Journal: PLoS ONE

Article Title: Linagliptin Limits High Glucose Induced Conversion of Latent to Active TGFß through Interaction with CIM6PR and Limits Renal Tubulointerstitial Fibronectin

doi: 10.1371/journal.pone.0141143

Figure Lengend Snippet: A) Diabetic mice demonstrated increased pSmad2/3 nuclear expression with immunohistochemistry, which was reduced by linagliptin and telmisartan. Representative photographs for control (ctrl), control + linagliptin (ctrl + lina), diabetic (dm), diabetic + linagliptin (dm + lina) and diabetic + telmisartan (dm + tel) groups are shown (Magnification = original X 200). Quantification was done by counting the number of positive nuclei at X200 magnification. Data are expressed as mean ± SEM with ** = P<0.01 vs ctrl, ## = P<0.01 vs dm. B) Diabetic mice showed a trend towards increase in pSmad 2/total smad2 expression compared to control mice and a reduction with both linagliptin and telmisartan. This trend was consistent with the immunohistochemistry findings but did not reach statistical significance. Quantification was done using Image J. Data are expressed as mean ± SEM, n = 6.

Article Snippet: Immunohistochemistry for nuclear pSmad 2/3, was done on 4 micron paraffin embedded sections using goat anti-mouse pSmad 2/3 (SC11769-G, Santacruz Biotechnology, USA), after an overnight incubation at a concentration of 1:100, followed by donkey anti goat HRP tagged secondary antibody (Santacruz Biotechnology, USA at a concentration of 1:100).

Techniques: Expressing, Immunohistochemistry, Control

Journal: Scientific Reports

Article Title: Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice

doi: 10.1038/s41598-018-35716-6

Figure Lengend Snippet: Multiple types of analyses show that GDF11 does not cross the BBB. ( a ) GDF11 levels in the serum of 24-month-old mice following acute GDF11 treatment. Full-length blot is presented in Supplementary Fig. . ( b ) ELISA of SMAD2/3 phosphorylation in whole tissue lysates of 24-month-old mice following acute GDF11 treatment (1 mg/kg). n = 6 for each experimental group. Data plotted as the background-subtracted absorbance and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, *p = 0.03 (heart), ***p = 0.0003 (kidney), ***p = 0.0006 (liver), **p = 0.01 (spleen), # p = 0.06 (muscle), not significant (ns) (brain) compared to vehicle controls of each tissue type. ( c ) ELISA of SMAD2/3 phosphorylation in primary mouse cortical neurons and primary mouse astrocytes following 1-hour treatment with GDF11 (50 ng/ml) or vehicle. n = 6 for each neuron condition, n = 4 for each astrocyte condition. Data calculated as fold change from vehicle controls and shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t- test, ***p = 0.0001 (neuron), # p = 0.06 (astrocyte), compared to vehicle controls of each cell type. ( d ) Number of pSMAD2/3 + nuclei in SVZ-derived, dissociated neurospheres following 90-minute treatment with GDF11 (50 ng/ml) or vehicle. n = 3 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.01 compared to vehicle control. ( e ) Average colony size (number of cells per sphere) of young and old SVZ-derived neurospheres following 10-day treatment with GDF11 (50 ng/ml) or vehicle, in a clonal assay. n = 11 for young, n = 3 for old condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.02 compared to vehicle control. ( f ) Number of Tuj1+ cells differentiated from young and old SVZ-derived neurospheres following 7-day treatment with GDF11 (50 ng/ml) or vehicle, in a differentiation assay. n = 4 for each condition. Data shown as mean ± s.e.m., statistical analysis by unpaired, two-tailed Student’s t -test, *p = 0.04, **p = 0.006 compared to vehicle control. ( g ) Detection of biotinylated recombinant GDF11 with streptavidin-HRP (left) or Coomassie staining (right). Full-length blot and gel are presented in Supplementary Fig. 7b. ( h ) Biotinylated GDF11 levels in the brain parenchyma (left) and the spleen (right) of 3–4-month-old mice following acute GDF11 treatment (8 mg/kg). Biotinylated recombinant GDF11 protein was loaded to help detect the biotinylated protein in tissue samples. Tubulin was used as a loading control. Full-length blots are presented in Supplementary Fig. 7c.

Article Snippet: Neurosphere-derived cells were permeabilized and blocked in 10% normal goat or donkey serum, 5% BSA and 0.1% Triton™ X-100 in PBS for 1 hour at room temperature and incubated overnight at 4 °C with mouse monoclonal anti-pSMAD2/3 (610842, BD Biosciences) primary antibody at 1:5,000 dilution or mouse monoclonal anti-Tuj1 (MMS-435P, BioLegend) primary antibody at 1:2,000 dilution in blocking solution.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Derivative Assay, Clone Assay, Differentiation Assay, Recombinant, Staining