anti pex5 antibody  (Proteintech)

 
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    Proteintech anti pex5 antibody
    Validation of <t>PEX5</t> KO HeLa cells. A. PEX5 KO HeLa cells were made by the CRISPR/Cas9 system. PEX5 Knock out efficiency was confirmed using western blot. B. Representative microscopy images of WT and PEX5 KO HeLa cells which were stained with LAMP1(red) and Pex14(green). The mean fluorescent intensity (MFI) quantification was presented.; scale bar 10 um. C. Graph represented the change in number of SCVs per cells after 6 hrs of infection with STM in wild type and PEX5 KO HeLa cells. D. Pex14 expression was quantified by RT-PCR. E. Cell viability was checked by MTT assay in PEX5 knockout cells F. Cell viability was checked by MTT assay the cells treated with 4-PBA. Experiment was repeated atleast two times. Individual data points represent mean ±SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non-significant ****p<0.0001, ***p<0.001, *p<0.05, unpaired two-tailed Student T-test.
    Anti Pex5 Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti pex5 antibody/product/Proteintech
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti pex5 antibody - by Bioz Stars, 2023-05
    86/100 stars

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    1) Product Images from "Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth"

    Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

    Journal: bioRxiv

    doi: 10.1101/2023.02.27.530266

    Validation of PEX5 KO HeLa cells. A. PEX5 KO HeLa cells were made by the CRISPR/Cas9 system. PEX5 Knock out efficiency was confirmed using western blot. B. Representative microscopy images of WT and PEX5 KO HeLa cells which were stained with LAMP1(red) and Pex14(green). The mean fluorescent intensity (MFI) quantification was presented.; scale bar 10 um. C. Graph represented the change in number of SCVs per cells after 6 hrs of infection with STM in wild type and PEX5 KO HeLa cells. D. Pex14 expression was quantified by RT-PCR. E. Cell viability was checked by MTT assay in PEX5 knockout cells F. Cell viability was checked by MTT assay the cells treated with 4-PBA. Experiment was repeated atleast two times. Individual data points represent mean ±SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non-significant ****p<0.0001, ***p<0.001, *p<0.05, unpaired two-tailed Student T-test.
    Figure Legend Snippet: Validation of PEX5 KO HeLa cells. A. PEX5 KO HeLa cells were made by the CRISPR/Cas9 system. PEX5 Knock out efficiency was confirmed using western blot. B. Representative microscopy images of WT and PEX5 KO HeLa cells which were stained with LAMP1(red) and Pex14(green). The mean fluorescent intensity (MFI) quantification was presented.; scale bar 10 um. C. Graph represented the change in number of SCVs per cells after 6 hrs of infection with STM in wild type and PEX5 KO HeLa cells. D. Pex14 expression was quantified by RT-PCR. E. Cell viability was checked by MTT assay in PEX5 knockout cells F. Cell viability was checked by MTT assay the cells treated with 4-PBA. Experiment was repeated atleast two times. Individual data points represent mean ±SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non-significant ****p<0.0001, ***p<0.001, *p<0.05, unpaired two-tailed Student T-test.

    Techniques Used: CRISPR, Knock-Out, Western Blot, Microscopy, Staining, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, MTT Assay, Two Tailed Test

    Peroxisomes are required for efficient intracellular replication of Salmonella Typhimurium. A. STM proliferation in WT and PEX5 -/- HeLa cells after 16 hr of infection with STM (MOI=10). CFU was shown as fold change compared to 2 hr. The graph is representative of three independent experiments with similar results. B. HeLa cells were treated with 5 mM 4-PBA for 10 days. Those cells were infected with STM (MOI=10) and CFU was shown as fold change compared to 2 hr. The experiment was repeated 3 times. C. WT and PEX5 -/- HeLa cells infected with STM for 12 hours. Cells were fixed and stained with LAMP1 (red), Pex14 (green) and STM (blue). D. Graph represented the area occupied by SCVs with SIFs in WT and PEX5 -/- HeLa cells after 12 hours of STM infection (MOI=50). Cell number, n=20; scale bar 10 um. Individual data points represent mean ±SD. ***p<0.001, **p<0.01, unpaired two-tailed Student T-test.
    Figure Legend Snippet: Peroxisomes are required for efficient intracellular replication of Salmonella Typhimurium. A. STM proliferation in WT and PEX5 -/- HeLa cells after 16 hr of infection with STM (MOI=10). CFU was shown as fold change compared to 2 hr. The graph is representative of three independent experiments with similar results. B. HeLa cells were treated with 5 mM 4-PBA for 10 days. Those cells were infected with STM (MOI=10) and CFU was shown as fold change compared to 2 hr. The experiment was repeated 3 times. C. WT and PEX5 -/- HeLa cells infected with STM for 12 hours. Cells were fixed and stained with LAMP1 (red), Pex14 (green) and STM (blue). D. Graph represented the area occupied by SCVs with SIFs in WT and PEX5 -/- HeLa cells after 12 hours of STM infection (MOI=50). Cell number, n=20; scale bar 10 um. Individual data points represent mean ±SD. ***p<0.001, **p<0.01, unpaired two-tailed Student T-test.

