Journal: International Journal of Molecular Medicine

Article Title: Identification of differentially expressed genes and preliminary validations in cardiac pathological remodeling induced by transverse aortic constriction

doi: 10.3892/ijmm.2019.4291

Figure Lengend Snippet: Forward and reverse primer pairs.

Article Snippet: The sections were incubated with anti-integrin subunit beta-like 1 (Itgbl1; Santa Cruz Biotechnology, Inc.; sc-365162, 1:50) and anti-Asporin (Abcam; ab58741, 1:100), and developed using peroxidase-coupled secondary antibodies and 3,3′-diaminobenzidine as a substrate.

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: Identification of differentially expressed genes and preliminary validations in cardiac pathological remodeling induced by transverse aortic constriction

doi: 10.3892/ijmm.2019.4291

Figure Lengend Snippet: DEGs identified from the four profiles (n=24).

Article Snippet: The sections were incubated with anti-integrin subunit beta-like 1 (Itgbl1; Santa Cruz Biotechnology, Inc.; sc-365162, 1:50) and anti-Asporin (Abcam; ab58741, 1:100), and developed using peroxidase-coupled secondary antibodies and 3,3′-diaminobenzidine as a substrate.

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: Identification of differentially expressed genes and preliminary validations in cardiac pathological remodeling induced by transverse aortic constriction

doi: 10.3892/ijmm.2019.4291

Figure Lengend Snippet: Reverse transcription-quantitative polymerase chain reaction analysis validation of mRNA expression levels of the selected common differentially expressed genes. (A-O) mRNA levels of Aqp8, Aspn, Cnksr1, Col8a1, Frzb, Fstl1, Gdf15, Itgbl1, Mfap4, Mfap5, Meox1, Ltbp2, Retnla and Pamr1 in heart tissues of mice in the TAC and sham-operated groups. Values shown are normalized to GAPDH (n=6); ** P<0.01, *** P<0.001; NS, not significant; TAC, transverse aortic constriction.

Article Snippet: The sections were incubated with anti-integrin subunit beta-like 1 (Itgbl1; Santa Cruz Biotechnology, Inc.; sc-365162, 1:50) and anti-Asporin (Abcam; ab58741, 1:100), and developed using peroxidase-coupled secondary antibodies and 3,3′-diaminobenzidine as a substrate.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Biomarker Discovery, Expressing

Journal: International Journal of Molecular Medicine

Article Title: Identification of differentially expressed genes and preliminary validations in cardiac pathological remodeling induced by transverse aortic constriction

doi: 10.3892/ijmm.2019.4291

Figure Lengend Snippet: Western blotting and immunohistochemistry validation of protein expression levels of Itgbl1 and Aspn. (A) Immunohistochemistry images of protein expression of Itgbl1. (B) Immunohistochemistry images of protein expression of Itgbl1. (C) Western blots of protein expression of Itgbl1 and Aspn. (D-G) Immunohistochemistry and western blot bar graph (n=6). Values shown are normalized to GAPDH; * P<0.05, ** P<0.01, *** P<0.001. ASPN, Asporin; TAC, transverse aortic constriction.

Article Snippet: The sections were incubated with anti-integrin subunit beta-like 1 (Itgbl1; Santa Cruz Biotechnology, Inc.; sc-365162, 1:50) and anti-Asporin (Abcam; ab58741, 1:100), and developed using peroxidase-coupled secondary antibodies and 3,3′-diaminobenzidine as a substrate.

Techniques: Western Blot, Immunohistochemistry, Biomarker Discovery, Expressing

Journal: International Journal of Molecular Medicine

Article Title: Identification of differentially expressed genes and preliminary validations in cardiac pathological remodeling induced by transverse aortic constriction

doi: 10.3892/ijmm.2019.4291

Figure Lengend Snippet: Expression of Itgbl1 and Asporin in cardiomyocytes (CMs) and cardiac fibroblasts (CFs). (A) Immunofluorescence staining of CMs with anti-α-actinin. (B) Immunofluorescence staining of CFs with anti-α-SMA. (C) The cell surface area of CMs in the indicated groups (n=6 samples, with 100+ cells per group). (D) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis validation of mRNA expression levels of Itgbl1 and ASPN in CMs and CFs (n=6 sample). (E) RT-qPCR analysis validation of mRNA expression levels of Itgbl1 and ASPN in TGFβ-treated CFs (n=6 sample). (F) RT-qPCR analysis validation of mRNA expression levels of Itgbl1 and ASPN in Ang II-treated CMs (n=6 sample) * P<0.05, # P<0.05, NS, not significant. ASPN, Asporin; SMA, smooth muscle actin; TGF, transforming growth factor; Ang, angiotensin.

