Journal: Oncotarget

Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

doi: 10.18632/oncotarget.22287

Figure Lengend Snippet: ( A ) G-TPP treatment leads to PINK1 stabilization and pS65-Ub induction in HeLa cells. HeLa cells stably expressing untagged Parkin were treated with 10 µM G-TPP for the indicated times. Western blots were prepared with cell lysates and probed with antibodies against PINK1 and pS65-Ub. GAPDH served as a loading control. ( B ) pS65-Ub is induced in G-TPP treated cells and co-localizes with EGFP-Parkin and mitochondria. HeLa cells stably expressing EGFP-Parkin (green) were treated with 10 µM G-TPP for the indicated times and fixed. Cells were stained with antibodies against pS65-Ub (red) and the mitochondrial marker TOM20 (cyan). Scale bars correspond to 10 µM. ( C ) Quantification of Parkin translocation using High Content Imaging. HeLa EGFP-Parkin cells were treated for 4 or 8 h with or without 10 µM G-TPP. CCCP treatment (10 µM for 2 h) was used as a positive control. Cells were fixed, counterstained with Hoechst dye to visualize nuclei, imaged and analyzed using the ratio of cytoplasmic to nuclear EGFP signal . Data was normalized to positive (2 h 10 µM CCCP treatment) and negative (2 h DMSO) controls. G-TPP significantly induced Parkin re-localization to levels similar to or beyond 2 h CCCP treatment. Shown are the mean values of three independent experiments with triplicate wells each ± SEM (one-way ANOVA with Tukey’s posthoc, *** p < 0.0005).

Article Snippet: The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).

Techniques: Stable Transfection, Expressing, Western Blot, Control, Staining, Marker, Translocation Assay, Imaging, Positive Control

Journal: Oncotarget

Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

doi: 10.18632/oncotarget.22287

Figure Lengend Snippet: ( A , B ) HeLa cells stably expressing EGFP-Parkin were treated with 12 different concentrations of G-TPP or 17-AAG in a dose-response format for 2 or 6 h before adding (A) DMSO or (B) CCCP (final assay concentration = 10 µM) for an additional 2 h. Cells were fixed and analyzed for Parkin translocation (black line). Cell number was assessed by counting the number of Hoechst-positive nuclei in each well (gray line). Values were normalized to positive (2 h 10 µM CCCP) and negative (2 h DMSO) controls. In absence of CCCP, only G-TPP but not 17-AAG induced Parkin translocation. In combination with CCCP, increasing doses of 17-AAG led to inhibition of Parkin translocation. Very high concentrations of G-TPP also inhibited Parkin translocation and resulted in cell toxicity. ( C ) HeLa cells expressing untagged Parkin were treated with 1 µM 17-AAG, 10 µM G-TPP or DMSO as a control for 6 h before CCCP (10 µM) or medium containing DMSO was added for 4 h. Cells were harvested and western blots probed with antibodies against PINK1, pS65-Ub, the mitochondrial phosphatase PGAM5. Vinculin was used as a loading control. Compared to controls, 17-AAG pre-treated cells showed lower PINK1 levels that were accompanied by reduced pS65-Ub induction upon CCCP treatment, while G-TPP pre-treatment led to induction of pS65-Ub in the absence of CCCP, as expected.

Article Snippet: The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).

Techniques: Stable Transfection, Expressing, Concentration Assay, Translocation Assay, Inhibition, Control, Western Blot

Journal: Oncotarget

Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

doi: 10.18632/oncotarget.22287

Figure Lengend Snippet: ( A , C ) Fibroblasts were treated with 15 µM G-TPP for the indicated time points. Cells were harvested and western blots were probed with antibodies against (A) PINK1, pS65-Ub and total Ub or (C) autophagy adapter proteins. GAPDH and Vinculin served as loading control. G-TPP treatment led to PINK1 stabilization and pS65-Ub induction in primary skin fibroblasts. p62 levels were induced upon G-TPP treatment, while other adapters seemed decreased. ( B , D ) Human fibroblasts were treated with 15 µM G-TPP for 16 h and fixed and stained with antibodies against (B) pS65-Ub (green) or (D) the autophagy adapters NBR1, NDP52, p62, OPTN and TAX1BP1 (green). Mitochondria were stained with antibodies against TOM20 (red), nuclei were visualized with Hoechst (blue). Scale bars indicate 10 µM. A magnified image of the boxed region, the fluorescence profile along the arrow and the Pearson’s correlation coefficient of adapter protein and mitochondrial stainingare shown to the right. Shown is the mean ± SEM of at least five randomly selected images (unpaired, two-sided t -test, *** p < 0.0005).

