JCS087452 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Several PUFAs play an auxiliary role in peroxisome proliferation. (A) Several different PUFAs augment peroxisomal proliferation. AOx-deficient fibroblasts were cultured for 48 h in the presence of 50 µM DHA, EPA, AA, α-LA, OA, or stearic acid (SA). Peroxisome abundance per cell was determined as in Fig. 2B. *P<0.01 vs mock treatment. (B) Several PUFAs induce hyper-oligomerization of Pex11pβ. AOx-deficient fibroblasts were cultured for 20 h in the absence or presence of 150 µM DHA, EPA, or α-LA and then treated for 30 min with 1 mM DSP. Cell lysates were fractionated by sucrose density-gradient ultracentrifugation and each fraction was analyzed by immunoblot using an anti-Pex11pβ antibody. Upward arrowheads indicate the higher molecular-mass oligomer of Pex11pβ. (C) Dysfunctional peroxisomal β-oxidation does not affect intracellular levels of EPA and AA. Lipids extracted from AOx- and D-BP-deficient fibroblasts were analyzed by LC/MS. Values are the mole % of total DHA in control fibroblasts and represent means ± s.d.; n=3.

• Supplemental Figure S2 -

  Fig. S2. DHA-induced peroxisome division is independent of the activation of PPARα. (A) AOx-deficient fibroblasts were treated with a control dsRNA or dsRNAs each for PPARα (#1 and #2) for 48 h and then cultured for an additional 24 h in the absence or presence of 150 µM DHA. Left, peroxisome abundance per cell was determined as in Fig. 2B. Data represent the means ± s.d. of three independent experiments. Right, PPARα mRNA levels were assessed by RT-PCR using total RNA. Actin was assessed as a loading control. (B) MEFs and human fibroblasts from a normal control or a patient with AOx-deficiency were cultured in the absence or presence of 50 µM DHA or 100 µM Wy-14,643 for 48 h. Peroxisome abundance per cell was determined as in Fig. 2B. Data represent the means ± s.d. of three independent experiments. *P<0.01. (C) mRNA levels of PEX11α and PEX11β were assessed by RT-PCR using total RNA extracted from AOx- and D-BP-deficient fibroblasts cultured in the absence or presence of 50 µM DHA for 24 h. Actin was assessed as a loading control.

• Supplemental Figure S3 -

  Fig. S3. Dynasore inhibits DLP1 in vivo. Control fibroblasts were cultured for 30 min in the presence of DMSO or 40 µM dynasore and then treated for 4 h with 0.3 mM or 1 mM H₂O₂. The morphology of the mitochondria was verified using an anti-Tom20 antibody (left panels). Mitochondrial morphology was classified into two types, tubular (right panel, open bar) and fragmented (right panel, solid bar) in 150 randomly selected cells. Scale bar: 20 µm. Data represent the means ± s.d. of three independent experiments. *P<0.01.

• Supplemental Figure S4 -

  Fig. S4. Knockdown of PEX11α inhibits DHA-mediated peroxisome division. (A) AOx-deficient fibroblasts were treated with control dsRNA (upper panel) or a dsRNA (#2) for PEX11α (lower panel) for 48 h. Cells were cultured for an additional 12 h (b and e) and 24 h (a, c, d, and f) in the absence (a and d) or presence (b, c, e, and f) of 150 µM DHA and then stained with an anti-Pex14p antibody. Scale bar: 10 µm. Insets, higher magnification images of the boxed regions. Scale bar: 2 µm. (B) AOx-deficient fibroblasts were treated as in (A) with PEX11α #1 and PEX11α #2 dsRNAs. PEX11α mRNA level was assessed by RT-PCR using total RNA. Actin was assessed as a loading control. (C) Peroxisome abundance per cell was determined as in Fig. 2B. Data represent the means ± s.d. of three independent experiments. *P<0.01.

• Supplemental Figure S5 -

  Fig. S5. Knockdown of PEX16 does not affect peroxisomal morphogenesis. (A) AOx-deficient fibroblasts were treated with control dsRNA (a and b) or PEX16 #2 dsRNA (c and d) for 72 h and then stained with antibodies to Pex14p (a and c) and Pex16p (b and d). Scale bar: 10 µm. (B) AOx-deficient fibroblasts were treated with control dsRNA or two different dsRNAs for PEX16 (#1 and #2) for 72 h. Pex16p levels were verified by immunoblot analysis using an antibody to Pex16p. Actin was assessed as a loading control. (C) Peroxisome abundance per cell was determined as in Fig. 2B. Data represent the means ± s.d. of three independent experiments.

• Supplemental Table S1 -

  Table S1. Sequences of complementary antisense oligonucleotides used in this study

• Movie 1 -

  Movie 1. Peroxisome division is highly elevated in AOx-deficient fibroblasts in the presence of DHA. Frames (58 total) were taken at 0.8-second intervals. The video runs at 15 frames/second and corresponds to Fig. 8A.

• Movie 2 -

  Movie 2. Peroxisomal elongation occurs in one direction. Frames (39 total) were taken at 0.8-second intervals. The video runs at 20 frames/second and corresponds to Fig. 8B.