eb2 kd stable cell lines  (Genechem)

Journal: Scientific Reports

Article Title: Endophilin B2 promotes inner mitochondrial membrane degradation by forming heterodimers with Endophilin B1 during mitophagy

doi: 10.1038/srep25153

Figure Lengend Snippet: Alignment of protein sequences is exhibited using ESPript 3.0. Conserved residues (red) and similar residues (yellow) are indicated. The amphipathic helix 0 (light blue spirals) that precedes the BAR domain, and the helix 1 insertion (green spirals) were conserved between EB1 and EB2, whereas the coiled-coil domains (yellow spirals) were not conserved. Dimer motifs in the coiled-coil domain are marked with black boxes.

Article Snippet: For the establishment of EB1 and EB2 KD stable cell lines, HEK 293T cells were transfected with lentiviral packaging plasmids and related shRNA vectors (GV112, Genechem).

Techniques:

Journal: Scientific Reports

Article Title: Endophilin B2 promotes inner mitochondrial membrane degradation by forming heterodimers with Endophilin B1 during mitophagy

doi: 10.1038/srep25153

Figure Lengend Snippet: ( A – C ) EB2 aggregated into foci and partially translocated to mitochondria in response to CCCP treatment. HeLa (Parkin-flag expressed) cells expressing mitochondria-DsRed (red) and CFP-EB1 or CFP-EB2 (green) were treated with 20 μM CCCP for the indicated times and analyzed using confocal laser scanning microscope. Magnified images are shown in the insets. Scale bar, 5 μm. The number of GFP positive dots per cell and the number of co-localization with mitochondria (the number of yellow dots) per cell are shown in panels ( B , C ), respectively (n = 19, mean ± SD). ( D ) Single foci of EB2 translocated to fragmented mitochondria under CCCP treatment. HeLa (Parkin-flag expressed) cells co-expressing DsRed-EB2 (red) and mitochondria-GFP (green) were treated with 20 μM CCCP for 12 h and then analyzed by time-lapse fluorescent microscopy at 1-s intervals. Red, green and yellow arrows indicate EB2, fragmented mitochondria, and double-positive signals, respectively.

Article Snippet: For the establishment of EB1 and EB2 KD stable cell lines, HEK 293T cells were transfected with lentiviral packaging plasmids and related shRNA vectors (GV112, Genechem).

Techniques: Expressing, Laser-Scanning Microscopy, Microscopy

Journal: Scientific Reports

Article Title: Endophilin B2 promotes inner mitochondrial membrane degradation by forming heterodimers with Endophilin B1 during mitophagy

doi: 10.1038/srep25153

Figure Lengend Snippet: ( A , B ) Only EB2-positive EB1 signals were found to co-localize with fragmented mitochondria. HeLa (Parkin-flag expressed) co-expressing YFP-EB2 (green), DsRed-EB1 (red) and mitochondria-CFP (blue) were treated with 20 μM CCCP or DMSO for 24 h. Triple-positive signals are indicated by arrowheads. Magnified images are shown in the insets. Scale bar, 5 μm. The fluorescence intensities along the dotted arrow were quantified using NIS-Elements AR-Analysis software and presented in panel ( B ). The length of horizontal dotted arrow in panel B represents distance of dotted arrow in panel ( A ). ( C ) Low expression of EB1 and EB2 in EB1 and EB2 knockdown cell lines were confirmed by western blotting respectively. ( D – F ) Loss of one of EB protein altered the number of foci and percentages of translocation to mitochondria of the other EB proteins in response to CCCP treatment. Cells co-expressing with either CFP-EB1 (green) or CFP-EB2 (green) and mitochondria-DsRed (red) were treated with 20 μM CCCP for 24 h. Scale bar, 5 μm. The number of green and yellow dots per cell and percentages of co-localization with damaged mitochondria (the number of yellow dots/green dots) are shown in panels E and F, respectively. Statistical significance was determined using t tests (n = 19, mean ± SD, * P < 0.05). ( G – I ) Knocking down of EB2 or EB1 alleviated Timm23 degradation in response to CCCP treatment. The cells were treated with 20 μM CCCP for 24 h and subjected to immunoblot analysis using the indicated antibodies. NC: negative control, EB1 KD: EB1 knockdown cells, EB2 KD: EB2 knockdown cells, Double KD: EB1- and EB2-knockdown cells, EB1 KD + EB1: CFP-EB1-expressing cells with knockdown of endogenous EB1, EB2 KD + EB2: CFP-EB2-expressing cells with knockdown of endogenous EB2.

Article Snippet: For the establishment of EB1 and EB2 KD stable cell lines, HEK 293T cells were transfected with lentiviral packaging plasmids and related shRNA vectors (GV112, Genechem).

Techniques: Expressing, Fluorescence, Software, Western Blot, Translocation Assay, Negative Control

Journal: Scientific Reports

Article Title: Endophilin B2 promotes inner mitochondrial membrane degradation by forming heterodimers with Endophilin B1 during mitophagy

doi: 10.1038/srep25153

Figure Lengend Snippet: ( A ) Schematic diagram summarizing interactions between EB1 and EB2. 293T cells were co-transfected with full-length and truncated EB1 or EB2 tagged with flag or CFP. Cell lysates were then subjected to immunoprecipitation with anti-flag monoclonal antibodies and then analyzed by immunoblotting. Immunoprecipitation results were marked by “+”, “−” and “n.d.”, which means “interaction”, “no interaction”, and “not determined”. ( B ) The roles of dimer domains of EB1 and EB2 in hetero-interaction between EB2 and EB1. ( C ) The roles of dimer domains of EB1 and EB2 in self-interaction of EB1 and EB2. ( D ) Effects of CCCP treatment on the hetero-interactions between EB1 and EB2.

Article Snippet: For the establishment of EB1 and EB2 KD stable cell lines, HEK 293T cells were transfected with lentiviral packaging plasmids and related shRNA vectors (GV112, Genechem).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: Endophilin B2 promotes inner mitochondrial membrane degradation by forming heterodimers with Endophilin B1 during mitophagy

doi: 10.1038/srep25153

Figure Lengend Snippet: ( A , B ) Co-localization of EB1 and EB2 foci was not mediated by the dimer domain of EB2. HeLa (Parkin-flag expressed) cells co-expressing with RFP-EB1 (red) and CFP-EB2 or CFP-EB2Δdimer (green) were treated with 20 μM CCCP for 20 h. Scale bar, 5 μm. Co-localization of RFP-EB1 with EB2 or EB2Δdimer is shown in the panel ( A ) and the number of dots (green column represent green dots and yellow column represent yellow dots) and the percentage of co-localization is shown in panel ( B ). Statistical significance was determined using t tests (n = 19, mean ± SD). n.s. represents for no significant difference. ( C–E ) Loss of the EB2 dimer domain reduced the translocation of EB2 foci. HeLa (Parkin-flag expressed) cells expressing the full-length or truncated EB1 or EB2 were treated with 20 μM CCCP for 20 h. Images from microscopic analysis are shown in panel ( A ). Scale bar, 5 μm. The number of green dots and yellow dots and the percentages of foci translocation to mitochondria are shown in panels ( D,E ), respectively. Statistical significance was determined using t test (n = 19, mean ± SD, *P < 0.05). ( F–G ) Disruption of heterodimerization alleviated Timm23 degradation in response to CCCP treatment. Negative control (NC), EB1-knockdown (EB1 KD) and EB2-knockdown (EB2 KD) cells expressing the indicated proteins were treated with or without 20 μM CCCP for 20 h as shown. Cells were then subjected to immunoblot analysis using the indicated antibodies.

Article Snippet: For the establishment of EB1 and EB2 KD stable cell lines, HEK 293T cells were transfected with lentiviral packaging plasmids and related shRNA vectors (GV112, Genechem).

Techniques: Expressing, Translocation Assay, Negative Control, Western Blot

Journal: Scientific Reports

Article Title: Endophilin B2 promotes inner mitochondrial membrane degradation by forming heterodimers with Endophilin B1 during mitophagy

doi: 10.1038/srep25153

Figure Lengend Snippet: ( A,B ) The formation of EB family protein foci was abolished in Pink1- and Parkin-deficient cells. The indicated proteins were co-expressed in Parkin +/+ (wild type [WT]), Parkin −/− (knockout [KO]), and PINK1-knockdown MEFs, and cells were treated with 30 μM CCCP for 12 h. Microscopic images are shown in panel ( A ), red, blue and purple column represent EB1, EB2 and EB1EB2 respectively. Scale bar, 10 μm. The number of foci is shown in panel ( B ). Statistical significance was determined using t test (n = 19, mean ± SD, ** P < 0.01). (C–D) Parkin translocation to fragmented mitochondria in EB2 KD cells in response to CCCP treatment. Parkin-GFP and mitochondria-DsRed were co-expressed in NC and EB2 KD cells, and cells were treated with CCCP for 9 h. Microscopic images are shown in panel ( C ). Scale bar, 10 μm. Histogram shows Pearson’s coefficient between Parkin and damaged mitochondria in WT or EB2 KD cells in panel ( D ). Statistical significance was determined by using t test. n.s. represents for no significant difference. ( E ) Timm23 degradation was significantly limited in Parkin −/− MEFs and PINK1 KD MEFs in response to CCCP treatment. Indicated cells were treated with or without CCCP as shown. Cells were then subjected to immunoblot analysis using the indicated antibodies. ( F ) Parkin and PINK1 expression levels in Parkin −/− and PINK1 KD cells were examined by immunoblotting.

Article Snippet: For the establishment of EB1 and EB2 KD stable cell lines, HEK 293T cells were transfected with lentiviral packaging plasmids and related shRNA vectors (GV112, Genechem).

Techniques: Knock-Out, Translocation Assay, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Endophilin B2 promotes inner mitochondrial membrane degradation by forming heterodimers with Endophilin B1 during mitophagy

doi: 10.1038/srep25153

Figure Lengend Snippet: Mitochondria and different forms of EB1 and EB2 diffused in cytoplasm are shown. In response to mitochondrial damage, Parkin was recruited by PINK1 on OMM. Subsequently, heterodimers in the cytoplasm aggregated into foci and fused with the EB1 positive phagophore to induce mitochondria sequestration. Without heterodimerization, homodimers aggregated into more foci to compensate for loss of heterodimers, but mitochondria sequestration and IMM degradation were significantly limited.

Article Snippet: For the establishment of EB1 and EB2 KD stable cell lines, HEK 293T cells were transfected with lentiviral packaging plasmids and related shRNA vectors (GV112, Genechem).

Techniques: