A-mediated fatty acid oxidation

doi: 10.3389/fonc.2023.1105117

Figure Lengend Snippet: The effect of CPT1 inhibitor etomoxir on primary and endocrine resistant ER+ breast cancer cell growth. (A–D) Cell proliferation (A, B) and 2D colony formation (C, D) from T47D or MCF7 cells with or without the indicated doses of etomoxir treatment for 4 days and 10 days, respectively.

Article Snippet: Rabbit FDXR (15584-1-AP) and CPT1A antibodies (15184-1-AP) were purchased from Proteintech.

Techniques:

Journal: Frontiers in Oncology

Article Title: FDXR drives primary and endocrine-resistant tumor cell growth in ER+ breast cancer via CPT1A-mediated fatty acid oxidation

doi: 10.3389/fonc.2023.1105117

Figure Lengend Snippet: The effect of combined CPT1 inhibitor and fulvestrant treatment on primary and endocrine resistant ER+ breast tumor cell growth. (A–F) Cell survival analysis of the indicated T47D or MCF7 cell lines under the indicated treatments for 4 days. **, and *** denote P-value of < 0.05, 0.01, and 0.005, respectively. NS denotes not significant.

Article Snippet: Rabbit FDXR (15584-1-AP) and CPT1A antibodies (15184-1-AP) were purchased from Proteintech.

Techniques:

Journal: Journal of Thoracic Disease

Article Title: SH3BGRL2 as a vital tumor suppressor and prognostic factor in human esophageal squamous cell carcinoma

doi: 10.21037/jtd-2025-1878

Figure Lengend Snippet: mRNA and protein expression levels and prognostic significance of SH3BGRL2 in ESCC. (A) Differentially expressed mRNAs between ESCC tumor samples and adjacent normal tissues identified by RNA sequencing (T: tumor tissue; N: normal tissue). (B,C) SH3BGRL2 expression levels in ESCC as analyzed via the GEPIA database (P<0.01) and GEO database (both P<0.05, GSE23400, GSE17351, and GSE45670). (D) Representative tissue microarray images of SH3BGRL2 staining via immunohistochemistry (×200): positive SH3BGRL2 expression in tumor tissues (a) and normal tissues (b) and negative SH3BGRL2 expression in tumor tissues (c) and normal tissues (d). (E) Percentages of SH3BGRL2-positive samples in tumor and nontumor esophageal tissues (31.2% vs. 51.0%; P<0.001). (F,G) Kaplan-Meier curves showing the disease-free survival (F) or overall survival (G) of patients with ESCC and higher SH3BGRL2 expression and in those with lower SH3BGRL2 expression. Error bars represent the standard deviation of the mean. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 (Student t -test, one-way ANOVA). ANOVA, analysis of analysis; DFS, disease-free survival; ESCC, esophageal squamous cell carcinoma; GEO, Gene Expression Omnibus; GEPIA, Gene Expression Profiling Interactive Analysis; OS, overall survival; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2.

Article Snippet: Standard IHC was performed with a primary antibody against human SH3BGRL2 (#21944-1-AP; Proteintech, Rosemont, IL, USA) at a dilution of 1:200.

Techniques: Expressing, RNA Sequencing, Microarray, Staining, Immunohistochemistry, Standard Deviation, Gene Expression, Binding Assay

Journal: Journal of Thoracic Disease

Article Title: SH3BGRL2 as a vital tumor suppressor and prognostic factor in human esophageal squamous cell carcinoma

doi: 10.21037/jtd-2025-1878

Figure Lengend Snippet: SH3BGRL2 inhibited the proliferation of ESCC PDCs. (A) RNA sequencing and western blot analysis of SH3BGRL2 expression levels in different ESCC PDCs. Western blot assays validated the efficiencies of SH3BGRL2 knockdown in ZEC043, ZEC056, and ZEC145 cells (B,C) and overexpression in ZEC014 cells (D). CCK-8 and colony formation with crystal violet staining assays analyzed cell proliferation in ZEC043, ZEC056, ZEC145 cells (E-H), and ZEC014 cells (I). Each picture represents a well of a 6-well plate. Data are presented as the mean ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001 (Student t -test). The grayscale analysis values of western blot bands are listed below the bands. CCK-8, Cell Counting Kit-8; ESCC, esophageal squamous cell carcinoma; OD, optical density; PDC, patient-derived cell line; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2.

