Journal: Molecular Medicine Reports

Article Title: Overexpressed p32 localized in the endoplasmic reticulum and mitochondria negatively regulates calcium-dependent endothelial nitric oxide synthase activity

doi: 10.3892/mmr.2020.11307

Figure Lengend Snippet: Overexpression of p32 in the ER and mitochondria. (A) Electron microscopic analysis of the p32 localization using tagged APEX2-generated 3,3-diaminobenzidine labeling pattern. Untransfected cells exhibited constant staining of mitochondria and the ER membrane (a and b). HeLa cells expressing the p32-APEX2 constructs exhibited clear densities in targeted areas. The APEX2 was targeted to the ER membrane and mitochondria membrane (black arrow, c and d). In adp32-treated HUVECs, (B) p32 protein levels were increased and (C) p32 expression was induced in a time-dependent manner. Confocal microscopic images of adp32-treated HUVECs indicated that overexpressed p32 localized to both (D) mitochondria and (E) ER. Mitotracker green and concanavalin A-594 were used as indicators of mitochondria and the ER, respectively. n=3. *P<0.05, vs. untreated. HUVEC, human umbilical vein endothelial cell; ER, endoplasmic reticulum; ad, adenovirus; AU, arbitrary units.

Article Snippet: The p32 gene was cloned into the AfI I and Bam HI restriction sites of the pcDNA3 connexin43-GFP-APEX2 plasmid (Addgene) for EM analysis.

Techniques: Over Expression, Generated, Labeling, Staining, Expressing, Construct

Journal: The EMBO Journal

Article Title: Torsin and NEP1R1‐CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid‐dependent and lipid‐independent mechanisms

doi: 10.15252/embj.2020106914

Figure Lengend Snippet: A dTor mutants and chimeras. Sig Seq, ER targeting signal sequence. B Wild‐type and mutant dTor mGFP proteins expressed in dTor KO fat body cells. C, D Size of individual fat body cells (C) and LD density of individual fat body cells (D). Bars show the group mean, light gray highlights 2× SD of the control mean (animals expressing the Sec61b control ER protein), and dotted red lines show dTor KO mean. These are results of n ≥ 3 larvae (of 3 independent crosses). One‐way ANOVA, with Bonferroni’s post hoc test. ** P < 0.01, *** P < 0.001, and **** P < 0.0001. E–M GFP, Phalloidin, and LipidTOX detect the expression of dTor mGFP chimeras (green), cell boundaries (red), and LD (red), respectively, in dTor KO fat body cells. Source data are available online for this figure.

Article Snippet: For dTor membrane domain chimeras, these again used Gibson Assembly to integrate 2 PCR‐derived fragments into pUAST: PCR 1 generated sequence encoding the TMD of Sec61b or dLap1 using pcDNA3 Sec61b‐V5‐APEX (Addgene #83411 (Lee et al , )) and dLap1 (Grillet et al , ) as templates, respectively.

Techniques: Sequencing, Mutagenesis, Expressing

Journal: The EMBO Journal

Article Title: Torsin and NEP1R1‐CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid‐dependent and lipid‐independent mechanisms

doi: 10.15252/embj.2020106914

Figure Lengend Snippet: A–C Fat body cells expressing GFP tagged UAS transgenes with r4‐Gal4. D TAG (nmol/ µg protein) in the 4 DO fat body expressing UAS transgenes with the r4‐Gal4 driver. Bars show group mean ± SEM ( n = 6), and points show values from individual MS runs. Unpaired t ‐test. ** P < 0.01. E–I NE ultrastructure of 5DO fat body cells overexpressing UAS transgenes, or from dLap1 −/− animals. Yellow dotted lines highlight NE regions with subtly abnormal membrane architecture. Red arrows indicate nuclear pores. R1, region 1; R2, region 2; Nuc, nucleus; Cyto, cytosol. J–M Data from FIB‐SEM imaging of fat body nuclei expressing Sec61b (control) or CTDNEP1 mGFP . (J–L) 3D reconstructions, (M) distance between the INM and ONM at NPC in wild‐type cells versus CTDNEP1 mGFP ‐induced structures. Kolmogorov–Smirnov non‐parametric t ‐test. **** P < 0.0001. N, O TEM analysis of the NE in 5DO fat body cells expressing the CTDNEP1 cDNA with DAGK or Sec61 cDNAs (control). Red arrows indicate nuclear pores.

