Journal: Investigative Ophthalmology & Visual Science

Article Title: Essential Role of CD63 in Maintaining Corneal Epithelial Identity in the Human Limbus

doi: 10.1167/iovs.67.3.25

Figure Lengend Snippet: CD63 is expressed in the human limbal epithelium and regulates corneal epithelial gene expression. ( A ) Representative KRT12 (corneal epithelial cell marker, green ), KRT13 (conjunctival epithelial marker, yellow ), and CD63 ( red ) immunostaining in the human conjunctiva, limbus, and central cornea. Hoechst 33342 ( blue ) was used for the nuclei staining ( n = 3 donors). Scale bar = 50 µm. ( B ) The bar graph represents CD63 RNA expression in CD63 siRNA-treated cultured limbal epithelial cells ( n = 4 donors, mean ± SD; *** P < 0.001, **** P < 0.0001). ( C , left panel ) Representative flow cytometry analyses of CD63 protein expression in CD63 siRNA-treated cultured limbal epithelial cells detected by flow cytometry. ( C , right panel ) The bar graph represents quantitative analyses of CD63 protein expression determined by flow cytometry ( n = 3 donors, mean ± SD; * P < 0.05). ( D , left panel ) Representative macroscopic images of colonies formed by CD63 KD cells compared to the control siRNA-transfected cells. ( D , right panel ) The bar graph represents a comparative analysis of the colony-forming efficiency ( n = 6 donors, mean ± SD; * P < 0.05, ** P < 0.01). ( E ) Comparative analyses of the percentage of EdU-positive proliferating cells by CD63 KD in cultured human limbal epithelial cells ( n = 5 donors, mean ± SD; ** P < 0.01). ( F ) A Venn diagram illustrates the overlap of differentially expressed genes identified in both CD63 KD#1 and CD63 KD#2 conditions. ( G ) Bar graphs represent normalized counts of CD63 , KRT12 , CLU , ALDH1A1 , ALDH3A1 , TGFBI , and MYEOV RNA expression in CD63 siRNA-treated cultured limbal epithelial cells ( n = 3 donors, mean ± SD, *adjusted P < 0.05, **adjusted P < 0.01, ***adjusted P < 0.001, ****adjusted P < 0.0001). ( H , left panel ) Western blot analyses of KRT12, CLU, ALDH1A1, ALDH3A1, and TGFBI expression in control and CD63 siRNA-treated limbal epithelial cells. ( H , right panel ) Bar graphs represent quantitative analyses of protein expression ( n = 4 donors, mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.01, **** P < 0.0001).

Article Snippet: GAPDH (Hs99999905_m1), CD63 (Hs01041237_g1), PAX6 (Hs01088114_m1), KRT12 (Hs00165015_m1), CLU (Hs00971656_m1), ALDH1A1 (Hs00946916_m1), ALDH3A1 (Hs00964880_m1), and TGFBI (Hs00932747_m1) TaqMan probes were used.

Techniques: Gene Expression, Marker, Immunostaining, Staining, RNA Expression, Cell Culture, Flow Cytometry, Expressing, Control, Transfection, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Essential Role of CD63 in Maintaining Corneal Epithelial Identity in the Human Limbus

doi: 10.1167/iovs.67.3.25

Figure Lengend Snippet: CD63 regulates corneal epithelial phenotype by maintaining PAX6 expression. ( A , left panel ) Western blot analysis of PAX6 expression in control and CD63 siRNA-treated limbal epithelial cells. ( A , right panel ) The bar graph represents quantitative analyses of PAX6 protein expression ( n = 4 donors, mean ± SD; * P < 0.05). ( B , left panel ) Western blot analysis of PAX6, KRT12, CLU, ALDH1A1, ALDH3A1, and TGFBI expressions in control and PAX6 siRNA-treated limbal epithelial cells. ( B , right panel ) Bar graph represents quantitative analyses of protein expression ( n = 3 donors, mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). ( C ) Bar graph represents miR-184 expression by CD63 siRNA-treated cultured limbal epithelial cells ( n = 3 donors, mean ± SD; ** P < 0.01, *** P < 0.001). ( D ) Bar graphs represent normalized counts of FOG2 and NUS1 RNA expression in CD63 siRNA-treated cultured limbal epithelial cells ( n = 3 donors, mean ± SD).