Article Snippet: Standard IHC was performed with a primary antibody against human SH3BGRL2 (#21944-1-AP; Proteintech, Rosemont, IL, USA) at a dilution of 1:200.

Techniques: RNA Sequencing, Western Blot, Expressing, Knockdown, Over Expression, CCK-8 Assay, Staining, Standard Deviation, Cell Counting, Derivative Assay, Binding Assay

Journal: Journal of Thoracic Disease

Article Title: SH3BGRL2 as a vital tumor suppressor and prognostic factor in human esophageal squamous cell carcinoma

doi: 10.21037/jtd-2025-1878

Figure Lengend Snippet: SH3BGRL2 suppressed the growth of ESCC PDCs in vivo . (A) Representative images of BALB/c nude mice subcutaneously injected with vector control (upper row) or SH3BGRL2 knockdown ZEC-145 cells (lower row). (B) Analysis of tumor volume of mice measured weekly (n=8 per group). (C) Analysis of tumor weight of xenograft tumors 4 weeks after tumor inoculation (n=8 per group). Data are presented as the mean ± standard deviation. *, P<0.05 (Student t -test and Chi-squared test). ESCC, esophageal squamous cell carcinoma; PDC, patient-derived cell line; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2.

Article Snippet: Standard IHC was performed with a primary antibody against human SH3BGRL2 (#21944-1-AP; Proteintech, Rosemont, IL, USA) at a dilution of 1:200.

Techniques: In Vivo, Injection, Plasmid Preparation, Control, Knockdown, Standard Deviation, Derivative Assay, Binding Assay

Journal: Journal of Thoracic Disease

Article Title: SH3BGRL2 as a vital tumor suppressor and prognostic factor in human esophageal squamous cell carcinoma

doi: 10.21037/jtd-2025-1878

Figure Lengend Snippet: SH3BGRL2 inhibited the EGR1 expression of ESCC cells. (A) Differentially expressed genes between SH3BGRL2-silenced and vector control ZEC145 cells. (B) Elevated mRNA expression of EGR1 in SH3BGRL2-silenced ZEC145 and vector control cells, as indicated in orange borders. (C) Transcription factors associated with differentially expressed genes between SH3BGRL2-silenced and vector control ZEC145 cells. Results show the significant enrichment of the C2H2 zinc finger transcription factor family (zf-C2H2). X-axis: the number of genes in each transcription factor family. (D) The significant negative correlation between EGR1 and SH3BGRL2 expression in ESCC tissues as analyzed via TCGA database (P<0.001). (E) Quantitative reverse transcription-PCR confirmed that EGR1 mRNA expression was increased in SH3BGRL2-knockdown cells (P<0.001). (F,G) Protein expression of EGR1 in SH3BGRL2-silenced ZEC145 cells and SH3BGRL2-overexpressing ZEC014 cells and corresponding vector control cells. Data are presented as the mean ± standard deviation. The grayscale analysis values of western blot bands are listed below the bands. **, P<0.01; ***, P<0.001 (Student t -test). EGR1, early growth response 1; ESCC, esophageal squamous cell carcinoma; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2; TCGA, The Cancer Genome Atlas; TPM, transcripts per million.

Article Snippet: Standard IHC was performed with a primary antibody against human SH3BGRL2 (#21944-1-AP; Proteintech, Rosemont, IL, USA) at a dilution of 1:200.

Techniques: Expressing, Plasmid Preparation, Control, Reverse Transcription, Knockdown, Standard Deviation, Western Blot, Binding Assay

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Analysis of the correlation between FUS protein and myocardial infarction. (a) Enrichment Analysis Bar Plot based on differential gene expression profiles in lncRNA microarray analysis.(b) Detection information about lncRNA LOC101928697 binding to FUS proteins in AnnoLnc2 database. (c) Detection information about lncRNA LOC101928697 binding to FUS protein in RBPDP database. (d) Scores in the RPISeq database on the model of lncRNA LOC101928697 binding to FUS protein. (e-g) Prediction information about lncRNA LOC101928697 binding to FUS protein in catRAPID website, (e) Statistical map information about protein and RNA binding sites, (f) Total scoring information, and (g) Interaction map showing the interaction region between protein and RNA. (h-i) Analyses about bioinformatics techniques based on GSE163772 in the GEO database, where (h) is a statistical map of FUS gene expression in endothelial cells of a mouse model of myocardial infarction, and (i) A scatter plot about the correlation between the level of FUS gene expression and the disease state (control vs. myocardial infarction).