Article Snippet: The sections were incubated with anti-integrin subunit beta-like 1 (Itgbl1; Santa Cruz Biotechnology, Inc.; sc-365162, 1:50) and anti-Asporin (Abcam; ab58741, 1:100), and developed using peroxidase-coupled secondary antibodies and 3,3′-diaminobenzidine as a substrate.

Techniques: Expressing, Immunofluorescence, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome.

doi: 10.1016/j.bbadis.2023.166991

Figure Lengend Snippet: Fig. 2. ATP5PO is expressed in the myenteric plexus of the human colon. A. Intestine of a healthy individual showing ATP5PO staining in the myenteric plexus (arrowheads) and the epithelium (arrow). Scale bars represent 100 μm. B. Representative images of the (ganglionic) intestines of a HSCR patient without DS, and C. a HSCR patient with DS. Similar expression patterns of ATP5PO were detected. Scale bars represent 100 μm (left panels) and 50 μm (right panels); n = 3 per group.

Article Snippet: Primary antibodies specific for ATP5PO (HPA041394 Atlas antibodies, 1:100) and B-actin (sc-69879 Santa Cruz, 1:1000) were used, followed by incubation with the secondary antibodies, IRDye 800CW Goat anti-mouse and IRDye 680RD Goat anti-Rabbit (Li-Cor).

Techniques: Staining, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome.

doi: 10.1016/j.bbadis.2023.166991

Figure Lengend Snippet: Fig. 3. ATP5PO RNA overexpression leads to reduced ATP5PO protein. A. RT-qPCR data showing ATP5PO RNA expression levels in SH-SY5Y and SH-SY5Y-ATP5PO stable cells. Mean of two technical replicates is visualized. Error bar represents standard deviation; *p < 0.05. Experiment was performed in triplicate. B. Representative image of western blot. C. Western blot quantification showing reduced ATP5PO protein expression in SH-SY5Y-ATP5PO cells, normalized to b-actin. Mean of three independent experiments is plotted. Error bar represents standard deviation; **p < 0.01.

Article Snippet: Primary antibodies specific for ATP5PO (HPA041394 Atlas antibodies, 1:100) and B-actin (sc-69879 Santa Cruz, 1:1000) were used, followed by incubation with the secondary antibodies, IRDye 800CW Goat anti-mouse and IRDye 680RD Goat anti-Rabbit (Li-Cor).

Techniques: Over Expression, Quantitative RT-PCR, RNA Expression, Standard Deviation, Western Blot, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome.

doi: 10.1016/j.bbadis.2023.166991

Figure Lengend Snippet: Fig. 4. Overexpression of ATP5PO impairs neuronal differentiation and production of ATP levels. A. Representative images of undifferentiated (T0) and differentiated (T72) control SH-SY5Y and SH-SY5Y-ATP5PO cells. Phalloidin in red, TUJ1 in green and DAPI in blue. A differentiated neuronal morphology was only observed in control cells. Moreover, TUJ1 expression was reduced in SH-SY5Y-ATP5PO cells when compared to SH-SY5Y. Scale bar represents 50 μm. B. RT-qPCR data showing increased expression of neuronal markers (TUJ1 and GAP43) in control SH-SY5Y cells 72 h after differentiation, but not in SH-SY5Y-ATP5PO cells. Mean of two technical replicates is visualized. (two-way ANOVA TUJ1: effect of ATP5PO overexpression p < 0.01, effect of differentiation p < 0.05, and interaction effect between genotype and differentiation p < 0.01; GAP43: effect of ATP5PO overexpression p < 0.0001, effect of differentiation p < 0.0001, and interaction effect between genotype and differentiation p < 0.0001) C. HuC/D stainings performed at 4 dpf showed that over expression of ATP5PO in zebrafish, leads to reduced neuronal differentiation. D. ATP levels in control SH-SY5Y cells increase, as expected, upon induction of dif ferentiation, whereas in SH-SY5Y-ATP5PO cells there is a decrease in ATP levels; **p < 0.01, ****p < 0.0001. A–B and D Average of three independent experiments is visualized. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies specific for ATP5PO (HPA041394 Atlas antibodies, 1:100) and B-actin (sc-69879 Santa Cruz, 1:1000) were used, followed by incubation with the secondary antibodies, IRDye 800CW Goat anti-mouse and IRDye 680RD Goat anti-Rabbit (Li-Cor).