Article Snippet: GAPDH (Hs99999905_m1), CD63 (Hs01041237_g1), PAX6 (Hs01088114_m1), KRT12 (Hs00165015_m1), CLU (Hs00971656_m1), ALDH1A1 (Hs00946916_m1), ALDH3A1 (Hs00964880_m1), and TGFBI (Hs00932747_m1) TaqMan probes were used.

Techniques: Expressing, Western Blot, Control, Cell Culture, RNA Expression

Journal: Molecular cancer

Article Title: TGFBI expression is associated with a better response to chemotherapy in NSCLC.

doi: 10.1186/1476-4598-9-130

Figure Lengend Snippet: Figure 3 TGFBI sensitizes NSCLC cells to chemotherapy. A549 cells transfected with TGFBI siRNA plasmid (TGFBIsi) (A), and H1299 cells transfected with TGFBI expression vector (TGFBIve) (B), were exposed to increasing amounts of different cytotoxic agents for 48 h and cell viability was measured. Three independent experiments were performed. Comparisons to analyze statistical differences between control and transfected cells were per- formed using Student t-test (* p < 0.05; ** p < 0.01). IC50 for each experimental condition is indicated. (C, D) PARP-1 cleavage was detected by Western blot analysis of total protein extracts derived from A549-TGFBIsi (C) and H1299-TGFBIve (D) transfected cells treated with etoposide for 48 h. One rep- resentative experiment out of three is shown.

Article Snippet: To over-express TGFBI in H1299 cells (H1299 TGFBIve), 2 × 105 cells/well were transfected with 1 μg of the pCMV6-XL4 plasmid vector containing TGFBI cDNA (Origene, MD, USA) using 2 or 3 μl of FuGENE 6 Transfection Reagent (Roche, IN, USA) in 97 μl of Opti-MEM medium (Invitrogen, Barcelona, Spain) following manufacturer's instructions.

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Western Blot, Derivative Assay

Journal: Molecular cancer

Article Title: TGFBI expression is associated with a better response to chemotherapy in NSCLC.

doi: 10.1186/1476-4598-9-130

Figure Lengend Snippet: Figure 4 High concentration of rh-TGFBI protein increases cell death and potentiates NSCLC responses to chemotherapy. Caspase 3/7 ac- tivity was measured in A549 (white histograms) and H1299 (black histograms) NSCLC cells seeded onto non coated 96-well plates and further treated with increasing amounts of rh-TGFBI (A), or seeded onto 96 well plates coated with increasing amounts of rh-TGFBI (B), using the caspase 3/7 Glo- assay from Promega. (C and D) Caspase 3/7 activity of 24 h etoposide-treated A549 (5 μM) and H1299 (50 μM) cells seeded onto non coated 96-well plates and further exposed for 24 h to increasing amounts of rh-TGFBI previous to etoposide exposure (C) or seeded onto 96-well plates coated with increasing amounts of rh-TGFBI before exposure to etoposide (D). Statistical analyses were performed using Student t-test to compare caspase 3/7 activity in treated cells to non-treated cells (* p < 0.05; **p < .01).

Article Snippet: To over-express TGFBI in H1299 cells (H1299 TGFBIve), 2 × 105 cells/well were transfected with 1 μg of the pCMV6-XL4 plasmid vector containing TGFBI cDNA (Origene, MD, USA) using 2 or 3 μl of FuGENE 6 Transfection Reagent (Roche, IN, USA) in 97 μl of Opti-MEM medium (Invitrogen, Barcelona, Spain) following manufacturer's instructions.

Techniques: Concentration Assay, Glo Assay, Activity Assay

Journal: Molecular cancer

Article Title: TGFBI expression is associated with a better response to chemotherapy in NSCLC.

doi: 10.1186/1476-4598-9-130

Figure Lengend Snippet: Figure 5 TGFBI influences NSCLC susceptibility to chemotherapy through binding to the αvβ3 integrin. Adhesion of calcein-labeled A549 (A) and H1299 (B) NSCLC cells to rh-TGFBI coated (1 μg/mL) 96-well plates was evaluated in the presence of antibodies against several integrins. Statistical analyses were performed using Student t-test and comparing treated cells to control cells (**p < 0.01). (C) Flow cytometry analysis of basal αvβ3 inte- grin expression in A549 cells (dashed histogram), H1299 cells (line histrogram) or isotype control (dotted histogram). (D) Caspase 3/7 detection in A549 and H1299 cells seeded onto non-coated 96 well-plates, pre-incubated for 1 h with anti-αvβ3 monoclonal blocking antibody, exposed to rh-TGFBI (20 μg/mL) for 24 h and further treated with etoposide at the IC50 (5 μM for A549 cells and 50 μM for H1299 cells) for additional 24 h. Statistical comparisons were performed using Student t-test. **p < 0.01 for the comparison of the activity control cells versus integrin blocked cells. ##p < 0.01 for the com- parison of the activity of etoposide-treated cells versus cells treated in the presence of the anti-integrin blocking antibody.