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Gene Expression, Microarray, Binding Assay, RNA Binding Assay, Control

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Interaction of exosomal lncRNA LOC101928697 with FUS proteins. (a and b) The western blot detection of FUS protein expression in each group of cells and the statistical graph. (c) Statistical graph of RT-qPCR to detect the expression of FUS at the mRNA level in each group of cells. (d) The fluorescence graph of fluorescence in situ hybridization (FISH) experiment. In which FUS was labeled with green fluorescence, lncRNA LOC101928697 was labeled with red fluorescence, and the nucleus was labeled with blue fluorescence (20×). (e) Western blot detection of FUS protein following RNA pull-down using sense or antisense LOC101928697 transcripts. (f) Quantification of FUS protein enrichment in sense RNA pull-down versus antisense control, based on densitometric analysis. (g-h) Western blot detection of FUS protein expression in each group of cells after knockdown or overexpression of lncRNA LOC101928697 and the statistical graphs. (i) Statistical graph of mRNA level expression of FUS in each group of cells after knockdown or overexpression of lncRNA LOC101928697 by RT-qPCR assay. a p < 0.05 compared to control group. b p < 0.05 compared to exosome group. c p < 0.05 compared to siRNA + exosome group.

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Labeling, Protein Enrichment, Control, Knockdown, Over Expression

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Fold changes in the microarray for all 35 genes were found to be differentially regulated by C. pneumoniae infection and satisfied the comparison filtering criteria

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Microarray, Infection, Comparison, Binding Assay, Coagulation

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: C. pneumoniae stimulates TLR2 expression in rVSMCs. (A) Validation of TLR2 gene expression profiling using quantitative real-time RT-PCR normalized to GAPDH expression. Data shown are mean values of PCR replicates from individual groups. (B) TLR2 mRNA expression at the indicated time points after C. pneumoniae infection. rVSMCs infected with C. pneumoniae (5 × 105 IFU) for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h or control cells were lysed to prepare the total RNA. cDNA was amplified for 30 cycles, and PCR products were separated by agarose gel electrophoresis. Relative TLR2 mRNA expression levels determined by standard PCR (n = 3 replicates per group). (C) C. pneumoniae infection stimulates TLR2 protein expression in rVSMCs. Cells were infected with C. pneumoniae for 0 h, 12 h, or 24 h. Cell lysates were separated by SDS-PAGE, and blots were probed with anti-TLR2 and anti-β-actin antibodies, followed by donkey anti-goat IgG-HRP and goat anti-mouse IgG-HRP antibodies, and developed with enhanced chemiluminescence (ECL).

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing, Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Infection, Control, Amplification, Agarose Gel Electrophoresis, SDS Page

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Confocal microscopy analysis of the TLR2 expression pattern in C. pneumoniae-infected rVSMCs. rVSMCs were grown in coverslips and infected with C. pneumoniae for 60 h and then were fixed, permeabilized, and stained using specific antibodies. C. pneumoniae inclusions were observed using a mouse polyclonal antibody to C. pneumoniae. Fluorescence micrographs were stained with TLR2-specific antibody. Cell nucleus was stained with DAPI (4′,6-diamidino-2-phenylindole).