Article Snippet: For dTor membrane domain chimeras, these again used Gibson Assembly to integrate 2 PCR‐derived fragments into pUAST: PCR 1 generated sequence encoding the TMD of Sec61b or dLap1 using pcDNA3 Sec61b‐V5‐APEX (Addgene #83411 (Lee et al , )) and dLap1 (Grillet et al , ) as templates, respectively.

Techniques: Expressing, Imaging

Journal: The EMBO Journal

Article Title: Torsin and NEP1R1‐CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid‐dependent and lipid‐independent mechanisms

doi: 10.15252/embj.2020106914

Figure Lengend Snippet: A The NPC is comprised of several subcomplexes. B–G Confocal imaging of 5DO fat body nuclei expressing UAS transgenes and labeled by anti‐Megator/Tpr, Nup214, Nup35, or mAb414, or expressing Nup107 RFP . H mAb414 localization in 5DO fat body cells expressing the indicated UAS transgenes or from a dLap1 −/− animal. *** and **** indicate a significant difference compared with control mGFP Sec61b‐expressing cells. Chi‐square test followed by individual post hoc chi‐square tests. *** P < 0.001 and **** P < 0.0001. I–K NE ultrastructure of 5DO fat body cells from a (I) control animal, (J) homozygous Nup35 BG01311 , and (K) homozygous Nup35 MB02683 animals. Nuc, nucleus; Cyto, cytosol. L Model of the relationship between nucleocytoplasmic channels, PA metabolism, and NPC maturation.

Article Snippet: For dTor membrane domain chimeras, these again used Gibson Assembly to integrate 2 PCR‐derived fragments into pUAST: PCR 1 generated sequence encoding the TMD of Sec61b or dLap1 using pcDNA3 Sec61b‐V5‐APEX (Addgene #83411 (Lee et al , )) and dLap1 (Grillet et al , ) as templates, respectively.

Techniques: Imaging, Expressing, Labeling

Journal: eLife

Article Title: The role of sigma 1 receptor in organization of endoplasmic reticulum signaling microdomains

doi: 10.7554/eLife.65192

Figure Lengend Snippet: ( A ) Proximity labeling of IRE1α. HEK293 cells expressed S1R-APEX2 and Sec61β-APEX2 or APEX2-KDEL as controls. Cells were incubated with biotin-phenol, and proteins were labeled by a 1 min addition of H 2 O 2 . Biotinylated proteins were pulled down and analyzed by western blot. ( B ) Western blot analysis of lysates prepared from control HEK293 cells, S1R KO HEK293 cells, and both cell lines transfected with S1R-6His construct as indicated. ( C ) Time-dependence of the thapsigargin (Tg)-induced IRE1α response in WT and S1R KO HEK293 cells. Western blot is representative of n = 3 independent experiments. ( D ) Quantification results of the western blot shown above. p-IRE1α levels were normalized to the total level of IRE1α and plotted as mean (solid line) ± SEM (shaded) from n = 3 independent experiments for WT (black) and S1R KO cells (red). ( E ) Quantification results for area under the curve of the Tg-induced p-IRE1α response. p-value=0.015 based on t-test. ( F ) Quantification results of the western blot results for XBP1s. Intensities of protein bands were normalized to tubulin and plotted as mean ± SEM from n = 3 independent experiments for WT (black) and S1R KO cells (red).

Article Snippet: Recombinant DNA reagent , Sec61b-APEX2 , This study , , Generated by cloning Sec61b-APEX fusion gene (RRID: Addgene_83411 ) into lentivector.

Techniques: Labeling, Incubation, Western Blot, Transfection, Construct

Journal: eLife

Article Title: The role of sigma 1 receptor in organization of endoplasmic reticulum signaling microdomains

doi: 10.7554/eLife.65192

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , Sec61b-APEX2 , This study , , Generated by cloning Sec61b-APEX fusion gene (RRID: Addgene_83411 ) into lentivector.