Article Snippet: The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).

Techniques: Western Blot, Control, Staining, Fluorescence

Journal: Oncotarget

Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

doi: 10.18632/oncotarget.22287

Figure Lengend Snippet: ( A ) Control cells or PINK1 Q456X fibroblasts were converted to induced neurons (iNeurons). Cells were treated for the indicated times with 10 µM G-TPP and harvested. Western blots were prepared and probed with antibodies against PINK1, pS65-Ub and Parkin. Beta III tubulin served as a control for successful conversion to neuronal cells, GAPDH as a loading control. ( B ) iNeurons were treated with 15 µM G-TPP for 8 h and fixed. Cells were stained with antibodies against pS65-Ub (green) and the mitochondrial marker TOM20 (red) and the neuronal marker Beta III tubulin (cyan). Nuclei were stained with Hoechst 33342 (blue). Scale bars correspond to 10 µM. A magnified image of the boxed region and the fluorescence profile along the arrow are shown to the right.

Article Snippet: The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).

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

Journal: Biochemical Journal

Article Title: α-Lipoic acid promotes α-tubulin hyperacetylation and blocks the turnover of mitochondria through mitophagy

doi: 10.1042/bcj20160281

Figure Lengend Snippet: Figure 6 αLA treatment prevents the removal of depolarized mitochondria through PINK1-mediated mitophagy

Article Snippet: Protein expression was analysed using the following antibodies: mouse monoclonal acetyl-lysine (Ac-K), mouse α-tubulin, rabbit glyceraldehyde 3-phosphate dehydrogenase (GAPDH), rabbit glutamate dehydrogenase (GDH), rabbit acetyl-tubulin K40, rabbit HDAC6, rabbit LC3A/B, rabbit COX IV, rabbit succinate dehydrogenase A (SDHA), rabbit phospho-ACC from Cell Signaling Technologies; rabbit PINK1 from Novus Biochemicals.

Techniques:

Journal: Life

Article Title: Spermine Ameliorates DSS-Induced Ulcerative Colitis in Mice by Improving Mitophagy and Intestinal Microbiota

doi: 10.3390/life16030417

Figure Lengend Snippet: Spermine treatment inhibited NLRP3-mediated inflammatory response in DSS-treated mice. ( A – E ) Genes expressions related to NLRP3-mediated inflammatory response in the colon were measured. ( F – I ) The levels of pro-inflammatory cytokines in the serum. ( J ) Detection of NLRP3 inflammasome protein expression levels in the colon using immunohistochemistry. Data are presented as mean ± SEM (* p < 0.05, *** p < 0.001 vs. control group. ## p < 0.01, and ### p < 0.001 vs. DSS group).

Article Snippet: The sections were then incubated at 4 °C overnight with primary antibodies targeting NLRP3 (1:500, Proteintech, 30109-1-AP, Chicago, IL, USA), PINK1 (1:500, Proteintech, 23274-1-AP, Chicago, IL, USA), and LC3 (1:500, Proteintech, 14600-1-AP, Chicago, IL, USA).

Techniques: Expressing, Immunohistochemistry, Control

Journal: Life

Article Title: Spermine Ameliorates DSS-Induced Ulcerative Colitis in Mice by Improving Mitophagy and Intestinal Microbiota

doi: 10.3390/life16030417

Figure Lengend Snippet: Potential mechanism of Spermine treatment alleviating DSS-induced colitis. First, spermidine enhances mitochondrial autophagy by upregulating the expression of PINK1/Parkin, thereby eliminating damaged mitochondria and reducing the production of reactive oxygen species. This, in turn, inhibits the activation of the NLRP3 inflammasome and the subsequent release of IL-1β/IL-18, thereby alleviating the inflammatory response. Secondly, spermidine can reverse the intestinal flora imbalance induced by DSS, manifested as a decrease in the ratio of Firmicutes to Bacteroidetes, inhibition of Blautia enrichment, and restoration of the abundance of beneficial Muribaculaceae. These two pathways work together in synergy, jointly forming an integrated mechanism by which spermidine inhibits the NLRP3-mediated inflammatory response, promotes mitochondrial autophagy, and improves intestinal flora imbalance to alleviate DSS-induced colitis.

