Journal: Theranostics

Article Title: A thermosensitive, reactive oxygen species-responsive, MR409-encapsulated hydrogel ameliorates disc degeneration in rats by inhibiting the secretory autophagy pathway

doi: 10.7150/thno.47723

Figure Lengend Snippet: Activation of secretory autophagy in degenerated human and rat discs. (A, a-h) Safranin-O-stained sections showing reduced proteoglycan-rich matrix (red), fewer chondrocyte-like cells (black arrows), and more numerous infiltrating inflammation cell clusters (red arrows) in degenerated human and rat discs compared to non-degenerated discs. (A(i-x), B) Immunofluorescence images demonstrating significantly greater expression of the secretory autophagy-related proteins TRIM16 (red) and LC3B (green) as well as greater TRIM16-LC3B co-localization (merged yellow) in human and rat degenerated discs. (C and D) Immunoblot showing higher LC3B and TRIM16 proteins levels in human (macrophage-like) THP-1 cells under oxidative stress (TBHP, 400 µM, 2 h) (n = 3). Data are expressed as the mean + SD. Group means were compared with independent samples t -test. ** p < 0.01; *** p < 0.001.

Article Snippet: Membranes were blocked with 5% (w/v) non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against one or more of the following proteins: TRIM16 (1:500, Santa Cruz, sc-398851), LC3B (1:1000, CST, #3868), ACAN (1:1000, Proteintech, 13880-1-AP), MMP13 (1:3000, abcam, 39012), SOX9 (1:1000, abcam, ab185966), NBR1 (1:400, Santa Cruz, sc-130380), GAPDH (1:5000, CST, #5174S), and (or) actin (1:5000, MultiSciences, 70-ab008-040).

Techniques: Activation Assay, Staining, Immunofluorescence, Expressing, Western Blot

Journal: Theranostics

Article Title: A thermosensitive, reactive oxygen species-responsive, MR409-encapsulated hydrogel ameliorates disc degeneration in rats by inhibiting the secretory autophagy pathway

doi: 10.7150/thno.47723

Figure Lengend Snippet: Oxidative stress induced TRIM16-mediated, secretory autophagy-dependent IL-1β secretion from THP-1 cells. THP-1 cells were transfected with a TRIM16 overexpression plasmid or siRNA, and then treated with 400 µM TBHP for 2 h. (A) TRIM16 knockdown suppressed IL-1β secretion under oxidative stress as measured by ELISA. (B) Immunofluorescence demonstrated that knockdown also decreased LC3B (green) and TRIM16 (red) co-localization (merged yellow) in THP-1 cells under oxidative stress. Magnification showed the representative co-localization of LC3B and TRIM16. (C and D) TRIM16 knockdown and concomitant LC3B down-regulation under oxidative stress were confirmed in immunoblotting. (E-H) Conversely, TRIM16 overexpression enhanced IL-1β secretion (E) and expression of LC3B under oxidative stress (F-H). Data are expressed as the mean + SD. Group means were compared by independent samples t-test, n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Membranes were blocked with 5% (w/v) non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against one or more of the following proteins: TRIM16 (1:500, Santa Cruz, sc-398851), LC3B (1:1000, CST, #3868), ACAN (1:1000, Proteintech, 13880-1-AP), MMP13 (1:3000, abcam, 39012), SOX9 (1:1000, abcam, ab185966), NBR1 (1:400, Santa Cruz, sc-130380), GAPDH (1:5000, CST, #5174S), and (or) actin (1:5000, MultiSciences, 70-ab008-040).

Techniques: Transfection, Over Expression, Plasmid Preparation, Knockdown, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Western Blot, Expressing

Journal: Theranostics

Article Title: A thermosensitive, reactive oxygen species-responsive, MR409-encapsulated hydrogel ameliorates disc degeneration in rats by inhibiting the secretory autophagy pathway