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Confocal Microscopy, Expressing, Infection, Staining, Fluorescence

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: Effects of TLR2 on rVSMC migration induced by C. pneumoniae infection. The TLR2-neutralizing antibody was added 1 h before C. pneumoniae infection. (A) Wound healing assay. “Scratch wounds” were created by scraping the confluent cell monolayer with a sterile pipette tip, and then cells were infected with C. pneumoniae at an infectious dose of 5 × 105 IFU. Photographs were taken of the same wounded area of each well at 0 h and 24 h. The scratched regions were photographed under an inverted Nikon microscope (×100 magnification) at 24 h after C. pneumoniae infection. Migration velocity is presented as a ratio of the cellular recoverage area to the whole wound area. *, P < 0.05 versus control; **, P < 0.05 versus C. pneumoniae infection group. (B) Transwell migration assay. Cell morphology was observed by staining with 0.1% crystallin violet. The number of cells that had migrated through the pores was quantified by counting nine independent visual fields using a microscope (×200 magnification). *, P < 0.05 versus control; **, P < 0.05 versus the C. pneumoniae infection group.

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Migration, Infection, Wound Healing Assay, Sterility, Transferring, Microscopy, Control, Transwell Migration Assay, Staining

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

doi: 10.1128/IAI.01087-13

Figure Lengend Snippet: The TLR2-neutralizing antibody suppresses Akt phosphorylation induced by C. pneumoniae infection. rVSMCs cultured for 24 h in 6-well plates were incubated with the TLR2-neutralizing antibody (10 μg/ml) and then infected with C. pneumoniae. Equal amounts of protein lysates were subjected to SDS-PAGE, and blots were probed with anti-Akt, anti-phospho-Akt (Ser 473), and anti-β-actin antibodies, followed by the corresponding horseradish peroxidase-conjugated secondary antibodies, and developed with ECL. P-Akt indicates phosphorylated Akt. *, P < 0.05 versus control; **, P < 0.05 versus the C. pneumoniae infection group.

Article Snippet: The following antibodies were used: primary mouse polyclonal anti- C. pneumoniae (CPN0308), which was kindly provided by Guangming Zhong (San Antonio, TX), goat polyclonal antibodies to TLR2 (Santa Cruz, CA), TLR2-neutralizing antibody (AbD Serotec, Kidlington, United Kingdom), rabbit anti-Akt and anti-phospho-Akt monoclonal antibodies (Ser 473) (Cell Signaling Technology, Beverly, MA), and mouse anti-β-actin monoclonal antibody (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China).

Techniques: Phospho-proteomics, Infection, Cell Culture, Incubation, SDS Page, Control

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Cyclin D1-Dependent and Independent Function in Estrogen-Regulated Development in Vivo. A, Schematic depicting experimental procedure for ovariectomy and estrogen pellet implantation (n = 16 female mice). Mice were implanted with an estrogen pellet or placebo pellet 14 days after ovariectomy. Tissues were harvested at day 21. B, The representative images of uterus from cyclin D1+/+ and cyclin D1−/− mice with or without estrogen treatment. Graph depicts uterus weights as a percentage of body weight in cyclin D1+/+ and cyclin D1−/− mice with or without estrogen treatment. C, Mouse mammary gland whole mounts stained with Carmine dye.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: In Vivo, Staining

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Genome-Wide Profiling of Cyclin D1-Dependent Estrogen-Regulated Genes in Vivo. A, Venn diagram displays the number of genes that were differentially regulated by estrogen in cyclin D1+/+ mouse mammary glands compared with cyclin D1−/− mouse mammary glands. The directionality of change is depicted by up and down arrows. B, Cyclin D1-dependent estrogen-regulated genes were grouped by hierarchical clustering via complete linkage (Cluster 3.0) and visually depicted using Treeview (left). The up-regulated genes are in red and down-regulated genes are in green (P < .05). Chart to the right of heat map depicts Log2 fold change of E2-induced and -repressed genes comparing cyclin D1+/+ with cyclin D1−/− mouse mammary glands. C–E, DAVID analysis was used to classify the pathways differentially regulated by cyclin D1 in E2-treated mice. Pathways depicted represent member genes from microarray analysis of estrogen-treated cyclin D1+/+ mouse mammary glands vs cyclin D1−/− mouse mammary glands, growth factors (C), growth factor receptors (D), and peptidases (E). V, vehicle; Veh., vehicle.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Genome Wide, In Vivo, Microarray