Article Snippet: To over-express TGFBI in H1299 cells (H1299 TGFBIve), 2 × 105 cells/well were transfected with 1 μg of the pCMV6-XL4 plasmid vector containing TGFBI cDNA (Origene, MD, USA) using 2 or 3 μl of FuGENE 6 Transfection Reagent (Roche, IN, USA) in 97 μl of Opti-MEM medium (Invitrogen, Barcelona, Spain) following manufacturer's instructions.

Techniques: Binding Assay, Labeling, Control, Flow Cytometry, Expressing, Incubation, Blocking Assay, Comparison, Activity Assay

Journal: Molecular cancer

Article Title: TGFBI expression is associated with a better response to chemotherapy in NSCLC.

doi: 10.1186/1476-4598-9-130

Figure Lengend Snippet: Figure 6 Peptides derived from TGFBI mediate NSCLC cells response to chemotherapy and the induction of caspase 8 and caspase 3/7 ac- tivation. (A) Western blot detection of proteolytic fragments of TGFBI in H1299 and A549 cells transiently transfected with the TGFBI expression vector. (B) Detection of caspase 3/7 activity in A549 and H1299 cells exposed for 24 h to cell supernatants derived from cultures of A549 and H1299 cells tran- siently transfected with the TGFBI expression vector. Control: non-treated cells; Control > 3 kDa: NSCLC cells exposed to supernatants from non- transfected cell cultures containing fragments larger than 3 kDa in size; Control < 3 kDa: NSCLC cells exposed to supernatants from non-transfected cell cultures containing fragments smaller than 3 kDa in size; TGFBI > 3 kDa: NSCLC cells exposed to supernatants from TGFBI-transfected cells con- taining fragments larger than 3 kDa in size; TGFBI < 3 kDa: NSCLC cells exposed to supernatants from TGFBI-transfected cells containing fragments smaller than 3 kDa in size; αvβ3: cells blocked with anti-αvβ3 blocking antibody 1 h before the addition of the supernatant; Et: Cells exposed to IC50 etoposide for 24 h. Statistical analyses were performed using Student t-test to compare caspase 3/7 activity in control cells (** p < 0.01) or etoposide treated cells (## p < 0.01) to the caspase activity in cell cultures treated with supernatants. (C) Caspase 8 and (D) caspase 3/7 detection in A549 and H1299 cells maintained for different time-periods in the presence of TGFBI < 3 kDa supernatant. Statistical analyses were performed using Student t- test and the caspase activity of treated cells was compared to that of untreated cells. (*p < 0.05; **p < 0.01).

Article Snippet: To over-express TGFBI in H1299 cells (H1299 TGFBIve), 2 × 105 cells/well were transfected with 1 μg of the pCMV6-XL4 plasmid vector containing TGFBI cDNA (Origene, MD, USA) using 2 or 3 μl of FuGENE 6 Transfection Reagent (Roche, IN, USA) in 97 μl of Opti-MEM medium (Invitrogen, Barcelona, Spain) following manufacturer's instructions.

Techniques: Derivative Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Activity Assay, Control, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Lactate Promotes Endothelial-Mesenchymal Transition via Mediating Twist1 Lactylation in Hypoxic Pulmonary Hypertension

