Journal: The Journal of Biological Chemistry

Article Title: Cell type–specific labeling of newly synthesized proteins by puromycin inactivation

doi: 10.1016/j.jbc.2023.105129

Figure Lengend Snippet: PAT effectively quenches incorporation of PMY and OPP into newly synthesized proteins . A , immunocytochemistry of HEK 293 cells transfected with mCherry-PAT ( magenta ) mixed with nontransfected cells (no magenta signal), after 10 min labeling with 5 μM PMY. Anti-PMY is shown in white and cell territories delineated by phalloidin staining are shown in teal . The scale bar represents 20 μm. B , quantification of ( A ). Signal intensities from the anti-mCherry and anti-PMY channels were quantified under a phalloidin mask for transfected ( magenta dots ) and untransfected cells ( gray dots ) and plotted against each other. Ten different images were used for quantification. C , Western blot analysis of puromycinylated proteins (anti-PMY) in regular HEK 293 or HEK 293 cells stably expressing PAT (HEK-PAT) labeled for the indicated time periods with 20 μM OPP. The corresponding Coomassie-stained membrane is presented on the right panel. D , quantification of ( C ). Graph depicts mean ± SD; n = 3 independent labeling experiments. E , streptavidin pull-down of newly synthesized proteins from HEK 293 and HEK-PAT cells. (Western blot analysis with indicated antibodies and HRP-conjugated streptavidin, Strep-HRP). HEK 293 and HEK-PAT cells were labeled for 2 h with 20 μM OPP (+) or DMSO as a control and biotinylated by click-reaction. Biotinylated proteins were isolated by streptavidin pull-down and analyzed by Western blot alongside input samples. DMSO, dimethyl sulfoxide; OPP, O-propargyl puromycin; PAT, puromycin N-acetyltransferase; PMY, puromycin.

Article Snippet: For streptavidin pull-downs, equal amounts of protein (200–300 μg) were incubated with 20 to 30 μl of Pierce Streptavidin Magnetic Beads (Thermo Fisher Scientific, 88817) in PBS-0.2% SDS-1% NP-40 for 2 h, at RT with gentle rotation.

Techniques: Synthesized, Immunocytochemistry, Transfection, Labeling, Staining, Western Blot, Stable Transfection, Expressing, Membrane, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Cell type–specific labeling of newly synthesized proteins by puromycin inactivation

doi: 10.1016/j.jbc.2023.105129

Figure Lengend Snippet: Mixed-species mass spectrometry experiment shows proteome-wide efficiency of PICSL . A , schematic of the experiment. Neuro2a cells (N2a, mouse) were mixed at a 1:1 ratio with either regular HEK 293 cells (human) or HEK 293 cells stably expressing PAT (HEK-PAT). After cell attachment (4 h), cells were labeled for 2 h with 20 μM OPP or DMSO, lysed, and clicked with biotin azide. Newly synthesized proteins were pulled down using streptavidin beads and analyzed by LC-MS/MS. PAT-expressing cells are shown in blue and OPP-incorporating cells in orange . B , peptides from de novo –synthesized proteome pull-downs (four samples as indicated) were mapped to UniProt taxonomy identifiers. Shown are spectral counts for all peptides, peptides mapping to both mouse and human proteins, and peptides unique to mouse or human proteins. Bovine serum proteins and streptavidin account for the difference in counts in “All” and combined counts of mouse and human peptides. C – E , distribution of intensity values for individual peptides common to mouse and human ( C ), unique to mouse ( D ), or human ( E ). Number of peptides in log(2) intensity value bins ( dots , bin width 0.2) are shown with fitted curves ( lines ). The difference in mouse-specific peptides identified in the HEK-PAT+N2a/OPP compared to HEK293 + N2a/OPP may be caused by more efficient pull-down of mouse proteins from the click-conjugated HEK-PAT+N2a/OPP lysate due to better availability of streptavidin-binding sites. DMSO, dimethyl sulfoxide; OPP, O-propargyl puromycin; PAT, puromycin N-acetyltransferase; PICSL, puromycin inactivation for cell-selective proteome labeling.

Article Snippet: For streptavidin pull-downs, equal amounts of protein (200–300 μg) were incubated with 20 to 30 μl of Pierce Streptavidin Magnetic Beads (Thermo Fisher Scientific, 88817) in PBS-0.2% SDS-1% NP-40 for 2 h, at RT with gentle rotation.