Techniques: Generated, Selection, Isolation, Recombinant, Plasmid Preparation, Mutagenesis, Construct, Clone Assay, Sequencing, Software

Journal: Nature Communications

Article Title: The CHD6 chromatin remodeler is an oxidative DNA damage response factor

doi: 10.1038/s41467-018-08111-y

Figure Lengend Snippet: Cells lacking CHD6 display elevated PAR, oxidative stress and have diminished antioxidant responses. a A549 were subjected to CRISPR-mediated CHD6 gene ablation (ΔCHD6) or treated with CHD6 siRNA (siCHD6) before being immunoblotted (left) or immunostained (right) as indicated. Parental refers to the wild-type (WT) A549 used to derive ΔCHD6. Scale bars = 20 µm. b WT and A549 ΔCHD6 were treated ±5 µM PARGi and indicated H 2 O 2 doses for 1 h before immunoblotting for PAR, CHD6 and actin. Representative blot shown, n = 3. c WT and ΔCHD6 A549 were complemented with GFP, CHD6 GFP or CHD4 GFP and treated with 5 µM PARGi ± 1 mM H 2 O 2 in media for 1 h before immunostaining for PAR (red) and DAPI (blue). Lower panels highlight GFP-expressing cells. Scale bars = 10 µm. d WT and A549 ΔCHD6 were transfected with GFP, or GFP-tagged CHD6 wildtype , CHD6 Δchromo or CHD6 K492Q before treatment with 5 µM PARGi ± 0–1 mM H 2 O 2 for 1 h and immunostaining for PAR and GFP as in ( c ). Nuclear PAR in >1000 GFP-positive cells ( n = 3) was quantified using ImageJ. Dot colors represent: light blue = GFP alone, WT cells; dark blue = WT CHD6 GFP , WT cells; cyan = K492Q CHD6 GFP , WT cells; purple = ΔCD1+2 CHD6 GFP , WT cells; light green = GFP alone, ΔCHD6 cells; dark green = WT CHD6 GFP , ΔCHD6 cells; dark red = K492Q CHD6 GFP , ΔCHD6 cells; orange = ΔCD1+2 CHD6 GFP , ΔCHD6 cells. Error bars = s.e.m.; n = 3. e WT, A549 ΔCHD6 and A549 ΔCHD6 complimented with CHD6 mPLUM , incubated with H 2 DCFA then exposed ± 0.5 mM H 2 O 2 for 1 h and FACS analysis of green fluorescent DCF signal. Data represent an average of the percent DCF-positive cells within a population, expressed relative to the PBS-treated wild-type control. Error bars = s.e.m.; n > 3. f , g WT and A549 ΔCHD6 were exposed to ±500 µM H 2 O 2 in media for up to 1 day and analyzed by qPCR to ascertain HMOX1, TXNRD1, NQO1, G6PD and TBP mRNA expression. Error bars = s.e.m.; n = 3. In all cases, P values are for Student’s t -test: ns not significant ( P > 0.05); * P < 0.05; ** P < 0.01; *** P < 0.001. In all cases, ± refers to with and without

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was a gift from Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: CRISPR, Western Blot, Immunostaining, Expressing, Transfection, Incubation

Journal: Nature Communications

Article Title: The CHD6 chromatin remodeler is an oxidative DNA damage response factor

