Journal: International Journal of Molecular Sciences

Article Title: Hyaluronan Synthases’ Expression and Activity Are Induced by Fluid Shear Stress in Bone Marrow-Derived Mesenchymal Stem Cells

doi: 10.3390/ijms22063123

Figure Lengend Snippet: Next-generation RNA sequencing analyses of hMSCs exposed to fluid shear stress. ( A ) Principal component analysis (PCA) for fluid shear stress (PC1) and donors (PC2). ( B ) Distribution of genes with more than ten reads; genes are grouped according to significance (adjusted p ≤ 0.05) and log 2 -fold change (≤−2 or ≥2). ( C ) Heatmap of the 50 most significantly regulated genes with the highest and lowest log 2 -fold changes. ( D ) Volcano plot of genes with more than ten reads. Dashed lines show thresholds for significance ( p ≤ 0.05) and log 2 -fold change (≤−2 or ≥2). The top significantly regulated genes with the highest and lowest log 2 -fold change are shown in blue; the significantly changed genes of the hyaluronan biosynthetic pathway (GO:0030213) and the significantly changed hyaluronan (HA) receptors CD44 , HMMR , ICAM1 and LAYN are shown in red.

Article Snippet: Following this, a rabbit anti-HAS2 antibody (1:100, orb157430, Biorbyt, Cambridge, UK) and a mouse anti-CD44 antibody (1:400, #3570, Cell Signaling Technology, Cambridge, UK) were diluted in 1% BSA in DPBS.

Techniques: RNA Sequencing, Shear

Journal: International Journal of Molecular Sciences

Article Title: Hyaluronan Synthases’ Expression and Activity Are Induced by Fluid Shear Stress in Bone Marrow-Derived Mesenchymal Stem Cells

doi: 10.3390/ijms22063123

Figure Lengend Snippet: Hyaluronan biosynthetic process (GO:0030213) and five HA receptors ( CD44 , HMMR , ICAM1 , LAYN and LYVE1 ) in hMSCs exposed to fluid shear stress. ( A ) Heatmap of the gene set. ( B ) Change in expression of the gene set. Black bars show an upregulation of expression by fluid shear stress; white bars show downregulation; dashed lines indicate the thresholds for the log 2 -fold change of ≤−2 and ≥2; asterisks indicate an adjusted p ≤ 0.05. Error bars represent SE.

Article Snippet: Following this, a rabbit anti-HAS2 antibody (1:100, orb157430, Biorbyt, Cambridge, UK) and a mouse anti-CD44 antibody (1:400, #3570, Cell Signaling Technology, Cambridge, UK) were diluted in 1% BSA in DPBS.

Techniques: Shear, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Hyaluronan Synthases’ Expression and Activity Are Induced by Fluid Shear Stress in Bone Marrow-Derived Mesenchymal Stem Cells

doi: 10.3390/ijms22063123

Figure Lengend Snippet: Formation of cell protrusions after the application of 20 h of fluid shear stress on primary hMSCs and HAS2-overexpressing immortalized hMSCs (SCP1-HAS2-eGFP). Cells were stained for confocal microscopy with phalloidin for F-actin (white); immunostained for CD44 (red) and HAS2 (green); and counterstained with DAPI for the nuclei (blue). ( A ) Z-stack maximum projection of fluid shear stress-stimulated and static (control) cells. ( B ) Y-stack maximum projection of shear stressed and control cells. Note the increased number of actin-containing membrane protrusions upon shear stress. ( C ) Length of protrusions. ( D ) Density of protrusions. Asterisks indicate a p -value < 0.05. Error bars represent SD.

Article Snippet: Following this, a rabbit anti-HAS2 antibody (1:100, orb157430, Biorbyt, Cambridge, UK) and a mouse anti-CD44 antibody (1:400, #3570, Cell Signaling Technology, Cambridge, UK) were diluted in 1% BSA in DPBS.