Techniques: Mass Spectrometry, Stable Transfection, Expressing, Cell Attachment Assay, Labeling, Synthesized, Liquid Chromatography with Mass Spectroscopy, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Cell type–specific labeling of newly synthesized proteins by puromycin inactivation

doi: 10.1016/j.jbc.2023.105129

Figure Lengend Snippet: Neuronal-targeted expression of PAT in mixed rat cortical cell cultures . A , mixed cortical neuron-glia cultures from embryonic day 21 (E21) rat pups were transduced at seeding with AAV-[Syn]-PAT-mCherry to direct expression of PAT to neurons. At 7 days in vitro , cells were pulse-labeled for 10 min with 5 μM PMY and costained with anti-mCherry (PAT, in magenta ), anti-NeuN (neuronal marker, in green ), anti-Aldh1L1 (astrocyte marker, in green ), or anti-PMY (in white ) in the indicated combinations. Bottom right image shows an enlargement of the area indicated above. Full images with separate channels are shown in Fig. S5 . The scale bar represents 50 μm. B , untransduced or AAV-[Syn]-PAT–transduced cocultures were labeled for 2 h with 20 μM OPP (“OPP”, “OPP + [Syn]-PAT”) or DMSO as a control (“DMSO”), and newly synthesized proteins were biotinylated by click-reaction and isolated by streptavidin pull-down. Western blot analysis of three neuronal markers (NeuN, calbindin 2, and β-III-tubulin) and overall biotinylation (detected by streptavidin-HRP) showed signal reduction in cultures transduced with AAV-[Syn]-PAT-mCherry. C and D , quantification of ( B ). C . signal intensities of the three tested neuronal markers were determined in the pull-down fractions of OPP-labeled untransduced ( blue ) or AAV-[Syn]-PAT–transduced ( red ) samples. Bars indicate mean ± SD of 4 to 5 biological replicates. Statistical differences were determined by multiple paired t-tests and the Holm-Šídák correction for multiple comparisons. D , streptavidin-HRP signal intensity (representing biotinylated proteins) was quantified for pull-down and input fractions in OPP-labeled untransduced ( blue ) or OPP-labeled AAV-[Syn]-PAT–transduced ( red ) samples. Strep-HRP signal intensity was not quantified for DMSO-treated samples since this background signal arises from endogenous biotinylated proteins. Bars show mean ± SD of 3 to 4 biological replicates. One-way ANOVA and the Holm-Šídák’s multiple comparisons test was used to determine statistically significant differences. ∗adjusted p -value ≤ 0.05, ∗∗adjusted p -value ≤ 0.01. AAV, adeno-associated virus; DMSO, dimethyl sulfoxide; HRP, horseradish peroxidase; OPP, O-propargyl puromycin; PAT, puromycin N-acetyltransferase; PMY, puromycin; Syn, synapsin.

Article Snippet: For streptavidin pull-downs, equal amounts of protein (200–300 μg) were incubated with 20 to 30 μl of Pierce Streptavidin Magnetic Beads (Thermo Fisher Scientific, 88817) in PBS-0.2% SDS-1% NP-40 for 2 h, at RT with gentle rotation.

Techniques: Expressing, In Vitro, Labeling, Marker, Synthesized, Isolation, Western Blot, Transduction, Virus

Journal: The Journal of Biological Chemistry

Article Title: Cell type–specific labeling of newly synthesized proteins by puromycin inactivation