Article Snippet: The sections were then incubated at 4 °C overnight with primary antibodies targeting NLRP3 (1:500, Proteintech, 30109-1-AP, Chicago, IL, USA), PINK1 (1:500, Proteintech, 23274-1-AP, Chicago, IL, USA), and LC3 (1:500, Proteintech, 14600-1-AP, Chicago, IL, USA).

Techniques: Expressing, Activation Assay, Inhibition

Journal: The Journal of Cell Biology

Article Title: Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin

doi: 10.1083/jcb.201007013

Figure Lengend Snippet: Mitofusin degradation requires PINK1 expression. (a) M17 cells expressing control or PINK1 shRNA were treated with CCCP or CCCP plus MG132. Lysates were immunoblotted with anti-Mfn1, Mfn2, PINK1, and Tim23. CCCP, 20 µM for 4 h; MG132, 30 µM, 30 min prior and with CCCP. (b) The average of Mfn1 or Mfn2 levels normalized to the level of Tim23 is presented with the standard deviation indicated by the error bars. Each graph was generated from three independent experiments from panel a. The protein levels in control (nontreated, control shRNA) cells were set as 1. (c) HeLa cells stably transfected with YFP-Parkin were pretreated with CHX before treatment with CCCP, as indicated on top. Lysates were immunoblotted with anti-Mfn1, Mfn2, PINK1, and Tim23. CCCP, 10 µM for 90 min; CHX, 100 µM, 30 min prior as a pretreatment and during incubation with or without CCCP. (d) The average of the Mfn1 or Mfn2 level normalized to the level of Tim23 is presented with the standard deviation indicated by the error bars. Each graph was generated from three independent experiments from c. The protein levels in control (nontreated) cells were set as 1. (e) SH-SY5Y cells were pretreated with CHX before the treatments of CCCP, as indicated on top. Lysates were immunoblotted with anti-Mfn1, Mfn2, PINK1, and Tim23. CCCP, 20 µM for 2 h; CHX, 100 µM, 30 min prior as a pretreatment and during incubation with or without CCCP. Molecular mass is indicated in kilodaltons next to the gel blots. (f) The average of the Mfn1 or Mfn2 level normalized to the level of Tim23 is presented with the standard deviation indicated by the error bars. Each graph was generated from three independent experiments from e. The protein levels in control (nontreated) cells were set as 1.

Article Snippet: Commercial antibodies were purchased as follows: Opa1 (BD), Fis1 (Enzo Life Sciences, Inc.), Drp1 (BD), VDAC (EMD), Tim23 (BD), Hsp60 (Stressgen), Bip (BD), Tom 20 (BD), Tom40 (Santa Cruz Biotechnology, Inc.), cytochrome c (BD), Ub (P4D1 [Santa Cruz Biotechnology, Inc.] or Fk1 [Enzo Life Sciences, Inc.]), PINK1 (BD), Parkin (PRK8; Santa Cruz Biotechnology, Inc.), and p97 (Thermo Fisher Scientific).

Techniques: Expressing, Control, shRNA, Standard Deviation, Generated, Stable Transfection, Transfection, Incubation

Journal: The Journal of Cell Biology

Article Title: Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin

doi: 10.1083/jcb.201007013

Figure Lengend Snippet: p97 recruitment to ubiquitinated mitochondria mediates PINK1–Parkin-induced mitochondrial elimination. (a) MEFs from PINK1 +/+ or PINK1 −/− mice were transiently transfected with YFP-Parkin and Myc-p97. Cells were treated with DMSO or CCCP, then immunostained with anti-Myc and anti–cytochrome c . CCCP, 20 µM for 2 h. Bar, 10 µm. (b) HeLa cells transiently expressing Myc-p97 or YFP-Parkin/Myc-p97 were treated with DMSO or CCCP. Cells were immunostained with anti-Myc and anti-Ub (fk1). CCCP, 10 µM for 90 min. The boxed regions are magnified in the bottom panels. (c) HeLa cells were transiently transfected with YFP-Parkin and wild-type Myc-p97 or Myc-p97 QQ . After treatment with CCCP for 24 h, cells were fixed and immunostained with anti-Myc and anti-Tom20 antibodies. CCCP, 10 µM. The boxed regions are magnified in the bottom panels. Bars: (top panels) 10 µm; (bottom enlarged panels) 1 µm. (d) Parkin-induced mitophagy in cells shown as in c was quantified ( n > 50). CCCP, 10 µM for 24 h. Data represent the mean ± SD with P values of at least three replicates. (e) HeLa cells or HeLa cells expressing YFP-Parkin were treated with CCCP or CCCP plus MG132. Mitochondria were immunostained with anti-Tom20. Cells expressing YFP-Parkin are indicated with asterisks. CCCP, 10 µM for 24 h; MG132, 30 µM 30 min prior and with CCCP. Bar, 10 µm. (f) MG132 blocks Parkin-induced mitophagy in HeLa cells as in d. A quantification is shown. Data represent the mean ± SD with P values of at least three replicates.

Article Snippet: Commercial antibodies were purchased as follows: Opa1 (BD), Fis1 (Enzo Life Sciences, Inc.), Drp1 (BD), VDAC (EMD), Tim23 (BD), Hsp60 (Stressgen), Bip (BD), Tom 20 (BD), Tom40 (Santa Cruz Biotechnology, Inc.), cytochrome c (BD), Ub (P4D1 [Santa Cruz Biotechnology, Inc.] or Fk1 [Enzo Life Sciences, Inc.]), PINK1 (BD), Parkin (PRK8; Santa Cruz Biotechnology, Inc.), and p97 (Thermo Fisher Scientific).

Techniques: Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin

doi: 10.1083/jcb.201007013

Figure Lengend Snippet: Mitochondrial fission is required for Parkin-mediated mitophagy. (a) MEFs from DRP1 +/+ or DRP1 −/− mice were transiently transfected with YFP-Parkin. Cells were treated with CCCP and then immunostained with anti-Tom20. Cells expressing YFP-Parkin are indicated with asterisks. CCCP, 20 µM. Bar, 10 µm. (b) Whole cell lysates from DRP1 +/+ or DRP1 −/− MEFs with or without transient expression of Parkin were subjected to SDS-PAGE. Molecular mass is indicated in kilodaltons next to the gel blots. (c) The average of the Mfn1 or Mfn2 level normalized to the level of Tim23 is presented with the standard deviation indicated by the error bars. Each graph was generated from three independent experiments from b. The protein levels in control (nontreated, nontransfected) cells were set as 1. (d) Clumping mitochondria in DRP1 +/+ or DRP1 −/− MEFs transiently expressing mCherry-Parkin and Mito-YFP were subjected to the FRAP assay. The curve represents an average of 30 individual FRAP curves. CCCP, 20 µM for 8 h. (e) The mobile fractions of Mito-YFP in d. (f and g) YFP-Parkin translocation (f) and YFP-Parkin–induced mitophagy (g) in DRP1 +/+ or DRP1 −/− MEFs treated with CCCP as in panel a were quantified ( n > 50). CCCP, 20 µM for 24 h. Data represent the mean ± SD with P values of at least three replicates. (h) HeLa cells treated with DRP1 shRNA or control HeLa cells were transiently transfected with YFP-Parkin and treated with or without CCCP. Mitochondria were immunostained with anti-Tom20. Cells expressing YFP-Parkin are indicated with asterisks. CCCP, 10 µM for 24 h. Bar, 10 µm. (i) YFP-Parkin–induced mitophagy in HeLa cells with or without DRP1 RNAi as in h was quantified ( n > 50). CCCP, 10 µM for 24 h. Data represent the mean ± SD with P values of at least three replicates. (j and k) YFP-Parkin–induced mitophagy in MFN1 +/+ 2 +/+ MEFs or MFN1 −/− 2 −/− MEFs was observed (j) and quantified (k) ( n > 50). CCCP, 20 µM for 24 h. Data represent the mean ± SD with P values of at least three replicates. Cells expressing YFP-Parkin are indicated with asterisks. (l) A model for Parkin-mediated Mfn degradation and mitophagy. Depolarized mitochondria (orange) that accumulated PINK1 (gray) are sensed by Parkin (green). After Parkin recruitment to the damaged mitochondria, mitochondrial Parkin induces mitofusin (blue) ubiquitination and degradation. Degradation of mitofusin is regulated by p97 (purple) and is important to prevent impaired mitochondria from fusing with the vital mitochondrial network. Damaged mitochondria isolated by Parkin are engaged with autophagosomes and eliminated by auto-lysosomes.

