Mitochondrial degradation during autophagy in control and patients´ fibroblasts by immunofluorescence analysis.

<p>Cultured fibroblasts were incubated with lysotraker (lysosomal marker; red); inmunostained with anti-cytochrome <i>c</i> (mitochondrial marker; green) and examined in a fluorescence microscope (63X magnifications) as described in Material and Methods. Colocalization of both markers was assessed by Fiji program (Bar = 16 μm).</p

Quantitative gene expression analysis of ATF4, CHOP, asparagine synthase and GADD45 by qRT-PCR.

<p>(A, B, C and D) Quantities are shown relative to the expression levels of ATF4, CHOP, asparagine synthase and GADD45 mRNA in control sample. The data represent mean ± SD of three different experiments. *<i>P</i><0.05; **<i>P</i><0.01, ***<i>P</i><0.001.</p

Analysis of autophagy process in patients-derived fibroblasts with homocystinuria.

<p>(A) Analysis of protein levels by immunoblotting. Equal amounts from control and the five patients were loaded (50 μg of total cell lysates) and subjected to Western Blot with anti-LAMP1 antibody. We used anti-GAPDH antibody to ensure equal amounts of protein loaded in each lane. This result is representative of three independent experiments. (B) Protein quantification was performed by laser densitometry. The ratios between proteins/GAPDH for each cell line were calculated to determine the expression fold-change relative to control. Data represent mean ± standard deviation of three independent experiments. (C) Analysis of autophagosomes formation by fluorescence microscopy. Representative images of the autophagosome monitorization in control and patients-derived fibroblasts. (D) Quantification of autophagosome number per area in patients´cells compared to control. Results were obtained from three independent experiments. Data represent mean ± SEM (standard error of the mean = SD/) of positive fluorescence points of LC3B in each mm² of 80 cells analysis. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

Levels of intracellular Ca²⁺ in control and patients´ fibroblasts.

<p>(A) Representative recording showing levels of intracellular Ca²⁺ prior to and after exposure of patients and control to 1 μM thapsigargin (Thap). (B) Quantitative data from three different experiments showing the peak amplitude of the Thap-induced rise in cytosolic Ca²⁺ in patients and control fibroblasts. Data represent mean ± SEM (standard error of the mean = SD/) (***<i>P</i><0.001).</p

Mitochondrial ultrastructure analysis.

<p>Representative electron micrographs of control- and patients-derived fibroblasts. Control fibroblasts showed mitochondria with typical ultrastructure. Laminar bodies and autophagosomes with mitochondria were observed in patients´ fibroblasts. (Bar = 1μm).</p

Splicing factors binding sites in the <i>PAH</i> intronic region surrounding the c.1199+17G>A and c.1199+20G>C variants, predicted with ESEFinder (http://rulai.cshl.edu) and HSF (http://www.umd.be/HSF3/) programs.

<p>Effects of variants and deletions introduced in minigenes. WT; wild type.</p

Analysis of MAM-associated protein levels in control and patients´ fibroblasts.

<p>(A) Equal amounts from one control and the five patients were loaded (50 μg of total cell lysates) and subjected to Western Blot with anti-Grp75, anti-σ-1R and anti-Mfn2 antibodies. We used anti-GAPDH antibody to ensure equal amounts of protein loaded in each lane. This result is representative of three independent experiments. (B) Protein quantification was performed by laser densitometry. The ratios between proteins/GAPDH for each cell line were calculated to determine the expression fold-change relative to control. Data represent mean ± standard deviation of three independent experiments (*<i>P</i><0.05; **<i>P</i><0.01).</p

Analysis of trolox effect in ROS levels and autophagy marker´s levels in control and patients´ fibroblasts.

<p>(A) ROS content was determined by flow cytometry using H₂DCFDA as fluorescence probe after cells were incubated for 72 h with 1 mM of trolox. Results are expressed as DCF fluorescence relative to untreated cells. Data represent mean values ± standard deviation from two experiments performed by triplicate (*<i>P</i><0.05; **<i>P</i><0.01). A representative overlap of ROS levels in untreated P1 cells and trolox-treated P1 fibroblasts is shown. (B) Cells were incubated with 1mM of trolox for 72 h. Equal amounts of protein from control and the five patients´ fibroblasts were loaded (50 μg of total cell lysates) and subjected to immunoblotting with anti-LAMP1 antibody. We used anti-GAPDH antibody to ensure equal amounts of protein loaded in each lane. This result is representative of three independent experiments.</p

Analysis of protein levels involved in ER stress and Ca²⁺ homeostasis and processing of mRNA XBP1 in control and patients-derived fibroblasts.

<p>(A) Equal amounts from controls and patients were loaded (50 μg of total cell lysates) and subjected to Western Blot with anti-Herp, anti-Grp78 and anti-IP₃R1 antibodies. We used anti-Hsp60 antibody to ensure equal amounts of protein loaded in each lane. This result is representative of three independent experiments. Protein quantification was performed by laser densitometry. The ratios between proteins/Hsp60 for each cell line were calculated to determine the expression fold-change relative to control. (B) Data represent mean ± standard deviation of three independent experiments. (C) Equal amounts from controls were loaded (50 μg of total cell lysates) and subjected to Western Blot with anti-Herp and anti-Grp78 antibodies. We used anti-GAPDH antibody to ensure equal amounts of protein loaded in each lane. This result is representative of two independent experiments. (D) Equal amounts from control and patients were loaded (50 μg of total cell lysates) and subjected to Western Blot with anti-phospho-PERK antibody. We used anti-GAPDH antibody to ensure equal amounts of protein loaded in each lane. This result is representative of two independent experiments. (E) RT-PCR analysis of the processing of mRNA XBP1 transcription factor. Tm: tunicamycin; u: XBP1 unspliced form; s: XBP1 spliced form.</p

Autophagy inhibition and analysis of apoptosis in control and patients´ fibroblasts by flow cytometry.

<p>Cells were incubated for 48h with 20 mM of 3-MA. Data represent mean values ± standard deviation from two experiments performed by triplicate (*<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001). (A) Percentage of annexinV-positive apoptotic cells in patients and control cells relative to total cell number per sample. (B) Percentage of viability in patients and control cells relative to total cell number per sample. Dot blots from a representative experiment of apoptosis levels are shown.</p