    Techniques Used: Infection, Staining, Two Tailed Test

    SseI interacts with host GTPase ARF1 on peroxisome. A. HeLa cells were transfected with HA: SseI and GFP: Arf1 plasmids and then immunoprecipitated as described in the experimental procedures. Blot was incubated with anti-HA for SseI, anti-GFP for ARF1 and anti-PEX5. Input indicates the total cell lysate of the cells mentioned above. B. Total cell lysates from HeLa cells were transfected with HA: ARF1 was collected and incubated with purified his tagged SseI for 2 hr. Then his tagged SseI was pulled down by Ni-NTA beads and the eluted sample was run on SDS-PAGE. Immunoblot analysis for His-tagged SseI and HA-tagged ARF1 is shown. Input indicates total cell lysate from the ARF1: HA transfected cells. C. Representative microscopy images of HeLa cells infected with STM (MOI=50) for 6 hours; Cells were fixed; stained for endogenous ARF1(red), Pex14(green), and STM-DAPI (blue); and Zoom part of image are shown ARF1(red), Pex14(green), and STM-DAPI (blue) in both infected and uninfected cells; scale bar=5um. D. Equal number of HeLa Cells were infected with either the WT or STM ΔsseI (MOI=10) for 6 hr. Then the active ARF1 was pulled down and were run on SDS page. Densitometry graph from 3 independent experiment representing the relative ARF1 activation in the WT and ΔsseI infected cells compared to that of uninfected HeLa cells is shown (MOI=10). E. HeLa cell were transfected with either full length or mutated (Q71L) ARF-1 or silenced ARF-1. Then infected with the WT or ΔsseI STM and changes in the fold proliferation were plotted. Experiments were repeated three times. Individual data points represent mean ±SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non-significant ***p<0.001, *p<0.05, **p<0.01, unpaired two-tailed Student T-test.
    Figure Legend Snippet: SseI interacts with host GTPase ARF1 on peroxisome. A. HeLa cells were transfected with HA: SseI and GFP: Arf1 plasmids and then immunoprecipitated as described in the experimental procedures. Blot was incubated with anti-HA for SseI, anti-GFP for ARF1 and anti-PEX5. Input indicates the total cell lysate of the cells mentioned above. B. Total cell lysates from HeLa cells were transfected with HA: ARF1 was collected and incubated with purified his tagged SseI for 2 hr. Then his tagged SseI was pulled down by Ni-NTA beads and the eluted sample was run on SDS-PAGE. Immunoblot analysis for His-tagged SseI and HA-tagged ARF1 is shown. Input indicates total cell lysate from the ARF1: HA transfected cells. C. Representative microscopy images of HeLa cells infected with STM (MOI=50) for 6 hours; Cells were fixed; stained for endogenous ARF1(red), Pex14(green), and STM-DAPI (blue); and Zoom part of image are shown ARF1(red), Pex14(green), and STM-DAPI (blue) in both infected and uninfected cells; scale bar=5um. D. Equal number of HeLa Cells were infected with either the WT or STM ΔsseI (MOI=10) for 6 hr. Then the active ARF1 was pulled down and were run on SDS page. Densitometry graph from 3 independent experiment representing the relative ARF1 activation in the WT and ΔsseI infected cells compared to that of uninfected HeLa cells is shown (MOI=10). E. HeLa cell were transfected with either full length or mutated (Q71L) ARF-1 or silenced ARF-1. Then infected with the WT or ΔsseI STM and changes in the fold proliferation were plotted. Experiments were repeated three times. Individual data points represent mean ±SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non-significant ***p<0.001, *p<0.05, **p<0.01, unpaired two-tailed Student T-test.

    Techniques Used: Transfection, Immunoprecipitation, Incubation, Purification, SDS Page, Western Blot, Microscopy, Infection, Staining, Activation Assay, Two Tailed Test