Article Snippet: Commercial antibodies were purchased as follows: Opa1 (BD), Fis1 (Enzo Life Sciences, Inc.), Drp1 (BD), VDAC (EMD), Tim23 (BD), Hsp60 (Stressgen), Bip (BD), Tom 20 (BD), Tom40 (Santa Cruz Biotechnology, Inc.), cytochrome c (BD), Ub (P4D1 [Santa Cruz Biotechnology, Inc.] or Fk1 [Enzo Life Sciences, Inc.]), PINK1 (BD), Parkin (PRK8; Santa Cruz Biotechnology, Inc.), and p97 (Thermo Fisher Scientific).

Techniques: Transfection, Expressing, SDS Page, Standard Deviation, Generated, Control, FRAP Assay, Translocation Assay, shRNA, Ubiquitin Proteomics, Isolation

Journal: Molecular Medicine

Article Title: Complement protein C1q induces endothelial apoptosis through C1qBP regulation of mitochondrial function in lung endothelial cells in systemic sclerosis

doi: 10.1186/s10020-026-01445-0

Figure Lengend Snippet: C1qBP affected PHB2 phosphorylation and cytoplasmic translocation as well as mitochondrial dysfunction in endothelial cells. BLM-induced endothelial cells were treated with si-C1qBP. A - B Western blot analysis was utilized to examine the effects of si-C1qBP on C1qBP levels and the phosphorylation and cytoplasmic translocation of PHB2. C - D The binding and co-localization of C1qBP with PHB2 were observed through co-immunoprecipitation and immunofluorescence. E - F Western blot was performed to evaluate the impact of C1qBP on the expression of apoptosis-related proteins (BAX and Bcl-2), mitophagy-related proteins (Beclin1, Parkin, Pink1), as well as UPRmt-related markers (mtDnaJ, ClpP, LonP1, Hsp10). G Red CMXRos staining was used to assess the effect of C1qBP on mitochondrial activity in endothelial cells. H The influence of C1qBP on intracellular Ca 2+ levels in endothelial cells was measured by Fluo-4 AM fluorescent probe detection. I - J MMP detection was conducted to observe the effect of C1qBP on the MMP of endothelial cells. K - L The impact of C1qBP on ATP levels and electron transport chain (complexes I-IV) activity in endothelial cells was determined by ATP colorimetric/fluorescence assay and mitochondrial electron transport chain activity detection. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Con/ BLM + si-NC

Article Snippet: Antibodies used included: C1qA Primary Antibody (1:1,000, A1821, Abclonal), C1qB Primary Antibody (1:1,000, BD-PB4657, Biodragon), C1qBP Primary Antibody (1:1,000, 68084-1-Ig, Proteintech), COV IV Primary Antibody (1:1, 000, 4850 S, Cellular), PHB2 primary antibody (1:1,000, 66424-1-Ig, Proteintech), Cleaved caspase-3 primary antibody (1:1,000, 68773-1-Ig, Proteintech), ICAM1 primary antibody (1:1,000, A20472, Abclonal), ZO1 primary antibody (1:1,000, 21773-1-AP, Proteintech), ZO2 primary antibody (1:1,000, 18900-1-AP, Proteintech), OCLN primary antibody (1:1,000, 27260-1-AP, Proteintech), JAM3 primary antibody (1:1,000, BD-PN0240, Biodragon), BAX primary antibody (1:1,000, CY5059, Abways), Bcl-2 primary antibody (1:1,000, ab182858, Abcam), Beclin1 primary antibody (1:1,000, 3738 S, Cell signalling), Parkin primary antibody (1:1,000, CY6641, Abways), Pink1 primary antibody (1:1,000, BY0130, Abways), ClpP primary antibody (1:1,000, ab124822, Abcam), mtDnaJ primary antibody (1:1,000, ab124822, Abcam), LonP1 primary antibody (1:1,000, A03808-2, BOSTER), Hsp10 primary antibody (1:1,000, CY8473, Abways), and β-actin primary antibody (1:1,000, 66009-1-Ig, Proteintech).