doi: 10.1016/j.jbc.2023.105129

Figure Lengend Snippet: Application of PICSL for cortical coculture proteomics . A , LC-MS/MS analysis of de novo –synthesized proteins isolated from cortical cocultures with high glial content (see main text for details) with or without AAV-[Syn]-PAT transduction upon seeding. After 7 days in vitro , cells were pulse-labeled for 2 h with 20 μM OPP ( blue and red for untransduced and AAV-[Syn]-PAT transduced, respectively) or DMSO ( black ) and processed for streptavidin-mediated pull-down. Shown are log2 intensity distributions of identified proteins. Bars indicate mean ± SD of two biological replicates. The distribution for DMSO-treated samples indicates the background binding of unlabeled proteins to beads in affinity purification. B . Venn diagram showing the overlap of proteins identified in the DMSO-treated samples ( gray ) and OPP-treated samples ( blue and red for untransduced and AAV-[Syn]-PAT transduced, respectively). Only proteins with an intensity > 0 for each condition in at least one replicate were included. The diagram was generated using the BioVenn web application tool . C , top 10 cellular compartment gene ontology (GO) enriched terms for proteins detected only in the untransduced, OPP-labeled samples. Genes in the GO term “synapse” are shown in the fly out. The list of proteins identified in the MS analysis was refined by including only those that showed no intensity in the DMSO-treated or the OPP-labeled PAT-expressing samples but did so in the untransduced, OPP-labeled samples (see list of “Proteins in OPP only” in Table S2 ). Enriched GO terms for this subset of proteins were determined using g:Profiler . Shown here are GO cellular compartment terms and their corresponding adjusted p -values (in –log 10 scale). D , list of the top 10 proteins (ranked by signal intensity) detected only in PAT negative, OPP-labeled samples ( i.e. , neuron-enriched proteins). Highlighted in blue are those that were selected for further validation. E , schematic of the RiboTag assay for validation of neuron-enriched candidate proteins. Neuron-glia cocultures transduced with AAV-[Syn]-Rpl22-HA express HA-tagged ribosomes only in neurons, allowing neuron-specific immunopurification of ribosome-associated mRNA. F , RiboTag analysis of the top five neuron-specific proteins from the OPP mass spectrometry experiment (shown in D ). Cortical cocultures were transduced with AAV-[Syn]-Rpl22-HA on the day of seeding. At 7 days in vitro , neuronal ribosome-associated mRNAs were immunoprecipitated with anti-HA antibody (RiboTag +), using untransduced cultures (RiboTag -) as negative controls. For reference, glia-enriched marker genes Aldh1L1 (astrocyte), Iba1 (microglia), Cspg4 (oligodendrocyte precursor cells), Pdgfra (oligodendrocyte precursor cells), and Cnp (oligodendrocytes) were analyzed. Bars depict mean ± SD of n = 3 independent biological replicates. Statistical significance between RiboTag + and RiboTag - groups was determined by multiple unpaired t tests and the Holm-Šídák correction for multiple comparisons. ∗∗∗∗adjusted p -value ≤ 0.001, ns: not significant. AAV, adeno-associated virus; DMSO, dimethyl sulfoxide; MS, mass spectrometry; OPP, O-propargyl puromycin; PAT, puromycin N-acetyltransferase; PICSL, puromycin inactivation for cell-selective proteome labeling; Syn, synapsin.

Article Snippet: For streptavidin pull-downs, equal amounts of protein (200–300 μg) were incubated with 20 to 30 μl of Pierce Streptavidin Magnetic Beads (Thermo Fisher Scientific, 88817) in PBS-0.2% SDS-1% NP-40 for 2 h, at RT with gentle rotation.

Techniques: Liquid Chromatography with Mass Spectroscopy, Synthesized, Isolation, Transduction, In Vitro, Labeling, Binding Assay, Affinity Purification, Generated, Expressing, Immu-Puri, Mass Spectrometry, Immunoprecipitation, Marker, Virus

Journal: Scientific Reports

Article Title: A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells

doi: 10.1038/s41598-024-64195-1

Figure Lengend Snippet: Rapid process to differentiate iPSCs into iEPCs. ( A ) Schematic representation of the protocol for the differentiation of hiPSCs to iEPCs. ( B ) The cell morphology from day 1 to day 4 during differentiation. Scale bar, 50 μm. ( C ) The cells were treated with different concentrations of VEGF (0 ng/ml, 25 ng/ml, 50 ng/ml, or 100 ng/ml) from days 2–3. Representative FACS plots for iPSC-derived cells on day 4 are shown. ( D ) Changes in the gene expression of OCT4, NANOG, T, SCL, KDR, PECAM1, CD34 and CDH5 during differentiation. The results are shown as the mean ± SD of 3 independent experiments. ( E ) Immunofluorescence staining for CD31, CD34, and VE-CADHERIN on day 4. Nuclei were counterstained with DAPI. Scale bar, 50 μm. *** p < 0.001 and **** p < 0.0001.

Article Snippet: EPCs were purified based on CD34 expression using mouse anti-human CD34 antibody-conjugated magnetic beads according to the manufacturer’s instructions (Miltenyi, 130046702).

Techniques: Derivative Assay, Gene Expression, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells

doi: 10.1038/s41598-024-64195-1

Figure Lengend Snippet: Generation of iLSECs from iEPCs. ( A ) Schematic of the differentiation from hiPSC-derived EPCs into LSECs. ( B ) Expression levels of LSEC gene markers in CD34 sorted and no-sorted cells at D8. The results are shown as the mean ± SD of 3 independent experiments. **p < 0.01, ***p < 0.001. ( C ) FACS analysis of differentiated LSECs. Representative plots are shown. ( D ) Gene expression of STAB2, LYVE1, FCGR2B, and CD36 in hiPSC-derived LSEC-like cells cultured in differentiation medium A, B and C under hypoxic (5% O2) or normoxic conditions (21% O2). The results are shown as the mean ± SD of 3 independent experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns not significant. ( E ) Percentages of LYVE1 + cells at D4 and D8 (n = 5). The results are shown as representative plots for purified D8 cells. On D4, 0.92 ± 0.57% of the cells were LYVE1 + cells, and on D8, 76.94 ± 5.16% were LYVE1 + cells.

Article Snippet: EPCs were purified based on CD34 expression using mouse anti-human CD34 antibody-conjugated magnetic beads according to the manufacturer’s instructions (Miltenyi, 130046702).

Techniques: Derivative Assay, Expressing, Gene Expression, Cell Culture, Purification

Journal: Scientific Reports

Article Title: A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells

doi: 10.1038/s41598-024-64195-1

Figure Lengend Snippet: Evaluation of iLSECs. ( A ) The morphology of iLSEC-like cells after CD34 sorting. Scale bar, 100 μm. ( B ) qPCR analysis of canonical LSEC gene markers in iLSEC-like cells and HUVECs. The results are shown as the mean ± SD of 3 independent experiments. ** p < 0.01, *** p < 0.001 and **** p < 0.0001. ( C ) qRT‒PCR analysis of novel LSEC-specific marker genes (screened by ScRNA-seq analyses) in iLSECs and HUVECs. The results are shown as the mean ± SD of 3 independent experiments. * p < 0.05, **p < 0.01, ***p < 0.001 and **** p < 0.0001. ( D ) Immunostaining of LYVE-1 (green) in the iLSECs revealed irregular round or oval-shaped perforations. The nuclei were counterstained with DAPI. Scale bar, 50 μm. E Fluorescence microscopy analysis of Dil-AcLDL and CyTM5-conjugated IgG uptake. hiPSCs, purified iLSEC-like cells and HUVECs were cultured in medium supplemented with DiI-ac-LDL or CyTM5-conjugated IgG. Scale bar, 100 μm.

Article Snippet: EPCs were purified based on CD34 expression using mouse anti-human CD34 antibody-conjugated magnetic beads according to the manufacturer’s instructions (Miltenyi, 130046702).

Techniques: Marker, Immunostaining, Fluorescence, Microscopy, Purification, Cell Culture

Journal: Microbiology Spectrum

Article Title: Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes

doi: 10.1128/spectrum.02452-21

Figure Lengend Snippet: Immune recognition of various EV-D68 densities and characterization of membrane-associated virus. (A) The y axis represents average dilutions of anti-EV-D68 mouse serum required to neutralize virus, divided by the average TCID 50 for respective viral densities (ANOVA post hoc Student'’s t test, P = 0.42, P = 0.68, P = 0.70). (B) Three viral density isolates (1.11, 1.20, and 1.24 g/cm 3 ) were treated with 0.01 mg/mL 15C5-Chmra antibody for 1 h, then mix was put onto TCID 50 plates to assess the viral titer of each isolate. Gray highlight represents detection limit. Asterisks (*) indicate statistical significance (1.11 g/cm 3 , P = 0.0003; 1.20 g/cm 3 , P < 0.0001; 1.24 g/cm 3 , P = 0.0064), all versus respective control, determined by Dunnett’s Method. (C) 15C5-Chmra antibody bound to magnetic beads was added to membrane-associated and naked virus. After 1 h, a magnet was used to remove antibody and the supernatant was added to a TCID 50 plate to assess viral titer (15C5-Chmra versus control: *, P < 0.0005 for both membrane-associated and naked virus; Dunnett’s Method). (D) ICAM-5 or N -acetylneuraminic acid (sialic acid) were attached to magnetic beads and the antibody/bead complex was incubated with membrane-associated or naked virus samples for 1 h. Beads were rinsed twice in excess PBS and viral titer was assessed to determine how much virus was immunoprecipitated from the supernatant (control versus ICAM5 and control versus sialic acid for membrane-associated and naked virus; *, P = 0.0001 determined by Dunnett’s Method). (E) Exosome antibody array on 1.11 g/cm 3 fraction, examining cytosolic proteins (FLOT1, ALIX, TSG101), transmembrane proteins (CD63, CD81, ANXA5), and cis -golgi matrix protein as markers for cellular contamination (GM130). Example blot is shown on the right and chart represents average intensity across three biological replicates. Positive control indicates detection reagents are working correctly, and do not represent an exosome-specific control. Error bars represent standard deviation. Statistics: comparison with control (blank) using Dunnett’s Method ( P = 0.999 for GM130; *, P = 0.027 for FLOT1; P = 0.218 for ICAM; *, P = 0.005 for ALIX; P = 0.086 for CD81; *, P < 0.0001 for CD63; P = 0.305 for EpCAM; *, P < 0.0001 for ANXA5; *, P = 0.0008 for TSG101). Asterisks indicate statistical significance. (F) Anti-CD81 or anti-CD63 antibodies were attached to magnetic beads and incubated with membrane-associated virus. Supernatant was discarded, and beads were rinsed and treated with 0.01% NP-40 (to dissolve exosomes and release virus from bead) before TCID 50 measurement. CD81 versus control: *, P = 0.0178; CD63 versus control: *, P = 0.0180 as determined by Dunnett’s Method. Gray highlight represents detection limit. (G) RD or SH-SY5Y cells in TCID 50 plate were infected with MO47 with or without exosomes in the medium. The “A549 exosomes added” bar represents exosome-depleted media to which purified A549 exosomes were added. Gray panel represents TCID 50 plates containing SH-SY5Y cells. Each condition represents 3 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05. Green panel represents TCID 50 plates containing RD cells. Each condition represents 4 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05.

Article Snippet: We followed the coupling protocol from the Dynabeads Antibody Coupling Kit (Thermo Fisher, cat no. 14311D) to covalently attach magnetic beads to the following antibodies: anti-CD81 (1D6) monoclonal antibody (Novus Biologicals NB100-65805), anti-CD63 (H5C6) monoclonal antibody (Novus Biologicals NBP2-42225), 15C5-chimeric monoclonal antibody (generous gift from Michael Pauly at ZabBio), and anti-HSV negative control (generous gift from Michael Pauly at ZabBio).

Techniques: Membrane, Virus, Control, Magnetic Beads, Incubation, Immunoprecipitation, Ab Array, Positive Control, Standard Deviation, Comparison, Infection, Purification

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Hela Flp-in T-Rex cells were treated with 1 μM Btz for 0, 4, 6, 8, or 18 hrs. Cells were stained with UBXN1 and ubiquitin and imaged (B) Hela Flp-in T-Rex cells were treated with 1 μM Btz for 0, 8, or 18 hrs. Cells were stained with ubiquitin and perinuclear aggregate size was quantified using AggreCount. The mean area of the largest perinuclear aggregate for each time point (2μm 2 or 4μm 2 respectively) was used for all subsequent analysis to quantify perinuclear aggresomes. (C) Levels of p97 depletion in doxycycline-inducible shRNA Hela Flp-in T-Rex cell lines. Cells were treated with doxycycline for 72 hours. (D) sh-p97 cell lines were treated with 1 μM Btz for 18hrs and released into drug-free media for a further 24 hours in the presence or absence of p97 depletion. Cells were stained with ubiquitin and Hoechst. The number of cells containing ubiquitin-positive aggresomes was quantified using AggreCount. (E) Quantification of data in (D). The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show mean +/- standard deviation. *: p<=0.1, **: p<=0.05, ***: p<=0.001 as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Staining, Ubiquitin Proteomics, shRNA, Standard Deviation

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Hela Flp-in T-Rex cells were treated with 1 μM Btz for 18 hrs. Cells were stained for p97, ubiquitin (FK2), and nuclei (Hoechst dye). (B) Hela Flp-in T-Rex cells were treated with 1 μM Btz, 2 μM CB-5083, or both for 8 hrs. Cells were released into drug-free media or media containing 1 μM CB-5083 for 24 hours. Cell lysates were probed for ubiquitin. (C) Cells were treated as in (B) and imaged for ubiquitin-positive aggregates and aggresomes. (D) Total cellular aggregates (encompassing cytosolic and perinuclear aggregates) were quantified using AggreCount for images in (C). (E) Perinuclear aggresomes (minimum size cutoff 2 μm 2 ) were quantified using AggreCount for the images in (C). (F) Total cellular aggregates (encompassing cytosolic and perinuclear aggregates) in the release samples were quantified using AggreCount for images in (C). (G) Perinuclear aggresomes in release samples were quantified using AggreCount for the images in (C). The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show the mean +/- s.e.m. *: p<=0.1, **: p<=0.05, ***: p<=0.001 as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Staining, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Hela Flp-in T-Rex cells were treated with 1 μM Btz for 18 hrs and cells were stained for UBXN1 and ubiquitin. Co-localization was determined by the Mander’s overlap coefficient for 25 cells in 3 replicate experiments. (B) NPL4, UFD1 and p47 localization to aggresomes labeled with ubiquitin. Co-localization was determined by the Mander’s overlap coefficient for 25 cells in 3 replicate experiments. (C) GFP UBXN1 co-localizes with aggresome markers: Proteostat, HDAC6, and 20S proteasomes in Btz treated cells. (D) Microtubules are required for GFP-UBXN1 localization to aggresomes. Nocodazole co-incubation in Btz treated cells prevents aggresome formation. Lower panel: The number of aggresomes was quantified. (E) Hela Flp-in T-Rex cells were treated with 0.1 mM sodium arsenite for 2 h. Cells were stained for stress granule marker G3BP1 and p97 adaptors (UBXN1 or p47, used here as a positive control. (F) Stable mCherry-Dcp1a cells (labeling P bodies) were stained with UBXN1. The indicated number of cells was analyzed from the three independent biological replicates. Graphs show the mean +/- s.e.m. ***: p<=0.001 as determined by unpaired Students t-test. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Staining, Ubiquitin Proteomics, Labeling, Incubation, Marker, Positive Control

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) HeLa Flp-in TRex cells were treated with 1 μM Btz for 18hrs. Cells were stained for p97 adaptors (UBXD1, UBXD2, FAF1, UBXD8, and ASPSCR1) and ubiquitin. Note that UBXD2 and UBXD8 are known to be ER-tethered adaptors and localize to both ER tubules at the cell periphery and ER sheets near the nuclear envelope. They do not co-localize with aggresomes and can be seen to occupy a greater cellular area (corresponding to ER sheets) than the aggresome. (B) Neuroblastoma cells SH-SY5Y were treated with 1 μM Btz for 18 hrs. Cells were stained for UBXN1 and ubiquitin. (C) Multiple Myeloma cell line MM1.S was treated with 1 μM Btz for 18 hrs. Cells were stained for UBXN1 and ubiquitin. (D) Hela Flp-in T-Rex cells were treated with 5 μg/ml of puromycin for 4 hrs. Cells were stained for UBXN1 and ubiquitin. ALIS: Aggresome-like induced structures. (E) Aggresomes formed by Btz treatment contain primarily newly synthesized ubiquitylated proteins. Co-treatment with the translational inhibitor cycloheximide results in the loss of aggresomes in Btz treated cells. Cells were stained for ubiquitin and nuclei (Hoechst dye). (F) Immunoblot of global ubiquitin conjugate levels corresponding to samples in (E). Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Staining, Ubiquitin Proteomics, Synthesized, Western Blot

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Immunoblot showing loss of UBXN1 in CRISPR-Cas9 generated knockout cells and re-expression of wildtype GFP-UBXN1 by doxycycline induction. (B) Wildtype (WT) and UBXN1 knock-out (KO) HeLa Flp-in TRex cell lines were treated with 1 μM Btz for 18 hrs. Cells were stained for ubiquitin. (C) Cell lysates from wildtype and KO celss treated with 1 μM Btz for 18 hrs were probed for ubiquitin. (D) GFP-UBXN1 expression in UBXN1 KO cells was induced by doxycycline. Cells were treated with Btz and stained for ubiquitin. Expression of GFP-UBXN1 reinstated aggresome formation in UBXN1 KO cells. (E) Quantification of data in panels (B and D). (F) WT and UBXN1 KO lines were treated with Btz for the indicated times and stained for ubiquitin. The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show the mean +/- s.e.m. *: p<=0.1, **: p<=0.05, as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Western Blot, CRISPR, Generated, Knock-Out, Expressing, Staining, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Parental or UBXN1 knock-out (KO) clone 6239 in HeLa Flp-in TRex cells were treated with 1 μM Btz for 18 hrs. Cells were stained for endogenous ubiquitin and nuclei. Bottom Panel: immunoblot showing loss of UBXN1 in CRISPR-Cas9 generated knockout clonal cell lines (Clone 9 is 6239 in panel A). (B) Quantification of aggresome formation upon transient depletion of UBXN1 with two separate siRNAs. Graphs show mean +/- standard deviation. Bottom Panel: immunoblot of transient depletion of UBXN1. The indicated number of cells was analyzed from the three independent biological replicates. *: p<=0.1, **: p<=0.05, as determined by unpaired students t-test. Scale bar: 10μm

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Knock-Out, Staining, Ubiquitin Proteomics, Western Blot, CRISPR, Generated, Standard Deviation

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Immunoblot validation of CRISPR-Cas9 gene-edited knockout (p47) or knockdown (NPL4) cell lines. (B) Aggresome formation in CRISPR-Cas9 gene-edited knockout cell lines for the indicated UBXD adaptors. (C) Quantification of data in (B). (D) HEK293T cells were transfected with the indicated FLAG or Myc-tagged constructs. NPL4 was affinity purified using FLAG magnetic beads and associated proteins were eluted with FLAG peptide. Endogenous p97 in the eluate was immunopurified and probed for UBXN1, NPL4, and ubiquitin. UBXN1 associates with the same p97 hexamer associated with NPL4. The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show the mean +/- s.e.m. *: p<=0.1, **: p<=0.05 as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Western Blot, Biomarker Discovery, CRISPR, Knock-Out, Knockdown, Transfection, Construct, Affinity Purification, Magnetic Beads, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Aggresome formation in Hela Flp-in T-Rex cells upon transient depletion of the indicated p97 adaptors. Cells were stained with anti-ubiquitin. (B) Levels of p97 adaptor depletion in Hela Flp-in TRex cell lines. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Staining, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Domain organization of UBXN1 showing N-terminal ubiquitin associated domain (UBA), coiled coil domain (cc) and C-terminal ubiquitin X domain (UBX). (B) Expression of GFP-UBXN1, GFP-UBXN1-UBA mut and GFP-UBXN1-UBX mut in the UBXN1 KO cell line by the addition of doxycycline for 72 hrs. (C) UBXN1 KO cells expressing GFP-UBXN1 wildtype, GFP-UBXN1-UBA mut or GFP-UBXN1-UBX mut were treated with Btz for 18 hours and stained for ubiquitin. Re-expression of wildtype UBXN1 but not the UBX mut rescued aggresome formation. The UBA mut has smaller aggresomes but did not reach significance (D) Total cellular aggregates (encompassing cytosolic and perinuclear aggregates) were quantified for images in (C). (E) Perinuclear aggresomes were quantified for the images in (C). (F) Wildtype, UBXN1 KO (and indicated rescue lines), NPL4, and p97 depletion cell lines were plated in triplicate into 96-well plates and treated with 1 μM bortezomib for 18 hrs. Cell viability was measured and normalized to the value of untreated controls for each cell line. The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show the mean +/- s.e.m. *: p<=0.1, **: p<=0.05 as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Ubiquitin Proteomics, Expressing, Staining

