Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Co-immunoprecipitation (co-IP) of ChAT with heat shock proteins HSC70, HSP70, and HSP90 is altered by mutation of N-terminal proline-rich motif in ChAT. (A) Immunoblots showing co-IP of ChAT with endogenous HSC70, HSP70 and HSP90 from HEK293 cells expressing either wild-type or P17A/P19A-ChAT. Control cells were transfected with empty vector. Using HEK293 cells, co-IP of P17A/P19A-ChAT with HSP70 (B) , HSP90 (C) and HSC70 (D) , respectively, is greater than that of wild-type ChAT ( *** p ≤ 0.001, Student's t -test, mean ± SEM, n = 4). (E) Co-IP of ChAT with endogenous HSC70 and HSP90 from mouse cholinergic SN56 cells expressing either wild-type or P17A/P19A-ChAT or CMS-related mutant proteins V18M- or A513T-ChAT. Control cells were transfected with empty vector. (F) Using SN56 cells, Co-IP of P17A/P19A-ChAT ( *** p ≤ 0.001) and V18M-ChAT ( * p ≤ 0.05), but not A531T-ChAT, with HSC70 is greater than that of wild-type ChAT (mean ± SEM, n = 5). (G) While there was a trend toward increased HSP90 interaction with P17A/P19A-ChAT ( p = 0.09), no significant differences were observed for HSP90 interaction with mutant ChAT compared to wild-type ChAT in SN56 cells (mean ± SEM, n = 5). Statistical analysis for (F) and (G) was performed by one-way ANOVA with Dunnett's post-hoc test. (H) Detection of in situ interactions of wild-type ChAT with endogenous HSC70 and HSP90 by proximity ligation assay (PLA) in SN56 cells. Formalin-fixed cells were first co-labeled with goat anti-ChAT together with either mouse anti-HSC70 or mouse anti-HSP90 primary antibodies, then incubated with oligonucleotide-linked secondary antibodies. Following DNA ligation and DNA amplification using the Duolink in Situ Orange Kit (Sigma), in situ ChAT-HSP interactions were imaged by confocal microscopy. Positive in situ ChAT-HSP interactions where visualized as fluorescent red dots while nuclei were stained with DAPI (blue). Control cells were either transfected with empty vector or had primary antibodies omitted from the assay (No 1° antibodies). Images are representative of 3 independent experiments; scale bars are 10 μm.

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis, Western Blot, Expressing, Transfection, Plasmid Preparation, In Situ, Proximity Ligation Assay, Labeling, Incubation, DNA Ligation, Amplification, Confocal Microscopy, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: ChAT interacts with the HSP-associated E3 ubiquitin ligase C-terminus of HSC70-interaction protein (CHIP). (A) Immunoblots showing co-IP of ChAT with FLAG-CHIP from SN56 cells co-expressing either wild-type or mutant ChAT protein with FLAG-tagged CHIP. Control cells were transfected with either empty vector or to express either wild-type ChAT or FLAG-CHIP alone. (B) Co-IP of ChAT with FLAG-CHIP is enhanced for P17A/P19A- ( *** p ≤ 0.001), V18M- ( *** p ≤ 0.001), and A513T-ChAT ( * p ≤ 0.05) as compared to wild-type ChAT (one-way ANOVA with Dunnett's post-hoc test, mean ± SEM, n = 5). (C) Co-IP of wild-type and mutant ChAT with endogenous CHIP following anti-ChAT co-IP from ChAT-expressing SN56 cells ( n = 3). (D) Detection of in situ ChAT interactions with endogenous CHIP by proximity ligation assay (PLA) in SN56 cells expressing wild-type ChAT. Formalin-fixed cells were first co-labeled with goat anti-ChAT together with rabbit anti-CHIP primary antibodies, then incubated with oligonucleotide-linked secondary antibodies. Following DNA ligation and DNA amplification using the Duolink In Situ Orange Kit (Sigma), in situ ChAT-CHIP interactions were imaged by confocal microscopy. Positive in situ ChAT-CHIP interactions were visualized as fluorescent red dots while nuclei were stained with DAPI (blue). Control cells were either transfected with empty vector or primary antibodies omitted from the assay (No 1° antibodies). Images are representative of 4 independent experiments; scale bars are 10 μm. (E) siRNA-mediated knock-down of CHIP has no effect on the steady-state protein levels of either wild-type or mutant ChAT. ChAT-expressing SN56 cells were co-transfected with 25 nM of either anti-CHIP siRNA or scramble-control siRNA for 72 h. Control cells were mock-transfected ( n = 4).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Expressing, Mutagenesis, Transfection, Plasmid Preparation, In Situ, Proximity Ligation Assay, Labeling, Incubation, DNA Ligation, Amplification, Confocal Microscopy, Staining