    LDL-derived cholesterol routed via lysosome-peroxisome is essential for Salmonella growth A. Graph representing percentage of peroxisome-lysosome co-localization in the HeLa cells after infection with the WT STM (MOI=10) for different time points. B. WT HeLa cells were infected with WT STM or STM ΔsseI for 6 h with MOI= 50. pex5 -/- HeLa cell were infected with WT STM for 6 h with MOI= 50. Cells were fixed and immunostained with LAMP1(red), cholesterol using filipin(blue) and STM (green). Zoom part represented SCV and Lysosome cholesterol (blue) level in the cells. Graph representing change in the filipin fluorescent intensity is shown. (n=50 cells). Scale bar 10 uM. C. PEROXO- tagged stable cell was generated using transfection with lentivirus overexpressing 3X myc-EGFP-Pex26. Stable PEROXO tag HeLa cells were next infected with the WT and ΔsseI STM with MOI=10 for 6 hr. Isolated peroxisomal cholesterol amount was measured using cholesterol assay kit. Graph representing the fold proliferation of STM silencing D. NPC1 E. ABCD1 in HeLa cells. Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. F. Cells were kept untreated or treated with lovastatin (10uM). Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. G. Cells were kept untreated or treated with LDL/U18666A in the media containing 1% FBS in HeLa cells. Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. Individual data points represent mean± SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘src’ denoted scrambled siRNA. ‘UI’ denotes uninfected, ‘UT’ denotes untreated. ‘ns’ denotes non- significant ****p<0.0001, ***p<0.001, *p<0.05, **p<0.01, unpaired two-tailed Student T-test.
    Figure Legend Snippet: LDL-derived cholesterol routed via lysosome-peroxisome is essential for Salmonella growth A. Graph representing percentage of peroxisome-lysosome co-localization in the HeLa cells after infection with the WT STM (MOI=10) for different time points. B. WT HeLa cells were infected with WT STM or STM ΔsseI for 6 h with MOI= 50. pex5 -/- HeLa cell were infected with WT STM for 6 h with MOI= 50. Cells were fixed and immunostained with LAMP1(red), cholesterol using filipin(blue) and STM (green). Zoom part represented SCV and Lysosome cholesterol (blue) level in the cells. Graph representing change in the filipin fluorescent intensity is shown. (n=50 cells). Scale bar 10 uM. C. PEROXO- tagged stable cell was generated using transfection with lentivirus overexpressing 3X myc-EGFP-Pex26. Stable PEROXO tag HeLa cells were next infected with the WT and ΔsseI STM with MOI=10 for 6 hr. Isolated peroxisomal cholesterol amount was measured using cholesterol assay kit. Graph representing the fold proliferation of STM silencing D. NPC1 E. ABCD1 in HeLa cells. Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. F. Cells were kept untreated or treated with lovastatin (10uM). Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. G. Cells were kept untreated or treated with LDL/U18666A in the media containing 1% FBS in HeLa cells. Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. Individual data points represent mean± SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘src’ denoted scrambled siRNA. ‘UI’ denotes uninfected, ‘UT’ denotes untreated. ‘ns’ denotes non- significant ****p<0.0001, ***p<0.001, *p<0.05, **p<0.01, unpaired two-tailed Student T-test.

    Techniques Used: Derivative Assay, Infection, Stable Transfection, Generated, Transfection, Isolation, Cholesterol Assay, Two Tailed Test

    Schematic diagram. Mature SCVs secretes SPI-2 effector proteins in the host cell cytosol among which SseI contains host PTS1 signal. The effector protein, SseI interacts with GTPase ARF1 on host peroxisome. And recruits specific PIP4K2A which is essential for PIP2 synthesis on peroxisome. Simultaneously, cells receiving LDL from outside is further metabolized in lysosome and results in the release of free cholesterol. The lysosomal cholesterol is rerouted to the pathogenic bacteria using peroxisome as a bridge. This helps in SCV maturation and SIF formation resulting in the growth and proliferation of Salmonella. On the other hand, in the Pex5 -/- HeLa cells, dysfunctional peroxisome prevents its interaction with lysosome and SCV both. This results in the decreased proliferation of STM within the host cell.
    Figure Legend Snippet: Schematic diagram. Mature SCVs secretes SPI-2 effector proteins in the host cell cytosol among which SseI contains host PTS1 signal. The effector protein, SseI interacts with GTPase ARF1 on host peroxisome. And recruits specific PIP4K2A which is essential for PIP2 synthesis on peroxisome. Simultaneously, cells receiving LDL from outside is further metabolized in lysosome and results in the release of free cholesterol. The lysosomal cholesterol is rerouted to the pathogenic bacteria using peroxisome as a bridge. This helps in SCV maturation and SIF formation resulting in the growth and proliferation of Salmonella. On the other hand, in the Pex5 -/- HeLa cells, dysfunctional peroxisome prevents its interaction with lysosome and SCV both. This results in the decreased proliferation of STM within the host cell.

    Techniques Used:

    pex5  (Proteintech)

     
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    Proteintech pex5
    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 <t>PEX5</t> 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).
    Pex5, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pex5/product/Proteintech
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pex5 - by Bioz Stars, 2023-05
    93/100 stars

    Images

    1) Product Images from "Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance"

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

    Journal: Autophagy

    doi: 10.1080/15548627.2021.1936932

    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).
    Figure Legend 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).

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

    Techniques Used: Functional Assay, Inhibition

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    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 <t>PEX5</t> 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).
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    1) Product Images from "Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance"

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

    Journal: Autophagy

    doi: 10.1080/15548627.2021.1936932

    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).
    Figure Legend 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).

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

    Techniques Used: Functional Assay, Inhibition

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    Proteintech pex5
    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 <t>PEX5</t> 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).
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    1) Product Images from "Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance"

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

    Journal: Autophagy

    doi: 10.1080/15548627.2021.1936932

    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).
    Figure Legend 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).

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

    Techniques Used: Functional Assay, Inhibition

    pex5 antibody  (Proteintech)

     
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    Proteintech pex5 antibody
    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 <t>PEX5</t> 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).
    Pex5 Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86/100 stars

    Images

    1) Product Images from "Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance"

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

    Journal: Autophagy

    doi: 10.1080/15548627.2021.1936932

    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).
    Figure Legend 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).