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A and B) HEK-293T cells were transfected with the indicated Myc-tagged UBXN1 constructs. Cells were treated with Btz (A) and Myc affinity purifications were probed for endogenous ubiquitin (A) or p97 (B). (C) Cumulative frequency distribution of largest perinuclear aggregate areas in UBXN1 KO cells expressing GFP-UBXN1 wildtype, UBA, or UBX mutants. The left-ward shift of the curve indicates decrease in perinuclear aggregate area in the mutants (D) Wildtype, UBXN1 KO, or NPL4 KD cells were treated with Btz for 18 hrs and released into drug-free media to induce clearance of aggregates. The loss of UBXN1 or NPL4 did not impact aggregate clearance. (E) p97 interacts with HDAC6. HEK-293T cells were transfected with the indicated cDNAs and treated with Btz for 18 hrs, Myc affinity purifications were performed and probed for the indicated tagged proteins. (F) The depletion of HDAC6 inhibits aggresome formation and clearance in HeLa Flp-in TRex cells treated with Btz for 18 hrs. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Transfection, Construct, Ubiquitin Proteomics, Expressing

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) Hela Flp-in TRex cells were treated with the indicated concentration of the ubiquitin E1 inhibitor TAK273 or Btz for 8 hours. Cell lysates were probed for endogenous ubiquitin. (B) U2OS cells were treated with doxycycline to induce the expression of HTT Q91-mCherry. Cells were treated with 1 μM TAK273 for 8 hours, fixed and stained for ubiquitin. Inclusion bodies still form but are devoid of ubiquitin staining. (C) Quantification of inclusion bodies observed by imaging in cells treated with 1 μM TAK274, 1 μM Btz, and or 10 μM CB-5083 for 8 hours. (D) Depletion of UBXN1 leads to an increase in HTT Q91-mCherry inclusion bodies. Quantification is provided in . (E) PuLSA analysis of HTT Q91-mCherry aggregates in UBXN1 and p97 depleted cells. The gates show the distribution of polyQ aggregates of various sizes based on pulse height. The greater the pulse height, the larger the inclusion body and vice versa. Note that this population represents only the HTT aggregates and not the complete mCherry signal. Total mCherry signal is represented in . The indicated number of cells was quantified in three biological replicates shown by the black dot. Graphs show the mean and standard deviation. *: p<=0.1, **: p<=0.05, ***: p<=0.001 as determined by One-way ANOVA with Bonferroni correction. Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Concentration Assay, Ubiquitin Proteomics, Expressing, Staining, Imaging, Standard Deviation

Journal: bioRxiv

Article Title: The p97-UBXN1 complex regulates aggresome formation

doi: 10.1101/2020.09.03.281766

Figure Lengend Snippet: (A) U2OS cells were treated with doxycycline to induce the expression of HTT Q91-mCherry. Cells were fixed and stained for ubiquitin and UBXN1 to demonstrate co-localization with HTT Q91-mCherry. (B) UBXN1 was transiently depleted with siRNAs in U2OS HTT Q91-mCherry and imaged for inclusion body formation. The number of HTT Q91-mCherry inclusion bodies was quantified. (C) PuLSA analysis of HTT Q91-mCherry aggregates in UBXN1 and p97 depleted cells. (D and E) Representative fluorescent images of L4 larvae stage wild type, cdc-48.1(tm544) (D), ubxn-1(tm2759) and ubxn-4(ok3343) (E) loss-of-function mutants expressing polyQ40::YFP in body wall muscle. Images were taken of worms precisely age-matched at the L4.4 vulva developmental stage. Bottom panels in each show quantification of visible fluorescent aggregates in L4 larvae animals expressing polyQ40::YFP in either wildtype, cdc-48.1, ubxn-1 and ubxn-4 mutant animals. Quantification was only performed on worms at the L4.4 vulva development stage based on vulva morphology. The indicated number of cells was analyzed from the three independent biological replicates indicated by the black dot. Graphs show mean +/- s.e.m. *: p<=0.1, **: p<=0.05, ***: p<=0.001 as determined by One-way ANOVA with Tukey (B) or Dunnett’s Test (D and E). Scale bar: 10μm.

Article Snippet: Rabbit p97 antibody for immunopurification and immunoblotting is from Bethyl (A300-589A), mouse FLAG-M2 antibody was from Sigma; mouse ubiquitin (FK2) antibody used for immunofluorescence was from EMD Millipore; ubiquitin antibody (clone P4D1) for immuno-blotting, GAPDH (sc-47724), and mouse Myc antibody was from Santa Cruz (sc-40).

Techniques: Expressing, Staining, Ubiquitin Proteomics, Mutagenesis