Journal: Plant Physiology

Article Title: The PEX1 ATPase Stabilizes PEX6 and Plays Essential Roles in Peroxisome Biology

doi: 10.1104/pp.17.00548

Figure Lengend Snippet: PEX5 overexpression did not rescue pex1 mutants. A, Seedlings were grown in the absence of hormone for 4 d and then transferred to either medium without hormone or supplemented with IBA for an additional 4 d. The numbers of lateral roots and root lengths were measured, and the ratio is shown. Error bars show se (n ≥ 20). Significant differences (one-way ANOVA, P < 0.001) for each growth condition are marked with different letters above the bars. B and C, Eight-day-old seedlings (B) or 21-d-old plants (C) were prepared for immunoblot analysis and serially probed with antibodies recognizing the indicated proteins. PMDH is translated as a precursor (p) with a PTS2 that is cleaved inside the peroxisome to yield mature (m) protein. HSC70 is a loading control. Wt, Wild type.

Article Snippet: After blocking, membranes were incubated overnight at 4°C with primary antibodies in blocking solution with 0.1% sodium azide at the indicated dilutions: rabbit antibodies against PMDH2 (1:1,500; Pracharoenwattana et al., 2007 ), the PED1 isoform of thiolase (1:10,000; Lingard et al., 2009 ), PEX1 (1:200; raised to the first 400 amino acids of Arabidopsis PEX1 and affinity purified by Proteintech Group), PEX5 (1:100; Zolman and Bartel, 2004 ), and PEX6 (1:1,000; Ratzel et al., 2011 ); mouse antibodies against HSC70 (1:50,000; Stressgen SPA-817) and GFP (1:100; sc-9996; Santa Cruz Biotechnology); or a rat antibody against HA (1:100; Roche 3F10).

Techniques: Over Expression, Western Blot

Journal: Plant Physiology

Article Title: The PEX1 ATPase Stabilizes PEX6 and Plays Essential Roles in Peroxisome Biology

doi: 10.1104/pp.17.00548

Figure Lengend Snippet: PCR-based markers used to genotype identified mutants, reference lines, and transgenes

Article Snippet: After blocking, membranes were incubated overnight at 4°C with primary antibodies in blocking solution with 0.1% sodium azide at the indicated dilutions: rabbit antibodies against PMDH2 (1:1,500; Pracharoenwattana et al., 2007 ), the PED1 isoform of thiolase (1:10,000; Lingard et al., 2009 ), PEX1 (1:200; raised to the first 400 amino acids of Arabidopsis PEX1 and affinity purified by Proteintech Group), PEX5 (1:100; Zolman and Bartel, 2004 ), and PEX6 (1:1,000; Ratzel et al., 2011 ); mouse antibodies against HSC70 (1:50,000; Stressgen SPA-817) and GFP (1:100; sc-9996; Santa Cruz Biotechnology); or a rat antibody against HA (1:100; Roche 3F10).

Techniques: Sequencing, Mutagenesis, Amplification

Journal: JCI Insight

Article Title: Resolving monocytes generated through TRAM deletion attenuate atherosclerosis

doi: 10.1172/jci.insight.149651

Figure Lengend Snippet: BM cells from WT C57 BL/6 mice and Tram −/− mice were cultured with M-CSF (10 ng/mL) in the presence of PBS or super-low-dose LPS (100 pg/mL) for 5 days. ( A and B ) Protein levels of PPARγ ( A ) and PEX5 ( B ) were examined by Western blotting, and relative expressions were normalized to β-actin. ( C ) BMMs were stained with anti-PMP70 and anti-LAMP1 antibodies, and the localization of peroxisomes and lysosomes was examined by confocal microcopy. Scale bars: 10 μm. Inset original magnification, 400×. ( D ) BMMs were labeled with CellROX, and ROS levels were quantified by flow cytometry. Data are representative of 3 independent experiments, and error bars represent means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001; 1-way ANOVA ( n = 3 for each group).

Article Snippet: For detecting PEX5, the fixed and permeabilized cells were stained with primary rabbit anti–mouse PEX5 antibody (1:100 dilution, Proteintech, no. 12545-1-AP) followed by Alexa Fluor 647–conjugated goat anti–rabbit IgG (1:500 dilution, Thermo Fisher Scientific, no. A32733).