Journal: Redox Biology

Article Title: Inactivation of RIP3 kinase sensitizes to 15LOX/PEBP1-mediated ferroptotic death

doi: 10.1016/j.redox.2022.102232

Figure Lengend Snippet: PEBP1 associates with RIP3. ( A ) Structures of RAF (pdbid: 3c4c ) and RIP3 (pdbid: 4m66 ) are highly conserved in sequence and tertiary structure. Catalytic regions shown enclosed in dashed region. ( B ) Lowest energy binding pose of PEBP1 ( cyan ) on RIP3 ( grey ) from docking simulations. Key interfacial interactions between PEBP1’s heterodimerization loop region (residues 127–150, including D144-H145) and RIP3’s α-helix C (αC), specifically R69 (spacefilling model) are highlighted. ( C–F ) Full Atomistic Molecular Dynamics simulations between PEBP1 with RAF, RIP3, RIP1, or RIP3 K51A . Grey dotted line indicates the threshold maximum distance positive protein-protein interaction (≤0.5 nm). Favorable interaction is predicted between ( C ) PEBP1 and RAF or ( D ) RIP3 WT , but not ( E ) RIP1 or ( F ) RIP3 K51A mutant. N = 3–4 independent simulations. ( G – H ) Colocalization between RIP3 and PEBP1 is reduced in Rip3 K51A/K51A vs. Rip3 +/+ primary bone marrow cells. ( G ) PEBP1_RIP3 colocalization normalized to cell number, Mean ± SD, *p < 0.05. ( H ): “Merge” [ left panels ]: PEBP1 ( red ), RIP3 ( green ), and hoechst ( blue ). “Colocalized objects” [ right panels ]: PEBP1_RIP3 colocalized objects ( yellow ), n = 3 independent experiments, scale: 20 μm. ( I-J ) FRET analysis showing close physical proximity (≤10 nm) of RIP3 with PEBP1 in HT22 cells. ( I ) FRET effect was confirmed through acceptor (cy5) photobleaching ( white arrows ) and reciprocal Cy3 donor fluorophore unquenching. FRET ratio (donor/acceptor relative fluorescent intensity (RFU)) is pseudo-colored (range 0–10, violet-red); ( J ) Representative RFU vs. single excitation (Ex) wavelength. Cy3 vs. Cy5 emission (Em) wavelengths are indicated. N = 3 independent experiments. ( K ) Far western blotting demonstrating specific interaction of recombinant human PEBP1 with RIP3. Representative non-denaturing immunoblots showing PEBP1 (0.5 μg protein per incubation) binds to membrane-immobilized RIP3 ( left ) but not GST or BSA control proteins. No PEBP1 signal is detected if the blot is not incubated with recombinant PEBP1 ( middle ). Protein loading was verified by Ponceau S prior to incubation with PEBP1 ( right ). N = 3 independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Activity of recombinant human RIP1 (SignalChem, R07-10G-10) and MAPK14 (ThermoFisher, PV3304) in the presence or absence of PEBP1 was also assessed.

Techniques: Sequencing, Binding Assay, Mutagenesis, Far Western Blot, Recombinant, Western Blot, Incubation, Membrane, Control

Journal: Redox Biology

Article Title: Inactivation of RIP3 kinase sensitizes to 15LOX/PEBP1-mediated ferroptotic death

doi: 10.1016/j.redox.2022.102232

Figure Lengend Snippet: PEBP1 regulates necroptotic death. ( A ) Human recombinant RIP3 kinase activity is specifically and potently inhibited by equimolar PEBP1 or the small molecule inhibitor, GSK’872. Human recombinant RIP1 kinase and another canonical serine/threonine kinase, MAPK14 (p38α), are not inhibited by equimolar PEBP1. PEBP1 alone has no effect on ATP concentration. Mean ± SD, *p < 0.05 vs. uninhibited enzyme, n = 3 independent experiments. ( B ) Representative immunoblots showing reduced PEBP1 expression is associated with greater pRIP3 (pS231/pT232) and pMLKL (pS345) levels 12 h following TNFα, z-VAD-fmk, and SM-164 (T/Z/S) necroptosis induction. L929 cells were transfected with PEBP1 or non-targeted (NT) siRNA for 48 h prior to T/Z/S treatment. Representative of 3 independent experiments. ( C ) PEBP1 siRNA knockdown L929 cells experienced a greater increase in necroptosis at higher TNFα (1, 5, 10 ng/mL) doses compared to NT siRNA cells. Cell death was specifically rescued by Nec-1s (see ). Z/S: z-VAD-fmk + SM164; cell death measured by LDH release at 16–20 h, Mean ± SD, *p < 0.05 vs. NT siRNA, n = 3 independent experiments. ( D ) PEBP1 knockdown CRISPR sensitizes L929 cells to necroptotic death. T/Z: TNFα (10 ng/mL) + z-VAD-fmk. Cell death measured by LDH release at 18 h, Mean ± SD, *p < 0.05 vs. non-targeted (NT) CRISPR, n = 3 independent experiments.