Techniques: Over Expression, Control, Expressing, Quantitative RT-PCR

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome.

doi: 10.1016/j.bbadis.2023.166991

Figure Lengend Snippet: Fig. 5. ATP5PO overexpression shows epistasis with RET. Quantification of the percentage of larvae presenting with ENS phenotypes reminiscent of HSCR in the different experimental groups. Due to ATP5PO overexpression in the retSA2684/+ background, more larvae significantly present with a phenotype reminiscent of total colonic HSCR (black bars), compared to retSA2684/+ alone. ***p < 0.001, **p < 0.01.

Article Snippet: Primary antibodies specific for ATP5PO (HPA041394 Atlas antibodies, 1:100) and B-actin (sc-69879 Santa Cruz, 1:1000) were used, followed by incubation with the secondary antibodies, IRDye 800CW Goat anti-mouse and IRDye 680RD Goat anti-Rabbit (Li-Cor).

Techniques: Over Expression

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: Detection of ITGBL1 mRNA in different ovarian cancer cell lines. ( A ) Schematic representation of four predicted ITGBL1 mRNA variants. Location of primers used for unambiguous detection is indicated by boxes below each mRNA variant. Primers used for concurrent detection of all four isoforms are marked in pink (F-PCR, R-PCR); primers used for cloning of the whole ITGBL1 coding sequence (CDS) are marked in violet (F-cloning and R-cloning); as a reminder, we show location of primers used for quantitative RT-PCR (F-qPCR, R-qPCR; marked in blue). ( B ) Primer sets used for different purposes and PCR product sizes. ( C ) RT-PCR detection of different ITGBL1 mRNA isoforms in wild-type and genetically modified cell lines. The ribosomal 18S rRNA served as the reference. Full gel electrophoresis images from ( C ) are shown in .

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques: Variant Assay, Cloning, Sequencing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Genetically Modified, Nucleic Acid Electrophoresis

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: Construction and verification of cellular models. ( A ) RT-PCR amplified ITGBL1 CDS (variant 1 mRNA) was cloned into expression vector pLNCX2. ( B ) RT-PCR revealed that ITGBL1 mRNA (concurrent amplification of all isoforms) is detectable in the wild-type ES2 cell line (positive control) and in OAW42 and SKOV3 cell lines stably transduced with pLNCX1-ITGBL1 construct, while absent in wild-type and empty pLNCX2-transduced OAW42 and SKOV3 cells. No ITGBL1 signal could be detected by WB in the protein extracts from each analyzed cell line using Sigma-Aldrich HPA005676 antibody. ( C ) WB analysis with the same antibody confirmed presence of ITGBL1 in the culture media from indicated cell cultures (upper panel). Ponceau stained blot is shown to demonstrate protein loading uniformity (lower panel). Full blots from ( C ) are shown in . Full gel electrophoresis image from ( B ) is shown in .

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Variant Assay, Clone Assay, Expressing, Plasmid Preparation, Positive Control, Stable Transfection, Transduction, Construct, Staining, Nucleic Acid Electrophoresis

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: Evaluation of cellular attachment to the uncoated plastic surface. The amount of surface-attached cells was measured 5 min (OAW42) and 15 min (SKOV3) after seeding by crystal violet staining ( A , C ) and by MTS ( B , D ). Statistical significance was assessed with Student’s t -Test; * indicates p < 0.01, *** p < 0.001. Y -axis represents percentage of crystal violet/formazan released from fixed attached cells (mean ± SD, n = 3, each in 12 technical repeats). The amount obtained from control cells was taken as the reference value (100%). ( E , F ) Representative images of surface-attached cells, ITGBL1 -overexpressing and control (crystal violet staining).