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Genes Regulated by E2 in Vivo Are Bound by Cyclin D1 by ChIP-Seq. A, Comparison of genes occupied by cyclin D1 in ChIP-Seq to those regulated by E2 in cyclin D1+/+ mouse mammary gland and (B) those E2-responsive genes that are regulated in a cyclin D1-dependant manner. C, Location of and (D) representative tag density profiles for, cyclin D1-occupied genes that are regulated by E2. Vertical axis shows average peak height and horizontal axis depicts chromosomal location of cyclin D1-associated interval sequence. TSS, transcription start site.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: In Vivo, ChIP-sequencing, Comparison, Sequencing

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: ERα Induces AREG Gene Expression in a Cyclin D1-Dependent Manner. A and B, MCF7 cells were transfected with siRNAs targeting cyclin D1 and treated with vehicle or E2 (10−8 M) for 24 hours. CCND1 and AREG mRNA abundance was determined by quantitative RT-PCR. C, Western blot was performed to determine the cellular levels of AREG expression in cyclin D1 knockdown cells compared with scramble siRNA control. β-tubulin was included as loading control for protein abundance. D, The concentration of AREG in cell culture medium was measured by ELISA. Concentration of AREG in the conditioned media was normalized to total protein. Data are mean ± SEM. E, AREG promoter luciferase reporter plasmids were transfected into MCF7 cells with a cyclin D1 expression vector. Relative luciferase activity is shown as mean ± SEM normalized to β-galactosidase activity of a cotransfected vector and as (F) fold-induction by cyclin D1. G, Promoter sequence alignment of mouse and human amphiregulin promoter. Homologous nucleotides (:) and regions of discontinuity (−) are indicated. Predicted BRCA1 sites are highlighted for human (− strand), and mouse (2 sites on + strand) with predicted confidence values of 86%, 87%, and 94% respectively. Ctrl., control.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Gene Expression, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Control, Quantitative Proteomics, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Luciferase, Plasmid Preparation, Activity Assay, Sequencing

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Cyclin D1 Is Recruited to a BRCA1 Binding Site. A, ChIP assay in MCF7 cells treated with E2 (10 nM) for ERα at the AREG gene promoter. B. The pS2 gene was included as a positive control. C, Western blot shows FLAG-cyclin D1 expression in transduced MCF7 cells. D, ChIP assay to determine cyclin D1 occupancy at the AREG gene promoter. Using either MCF7 cells transduced with FLAG-cyclin D1 or (E) MCF7 cells ChIP analysis with antibodies directed to endogenous cyclin D1, BRCA1 or ERα (E2 10 nM 24 hours). F and G, GST pulldown was performed to determine the minimal region of BRCA1 required for cyclin D1 binding. H and I, GST-cyclin D1 or mutants were incubated with in vitro translated BRCA1. The N terminus (1–100 amino acids) of cyclin D1 was required for BRCA1 binding. IB, immunoblot; IP, immunoprecipitation; IVT, in vitro translation.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Binding Assay, Positive Control, Western Blot, Expressing, Transduction, Incubation, In Vitro, Immunoprecipitation

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: E2 Induces Cyclin D1 Distribution within a LMW Complex with AIB1. A,. Western blot analysis of Superose 6 chromatography from asynchronously cycling MCF7 cell lysates using antibodies as indicated to the left of the figure. The molecular weight of the fractions is indicated at the bottom of the figure. Cells were treated with (+) E2 (10−8 M). The coeluting fractions > 4 mDa (HMC) and 670 kDa (LMC) are indicated by the boxes. B, Western blot analysis of MCF7 cell extracts after Superose 6 chromatographic fractionation. The antibodies are as indicated. Extracts were treated with E2 (10−8M) for 30 minutes. Molecular weight markers are shown below panel B, indicating the HMC (4 MDa) or LMC (670 kDa). C, Hormone-deprived MCF7 cells infected with shCCND1 or shControl were treated for 1 hour with 10 nM E2 or vehicle control; ERα was immunoprecipitated followed by Western blotting for AIB1, cyclin D1, and ERα. IP, immunoprecipitation; V, vehicle.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Western Blot, Chromatography, Molecular Weight, Fractionation, Infection, Control, Immunoprecipitation

