hspa5 p495l  (Addgene inc)

Journal: PLoS Biology

Article Title: Role of Mitochondrial Dynamics in Neuronal Development: Mechanism for Wolfram Syndrome

doi: 10.1371/journal.pbio.1002511

Figure Lengend Snippet: (A) Neurons were transfected with plasmids expressing scrambled shRNA or Wfs1 shRNA, firefly luciferase constructs containing ATF6 or ATF4 binding sites or a XBP-1 splicing reporter, and Renilla luciferase. Firefly luciferase signal normalized to Renilla signal demonstrates a moderate increase in ATF6 and ATF4 reporter activity. (B) Positive control experiments in which the above-mentioned reporter systems and Renilla luciferase were co-transfected with ATF4, ATF6, or IRE1. (C) The mitochondrial fusion rate is reduced by Wfs1 shRNA and is restored by co-expressing wt HSPA5 ( p = 0.001 for interaction, two-way ANOVA). (D) ATPase-deficient HSPA5 mutant (T37G) but not peptide binding-deficient mutant (P495L) restores the fusion rate reduced by Wfs1 shRNA. (E) HSPA5 overexpression attenuates mitophagy activated by Wfs1 silencing ( p = 0.012 for interaction). (F–H) Activation of the primary ER stress pathways by overexpression of ATF6, ATF4, or IRE1 modulates neither fusion rate (F), mitochondrial length (G), nor mitophagy (H). (I–L) Silencing of ATF6 or ATF4 modulates neither fusion rate (I, J) nor mitophagy (K, L). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with respective control groups, or ### p < 0.001 compared with the Wfs1 shRNA-transfected control group and ns non-significant compared with the Wfs1 shRNA-transfected control group. Underlying data is shown in .

Article Snippet: ATeam (51958), ATF6 (11975), ATF6-GL3 (11976), ATF4 (26114), ATF4-luc (21850), WFS1 wt (13011), WFS1 P724L (13012), IRE1α (13009), D1ER (36325), D3cpv (36323), DRP1 K38A, EGFP-LC3 (24920), HSPA5 wt (27164), HSPA5 T37G (27165), HSPA5 P495L (27166), NRF2 (21555), ORAI1 (21638), PSD-95 (15463), and pAAV-hSyn-DsRedExpress (22907) were obtained from Addgene (Cambridge, MA).

Techniques: Transfection, Expressing, shRNA, Luciferase, Construct, Binding Assay, Activity Assay, Positive Control, Mutagenesis, Over Expression, Activation Assay

Journal: PLoS Biology

Article Title: Role of Mitochondrial Dynamics in Neuronal Development: Mechanism for Wolfram Syndrome

doi: 10.1371/journal.pbio.1002511

Figure Lengend Snippet: (A) Neurons were transfected with plasmids expressing scrambled shRNA or Wfs1 shRNA, firefly luciferase constructs containing ATF6 or ATF4 binding sites or a XBP-1 splicing reporter, and Renilla luciferase. Firefly luciferase signal normalized to Renilla signal demonstrates a moderate increase in ATF6 and ATF4 reporter activity. (B) Positive control experiments in which the above-mentioned reporter systems and Renilla luciferase were co-transfected with ATF4, ATF6, or IRE1. (C) The mitochondrial fusion rate is reduced by Wfs1 shRNA and is restored by co-expressing wt HSPA5 ( p = 0.001 for interaction, two-way ANOVA). (D) ATPase-deficient HSPA5 mutant (T37G) but not peptide binding-deficient mutant (P495L) restores the fusion rate reduced by Wfs1 shRNA. (E) HSPA5 overexpression attenuates mitophagy activated by Wfs1 silencing ( p = 0.012 for interaction). (F–H) Activation of the primary ER stress pathways by overexpression of ATF6, ATF4, or IRE1 modulates neither fusion rate (F), mitochondrial length (G), nor mitophagy (H). (I–L) Silencing of ATF6 or ATF4 modulates neither fusion rate (I, J) nor mitophagy (K, L). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with respective control groups, or ### p < 0.001 compared with the Wfs1 shRNA-transfected control group and ns non-significant compared with the Wfs1 shRNA-transfected control group. Underlying data is shown in .