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques: Cell Attachment Assay, Staining, Control

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: The effect of ITGBL1 overexpression on the adhesiveness of OAW42 and SKOV3 cells. ( A , B ) comparison of cellular attachment to uncoated and fibronectin or collagen coated plastic surface (crystal violet assay), 5 min (OAW42) and 15 min (SKOV3) after seeding. Y -axis represents percentage of crystal violet released from fixed adherent cells (mean ± SD, n = 3, each in 12 technical repeats). The amount obtained from control cells was taken as the reference value (100%). Statistical significance was determined by one-way analysis of variance (ANOVA) with Scheffe’s adjustment for pairwise comparisons; ** indicates p < 0.01, *** p < 0.001. ( C ) Spreading assay. Images taken at the indicated time points after seeding show that ITGBL1 -overexpressing cells tend to adopt a spread morphology (flatten) quicker than control cells. Flattened cells are indicated by arrowheads.

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques: Over Expression, Comparison, Cell Attachment Assay, Crystal Violet Assay, Control

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: Scratch assay. ( A ) Comparison of scratch area filling time required for OAW42-ITGBL1 and OAW42-PLNCX2 cells. ( C ) Comparison of scratch area filling time by SKOV3-ITGBL1 and SKOV3-PLNCX2 cells; X -axis represents observation time points, Y -axis represents size of the remaining scratch area (mean ± SD, n = 3, each in 10 technical repeats). The initial size of scratch area was assumed as 100%. Statistical significance was assessed with Student’s t -Test with Bonferroni correction for multiple testing; * indicates p < 0.05; ** p < 0.01. ( B , D ) Representative images showing scratch filing by ITGBL1 -overexpressing and control cells at the indicated time points.

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques: Wound Healing Assay, Comparison, Control

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: Comparison of trans-well migration rate and Matrigel invasiveness of ITGBL1 overexpressing and control cell lines. ( A , B ) Trans-well migration assay. Y -axis represents percentage of cells that migrated through the membrane with 8 μm pores. ( C , D ) Matrigel invasion assay. Y -axis represents the percentage of cells that migrated through Matrigel coated trans-well inserts with 8 μm pores (mean ± SD, n = 3, each in 9 technical repeats). The result obtained with control cells was taken as the reference value (100%). Statistical significance was assessed with Student’s t -Test; ** indicates p < 0.01; *** p < 0.001.

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques: Comparison, Migration, Control, Membrane, Invasion Assay

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: IC50 values of Cisplatin and Paclitaxel.

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques:

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: Unsupervised analysis of gene expression profiles in the analyzed cell lines. ( A ) Principal Component Analysis performed on all samples. Samples tend to group by the cell line. This difference is most prominent (74% of variance) and is described by first principal component (PC1). ( B ) Hierarchical clustering of all samples according to PC1. Heat map demonstrates that genes from PC1 have distinct expression pattern in OAW42 and SKOV3 cells. ( C ) Hierarchical clustering of OAW42 samples according to PC2. PC2 differentiates ITGBL1 -overexpressing OAW42 samples from all control OAW42 samples (wild-type and with an empty pLNCX2). ( D ) Hierarchical clustering of OAW42 samples (only empty pLNCX2 controls and samples with ITGBL1 overexpression).

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques: Gene Expression, Expressing, Control, Over Expression

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: Signaling pathways affected by changed expression of the genes from PC1 (PCA performed on OAW42-PLNCX2 and OAW42-ITGBL1 samples). Only 22 arbitrarily chosen pathways are shown (those related with ECM, integrin signaling, focal adhesion, cellular motility, etc.). Full list of 76 significant pathways is shown in Supplementary Material 5K .

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques: Protein-Protein interactions, Expressing

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: Signaling pathways affected by changed expression of the genes from PC1 (PCA performed on SKOV3-PLNCX2 and SKOV3-ITGBL1 samples). Only 44 arbitrarily chosen pathways which are related to ECM, cell junction, cellular motility, ERBB2/ERBB4 signaling, etc. are shown. Full list of 146 significant pathways is shown in Supplementary Material 5O .

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques: Protein-Protein interactions, Expressing

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: Primers used in this study.

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques: Sequencing, Cloning, Control, Variant Assay

Journal: Cancers

Article Title: Evaluation of the Role of ITGBL1 in Ovarian Cancer

doi: 10.3390/cancers12092676

Figure Lengend Snippet: List of antibodies used in Western blot analysis.

Article Snippet: ITGBL1 , Rabbit/Polyclonal , Exon: 3 (fragment), 4, 5 (fragment) , CSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKC , ProSci (29-712) , 1:300.

Techniques: Western Blot, Sequencing