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Binding Assay, Immunoprecipitation, Immunofluorescence, Expressing, Staining, Activity Assay, Fluorescence

Journal: Molecular Medicine

Article Title: Complement protein C1q induces endothelial apoptosis through C1qBP regulation of mitochondrial function in lung endothelial cells in systemic sclerosis

doi: 10.1186/s10020-026-01445-0

Figure Lengend Snippet: C1qBP promoted endothelial cell apoptosis by causing mitochondrial dysfunction through affecting the cytosolic translocation of PHB2. A PHB2-S293A phosphorylation-deficient mutant was constructed. BLM-induced endothelial cells were co-transfected with PHB2-S293A (WT or MUT) and siRNA (si-NC or si-C1qBP), respectively. A Western blot analysis of C1qBP, PHB2 and p-PHB2 expression. B Western blot analysis of apoptosis- and mitophagy-related proteins BAX, Bcl-2, Beclin1, Parkin, and Pink1. C - D Immunofluorescence detection of BAX, Bcl-2, Beclin1, Parkin and Pink1 expression. E Western blot analysis of mitochondrial unfolded protein response (UPRmt)-related markers mtDnaJ, ClpP, LonP1 and Hsp10. F Red CMXRos staining to detect mitochondrial activity. G Fluo-4 AM fluorescent probe to measure intracellular Ca 2+ levels. H Evaluation of mitochondrial electron transport chain (ETC) activity. I JC-1 assay to detect mitochondrial membrane potential (MMP). J ATP colorimetric/fluorometric assay kit to measure ATP levels. K Flow cytometry analysis of the number of viable endothelial cells. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. BLM + si-NC+PHB2-S293A WT/ BLM + si-C1qBP+PHB2-S293A WT/ BLM + si-NC+PHB2-S293A MUT. ns means no significant difference vs. BLM + si-NC+PHB2-S293A WT/ BLM + si-C1qBP+PHB2-S293A WT

Article Snippet: Antibodies used included: C1qA Primary Antibody (1:1,000, A1821, Abclonal), C1qB Primary Antibody (1:1,000, BD-PB4657, Biodragon), C1qBP Primary Antibody (1:1,000, 68084-1-Ig, Proteintech), COV IV Primary Antibody (1:1, 000, 4850 S, Cellular), PHB2 primary antibody (1:1,000, 66424-1-Ig, Proteintech), Cleaved caspase-3 primary antibody (1:1,000, 68773-1-Ig, Proteintech), ICAM1 primary antibody (1:1,000, A20472, Abclonal), ZO1 primary antibody (1:1,000, 21773-1-AP, Proteintech), ZO2 primary antibody (1:1,000, 18900-1-AP, Proteintech), OCLN primary antibody (1:1,000, 27260-1-AP, Proteintech), JAM3 primary antibody (1:1,000, BD-PN0240, Biodragon), BAX primary antibody (1:1,000, CY5059, Abways), Bcl-2 primary antibody (1:1,000, ab182858, Abcam), Beclin1 primary antibody (1:1,000, 3738 S, Cell signalling), Parkin primary antibody (1:1,000, CY6641, Abways), Pink1 primary antibody (1:1,000, BY0130, Abways), ClpP primary antibody (1:1,000, ab124822, Abcam), mtDnaJ primary antibody (1:1,000, ab124822, Abcam), LonP1 primary antibody (1:1,000, A03808-2, BOSTER), Hsp10 primary antibody (1:1,000, CY8473, Abways), and β-actin primary antibody (1:1,000, 66009-1-Ig, Proteintech).