Techniques: Cell Culture, Western Blot, Staining, Labeling, Flow Cytometry

Journal: Autophagy

Article Title: Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance

doi: 10.1080/15548627.2021.1936932

Figure Lengend Snippet: Pexophagy occurs in proportion to whole-cell peroxisome levels. (A) Immunoblot validation of Fr.1 from all conditions, alongside respective whole-cell extracts (inputs). Peroxisomal proteins: CAT, ACSL1, PEX11B, PEX19 and PEX3. LAMP2 and LC3B-II are lysosome and autophagosome protein, respectively. (B) Whole-cell extract peroxisomal protein profile. Shown are acute Vor-treated U937 cells (6,12,18 h), chronically-treated B8 (Vor) and B8 cells washed-off from Vor for one week, referred to as B8 washoff (WO). All samples were treated with CQ. ACTB is a loading control. (C) Relative mRNA expression of PEX genes in vehicle and Vor (2 μM)-treated U937 cells, Vor-maintained B8 (B8 [Vor]), and vehicle-cultured B8 (WO) cells. MAP1LC3B and FOXO1 are shown as positive controls for HDACi-induced transcriptional upregulation. All samples were normalized to the housekeeping gene ACTB. (D) Schematic of pexophagy. Ubiquitinated PEX5 is attached to the outer surface of the peroxisome and binds the cargo receptors SQSTM1 or NBR1, which facilitates peroxisomal engulfment into an expanding autophagosome (AP). (E) PEX5-Ubiquitin colocalization (+CQ) in vehicle (DMSO)-treated U937, Vor-treated U937, and B8 cells chronically maintained in Vor, with quantitation show below. (F) PEX5 and SQSTM1 colocalization, and (G) PEX5 and NBR1 colocalization (+CQ) in vehicle (DMSO)-treated U937, Vor-treated U937, and B8 cells chronically maintained in Vor. Insets shown for each condition (scale bar for D and E: 10 μm). (H) Number of colocalized puncta per condition (merge) from analyses of (F) PEX5 with SQSTM1, and (G) PEX5 with NBR1 are shown below respective images. (I) Co-immunoprecipitation of PEX5 with SQSTM1 (top), and corresponding inputs (below). All Vor treatments are 2 μM (18 h U937, chronic treatment B8), and CQ 25 μM (18 h).

Article Snippet: For PEX5 immunoprecipitations, 2.5 μg of PEX5 antibody (Proteintech Group, 12,545-1-AP) was added to sample tubes containing 1 mg of protein extract in 1 mL lysis buffer, while 2.5 μg of IgG control antibody (Proteintech Group, 3000-0-AP) was added to separate tubes containing 1 mg of respective protein extracts in 1 mL lysis buffer.

Techniques: Western Blot, Expressing, Cell Culture, Quantitation Assay, Immunoprecipitation

Journal: Autophagy

Article Title: Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance

doi: 10.1080/15548627.2021.1936932

Figure Lengend Snippet: Silencing of the peroxisomal exportomer complex promotes pexophagy and induces apoptosis in Vor-resistant cells. (A, left-schematic) PEX5 (represented by ‘5ʹ) delivers proteins with a peroxisome-targeting signal-1 (PTS1) sequence (not shown) to the peroxisomal matrix. PEX5 is then recycled back to the cytosol via the exportomer complex, consisting of PEX1, PEX6 and PEX26 (blue circles). When the exportomer complex is compromised, PEX5 cannot be efficiently exported to the cytosol. PEX5 then accumulates on the peroxisomal membrane, is ubiquitinated (yellow circles), and interacts with the pexophagy receptor, SQSTM1. The peroxisome then undergoes pexophagy and enters an expanding autophagosome (AP). Peroxisomes within autophagosomes are shown as small purple circles. We hypothesize that pexophagy promotes apoptosis. (A, right) Immunoblots confirming knockdown of PEX1, PEX6, and PEX26 in Vor (2 μM)-maintained B8 (Vor) cells. ACTB is a loading control. (B) Immunofluorescence colocalization in B8 (Vor cells) of ubiquitin with PEX5, and (C) SQSTM1 with PEX5; respective quantifications are shown below. Scale bars for B and C: 7.5 μm. (D) ABCD3 puncta upon silencing of PEX1, PEX6 and PEX26 in B8 (Vor) cells with quantification shown below. Scale bar: 5 μm. (E, left) Representative flow cytometry scatter plots of ANXA5-Cy5/PI co-stained B8 (Vor) cells. (E, right) Apoptosis measurements 72 h post-transfection, detected by ANXA5-Cy5/PI co-staining. For all statistics: *p < 0.01, **p < 0.001, ***p < 0.0001 (One-way ANOVA, Tukey’s test).