doi: 10.3390/ijms27052255

Figure Lengend Snippet: Lactate promotes TGF-β/Smad2 signaling after hypoxia; ( a – c ) Mice were subjected to hypoxia or normoxia followed by supplemental lactate administration. The mRNA expression of Tgfb1 ( a ), Smad2 ( b ), and Smad3 ( c ) was examined by qRT-PCR; ( d – f ) In separate experiments, mice were subjected to hypoxia or normoxia with 2-DG or vehicle administration. The mRNA expression of Tgfb1 ( d ), Smad2 ( e ), and Smad3 ( f ) was examined by qRT-PCR; ( g – h ) HPAECs were treated with lactate (10 mM) followed by normoxic or hypoxic challenge. The mRNA levels of TGFB1 and SMAD2 were examined by qRT-PCR; ( i , i1 – i3 ) Representative images and quantification of Tgfb1, p-Smad2, p-Smad3, and Smad2/3 protein levels in HPAECs by Western blot analysis after treatment of lactate during hypoxia; ( j , j1 , j2 ) Representative images and quantification of Tgfb1, p-Smad2, and Smad2 protein levels in HPAECs by Western blot analysis after treatment of lactate and 2-DG. Data were presented as Mean ± SD. n = 3–6, ns: no statistical signification, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The membranes were then incubated overnight at 4 °C with the following specific primary antibodies: Endothelial cell markers: CD31 (1:1000, 11265-1-AP, Proteintech), VE-Cadherin (1:1000, 2500, Cell Signaling Technology, Danvers, MA, USA); Mesenchymal cell markers: α-SMA (1:2000, ab21027, Abcam), FSP1 (1:1000, ab27957, Abcam); SM22-α (1:1000, 10493-1-AP, Proteintech); Vimentin (1:1000, 5741, Cell Signaling Technology, Danvers, MA, USA); TGF-β signaling pathway-related proteins: TGF-β1 (1:1000, 10188-1-AP, proteintech), p-Smad2 (1:1000, 18338, Cell Signaling Technology), Smad2 (1:1000,12570-1-AP, proteintech), Total-Smad2/3 (1:1000, 3102, Cell Signaling Technology); Twist1 (1:1000, 25465-1-AP, Proteintech), β-actin (1:500; SC-130657, Santacruz), H3 (1:1000, 17168-1-AP, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Lactate Promotes Endothelial-Mesenchymal Transition via Mediating Twist1 Lactylation in Hypoxic Pulmonary Hypertension

doi: 10.3390/ijms27052255

Figure Lengend Snippet: Twist1 knockdown reverses lactate-induced EndoMT and TGF-β/Smad2 signaling. HPAECs were transfected with siRNA specific for Twist (siTwist). Scrambled siRNA served as control (siNC). 24 h after transfection, cells were treated with lactate (10 mM) followed by hypoxic challenge; ( a , a1 – a3 ) Representative images ( a ) and quantification ( a1 – a3 ) of the endothelial markers and mesenchymal markers was measured by Western blot; ( b , b1 , b2 ) Representative images ( b ) and quantification ( b1 , b2 ) of TGF-β/Smad2 pathway activation by Western blot; ( c , d ) Representative images and quantification of EdU incorporation ( c ) and migration ( d ) in each group; ( e , f ) Endothelial barrier function assessed by transendothelial electrical resistance (TER) ( e ) and BSA permeability assay ( f ). Data were presented as Mean ± SD. n = 4–6, ns: no statistical signification, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The membranes were then incubated overnight at 4 °C with the following specific primary antibodies: Endothelial cell markers: CD31 (1:1000, 11265-1-AP, Proteintech), VE-Cadherin (1:1000, 2500, Cell Signaling Technology, Danvers, MA, USA); Mesenchymal cell markers: α-SMA (1:2000, ab21027, Abcam), FSP1 (1:1000, ab27957, Abcam); SM22-α (1:1000, 10493-1-AP, Proteintech); Vimentin (1:1000, 5741, Cell Signaling Technology, Danvers, MA, USA); TGF-β signaling pathway-related proteins: TGF-β1 (1:1000, 10188-1-AP, proteintech), p-Smad2 (1:1000, 18338, Cell Signaling Technology), Smad2 (1:1000,12570-1-AP, proteintech), Total-Smad2/3 (1:1000, 3102, Cell Signaling Technology); Twist1 (1:1000, 25465-1-AP, Proteintech), β-actin (1:500; SC-130657, Santacruz), H3 (1:1000, 17168-1-AP, Proteintech).

