Silencing nucleolin does not alter the ECM-promoting effects of IGFBP-5.

<p>(A) Nucleolin is efficiently silenced using sequence-specific siRNA. Silencing nucleolin has no effect on IGFBP-5 production. (B) Silencing nucleolin inhibits translocation of IGFBP-5 to the nucleus as detected by immunoblotting of nuclear extracts. Histone H3 is used to confirm nuclear localization. (C) Silencing nucleolin does not alter the ECM-promoting effects of IGFBP-5 as detected using immunoblotting.</p

Nucleolin is an IGFBP-5 binding partner.

<p>Following identification of nucleolin using mass spectrometry, interaction with IGFBP-5 was confirmed using co-precipitation. (A) Wild type IGFBP-5 binds nucleolin in fibroblasts from three different donors. (B) Reverse pull-down with nucleolin antibody confirms the interaction with IGFBP-5. GAPDH in input lysate was detected to confirm that lysate fractions with equivalent protein levels were used for the IP. (C) Co-localization of nucleolin and IGFBP-5 is detected using immunofluorescence (IGFBP-5: red; nucleolin: green).</p

Mutating the NLS domain of IGFBP-5 reduces its ability to bind nucleolin.

<p>The interaction of Flag-tagged IGFBP-5 and nucleolin was detected using immunoprecipitation using anti-Flag antibody followed by immunoblotting to detect nucleolin. Input nucleolin was detected in lysates prior to co-precipitation to ensure that samples contained equivalent amounts of protein.</p

Generation of IGFBP-5 with mutated IGF binding and NLS domains and validation of the loss of function imparted by the mutations.

<p>(A) Schematic of wild type, IGF binding site-mutant, and NLS-mutant IGFBP-5 expressing constructs used for the generation of adenoviruses. (B) Confirmation that all three constructs result in IGFBP-5 production by western blotting and validation of the loss of binding to IGF of the IGF-binding site mutant using western ligand blotting. (C) The NLS-mutant IGFBP-5 does not translocate to the nucleus of primary human fibroblasts as detected using immunofluorescence. (D) The NLS-mutant IGFBP-5 is not detected in nuclear extracts of primary human fibroblasts expressing the indicated constructs, but its levels were higher in cytoplasmic fractions as detected following cellular fractionation and immunoblotting.</p

Mutant IGFBP-5 is pro-fibrotic <i>ex vivo</i> in human skin.

<p>Human skin from four different donors was injected with the indicated IGFBP-5 expressing adenoviruses and maintained in organ culture for 10 days. (A) Skin was harvested and examined histologically. (B) Dermal thickness was measured in human skin explants. (C) ECM was quantified using hydroxyproline assay. Data in (B) and (C) represent mean and standard deviation.</p

DataSheet1_Supporting clinical research professionals through educational innovations.PDF

Clinical Research Professionals (CRPs) are essential members of the Clinical and Translational Research Workforce. Many academic medical institutions struggle to recruit and retain these vital team members. One strategy to increase job satisfaction and promote the retention of CRPs is through educational initiatives that provide training and professional development. The South Carolina Clinical and Translational Research (SCTR) Institute Workforce Development (WD) team at the Medical University of South Carolina (MUSC) developed several trainings as part of our larger educational portfolio for CRPs. In 2022 WD implemented a digital badge micro-credential for SCTR’s Core Clinical Research Training (CCRT) course in collaboration with institution-wide education and technology offices. Beginning in January 2023, individuals were able to earn the CCRT Certified Digital Badge upon successful completion of the CCRT course.</p

Characterization of human lung myofibroblasts by immunofluorescence.

<p>Primary human lung myofibroblast cultures between passages 4 and 5 were stained with myofibroblast markers. Representative images are shown for: anti-fibroblast surface protein (FSP) and the mouse isotype control IgM; anti-fibroblast antigen which recognises the fibroblast antigen (thy-1/CD90) and the mouse isotype control IgG1; α-smooth muscle actin, and the isotype control IgG2a; collagen type 1 antibody and rabbit isotype control IgG; CD68 cell staining was negative; and corresponding isotype control IgG3, indicating that there is no contamination of monocytes or macrophage cells; CD34 antibody shows negative staining as does the appropriate isotype control IgG1. Nuclei are stained with DAPI. </p

Myofibroblast proliferation is inhibited by K_Ca3.1 channel block.

<div><p>Constitutive unstimulated wound healing is not altered by K_Ca3.1 blockade, and growth factor-stimulated healing is not inhibited by TRAM-7 or TRAM-85 .</p> <p><b>a</b>) Myofibroblast proliferation was increased following 48h of stimulation with FBS and significantly reduced by TRAM-34 (200 nM). <b>b</b>) An example of the wound created in a confluent monolayer of myofibroblasts in the wound healing assay and how it heals over the 48 hours. <b>c</b>) This graph displays that over the time course of the wound healing assay no significant differences were found between NFC and IPF donors in response bFGF, similar results were seen with FBS but results are not shown. <b>d</b>) TRAM-34 does not inhibit wound healing in the absence of mitogenic stimulation. <b>e</b>) FBS significantly increases wound healing (*<i>P</i>=0.0168, Paired t-test) but the molecules TRAM-7 and TRAM-85 which are structurally related to TRAM-34 do not have K_Ca3.1 channel-blocking activity and do not inhibit mitogen-dependent wound healing. Data represent mean±SEM for all figures a, c, d and e.</p></div

The effect of low dose BLM on dermal and pulmonary fibrosis.

<p>Mice treated with BLM (1 U/kg) were sacrificed on days 7 (N = 7), 10 (N = 4), 14 (N = 4), 21 (N = 4), and 28 (N = 4). (A) Hydroxyproline levels were measured in lungs using hydroxyproline assay. (B) Dermal thickness was measured in dorsal skin. (C) Subcutaneous adipose layer thickness was measured in dorsal skin.</p

Blocking K_Ca3.1 with TRAM-34 and ICA-17043 induces a dose-dependent attenuation of myofibroblast wound healing.

<p><b>a</b>) and <b>b</b>) Myofibroblasts stimulated with 10% FBS and 0.1% DMSO vehicle control, showed accelerated wound healing in comparison to 0.1% DMSO alone (<i>P</i>=0.002, and P=0.004 respectively, paired t-test). There was a dose-dependent decrease in FBS-induced wound healing over 48h in the presence of either <b>a</b>) TRAM-34 (20 nM and 200 nM) (<i>P</i><0.0001, repeated measures ANOVA) or <b>b</b>) ICA-17043 (10 nM and 100 nM) (<i>P</i>=0.0095, repeated measures ANOVA). <b>c</b>) Myofibroblast wound healing in response to 10 ng/ml bFGF and 0.1% DMSO stimulation at 48 h was increased in comparison to media alone (*<i>P</i>=0.002, paired t-test). There was a dose-dependent decrease in wound healing over the 48 h in the presence ICA-17043 (<i>P</i>=0.0076, Repeated measures ANOVA). Data represented as mean±SEM for all the above figures.</p