Techniques: Shear, Staining, Confocal Microscopy, Control, Membrane

Journal: Scientific Reports

Article Title: Nexilin/ NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP

doi: 10.1038/s41598-018-31328-2

Figure Lengend Snippet: Nexilin/ NEXN is expressed in striated and smooth muscle cells (SMCs) and correlates at the mRNA level with the smooth muscle myosin. Panel A shows RNA-Seq data (GTExPortal.org) for the top-twelve NEXN -expressing human tissues (N = 6–430, Esophagus-M: muscular layer, -GEJ: Gastroesophageal Junction). Panel B shows immunohistochemical staining for NEXN in human heart (top left), skeletal muscle (top right), esophagus (bottom left) and gall bladder (bottom right), all from the Human Protein Atlas (proteinatlas.org). Panel C shows correlation between smooth muscle myosin ( MYH11 ) and NEXN in human coronary artery (N = 133, data from the GTExPortal). Panel D shows our own double staining for Nexilin (green, ab213628) and CAV1 (red) in cross-sectioned human urinary bladder SMCs (white scale bar represents 10 µm, N = 2) imaged in a conventional fluorescence microscope.

Article Snippet: Sections were blocked with 3% BSA for 60 min at room temperature before incubation with the following primary antibodies overnight: Nexilin (1:200 in 3% BSA, Abcam, ab213628 or HPA011185, Sigma), CAV1 (1:400 in 3% BSA, Cell Signaling Technology, #3267) and YAP1 (1:200 in 3% BSA, Cell Signaling Technology, #4912).

Techniques: RNA Sequencing, Expressing, Immunohistochemical staining, Staining, Double Staining, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Nexilin/ NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP

doi: 10.1038/s41598-018-31328-2

Figure Lengend Snippet: Nexilin localizes to puncta in the cytosol and at the membrane and is excluded from caveolae domains at the membrane. Nexilin ( NEXN ) and CAV1 immunofluorescence was imaged by confocal microscopy and using two different Nexilin antibodies (ab213628 in ( A) and HPA011185 in ( B ). Both show punctate/granular distribution of Nexilin (green) in the cytoplasm and at the membrane in human bladder SMCs that overlaps poorly with CAV1 (red) staining. Correlation of the two labels (CAV1 and NEXN ) indicated poor co-localization (panel C, R = 0.073 and 0.18, for the respective NEXN antibodies). The fluorescence intensity ratio for the two labels was also plotted for three membrane profiles ( D ). Panel E shows immuno-electron microscopy for Nexilin. The gold particle on the secondary antibody appears as a black sphere with a diameter of 10 nm, and examples of dense bodies (dbo) and dense bands (dba) are shown. White arrowheads highlight gold particles.

Article Snippet: Sections were blocked with 3% BSA for 60 min at room temperature before incubation with the following primary antibodies overnight: Nexilin (1:200 in 3% BSA, Abcam, ab213628 or HPA011185, Sigma), CAV1 (1:400 in 3% BSA, Cell Signaling Technology, #3267) and YAP1 (1:200 in 3% BSA, Cell Signaling Technology, #4912).

Techniques: Membrane, Immunofluorescence, Confocal Microscopy, Staining, Fluorescence, Immuno-Electron Microscopy

Journal: Scientific Reports

Article Title: Nexilin/ NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP

doi: 10.1038/s41598-018-31328-2

Figure Lengend Snippet: NEXN correlates with gene products that control and respond to changes in actin polymerization and Nexilin is reduced by depolymerization of actin. Correlations of NEXN versus all other RNAs in the top-ten NEXN expressing tissues were examined (data from GTExPortal). The sum of correlation coefficients for individual RNAs across tissues was calculated (R sum ) and the positive extreme of this distribution was plotted ( A ). Actin controlling and responding gene products represented in the extreme are highlighted in blue colors. Examples of NEXN correlations in the human coronary artery (N = 133) are shown in panels B through ( D ). P-values and Spearman Rho values are given in the respective panels. Panels E and F show mRNA data for NEXN in cultured human bladder (HBSMCs, N = 8) and coronary artery (HCASMCs, N = 9) SMCs after treatment with Latrunculin B (LatB). Panels H and I show protein data for Nexilin/ NEXN in the presence and absence of LatB (HBSMCs, 300 nM, N = 12; HCASMCs, 100 nM, N = 10). The top micrographs in panel J shows confocal imaging of YAP (red) on the left, and YAP (green) and CAV1 (red) on the right. The bottom row shows YAP (red) and Nexilin (green). The high magnification overlay at the bottom right shows partial colocalization of YAP and Nexilin at the cell membrane in yellow. All micrographs are from cross-sectioned HBSMCs and white scale bars represent 5 μm throughout.

Article Snippet: Sections were blocked with 3% BSA for 60 min at room temperature before incubation with the following primary antibodies overnight: Nexilin (1:200 in 3% BSA, Abcam, ab213628 or HPA011185, Sigma), CAV1 (1:400 in 3% BSA, Cell Signaling Technology, #3267) and YAP1 (1:200 in 3% BSA, Cell Signaling Technology, #4912).

Techniques: Control, Expressing, Cell Culture, Imaging, Membrane

Journal: International journal of molecular sciences

Article Title: Bone Morphogenetic Protein 13 Has Protumorigenic Effects on Hepatocellular Carcinoma Cells In Vitro.

doi: 10.3390/ijms241311059

Figure Lengend Snippet: Figure 1. Expression of BMP13 in hepatocellular carcinoma (HCC). (A) Analysis of mRNA expression of BMP13 in activated hepatic stellate cells (HSCs) and four human HCC cell lines. (B) Western blot analysis of BMP13 protein expression in HSCs and four human HCC cell lines. GAPDH was used as a housekeeper. Densitometric analysis (BMP13/GAPDH) revealed that, compared to the BMP13 signal in HSCs, the immunosignal in HCC cells was 20 to 210-fold lower (20-fold in Hep3B cells, 23-fold in HepG2 cells, 210-fold in PLC/PRF/5 cells, and 30-fold in Huh7 cells). (C) Correlation of BMP13 and α-smooth muscle actin (α-SMA) RNA expression levels (log2(transcript per million)) in 361 human HCC tissues. The cancer genome atlas (TCGA)-derived data were used, applying the gene expression profiling interactive analysis (GEPIA) database. (D) Representative image of immunofluorescence staining for BMP13 (red) and α-SMA (green) in human HCC tissue section. (E) Hematoxylin and eosin staining of the same tissue section area as shown in panel (D) (*: p < 0.05).

Article Snippet: After cooling to room temperature, slides for immunofluorescence staining were washed with PBS and permeabilized with 0.5% Triton X 100/1% PBS for 1 h. After that, tissues were incubated with anti-BMP13 antibodies (HPA045206, 1:400, Atlas Antibodies, SE-168 69, Bromma, Sweden) and anti-α smooth muscle actin antibodies (NB300-978SS, 1:500, Bio-Techne GmbH, Wiesbaden Nordenstadt, Germany) overnight at 4 ◦C and washed two times with PBS.

Techniques: Expressing, Western Blot, RNA Expression, Derivative Assay, Gene Expression, Staining

Journal: International journal of molecular sciences

Article Title: Bone Morphogenetic Protein 13 Has Protumorigenic Effects on Hepatocellular Carcinoma Cells In Vitro.

doi: 10.3390/ijms241311059

Figure Lengend Snippet: Figure 2. Effect of BMP13 on tumorigenicity of HCC cells in vitro. (A) Analysis of the proliferation of HCC cell lines Hep3B, HepG2, and PLC/PRF/5 after 24 h stimulation with rBMP13. The proliferation rate of control cells has been set as 1 and the effect of BMP13 is shown as the relative induction of proliferation in stimulated cells, compared to control cells. (B) Representative images of Hep3B cells stimulated without (ctr) and with recombinant BMP13 for 24 h (40-fold magnification). Quantification of (C) colony size, (D) colony number, and (E) representative images (1-fold magnification) in anchorage-dependent clonogenic assays with Hep3B treated without (ctr) or with rBMP13 for 9 days. The size and number of colonies of control cells have been set as 1 and the effect of BMP13 is shown as relative induction compared to control cells (*: p < 0.05).