doi: 10.1038/s41467-018-08111-y

Figure Lengend Snippet: CHD6-deficient cells display increased single-strand breakage, DNA damage signaling but normal DNA repair capacity. a WT and ΔCHD6 A549 were transfected with GFP or HMOX1 GFP and treated with 5 µM PARGi ± 1 mM H 2 O 2 in media for 1 h before immunostaining for PAR. Nuclear PAR and GFP signal was quantified by ImageJ. Right panels = representative images indicating GFP (green) and/or DAPI (blue) signal in each condition. b – d WT and ΔCHD6 A549 in suspension were treated with 50 µM H 2 O 2 and analyzed by alkaline comet assay as in Methods; b raw data from single experiment; c mean comet tail moment for n = 3; error bars = s.e.m.; d data normalized to induced damage (0 min time point). Error bars = s.e.m. e WT and A549 ΔCHD6 were exposed to 0–300 µM H 2 O 2 in PBS for 20 min, washed, returned to media for 0.5–8 h, fixed and immunostained for 53BP1. 53BP1 foci per cell were enumerated for >50–100 cells per n , n = 3. Graph indicates scatters of foci per cell across population; average number of foci per cell are indicated. f Cells from ( a ) were irradiated and harvested at indicated time points, then processed and analyzed as in ( a ). g (Top) WT, A549 ΔCHD6 were incubated for 4 h at 3% O 2 for acclimatization and transfected with FM-HCR reporter plasmids (see Methods). A549 ΔCHD6 repair capacity for each substrate was normalized to WT values, and is represented as relative repair capacity. (Bottom) A549 were transfected (48 h earlier) with scrambled, CHD2 or Ku80 siRNA, and transfected 48 h later with FM-HCR plasmids. Error bars = s.e.m.; n = 3. h WT and A549 ΔCHD6 were exposed to 0–300 µM H 2 O 2 in PBS for 20 min, washed, returned to media for 0.5 h and immunoblotted for ATM S1981p , ATM and actin. Average signal from n = 3 is indicated in lower panel, error bars = s.d. In all cases, P values are for Student’s t -test: ns not significant ( P > 0.05); * P < 0.05. In all cases, ±refers to with and without, blue refers to WT cells; green refers to ΔCHD6 cells

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was a gift from Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: Transfection, Immunostaining, Alkaline Single Cell Gel Electrophoresis, Irradiation, Incubation

Journal: Nature Communications

Article Title: The CHD6 chromatin remodeler is an oxidative DNA damage response factor

doi: 10.1038/s41467-018-08111-y

Figure Lengend Snippet: CHD6 loss causes failure to thrive after oxidative stress and perturbed oxidative base excision repair initiation. a WT and A549 ΔCHD6 cells were exposed to H 2 O 2 in PBS for 20 min, washed, plated and scored for colony formation 10 days later. Error bars = s.e.m.; n = 3; P value determined by two-way ANOVA. b The 1.0E + 05 WT and A549 ΔCHD6 were plated and, 24 h later, exposed to ±300 µM H 2 O 2 in PBS for 20 min, washed and returned to media for 8 days. Viable cells were counted daily. Error bars = s.e.m.; n = 3. P values represent unpaired t -test between WT and A549 ΔCHD6 at indicated time points. c The 300× WT and A549 ΔCHD6 cells were exposed to H 2 O 2 in media for 48 h, plated and scored for colony formation 10 days later. Error bars = s.e.m.; n = 5; P value determined by two-way ANOVA. d The 1.0E + 05 WT and A549 ΔCHD6 were plated and, 24 h later, 0–500 µM H 2 O 2 was added to media. Fresh H 2 O 2 was added daily to maintain chronic exposure. Cells were analyzed as in ( b ). Error bars = s.e.m.; n = 3. e WT and A549 ΔCHD6 were seeded at 2.0E + 05 and, 24 h later, transfected with either GFP or HMOX1 GFP . After 24 h, cells were exposed to 500 µM H 2 O 2 in media and counted as in ( d ). For unpaired t -test at indicated points relative to 0 µM H 2 O 2 control; * p < 0.05. In all cases, ±refers to with and without, blue refers to WT cells; green refers to ΔCHD6 cells. f A model for CHD6 function. (1) ROS such as hydroxyl radicals react with DNA to produce oxidative damage. (2) Oxidative stress suppresses proteasomal degradation of CHD6, stabilizing protein levels, while oxidative DNA breaks elicit PARP enzyme activation, generating PAR polymers in the vicinity of DNA lesions that enable CHD6 relocalization to oxidative DNA lesions. (3) CHD6 contributes to nucleosome compaction and transcriptional responses, which potentially modulate the magnitude of ATM-dependent signaling. (4) CHD6 promotes oxidative stress transcriptional responses (potentially via Nrf2) and, as a consequence, antioxidant proteins such as HMOX1 and TXNRD1 suppress oxidative stress and DNA damage

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was a gift from Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: Transfection, Activation Assay