Journal: Molecular Metabolism

Article Title: P2Y2R contributes to the development of diabetic nephropathy by inhibiting autophagy response

doi: 10.1016/j.molmet.2020.101089

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Wilms Tumor-1 (WT1) , Boster Biological Technology , #M00199-1 , Rabbit , 1 μg/ml (IHC).

Techniques:

Journal: Molecular Metabolism

Article Title: P2Y2R contributes to the development of diabetic nephropathy by inhibiting autophagy response

doi: 10.1016/j.molmet.2020.101089

Figure Lengend Snippet: P2Y2R deficiency protects against podocyte loss and glomerular injury in DN. (A) Relative mRNA levels of Nphs1 (nephrin) and Nphs2 (podocin) were determined by real-time PCR analysis (n = 3–5). (B) Localisation of WT1, a podocyte marker, was analysed by immunohistochemistry, and representative images are shown. The number of stained podocytes per glomeruli was counted by using ImageJ software (n = 3). (C) Kidney sections were stained with PAS staining and glomerular morphological changes were scored as described in the method section (n = 3). Data are presented as mean ± SEM. One-way ANOVA was used for statistical analysis followed by Bonferroni's multiple comparisons test. ∗∗∗p < 0.001 vs WT control mice; and ### p < 0.001 vs WT DN mice. Scale bar, 50 μm.

Article Snippet: Wilms Tumor-1 (WT1) , Boster Biological Technology , #M00199-1 , Rabbit , 1 μg/ml (IHC).

Techniques: Real-time Polymerase Chain Reaction, Marker, Immunohistochemistry, Staining, Software, Control

Journal: Physiological Reports

Article Title: Renal stromal miRNAs are required for normal nephrogenesis and glomerular mesangial survival

doi: 10.14814/phy2.12537

Figure Lengend Snippet: Mesangial defects in FoxD1 GC ;Dicer fl/fl mature glomeruli at E18.5. (A–D) Immunofluorescence staining for Desmin (red) showed evidence for early mesangial cell migration in both control ( n = 3 embryos, 25 comma-/s-shaped bodies) and FoxD1 GC ;Dicer fl/fl ( n = 3 embryos, 16 comma-/s-shaped bodies) developing WT1 + (red) comma- and s-shaped bodies. (E, F) Developing capillary loop nephrons as marked by expression of Lef1 (red) in early podocytes showed evidence for Pdgfr β + (green) mesangial formation in FoxD1 GC ;Dicer fl/fl kidneys ( n = 3 embryos, 49 glomeruli), although the mesangial network appeared simplified when compared to controls ( n = 3 embryos, 52 glomeruli). (G–L) Mature FoxD1 GC ;Dicer fl/fl glomeruli displayed overt mesangial abnormalities as evident by a significant reduction in Pdgfr β + (green) and Desmin + (red) immunoreactivity. (M–O) Semiquantitative analysis revealed no significant difference in developing WT1 + comma- and s-shaped bodies (M) or Lef1 + early glomeruli (N) in control and FoxD1 GC ;Dicer fl/fl embryos. However, there were significantly fewer WT1 + mature glomeruli with a Desmin + mesangial network in FoxD1 GC ;Dicer fl/fl kidneys ( n = 3 embryos, 29 glomeruli, **** P < 0.0001), when compared to controls ( n = 3 embryos, 19 glomeruli) (O). The presence of WT1 + (red) podocytes and intussuscepted Pecam + (red) capillaries in the FoxD1 GC ;Dicer fl/fl glomeruli suggest that the mesangial defects are likely to be a primary consequence of loss of stromal miRNAs.

Article Snippet: Primary antibodies were used at 1:100 dilution and include: Six2 (Proteintech #11562-1-AP, Chicago, IL), Forkhead box D1 (FoxD1) (Santa Cruz #sc-47585, Dallas, TX), Calbindin (Sigma #C9848, St. Louis, MO), annexin A2 (Anxa2) (Cell Signaling #8235, Danvers, MA), Neural cell adhesion molecule (Ncam) (Sigma #C9672), Tenascin C (Millipore #AB19011, Billerica, MA), Yes-associated protein 1 (Yap) (Cell Signaling #4912), phospho-Yap (pYap) (Cell Signaling #4911), Desmin (Dako #M076029-2, Carpinteria, CA), Wilm’s tumor-1 (WT1) (Santa Cruz #sc-192), Platelet-derived growth factor receptor- β (Pdgfr β ) (eBiosciences, Millipore #14-1402), α -smooth muscle actin (SMA)-FITC (Sigma #F3777), Renin (Santa Cruz #sc-27318), Lef1 (Cell Signaling #2230), Pecam (BD Pharmingen #BDB550274, San Jose, CA), Bcl2-like 11 (Bim) (Cell Signaling #2819), Meis1/2/3 (Millipore #05-779), aCaspase3 (Promega #PRG7481, Madison, WI), and phospho-histone H3 (Sigma #H9161).

Techniques: Immunofluorescence, Staining, Migration, Control, Expressing

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: A preclinical mouse model of glioma with an alternative mechanism of telomere maintenance (ALT)

doi: 10.1002/ijc.29171

Figure Lengend Snippet: Absence of hTERT expression in TG20 ALT cells. ( a ) hTERT mRNA expression in telomerase-positive GSCs (TG16, TG1N, OB1 and TG10) and ALT cells (TG20 and SAOS-2). Values are presented as the mean ± SEM of two independent experiments performed in duplicate. ( b ) The same cell lines as in ( a ) were evaluated but for hTERC mRNA expression. ( c ) Representative methylation-specific PCR (MSP) results for the proximal region and WT1 binding site of the TERT promoter in SAOS-2 and TG20 ALT cells and in TG1N and TG16 telomerase-positive cells. Bands in the M and U lanes represent methylated and unmethylated PCR products, respectively. ( d ) mRNA expression of hTERT, TES, and RUNX3 in TG20 cells after treatment with 2.5 or 5 µM 5-azacytidine (5-aza), relative to their expression in control DMSO-treated cells. ( e ) WT1 protein levels determined by Western blot analysis for the indicated cell lines. β-Actin was used as a loading control. ( f ) Comparison of expression ratios of WT1 protein vs . β-actin in the different cell lines. Experiment was repeated twice using samples from independent cell cultures.

Article Snippet: WT1 (Wilm's tumor protein 1) antibody (1:400, NB-110–60011; Novus Biologicals) was used to detect the WT1 protein.

Techniques: Expressing, Methylation, Binding Assay, Control, Western Blot, Comparison