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: ERα Recruitment to the LMW Complex Requires Cyclin D1. A, Western blot analysis of superose 6 chromatographic fractions from female cyclin D1−/− or cyclin D1+/+ mice cell lysates (liver) using antibodies as indicated to the left of the figure. The molecular weight markers are shown below. B, The relative abundance of ERα in the HMC or LMC (670 kDa) is shown graphically indicating increased ERα in the HMC in cyclin D1−/− mice. C, Schematic presentation of cyclin D1 regulation of ERα activation proposes a model in which cyclin D1 participates in ERα signaling by binding to BRCA1 and in the presence of E2 facilitates an LMC that includes ERα and AIB1. Cyclin D1 binding to BRCA1 antagonized BRCA1 action, including BRCA1 repression of Areg expression.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Western Blot, Molecular Weight, Activation Assay, Binding Assay, Expressing

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 3. Generation of Tg-MPP1 mice. A, Upper panel: scheme of the plasmid used for the generation of Tg-MPP1 mice. Lower panel: PCR genotyping of ear- punch biopsies from 11 Tg-MPP1-positive mice with stable integration of the transgenic MPP1 cDNA into the genomic DNA. The negative control (-) did not contain genomic DNA, and the linearized MPP1 plasmid DNA (P) was used as a positive control. The lane marked with M, is the DNA marker. B, Immunoblot detection of the MPP1 protein in heart protein extracts from Tg-MPP1 mice and non-transgenic B6 mice. The left panel is a representative immunoblot, and the right panel shows quantitative data (mean ± s.d., n = 4 mice per group). The p- value is indicated and was determined by the unpaired, two-tailed, t-test. The lower panel is a control immunoblot detecting α-tubulin. C, As a specificity control of the monoclonal anti-MPP1 antibody, immunoblot detection of MPP1 in MPP1-transfected HEK cells was performed in comparison to mock- transfected HEK cells. The lower blot shows a loading control detecting GAPDH.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Plasmid Preparation, Transgenic Assay, Negative Control, Positive Control, Marker, Western Blot, Two Tailed Test, Control, Transfection, Comparison

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 2. Upregulation of the MAGUK family protein, MPP1, in three different heart failure models. A,B, Probe set intensities of cardiac Mpp iso forms were determined by whole genome microarray gene expression profiling of the AAC-induced heart failure model in comparison to sham-operated con trols (A), and of Apoe−/− mice with long-term atherosclerosis-induced heart failure in comparison to age-matched non-transgenic B6 mice (B). Affymetrix IDs of probe sets detecting Mpp1, Mpp2, Mpp3, Mpp4, Mpp5, Mpp6, and Mpp7 are indicated. Data are mean values ± s.d. (four hearts per microarray chip with two microarray chips per group). Probe set intensities are taken from NCBI GEO dataset GSE25765. C, Cardiac transcript levels of Mpp isoforms in 8-month-old, male Tg-RKIP mice were determined by NGS in comparison to age- and sex- matched, non-transgenic FVB controls (NCBI GEO dataset GSE191316) (mean ± s.d., n = 3 mice per group). Statistically significant differences between transcript levels of the heart failure groups and the respective control group were determined by Tukey’s test, and are indicated for each individual MAGUK gene (A,B,C). P-values for statistically different MAGUK genes are indicated. All other MAGUK genes were not significantly different (n.s.) between the heart failure and control groups.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Microarray, Gene Expression, Comparison, Transgenic Assay, Control

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 4. Tg-MPP1 mice develop features of heart failure with cardiac enlarge ment at an age of 8 months. A, Echo cardiographic measurement of the left ventricular ejection fraction (LVEF, %), the fractional shortening (FS, %), the left ventricular internal diameter in diastole (LVIDd), and the left ventricular internal diameter in systole (LVIDs) of 8-month- old, male Tg-MPP1 mice, and sex- and age-matched, non-transgenic B6 mice. Echocardiographic measurements were performed under anesthesia. B, Determi nation of the body weights (BW), heart weights (HW), and the heart weight to body weight ratios (HW/BW) of 8-month- old, male Tg-MPP1 mice, and of sex- and age-matched, non-transgenic B6 mice. Data (A,B) are the mean ± s.d., n = 6 mice per group. P-values were determined by the unpaired, two-tailed t-test. C, Immu nohistological detection of MPP1 on heart sections of Tg-MPP1 mice in comparison to those of non-transgenic B6 mice (n = 4 mice/group; bar: 2 mm). Sections were stained with the anti-MPP1 antibody (MPP1) and counterstained with hema toxylin (HE). The right panels show higher magnification images of representative sections from a Tg-MPP1 mouse and a non- transgenic B6 control (bar: 20 μm).