Article Snippet: Activity of recombinant human RIP1 (SignalChem, R07-10G-10) and MAPK14 (ThermoFisher, PV3304) in the presence or absence of PEBP1 was also assessed.

Techniques: Recombinant, Activity Assay, Concentration Assay, Western Blot, Expressing, Transfection, Knockdown, CRISPR

Journal: EBioMedicine

Article Title: Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice

doi: 10.1016/j.ebiom.2021.103803

Figure Lengend Snippet: Nlrp3 deficiency alleviates COVID-19 like pathology in the mouse model. (A and B) Quantification of viral loads in tissues of SARS-CoV-2 infected animals. C57BL/6J and Nlrp3 -KO mice were intranasally incubated with hACE2-expressing AAV (AAV-hACE2) at a total of 40 μl containing 4 × 10 12 viral genome copies (GC) (20 μl per nostril). All mice were monitored daily until SARS-CoV-2 infection. Viral loads in lungs (A) and nasal turbinates (B) at 4 dpi of C57BL/6J and Nlrp3 -KO mice pre-injected with AAV-hACE2 2 weeks before SARS-CoV-2 nasal infection were quantified by using quantitative real-time PCR. Mean ± SD; n = 5; * p < 0.05 and *** p < 0.001, Student's t -test. (C) Immunofluorescence of anti-SARS-CoV-2 N protein in lung tissues from C57BL/6J and Nlrp3 -KO mice at 4 dpi. Scale bar, 30 μm. (D and E) Histopathology of lung tissues from SARS-CoV-2 infected C57BL/6J and Nlrp3 -KO mice at 4 dpi (D). Histopathological observations indicated the presence of moderate interstitial pneumonia with thickened alveolar septa (black arrow) and infiltration of lymphocytes (red frames; magnification). The swollen and degenerative mononuclear cells (green frames; magnification) are scattered within the alveolar cavities. Scale bar, 50 μm. The pathology severity was scored (E). Mean ± SD; n = 5; * p < 0.05, Student's t -test. (F-H) Immunofluorescence of macrophage marker Iba1 (F), neutrophil marker Ly6G (G), and cell death marker cleaved caspase-3 (cCaspase-3) (H) in lung tissues from non-infected or SARS-CoV-2 infected C57BL/6J and Nlrp3 -KO mice at 4 dpi. Scale bar, 30 μm.

Article Snippet: However, there is so far no fully accepted drug available to combat COVID-19 in patients, albeit there were reports for the controversial efficacy of remdesivir, chloroquine and hydroxychloroquine for the treatment of COVID-19.

Techniques: Infection, Incubation, Expressing, Injection, Real-time Polymerase Chain Reaction, Immunofluorescence, Histopathology, Marker

Journal: EBioMedicine

Article Title: Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice

doi: 10.1016/j.ebiom.2021.103803

Figure Lengend Snippet: Decrease of COVID-19 related inflammatory response in Nlrp3 -KO mice. (A) Immunoblot of NLRP3 inflammasome molecules including NLRP3, Asc, Pro-caspase-1, caspase-1, and IL-1β in lung tissues of C57BL/6J and Nlrp3 -KO mice without (Mock) or with SARS-CoV-2 infection at 4 dpi. (B-D) Quantification of protein expression levels of NLRP3 (B), caspase-1 (C), and IL-1β (D) relative to β-actin in (A). Mean ± SD; n = 3; ** p < 0.01, *** p < 0.001, one-way ANOVA with Bonferroni's post hoc test. (E) Relative mRNA levels of pro-inflammatory cytokines Il1β, Il2, Il6, Il8, Il18 and Tnfα in lung tissues of SARS-CoV-2 infected C57BL/6J and Nlrp3 -KO mice at 4 dpi. β-actin was used as a control for normalization during the qRT-PCR. Mean ± SD; n = 5; ** p < 0.01, *** p < 0.001, Student's t -test. (F) Relative mRNA levels of pro-inflammatory chemokines Mcp1, Ccl3, Ccl5, Ccl8, Cxcl9 and Cxcl16 in lung tissues of SARS-CoV-2 infected C57BL/6J and Nlrp3 -KO mice at 4 dpi. Procedure and statistical analysis were same to (E).

