Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A) Quantitative real-time PCR of miR-145 expression in glioma samples (n = 19) compared with normal samples (n = 10). (B) quantitative real-time PCR of miR-145 expression in rat glioma tissues (n = 6) and U87, U251 glioma cells compared to normal tissues (n = 6). (C) Quantitative real-time PCR of BNIP3 mRNA expression in glioma samples (n = 19) compared with normal samples (n = 10). (D) quantitative real-time PCR of BNIP3 mRNA expression in rat glioma tissues and U87, U251 glioma cells compared to normal tissues. (E) Pathology observation of mice brain tissues sections stained with IHC (×100, ×200, ×400). (F) Immunofluorescence with BNIP3 (green) in rat normal tissues and glioma tissues (×200).*p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Immunofluorescence, Control

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A) Western analysis of BNIP3 and control β-actin in glioma samples compared with normal samples. (B) Western analysis of BNIP3 and control β-actin in rat glioma tissues compared to normal tissues. *p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Western Blot, Control

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A) Bioinformatics analysis shows the seed sequence of miR-145 binding to the 3′-UTR of BNIP3 mRNA. (B) , (C) Quantitative real-time PCR analysis of mRNA expression of BNIP3 in U87 cells treated with miR-145 mimics and inhibitor for 48 h. (D) Western analysis of protein expression of BNIP3 in U87 and U251 cells treated with miR-145 mimics and inhibitor for 48 h. (E) Immunofluorescence with BNIP3 (green) in U87 and U251 cells after miR-145 mimics or mimics NC treatment.*p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Sequencing, Binding Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Control

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A) Western analysis of BNIP3, which is localized in the nucleus or the cytoplasm in U87 and U251 cells treated with miR-145 mimics and inhibitor for 48 h. (B) Wild-type 3′-UTR of BNIP3 gene was cloned into the firefly and Renilla reporter plasmid. The BNIP3-3′UTR constructs or blank plasmid were transfected into U87 and U251 cells with control or miR-145 mimics, followed by dual luciferase assays. *p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Western Blot, Clone Assay, Plasmid Preparation, Construct, Transfection, Control, Luciferase

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A, B) Transfection effect of BNIP3-siRNA or BNIP3-vector was confirmed by quantitative real-time PCR. (C) U87 cells and U251 cells were stained with Hoechst 33342 dye after BNIP3-siRNA or control treatment. (D) U87 and U251 cells were stained with Tunel after BNIP3-siRNA or control treatment. (E) U87 cell apoptosis after BNIP3-siRNA or control treatment was determined by FACS. *p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Staining, Control, TUNEL Assay

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A, B) Protein expression of Notch1, p21 and Hes1 was determined by western blot analysis in U87 and U251 cells transfected with miR-145 mimics and mimics-NC, or miR-145 inhibitor and inhibitor-NC. (C, D) Western analysis of Notch1-related proteins in U87 and U251 cells transfected with Bnip3 siRNA and siRNA control, or BNIP3 expression vector and blank vector. *p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Expressing, Western Blot, Transfection, Control, Plasmid Preparation

Journal: Oncotarget

Article Title: MicroRNA-145 induces apoptosis of glioma cells by targeting BNIP3 and Notch signaling

doi: 10.18632/oncotarget.18604

Figure Lengend Snippet: (A) Protein expression of Notch1, p21, Hes1 was determined by western blot analysis in U87 and U251 cells co-transfected with miR-145 inhibitor and BNIP3-siRNA, or with miR-145 inhibitor. *p < 0.05, **p < 0.01 versus control group.

Article Snippet: Rabbit monoclonal antibodies against Bax, Bcl-2, Caspase-3, and p21 (Abcam, USA) were used at 1:600 dilution, rabbit monoclonal antibody against Notch 1 Hes1 (Cell Signaling Technology, USA) was used at 1:600, anti-BNIP3 antibodies (Boster, China), anti-β-actin and anti-PCNA (ZSGB-BIO, China) were diluted 1:300

Techniques: Expressing, Western Blot, Transfection, Control

Journal: eLife

Article Title: NHE6 depletion corrects ApoE4-mediated synaptic impairments and reduces amyloid plaque load

doi: 10.7554/eLife.72034

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , γ-Secretase inhibitor L-685458 , Tocris Bioscience , 2627 , .

Techniques: Staining, Plasmid Preparation, Transfection, Construct, Expressing, shRNA, Recombinant, Sequencing, Software, Imaging

Journal: bioRxiv

Article Title: Differential regulation of SHH signaling and the developmental control of species-specific jaw size through neural crest-mediated Gas1 expression

doi: 10.1101/2021.12.17.473230

Figure Lengend Snippet: (A) Relative levels of mRNA expression (on the y-axis in log 2 scale) for Gas1 , Ptch1 , and Gli1 in chick fibroblasts (i.e., DF-1) 24 hours (h) after treatment with five concentrations of recombinant (r) SHH protein. (B) Relative Gas1 mRNA levels in chick fibroblasts 24 hours after doxycycline (dox)-induction of a stably integrated chick Gas1 overexpression vector. Number of chick Gas1 -positive cells 72 hours after induction with dox. (C) Relative Gas1 mRNA levels in chick fibroblasts 24 hours after dox-induction of a stably integrated quail Gas1 overexpression vector. Number of quail Gas1 -positive cells 72 hours after induction with dox. (D) Relative levels of mRNA expression for Gas1 , Ptch1 , and Gli1 in chick fibroblasts 48 hours after treatment with rSHH protein. Relative levels of mRNA expression for Gas1 , Ptch1 , and Gli1 in chick fibroblasts 48 hours after dox-induction of a stably integrated Gas1 overexpression vector and treatment with rSHH protein (control 0 ng/mL in white and 100 ng/mL dark grey). Expression levels were assayed by qRT-PCR and normalized to r18s . Significance is shown for comparisons between control and treatment groups as denoted by brackets and p-values are as indicated. P-values ≥ 0.05 are considered not significant (ns). n ≥ 2 for each group and data point, and error bars represent SEM.

