Conditionally immortalized human podocytes were generated by Prof. Dr. Moin A. Saleem (University of Bristol, South Mead Hospital, Bristol, UK) . Culture conditions were described previously .
Experimental Design and Drug Treatment
In order to examine the non-immunological effects of RTX on PAN induced cytoskeletal defects, podocytes were grown under growth restrictive conditions for 12 days and subsequently incubated with media containing 10 % FBS in the presence of 30 µg / ml PAN (Sigma, Munich, Germany), 100 µg / ml MabThera® (Roche, Basel, Switzerland) or the combination of both for 48 hours. All experiments were performed at least three times starting on growth-restricted days 12–14 (methodology previously described in ).
RNA isolation from cells
Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions including DNase digestion.
1–2 μg of total RNA was reverse transcribed with random hexamers and the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions.
RT-PCR for MS4A1 was performed according to the manufacturer’s instructions using the TaqMan Gene Expression Assay HS00544819_m1 (Applied Biosystems, Darmstadt, Germany) in combination with the TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Darmstadt, Germany). RT-PCR was performed with a StepOnePlus engine (Applied Biosystems, Darmstadt, Germany) (methodology previously described in ).
Hoechst 33342-staining of podocytes was performed as previously described . Images were obtained by a Zeiss Axio Imager A1 fluorescence microscope and Axio Vision SE64 Rel. 4.9.1 software (Zeiss, Jena, Germany). Apoptosis was defined as percentage of cells with nuclear fragmentation. For each sample in a given experiment, at least 200 randomly chosen cells were analyzed.
Immunofluorescence and cell imaging
For immunofluorescence, podocytes were plated on glass coverslips. After treatment, cells were fixed with 4 % formaldehyde (Fischar, Saarbrücken, Germany) for 15 min at 37°C, washed with PBS and permeabilized with PBS / 0.5 % Triton X-100 / 3 % BSA for 45 min at room temperature. After washing with blocking buffer (PBS / 0.5 % BSA), cells were incubated with Alexa FluorTM 488 phalloidin (1∶1000, Thermo Fisher Scientific, Waltham, USA) for actin staining in blocking buffer for 1 hour at room temperature (methodology previously described in ). In parallel, nuclei were stained with 4.6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma, Munich, Germany).
Fluorescence imaging was performed on a Zeiss Axio Imager A1 Fluorescence microscope with Axio Vision SE64 Rel. 4.9.1 software (Zeiss, Jena, Germany). Images were acquired using 10× and 20× phase contrast objectives with appropriate filter sets. Image processing and analysis was performed with ImageJ (https://imagej.nih.gov/ij/) software. All images were acquired at random positions.
Cell Adhesion Assay
After 48 hours of pharmacological treatment human podocytes were detached using Trypsin-EDTA (Biochrom, Berlin, Germany) and seeded on glass-coverslips in a 24-well plate for adhesion tests. After 1 and 6 hours, cells were fixed with 4 % formaldehyde (Fischar, Saarbrücken, Germany) and staining of the actin cytoskeleton was carried out as described above. For each condition the degree of spreading of 300 randomly chosen cells was measured using region measurement tools in ImageJ software (https://imagej.nih.gov/ij/).
Western Blot Analysis
Cells were harvested using CelLytic MT-buffer (Sigma-Aldrich, Hamburg, Germany) according to manufacturer’s instructions. Lysis-buffer was supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich, Hamburg, Germany), 10 µg / ml aprotinin (Roche, Mannheim, Germany), 10 µg / ml leupeptin (Roche, Mannheim, Germany) and 2 mM phenylmethanesulfonylfluoride (PMSF, Sigma, Munich, Germany). Isolation was performed at 4°C. Total protein content was measured by Bio-Rad protein assay (Bio.Rad, Munich, Germany). Samples (each 15 µg protein) were supplemented with Laemmli sample buffer (Bio-Rad, Munich, Germany) and boiled for 10 min at 95°C (methodology previously described in ).
Proteins were separated using 12 % Mini-PROTEAN TGX Precast Gels (Bio-Rad, Munich, Germany) and transferred on 0.45 µm PVDF Transfer Membranes (Thermo Scientific, Schwerte, Germany) with a MiniProtean Tetra Cell electrophoresis system (Bio-Rad, Munich, Germany) and a Biometra fastblot B34 blotting device (Biometra, Göttingen, Germany). 15 µl Precision Plus Protein All Blue Standard (Bio-Rad, Munich, Germany) was used as marker. Membranes were incubated with primary antibodies against CD20 (MabThera®: 1∶100; Roche, Basel, Switzerland), Caspase-3 (1∶5000; Cell Signaling, Danvers, USA), cleaved Caspase-3 (1∶5000; Cell Signaling, Danvers, USA), Caspase 8 (1∶5000; Cell Signaling, Danvers, USA) or cleaved Caspase-8 (1∶1000; Cell Signaling, Danvers, USA). Secondary antibodies used were horseradish peroxidase-conjugated goat anti-human IgG (Santa Cruz, Heidelberg, Germany: 1:10000 against MabThera®), goat anti-rabbit IgG (Santa Cruz, Heidelberg, Germany; 1∶10000 against Caspase-3 / cleaved Caspase-3 and cleaved Caspase-8) and goat anti-mouse IgG (Santa Cruz, Heidelberg, Germany; 1∶10000 against Caspase-8). Signal detection was performed with SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific, Schwerte, Germany) and visualized by the FUSION FX7 chemiluminescence-system (PEQLAB, Erlangen, Germany) und Fusion-software (PEQLAB, Erlangen, Germany). Intensity of signals was determined using ImageJ software (https://imagej.nih.gov/ij/). Densitometric data of the cleaved Caspase antibodies were normalized to full-length Caspase proteins (methodology previously described in ).
Values from multiple experiments were expressed as means ± SD. Statistical analysis was performed with GraphPad Prism 6.0 using Kruskal-Wallis test (non-parametric one-way ANOVA) with Dunn’s multiple comparisons test or an ordinary one-way ANOVA with Tukey’s multiple comparisons test. Statistical significance was defined as p < 0.05.