doi: 10.7150/thno.47723

Figure Lengend Snippet: MR409 attenuated secretory autophagy-mediated IL-1β secretion from THP-1 cells and suppressed the activation of IL-1β-induced pro-inflammatory pathway and matrix catabolism in disc nucleus pulposus cells. (A) MR409 significantly inhibited tert-butyl hydroperoxide (TBHP)-induced IL-1β secretion into the supernatant as measured by ELISA. (B) MR409 suppressed TRIM16 and LC3B protein expression levels in THP-1 cells with immunoblotting. GAPDH expression was used as the gel loading control. (C) MR409 attenuated TBHP-induced expression and co-localization of LC3B in THP-1 cells as evidenced by immunofluorescence staining. Magnification showed the representative co-localization of LC3B and TRIM16. (D) IL-1β-induced downregulation of anabolic factor genes (ACAN and SOX9) and upregulation of both catabolic factor genes (MMP13 and ADATMS5) and inflammatory mediator genes (COX-2, IL-6, iNOS and TNF-α) in rat NP cells were effectively reversed by MR409. mRNA expression levels were measured with qRT-PCR. (E) MR409 reversed IL-1β-mediated induction of ACAN, MMP13, and SOX9 protein expression in rat NP cells. (F and G) Immunofluorescence images showed that MR409 abrogated the IL-1β-induced decrease in ACAN expression (green) and increase in MMP13 expression (red) in rat NP cells. Data are expressed as the mean + SD of three independent cultures. Means were compared by one-way ANOVA with post-hoc Tukey tests. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Membranes were blocked with 5% (w/v) non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against one or more of the following proteins: TRIM16 (1:500, Santa Cruz, sc-398851), LC3B (1:1000, CST, #3868), ACAN (1:1000, Proteintech, 13880-1-AP), MMP13 (1:3000, abcam, 39012), SOX9 (1:1000, abcam, ab185966), NBR1 (1:400, Santa Cruz, sc-130380), GAPDH (1:5000, CST, #5174S), and (or) actin (1:5000, MultiSciences, 70-ab008-040).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Theranostics

Article Title: A thermosensitive, reactive oxygen species-responsive, MR409-encapsulated hydrogel ameliorates disc degeneration in rats by inhibiting the secretory autophagy pathway

doi: 10.7150/thno.47723

Figure Lengend Snippet: Subcutaneous administration of MR409 delayed age-related disc degeneration in mice through inhibiting secretory autophagy. (A and B) Representative immunofluorescence images of the nucleus pulposus (NP) region (A) and analysis (B) of TRIM16 (red) and ACAN (green) immunoexpression in lumbar disc sections from 23-month-old mice receiving daily subcutaneous injection of saline (control group) or MR409 for 8 months. MR409 inhibited secretory autophagy as evidenced by TRIM16 downregulation and promoted expression of matrix proteoglycan ACAN. (C and D) Representative X-ray images (C) and disc height index (DHI) measurements (D) of control and MR409 group mice demonstrated that MR409 treatment alleviated disc height loss. (E) Representative micro-CT images of the lumbar spine revealed that administration of MR409 decreased osteophyte formation. (F) Representative images of HE-stained, Safranin-O-stained, and ACAN/MMP13-immunostained disc sections from control and MR409 mice. (G and H) Histological evaluation of Safranin O-stained sections based on cellularity, morphology, and structure of the annulus fibrosus and NP (G) and quantitation of MMP13-positive cells (H) demonstrated that MR409 treatment protected against age-related disc degeneration in mice. Data are expressed as the mean + SD of five mice per treatment group. Means were compared with independent samples t -tests. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Membranes were blocked with 5% (w/v) non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against one or more of the following proteins: TRIM16 (1:500, Santa Cruz, sc-398851), LC3B (1:1000, CST, #3868), ACAN (1:1000, Proteintech, 13880-1-AP), MMP13 (1:3000, abcam, 39012), SOX9 (1:1000, abcam, ab185966), NBR1 (1:400, Santa Cruz, sc-130380), GAPDH (1:5000, CST, #5174S), and (or) actin (1:5000, MultiSciences, 70-ab008-040).

Techniques: Immunofluorescence, Injection, Saline, Control, Expressing, Micro-CT, Staining, Quantitation Assay

Journal: Theranostics

Article Title: A thermosensitive, reactive oxygen species-responsive, MR409-encapsulated hydrogel ameliorates disc degeneration in rats by inhibiting the secretory autophagy pathway

