Journal: bioRxiv

Article Title: Triple A patient cells suffering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fibers

doi: 10.1101/381541

Figure Lengend Snippet: ( A ) Human adrenocortical NCI-H295R GFP and GFP-ALADIN over-expressing cells, NCI-H295R1-TR scrambled shRNA and AAAS shRNA ( AAAS knock-down) cells and human skin fibroblasts of healthy wild-type donors and triple A syndrome patients were stained with anti-PGRMC1 (green), anti-Aurora kinase A (AURKA) (red) and DAPI (blue). The different mutations in the human ALADIN protein are denoted as IVS14, S263P and W295X. Scale bars 5 μm, but for NCI-H295R GFP over-expressing cells: 10 μm. ( B ) Schematic drawing of the immunofluorescence staining of PGRMC1 and Aurora kinase A in (A).

Article Snippet: Here we show that fibroblasts from triple A patients carrying the ALADIN missense mutation S263P or the nonsense mutation W295X had about two-fold increased levels of PGRMC2 on mRNA and protein level compared to anonymized healthy control fibroblasts ( ).

Techniques: Expressing, shRNA, Knockdown, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Triple A patient cells suffering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fibers

doi: 10.1101/381541

Figure Lengend Snippet: ( A ) Total RNA was isolated from human skin fibroblasts of healthy wild-type donors and patients with triple A syndrome. The different mutations in the human ALADIN protein are denoted on the x-axis of the diagram: IVS14, S263P and W295X. WT, wild-type. Significant differences were measured with unpaired Wilcoxon–Mann–Whitney U-test. Boxplot widths are proportional to the square root of the samples sizes. Whiskers indicate the range outside 1.5 times the inter-quartile range (IQR) above the upper quartile and below the lower quartile. Outliers were plotted as dots. ( B ) Total protein was isolated from human skin fibroblasts of healthy wild-type donors and triple A patients followed by western blot with indicated antibodies.

Article Snippet: Here we show that fibroblasts from triple A patients carrying the ALADIN missense mutation S263P or the nonsense mutation W295X had about two-fold increased levels of PGRMC2 on mRNA and protein level compared to anonymized healthy control fibroblasts ( ).

Techniques: Isolation, MANN-WHITNEY, Western Blot

Journal: bioRxiv

Article Title: Triple A patient cells suffering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fibers

doi: 10.1101/381541

Figure Lengend Snippet: Cells were cold-treated and stained with anti-PGRMC1 (green), anti-α-tubulin (TUBA) (red), anti-CENPB (red), anti-NDC80 (red), anti-TACC3 (red) and DAPI (blue). ( A ) Human adrenocortical NCI-H295R cells. ( B ) Human skin fibroblasts of healthy wild-type donors. ( C ) Human skin fibroblasts of triple A patients. The different mutations in the human ALADIN protein are denoted as IVS14, S263P and W295X. Scale bars 5 μm.

Article Snippet: Here we show that fibroblasts from triple A patients carrying the ALADIN missense mutation S263P or the nonsense mutation W295X had about two-fold increased levels of PGRMC2 on mRNA and protein level compared to anonymized healthy control fibroblasts ( ).

Techniques: Staining

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: Partial loss of phenotype in seed-sequence variant of gFA11A. ( a ) Primary FRDA fibroblasts GM3816 were transfected with gFA11, Mut1, and Mut2 siRNAs (Fig. ). The cells were transfected twice over 7 days. Cells were kept in DMEM + 5 mM BHB after the first transfection. **p < 0.01 by ANOVA with Tukey pair-wise comparisons. ( b ) Primary FRDA fibroblasts GM3816 were transfected with gFA11, Mut1, or control (C3) siRNA, or mock transfected, every 3–4 days for a total of four times over 14 days. Cells were kept in DMEM + 5 mM glucose throughout. **p < 0.01 and ***p < 0.005 by ANOVA with Tukey pair-wise comparisons. Error bars represent means ± 1 SD. The averages shown were calculated on three independent replicates and the results are representative of at least two independent experiments.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Sequencing, Variant Assay, Transfection, Control

