Journal: International Journal of Molecular Sciences

Article Title: Cadmium Complexed with β2-Microglubulin, Albumin and Lipocalin-2 rather than Metallothionein Cause Megalin:Cubilin Dependent Toxicity of the Renal Proximal Tubule

doi: 10.3390/ijms20102379

Figure Lengend Snippet: Expression of megalin and cubilin in cultured low and high passage rat renal proximal tubule cells (WKPT-0293 Cl.2). ( A ) Expression levels of megalin ( Lrp2 ) and cubilin ( Cubn ) mRNA by qPCR in means ± SD of 4 experiments are shown. The C T values in low passage cells were 20.6 ± 0.6 and 29.6 ± 0.1 for megalin and cubilin, respectively. Data obtained were normalized to the expression of the reference gene glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ). Statistical analysis compares the low and high passage cells by unpaired t -test. ( B ) Expression of megalin protein in low and high passage WKPT-0293 Cl.2 cells. Megalin was detected by immunofluorescence microscopy of permeabilized cells (green). Nuclei were counterstained with Hoechst 33342 (blue). The experiment is representative of four similar ones. Scale bar = 20 µm. ( C ) Fluorescence intensity was analyzed in 188–705 cells (dots) from 4 different experiments (colors); for details of box and whisker plot statistics, see . Statistical analysis compares the low and high passage cells by non-parametric one-way ANOVA (Kruskal-Wallis-Test, using Dunn’s post-hoc analysis). ( D ) Expression of cubilin protein was detected by immunoblotting. β-actin was used as loading control. A representative experiment is shown. MM = molecular mass.

Article Snippet: Briefly, cells were fixed in 4% paraformaldehyde/PBS, permeabilized with 0.1% Triton X-100/PBS, blocked in 1% bovine serum albumin/PBS, incubated with primary mouse LRP2 antibody (1:200, cat. # NB110-96417, Novus Biologicals, Wiesbaden, Germany) followed by secondary donkey anti-mouse antibody conjugated to Alexa488 (1:500 cat. # 715-545-202, Dianova, Hamburg, Germany) together with the nuclear dye Hoechst 33342 (0.8 μg/mL).

Techniques: Expressing, Cell Culture, Immunofluorescence, Microscopy, Fluorescence, Whisker Assay, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Cadmium Complexed with β2-Microglubulin, Albumin and Lipocalin-2 rather than Metallothionein Cause Megalin:Cubilin Dependent Toxicity of the Renal Proximal Tubule

doi: 10.3390/ijms20102379

Figure Lengend Snippet: qPCR primers.

Article Snippet: Briefly, cells were fixed in 4% paraformaldehyde/PBS, permeabilized with 0.1% Triton X-100/PBS, blocked in 1% bovine serum albumin/PBS, incubated with primary mouse LRP2 antibody (1:200, cat. # NB110-96417, Novus Biologicals, Wiesbaden, Germany) followed by secondary donkey anti-mouse antibody conjugated to Alexa488 (1:500 cat. # 715-545-202, Dianova, Hamburg, Germany) together with the nuclear dye Hoechst 33342 (0.8 μg/mL).

Techniques:

Journal: STAR Protocols

Article Title: Differentiation and single-cell RNA-seq analyses of human pluripotent-stem-cell-derived renal organoids

doi: 10.1016/j.xpro.2023.102314

Figure Lengend Snippet:

Article Snippet: LRP2 (Mouse mAb) 1:100 dilution for immunofluorescence , Novus Biologicals , Cat# NB110-96417.

Techniques: Immunofluorescence, Recombinant, Saline, Transfection, Staining, Gene Expression, Sequencing, CRISPR, Plasmid Preparation, Software, Cell Culture, Nucleic Acid Electrophoresis, Transferring

Journal: International journal of molecular sciences

Article Title: Autophagy Deficiency in Renal Proximal Tubular Cells Leads to an Increase in Cellular Injury and Apoptosis under Normal Fed Conditions.

doi: 10.3390/ijms21010155

Figure Lengend Snippet: Figure 1. Impairment of autophagy in Atg7-deficient renal proximal tubular cells. (A) Decrease of Atg7 and increase of LC3-I in 2-month-old Atg7flox/flox;KAP-Cre+ mouse kidney. Atg7 and LC3 Western blot (LC3-I/unconjugated LC3 and LC3-II/lipidated LC3) using whole kidney lysate of Atg7flox/flox;KAP-Cre+

Article Snippet: Rabbit anti-LC3 and anti-Atg7 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA, cat#12741 and #8540), guinea pig anti-p62 antibody was form PROGEN Biotech GmbH (Heidelberg, Germany, cat#GP62-C), mouse anti-Megalin antibody was from NOVUS (Littleton, CO, USA, cat#NB110-96417), goat anti-Kim-1 antibody was from R&D systems (Minneapolis, MN, USA, cat#AF1817), and rabbit anti-Lamp1 was from Abcam (Tokyo, Japan, cat# ab24170).

Techniques: Western Blot

Journal: International journal of molecular sciences

Article Title: Autophagy Deficiency in Renal Proximal Tubular Cells Leads to an Increase in Cellular Injury and Apoptosis under Normal Fed Conditions.

doi: 10.3390/ijms21010155

Figure Lengend Snippet: Figure 5. Electron microscopic analyses of Atg7 deficient renal proximal tubular cells. The renal proximal tubular cells of 2-month-old Atg7flox/flox;KAP-Cre+ (A,B,D–F) and Atg7flox/flox (C) mice were investigated with a transmission electron microscopy. (B) is higher magnification of area. Black asterisks indicated degenerated mitochondria. (A) (white box). (E,F) are higher magnification of areas (white boxes 1 and 2, respectively) in (D). Asterisks in (E) indicated peroxisome-like structures in the multi-lamellar bodies. Arrows indicate lysosome like structures in the multi-lamellar bodies.

Article Snippet: Rabbit anti-LC3 and anti-Atg7 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA, cat#12741 and #8540), guinea pig anti-p62 antibody was form PROGEN Biotech GmbH (Heidelberg, Germany, cat#GP62-C), mouse anti-Megalin antibody was from NOVUS (Littleton, CO, USA, cat#NB110-96417), goat anti-Kim-1 antibody was from R&D systems (Minneapolis, MN, USA, cat#AF1817), and rabbit anti-Lamp1 was from Abcam (Tokyo, Japan, cat# ab24170).

Techniques: Transmission Assay, Electron Microscopy