doi: 10.7150/thno.47723

Figure Lengend Snippet: The thermosensitive hydrogel with ROS-responsive MR409-loaded vesicles attenuated needle puncture-induced disc degeneration in rats. (A and B) Representative spine X-ray (A) and MR images (B) from the 5 experimental groups at 8 and 12 weeks. (C and D) Disc height index measured on X-ray images (C) and Pfirrmann score assessed on MR images (D) showed that the MR409-encapsulated hydrogel significantly alleviated disc height loss and attenuated disc degeneration at both 8 and 12 weeks after disc puncture. (E) Representative immunofluorescence staining of ACAN (green) and TRIM16 (red), Safranin O staining, and immunohistochemical staining of MMP13 in experimental discs at postoperative weeks 8 and 12. Arrows indicate residual hydrogel. (F) Reduced numbers of TRIM16-positive cells in discs, indicating that MR409-encapsulated hydrogel inhibited secretory autophagy. (G and H) Histological score (G) and quantitation of MMP13-positive cells (H) showed that MR409-encapsulated hydrogel alleviated disc degeneration at both 8 and 12 weeks after puncture surgery. Data are expressed as the mean + SD of 5 rats per treatment group. Group means were compared by one-way ANOVA with post-hoc Tukey tests. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Membranes were blocked with 5% (w/v) non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against one or more of the following proteins: TRIM16 (1:500, Santa Cruz, sc-398851), LC3B (1:1000, CST, #3868), ACAN (1:1000, Proteintech, 13880-1-AP), MMP13 (1:3000, abcam, 39012), SOX9 (1:1000, abcam, ab185966), NBR1 (1:400, Santa Cruz, sc-130380), GAPDH (1:5000, CST, #5174S), and (or) actin (1:5000, MultiSciences, 70-ab008-040).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Quantitation Assay

Journal: bioRxiv

Article Title: Stress-primed secretory autophagy drives extracellular BDNF maturation

doi: 10.1101/2020.05.13.090514

Figure Lengend Snippet: a) Results of automated literature mining of FKBP51 interactors in association to “autophagy”, “proteostasis” and “ubiquitin proteasome system”. b) Western blotting for FKBP51 and SEC22B in GFP-tagged SEC22B co-IP (GFP-IP) and whole cell extract (WCE) as control; c) Western blotting for FKBP51 and SEC22B in FLAG-tagged FKBP51 co-IP (FLAG-IP) and WCE as control. d) Western blotting for FKBP51, TRIM16, LC3B and CTSD in FLAG-tagged FKBP51 co-IP (FLAG-IP) and WCE as control. e) Western blotting for TRIM16, CTSD and SEC22B in FLAG-tagged TRIM16 co-IP (FLAG-IP) and WCE as control performed in WT and FKBP5 KO cells. f) Quantifications of e with n = 3 per group. g) Western blotting for TRIM16, CTSD and SEC22B in FLAG-tagged TRIM16 co-IP (FLAG-IP) and WCE as control performed in cells treated with 100 nM dexamethasone or vehicle for 4 hours. h) Quantifications of g with n = 3 per group. i) Western blotting for FKBP51, SEC22B, SNAP23, SNAP29, STX3 and STX4 in FLAG-tagged FKBP51 co-IP (FLAG-IP) and WCE as control. j) Western blotting for, SEC22B, SNAP23, SNAP29, STX3 and STX4 in GFP-tagged SEC22B co-IP (GFP-IP) and WCE as control performed in WT and FKBP5 KO cells treated with 100 nM dexamethasone or vehicle for 4 hours. k) Quantifications of j with n = 3 per group. l) Schematic overview of the interactions of FKBP51 in the secretory autophagy pathway. Unpaired t-tests were performed on all quantifications; ns= not significant, **P < 0.01, ***P < 0.001. Data shown as mean ± s.e.m. Ab, antibody; Fc, fold change.

Article Snippet: LC3B-II/I (1:1000, Cell Signaling, #2775), FLAG (1:7000, Rockland, 600-401-383), FKBP51 (1:1000, Bethyl, A301-430A), ACTIN (1:5000, Santa Cruz Biotechnology, sc-1616), GAPDH (1:8000, Millipore CB1001), TRIM16 (1:1’000, Bethyl A301-160A), CTSD (for Mouse) (1:50, Abcam, ABCAAB6313-100), SNAP29 (1:1000, Sigma, SAB1408650), SNAP23 (1:1000, Sigma, SAB2102251), STX3 (1:1000, Sigma, SAB2701366), STX4 (1:1000, Cell Signalling.

Techniques: Ubiquitin Proteomics, Western Blot, Co-Immunoprecipitation Assay, Control

Journal: bioRxiv

Article Title: Stress-primed secretory autophagy drives extracellular BDNF maturation

doi: 10.1101/2020.05.13.090514

Figure Lengend Snippet: a) Glucocorticoid-mediated stress induces lysosomal damage, which leads to the assembly of FKBP51 with the cargo receptor TRIM16 and the cargo protein (e.g., MMP9). This complex is transported on the autophagosome membrane via the interaction of FKBP51 with SEC22B. The cargo protein is internalized into the autophagosome which is transported to the plasma membrane. The vesicle-plasma membrane fusion is mediated via the SNARE-protein complex assembly, which is regulated by FKBP51 and sensitive to glucocorticoids. The membrane fusion leads to the release of the cargo proteins into the extracellular milieu. The increased secretion of extracellular MMP9 induces the cleavage of proBDNF to its mature form, which becomes the prevailing form in the extracellular space; b) A first response to stress triggers lytic autophagy . When the stress persists, a second defense line is activated, which switches the stress response from lytic to secretory autophagy. If stress further persists (e.g., chronic), the initially beneficial proteins secreted in response to stress, might lead to neuroinflammation and psychiatric disorders.

Article Snippet: LC3B-II/I (1:1000, Cell Signaling, #2775), FLAG (1:7000, Rockland, 600-401-383), FKBP51 (1:1000, Bethyl, A301-430A), ACTIN (1:5000, Santa Cruz Biotechnology, sc-1616), GAPDH (1:8000, Millipore CB1001), TRIM16 (1:1’000, Bethyl A301-160A), CTSD (for Mouse) (1:50, Abcam, ABCAAB6313-100), SNAP29 (1:1000, Sigma, SAB1408650), SNAP23 (1:1000, Sigma, SAB2102251), STX3 (1:1000, Sigma, SAB2701366), STX4 (1:1000, Cell Signalling.

Techniques: Membrane, Clinical Proteomics

Journal: Journal of Diabetes Investigation

Article Title: Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR ‐30a‐3p/ TRIM16 axis

doi: 10.1111/jdi.14116

Figure Lengend Snippet: PCR primers used in this study

Article Snippet: The renal sections, after being deparaffinized and rehydrated, were incubated overnight at 4°C with TRIM16 primary antibodies (A09514‐4, 1:500; Boster Biological Engineering Co., International Enterprise Center, Wuhan, China), Bcal‐2 (ab182858, 1:500; Abcam), and bcl‐2‐associated X protein (BAX) (ab53154, 1:500; Abcam); and P‐cadherin antibodies (ab242060, 1:500; Abcam) and N‐cadherin (ab76011, 1:500; Abcam); then incubated with Alexa Fluor 594‐conjugated (red) goat anti‐mouse IgG and Alexa Fluor 488‐conjugated (green) goat anti‐rabbit IgG (1:1000; Invitrogen, Carlsbad, CA, USA) secondary antibodies at 25°C for 1 h. Nuclei were counterstained with a Hoechst solution (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques:

Journal: Journal of Diabetes Investigation

Article Title: Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR ‐30a‐3p/ TRIM16 axis

doi: 10.1111/jdi.14116

Figure Lengend Snippet: Cordyceps cicadae polysaccharides administration attenuated cell apoptosis and epithelial–mesenchymal transition in the kidneys of diabetic nephropathy mice. (a, b) At the end of 12 weeks of treatment, the expression of miR ‐30a‐3p and TRIM16 was determined in the kidney tissues of mice in each group by RT‐PCR , n = 8. (c) The protein expression level of TRIM16 in kidney tissues of mice was analyzed by western blot, n = 5. (d, e) Protein expression of TRIM16 in the kidney tissues using IHC staining, scale bar = 50 μm, n = 5. (f) TUNEL staining was used to detect cell apoptosis in the kidney tissues, scale bar, 20 μm, n = 5. (g) Protein expression of Bcl‐2 and Bax in the kidney tissues were detected by IHC staining, scale bar = 50 μm, n = 5. (h) Representative images of immunofluorescence staining for P‐cadherin (red), N‐cadherin (green), and DAPI (blue), scale bar, 50 μm, n = 5, scale bar = 20 μm. Data are expressed as mean ± SD . * P < 0.05, ** P < 0.01.

Article Snippet: The renal sections, after being deparaffinized and rehydrated, were incubated overnight at 4°C with TRIM16 primary antibodies (A09514‐4, 1:500; Boster Biological Engineering Co., International Enterprise Center, Wuhan, China), Bcal‐2 (ab182858, 1:500; Abcam), and bcl‐2‐associated X protein (BAX) (ab53154, 1:500; Abcam); and P‐cadherin antibodies (ab242060, 1:500; Abcam) and N‐cadherin (ab76011, 1:500; Abcam); then incubated with Alexa Fluor 594‐conjugated (red) goat anti‐mouse IgG and Alexa Fluor 488‐conjugated (green) goat anti‐rabbit IgG (1:1000; Invitrogen, Carlsbad, CA, USA) secondary antibodies at 25°C for 1 h. Nuclei were counterstained with a Hoechst solution (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, TUNEL Assay, Staining, Immunofluorescence

Journal: Journal of Diabetes Investigation

Article Title: Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR ‐30a‐3p/ TRIM16 axis

doi: 10.1111/jdi.14116

Figure Lengend Snippet: Expression of miR‐30a‐3p and TRIM16 in MPC5 podocytes treated with high glucose. The MPC5 podocytes were treated with normal glucose, mannitol, and high glucose conditions for 6, 12, and 24 h. (a) The expression level of miR‐30a‐3p in mouse podocyte was detected by RT‐qPCR. (b, c) The mRNA and protein expression of TRIM16 was determined by RT‐PCR and western blotting. * P < 0.05, ** P < 0.01.

Article Snippet: The renal sections, after being deparaffinized and rehydrated, were incubated overnight at 4°C with TRIM16 primary antibodies (A09514‐4, 1:500; Boster Biological Engineering Co., International Enterprise Center, Wuhan, China), Bcal‐2 (ab182858, 1:500; Abcam), and bcl‐2‐associated X protein (BAX) (ab53154, 1:500; Abcam); and P‐cadherin antibodies (ab242060, 1:500; Abcam) and N‐cadherin (ab76011, 1:500; Abcam); then incubated with Alexa Fluor 594‐conjugated (red) goat anti‐mouse IgG and Alexa Fluor 488‐conjugated (green) goat anti‐rabbit IgG (1:1000; Invitrogen, Carlsbad, CA, USA) secondary antibodies at 25°C for 1 h. Nuclei were counterstained with a Hoechst solution (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Diabetes Investigation

Article Title: Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR ‐30a‐3p/ TRIM16 axis

doi: 10.1111/jdi.14116

Figure Lengend Snippet: miR‐30a‐3p regulates TRIM16 in MPC5 podocytes. (a) TRIM16 is a predicted target of miR‐30a‐3p by TargetScan. (b) A luciferase reporter plasmid containing wildtype or mutant TRIM16 was cotransfected into MPC5 podocytes with miR‐30a‐3p or miR‐NC. Luciferase activity was determined at 48 h after transfection using the dual‐luciferase assay and was normalized to Renilla activity. (c–e) RT‐PCR and western blot analysis of TRIM16 in podocytes transfected with miR‐30a‐3p or miR‐NC or miR‐30a‐3p inhibitor or inhibitor of negative control. Data are presented as mean ± SD, n = 3. ** P < 0.01, *** P < 0.001. ## P < 0.001.

Article Snippet: The renal sections, after being deparaffinized and rehydrated, were incubated overnight at 4°C with TRIM16 primary antibodies (A09514‐4, 1:500; Boster Biological Engineering Co., International Enterprise Center, Wuhan, China), Bcal‐2 (ab182858, 1:500; Abcam), and bcl‐2‐associated X protein (BAX) (ab53154, 1:500; Abcam); and P‐cadherin antibodies (ab242060, 1:500; Abcam) and N‐cadherin (ab76011, 1:500; Abcam); then incubated with Alexa Fluor 594‐conjugated (red) goat anti‐mouse IgG and Alexa Fluor 488‐conjugated (green) goat anti‐rabbit IgG (1:1000; Invitrogen, Carlsbad, CA, USA) secondary antibodies at 25°C for 1 h. Nuclei were counterstained with a Hoechst solution (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Negative Control

Journal: Journal of Diabetes Investigation

Article Title: Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR ‐30a‐3p/ TRIM16 axis

doi: 10.1111/jdi.14116

Figure Lengend Snippet: Cordyceps cicadae polysaccharides protect mouse podocyte from high glucose induced apoptosis and epithelial–mesenchymal transition by controlling miR‐30a‐3p expression. (a) Relative miR‐30a‐3p and TRIM16 expression were detected by RT‐PCR and western blotting analysis. (b) Cell viability was determined by MTT assay. (c, d) The apoptosis of cells as detected by flow cytometry. (e) Representative images of TUNEL staining to illustrate apoptotic MPC5 podocytes in each group, cell nuclei were labeled in blue by DAPI, scale bar, 50 μm. (f) Representative immunofluorescence images of E‐cadherin (E‐cad, red) and N‐cadherin (N‐cad, green), cell nuclei were labeled in blue by DAPI, scale bar, 20 μm. Data are presented as mean ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The renal sections, after being deparaffinized and rehydrated, were incubated overnight at 4°C with TRIM16 primary antibodies (A09514‐4, 1:500; Boster Biological Engineering Co., International Enterprise Center, Wuhan, China), Bcal‐2 (ab182858, 1:500; Abcam), and bcl‐2‐associated X protein (BAX) (ab53154, 1:500; Abcam); and P‐cadherin antibodies (ab242060, 1:500; Abcam) and N‐cadherin (ab76011, 1:500; Abcam); then incubated with Alexa Fluor 594‐conjugated (red) goat anti‐mouse IgG and Alexa Fluor 488‐conjugated (green) goat anti‐rabbit IgG (1:1000; Invitrogen, Carlsbad, CA, USA) secondary antibodies at 25°C for 1 h. Nuclei were counterstained with a Hoechst solution (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, MTT Assay, Flow Cytometry, TUNEL Assay, Staining, Labeling, Immunofluorescence

Journal: RSC Advances

Article Title: LncRNA CASC2 inhibits autophagy and promotes apoptosis in non-small cell lung cancer cells via regulating the miR-214/TRIM16 axis

doi: 10.1039/c8ra09573f

Figure Lengend Snippet: The interaction between miR-214 and TRIM16 in NSCLC cells. (A) The predicted binding sites of miR-214 in the 3′UTR of TRIM16. (B) Luciferase activity was detected by luciferase reporter assay in 293T cells after co-transfection of miR-214 or miR-NC and TRIM16-WT or TRIM16-MUT. (C) Anti-Ago1 RIP assay was performed to confirm the association between miR-214 and TRIM16. (D and E) Expression and localization of TRIM16 in A549 cells transfected with miR-214, anti-miR-214, or matched controls by western blot and immunofluorescence staining. * P < 0.05.

Article Snippet: All cells were propagated in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 U ml −1 penicillin G/streptomycin (Sigma, St. Louis, MO, USA) at 37 °C in a humidified atmosphere containing 5% CO 2 . pcDNA-CASC2 (CASC2), pcDNA-tripartite motif-containing protein (TRIM) 16 (TRIM16), pcDNA empty vector (pcDNA), siRNA specifically targeting CASC2 (si-CASC2), siRNA specifically targeting TRIM16 (si-TRIM16), siRNA negative control (si-NC), miR-214 mimic (miR-214), mimic negative control (miR-NC), miR-214 inhibitor (anti-miR-214), inhibitor negative control (anti-miR-NC) were synthesized by GenePharma Co. Ltd., (Shanghai, China).

Techniques: Binding Assay, Luciferase, Activity Assay, Reporter Assay, Cotransfection, Expressing, Transfection, Western Blot, Immunofluorescence, Staining

Journal: RSC Advances

Article Title: LncRNA CASC2 inhibits autophagy and promotes apoptosis in non-small cell lung cancer cells via regulating the miR-214/TRIM16 axis

doi: 10.1039/c8ra09573f

Figure Lengend Snippet: TRIM16 knockdown significantly overturned anti-miR-214-mediated promotion of apoptosis and inhibition of autophagy in NSCLC cells. (A) The expression of TRIM16 mRNA in 21 NSCLC tissues and adjacent normal tissues was examined by RT-qPCR. (B) The protein level of TRIM16 in 16HBE, A549, and H1299 cells was determined by western blot. (C) Apoptosis of A549 and H1299 cells transfected with anti-miR-214 or anti-miR-NC along with si-NC or si-TRIM16 was evaluated by flow cytometry analysis. (D) A549 and H1299 cells were transfected with anti-miR-214 or anti-miR-NC along with si-NC or si-TRIM16 and GFP-LC3-positive punctate cells were visualized by confocal microscope and the percentage of GFP-LC3-positive cells were calculated. (E) Western blot was applied to detect the protein levels of p62, Atg-5, LC3-I and LC3-II in A549 and H1299 cells transfected with anti-miR-214 or anti-miR-NC along with si-NC or si-TRIM16. * P < 0.05.

Article Snippet: All cells were propagated in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 U ml −1 penicillin G/streptomycin (Sigma, St. Louis, MO, USA) at 37 °C in a humidified atmosphere containing 5% CO 2 . pcDNA-CASC2 (CASC2), pcDNA-tripartite motif-containing protein (TRIM) 16 (TRIM16), pcDNA empty vector (pcDNA), siRNA specifically targeting CASC2 (si-CASC2), siRNA specifically targeting TRIM16 (si-TRIM16), siRNA negative control (si-NC), miR-214 mimic (miR-214), mimic negative control (miR-NC), miR-214 inhibitor (anti-miR-214), inhibitor negative control (anti-miR-NC) were synthesized by GenePharma Co. Ltd., (Shanghai, China).

Techniques: Knockdown, Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Flow Cytometry, Microscopy

Journal: RSC Advances

Article Title: LncRNA CASC2 inhibits autophagy and promotes apoptosis in non-small cell lung cancer cells via regulating the miR-214/TRIM16 axis

doi: 10.1039/c8ra09573f

Figure Lengend Snippet: CASC2 positively regulated TRIM16 expression by suppressing miR-214 expression in NSCLC cells. (A) The negative correlation between miR-214 and CASC2 expressions in NSCLC tissues. (B) The negative correlation between miR-214 and TRIM16 expressions in NSCLC tissues. (C) The positive correlation between TRIM16 and miR-214 expressions in NSCLC tissues. (D) After 293T cells were co-transfected with miR-214, miR-214 + pcDNA, or miR-214 + CASC2 and TRIM16-WT or TRIM16-MUT, luciferase activity was measured by luciferase reporter assay. (E and F) Western blot was conducted to detect the protein level of TRIM16 in A549 and H1299 cells after transfection with CASC2, pcDNA, CASC2 + miR-214, CASC2 + miR-NC. * P < 0.05.

Article Snippet: All cells were propagated in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 U ml −1 penicillin G/streptomycin (Sigma, St. Louis, MO, USA) at 37 °C in a humidified atmosphere containing 5% CO 2 . pcDNA-CASC2 (CASC2), pcDNA-tripartite motif-containing protein (TRIM) 16 (TRIM16), pcDNA empty vector (pcDNA), siRNA specifically targeting CASC2 (si-CASC2), siRNA specifically targeting TRIM16 (si-TRIM16), siRNA negative control (si-NC), miR-214 mimic (miR-214), mimic negative control (miR-NC), miR-214 inhibitor (anti-miR-214), inhibitor negative control (anti-miR-NC) were synthesized by GenePharma Co. Ltd., (Shanghai, China).

Techniques: Expressing, Transfection, Luciferase, Activity Assay, Reporter Assay, Western Blot

Journal: PLoS ONE

Article Title: TRIM16 Acts as an E3 Ubiquitin Ligase and Can Heterodimerize with Other TRIM Family Members

doi: 10.1371/journal.pone.0037470

Figure Lengend Snippet: . (A) TRIM16-GFP domain deletion plasmids. (B) Co-transfection of TRIM16-GFP and TRIM16-myc-His in HEK293 cells and subsequent immunoprecipitation by anti-myc antibody (Ab) and Western blot with anti-GFP antibody. Whole cell extract (WCE) used as total input. (C) TRIM16 homodimerizes through its coiled-coil domain. GFP deletion mutants were co-transfected with the TRIM16-myc-His vector. Anti-GFP antibody was used to pull down proteins binding the GFP tagged proteins and the TRIM16-myc-His was used to detect self-association via its different tag (right panel). Transfection efficiency was confirmed (left panel). TRIM16-GFP mutants were efficiently pulled down (middle panel). * non-specific bands, # refer to text.

Article Snippet: In vitro ubiquitination assay containing 500 ng of recombinant TRIM16 (Abnova) and 10 μM ZnCl 2 was performed as described above.

Techniques: Cotransfection, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Binding Assay

Journal: PLoS ONE

Article Title: TRIM16 Acts as an E3 Ubiquitin Ligase and Can Heterodimerize with Other TRIM Family Members

doi: 10.1371/journal.pone.0037470

Figure Lengend Snippet: (A) Schematic structures of TRIM proteins used in heterodimerization studies. (B) TRIM16 binds MID1. Co-transfection of MID1-GFP and TRIM16-myc-His in HEK293 cells and subsequent immunoprecipitation by anti-myc antibody and Western blot with anti-GFP antibody. (C) MID1 was pulled down via its GFP antibody and a Western blot was performed to detect TRIM16-myc-His in the immunoprecipitated protein complex. (D) Whole cell lysates (WCL) of HEK293 cells transfected with empty vector (EV) or TRIM16-GFP were immunopreciptated with anti-GFP antibody. An anti-PML antibody was used to detect PML as a binding partner of TRIM16. (E) Lysates containing both TRIM16-GFP and TRIM24-His proteins were immunopreciptated with anti-GFP antibody. Anti-His antibody was used to detect the presence of TRIM24 in the TRIM16-associated complex.