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: Alterations in cytokine secretion induced by gFA11. ( a ) Primary FRDA GM3816 fibroblasts were transfected in triplicate with gFA11 siRNA or Mut1 siRNA four times over twelve days. After the first transfection, the cells were grown in DMEM plus 5 mM BHB. At day 12, after the fourth transfection, cells were incubated with DMEM with BHB but without FBS. The following day, the medium was collected, concentrated, and analyzed by Luminex assay. Cytokine concentrations were normalized by cell numbers. ( b ) Primary FRDA GM3816 fibroblasts were infected with a gFA11-encoding vector or empty vector (E). Infected cells were selected with puromycin and expanded in glucose-based medium. Cells were then seeded at low density and kept in medium without FBS for 24 hours before collecting, concentrating, and analyzing the medium. *p < 0.05; **p < 0.01; ***p < 0.005 by Student’s t test. Error bars represent means ± 1 SD. The averages shown were calculated on three ( a ) or four ( b ) independent replicates and the results are representative of two independent experiments.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Transfection, Incubation, Luminex, Infection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: Effects of gFA11 on cell cycle and morphology. ( a ) Morphology of primary FRDA GM3816 fibroblasts transfected with gFA11 siRNA or Mut1 siRNA, or not transfected. Arrows indicate senescent-appearing cells. ( b ) Flow-cytometric side scattering (SSC) of GM3816 and GM3665B cells infected with a gFA11-encoding or control-encoding (C3) vector. ***p < 0.005 by Student’s t test. ( c ) Cell-cycle analysis of GM3816 cells, untransfected or transfected 4 times with gFA11 siRNA over two weeks. Error bars represent means ± 1 SD.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Transfection, Infection, Control, Plasmid Preparation, Cell Cycle Assay

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: Activation of p38 MAP kinase in FRDA cells. ( a ) Frataxin protein levels (by ELISA) in normal control fibroblasts (6030) and primary FRDA fibroblasts (4675, 156, 4491, 203). ***p < 0.005 by ANOVA with Tukey pair-wise comparisons. (The aggregate p value comparing the normal control to the FRDA cells combined was less than 1 × 10 -8 ). ( b ) p38 phosphorylation in the same cells as in ( a ) *p < 0.05 by ANOVA with Tukey pair-wise comparisons. (156 and 4491 cells grew extremely slowly and only two replicates were possible; the aggregate p value comparing the normal control to the FRDA cells combined was 0.008). ( c ) Frataxin protein levels (by ELISA) in normal control fibroblasts (6030) transfected with a random siRNA, C3 (siC3), a known toxic siRNA, C5 (siC5), or an siRNA to frataxin (siFXN) after one transfection (left bar) or two transfections (right bar). The decrease with siFXN was associated with p < 0.005 in both cases by Student’s t test. ( d ) p38 phosphorylation in the same cells as in ( c ) *p < 0.05; ***p < 0.005 by Student’s t test. Error bars represent means ± 1 SD. The averages shown were calculated on three independent replicates and the results are representative of at least two independent experiments.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Control, Transfection

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: p38 inhibitors increase the growth of FRDA cells in a dose-dependent manner. ( a ) p38 phosphorylation status in control GM8399 fibroblasts and FRDA GM3816 fibroblasts. ***p < 0.005 by Student’s t test. ( b ) Primary FRDA GM3816 fibroblasts treated every 48 h with the p38 inhibitor BIRB796 at the concentrations indicated. Cells were counted at day 14. **p < 0.01 relative to carrier control (CC) by ANOVA with Tukey pair-wise comparisons. (100 nM reached a p value of 0.051 relative to carrier control.) Error bars represent means ± 1 SD. ( c ) Primary FRDA GM3816 fibroblasts treated every 48 h with the p38 inhibitor BIRB796, at the concentrations indicated. Cells were counted at day 7. (d) Primary DL156 primary FRDA fibroblasts were treated every 48 h with the p38 inhibitor BIRB796, at a concentration of 500 nM. *p < 0.05 relative to carrier control (CC) by Student’s t test. **p < 0.01; ***p < 0.005; relative to carrier control (CC) by ANOVA with Tukey pair-wise comparisons. Error bars represent means ± 1 SD. In all experiments, the averages shown were calculated on three independent replicates and the results are representative of at least two independent experiments.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Control, Concentration Assay

Journal: Scientific Reports

Article Title: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

doi: 10.1038/s41598-018-23168-x

Figure Lengend Snippet: p38 phosphorylation is not sustained by cytokines. Primary apparently healthy fibroblasts GM8399 were transfected with FXN siRNA or a random control clone. Two hours after transfection, cells were incubated with DMEM without FBS in presence of 500 nM BIRB796 or carrier control. The following day, ( a ) frataxin protein levels, ( b ) p38 phosphorylation levels, and ( c ) IL-6 levels were measured by ELISA. **p < 0.01; ***p < 0.005 by ANOVA with Tukey pair-wise comparisons. Error bars represent means ± 1 SD. In all experiments, the averages shown were calculated on three independent replicates and the results are representative of at least two independent experiments.

Article Snippet: We obtained primary FRDA fibroblasts, GM3816 and GM3665B; age- and sex-matched apparently healthy control fibroblasts, GM8400 and GM8399; and FRDA lymphocytes, 15850, from Coriell (Coriell Institute for Medical Research, Camden, NJ).

Techniques: Transfection, Control, Incubation, Enzyme-linked Immunosorbent Assay