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Transgenic Assay, Two Tailed Test, Comparison, Staining, Control

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 5. Co-localization of AGTR1 with MPP1 in vivo, and increased cardiac AGTR1 protein levels in Tg- MPP1 mice. A, Immunofluorescence detection of MPP1 and AGTR1 on cardiac cryosections from Tg-CMV- AGTR1-Cerulean mice shows co-localization of AGTR1 with MPP1 on sarcolemmal membranes (yellow). MPP1 was stained with mouse monoclonal anti-MPP1 antibody (red), AGTR1-Cerulean was stained with rabbit poly clonal anti-GFP antibodies (green), and nuclei were stained with DAPI (blue). The immunofluorescence co- localization study shows cryosections from four different mice (bar: 40 μm). B, Cardiac AGTR1-specific binding sites were determined on sarcolemmal mem branes of Tg-MPP1 mice and non-transgenic B6 mice by radioligand binding with Sar1,[125I]Tyr4,Ile8-angiotensin II. Data are shown as mean values ± s.d., n = 6 mice per group. The p-value was determined by the unpaired, two- tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: In Vivo, Immunofluorescence, Staining, Binding Assay, Transgenic Assay, Two Tailed Test

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 6. MPP1 increased the cellular contents of AGTR1eYFP in HEK cells. A,B, Cellular AGTR1eYFP levels were increased by co-transfection of HEK293 cells with an MPP1-encoding pcDNA3 expression plasmid (+). Control cells were transfected with the pcDNA3 plasmid without insert (-). Panel (A) shows cellular AGTR1eYFP fluorescence peak intensities at an emission wavelength of 527 nm, and panel (B) shows representative AGTR1eYFP fluorescence emis sion spectra without (grey) and with MPP1-encoding plasmid co-transfection (red). The black line shows a spectrum of control cells transfected with pcDNA3 without insert (Cont.). C,D, Co-transfection of the MPP1-encoding plasmid did not significantly alter cellular ADRB1eYFP levels. Control cells were trans fected with the pcDNA3 plasmid without insert (-). Panel (C) shows cellular ADRB1eYFP fluorescence peak intensities at an emission wavelength of 527 nm, and panel (D) shows representative fluorescence emission spectra of ADRB1eYFP-expressing cells without and with MPP1-encoding plasmid co- transfection. Data (A,C) show mean values ± s.d. (n = 8 biological replicates). P-values were determined by Tukey’s test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Cotransfection, Expressing, Plasmid Preparation, Control, Transfection, Fluorescence

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 7. AGTR1-(1–319)-eYFP with deletion of the carboxyl terminal tail is also enhanced by MPP1 in HEK cells. A, Cellular fluorescence peak intensities at an emission wavelength of 527 nm were deter mined of HEK cells with expression of the full-length AGTR1-(1–359)-eYFP without (-) and with (+) co- transfection of the MPP1-encoding plasmid, and of HEK cells with expression of the truncated AGTR1- (1–319)-eYFP without (-), and with (+) co- transfection of MPP1. Data are mean values ± s.d. (n = 10 biological replicates). P-values were deter mined by Tukey’s test. B, Topological scheme of the full-length AGTR1-(1–359) protein sequence. Trun cated residues of AGTR1-(1–319) are marked in red. The AGTR1 topology was derived from Uniprot (P30556 AGTR1_Human), and the scheme was drawn with Protter, version 1.0. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Fluorescence, Expressing, Cotransfection, Plasmid Preparation, Sequencing, Derivative Assay