Article Snippet: However, there is so far no fully accepted drug available to combat COVID-19 in patients, albeit there were reports for the controversial efficacy of remdesivir, chloroquine and hydroxychloroquine for the treatment of COVID-19.

Techniques: Western Blot, Infection, Expressing, Quantitative RT-PCR

Journal: EBioMedicine

Article Title: Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice

doi: 10.1016/j.ebiom.2021.103803

Figure Lengend Snippet: Targeting NLRP3 inflammasome by MCC950 treatment ameliorates immune overactivation and COVID-19 like pathology in lung tissues of hACE2 transgenic mice. (A) Diagram of animal experiments. hACE2 transgenic mice were intranasally infected with 1 × 10 5 TCID 50 of SARS-CoV-2 and followed by daily intraperitoneal administration of 10 mg/kg MCC950 (MCC950-treated group) or equal amount of 1 x PBS solution (PBS-treated group). All mice were euthanized at 4 dpi. (B) Viral loads in the five lung lobes of the MCC950-treated and PBS-treated mice at 4 dpi. Mean ± SD; n = 6; n.s. , not significant, * p < 0.05, ** p < 0.01, Student's t -test. (C) Immunofluorescence and quantification of anti-SARS-CoV-2 N protein in lung sections of hACE2 transgenic mice at 4 dpi. Scale bar, 30 μm. Mean ± SD; n = 6; *** p < 0.001, Student's t -test. (D) Histopathology of lungs from SARS-CoV-2 infected hACE2 transgenic mice at 4 dpi. Scale bar, 50 μm. (E and F) Immunofluorescence and quantification of macrophage marker Iba1 (E) and neutrophil marker Ly6G (F) in lung sections at 4 dpi. Scale bar, 30 μm. Mean ± SD; n = 6; *** p < 0.001, Student's t -test. (G) Relative mRNA levels of pro-inflammatory cytokines Il1β, Il2, Il6, Il8, Il18 and Tnfα in lung tissues at 4 dpi. β-actin was used for normalization in qRT-PCR. Mean ± SD; n = 6; * p < 0.05, ** p < 0.01, Student's t -test. (H) Relative mRNA levels of inflammatory chemokines Mcp1, Ccl3, Ccl5, Ccl8, Cxcl9 and Cxcl16 in lung tissues at 4 dpi. Mean ± SD; n = 6; * p < 0.05, ** p < 0.01, Student's t -test.

Article Snippet: However, there is so far no fully accepted drug available to combat COVID-19 in patients, albeit there were reports for the controversial efficacy of remdesivir, chloroquine and hydroxychloroquine for the treatment of COVID-19.

Techniques: Transgenic Assay, Infection, Immunofluorescence, Histopathology, Marker, Quantitative RT-PCR

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) Immunoblotting for β-catenin and IKKβ proteins in control or β-catenin–deficient C3H/10T1/2 cells infected with control or WT IKKβ virus. (B and C) Oil Red O staining (B) and qPCR analysis (C) of control and β-catenin–deficient C3H/10T1/2 cells induced by an adipogenic cocktails (n = 3). Scale bar: 100 μm. (D and E) ALP staining (D) and qPCR analysis (E) of control and β-catenin–deficient C3H/10T1/2 cells induced by an osteogenic cocktails (n = 3). Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by 2-way ANOVA (C and E). **P < 0.01, ***P < 0.001.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Western Blot, Infection, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) Control or IKKβ-deficient C3H/10T1/2 cells were treated with vehicle or 100 nM PS-341. β-Catenin proteins were immunoprecipitated with anti–β-catenin antibodies and then probed with anti-ubiquitin antibodies. The whole cell lysates were probed with anti–β-catenin antibodies as an internal control. (B and C) Immunoblotting for nuclear β-catenin proteins (B) and β-catenin reporter activity (C) in control or IKKβ-deficient C3H/10T1/2 cells. (D and E) Immunoblotting for ubiquitinated β-catenin proteins (D) and nuclear β-catenin proteins (E) in control or BMS-345541–treated C3H/10T1/2 cells. (F and G) Immunoblotting for ubiquitinated β-catenin proteins (F) and nuclear β-catenin proteins (G) in C3H/10T1/2 cells infected with control, IKKβ WT, or IKK KM virus. (H and I) Immunoblotting for ubiquitinated β-catenin levels (H) and nuclear β-catenin proteins (I) in control or FFA-treated C3H/10T1/2 cells. (J) Immunoblotting for ubiquitinated β-catenin proteins in control or IKKβ-deficient C3H/10T1/2 cells treated with vehicle or FFAs. Error bars represent ± SEM. Significance was determined by Student’s t test (C). ***P < 0.001.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Immunoprecipitation, Western Blot, Activity Assay, Infection