Article Snippet: For PEX5 immunoprecipitations, 2.5 μg of PEX5 antibody (Proteintech Group, 12,545-1-AP) was added to sample tubes containing 1 mg of protein extract in 1 mL lysis buffer, while 2.5 μg of IgG control antibody (Proteintech Group, 3000-0-AP) was added to separate tubes containing 1 mg of respective protein extracts in 1 mL lysis buffer.

Techniques: Sequencing, Western Blot, Immunofluorescence, Flow Cytometry, Staining, Transfection

Journal: Autophagy

Article Title: Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance

doi: 10.1080/15548627.2021.1936932

Figure Lengend Snippet: CRISPRi-mediated PEX26 knockdown decreases peroxisome levels via pexophagy. (A) qPCR analyses of PEX26 mRNA levels (normalized to ACTB) in A375 cell lines stably expressing dCAS9 (clone A4 clone) alongside either sgSCR, sgPEX26-2 and sgPEX26-4 RNAs (n = 3, *** p < 0.0001, One-way ANOVA). (B) Immunoblots of dCAS9, PEX1, PEX6, and ACTB in the A375 cell lines presented in panel (A). (C) Immunoblots of PEX5, SQSTM1, (LC3B-II and GAPDH controls) from PEX5 co-immunoprecipitation (co-IP) of sgSCR and sgPEX26-4, both treated with vehicle (water) or CQ (25 μM, 48 h). Left portion displays immunoblots for inputs (10% of protein from IP), middle portion, PEX5 co-IP. The densitometry (Gray Mean Value, G.M.V.) of SQSTM1 and PEX5 bands are indicated below. Right portion, IgG IP. Right bar graph: densitometry ratio of SQSTM1 and PEX5 from PEX5 co-IP. Results are from three independent experiments, **** p < 0.00001, Two-way ANOVA. (D) Immunofluorescence staining of PEX5, SQSTM1 (DAPI nucleus control) of sgSCR and sgPEX26-4 cells, both treated with vehicle and CQ. Scale bar: 7.5 μm. Insets are shown below respective merge images. (E) Top: Immunofluorescence staining for ABCD3 (purple) of sgSCR and sgPEX26-4 cells, both treated with vehicle and CQ. Scale bar: 7.5 μm. Bottom: ABCD3 puncta quantitation of conditions normalized to sgSCR vehicle. n = 3, total of approximately 100 cells counted per condition, *** p < 0.0001, One-way ANOVA. (F) Top: Immunofluorescence staining of ABCD3 (green) and CAT (red), with DAPI (nucleus control, blue) of sgSCR, sgPEX26-2, and sgPEX26-4 cells. Bottom: Pearson values corresponding to co-localization of ABCD3 and CAT (*** p < 0.0001). Fifty cells were analyzed per condition. Scale bar: 7.5 μm. (G) Images: Immunofluorescence staining of GFP-PTS1 (green) and ABCD3 (red), with DAPI (nucleus control, blue) of sgSCR, sgPEX26-2, and sgPEX26-4 cells, and M2H PEX1G843D/null cell line. Bottom right: Pearson values corresponding to co-localization of GFP-PTS1 and ABCD3 (*** p < 0.0001). Fifty cells were analyzed per condition. Scale bar: 7.5 μm. (H) Immunoblot of ACOX1 components “a”, “b”, and “c”, representing the mature 71 kDa polypeptide “a” and peroxisomal proteolytically converted forms “b” and “c” with molecular weights of 50 and 21 kDa. (I) 26:0 LPC measurements (pmol) in sgSCR, sgPEX26-2, and sgPEX26-4 A375 cells, *** p < 0.0001, One-way ANOVA. (J) Total plasmalogen levels (pmol) in sgSCR, sgPEX26-2, and sgPEX26-4 A375 cells, *** p < 0.0001, One-way ANOVA.

Article Snippet: For PEX5 immunoprecipitations, 2.5 μg of PEX5 antibody (Proteintech Group, 12,545-1-AP) was added to sample tubes containing 1 mg of protein extract in 1 mL lysis buffer, while 2.5 μg of IgG control antibody (Proteintech Group, 3000-0-AP) was added to separate tubes containing 1 mg of respective protein extracts in 1 mL lysis buffer.

Techniques: Stable Transfection, Expressing, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Quantitation Assay

Journal: Autophagy

Article Title: Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance

doi: 10.1080/15548627.2021.1936932

Figure Lengend Snippet: CRISPRi-mediated PEX26 silencing attenuates tumor relapse in a xenograft melanoma mouse model. (A) Schematic of in vitro vemurafenib treatment of A375 dCAS9 sgSCR and A375 dCAS9 sgPEX26-4 cells. (B) in vitro growth curve of sgSCR and sgPEX26-4 cells in response to 1 μM vemurafenib treatment, maintained in media containing DMSO or 2 μM KU55933. (n = 3, Two-way RM ANOVA with Tukey’s multiple comparisons test). (C) Measurements of A375 dCAS9 sgSCR and A375 dCAS9 sgPEX26-4 tumor volume (mm3) versus days post subcutaneous-injection into 6–10 week old female NOD/SCID female mice (5 mice per group) fed control chow. At day 25, once a critical tumor volume (approximately 1200 mm3) was reached by at least one mouse, both groups were sacrificed. Tumor volumes were measured every 2–3 d until the endpoint was reached. No significant differences in tumor volume were observed. (D) Tumor volume versus time (days) plot of sgSCR and sgPEX26-4 injected mice. Mice were fed control chow until day 14 (gray dashed vertical line), then chow was switched to the Vemu analog, PLX4720. (E) Recurrence-free survival plot of PLX4720-chow-fed mice injected with A375 dCAS9 sgSCR or A375 dCAS9 sgPEX26-4 cells (p = 0.0496). (F) Top: PEX26 immunohistochemistry (IHC) 3,3′-Diaminobenzidine (DAB) staining of control chow and PLX4720-fed mice, bearing either sgSCR or sgPEX26-4 tumors. Nuclei are stained blue with hematoxylin. Scale bar: 20 μm. Bottom: comparative percentages of PEX26-positive stained (≥ 0.2 mean DAB intensity/cell) sgSCR versus sgPEX26-4 tumors from mice fed control chow (*p = 0.0373, Student’s t-test) and PLX4720 chow (*p = 0.0343, Student’s t-test). (G) Representative immunoblots from sgSCR and sgPEX26-4 tumors from mice fed control chow, or PLX4720. Shown are PEX26, PEX1, PEX6, and PEX5 (peroxisomal proteins), p-MAPK1-MAPK3 (control for MAPK/ERK reactivation upon chronic PLX4720 treatment), MAPK1, and ACTN1 as a loading control).

Article Snippet: For PEX5 immunoprecipitations, 2.5 μg of PEX5 antibody (Proteintech Group, 12,545-1-AP) was added to sample tubes containing 1 mg of protein extract in 1 mL lysis buffer, while 2.5 μg of IgG control antibody (Proteintech Group, 3000-0-AP) was added to separate tubes containing 1 mg of respective protein extracts in 1 mL lysis buffer.

Techniques: In Vitro, Injection, Immunohistochemistry, Staining, Western Blot

Journal: Autophagy

Article Title: Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance

doi: 10.1080/15548627.2021.1936932

Figure Lengend Snippet: PEX26 silencing mislocalizes peroxisomal matrix proteins, promotes pexophagy and enhances therapy sensitivity. (Top) Drug-resistant cells exhibit increased levels of peroxisomes compared to therapy-sensitive cancer cells. (Middle) When the exportomer complex (PEX1, PEX6, PEX26 – abbreviated with numbered circles) is functional, PEX5 can be recycled back to the cytosol to shuttle PTS1-bearing proteins to the peroxisomal matrix. When the exportomer complex is downregulated via PEX26 silencing or inhibition, ubiquitinated PEX5 interacts with the pexophagy receptor SQSTM1 (or NBR1), which mediates increased engulfment by autophagosomes. ATM localizes PEX5 to the peroxisomal membrane to facilitate pexophagy [9]. (Bottom) Peroxisomes are generally elevated in therapy resistance, while inducing peroxisomal matrix protein import deficiencies and/or pexophagy promotes therapy sensitivity.

Article Snippet: For PEX5 immunoprecipitations, 2.5 μg of PEX5 antibody (Proteintech Group, 12,545-1-AP) was added to sample tubes containing 1 mg of protein extract in 1 mL lysis buffer, while 2.5 μg of IgG control antibody (Proteintech Group, 3000-0-AP) was added to separate tubes containing 1 mg of respective protein extracts in 1 mL lysis buffer.

Techniques: Functional Assay, Inhibition