Article Snippet: In vitro ubiquitination assay containing 500 ng of recombinant TRIM16 (Abnova) and 10 μM ZnCl 2 was performed as described above.

Techniques: Cotransfection, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Binding Assay

Journal: PLoS ONE

Article Title: TRIM16 Acts as an E3 Ubiquitin Ligase and Can Heterodimerize with Other TRIM Family Members

doi: 10.1371/journal.pone.0037470

Figure Lengend Snippet: The conserved residues in the zinc binding regions are in bold underlined type and are; cysteine; C, histidine; H, alanine; A aspartic acid; D. ( A ) TRIM16 and MID1 share the zinc binding consensus sequence for B-box1. (B) TRIM16 and MID1 share the Zinc binding consensus sequence for B-box2. (C/D) Modeling of B-boxes reveals zinc binding capability. Superimposition of the alpha-carbon backbone of the B-boxes from the MDM1 NMR structure (purple) and the homology model of TRIM16 (blue-grey). These two structures overlay with an average root-mean-square deviation of 0.4 Å.

Article Snippet: In vitro ubiquitination assay containing 500 ng of recombinant TRIM16 (Abnova) and 10 μM ZnCl 2 was performed as described above.

Techniques: Binding Assay, Sequencing

Journal: PLoS ONE

Article Title: TRIM16 Acts as an E3 Ubiquitin Ligase and Can Heterodimerize with Other TRIM Family Members

doi: 10.1371/journal.pone.0037470

Figure Lengend Snippet: (A) In vivo degradation assay was performed with cells being incubated with and without 30 µM MG132 for 4 hours. Lysates were subjected to Western blot analysis of endogenous TRIM16 antibody. (B) The liable nature of TRIM16 was further evaluated by inhibition of protein synthesis using 100 µg/ml cycloheximide. At the specified time points the cells were harvested and the protein were extracted for analysis by Western blots.

Article Snippet: In vitro ubiquitination assay containing 500 ng of recombinant TRIM16 (Abnova) and 10 μM ZnCl 2 was performed as described above.

Techniques: In Vivo, Degradation Assay, Incubation, Western Blot, Inhibition

Journal: PLoS ONE

Article Title: TRIM16 Acts as an E3 Ubiquitin Ligase and Can Heterodimerize with Other TRIM Family Members

doi: 10.1371/journal.pone.0037470

Figure Lengend Snippet: (A) TRIM16 in vivo polyubiquitination assay. In HEK293 cells, HA-Ub was co-transfected with various TRIM16-GFP domain deletion expression plasmids. The protein lysate was subjected to immunoprecipitation by GFP antibody, and subjected to Western blot and probed with anti-HA antibody for Ub (right panel) and anti-GFP antibody for TRIM16 (left and middle panel). Two exposure times are shown. GFP antibodies detect both un-ubiquitinated and polyubiquitinated forms of TRIM16. Polyubiquitinated smear is present in the sample transfected with wild-type TRIM16 and shown by anti-GFP and anti-HA antibodies. (B) In vitro ubiquitination assay with myc-His tagged TRIM16 together with a panel of E2 enzymes, showing activity with the UbcH5 family. (C) In vitro ubiquitination assay with full-length TRIM16, TRIM16 domain deletion mutants or empty vector showing extensive polyubiquitination with full-length TRIM16 as detected by Western blot with anti-myc antibodies. Numbers indicate protein size in kDa. (D) Recombinant TRIM16 (Abnova) was evaluated for E3 activity in the presence of recombinant E1, UbcH5b, and HA-Ub as indicated. The capacity to catalyse auto-ubiquitination in vitro was observed only in the presence of ZnCl 2 and ATP. Western Blot (lower panel) with TRIM16 antibody showed amount of TRIM16 protein in each lane.

Article Snippet: In vitro ubiquitination assay containing 500 ng of recombinant TRIM16 (Abnova) and 10 μM ZnCl 2 was performed as described above.

Techniques: In Vivo, Transfection, Expressing, Immunoprecipitation, Western Blot, In Vitro, Ubiquitin Proteomics, Activity Assay, Plasmid Preparation, Recombinant