conditionally immortalized proximal tubular epithelial cells (ciptec)  (Radboud University)

Journal: International Journal of Molecular Sciences

Article Title: Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

doi: 10.3390/ijms21010192

Figure Lengend Snippet: Analysis of proteins involved in fission process of mitochondrial dynamics in untreated and cysteamine (MEA)-treated CTNS −/−compared to CTNS +/+. Cell cultures were treated with 100 μM cysteamine (MEA) or DMSO (vehicle) for 24 h as specified in the figure. ( a ) Representative immunoblotting analysis in cellular lysate of conditionally immortalized proximal tubular epithelial cells (ciPTEC) from a healthy subject ( CTNS +/+) and cystinotic patient ( CTNS −/−). ( b – e ) The histograms (Drp1 pS637 , panel ( b ), n = 8; mitochondrial fission factor (Mff), panel ( c ), n = 3; mitochondrial fission 1 protein (Fis1), panel ( d ), n = 4; ubiquitinated Fis1 (Ub-Fis1), panel ( e ), n = 3) represent the means values ± SEM of the relative expression normalized on actin level. Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. p -value less than 0.05 was considered as statistically significant, (Student’s t test, *** p < 0.001; ** p < 0.01; * p < 0.05). For further details see under “materials and methods” section.

Article Snippet: Conditionally immortalized proximal tubular epithelial cells (ciPTEC), from healthy donor and cystinotic patients were obtained from Radboud University Medical Center, Nijmegen, The Netherlands and cultured as described in [ ].

Techniques: Western Blot, Expressing, Imaging, Software

Journal: International Journal of Molecular Sciences

Article Title: Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

doi: 10.3390/ijms21010192

Figure Lengend Snippet: Processing and oligomerization of optic atrophy 1 (OPA1) fusion protein in untreated and MEA-treated CTNS −/− compared to CTNS +/+. ( a ) Representative immunoblotting analysis of ciPTEC obtained from CTNS + / + and CTNS − / −. Where indicated, the cells were treated with MEA or DMSO (vehicle) for 24 h. The histograms of OPA1 ( b ) represent the percentage of relative expression of L and S forms of OPA1 in each lane ( n = 3). The histograms of OMA1 ( c ) represent the means values ± SEM of the relative expression normalized on actin level ( n = 3). ( d ) The fresh collected cells were treated with the cross-linker 1,6-bismaleimidohexane (BMH) 1 mM or with vehicle (DMSO) for 30 min at 37 °C, then centrifuged and resuspended in sodium dodecyl sulfate (SDS) lysis buffer for western blotting analysis with the antibody against OPA1. ( e ) The histograms represent the means values ± SEM of the relative expression of OPA1 oligomers ( n = 3). Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. Student’s t test, *** p < 0.001; * p < 0.05. For further details see under “materials and methods” section.

Article Snippet: Conditionally immortalized proximal tubular epithelial cells (ciPTEC), from healthy donor and cystinotic patients were obtained from Radboud University Medical Center, Nijmegen, The Netherlands and cultured as described in [ ].

Techniques: Western Blot, Expressing, Lysis, Imaging, Software

Journal: International Journal of Molecular Sciences

Article Title: Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

doi: 10.3390/ijms21010192

Figure Lengend Snippet: Expression and ubiquitination of mitofusin 2 (MFN2) in untreated and MEA-treated CTNS +/+ and CTNS −/−. ( a ) Representative immunoblotting analysis of untreated and MEA-treated ciPTEC CTNS +/+ and CTNS −/−. ( b ) The histogram of MFN2, n = 3, and ( c ) the histogram of ubiquitinated MFN2 Ub-MFN2, n = 3, represent the mean values ± SEM of the relative expression normalized on actin level. Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. Student’s t test, *** p < 0.001.

Article Snippet: Conditionally immortalized proximal tubular epithelial cells (ciPTEC), from healthy donor and cystinotic patients were obtained from Radboud University Medical Center, Nijmegen, The Netherlands and cultured as described in [ ].

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Imaging, Software

Journal: International Journal of Molecular Sciences

Article Title: Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

doi: 10.3390/ijms21010192

Figure Lengend Snippet: Parkin and ubiquitin carboxyl-terminal hydrolase 30 (USP30) proteins levels and MEA effect in CTNS +/+ and CTNS −/−. ( a ) Immunoblotting analysis of untreated and MEA-treated ciPTEC CTNS + / + and CTNS − / −. ( b ) The histogram represents the means values ± SEM of the relative expression of Parkin normalized on actin level ( n = 3). ( c ) The histogram represents the means values ± SEM of the relative expression of USP30 normalized on actin level ( n = 3). Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. Student’s t test, *** p < 0.001; * p < 0.05.

Article Snippet: Conditionally immortalized proximal tubular epithelial cells (ciPTEC), from healthy donor and cystinotic patients were obtained from Radboud University Medical Center, Nijmegen, The Netherlands and cultured as described in [ ].

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Imaging, Software

Journal: International Journal of Molecular Sciences

Article Title: Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

doi: 10.3390/ijms21010192

Figure Lengend Snippet: Comparative ultrastructural analysis of mitochondria in ciPTEC. ( a ) Representative images of TEM with magnification 16,000× of untreated and MEA-treated ciPTEC CTNS +/+ and CTNS −/−; scale bar = 1 µm. As shown in high magnification cropped micrographs and in ad hoc schematic reconstruction, mitochondria kept preserved ultrastructure in ciPTEC CTNS +/+ and in MEA-treated ciPTEC CTNS +/+ and CTNS −/−, whereas ciPTEC CTNS −/− showed disruption of mitochondrial cristae and the disarrangement of the internal structures; scale bar = 200 nm. ( b – e ) Quantitative analysis was performed with ImageJ v.1.52p in n ≥ 5 double-blind acquisitions for each experimental condition, red lines represent median with interquartile range. ( b ) Evaluation of relative mitochondrial size measured as area of n ≥ 27 mitochondrial sections. ( c ) Average number of cristae per mitochondrion in each cell ( n ≥ 27 mitochondrial sections). ( d ) The measure of distance of cristae junction near the inner membrane boundary and ( e ) the measure of cristae lumen, assessed on cristae membranes that outline the lumen boundary, were assessed in n ≥ 100 cristae. Non-parametric Mann-Whitney test was applied, *** p < 0.001; * p < 0.05.

Article Snippet: Conditionally immortalized proximal tubular epithelial cells (ciPTEC), from healthy donor and cystinotic patients were obtained from Radboud University Medical Center, Nijmegen, The Netherlands and cultured as described in [ ].

Techniques: Disruption, Membrane, MANN-WHITNEY