Article Snippet: Gas1 was cloned into the pPIDNB plasmid, which integrates into the genome and is doxycycline (dox)-inducible , using AflII (R0520S, New England BioLabs, Ipswich, MA, USA), PstI (R3140S, New England BioLabs, Ipswich, MA, USA), and NEBuilder HiFi DNA Assembly Master Mix (E2621L, New England BioLabs, Ipswich, MA, USA).

Techniques: Expressing, Recombinant, Stable Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Differential regulation of SHH signaling and the developmental control of species-specific jaw size through neural crest-mediated Gas1 expression

doi: 10.1101/2021.12.17.473230

Figure Lengend Snippet: (A) Relative levels of mRNA expression (on the y-axis in log 2 scale) for Boc , Cdon , Smo , Ptch1 , Gli1 , Gli2 , and Gli3 in chick fibroblasts (i.e., DF-1) 24 hours (h) after treatment with five serial dilutions of doxycycline (dox) and induction of a stably integrated chick (yellow) or quail (green) Gas1 overexpression vector. (B) Relative levels of mRNA expression for Boc , Cdon , Smo , Gli2 , and Gli3 in chick fibroblasts 48 hours after dox-induction of a stably integrated Gas1 overexpression vector (serial dilution) and treatment with recombinant (r) SHH protein (control 0 ng/mL in white and 100 ng/mL dark grey). Expression levels were assayed by qRT-PCR and normalized to r18s . Significance is shown for comparisons between control (0 ng/mL of rSHH and/or dox) and treatment groups as denoted by brackets and p-values are as indicated. P-values ≥ 0.05 are considered not significant. n ≥ 2 for each group and data point, and error bars represent SEM.

Article Snippet: Gas1 was cloned into the pPIDNB plasmid, which integrates into the genome and is doxycycline (dox)-inducible , using AflII (R0520S, New England BioLabs, Ipswich, MA, USA), PstI (R3140S, New England BioLabs, Ipswich, MA, USA), and NEBuilder HiFi DNA Assembly Master Mix (E2621L, New England BioLabs, Ipswich, MA, USA).

Techniques: Expressing, Stable Transfection, Over Expression, Plasmid Preparation, Serial Dilution, Recombinant, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Differential regulation of SHH signaling and the developmental control of species-specific jaw size through neural crest-mediated Gas1 expression

doi: 10.1101/2021.12.17.473230

Figure Lengend Snippet: (A) Mesenchymal cell number in duck mandibular primordia following bilateral in ovo electroporation of a stably integrating and doxycycline (dox)-inducible Gas1 overexpression (OE) vector into NCM at HH8.5. Gas1 overexpression was induced with 50 ng/mL of dox at HH15 and mandibular primordia were collected at HH18, HH21, and HH24. (B) Dissected duck mandibular primordia in whole mount at HH18 following bilateral in ovo electroporation of a Gas1 overexpression vector into NCM at HH8.5. (C) Gas1 -positive NCM can be visualized by mScarlet fluorescence (RFP). Gas1 overexpression (D) in whole mount can be visualized (E) by RFP at HH21, and (F) in whole mount and (G) by RFP at HH24. (H) Mesenchymal cell number in quail mandibular primordia following bilateral in ovo electroporation of a Gas1 overexpression vector into NCM at HH8.5. Gas1 overexpression was induced with dox at HH15 and mandibular primordia were collected at HH18, HH21, and HH24. (I) Quail embryo in whole mount at HH27 following unilateral in ovo electroporation of a Gas1 overexpression vector into NCM at HH8.5 and induction with dox at HH15. The treated size appears smaller (white arrow) than the contralateral control side. (J) Unilateral distribution of Gas1 -positive NCM can be visualized by RFP on one side of the embryo. Higher magnification view of quail mandibular primordia (dashed lines) in (K) whole-mount and (L) with RFP showing a size reduction on the treated (arrow) versus control side. (M) Quail embryo in whole mount at HH27 following unilateral in ovo injection of a control anti-GFP morpholino (MO) at HH18. At higher magnification in (N) whole-mount and (O) in schematic view, the mandibular primordia (dashed lines) appear the same size on the treated ( striped) and control (green) sides. (P) Quail embryo in whole mount at HH27 following unilateral in ovo injection of an anti- Gas1 morpholino (MO) at HH18. At higher magnification in (Q) whole-mount and (R) in schematic view, the mandibular primordia (dashed lines) appear smaller on the treated ( striped) versus control (green) sides. Significance is shown for comparisons between control and treatment groups as denoted by brackets and p-values are as indicated. P-values ≥ 0.05 are considered not significant (ns). n ≥ 2 for each group and data point. Error bars represent SEM.

Article Snippet: Gas1 was cloned into the pPIDNB plasmid, which integrates into the genome and is doxycycline (dox)-inducible , using AflII (R0520S, New England BioLabs, Ipswich, MA, USA), PstI (R3140S, New England BioLabs, Ipswich, MA, USA), and NEBuilder HiFi DNA Assembly Master Mix (E2621L, New England BioLabs, Ipswich, MA, USA).

Techniques: In Ovo, Electroporation, Stable Transfection, Over Expression, Plasmid Preparation, Fluorescence, Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: EphA3-targeted chimeric antigen receptor T cells are effective in glioma and generate curative memory T cell responses

doi: 10.1136/jitc-2024-009486

Figure Lengend Snippet: EphA3 CAR T cells are functional against adult and pediatric glioma. ( A ) Schematic of second generation CAR constructs; ( B ) flow cytometry of cell surface expression of EphA3 (red) and HER2 (blue) CAR, as detected using anti-G4S protein tag antibody, and the percentage of CAR+ of T-cell population shown for three independent human donors; activated CD4 + EphA3-CAR T cells after 16 hour co-culture with U251CL tumor cells, or CD3/CD28 beads as positive control showing ( C ) %CD137 expression as detected using flow cytometry and ( D ) IFNγ and TNF secretion (pg/mL) into the culture. Transduction efficiencies of CD4+ GFP+ (empty vector) or CD4+ G4S+ CAR T cells shown in ( C ) and ( D ) for each group are as follows: EV 62%, HER2 94%, EphA3 79%. Shown is mean of triplicates (n=3± STDEV, *p<0.05 using two-way analysis of variance); ( E ) IncuCyte killing assay of 2.5:1 ratio of CD8 + CAR T cells co-cultured with adult U251CL glioma cells or pediatric SU-DIPG36GL tumor cells over 24 hours. Transduction efficiencies for CD8+ GFP+ (empty vector) or CD8+ G4S+ CAR T cells for each group are as follows: EV 41%, HER2 43%, EphA3 25%. CAR, chimeric antigen receptor; EphA3, Ephrin type-A receptor 3; HER2, Human epidermal receptor protein 2; IFNγ, interferon gamma; scFv, single-chain variable fragment; TNF, Tumour necrosis factor.