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) The sequence of a conserved 6–amino acid motif found in β-catenin and IκB family members. (B) Immunoblotting for FLAG-tagged IKKβ and HA-tagged β-catenin proteins after immunoprecipitation using control IgG or antibodies against FLAG or HA proteins in C3H10T1/2 cells and HEK 293T cells. (C) In vitro phosphorylation of purified GST–β-catenin proteins by IKKβ in the presence of γ-[32P]ATP. (D–F) GST–β-catenin proteins phosphorylated by IKKβ in vitro were tryptic digested and analyzed by mass spectrometry. Figures show the recovered phosphorylated 30-residue fragment of β-catenin (residues 20–49). Tandem mass spectrum of the recovered peptide phosphorylated at ser45 residue (D). The peptides phosphorylated at ser33 and ser37 residues were coeluted. Color codes are used to mark fragment ions that allows distinguishing phosphorylation of ser33 (blue) and ser37 (red) (E). Tandem MS ion traces (10 ppm extraction) for the nonphosphorylated peptide, ser45- and ser33/ser37-phosphorylated peptides, and AUCs shown in italics (F).

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Sequencing, Western Blot, Immunoprecipitation, In Vitro, Purification, Mass Spectrometry

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) GST–β-catenin and indicated mutant proteins were phosphorylated by IKKβ in vitro and analyzed by immunoblotting using anti–phospho-ser33, -ser37 or -ser45 β-catenin antibodies. (B) Immunoblotting for phosphorylated β-catenin proteins in C3H/10T1/2 cells infected with control, WT IKKβ, and IKKβ KM virus. (C) Immunoblotting for phosphorylated β-catenin proteins in C3H/10T1/2 cells treated with vehicle or FFAs. (D) Immunoblotting for phosphorylated β-catenin proteins in control or IKKβ-deficient C3H/10T1/2 cells treated with vehicle or LPS. (E) GST–β-catenin and indicated mutant proteins were phosphorylated by IKKβ in vitro. The reaction substrates were subjected for cell-free ubiquitination assays and analyzed by immunoblotting.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Mutagenesis, In Vitro, Western Blot, Infection