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

    Techniques Used: Functional Assay, Inhibition

    pex5 immunoprecipitations  (Proteintech)

     
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    Proteintech pex5 immunoprecipitations
    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 <t>PEX5</t> 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).
    Pex5 Immunoprecipitations, supplied by Proteintech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pex5 immunoprecipitations/product/Proteintech
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pex5 immunoprecipitations - by Bioz Stars, 2023-05
    86/100 stars

    Images

    1) Product Images from "Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance"

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

    Journal: Autophagy

    doi: 10.1080/15548627.2021.1936932

    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).
    Figure Legend 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).

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

    Techniques Used: Functional Assay, Inhibition

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    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 <t>PEX5</t> 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).
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    1) Product Images from "Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance"

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

    Journal: Autophagy

    doi: 10.1080/15548627.2021.1936932

    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).
    Figure Legend 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).

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

    Techniques Used: Functional Assay, Inhibition

    pex5 antibody  (Proteintech)

     
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    Proteintech pex5 antibody
    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 <t>PEX5</t> 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).
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    1) Product Images from "Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance"

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

    Journal: Autophagy

    doi: 10.1080/15548627.2021.1936932

    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).
    Figure Legend 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).

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

    Techniques Used: Functional Assay, Inhibition

    pex5 immunoprecipitations  (Proteintech)

     
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    Proteintech pex5 immunoprecipitations
    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 <t>PEX5</t> 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).
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    1) Product Images from "Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance"

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

    Journal: Autophagy

    doi: 10.1080/15548627.2021.1936932

    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).
    Figure Legend 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).

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

    Techniques Used: Functional Assay, Inhibition

    anti pex5  (Proteintech)

     
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    Anti Pex5, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech anti pex5 antibody
    Validation of <t>PEX5</t> KO HeLa cells. A. PEX5 KO HeLa cells were made by the CRISPR/Cas9 system. PEX5 Knock out efficiency was confirmed using western blot. B. Representative microscopy images of WT and PEX5 KO HeLa cells which were stained with LAMP1(red) and Pex14(green). The mean fluorescent intensity (MFI) quantification was presented.; scale bar 10 um. C. Graph represented the change in number of SCVs per cells after 6 hrs of infection with STM in wild type and PEX5 KO HeLa cells. D. Pex14 expression was quantified by RT-PCR. E. Cell viability was checked by MTT assay in PEX5 knockout cells F. Cell viability was checked by MTT assay the cells treated with 4-PBA. Experiment was repeated atleast two times. Individual data points represent mean ±SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non-significant ****p<0.0001, ***p<0.001, *p<0.05, unpaired two-tailed Student T-test.
    Anti Pex5 Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pex5  (Proteintech)
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    Proteintech pex5
    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 <t>PEX5</t> 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).
    Pex5, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pex5 antibody  (Proteintech)
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    Proteintech pex5 antibody
    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 <t>PEX5</t> 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).
    Pex5 Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pex5 immunoprecipitations  (Proteintech)
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    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 <t>PEX5</t> 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).
    Pex5 Immunoprecipitations, supplied by Proteintech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti pex5  (Proteintech)
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    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 <t>PEX5</t> 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).
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    Validation of PEX5 KO HeLa cells. A. PEX5 KO HeLa cells were made by the CRISPR/Cas9 system. PEX5 Knock out efficiency was confirmed using western blot. B. Representative microscopy images of WT and PEX5 KO HeLa cells which were stained with LAMP1(red) and Pex14(green). The mean fluorescent intensity (MFI) quantification was presented.; scale bar 10 um. C. Graph represented the change in number of SCVs per cells after 6 hrs of infection with STM in wild type and PEX5 KO HeLa cells. D. Pex14 expression was quantified by RT-PCR. E. Cell viability was checked by MTT assay in PEX5 knockout cells F. Cell viability was checked by MTT assay the cells treated with 4-PBA. Experiment was repeated atleast two times. Individual data points represent mean ±SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non-significant ****p<0.0001, ***p<0.001, *p<0.05, unpaired two-tailed Student T-test.

    Journal: bioRxiv

    Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

    doi: 10.1101/2023.02.27.530266

    Figure Lengend Snippet: Validation of PEX5 KO HeLa cells. A. PEX5 KO HeLa cells were made by the CRISPR/Cas9 system. PEX5 Knock out efficiency was confirmed using western blot. B. Representative microscopy images of WT and PEX5 KO HeLa cells which were stained with LAMP1(red) and Pex14(green). The mean fluorescent intensity (MFI) quantification was presented.; scale bar 10 um. C. Graph represented the change in number of SCVs per cells after 6 hrs of infection with STM in wild type and PEX5 KO HeLa cells. D. Pex14 expression was quantified by RT-PCR. E. Cell viability was checked by MTT assay in PEX5 knockout cells F. Cell viability was checked by MTT assay the cells treated with 4-PBA. Experiment was repeated atleast two times. Individual data points represent mean ±SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non-significant ****p<0.0001, ***p<0.001, *p<0.05, unpaired two-tailed Student T-test.

    Article Snippet: Antibodies used (Abs) used were as follows: anti-LAMP1 antibody (D401S from CST), PEX14 Antibody (A7336), ABCD1 (60153-1-Ig), GAPDH Antibody (T0004), ESYT1 antibody (A15410), anti-HA tag Antibody(2367), Anti-FLAG Antibody(8146 from CST), anti-myc (2276 from CST) anti-pex5 antibody (12545-1-AP from proteintech), ARF1 Antibody (10790-1-AP), Goat anti-mouse IgG Alexa fluor 594(A- 11032), Goat anti-Rabbit IgG Alexa Fluor 488(A-11034) IgG Alexa fluor 594(A- 11032) SYT7 antibody(NBP2-22420), PIP2 ANTIBODY(NBP2-76433), Cy5 goat anti-rabbit IgG (A10523 - Invitrogen), Control Mouse immunoglobulin (Cat#500-M00- 1mg - PEPROTECH), Control rabbit immunoglobulin (Cat#500-P00-500 ug - PEPROTECH) anti-rabbit IgG antibody and horseradish peroxidase (7074P2) were purchased from Cell Signalling Technology.

    Techniques: CRISPR, Knock-Out, Western Blot, Microscopy, Staining, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, MTT Assay, Two Tailed Test

    Peroxisomes are required for efficient intracellular replication of Salmonella Typhimurium. A. STM proliferation in WT and PEX5 -/- HeLa cells after 16 hr of infection with STM (MOI=10). CFU was shown as fold change compared to 2 hr. The graph is representative of three independent experiments with similar results. B. HeLa cells were treated with 5 mM 4-PBA for 10 days. Those cells were infected with STM (MOI=10) and CFU was shown as fold change compared to 2 hr. The experiment was repeated 3 times. C. WT and PEX5 -/- HeLa cells infected with STM for 12 hours. Cells were fixed and stained with LAMP1 (red), Pex14 (green) and STM (blue). D. Graph represented the area occupied by SCVs with SIFs in WT and PEX5 -/- HeLa cells after 12 hours of STM infection (MOI=50). Cell number, n=20; scale bar 10 um. Individual data points represent mean ±SD. ***p<0.001, **p<0.01, unpaired two-tailed Student T-test.

    Journal: bioRxiv

    Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

    doi: 10.1101/2023.02.27.530266

    Figure Lengend Snippet: Peroxisomes are required for efficient intracellular replication of Salmonella Typhimurium. A. STM proliferation in WT and PEX5 -/- HeLa cells after 16 hr of infection with STM (MOI=10). CFU was shown as fold change compared to 2 hr. The graph is representative of three independent experiments with similar results. B. HeLa cells were treated with 5 mM 4-PBA for 10 days. Those cells were infected with STM (MOI=10) and CFU was shown as fold change compared to 2 hr. The experiment was repeated 3 times. C. WT and PEX5 -/- HeLa cells infected with STM for 12 hours. Cells were fixed and stained with LAMP1 (red), Pex14 (green) and STM (blue). D. Graph represented the area occupied by SCVs with SIFs in WT and PEX5 -/- HeLa cells after 12 hours of STM infection (MOI=50). Cell number, n=20; scale bar 10 um. Individual data points represent mean ±SD. ***p<0.001, **p<0.01, unpaired two-tailed Student T-test.

    Article Snippet: Antibodies used (Abs) used were as follows: anti-LAMP1 antibody (D401S from CST), PEX14 Antibody (A7336), ABCD1 (60153-1-Ig), GAPDH Antibody (T0004), ESYT1 antibody (A15410), anti-HA tag Antibody(2367), Anti-FLAG Antibody(8146 from CST), anti-myc (2276 from CST) anti-pex5 antibody (12545-1-AP from proteintech), ARF1 Antibody (10790-1-AP), Goat anti-mouse IgG Alexa fluor 594(A- 11032), Goat anti-Rabbit IgG Alexa Fluor 488(A-11034) IgG Alexa fluor 594(A- 11032) SYT7 antibody(NBP2-22420), PIP2 ANTIBODY(NBP2-76433), Cy5 goat anti-rabbit IgG (A10523 - Invitrogen), Control Mouse immunoglobulin (Cat#500-M00- 1mg - PEPROTECH), Control rabbit immunoglobulin (Cat#500-P00-500 ug - PEPROTECH) anti-rabbit IgG antibody and horseradish peroxidase (7074P2) were purchased from Cell Signalling Technology.

    Techniques: Infection, Staining, Two Tailed Test

    SseI interacts with host GTPase ARF1 on peroxisome. A. HeLa cells were transfected with HA: SseI and GFP: Arf1 plasmids and then immunoprecipitated as described in the experimental procedures. Blot was incubated with anti-HA for SseI, anti-GFP for ARF1 and anti-PEX5. Input indicates the total cell lysate of the cells mentioned above. B. Total cell lysates from HeLa cells were transfected with HA: ARF1 was collected and incubated with purified his tagged SseI for 2 hr. Then his tagged SseI was pulled down by Ni-NTA beads and the eluted sample was run on SDS-PAGE. Immunoblot analysis for His-tagged SseI and HA-tagged ARF1 is shown. Input indicates total cell lysate from the ARF1: HA transfected cells. C. Representative microscopy images of HeLa cells infected with STM (MOI=50) for 6 hours; Cells were fixed; stained for endogenous ARF1(red), Pex14(green), and STM-DAPI (blue); and Zoom part of image are shown ARF1(red), Pex14(green), and STM-DAPI (blue) in both infected and uninfected cells; scale bar=5um. D. Equal number of HeLa Cells were infected with either the WT or STM ΔsseI (MOI=10) for 6 hr. Then the active ARF1 was pulled down and were run on SDS page. Densitometry graph from 3 independent experiment representing the relative ARF1 activation in the WT and ΔsseI infected cells compared to that of uninfected HeLa cells is shown (MOI=10). E. HeLa cell were transfected with either full length or mutated (Q71L) ARF-1 or silenced ARF-1. Then infected with the WT or ΔsseI STM and changes in the fold proliferation were plotted. Experiments were repeated three times. Individual data points represent mean ±SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non-significant ***p<0.001, *p<0.05, **p<0.01, unpaired two-tailed Student T-test.

    Journal: bioRxiv

    Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

    doi: 10.1101/2023.02.27.530266

    Figure Lengend Snippet: SseI interacts with host GTPase ARF1 on peroxisome. A. HeLa cells were transfected with HA: SseI and GFP: Arf1 plasmids and then immunoprecipitated as described in the experimental procedures. Blot was incubated with anti-HA for SseI, anti-GFP for ARF1 and anti-PEX5. Input indicates the total cell lysate of the cells mentioned above. B. Total cell lysates from HeLa cells were transfected with HA: ARF1 was collected and incubated with purified his tagged SseI for 2 hr. Then his tagged SseI was pulled down by Ni-NTA beads and the eluted sample was run on SDS-PAGE. Immunoblot analysis for His-tagged SseI and HA-tagged ARF1 is shown. Input indicates total cell lysate from the ARF1: HA transfected cells. C. Representative microscopy images of HeLa cells infected with STM (MOI=50) for 6 hours; Cells were fixed; stained for endogenous ARF1(red), Pex14(green), and STM-DAPI (blue); and Zoom part of image are shown ARF1(red), Pex14(green), and STM-DAPI (blue) in both infected and uninfected cells; scale bar=5um. D. Equal number of HeLa Cells were infected with either the WT or STM ΔsseI (MOI=10) for 6 hr. Then the active ARF1 was pulled down and were run on SDS page. Densitometry graph from 3 independent experiment representing the relative ARF1 activation in the WT and ΔsseI infected cells compared to that of uninfected HeLa cells is shown (MOI=10). E. HeLa cell were transfected with either full length or mutated (Q71L) ARF-1 or silenced ARF-1. Then infected with the WT or ΔsseI STM and changes in the fold proliferation were plotted. Experiments were repeated three times. Individual data points represent mean ±SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘ns’ denotes non-significant ***p<0.001, *p<0.05, **p<0.01, unpaired two-tailed Student T-test.

    Article Snippet: Antibodies used (Abs) used were as follows: anti-LAMP1 antibody (D401S from CST), PEX14 Antibody (A7336), ABCD1 (60153-1-Ig), GAPDH Antibody (T0004), ESYT1 antibody (A15410), anti-HA tag Antibody(2367), Anti-FLAG Antibody(8146 from CST), anti-myc (2276 from CST) anti-pex5 antibody (12545-1-AP from proteintech), ARF1 Antibody (10790-1-AP), Goat anti-mouse IgG Alexa fluor 594(A- 11032), Goat anti-Rabbit IgG Alexa Fluor 488(A-11034) IgG Alexa fluor 594(A- 11032) SYT7 antibody(NBP2-22420), PIP2 ANTIBODY(NBP2-76433), Cy5 goat anti-rabbit IgG (A10523 - Invitrogen), Control Mouse immunoglobulin (Cat#500-M00- 1mg - PEPROTECH), Control rabbit immunoglobulin (Cat#500-P00-500 ug - PEPROTECH) anti-rabbit IgG antibody and horseradish peroxidase (7074P2) were purchased from Cell Signalling Technology.

    Techniques: Transfection, Immunoprecipitation, Incubation, Purification, SDS Page, Western Blot, Microscopy, Infection, Staining, Activation Assay, Two Tailed Test

    LDL-derived cholesterol routed via lysosome-peroxisome is essential for Salmonella growth A. Graph representing percentage of peroxisome-lysosome co-localization in the HeLa cells after infection with the WT STM (MOI=10) for different time points. B. WT HeLa cells were infected with WT STM or STM ΔsseI for 6 h with MOI= 50. pex5 -/- HeLa cell were infected with WT STM for 6 h with MOI= 50. Cells were fixed and immunostained with LAMP1(red), cholesterol using filipin(blue) and STM (green). Zoom part represented SCV and Lysosome cholesterol (blue) level in the cells. Graph representing change in the filipin fluorescent intensity is shown. (n=50 cells). Scale bar 10 uM. C. PEROXO- tagged stable cell was generated using transfection with lentivirus overexpressing 3X myc-EGFP-Pex26. Stable PEROXO tag HeLa cells were next infected with the WT and ΔsseI STM with MOI=10 for 6 hr. Isolated peroxisomal cholesterol amount was measured using cholesterol assay kit. Graph representing the fold proliferation of STM silencing D. NPC1 E. ABCD1 in HeLa cells. Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. F. Cells were kept untreated or treated with lovastatin (10uM). Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. G. Cells were kept untreated or treated with LDL/U18666A in the media containing 1% FBS in HeLa cells. Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. Individual data points represent mean± SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘src’ denoted scrambled siRNA. ‘UI’ denotes uninfected, ‘UT’ denotes untreated. ‘ns’ denotes non- significant ****p<0.0001, ***p<0.001, *p<0.05, **p<0.01, unpaired two-tailed Student T-test.

    Journal: bioRxiv

    Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

    doi: 10.1101/2023.02.27.530266

    Figure Lengend Snippet: LDL-derived cholesterol routed via lysosome-peroxisome is essential for Salmonella growth A. Graph representing percentage of peroxisome-lysosome co-localization in the HeLa cells after infection with the WT STM (MOI=10) for different time points. B. WT HeLa cells were infected with WT STM or STM ΔsseI for 6 h with MOI= 50. pex5 -/- HeLa cell were infected with WT STM for 6 h with MOI= 50. Cells were fixed and immunostained with LAMP1(red), cholesterol using filipin(blue) and STM (green). Zoom part represented SCV and Lysosome cholesterol (blue) level in the cells. Graph representing change in the filipin fluorescent intensity is shown. (n=50 cells). Scale bar 10 uM. C. PEROXO- tagged stable cell was generated using transfection with lentivirus overexpressing 3X myc-EGFP-Pex26. Stable PEROXO tag HeLa cells were next infected with the WT and ΔsseI STM with MOI=10 for 6 hr. Isolated peroxisomal cholesterol amount was measured using cholesterol assay kit. Graph representing the fold proliferation of STM silencing D. NPC1 E. ABCD1 in HeLa cells. Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. F. Cells were kept untreated or treated with lovastatin (10uM). Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. G. Cells were kept untreated or treated with LDL/U18666A in the media containing 1% FBS in HeLa cells. Changes in fold proliferation of WT STM after 2 and 16 hr post-infection is plotted in these cells. Individual data points represent mean± SD. Result is representative of 3 independent experiments. ‘MOI’ denotes multiplicity of infection. ‘src’ denoted scrambled siRNA. ‘UI’ denotes uninfected, ‘UT’ denotes untreated. ‘ns’ denotes non- significant ****p<0.0001, ***p<0.001, *p<0.05, **p<0.01, unpaired two-tailed Student T-test.

    Article Snippet: Antibodies used (Abs) used were as follows: anti-LAMP1 antibody (D401S from CST), PEX14 Antibody (A7336), ABCD1 (60153-1-Ig), GAPDH Antibody (T0004), ESYT1 antibody (A15410), anti-HA tag Antibody(2367), Anti-FLAG Antibody(8146 from CST), anti-myc (2276 from CST) anti-pex5 antibody (12545-1-AP from proteintech), ARF1 Antibody (10790-1-AP), Goat anti-mouse IgG Alexa fluor 594(A- 11032), Goat anti-Rabbit IgG Alexa Fluor 488(A-11034) IgG Alexa fluor 594(A- 11032) SYT7 antibody(NBP2-22420), PIP2 ANTIBODY(NBP2-76433), Cy5 goat anti-rabbit IgG (A10523 - Invitrogen), Control Mouse immunoglobulin (Cat#500-M00- 1mg - PEPROTECH), Control rabbit immunoglobulin (Cat#500-P00-500 ug - PEPROTECH) anti-rabbit IgG antibody and horseradish peroxidase (7074P2) were purchased from Cell Signalling Technology.

    Techniques: Derivative Assay, Infection, Stable Transfection, Generated, Transfection, Isolation, Cholesterol Assay, Two Tailed Test

    Schematic diagram. Mature SCVs secretes SPI-2 effector proteins in the host cell cytosol among which SseI contains host PTS1 signal. The effector protein, SseI interacts with GTPase ARF1 on host peroxisome. And recruits specific PIP4K2A which is essential for PIP2 synthesis on peroxisome. Simultaneously, cells receiving LDL from outside is further metabolized in lysosome and results in the release of free cholesterol. The lysosomal cholesterol is rerouted to the pathogenic bacteria using peroxisome as a bridge. This helps in SCV maturation and SIF formation resulting in the growth and proliferation of Salmonella. On the other hand, in the Pex5 -/- HeLa cells, dysfunctional peroxisome prevents its interaction with lysosome and SCV both. This results in the decreased proliferation of STM within the host cell.

    Journal: bioRxiv

    Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

    doi: 10.1101/2023.02.27.530266

    Figure Lengend Snippet: Schematic diagram. Mature SCVs secretes SPI-2 effector proteins in the host cell cytosol among which SseI contains host PTS1 signal. The effector protein, SseI interacts with GTPase ARF1 on host peroxisome. And recruits specific PIP4K2A which is essential for PIP2 synthesis on peroxisome. Simultaneously, cells receiving LDL from outside is further metabolized in lysosome and results in the release of free cholesterol. The lysosomal cholesterol is rerouted to the pathogenic bacteria using peroxisome as a bridge. This helps in SCV maturation and SIF formation resulting in the growth and proliferation of Salmonella. On the other hand, in the Pex5 -/- HeLa cells, dysfunctional peroxisome prevents its interaction with lysosome and SCV both. This results in the decreased proliferation of STM within the host cell.

    Article Snippet: Antibodies used (Abs) used were as follows: anti-LAMP1 antibody (D401S from CST), PEX14 Antibody (A7336), ABCD1 (60153-1-Ig), GAPDH Antibody (T0004), ESYT1 antibody (A15410), anti-HA tag Antibody(2367), Anti-FLAG Antibody(8146 from CST), anti-myc (2276 from CST) anti-pex5 antibody (12545-1-AP from proteintech), ARF1 Antibody (10790-1-AP), Goat anti-mouse IgG Alexa fluor 594(A- 11032), Goat anti-Rabbit IgG Alexa Fluor 488(A-11034) IgG Alexa fluor 594(A- 11032) SYT7 antibody(NBP2-22420), PIP2 ANTIBODY(NBP2-76433), Cy5 goat anti-rabbit IgG (A10523 - Invitrogen), Control Mouse immunoglobulin (Cat#500-M00- 1mg - PEPROTECH), Control rabbit immunoglobulin (Cat#500-P00-500 ug - PEPROTECH) anti-rabbit IgG antibody and horseradish peroxidase (7074P2) were purchased from Cell Signalling Technology.

    Techniques:

    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).

    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: The following primary antibodies were used (manufacturer, product number; dilution): PEX5 (Proteintech Group, 12545-1-Fr.1; 1:400), ubiquitin (Santa Cruz Biotechnology, SC-8017; 1:100), GFP (Thermo Fisher Scientific, GF28R; 1:250), SQSTM1/p62 (Santa Cruz Biotechnology, SC-292; 1:100), ABCD3/PMP70 (Abcam, ab211533; 1:200), CAT/catalase (Cell Signaling Technology, 12980; 1:500).

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

    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).

    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: The following primary antibodies were used (manufacturer, product number; dilution): PEX5 (Proteintech Group, 12545-1-Fr.1; 1:400), ubiquitin (Santa Cruz Biotechnology, SC-8017; 1:100), GFP (Thermo Fisher Scientific, GF28R; 1:250), SQSTM1/p62 (Santa Cruz Biotechnology, SC-292; 1:100), ABCD3/PMP70 (Abcam, ab211533; 1:200), CAT/catalase (Cell Signaling Technology, 12980; 1:500).

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

    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.

    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: The following primary antibodies were used (manufacturer, product number; dilution): PEX5 (Proteintech Group, 12545-1-Fr.1; 1:400), ubiquitin (Santa Cruz Biotechnology, SC-8017; 1:100), GFP (Thermo Fisher Scientific, GF28R; 1:250), SQSTM1/p62 (Santa Cruz Biotechnology, SC-292; 1:100), ABCD3/PMP70 (Abcam, ab211533; 1:200), CAT/catalase (Cell Signaling Technology, 12980; 1:500).

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

    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).

    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: The following primary antibodies were used (manufacturer, product number; dilution): PEX5 (Proteintech Group, 12545-1-Fr.1; 1:400), ubiquitin (Santa Cruz Biotechnology, SC-8017; 1:100), GFP (Thermo Fisher Scientific, GF28R; 1:250), SQSTM1/p62 (Santa Cruz Biotechnology, SC-292; 1:100), ABCD3/PMP70 (Abcam, ab211533; 1:200), CAT/catalase (Cell Signaling Technology, 12980; 1:500).

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

    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.

    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: The following primary antibodies were used (manufacturer, product number; dilution): PEX5 (Proteintech Group, 12545-1-Fr.1; 1:400), ubiquitin (Santa Cruz Biotechnology, SC-8017; 1:100), GFP (Thermo Fisher Scientific, GF28R; 1:250), SQSTM1/p62 (Santa Cruz Biotechnology, SC-292; 1:100), ABCD3/PMP70 (Abcam, ab211533; 1:200), CAT/catalase (Cell Signaling Technology, 12980; 1:500).

    Techniques: Functional Assay, Inhibition

    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).

    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

    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).

    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

    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.

    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

    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).

    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

    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.

    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

    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).

    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

    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).

    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

    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.

    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

    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).

    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

    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.

    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