Article Snippet: Results were analyzed using IncuCyte software (Sartorius, V.2021A).

Techniques: Functional Assay, Construct, Flow Cytometry, Expressing, Co-Culture Assay, Positive Control, Transduction, Plasmid Preparation, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Pathogenic G2019S LRRK2, but not a kinase-inactive G2019S-K1906M variant, causes a deficit in EGF binding and degradation. A , HeLa cells were transfected with either pCMV or cotransfected with GFP and either myc-tagged G2019S LRRK2, FLAG-tagged G2019S, or G2019S-K1906M LRRK2 as indicated; incubated with Alexa555-EGF for 30 min at 4 °C; washed to remove unbound fluorescent EGF; and fixed and processed as described under “Materials and methods.” Scale bar , 10 μm. B , quantification of surface-bound fluorescent EGF ( t = 0 min) of cells transfected with the various constructs as indicated and normalized to EGF surface binding of pCMV-transfected cells ( ctrl ). n = 3 independent experiments. *, p < 0.05. C , HeLa cells transfected with the indicated constructs were allowed to bind Alexa555-EGF at 4 °C, washed to remove unbound fluorescent EGF, and then shifted to 37 °C for 10 min to allow for the internalization and degradation of fluorescent EGF. Scale bar , 10 μm. D , quantification of Alexa555-EGF was performed after 10 ( left ) and 30 min ( right ) upon internalization and normalized to the amount of Alexa555-EGF binding for each condition at t = 0 min, thus reflecting the percentage of internalized bound fluorescent EGF. n = 3 independent experiments. *, p < 0.05; ***, p < 0.005. E , quantification of the total number of fluorescent EGF-positive puncta per 250 μm 2 upon expression of distinct constructs as indicated after 10 ( left ) and 30 min ( right ) of internalization. n = 3 independent experiments. *, p < 0.05; ***, p < 0.005. F , because the immunofluorescence signal intensity directly correlates to the size of the individual structures, signal intensity per punctum was quantified at 10 ( left ) and 30 min ( right ) upon internalization, which revealed no change among the different conditions, further indicating a deficit in EGF degradation rather than an increase in the amount of internalized fluorescent EGF per cell. G , HEK293T cells were transfected with the indicated constructs followed by analysis of endogenous EGFR expression levels. H , HEK293T cells were transfected with either pathogenic G2019S LRRK2 or with kinase-inactive G2019S-K1906M variant and serum-starved for 1 h in the presence of cycloheximide to block novel protein synthesis, and EGFR internalization was stimulated with nonlabeled EGF for the indicated time points. Cell extracts were analyzed by Western blotting for EGFR levels, and tubulin was used as a loading control. I , quantification of EGFR degradation in HEK293T cells transfected with either G2019S LRRK2 or G2019S-K1906M, at distinct time points as indicated, and with values normalized to tubulin as a loading control. n = 4 independent experiments. *, p < 0.05; **, p < 0.01; ****, p < 0.001. All error bars represent S.E.M.

Article Snippet: Phosphorylation reactions were performed either in the presence or absence of 1 μ m LRRK2 kinase inhibitor PF 06447475 (Tocris Bioscience).

Techniques: Variant Assay, Binding Assay, Transfection, Incubation, Construct, Expressing, Immunofluorescence, Blocking Assay, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Active RAB8A and Rabin8 rescue the LRRK2-mediated deficit in EGF binding and degradation. A , HeLa cells were transfected with either empty pCMV vector ( ctrl ) or the indicated RAB8A constructs followed by quantification of the amount of surface-bound fluorescent EGF. n = 4 independent experiments. *, p < 0.05. B , cells were transfected as indicated followed by quantification of internalized Alexa555-EGF in transfected cells after 10 ( left ) and 30 min ( right ) of internalization. Values are normalized to the amount of Alexa555-EGF binding at t = 0. n = 4 independent experiments. *, p < 0.05. C , cells were cotransfected with G2019S LRRK2 and the indicated RAB8A constructs, and surface-bound fluorescent EGF was quantified. n = 8 independent experiments. *, p < 0.05. D , cells were cotransfected with G2019S LRRK2 and the indicated RAB8A constructs followed by quantification of internalized Alexa555-EGF after 10 ( left ) and 30 min ( right ) of internalization. n = 8 independent experiments. ****, p < 0.001. E , cells were transfected with either empty pCMV vector ( ctrl ) or with Rabin8, and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. F , cells were transfected as indicated followed by quantification of internalized fluorescent EGF at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. G , cells were transfected with either empty pCMV vector ( ctrl ) or cotransfected with G2019S pathogenic LRRK2 and either pCMV vector or Rabin8 as indicated, and surface-bound fluorescent EGF was quantified. n = 3 experiments. *, p < 0.05. H , cells were transfected as indicated followed by quantification of internalized fluorescent EGF as described above. n = 3 independent experiments. ***, p < 0.005. All error bars represent S.E.M.