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) Immunoblotting for IKKβ proteins in BMMSCs of IKKβF/F and Prrx1Cre+IKKβF/F mice. (B–G) BMMSCs were isolated from IKKF/F and Prrx1Cre+IKKβF/F mice. Oil Red O staining (B) and qPCR analysis (C) of BMMSCs induced by an adipogenic cocktail (n = 3). ALP staining (D) and qPCR analysis (E) of BMMSCs induced by an osteogenic cocktail (n = 3). Immunoblotting for ubiquitinated β-catenin (F) and nuclear β-catenin proteins (G) of isolated BMMSCs. Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by Student’s t test (C and E). *P < 0.05; **P < 0.01.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Western Blot, Isolation, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A–C) Adipose stem cells were isolated from s.c. adipose tissue of heathy human subjects. Immunoblotting for IKKβ and phosphorylated β-catenin proteins (A), ubiquitinated β-catenin proteins (B), and nuclear β-catenin proteins (C) in human adipose stem cells infected with control, WT IKKβ, and IKKβ KM virus. (D and E) Oil Red O staining (D) and qPCR analysis (E) of human adipose stem cells induced by an adipogenic cocktail (n = 3). Scale bar: 100 μm. (F and G) Alizarin Red S staining (F) and qPCR analysis (G) of human adipose stem cells induced by an osteogenic cocktail (n = 3). Scale bar: 100 μm. (H and I) Immunoblotting for phosphorylated β-catenin proteins (H) and ubiquitinated β-catenin proteins (I) in human adipose stem cells treated with vehicle control or FFAs. (J and K) Oil Red O staining (J) and qPCR analysis (K) of control or FFA-treated human adipose stem cells induced by an adipogenic cocktail (n = 3). Scale bar: 100 μm. (L and M) Alizarin Red S staining (L) and qPCR analysis (M) of control or FFA-treated human adipose stem cells induced by an osteogenic cocktail (n = 3). Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by Student’s t test (K and M) or 1-way ANOVA (E and G). *P < 0.05; **P < 0.01, ***P < 0.001.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Isolation, Western Blot, Infection, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) s.c. adipose tissues were isolated from a cohort of nondiabetic human subjects. Correlation between adipose IKKβ mRNA levels and BMI (n = 27). The correlation was analyzed by Pearson correlation coefficient. (B) IKKβ mRNA levels in adipose tissue of nonobese and obese human subjects (n = 12–15). (C and D) Immunoblotting (C) and densitometric quantification (D) of proteins in adipose tissue of nonobese and obese human subjects (n = 7). Error bars represent ± SEM. Significance was determined by Student’s t test (B and D). *P < 0.05; **P < 0.01, ***P < 0.001. (E) Schematic representation of the mechanism through which IKKβ reciprocally regulates adipogenesis and osteogenesis in MSCs. Activation of IKKβ by stimuli such as FFAs or inflammation cytokines phosphorylates serine-33, -37, and -45 of β-catenin to prime it for β-TrCP–mediated ubiquitination and degradation, leading to increased adipogenic differentiation and reduced osteogenic differentiation of MSCs.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Isolation, Western Blot, Activation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: VEGF-induced vascular permeability is reduced upon CRISPR/Cas9-mediated knockout of Stat3 in zebrafish. (A) VEGF-inducible zebrafish were crossed to Stat3 +/− (heterozygous) zebrafish to generate VEGF-inducible; Stat3 +/− double transgenic fish, which were intercrossed to generate VEGF-inducible; Stat3 −/− (KO) zebrafish. (B) CRISPR/Cas9-generated Stat3 KO zebrafish (bottom) display no overt vascular defects relative to wild-type (WT) zebrafish (top). The vascular system of 3 days post-fertilization (dpf) zebrafish was visualized by microangiography with 2000 kDa FITC-dextran. Representative images of at least three zebrafish per group are shown. Scale bars: 100 μm. (C) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf Stat3 +/+ (negative controls without VEGF induction; left) , VEGF-induced, Stat3 +/+ (middle) and VEGF-induced, Stat3 −/− (right) zebrafish. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at room temperature (RT). Scale bars: 50 μm. (D) Quantitative analysis of vascular permeability upon VEGF stimulation in WT Stat3 +/+ ( n =30) and KO Stat3 −/− ( n =9) zebrafish. Mean±s.e.m., unpaired, two-tailed Student's t -test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, CRISPR, Knock-Out, Transgenic Assay, Generated, Marker, Microscopy, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Endothelial cell-specific STAT3 knockout mice exhibit decreased VEGF-induced permeability. (A) Images of footpads from WT and endothelial cell-specific STAT3 knockout (STAT3 ECKO ) mice following tail vein injection with 1% Evans Blue dye and human recombinant VEGF-165 protein (2.5 µg/ml; left footpads) or PBS vehicle (right footpads) being injected into the root of the footpad. (B,C) Quantitation of Evans Blue leakage in Tie2-Cre negative; STAT3 flox/flox (WT) and Tie2-Cre positive; STAT3 flox/flox (STAT3 ECKO ) mice. n =7 mice in WT group and n =6 mice in STAT3 ECKO group. Each mouse was injected with PBS on the right anterior and posterior footpads and VEGF on the left anterior and posterior footpads. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. A.U., arbitrary units.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Knock-Out, Permeability, Injection, Recombinant, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Pharmacological inhibition of STAT3 stabilizes endothelial barrier integrity following VEGF stimulation in human endothelial cells. (A) Serum-starved human umbilical vein endothelial cells (HUVECs) were pretreated with DMSO (vehicle control) for 1 h, 30 µM AQ for 4 h, or 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 2 or 5 min. Lysates were immunoblotted. Densitometry was performed, and the values below the rows of bands represent the ratio of phosphorylated protein to respective total protein. (B) Human VEGF-165 recombinant protein (VEGF; 25 ng/ml) stimulation of HUVECs promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows; left column; DMSO vehicle control pretreatment for 1 h prior to VEGF stimulation). ZO-1 organization is maintained upon pretreatment with 30 μM AQ for 4 h (magenta arrows; middle column) or 10 μM PYR for 1 h (magenta arrows; right column) prior to VEGF stimulation. Nuclei were stained with DAPI (blue). (C) Serum-starved human pulmonary artery endothelial cells (HPAECs) were pretreated with 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 5 or 30 min. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained when HPAECs were pretreated with PYR (magenta arrows). Nuclei were stained with DAPI (blue). (D) VEGF (25 ng/ml) stimulation of human lung microvascular endothelial cells (HMVEC-Ls) promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained upon pretreatment with 20 μM PYR for 6 h prior to VEGF stimulation (magenta arrows). Nuclei were stained with DAPI (blue). At least two biological replicates were performed for each experiment depicted in A-D. Scale bars: 20 µm.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Inhibition, Recombinant, Staining

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Suppression of STAT3 activity by pyrimethamine (PYR) inhibits VEGF-induced vascular permeability in zebrafish and mice. (A) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf zebrafish without induced VEGF pretreated with DMSO ( n =6) or 25 μM PYR ( n =5) or 3 dpf zebrafish with induced VEGF pretreated with DMSO ( n =4) or 25 μM PYR ( n =9) for 3 days. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at RT. Scale bars: 50 μm. (B) The quantitative analysis of vascular permeability without VEGF stimulation or upon VEGF stimulation in zebrafish pretreated with DMSO or PYR. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. (C) Representative images of footpads from mice treated with vehicle or PYR following tail vein injection with 1% Evans Blue and footpad injection of VEGF (2.5 μg/ml) or PBS vehicle. (D) Quantitation of Evans Blue dye leakage in C57BL/6 WT mice treated with vehicle or PYR. n =9 mice in the vehicle group and n =7 mice in the PYR group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Activity Assay, Permeability, Marker, Microscopy, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: JAK2 phosphorylates STAT3 to transduce VEGF/VEGFR-2 signaling and promote vascular permeability. (A) To perform a STAT3 GST pull-down of VEGFR-2 and JAK2, lysates of HUVECs stimulated with serum for 30 min were used as prey. GST fusion protein STAT3 expressed in 293F cells was used as bait. GST alone served as a negative control. Binding experiments were analyzed by SDS-PAGE and visualized by immunoblotting. GST-STAT3 and GST were each detected using an anti-GST antibody. Three biological replicates were performed and depicted findings are representative. (B) JAK2 phosphorylates STAT3 in vitro . In vitro kinase assays were performed using purified human STAT3 protein and kinase active JAK2 protein. The results shown here are representative of two independent experiments. (C) Representative images of footpads from C57BL/6 WT mice treated with vehicle or JAK2 inhibitor AG490. Following tail vein injection with 1% Evans Blue dye, human VEGF-165 protein (2.5 μg/ml) or PBS vehicle was injected into the root of the footpad. After 30 min, the mice were euthanized and the footpads were excised. (D) Quantitation of Evans Blue dye leakage in C57BL/6 mice treated with vehicle or AG490. n =4 mice per group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Two biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Transduction, Permeability, Negative Control, Binding Assay, SDS Page, Western Blot, In Vitro, Purification, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: STAT3 transcriptionally activates ICAM-1, a cell adhesion molecule that promotes vascular permeability. (A) Top: the pGL3-ICAM1-WT plasmid containing the human ICAM-1 promoter with a STAT3 binding site located at −115 to −107 bp. Bottom: the pGL3-ICAM1-SDM plasmid with a site-directed mutation (SDM) in the STAT3 binding site as indicated. (B) Dual luciferase assays were performed in HUVECs that were transfected with pGL3-ICAM1-WT or pGL3-ICAM1-SDM and empty vector or constitutively active STAT3. Firefly and Renilla luminescence was measured and plotted as a ratio. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. n =9 technical replicates. Depicted findings are representative of three independent experiments. (C) HUVECs that had been stably transduced with lentivirus encoding STAT3-specific shRNA or control shRNA were stimulated with human VEGF-165 protein (25 ng/ml) and the lysates were immunoblotted for ICAM1, p-STAT3 (Y705) and total STAT3. Depicted data are representative of three biological replicates. (D) RNA was harvested from VEGF; Stat3 +/+ or VEGF; Stat3 −/− 3 dpf embryos for quantitative PCR. stat3 transcripts are reduced in VEGF; Stat3 −/− ( n =5) compared to VEGF; Stat3 +/+ zebrafish ( n =7). Mean±s.e.m., unpaired, two-tailed Student's t -test. (E) The expression of icam-1 was assessed by real-time quantitative PCR using RNA derived from each zebrafish embryo in the absence of VEGF induction (Stat3 +/+ , n =3; Stat3 −/− , n =2) or 8 h following VEGF induction (Stat3 +/+ , n =4; Stat3 −/− , n =3) in the heat-inducible VEGF; Stat3 mutant zebrafish. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, Plasmid Preparation, Binding Assay, Mutagenesis, Luciferase, Transfection, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, Two Tailed Test, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

doi: 10.1101/2022.01.12.476115

Figure Lengend Snippet: (A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of Cdk1 were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).