Techniques: Knockdown, Transfection, Control, Western Blot, Activation Assay, Migration, Permeability

Journal: JACC: Basic to Translational Science

Article Title: Endothelial Overexpression of TGF-β-Induced Protein Impairs Venous Thrombus Resolution

doi: 10.1016/j.jacbts.2023.08.005

Figure Lengend Snippet: Validation of Selected Genes With the Use of RT 2 Profiler PCR Array and qPCR Analysis (A) Schematic representation of the experimental flow, including the cultivation of chronic thromboembolic pulmonary hypertension (CTEPH) endothelial cells (ECs) from pulmonary endarterectomy (PEA) specimens and preparation for further analysis. (B to I) Real-time qPCR analysis (using custom-designed RT 2 Profiler PCR assay or qPCR with custom oligonucleotides) of CTEPH-ECs (7 biological repeats) compared with human pulmonary arterial endothelial cells (HPAECs) (3 biological repeats) for genes (B) involved in extracellular matrix organization (ie, CD44 molecule [ CD44 ], collagen type 1 alpha 1 chain - ( COL1A1 ), collagen type 1 alpha 2 chain [ COL1A2 ], collagen type 3 alpha 1 chain [ COL3A1 ], collagen type 6 alpha 3 chain [ COL6A3 ], fibrillin 2 [ FBN2 ], matrix metalloproteinase 1 [ MMP1 ], and nidogen 1 [ NID1 ]) or belonging to the biological pathways (C) hemostasis (ie, ADAM metallopeptidase with thrombospondin type 1 motif 13 [ ADAMTS13 ], C-X-C motif chemokine ligand 8 [ CXCL8 or IL8 ], selectin E [ CD62E ], selectin P [ CD62P ], endothelial protein C receptor [ PROCR ], coagulation factor III [ F3 ], plasminogen activator inhibitor-1 [ PAI-1 ], serpin family G member 1 [ SERPING1 ], von Willebrand factor [ VWF ], transgelin [ TAGLN ], tissue factor pathway inhibitor [ TFPI ], tissue-type plasminogen activator [ TPA ], and thrombomodulin [ THBD ]), (D) signal transduction (ie, smooth muscle α-actin [ ACTA2 ], chemokine C-X-C motif ligand 6 [ CXCL6 ], Dickkopf-related protein 1 [ DKK1 ], Gata-binding protein 3 [ GATA3 ], neurogenic locus Notch homolog protein 3 [ NOTCH3 ], and thrombospondin [ THBS2 ]), (E) immune system (ie, C-C motif chemokine ligand 2 [ CCL2 ], cathepsin K [ CTSK ], cathepsin S [ CTSS ], intercellular adhesion molecule-1 [ ICAM1 ], interleukin-1β [ IL1B ], IL6 , tumor necrosis factor-α-induced protein 3 [ TNFAIP3 ], TNF receptor superfamily member 1B [ TNFRSF1B ], vascular cell adhesion molecule-1 [ VCAM1 ]), (F) metabolism (ie, lipase H [ LIPH ], lymphatic vessel endothelial hyaluronan receptor 1 [ LYVE1 ], prostaglandin 2 [ PTGIS ], prostaglandin endoperoxide synthase 1 [ PTGS1 ], and Shisa family member 3 [ SHISA3 ]), (G) metabolism of proteins (ie, ADAM metallopeptidase with thrombospondin type 1 motif 18 [ ADAMTS18 ], pappalysin 1 [ PAPPA ], syndecan 2 [ SDC2 ], stanniocalcin 2 [ STC2 ], and transforming growth factor-β-induced [ TGFBI ]), and (H) cell cycle control (ie, cyclin A1 [ CCNA1 ], cyclin D2 [ CCND2 ], and cyclin-dependent kinase 6 [ CDK6 ]), as well as (I) playing a role in TGFβ signaling (ie, bone morphogenetic protein and activin membrane bound inhibitor [ BAMBI ], follistatin like protein 3 [ FSTL3 ], periostin [POSTN ], transforming growth factor-β1 [ TGFB1 ], and TGFB2 ). Quantitative data are presented as mean ± SEM. P values were determined with the use of Student’s t -test for normally distributed values ( ACTA2 , ADAMTS13 , DKK1 , F3 , FBN2 , LIPH , NID1 , PAI-1 , PTGIS , PTGS1 , POSTN , SDC2 , THBS2 , TFPI , TNFRSF1B , and VWF ) and Mann-Whitney U -test for values that did not pass the normality test ( ADAMTS18 , BAMBI , BIGH3 , CCL2 , CCNA1 , CCND2 , CD44 , CD62E , CD62P , CDK6 , COL1A1 , COL1A2 , COL3A1 , COL6A3 , CTSK , CTSS , CXCL6 , CXCL8 , FSTL3 , GATA3 , ICAM1 , IL1B , IL6 , LYVE1 , MMP1 , NOTCH3 , PAPPA , PROCR , SERPING1 , SHISA3 , STC2 , TAGLN , TGFB1 , TGFB2 , THBD , TNFAIP3 , TPA , and VCAM1 ) and are shown within the graph: ∗ P < 0.05; ns = nonsignificant. Data points in blue are from male patients, green from female patients.