Article Snippet: ATeam (51958), ATF6 (11975), ATF6-GL3 (11976), ATF4 (26114), ATF4-luc (21850), WFS1 wt (13011), WFS1 P724L (13012), IRE1α (13009), D1ER (36325), D3cpv (36323), DRP1 K38A, EGFP-LC3 (24920), HSPA5 wt (27164), HSPA5 T37G (27165), HSPA5 P495L (27166), NRF2 (21555), ORAI1 (21638), PSD-95 (15463), and pAAV-hSyn-DsRedExpress (22907) were obtained from Addgene (Cambridge, MA).

Techniques: Transfection, Expressing, shRNA, Luciferase, Construct, Binding Assay, Activity Assay, Positive Control, Mutagenesis, Over Expression, Activation Assay

Journal: Oncotarget

Article Title: Molecular chaperone GRP78 enhances aggresome delivery to autophagosomes to promote drug resistance in multiple myeloma

doi:

Figure Lengend Snippet: A. Shown is the Fold-increase in the expression of individual HSP pathway genes in MM patient samples compared to MGUS samples (top panel). Also shown is the fold-increase in the expression of individual HSP pathway genes in bortezomib resistant RPMI8226 cells relative to drug-naïve cells (bottom panel). Shown is the fold-increase in relative expression determined by microarray-based profiling using Affymetrix 3.0 chips. B. Western blot comparing GRP78 levels in parental and bortezomib resistant cells. Parental and bortezomib resistant were grown in bortezomib for 18 hours prior to preparation of cell lysates. C. GRP78 staining of myeloma cells by IHC and confocal microscopy in RPMI8226 cells that had been treated with bortezomib (10nM), metformin (1mM) or both agents. Cells were treated for 18 hours under standard growth conditions. D. Quantitation of GRP78 levels based upon the relative level of fluorescent intensity detected by IHC staining. E. GRP78 staining RPMI8226 cells by IHC and confocal microscopy treated with various concentrations of bortezomib and metformin for 18 hours. Shown are representative images obtained from the same experiment performed multiple times.

Article Snippet: Plasmids pCMV-GRP78-myc-WT or pCMV-GRP78-myc-P495L were from Addgene (Cambridge, MA).

Techniques: Expressing, Microarray, Western Blot, Staining, Confocal Microscopy, Quantitation Assay, Immunohistochemistry

Journal: Oncotarget

Article Title: Molecular chaperone GRP78 enhances aggresome delivery to autophagosomes to promote drug resistance in multiple myeloma

doi:

Figure Lengend Snippet: A. Western blot of GRP78 levels in lysates from RPMI8226 cells transfected with control or HSPA5 -specific shRNA. Ponceau staining of the membrane used for the GRP78 blot is shown. B. RPMI8226 cells transfected with either scrambled control or HSPA5 -specific shRNA were treated with drugs as indicated and the level of GRP78 determined by IHC and confocal microscopy. C. RPMI8226 cells transfected with either scrambled control or HSPA5 -specific shRNA were treated with drugs as indicated for 18 hours and aggresomes visualized by IHC and confocal microscopy. D. Quantitation of aggresome levels based upon the relative level of fluorescent intensity detected by IHC and confocal microscopy. E. RPMI8226 cells transfected with scrambled control or shRNA to inactivate the stress transducers ATF6, IRE1α or PERK were treated with drugs as indicated and GRP78 levels determined by IHC and confocal microscopy. shRNA-mediated knockdown of the three stress transducers was validated by qRT-PCR. F. Western blot of myeloma cell lysate after treatment with bortezomib or metformin as indicted for 18 hours.

Article Snippet: Plasmids pCMV-GRP78-myc-WT or pCMV-GRP78-myc-P495L were from Addgene (Cambridge, MA).