Article Snippet: The in vitro kinase assay was performed using the Cyclin A2/Cdk1 Kinase Enzyme System (Promega, Madison, WI, USA), recombinant Cyclin B1/Cdk1 (SignalChem Biotech Inc., Richmond, BC, Canada), and ADP-Glo kinase assay (Promega), according to the manufacturers’ instructions.

Techniques: Blocking Assay, Sample Prep, Cytometry, Microscopy, Western Blot

Journal: bioRxiv

Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

doi: 10.1101/2022.01.12.476115

Figure Lengend Snippet: (A) Cells expressing Flag-p27* under the control of tetracycline were synchronized by a double thymidine block. The cells were treated with 1 μg/ml DOX at the same time as release from the double thymidine block. The cells were harvested at 8 h after release from the double thymidine block (G2/M phase) and then immunoprecipitation was performed using anti-DDDDK (Flag) antibodies. Flag-tagged and coimmunoprecipitated proteins were analysed by immunoblotting. (B, C) Purified Flag-tagged proteins were applied to an in vitro kinase assay reaction and kinase activities of Cyclin A2/Cdk1 (B) and Cyclin B1/Cdk1 (C) were measured. Statistical significance was assessed by the one-way ANOVA and Dunnett’s test (*: P < 0.05; **: P < 0.01; ***: P < 0.01). Error bars indicate s.d. (n = 3).

Article Snippet: The in vitro kinase assay was performed using the Cyclin A2/Cdk1 Kinase Enzyme System (Promega, Madison, WI, USA), recombinant Cyclin B1/Cdk1 (SignalChem Biotech Inc., Richmond, BC, Canada), and ADP-Glo kinase assay (Promega), according to the manufacturers’ instructions.

Techniques: Expressing, Blocking Assay, Immunoprecipitation, Western Blot, Purification, In Vitro, Kinase Assay

Journal: Oncotarget

Article Title: Exosomal survivin facilitates vesicle internalization

doi: 10.18632/oncotarget.26182

Figure Lengend Snippet: (A) Extracellular vesicle size and concentration as measured using the Nanosight NS300. Mode size was within the exosome size range (30-150nm) at 142.5nm. Mean size was 162.7nm and concentration was 4.51x10 10 particles/ml (±1.39x10 10 ). (B) Western blot analysis of isolated vesicles and the corresponding vesicle-free supernatant. LAMP1, TSG101, and HSP70 were used as positive markers for exosome identification. The double band on the Survivin blot corresponds to the endogenous size as well as the HA/FLAG tagged Survivin expressed by the cells from which the exosomes originated. Ponceau S stain was used to verify equal amounts of protein were loaded. Both Nanosight readings and western blot data are representative of several repeated experiments.

Article Snippet: For competition studies, full-length recombinant human survivin protein was obtained from Abcam (Cambridge MA).

Techniques: Concentration Assay, Western Blot, Isolation, Staining

Journal: Oncotarget

Article Title: Exosomal survivin facilitates vesicle internalization

doi: 10.18632/oncotarget.26182

Figure Lengend Snippet: HeLa cells co-incubated with PKH67 stained exosomes and soluble Survivin show a significant reduction in exosome uptake in a dose dependent manner. Data is representative of 3 independent experiments. Significance was determined by one-way ANOVA with ad hoc Tukey’s multiple comparison’s tests * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001.

Article Snippet: For competition studies, full-length recombinant human survivin protein was obtained from Abcam (Cambridge MA).

Techniques: Incubation, Staining

Journal: Oncotarget

Article Title: Exosomal survivin facilitates vesicle internalization

doi: 10.18632/oncotarget.26182

Figure Lengend Snippet: (A) HeLa cells pre-treated with antibodies to Survivin showed no change in uptake of PKH67 stained exosomes as assessed with flow cytometry. (B) PKH67 stained exosomes pre-incubated with Survivin antibody show a decreased amount of internalization by HeLa cells. Data is representative of 3 independent experiments. Significance was determined by one-way ANOVA with ad hoc Tukey’s multiple comparison’s tests * p<0.05, ** p<0.01, *** p<0.005 , **** p<0.001.

Article Snippet: For competition studies, full-length recombinant human survivin protein was obtained from Abcam (Cambridge MA).

Techniques: Staining, Flow Cytometry, Incubation