Article Snippet: Plasma levels of human transforming growth factor-β-induced protein (TGFBI) (OriGene EA100707; detection limit: <10 pg/mL), follistatin-like 3 (FSTL3) (OriGene EA100552; detection limit: <10 pg/mL), stanniocalcin 2 (STC2) (Abcam ab222880; calculated minimal detectable dose: 0.95 ng/mL), and transgelin (TAGLN) (LS Biosciences LS-F22522; detection limit: <3.75 ng/mL) were determined with the use of specific ELISAs.

Techniques: Coagulation, Transduction, Binding Assay, Control, Membrane, MANN-WHITNEY

Journal: JACC: Basic to Translational Science

Article Title: Endothelial Overexpression of TGF-β-Induced Protein Impairs Venous Thrombus Resolution

doi: 10.1016/j.jacbts.2023.08.005

Figure Lengend Snippet: Histologic Localization of Selected Genes in PEA Tissue Microarrays (A) Flowchart showing consecutive steps to histologically localize selected genes to specific areas in CTEPH PEA specimens. Laser microdissection and nCounter analysis of mRNA expression levels of (B) TGFBI , (C) FSTL3 , (D) STC2 , and (E) TAGLN in PEA specimens isolated from 4 CTEPH patients. Quantitative data are presented as mean ± SEM, and P values were determined by comparing findings in vessels area with all other areas (3 comparisons) by means of 1-way ANOVA followed by Bonferroni’s multiple comparisons test for normally distributed values (B, D, E). Values that did not pass the normality test are presented as median with IQR, and P values were determined by means of Kruskal-Wallis test (C). ∗ P < 0.05; ∗∗∗ P < 0.001; ns = nonsignificant. (F) Representative images after immunohistochemical staining of TGFBI, FSTL3, STC2, and TAGLN on tissue microarrays from patients with CTEPH (PEA material: TMA1 and TMA2), pulmonary arterial hypertension (PAH; lung biopsies) or idiopathic pulmonary fibrosis (IPF; lung biopsies). Insets show higher magnification of selected areas of interest. Scale bars represent 100 μm; scale bars in insets represent 10 μm. Semiquantitative analysis of (G) TGFBI and (H) STC2 expression in tissue. Quantitative data are presented as mean ± SEM, and P values were determined by comparing the mean of each group with the mean of every other group (3 comparisons) by means of 1-way ANOVA followed by Bonferroni’s multiple comparisons test. ∗∗∗ P < 0.001; ns = nonsignificant. (I) Representative confocal images of (left) CD31-positive cells (green) expressing TGFBI (red) and (right) STC2 (red) on cryopreserved CTEPH PEA tissue. Scale bars represent 50 μm. Abbreviations as in Figure 1 .

Article Snippet: Plasma levels of human transforming growth factor-β-induced protein (TGFBI) (OriGene EA100707; detection limit: <10 pg/mL), follistatin-like 3 (FSTL3) (OriGene EA100552; detection limit: <10 pg/mL), stanniocalcin 2 (STC2) (Abcam ab222880; calculated minimal detectable dose: 0.95 ng/mL), and transgelin (TAGLN) (LS Biosciences LS-F22522; detection limit: <3.75 ng/mL) were determined with the use of specific ELISAs.

Techniques: Laser Capture Microdissection, Expressing, Isolation, Immunohistochemical staining, Staining

Journal: JACC: Basic to Translational Science

Article Title: Endothelial Overexpression of TGF-β-Induced Protein Impairs Venous Thrombus Resolution