Techniques: Western Blot, Transfection, shRNA, Staining, Confocal Microscopy, Quantitation Assay, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Molecular chaperone GRP78 enhances aggresome delivery to autophagosomes to promote drug resistance in multiple myeloma

doi:

Figure Lengend Snippet: A. RPMI8226 cells were treated with either bortezomib (10nM), metformin (1mM) or both agents for 18 hours under standard growth conditions. GRP78 was detected by IHC and confocal microscopy. Aggresomes and autophagosomes were detected using the dye-based methods. Shown are representative images seen on in at least three different experiments. B. Relative level of fluorescent intensity of autophagosomes after treatment of RPMI8226 cells with the indicated drugs. C. RPMI8226 cells were transfected with scrambled (control) or HSPA5 -specific shRNA, treated with drugs as indicated and GRP78, aggresomes and autophagosomes detected as in Figure . Shown are representative images seen on in at least three different experiments. D. Relative level of fluorescent intensity of autophagosomes after treatment of RPMI8226 cells transfected with either scrambled control or HSPA5 shRNA and then treated with drugs as indicated. E. Co-localization of GRP78 with aggresomes as determined by IHC and confocal microscopy. RPMI8226 cells were treated with bortezomib (10nM), metformin (1mM) or both and stained using a GRP78-specirfic antibody, for aggresomes using dye-based reagent or both the GRP78 antibody and the dye-based reagent. Shown are representative images from multiple experiments. F. Co-localization of GRP78 with autophagosome as determined by IHC and confocal microscopy. RPMI8226 cells were treated with bortezomib (10nM), metformin (1mM) or both and stained using a GRP78-specirfic antibody, for autophagsomes using dye-based reagent or both the GRP78 antibody and the dye-based reagent. Shown are representative images from multiple experiments. G. Effect of bortezomib and metformin on aggresomes and autophagosomes in MM patient tumor cells. Patient bone marrow was obtained, CD138 + cells purified, treated with drugs as indicated and aggresomes and autophagosomes detected using the dye-based methods and confocal microscopy.

Article Snippet: Plasmids pCMV-GRP78-myc-WT or pCMV-GRP78-myc-P495L were from Addgene (Cambridge, MA).

Techniques: Confocal Microscopy, Transfection, shRNA, Staining, Purification

Journal: Oncotarget

Article Title: Molecular chaperone GRP78 enhances aggresome delivery to autophagosomes to promote drug resistance in multiple myeloma

doi:

Figure Lengend Snippet: A. U266 cells were transfected with plasmids that expressed either control (pcDNA3.1) or a GRP78 mutant (P495L). Cells were treated with bortezomib (10nM), metformin (1mM) or both for 18h. Aggresomes were detected by the by dye-based method. Shown are representative images from multiple experiments. B. U266 cells were transfected with plasmids that expressed either control (pcDNA3.1) or the GRP78 mutant. Cells were treated with bortezomib (10nM), metformin (1mM) or both for 18h. Autophagosomes were detected by dye-based methods. Shown are representative images. C. U266 cells were transfected with plasmids that expressed GRP78-WT or the GRP78 mutant. Cells were treated with bortezomib, lysates immunoprecipitated and probed by western blot to detect the association of aggresome (p62 and HDAC6) or autophagosome pathway (KDEL receptor and LC3B) effectors with GRP78. D. U266 cells were transfected with plasmids that expressed either shRNA to inactivate control (scrambled) or HSPA5. Cells were treated with bortezomib at indicated concentrations and the effect on viability determined using the XTT assay. Values represent the mean of triplicate measurements and error bars represent the standard deviation (SD). E. U266 cells were transfected with plasmids that expressed either GRP78-WT or the GRP78 mutant. Cells were treated with bortezomib as indicated and the effect on viability determined using the XTT assay. Values represent the mean of triplicate measurements and error bars represent the SD.

Article Snippet: Plasmids pCMV-GRP78-myc-WT or pCMV-GRP78-myc-P495L were from Addgene (Cambridge, MA).

Techniques: Transfection, Mutagenesis, Immunoprecipitation, Western Blot, shRNA, XTT Assay, Standard Deviation