Article Snippet: Phosphorylation reactions were performed either in the presence or absence of 1 μ m LRRK2 kinase inhibitor PF 06447475 (Tocris Bioscience).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Construct

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: RAB11 rescues the LRRK2-mediated delay in EGFR trafficking. A , HeLa cells were cotransfected with G2019S LRRK2 and the indicated RAB11 constructs, and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. *, p < 0.05. B , cells were transfected with either empty pCMV vector ( ctrl ) or cotransfected with G2019S LRRK2 and the indicated RAB11 constructs followed by quantification of internalized fluorescent EGF at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005. C , cells were transfected with either empty pCMV vector ( ctrl ) or the indicated RAB11 constructs, and the amount of surface-bound fluorescent EGF was quantified. n = 3 independent experiments. D , cells were transfected as indicated followed by quantification of internalized EGF at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. E , cells were transfected with either empty pCMV vector ( ctrl ) or cotransfected with G2019S LRRK2 and the indicated RAB18 constructs, and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. *, p < 0.05. F , cells were transfected as indicated followed by quantification of internalized fluorescent EGF at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. ***, p < 0.005. G , same as in E , but cells were transfected with either empty pCMV vector ( ctrl ) or the indicated RAB18 constructs. n = 3 independent experiments. H , same as in F , but cells were transfected with either empty pCMV vector ( ctrl ) or the indicated RAB18 constructs. n = 3 independent experiments. All error bars represent S.E.M.

Article Snippet: Phosphorylation reactions were performed either in the presence or absence of 1 μ m LRRK2 kinase inhibitor PF 06447475 (Tocris Bioscience).

Techniques: Construct, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Phosphodeficient RAB8A, but not WT or phosphomimetic RAB8A variants, revert the LRRK2-mediated effects on EGFR trafficking. A , HeLa cells were transfected with either empty pCMV vector ( ctrl ) or the indicated RAB8A constructs, and surface-bound fluorescent EGF was quantified. n = 4 independent experiments. *, p < 0.05. B , cells were transfected with the indicated constructs followed by quantification of internalized fluorescent EGF. n = 4 independent experiments. *, p < 0.05. C , cells were cotransfected with G2019S LRRK2 and the indicated RAB8A constructs, and surface-bound fluorescent EGF was quantified. n = 8 independent experiments. *, p < 0.05. D , cells were transfected with the indicated constructs, and internalized fluorescent EGF was quantified at 10 ( left ) and 30 min ( right ). n = 8 independent experiments. *, p < 0.05; ***, p < 0.005; ****, p < 0.001. All error bars represent S.E.M.

Article Snippet: Phosphorylation reactions were performed either in the presence or absence of 1 μ m LRRK2 kinase inhibitor PF 06447475 (Tocris Bioscience).

Techniques: Transfection, Plasmid Preparation, Construct

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Knockdown of RAB8A mimics the endolysosomal trafficking deficits mediated by G2019S LRRK2. A , HeLa cells were either nontransfected (−) or transfected with ctrl-siRNA or RAB8A-siRNA, and cell extracts (30 μg) were analyzed by Western blotting for RAB8A protein levels and tubulin as a loading control. B , quantification of the type of experiments depicted in A . RAB8A levels in the presence of RAB8A-siRNA were normalized to levels in the presence of ctrl-siRNA. n = 3 independent experiments. *, p < 0.05. C , cells were either left untreated (−) or transfected with ctrl-siRNA or RAB8A-siRNA, and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. *, p < 0.05. D , cells were either left untreated (−) or transfected with ctrl-siRNA or RAB8A-siRNA followed by quantification of internalized fluorescent EGF at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. **, p < 0.01; ****, p < 0.001. E , cells were either left untreated or cotransfected with ctrl-siRNA or RAB8A-siRNA in the absence or presence of GFP-tagged active RAB7A (RAB7A-Q67L), and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. *, p < 0.05. F , cells were either left untreated or cotransfected with ctrl-siRNA or RAB8A-siRNA in the absence or presence of RAB7A-Q67L, and internalized fluorescent EGF was quantified at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. *, p < 0.05; **, p < 0.01. G , cells were either treated with ctrl-siRNA or RAB8A-siRNA as indicated, and the RAB7-binding domain of RILP coupled to GST was used to pull down the GTP-bound form of RAB7 from cell lysates (300 μg). Input (10%) was run alongside pulldowns to demonstrate equal levels of total RAB7 protein in ctrl-siRNA– or RAB8A-siRNA–treated cells, and the levels of RAB8A and tubulin were analyzed on a separate gel. H , experiments of the type depicted in G were quantified, and the amount of RAB7 isolated by GST-RILP was expressed relative to input. n = 3 independent experiments. ***, p < 0.005. I , cells were either treated with ctrl-siRNA or RAB8A-siRNA as indicated, and a conformation-specific antibody was used to immunoprecipitate active RAB7 from cell lysates (2 mg). As a positive control, ctrl-siRNA–treated cell extracts were incubated with 100 μ m GTPγS to activate RAB7A before immunoprecipitation. Input (1%) was run alongside pulldowns to demonstrate equal levels of total RAB7 protein in ctrl-siRNA– or RAB8A-siRNA–treated cells, and the levels of RAB8A and tubulin were analyzed on a separate gel. All error bars represent S.E.M.

Article Snippet: Phosphorylation reactions were performed either in the presence or absence of 1 μ m LRRK2 kinase inhibitor PF 06447475 (Tocris Bioscience).

Techniques: Knockdown, Transfection, Western Blot, Control, Binding Assay, Isolation, Positive Control, Incubation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Pathogenic LRRK2 or knockdown of RAB8A causes accumulation of EGF in a RAB4-positive endocytic compartment. A , HeLa cells were transfected with either empty pCMV vector or pathogenic LRRK2 or cotransfected with GFP-tagged RAB4, and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. *, p < 0.05. B , cells were transfected as indicated followed by quantification of internalized fluorescent EGF at 10 ( left ) and 30 min ( right ). n = 3 independent experiments. *, p < 0.05; **, p < 0.01. C , example of HeLa cells cotransfected with GFP-RAB4 and either empty pCMV vector or pathogenic LRRK2. Live pictures were taken 20 min upon fluorescent EGF internalization, and arrows point to GFP-RAB4–positive vesicles containing Alexa647-EGF. Scale bar , 10 μm. D , quantification of colocalization of Alexa647-EGF with GFP-RAB4 (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 6 independent experiments. ***, p < 0.005. E , example of HeLa cells cotransfected with GFP-RAB4 and either ctrl-siRNA or RAB8A-siRNA. Live pictures were taken as described above. Arrows point to GFP-RAB4–positive vesicles containing Alexa647-EGF. Scale bar , 10 μm. F , quantification of colocalization of Alexa647-EGF with GFP-RAB4 (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 3 independent experiments. **, p < 0.01. All error bars represent S.E.M.