doi: 10.1016/j.jacbts.2023.08.005

Figure Lengend Snippet: Overexpression of TGFBI in HPAECs Promotes the Expression of Genes Similar to CTEPH-ECs and Results in Larger Human Blood Clots (A) Representative immunofluorescence images and (B) quantitative analysis of the mean fluorescent intensity of HPAECs in culture after treatment with control lentiviral particles (lentiviral control) or lentiviral particles containing human TGFBI (TGFBI lentiviral particles) and immunostaining for TGFBI (red) and DAPI (blue). Quantitative data are presented as mean ± SEM. ∗∗∗ P < 0.001 (Student’s t -test). Scale bars represent 10 μm. (C) Quantitative RT 2 Profiler PCR array analysis confirmed significantly increased expression of TGFBI and also revealed significant changes of genes belonging to the biological pathways signal transduction ( ACTA2 ), metabolism of proteins ( STC2 ), cell cycle control ( CCND2 ), hemostasi’ ( TAGLN ), and metabolism ( PTGIS ) and genes involved in TGFβ signaling ( FSTL3 ) and extracellular matrix organization ( COL1A1 ) in HPAECs overexpressing TGFBI (n = 3 experimental repeats; female donor). Data are expressed as fold change vs lentiviral control cells. Quantitative data are presented as mean ± SEM. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 (Student’s t -test). (D) Weight of blood clots generated in vitro followed by addition of HPAECs expressing lentiviral TGFBI or control and incubation for 14 days (n = 3 biological repeats and 2 experimental repeats). Quantitative data are presented as mean ± SEM. ∗ P < 0.05 (Student’s t -test). (E and G) Representative immunohistochemical images of human blood clots after immunostaining of the endothelial marker VE-cadherin (CDH5) and the results of the quantitative analysis of the total vessel area after addition of HPAECs (F) overexpressing TGFBI or (H) stimulated with recombinant TGFBI. Scale bars represent 10 μm. Quantitative data are presented as mean ± SEM. ∗ P < 0.05; ∗∗∗ P < 0.001 (Student’s t -test). (I) Representative immunohistochemical images and (J) results of the quantitative analysis of human blood clots mixed with human ECs overexpressing lentiviral TGFBI or control after immunostaining of the mesenchymal marker smooth muscle actin (SMA). Scale bars represent 10 μm. Quantitative data are presented as mean ± SEM. ∗∗ P < 0.01 (Student’s t -test). Real-time qPCR analysis to detect the transcription factors (K) SNAI2 , (L) TWIST , and (M) ZEB1 in HPAECs overexpressing TGFBI or in lentiviral control cells. Data were normalized to the housekeeping gene HPRT1. Quantitative data are presented as mean ± SEM and compared by means of Student’s t -test (K and M), or as median with IQR and compared by means of Mann-Whitney U -test (L). ∗ P < 0.05; ∗∗∗ P < 0.001; ns = nonsignificant. Abbreviations as in Figure 1 .

Article Snippet: Plasma levels of human transforming growth factor-β-induced protein (TGFBI) (OriGene EA100707; detection limit: <10 pg/mL), follistatin-like 3 (FSTL3) (OriGene EA100552; detection limit: <10 pg/mL), stanniocalcin 2 (STC2) (Abcam ab222880; calculated minimal detectable dose: 0.95 ng/mL), and transgelin (TAGLN) (LS Biosciences LS-F22522; detection limit: <3.75 ng/mL) were determined with the use of specific ELISAs.

Techniques: Over Expression, Expressing, Immunofluorescence, Control, Immunostaining, Transduction, Generated, In Vitro, Incubation, Immunohistochemical staining, Marker, Recombinant, MANN-WHITNEY

Journal: JACC: Basic to Translational Science

Article Title: Endothelial Overexpression of TGF-β-Induced Protein Impairs Venous Thrombus Resolution

doi: 10.1016/j.jacbts.2023.08.005

Figure Lengend Snippet: Plasma Levels of TGFBI, STC2, FSTL3, and TAGLN in Patients With CTEPH, Patients With PAH, and Healthy Control Subjects (A) Flowchart showing steps of blood analyses in patients with CTEPH enrolled in the CTEPH Registry Bad Nauheim, before and at 12-month follow-up after PEA and in patients with PAH enrolled in the Pulmonary Hypertension Registry Mainz. Results of the quantitative analysis using specific enzyme-linked immunosorbent assay (ELISA) of plasma levels of (B) TGFBI, (C) STC2, (D) FSTL3, and (E) TAGLN in patients with CTEPH (n = 27) compared with patients with PAH (n = 23) or healthy control individuals (n = 6). Quantitative data are presented as violin plot (showing all points). P values were determined by comparing the mean of each group with the mean of every other group (3 comparisons) by means of 1-way ANOVA followed by Bonferroni’s multiple comparisons test for normally distributed values (E) or Kruskal-Wallis test followed by Dunn’s multiple comparisons test for values which did not pass the normality test (B, C, D). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ns = nonsignificant. Results of the quantitative analysis using ELISA of plasma levels of (F) TGFBI (n = 18), (G) FSTL3 (n = 10), and (H) TAGLN (n = 10) in patients with CTEPH, before (CTEPH pre) and at 12-month follow-up after PEA surgery (CTEPH post). Quantitative data are presented as symbols and lines. P values were determined with the use of paired Student’s t -test (F) or Wilcoxon matched-pairs signed rank test (G, H). ∗∗∗ P < 0.001; ns = nonsignificant. Data points in blue are for male patients, in green for female.