Article Snippet: After cooling to room temperature, slides for immunofluorescence staining were washed with PBS and permeabilized with 0.5% Triton X 100/1% PBS for 1 h. After that, tissues were incubated with anti-BMP13 antibodies (HPA045206, 1:400, Atlas Antibodies, SE-168 69, Bromma, Sweden) and anti-α smooth muscle actin antibodies (NB300-978SS, 1:500, Bio-Techne GmbH, Wiesbaden Nordenstadt, Germany) overnight at 4 ◦C and washed two times with PBS.

Techniques: In Vitro, Control, Recombinant

Journal: International journal of molecular sciences

Article Title: Bone Morphogenetic Protein 13 Has Protumorigenic Effects on Hepatocellular Carcinoma Cells In Vitro.

doi: 10.3390/ijms241311059

Figure Lengend Snippet: Figure 3. Effect of BMP13 on ID1 and cell-cycle regulators in HCC cells. (A) Analysis of ID1 mRNA expression in Hep3B cells treated with different doses of recombinant BMP13 (rBMP13) for 2 h. (B) Analysis

Article Snippet: After cooling to room temperature, slides for immunofluorescence staining were washed with PBS and permeabilized with 0.5% Triton X 100/1% PBS for 1 h. After that, tissues were incubated with anti-BMP13 antibodies (HPA045206, 1:400, Atlas Antibodies, SE-168 69, Bromma, Sweden) and anti-α smooth muscle actin antibodies (NB300-978SS, 1:500, Bio-Techne GmbH, Wiesbaden Nordenstadt, Germany) overnight at 4 ◦C and washed two times with PBS.

Techniques: Expressing, Recombinant

Journal: Cell systems

Article Title: BraInMap elucidates the macromolecular connectivity landscape of mammalian brain

doi: 10.1016/j.cels.2020.03.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies used were (mouse) anti-TARDBP (Abnova; H00023435-M01; 1:500), (rabbit) anti-TDP-43 phosph-S409/410 (a gift from Leonard Petrucelli; Rb3655; 1:250), (rabbit) anti-DDX1 (ProteinTech; 11357-1-AP; 1:500), (rabbit) anti-DDX5 (Abcam; ab21696; 1:1000), (rabbit) anti-hnRNP-H (Bethyl; A300-511A; 1:500), (rabbit) anti-ILF3 (Bethyl; NF90/NF110, A303-121A), (rabbit) anti-U2AF2 (Novus; NBP2-04138), (rabbit) anti-Histone H3 (Abcam; ab18521), (chicken) anti-MAP2 (Aves; MAP2) and (chicken) anti-NeuN (EMD; ABN91; 1:500).

Techniques: Mutagenesis, Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Functional Assay, Expressing, RNA Binding Assay, Software, Sequencing, Mann-Whitney U-Test, Mass Spectrometry

Journal: Nature Communications

Article Title: Colonic epithelial-derived FGF1 drives intestinal stem cell commitment toward goblet cells to suppress inflammatory bowel disease

doi: 10.1038/s41467-025-58644-2

Figure Lengend Snippet: A Heatmap of differentially expressed Atoh1 and its downstream target genes in PBS and rFGF1-treated UC mice ( n = 4). The mRNA level of Atoh1 in PBS or rFGF1-treated UC mice (upper panel, n = 4) and CD mice (lower panel, n = 5) was detected by qRT-PCR. B , C IF staining of ATOH1 in distal colon sections of Fgf1 fl/fl and VilCre Fgf1 fl/fl mice challenged with DSS ( B ) or TNBS ( C ), and its semi-quantitation of IF intensity ( n = 4, the mean value of 2 fields in each mouse). D , E IF staining of ATOH1 in distal colon sections of DSS ( D ) or TNBS ( E )-induced two IBD mouse models, followed by PBS or rFGF1 treatment and its semi-quantitation of IF intensity ( n = 4, the mean value of 2 fields in each mouse). F Representative images (upper panel) and IF staining of Muc2 (lower panel) in sh Atoh1 or shNC-transfected colonic organoids stimulated with vehicle or rFGF1 ( n = 6). G , H Deletion of Atoh1 in VilCre ERT2 Atoh1 fl/fl mice was confirmed by qRT-PCR ( G ) and IF staining of colon tissues ( H ) ( n = 4). I – M VilCre ERT2 Atoh1 fl/fl mice were injected with tamoxifen for five consecutive days and then given drinking water containing 1.5% DSS to induce acute colitis, followed by vehicle or rFGF1 administration for 7 days. At end of the experiment, the distal colon tissues were harvested and examined. The schematic diagram shows the strategy of tamoxifen injection, DSS challenge and rFGF1 administration ( n = 4). J – L Weight loss ( J ), disease activity index ( K ), colonic length ( L ) were monitored (n = 4). M H&E (upper panel), PAS-AB staining (middle panel) and Muc2 IF staining (lower panel) of distal colon sections ( n = 4). Data was presented as mean ± SEM. ( A , D – E , H ) two-tailed unpaired t -test; ( B , C , F , J – M ) ordinary two-way ANOVA, followed by Sidak; ( G ) Non-parametric statistical method, two-tailed Mann-Whitney test. ns, not significance; nd, not detectable.

Article Snippet: The primary antibodies are listed below: rabbit anti-FGF1 (1:200, 17400, Proteintech), mouse anti-FGF1 (1:100, sc-55520, Santa Cruz Biotechnology), EpCAM (1:800, CST42515, Cell Signaling Technology), Col1α1 (1:100, CST72026, Cell Signaling Technology), ATOH1 (1:400, 21215-1-AP, Proteintech), TCF4 (1:100, ab217668, Abcam), Muc2 (1:400, ab272692, Abcam), Dclk1 (1:300, CST62257, Cell Signaling Technology), ChgA (1:200, sc-393941, Santa Cruz Biotechnology), Fabp1 (1:50, CST13368, Cell Signaling Technology), Ki67 (1:400, ab15580, Abcam), FGFR2 (1:200, CST23328S, Cell Signaling Technology) and Lgr5 (1:100, UM870104, ORIGENE).

Techniques: Quantitative RT-PCR, Staining, Quantitation Assay, Transfection, Injection, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Colonic epithelial-derived FGF1 drives intestinal stem cell commitment toward goblet cells to suppress inflammatory bowel disease

doi: 10.1038/s41467-025-58644-2

Figure Lengend Snippet: A Workflow to find the rFGF1-regulated potential transcription factors of Atoh1 , and heatmap of expression level of potential transcription factors ( n = 4). B The expression levels of potential transcription factors of Atoh1 ( Tcf4 , Nr4a1 , Nfya , Tec , Hoxa13 , Ncaph2 , Kdm1a ) in distal colon sections of PBS or rFGF1-treated UC mice were analyzed by qRT-PCR ( n = 4). C , D IF staining of TCF4 in distal colon sections of Fgf1 fl/fl and VilCre Fgf1 fl/fl mice challenged with DSS ( C ) or TNBS ( D ) and its semi-quantitation of IF intensity ( n = 4, the mean value of 2 fields in each mouse). E , F The expression levels of TCF4 in distal colon sections of DSS-treated UC mice ( E ) and TNBS-treated CD mice ( F ) after PBS or rFGF1 administration and IF staining ( n = 8). G Predicted TCF4 binding sites on the promoter of Atoh1 by the JASPAR database (left panel). Six binding sites with the most significant prediction scores were selected for pairwise deletion mutation analysis (right panel; M1, in purple; M2, in green; M3, in red). H , I Luciferase activity in HT-29 cells transfected with a human Atoh1 promoter reporter plasmid or the predicted TCF4 binding site mutant reporter along with a Tcf4 overexpression plasmid ( n = 4). J ChIP experiments. The data were normalized to the corresponding IgG ( n = 4). K Images of murine colonic organoids transfected small hairpin RNA targeting Tcf4 (sh Tcf4 ) or negative control hairpin (shNT) followed by treated with rFGF1 or vehicle ( n = 6). L IF staining of ATOH1 (upper panel) and Muc2 (lower panel) in colonic organoids in different groups and its semi-quantitation of fluorescent intensity ( n = 6). Data was presented as mean ± SEM. ( B , right panel of E – F , J ) two-tailed unpaired t -test ( Tec and Ncaph2 in penal B using non-parametric statistical method, two-tailed Mann-Whitney Test); ( H – I ) ordinary one-way ANOVA, followed by Dunnett; (right panel of C – D and L , lower panel of K ) ordinary two-way ANOVA, followed by Sidak. ns, no significance.

Article Snippet: The primary antibodies are listed below: rabbit anti-FGF1 (1:200, 17400, Proteintech), mouse anti-FGF1 (1:100, sc-55520, Santa Cruz Biotechnology), EpCAM (1:800, CST42515, Cell Signaling Technology), Col1α1 (1:100, CST72026, Cell Signaling Technology), ATOH1 (1:400, 21215-1-AP, Proteintech), TCF4 (1:100, ab217668, Abcam), Muc2 (1:400, ab272692, Abcam), Dclk1 (1:300, CST62257, Cell Signaling Technology), ChgA (1:200, sc-393941, Santa Cruz Biotechnology), Fabp1 (1:50, CST13368, Cell Signaling Technology), Ki67 (1:400, ab15580, Abcam), FGFR2 (1:200, CST23328S, Cell Signaling Technology) and Lgr5 (1:100, UM870104, ORIGENE).

Techniques: Expressing, Quantitative RT-PCR, Staining, Quantitation Assay, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression, Negative Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Colonic epithelial-derived FGF1 drives intestinal stem cell commitment toward goblet cells to suppress inflammatory bowel disease

doi: 10.1038/s41467-025-58644-2

Figure Lengend Snippet: A Single-cell RNA sequencing analysis (GSM4983265) showed that Fgfr2 is highly expressed in stem cells. B Representative image of colon distal stained with Lgr5 (green), FGFR2 (red) and DAPI (blue) from the C57BL/6 J mice ( n = 3). C Deletion of Fgfr2 in Lgr5-EGFP-Cre ERT2 Fgfr2 fl/fl mice treated with vehicle or tamoxifen was confirmed by IF staining ( n = 5). D Murine colonic organoids derived from vehicle or tamoxifen-treated Lgr5-EGFP-Cre ERT2 Fgfr2 fl/fl mice were stimulated with vehicle or rFGF1. Representative images, IF staining of TCF4, ATOH1 and Muc2 in colonic organoids were shown (n = 6). E – I Vehicle or tamoxifen-treated Lgr5-EGFP-Cre ERT2 Fgfr2 fl/fl mice were challenged with drinking water containing 2.5% DSS to induce UC, followed by colon tissues were harvested and examined (n = 4 for TAM + PBS group, n = 5 for other groups). Weight loss ( E ), disease activity index ( F ) and colon length ( G ) of different groups were monitored. H H&E staining of distal colon sections and histology scores (upper panel), PAS-AB staining of distal colon sections and mucin granules-positive cells per crypt (lower panel). I IF staining of TCF4 (upper panel), ATOH1 (middle panel) and Muc2 (lower panel) in distal colon sections, and its semi-quantification of IF intensity. Data was presented as mean ± SEM. (right panel of C ) two-tailed unpaired t -test; (right panel of D – F , right panel of G – I ) ordinary two-way ANOVA, followed by Sidak. ns, no significance.

Article Snippet: The primary antibodies are listed below: rabbit anti-FGF1 (1:200, 17400, Proteintech), mouse anti-FGF1 (1:100, sc-55520, Santa Cruz Biotechnology), EpCAM (1:800, CST42515, Cell Signaling Technology), Col1α1 (1:100, CST72026, Cell Signaling Technology), ATOH1 (1:400, 21215-1-AP, Proteintech), TCF4 (1:100, ab217668, Abcam), Muc2 (1:400, ab272692, Abcam), Dclk1 (1:300, CST62257, Cell Signaling Technology), ChgA (1:200, sc-393941, Santa Cruz Biotechnology), Fabp1 (1:50, CST13368, Cell Signaling Technology), Ki67 (1:400, ab15580, Abcam), FGFR2 (1:200, CST23328S, Cell Signaling Technology) and Lgr5 (1:100, UM870104, ORIGENE).

Techniques: RNA Sequencing, Staining, Derivative Assay, Activity Assay, Two Tailed Test