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 9. The AGTR1-enhancing effect mediated by MPP1 requires all functional domains of MPP1. A, Scheme of MPP1 functional domains, and of the two MPP1 fragments 1–267 and 268–466, which were tested. B, Cellular fluorescence peak intensities at an emission wavelength of 527 nm were determined of AGTR1eYFP-expressing HEK cells without (-) and with (+) co- transfection of MPP1-encoding plasmid, MPP1-(1–267)-encoding plasmid, MPP1-(268–466)-encoding plasmid, or MPP1-(1–267) and MPP1-(268–466)- encoding plasmids together. Data are presented as mean values ± s.d. (n = 4 biological replicates). P-values were determined by Tukey’s test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Functional Assay, Fluorescence, Expressing, Cotransfection, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 8. Deletion of a putative internal PDZ domain-binding motif in AGTR1-(1–319)-(Δ213-220)-eYFP abolishes the AGTR1-enhancing effect by MPP1 in HEK cells. A, Topological scheme of the AGTR1-(1–359) protein sequence, in which deletions made in construct AGTR1-(1–319)-(Δ213-220) are marked in red. The scheme was drawn with Protter, version 1.0. Residues 213–220 at the beginning of the third intracellular loop of AGTR1 include the sequence “Y-T-L-I”, which could be an internal PDZ domain-binding motif, which is defined by “X-S/T-X-ϕ“ where “X” can be any amino acid, and “ϕ“ is a hydrophobic amino acid. B, Cellular fluorescence peak intensities at an emis sion wavelength of 527 nm were determined of HEK cells without (-) and with stable MPP1 (+) expression, and transfection of AGTR1-(1–319)-eYFP, or AGTR1-(1–319)-(Δ213-220)-eYFP with deletion of a putative internal PDZ domain-binding motif (Δ213-220). Data are presented as mean values ± s.d. (n = 3 biological replicates). P-values were determined by Tukey’s test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Binding Assay, Sequencing, Construct, Fluorescence, Expressing, Transfection

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 10. Upregulation of cardiac Mpp1 transcript levels by diabetes- induced cardiac dysfunction and by Hdac3 deficiency in rodents. A, Car diac Mpp1 transcript levels were up-regulated in rats with diabetes-induced cardiac dysfunction. Data were retrieved from the GEO profile GDS3153 (31), probe set ID 1389963_at of the Affymetrix Rat Expression 230A Array. Hearts were obtained from 12-week-old rats with four weeks of streptozotocin-induced diabetes and from control rats (mean ± s.d., n = 3 hearts per group). B, Upregulation of cardiac Mpp1 in hearts from 6-week-old mice with Hdac3- deficiency (Hdac3 KO) in heart and skeletal muscle (HDAC3fl/fl/MCK-Cre), which develop a severe hypertrophic cardiomyopathy on a high fat diet (32). Control hearts were isolated from wild-type mice (HDAC3fl/fl). Data were taken from the GEO profile GDS4886, probe set ID 106447481 of the Affymetrix Mouse Gene 1.0 ST Array (mean ± s.d., n = 4 male mice per group). P-values were determined by the unpaired, two-tailed t-test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Expressing, Control, Isolation, Two Tailed Test

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 11. Detection of increased MPP1 transcript levels in peripheral blood mononuclear cells of old human research participants. A-F, Transcript levels of MPP1 (A), GRK2 (B), GRK3 (C), DUSP3 (D), LRRN3 (E), and CD27 (F) in PBMC from old (age: 75–89 years, y; n = 5) human research participants were determined by whole genome microarray gene expression profiling. PBMC isolated from middle-aged research participants (age: 35–50 years, y; n = 4) served as the control group. Data are shown as mean values ± s.d. P-values were determined by the two- tailed (A,B,D,E,F), or one-tailed (C), unpaired t-test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Microarray, Gene Expression, Isolation, Control, Two Tailed Test, One-tailed Test

Journal: Cancer cell

Article Title: Elevated CXorf67 Expression in PFA Ependymomas Suppresses DNA Repair and Sensitizes to PARP Inhibitors

doi: 10.1016/j.ccell.2020.10.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Normal mouse IgG , Santa Cruz Bio , Cat# sc-2025; RRID: AB_737182.

Techniques: Virus, Recombinant, Staining, Cloning, Transfection, Single Cell Gel Electrophoresis, Microarray, Gene Expression, Sequencing, Control, Software