Article Snippet: Plasma levels of human transforming growth factor-β-induced protein (TGFBI) (OriGene EA100707; detection limit: <10 pg/mL), follistatin-like 3 (FSTL3) (OriGene EA100552; detection limit: <10 pg/mL), stanniocalcin 2 (STC2) (Abcam ab222880; calculated minimal detectable dose: 0.95 ng/mL), and transgelin (TAGLN) (LS Biosciences LS-F22522; detection limit: <3.75 ng/mL) were determined with the use of specific ELISAs.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Time-resolved quantitative proteomic analysis of the developing Xenopus otic vesicle reveals putative congenital hearing loss candidates

doi: 10.1016/j.isci.2023.107665

Figure Lengend Snippet: Loss-of-function experiments for tgfbi in the context of otic development (A) Xenopus 16-cell blastomeres that are fated to primarily contribute to neural crest (NC) and pre-placodal ectoderm (PPE) are labeled in green, and were microinjected with antisense morpholino oligonucleotides (MO) with or without mRNA encoding human TGFBI to which the MOs do not bind. Left (L) and right (R) sides of embryo. Uninjected half of the embryo was used as control for phenotype assessment. In the right panel, sequences of the two alleles of Xenopus laevis tgfbi on the long (.L) and short (.S) chromosomes and human TGFBI are aligned. Red and blue regions indicate sites for MO binding, blue arrow marks the translation start site (ATG). hTGFBI 5′ sequence cannot bind the MOs and therefore could be used to rescue the effects of the MOs. (B and C) Targeted MS analysis shows Tgfbi protein is not detected in OVs dissected from the MO injected sides of stage 34 larvae. Abundance of the reference protein Gapdh does not change significantly following Tgfbi KD (paired Student’s t test, p > 0.05) (C) MO injected embryos cultured to stage 32 and processed by ISH for dlx5 and pax2 . While OV gene expression was normal on the control side (black arrow), it was greatly reduced on the MO-injected side (red arrow) of same larva in a large percentage of the cases (n = 63 larvae for dlx5 , n = 39 larvae for pax2 ). Embryos injected with Tgfbi MOs plus the MO-insensitive human TGFBI mRNA showed a significantly reduced percentage of reduced dlx5 expression (p < 0.05, Chi-Squared test), demonstrating rescue of the morphant phenotype. MO: morpholino injection; MO Rescue: MOs + hTGFBI mRNA injection. Bar indicates 200 μm. (D) Larvae were vibratome sectioned to measure OV and luminal volumes. In the image shown, dlx5 expression was reduced in the OV on the MO injected side (red arrow) compared to the control side (black arrow). hb, hindbrain. (E) Otic and luminal volumes of MO injected sides represented as percent change in otic volume compared to control (uninjected) sides of the same larvae (paired Wilcoxon signed rank test, ∗, p < 0.05, Data are represented as mean ± SEM).

Article Snippet: Human TGFBI , Origene , Cat#RC200411.

Techniques: Labeling, Binding Assay, Sequencing, Injection, Cell Culture, Expressing

Journal: iScience

Article Title: Time-resolved quantitative proteomic analysis of the developing Xenopus otic vesicle reveals putative congenital hearing loss candidates

doi: 10.1016/j.isci.2023.107665

Figure Lengend Snippet:

Article Snippet: Human TGFBI , Origene , Cat#RC200411.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Peptide Fractionation, Mass Spectrometry, Software

Journal: Frontiers in Nutrition

Article Title: Dietary intake of micronized avian eggshell membrane in aged mice reduces circulating inflammatory markers, increases microbiota diversity, and attenuates skeletal muscle aging

doi: 10.3389/fnut.2023.1336477

Figure Lengend Snippet: Applied biosystem’s primer/probe assays were used in this study.

Article Snippet: Tgfbi , , Mm01337605_m1.

Techniques: