Journal: Redox Biology

Article Title: KLF2 regulates dental pulp-derived stem cell differentiation through the induction of mitophagy and altering mitochondrial metabolism

doi: 10.1016/j.redox.2020.101622

Figure Lengend Snippet: Knockdown of KLF2 or ATG7 or BECN1 reduced autophagy-related molecules and OB differentiation-related markers. KLF2 or ATG7 or BECN1 was knocked down in DPSCs and cells were cultured in β-glycerophosphate plus l -ascorbic acid-containing medium. A. Western blot analysis for detection of autophagy-related molecules and OB differentiation-related markers after knockdown of KLF2. B. MDC staining for detection of autophagy vesicles after knockdown of KLF2. C. Western blot analysis for detection of autophagy-related molecules and OB differentiation-related markers after knockdown of ATG7. D. Western blot analysis for detection of autophagy-related molecules and OB differentiation-related markers after knockdown of BECN1.

Article Snippet: The si ATG7 (AM16708), si BECN1 (4457298), negative control si RNA (AM4611), si KLF2 (4392420) and DEPC-Treated Water (AM9922) were purchased from Ambion.

Techniques: Cell Culture, Western Blot, Staining

Journal: Autophagy

Article Title: Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

doi: 10.4161/15548627.2014.981792

Figure Lengend Snippet: RNF216 inhibits autophagy in macrophages stimulated with LPS. ( A and B ) RAW 264.7 cells were transfected with Rnf216 vector or empty vector, and then stimulated without or with LPS (100 ng/mL) for 16 h ( A ) or were on starvation for 4 h ( B ). Cell lysates were separated with SDS-PAGE and transferred to polyvinylidene difluoride membranes, following with MAP1LC3A antibody and proper HRP-conjugated secondary antibody. EV, empty vector. The band densitometry was quantified using ImageJ software. The quantitative data were calculated from 3 independent experiments, and were shown as mean ± SEM. ( C ) Cells grown on coverslips were transiently transfected with GFP-MAP1LC3A and either EV, Rnf216 , or si Becn1 overnight, followed by treatment with LPS (100 ng/ml) for 16 h or starvation for 4 h, and then fixed. Digital images were captured with confocal microscopy. Scale bar = 10 μm. ( D ) Cells with featured puncta were considered as autophagy-positive, and at least 100 cells were quantified. Puncta dots per cell were shown as mean ± SEM. (* P < 0.05).

Article Snippet: HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch (115–035–003, 111–035–003). si Becn1 targeting mouse Becn1 was obtained from Life Technologies (s80168).

Techniques: Transfection, Plasmid Preparation, SDS Page, Software, Confocal Microscopy

Journal: Autophagy

Article Title: Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

doi: 10.4161/15548627.2014.981792

Figure Lengend Snippet: Knockdown of RNF216 expression abrogates the inhibition of RNF216 on autophagy induction. ( A ) RAW 264.7 cells were infected with lentivirus with scrambled shRNA (shNC) or Rnf216 shRNA1 and 2 (sh Rnf 1, and 2) (MOI=10), and 48 h later, the cells were lysed and subject to SDS-PAGE followed by being transferred to nitrocellulose membrane. After blotting with RNF216 antibody, the membrane was incubated with HRP-conjugated secondary antibody, and visualized with an ECL chemiluminescence kit. ( B and C ) RAW 264.7 cells infected with lentivirus with shNC or sh Rnf 2 (MOI=10) were treated with LPS (100 ng/mL) for 16 h ( B ), or starvation for 4 h ( C ), then the cells were lysed and subjected to SDS-PAGE followed by being transferred to nitrocellulose membrane. After blotting with MAP1LC3A antibody, the membrane was incubated with HRP-conjugated secondary antibody, and visualized with an ECL chemiluminescence kit. The band densitometry was quantified using ImageJ software. The quantitative data were calculated from 3 independent experiments, and were shown as mean ± SEM. ( D ) RAW 264.7 cells infected with lentivirus containing scrambled shNC, or sh Rnf 2 (MOI=10) alone or combined with si Becn1 transfection were grown on coverslips, and transiently transfected with GFP-MAP1LC3A overnight, followed by treatment with LPS (100 ng/ml) for 16 h or starvation for 4 h, and then fixed. Digital images were captured with confocal microscopy. Scale bar = 10 μm. ( E ) Cells with featured puncta were considered as autophagy-positive, and at least 100 cells were quantified. Puncta dots per cell were shown as mean ± SEM. (* P < 0.05).

Article Snippet: HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch (115–035–003, 111–035–003). si Becn1 targeting mouse Becn1 was obtained from Life Technologies (s80168).

Techniques: Expressing, Inhibition, Infection, shRNA, SDS Page, Incubation, Software, Transfection, Confocal Microscopy

Journal: Autophagy

Article Title: Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

doi: 10.4161/15548627.2014.981792

Figure Lengend Snippet: Inhibition of autophagy by RNF216 was mediated through BECN1. ( A ) Flag- Rnf216 and Myc-tagged plasmid ( Atg5 , Atg14 , Tirap , Becn1 ) were used to transfect the 293T cells. Then the cells were lysed and subjected to immunoblotting, and the bands were visualized with an ECL chemiluminescence kit. ( B ) 293T cells were transfected with Becn1 combined with increased Rnf216 , and subjected to immunoblotting for the corresponding proteins. ( C ) 293T cells transfected with Becn1 and Rnf216 were treated with MG132 or E64d, and lysed before being subjected to SDS-PAGE, followed by transferring to nitrocellulose membrane. Then the membrane was blotted with anti-Flag or -Myc antibody, and then incubated with HRP-conjugated secondary antibody. The bands were visualized with an ECL chemiluminescence kit. The images displayed were representatives from 3 independent experiments.

Article Snippet: HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch (115–035–003, 111–035–003). si Becn1 targeting mouse Becn1 was obtained from Life Technologies (s80168).

Techniques: Inhibition, Plasmid Preparation, Western Blot, Transfection, SDS Page, Transferring, Incubation

Journal: Autophagy

Article Title: Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

doi: 10.4161/15548627.2014.981792

Figure Lengend Snippet: RNF216 interacts with BECN1 through the triad domain. ( A ) 293T cells transfected with plasmids of Flag- Rnf216 and Myc- Becn1 were immunoprecipitated with IgG, Myc (left panel) or Flag (right panel) antibodies. The precipitates were subjected to blotting with the indicated antibodies, and the bands were visualized with an ECL chemiluminescence kit. ( B ) RAW 264.7 cells subjected to starvation or LPS treatment were immunoprecipitated with an antibody against RNF216 (left panel) or BECN1 (right panel), followed by immunoblotting with the indicated antibodies, and the bands were visualized with an ECL chemiluminescence kit. The images displayed are representative of 3 independent experiments. The densitometry of the bands was quantified using ImageJ software, and is shown as mean ± SEM. ( C ) RAW 264.7 cells grown on coverslips were subjected to starvation or LPS (100 ng/mL) treatment, followed by staining with BECN1, RNF216, and DAPI. The images were captured with a confocal microscope. Colocalization of BECN1 and RNF216 was processed using LaserSharp 2000 software (Bio-Rad), is presented as a percentage of RNF216, and is shown as mean ± SEM calculated from 3 independent experiments (* P < 0.05). Scale bar = 10 μm. ( D ) Schematic diagram of RNF216 and truncation mutants used in this study. TIM, TRAF6 interacting domain; IBR, ‘in-between-RING’ domain. ( E ) Myc-tagged full-length RNF216 or truncates were transiently transfected into 293T cells. Cell lysates were analyzed by immunoblotting with anti-Myc antibody for the expression of RNF216 protein or by immunoprecipitation with anti-BECN1 antibody following by immunoblotting with the anti-Myc antibody for the association between RNF216 and BECN1 proteins.

Article Snippet: HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch (115–035–003, 111–035–003). si Becn1 targeting mouse Becn1 was obtained from Life Technologies (s80168).

Techniques: Transfection, Immunoprecipitation, Western Blot, Software, Staining, Microscopy, Expressing

Journal: Autophagy

Article Title: Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

doi: 10.4161/15548627.2014.981792

Figure Lengend Snippet: K48 ubiquitination mediated by RNF216 is necessary for BECN1 degradation. ( A ) RAW 264.7 cells were stimulated with LPS (100 ng/mL) for different periods of time, followed by immunoprecipitation with anti-RNF216 antibody, and then blotted with the indicated antibodies. Meanwhile, lysates were immunoblotted with different antibodies, followed by band visualization. ( B ) RAW 264.7 cells infected with lentivirus with scrambled shNC or sh Rnf 2 (MOI=10) were treated with or without LPS (at the indicated concentration) for 1 h, followed by immunoprecipitation with anti-RNF216 antibody, and then blotted with the indicated antibodies. Meanwhile, lysates were immunoblotted with different antibodies, followed by band visualization. ( C ) RAW 264.7 cells transfected with the indicated plasmids were subjected to treatments in the absence or presence of MG-132, and followed by immunoprecipitation with anti-Myc antibody, then blotted with anti-HA antibody. The bands were visualized with an ECL chemiluminescence kit. ( D ) RAW 264.7 cells transfected with the indicated plasmids (RING1Δ represents Rnf216 RING1Δ ) were subjected to immunoprecipitation with anti-Myc antibody, then blotted with anti-HA antibody. The bands were visualized with an ECL chemiluminescence kit. ( E ) Schematic illustration of HA-Ub wild type (WT) and mutants. ( F ) RAW 264.7 cells transfected with the indicated plasmids were subjected to immunoprecipitation with anti-Myc antibody, then blotted with anti-HA antibody. The bands were visualized with an ECL chemiluminescence kit. ( G ) RAW 264.7 cells transfected with the indicated plasmids were subjected to immunoblotting with indicated antibodies. The bands were visualized with an ECL chemiluminescence kit. The band densitometries were quantified using ImageJ software. The quantitative data were calculated from 3 independent experiments, and are shown as mean ± SEM. (* P < 0.05).

Article Snippet: HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch (115–035–003, 111–035–003). si Becn1 targeting mouse Becn1 was obtained from Life Technologies (s80168).

Techniques: Immunoprecipitation, Infection, Concentration Assay, Transfection, Western Blot, Software