Article Snippet: Phosphorylation reactions were performed either in the presence or absence of 1 μ m LRRK2 kinase inhibitor PF 06447475 (Tocris Bioscience).

Techniques: Knockdown, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Pathogenic G2019S LRRK2 causes a deficit in EGFR recycling. A , example of HeLa cells transfected with pCMV or cotransfected with mRFP and either G2019S or kinase-inactive G2019S-K1906M LRRK2 and stained with an antibody against the extracellular domain of the EGFR in the absence of permeabilization to visualize only surface EGFR. Scale bar , 10 μm. B , quantification of fluorescence intensity of surface levels of EGFR at t = 0 min ( steady-state ), upon triggering internalization of the EGFR ( pulse ), or upon chase for various time points to assess recycling rates ( chase ) as described under “Materials and methods.” n = 3 independent experiments. *, p < 0.05; ***, p < 0.005. C , HeLa cells were transfected as indicated, and cell extracts (30 μg) were analyzed by Western blotting for FLAG-tagged LRRK2 levels and tubulin as a loading control. A.U. , arbitrary units. All error bars represent S.E.M.

Article Snippet: Phosphorylation reactions were performed either in the presence or absence of 1 μ m LRRK2 kinase inhibitor PF 06447475 (Tocris Bioscience).

Techniques: Transfection, Staining, Fluorescence, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: The G2019S variant of leucine-rich repeat kinase 2 (LRRK2) alters endolysosomal trafficking by impairing the function of the GTPase RAB8A

doi: 10.1074/jbc.RA118.005008

Figure Lengend Snippet: Accumulation of EGF in a RAB4-positive endocytic compartment and deficits in EGFR recycling due to G2019S LRRK2 expression are rescued by active RAB7A expression. A , example of HeLa cells cotransfected with GFP-RAB4 and either empty pCMV vector or pathogenic LRRK2 with or without RAB7A-Q67L expression as indicated. Live pictures were taken 20 min upon fluorescent EGF internalization, and arrows point to GFP-RAB4–positive vesicles containing Alexa647-EGF. An independent picture (543 HeNe laser line) was acquired to confirm coexpression of the distinct mRFP-tagged RAB7A constructs in all cases. Scale bar , 10 μm. B , quantification of colocalization of Alexa647-EGF with GFP-RAB4 in cells coexpressing empty pCMV vector or G2019S LRRK2 in the presence or absence of mRFP-tagged RAB7A constructs as indicated (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 3 independent experiments. **, p < 0.01. C , HeLa cells were transfected with the indicated constructs, and cell extracts (30 μg) were analyzed by Western blotting for FLAG-tagged G2019S-LRRK2, mRFP-RAB7A protein levels (anti-RAB7 antibody), and GAPDH as a loading control. D , HeLa cells were transfected with either empty pCMV vector or pathogenic G2019S LRRK2 in the presence or absence of mRFP-tagged RAB7A constructs as indicated, and EGFR surface levels and EGFR recycling were determined at the indicated time points. n = 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001. A.U. , arbitrary units. All error bars represent S.E.M.

Article Snippet: Phosphorylation reactions were performed either in the presence or absence of 1 μ m LRRK2 kinase inhibitor PF 06447475 (Tocris Bioscience).

Techniques: Expressing, Plasmid Preparation, Construct, Transfection, Western Blot, Control

Journal: Cell

Article Title: Portable, On-Demand Biomolecular Manufacturing.

doi: 10.1016/j.cell.2016.09.013

Figure Lengend Snippet: Figure 6. FD-CF Combinatorial Small-Molecule Biosynthesis (A) Schematic of the biosynthetic violacein pathway. Violacein is produced through five enzymatic steps from two molecules of L-tryptophan. Various combi- nations of five enzymes yield different small molecules. CPA, chromopyrrolic acid; DV, deoxyviolacein; IPAI, indole-3-pyruvic acid imine; IPAID, IPAI dimer; L-trp, L-tryptophan; PDV, prodeoxyviolacein; PV, proviolacein; V, violacein. (B) Anti-FLAG WB confirming expression of each enzyme in the violacein pathway. (C–G) Combinatorial production of small molecules. FD-CF reaction pellets containing the substrate and cofactors were rehydrated with the indicated combi- nations of template DNA encoding VioA, VioB, VioC, VioD, or VioE. Samples taken from each reaction mixture every 2 hr up to 8 hr were analyzed by UHPLC-MS. Violacein and deoxyviolacein were quantified using a commercially available standard, and numbers in the parentheses of (C) and (G) indicate the measured concentrations in mM. AU, arbitrary units. Data represent the mean ± SD of three replicates.

Article Snippet: DNA constructs for enzymes of the violacein pathway [vioA, vioB, and vioC from Addgene plasmid #40782; vioD synthesized based on the sequence provided in Registry of Standard Biological Parts (BBa_K274002); vioE from pVio1-2 (Sánchez et al., 2006)] were cloned into T7 vector to yield pET15b-vioA, -vioB, -vioC, and -vioD, and pET21-vioE (GenBank: KX461959, KX461960, KX461961, KX461962, KX461963).

Techniques: Produced, Expressing

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 2 ｜Cellular ATP levels and F1F0-ATPase activity in isolated mitochondria in a in SH-SY5Y cell model of Parkinson’s disease. (A, B) Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells cultured in MEM-F12 medium; Flag-vector + MPP+: LV-Flag-vector- transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-Flag + MPP+: LV-CHCHD2-Flag-transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-T61I + MPP+: LV-CHCHD2-T61I-Flag-transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours). (A) Detection of ATP levels in control or MPP+-treated SH-SY5Y cells stably transfected with empty vector or constructs encoding WT or T61I-mutant CHCHD2. (B) Effect of WT or T61I-mutant CHCHD2 on ATP synthase specific activity in control or MPP+-treated cells. (C) Effect of WT or T61I-mutant CHCHD2 on ATP synthase specific activity in AVV-transfected mice with or without MPTP treatment. CHCHD2-Flag + MPTP: AVV-CHCHD2-Flag–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I + MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); Flag-vector: AVV-Flag (CMV- betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector + MPTP: AVV-Flag-transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). Data are expressed as the mean ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ATP: Adenosine triphosphate; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Activity Assay, Isolation, Plasmid Preparation, Transfection, Cell Culture, Control, Stable Transfection, Construct, Mutagenesis, Saline

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 3 ｜CHCHD2 T61I mutation promotes mitochondrial permeability transition pore (mPTP) opening in SH-SY5Y cells. CHCHD2-Flag: LV-CHCHD2-Flag-transfected SH-SY5Y cells; CHCHD2-T61I: LV-CHCHD2-T61I-Flag-transfected SH-SY5Y cells; Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells. (A) Calcein-AM and CoCl2 were administered to live cells expressing WT or T61I-mutant CHCHD2, and fluorescence intensity was detected by flow cytometry. (B) Effect of WT or the T61I-mutant CHCHD2 on mPTP opening as determined by detecting mitochondrial membrane potential using JC-1 fluorescent probes (white squares) in control or MPP+-treated SH-SY5Y cells. The fluorescence intensity of intracellular JC-1 aggregates (red) in the MPP+ + CHCHD2-Flag group was greater than that in the MPP+ + Flag-vector group. The CHCHD2-T61I group showed an increased amount of JC-1 monomers (green). Scale bars: 10 μm. (C) The mean fluorescence intensity of JC-1 aggregates or monomers in control or MPP+-treated SH-SY5Y cells expressing WT or T61I-mutant CHCHD2. Data are expressed as the mean ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). Calcein-AM: Calcein acetoxymethyl ester; MPP+: 1-methyl-4-phenylpyridinium; mPTP: mitochondrial permeability transition pore; WT: wild type.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Mutagenesis, Permeability, Transfection, Plasmid Preparation, Expressing, Fluorescence, Flow Cytometry, Membrane, Control

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 5 ｜CHCHD2 T61I mutation aggravates movement deficits and nigral DA neuron function in a mouse model of PD. (A) Experimental design. AAV-CHCHD2, AAV-CHCHD2 T61I, or AAV-Vector was stereotaxically injected into the substantia nigra pars compacta, and 4 weeks later mice were injected intraperitoneally with normal saline or MPTP for 5 weeks. One day after the last MPTP/normal saline injection, behavioral tests were performed, and then the mice were sacrificed. Flag-vector: AVV-Flag(CMV-betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector+MPTP: AVV-Flag– transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-Flag+MPTP: AVV-CHCHD2-Flag–infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I+MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). (B) The grasping test was used to examine grip strength. (C) The pole-climbing test was used to examine bradykinesia. (D) The rotarod test was used to examine motor coordination. (E) Representative immunohistochemical staining of TH-positive neurons in the substantia nigra (SN). There were fewer TH-positive neurons in the SN in the AAV-CHCHD2-T61I + MPTP group than in the AAV-Flag-vector + MPTP and AAV-CHCHD2-Flag + MPTP groups. Scale bars: 800 μm. (F) Quantification of the TH-positive cells shown in E. (G) Representative images of double-immunofluorescent staining for CHCHD2-Flag (green, Alexa Fluor 488) and TH (red, Alexa Fluor 555) (white squares) in the SN. There were significantly fewer TH-positive neurons in the SN in the AAV-CHCHD2-T61I + MPTP group than in the AAV-Flag-vector + MPTP and AAV-CHCHD2-Flag + MPTP groups. Scale bars: 50 μm. (H) Quantification of TH mean fluorescence in the SN as shown in G. (I) Representative immunoblot for TH in the SN. (J) TH expression in the SN. Results are expressed as the mean ± SD (n ≥ 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ACTB: β-Actin; AAV: adeno-associated virus; DA: dopaminergic; DAPI: 4′,6-diamidino-2-phenylindole; MPTP: 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine; TH: tyrosine hydroxylase; SN: substantia nigra.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Mutagenesis, Plasmid Preparation, Injection, Saline, Transfection, Infection, Immunohistochemical staining, Staining, Fluorescence, Western Blot, Expressing, Virus

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 8 ｜CHCHD2 modulates OSCP expression in MPP+-/MPTP-induced PD models. (A, B) Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells cultured in MEM-F12 medium; Flag-vector+MPP+: LV-Flag-vector–transfected SH- SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-Flag+MPP+: LV-CHCHD2-Flag–transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-T61I + MPP+: LV-CHCHD2-T61I-Flag–transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours). (A) Representative immunoblot of OSCP, CHCHD2, and ACTB in MPP+-treated SH-SY5Y cells. (B) Quantification of relative OSCP expression shown in A. (C, D) Flag-vector: AVV-Flag(CMV-betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector + MPTP: AVV-Flag-infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-Flag + MPTP: AVV-CHCHD2-Flag- infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I + MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). (C) Representative immunoblot of OSCP, CHCHD2, and ACTB in control or MPTP-treated mice. (D) Quantification of relative OSCP expression shown in C. Data normalized to SH-SY5Y cells overexpressing Flag-vector or C57BL/6J mice transfected with AAV-Flag are expressed as the mean ± SD (n = 3 independent experiments for cells, n = 6 for animals). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ACTB: β-Actin; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine; OSCP: oligomycin sensitivity conferral protein.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Expressing, Plasmid Preparation, Transfection, Cell Culture, Western Blot, Saline, Infection, Control

Journal: Arthritis and rheumatism

Article Title: Altered Mineralization of Human Osteoarthritic Osteoblasts Is Attributable to Abnormal Type I Collagen Production

doi: 10.1002/art.24489

Figure Lengend Snippet: Effect of transforming growth factor β1 (TGFβ1) on bone morphogenetic protein 2 (BMP-2)–induced alizarin red staining (ARS). A, Representative alizarin red staining for confluent normal osteoblasts (n = 5 separate experiments) incubated as described in Figure 1. B, TGFβ1 levels in normal and osteoarthritis (OA) osteoblasts as measured by selective enzyme-linked immunosorbent assay. Values are the mean and SEM results from 8 normal osteoblast preparations, 14 preparations of osteoblasts producing low levels of prostaglandin E2 (PGE2), and 14 preparations of OA osteoblasts producing high levels of PGE2. C, TGFβ1 mRNA levels, as determined by quantitative polymerase chain reaction, under basal conditions and following inhibition of TGFβ1 expression with short hairpin RNA (shRNA) plasmids. Values are the mean and SEM results from 7 OA osteoblast preparations. D, COL1A1- to -COL1A2 ratio of OA osteoblasts under basal conditions or following inhibition of TGFβ1 expression with shRNA. Values are the mean and SEM results from 7 OA osteoblast preparations. E, Top, Representative alizarin red staining of OA osteoblasts treated or not treated with TGFβ1 shRNA. Bottom, Quantification of alizarin red staining following BMP-2 treatment in OA osteoblasts treated or not treated with TGFβ1 shRNA. Values are the mean and SEM results from 6 preparations.

Article Snippet: Using the optimized conditions for transfection, 2 TGF β 1 short hairpin RNA (shRNA) constructs (Origen, Rockville, MD) were tested for their effect on mineralization, as described above, after 28 days of continuous treatment with BMP-2.

Techniques: Staining, Incubation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Inhibition, Expressing, shRNA

Journal: Journal of Biological Chemistry

Article Title: Cytokine Receptor Common β Subunit-mediated STAT5 Activation Confers NF-κB Activation in Murine proB Cell Line Ba/F3 Cells

doi: 10.1074/jbc.m109878200

Figure Lengend Snippet: FIG. 4. Nuclear translocation of NF-B is not involved in GM-CSF-de- pendent NF-B activation. Ba/F3-wild cells were stimulated with 5 ng/ml GM- CSF (left) or 20 ng/ml TNF (right) after 5 h of cytokine deprivation (A). After the indicated time periods, cells were fixed and processed for immunocytochemical analysis. Subcellular localization of p65 was analyzed by indirect immunocyto- chemical staining using anti-p65 anti- body as described under “Experimental Procedures.” FITC, subcellular localiza- tion of p65 with fluorescein isothiocya- nate; PI, nuclear staining with propidium iodide; Merged, the merged image of both. Ba/F3-wild cells were stimulated with GM-CSF (left) or TNF (right) as de- scribed above (B). After the indicated time periods, cells were harvested, and both cytosol and nuclear extracts were iso- lated. Ten g of each was separated on a 10% SDS-polyacrylamide gel and immu- noblotted with anti-p65 antibody. Ba/F3- wild cells were stimulated with GM-CSF (left) or TNF (right) and harvested at the indicated time points, and whole cell ex- tracts were prepared (C). Ten g of each was separated on a 10% SDS-polyacryl- amide gel and immunoblotted with anti- phosphorylated IB (p-IB) antibody, and the protein blot was reprobed with anti-IB antibody. As a control, whole cell extracts from cells treated with TNF for 5 min were loaded on the same gel (lane 8). Immunoblotting with anti-IB antibody was also performed. N.S., non-specific.

Article Snippet: In supershift assays, 2 g of antibodies against either p65 (sc-372), p50 (sc-1190), p52 (sc298), RelB (sc-226), c-Rel (sc-70), Bcl-3 (sc-185), or STAT5 (sc-836) (purchased from Santa Cruz Biotechnology, Santa Cruz, CA) was added to the nuclear extracts for 1 h prior to the addition of oligonucleotide probe.

Techniques: Translocation Assay, Activation Assay, Staining, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cytokine Receptor Common β Subunit-mediated STAT5 Activation Confers NF-κB Activation in Murine proB Cell Line Ba/F3 Cells

doi: 10.1074/jbc.m109878200

Figure Lengend Snippet: FIG. 5. Effects of STAT5 on constitutive DNA binding of NF-B in Ba/F3 cells. A and B, Ba/F3-wild and -Fall cells, grown in the culture medium containing IL-3, extensively washed with the depletion medium, deprived of IL-3, and then complemented with either IL-3 (lanes 2 and 6) or GM-CSF (lanes 3 and 7) or left untreated (lanes 4 and 8). Cells were harvested after 9 h of complementation as well as before substitution of cytokines (lanes 1 and 5). Cells were collected, and then cytosol and nuclear extracts or whole cell extracts were isolated. Nuclear extracts were subjected to EMSA using the oligonucleotide probes for STAT5 and NF-B (A, top part), and whole cell extracts were separated on a 10% SDS-polyacrylamide gel and immunoblotted with anti-p65 or anti-p50 antibody (A, bottom part). Cytosol and nuclear extracts of Ba/F3-wild (B, top panel) and -Fall (B, bottom panel) cells were also separated on a 10% SDS-polyacrylamide gel and immunoblotted with anti-p65 antibody. C, parental Ba/F3 cells, grown in the culture medium containing IL-3 were deprived of IL-3 and transiently transfected with 1 g of either an empty vector (lanes 1–4) or the expression plasmid encoding STAT5A1*6 (lanes 5–8). After transfection, cells were cultured in the depletion medium and harvested at the indicated time points. Nuclear extracts were prepared and subjected to EMSA for STAT5 (top panel) and NF-B (bottom panel). D, Ba/F3-wild cells, grown in the culture medium containing IL-3, were deprived of IL-3 and transiently transfected with either 1 g of empty vector (lanes 1 and 2) or the indicated amounts of expression plasmid encoding WT STAT5A (lanes 3–5). Cells transfected with empty vector were cultured in the absence (lane 1) or presence (lane 2) of 5 ng/ml GM-CSF, and those transfected with WT STAT5A expression plasmids were cultured in the presence of GM-CSF (lanes 3–5). After 9 h of culture, cells were harvested, and nuclear extracts were prepared and subjected to EMSA for STAT5 (top panel) and NF-B (bottom panel).

Article Snippet: In supershift assays, 2 g of antibodies against either p65 (sc-372), p50 (sc-1190), p52 (sc298), RelB (sc-226), c-Rel (sc-70), Bcl-3 (sc-185), or STAT5 (sc-836) (purchased from Santa Cruz Biotechnology, Santa Cruz, CA) was added to the nuclear extracts for 1 h prior to the addition of oligonucleotide probe.

Techniques: Binding Assay, Isolation, Transfection, Plasmid Preparation, Expressing, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Cytokine Receptor Common β Subunit-mediated STAT5 Activation Confers NF-κB Activation in Murine proB Cell Line Ba/F3 Cells

doi: 10.1074/jbc.m109878200

Figure Lengend Snippet: FIG. 6. STAT5 up-regulates transactivation potential of NF-B p65. A, schematic drawing of Gal4 fusion constructs. The indicated sequences of murine p65 were fused to the DNA binding domain (DBD) of Gal4 (amino acids 1–147), which is indicated as gray boxes. B, Ba/F3-wild cells transiently transfected with 3 g of Gal4-inducible luciferase reporter plasmid in combination with 1 g of expression vectors encoding various Gal4 fusion proteins. The indicated amounts of expression vectors encoding WT STAT5A or STAT5A749 were also cotransfected. Cells were cultured in the absence or presence of 5 ng/ml GM-CSF for 12 h and subjected to luciferase assays. Luciferase activities were normalized to RLU per g of protein and indicated as fold activation as compared with those of cells transfected with Gal4 alone and left nonstimulated. Results are the mean S.D. of 3 independent experiments.

Article Snippet: In supershift assays, 2 g of antibodies against either p65 (sc-372), p50 (sc-1190), p52 (sc298), RelB (sc-226), c-Rel (sc-70), Bcl-3 (sc-185), or STAT5 (sc-836) (purchased from Santa Cruz Biotechnology, Santa Cruz, CA) was added to the nuclear extracts for 1 h prior to the addition of oligonucleotide probe.

Techniques: Construct, Binding Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Cell Culture, Activation Assay

Journal: eLife

Article Title: N-acetylation of α-synuclein enhances synaptic vesicle clustering mediated by α-synuclein and lysophosphatidylcholine

doi: 10.7554/eLife.97228

Figure Lengend Snippet:

Article Snippet: Lipids used in this study were purchased from Avanti Polar Lipids as follows: 16:0 LPC (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine) (855675), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) (850375), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) (840035), POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) (850457), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) (850757), biotin-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)) (870277), cholesterol (70000).

Techniques: Transfection, Construct, Plasmid Preparation, Software

Journal: Carcinogenesis

Article Title: MicroRNA 345, a methylation-sensitive microRNA is involved in cell proliferation and invasion in human colorectal cancer.

doi: 10.1093/carcin/bgr114

Figure Lengend Snippet: Fig. 5. BAG3 was identified to be a target of mir-345. (A) Base pairing comparison between mature mir-345 and the WTor mutant (MUT) putative target site in the 3#-UTR of BAG3 mRNA. The mutant sequence is identical to the WT construct except for mutations highlighted in bold disrupting base pairing at the 5# end of mir-345. (B) Dual luciferase assay of HT29 cells co-transfected with vector containing the WT or mutant (MUT) putative BAG3 3#-UTR target site and pri-mir-345 or mir-345 ASO. The luciferase values of pri-mir-345 transfections were normalized to the average values for the corresponding mir-345 ASO transfections. P , 0.01. (C) Western blots of BAG3 in HT29 cells transfected with pri-mir-345. b-actin was used as a loading control. The density of the western blot bands was normalized to the amount of BAG3 protein as determined by the density of the band obtained by western blot for b-actin. P , 0.05.

Article Snippet: Slides were then incubated overnight at 4 C in a humidified chamber with rabbit polyclonal anti-BAG3 antibody (1:200; Abcam, Cambridge, UK) and subsequently with antirabbit IgG for 20 min.

Techniques: Comparison, Mutagenesis, Sequencing, Construct, Luciferase, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Carcinogenesis

Article Title: MicroRNA 345, a methylation-sensitive microRNA is involved in cell proliferation and invasion in human colorectal cancer.

doi: 10.1093/carcin/bgr114

Figure Lengend Snippet: Fig. 6. Western blots and qRT–PCR results of BAG3 in CRC patients. (A) qRT–PCR of BAG3 in CRC tissues (Ca), para-cancer tissues (P) and adjacent non-tumor mucosal tissues (N) of 31 patients. P , 0.001. (B) Western blots of BAG3 in CRC cases 1 and 8 with glyceraldehyde-3-phosphate dehydrogenase as a loading control (bottom panel). Western blots of BAG3 in all of the 31 CRC cases were shown in supplementary Figure 3, available at Carcinogenesis Online. The density of the western blot bands was normalized to the amount of BAG3 protein as determined by the density of the glyceraldehyde-3-phosphate dehydrogenase band. The three columns in the bar represent the mean density of BAG3 bands in the N, P and Ca groups. The amount of BAG3 protein was significantly higher in the Ca group. (C) Immunocytochemistry analysis of BAG3 expression in CRC case (Ca), its normal surrounding tissue (N) and adenoma (A).

Article Snippet: Slides were then incubated overnight at 4 C in a humidified chamber with rabbit polyclonal anti-BAG3 antibody (1:200; Abcam, Cambridge, UK) and subsequently with antirabbit IgG for 20 min.

Techniques: Western Blot, Quantitative RT-PCR, Control, Immunocytochemistry, Expressing