Techniques: Translocation Assay, Phospho-proteomics, Mutagenesis, Construct, Transfection, Western Blot, Expressing, Immunofluorescence, Staining, Activity Assay, Membrane, ATP Colorimetric Fluorometric Assay, Flow Cytometry

Journal: Molecular Medicine

Article Title: Complement protein C1q induces endothelial apoptosis through C1qBP regulation of mitochondrial function in lung endothelial cells in systemic sclerosis

doi: 10.1186/s10020-026-01445-0

Figure Lengend Snippet: C1qBP knockdown promoted mitophagy, alleviated endothelial cell apoptosis, and pulmonary fibrosis. A - B Immunohistochemical detection of BAX, Bcl-2, Beclin1, Parkin, and Pink1 expression. C TUNEL and CD31 co-staining assay was conducted to detect apoptosis. D Evaluation of pulmonary microvascular permeability in mice by Evans Blue extravasation assay. E Co-localization of pimonidazole with CD31 in fibrotic lung tissue. F Immunofluorescence co-staining of CD31 with HIF-1α

Article Snippet: Antibodies used included: C1qA Primary Antibody (1:1,000, A1821, Abclonal), C1qB Primary Antibody (1:1,000, BD-PB4657, Biodragon), C1qBP Primary Antibody (1:1,000, 68084-1-Ig, Proteintech), COV IV Primary Antibody (1:1, 000, 4850 S, Cellular), PHB2 primary antibody (1:1,000, 66424-1-Ig, Proteintech), Cleaved caspase-3 primary antibody (1:1,000, 68773-1-Ig, Proteintech), ICAM1 primary antibody (1:1,000, A20472, Abclonal), ZO1 primary antibody (1:1,000, 21773-1-AP, Proteintech), ZO2 primary antibody (1:1,000, 18900-1-AP, Proteintech), OCLN primary antibody (1:1,000, 27260-1-AP, Proteintech), JAM3 primary antibody (1:1,000, BD-PN0240, Biodragon), BAX primary antibody (1:1,000, CY5059, Abways), Bcl-2 primary antibody (1:1,000, ab182858, Abcam), Beclin1 primary antibody (1:1,000, 3738 S, Cell signalling), Parkin primary antibody (1:1,000, CY6641, Abways), Pink1 primary antibody (1:1,000, BY0130, Abways), ClpP primary antibody (1:1,000, ab124822, Abcam), mtDnaJ primary antibody (1:1,000, ab124822, Abcam), LonP1 primary antibody (1:1,000, A03808-2, BOSTER), Hsp10 primary antibody (1:1,000, CY8473, Abways), and β-actin primary antibody (1:1,000, 66009-1-Ig, Proteintech).

Techniques: Knockdown, Immunohistochemical staining, Expressing, TUNEL Assay, Staining, Permeability, Immunofluorescence

Journal: Neural Regeneration Research

Article Title: Differential distribution of PINK1 and Parkin in the primate brain implies distinct roles

doi: 10.4103/nrr.nrr-d-23-01140

Figure Lengend Snippet: Figure 1 ｜A newly developed PINK1 monoclonal antibody shows selective expression of PINK1 in primate brain. (A) Schematic diagram of human PINK1 protein structure and the recognition sites of monoclonal PINK1 antibody E7B6. (B) HEK293 cells were transfected with human PINK1 cDNA. Western blot analysis of lysates from the cortex of wild type (WT) and PINK1 mutant (M6) monkeys and PINK1-transfected HEK293 cell lysates showing that E7B6 recognizes the endogenous monkey PINK1 and transfected human PINK1. (C) E7B6 western blot of monkey brain tissue showing various expression levels of PINK1 and Parkin. (D) The ratios of PINK1 to vinculin and Parkin to vinculin in monkey brain tissue of C. (E) E7B6 western blot analysis of human brain and peripheral tissue from a 54-year-old individual showing the selective expression of PINK1 in the human brain. In C and E, Parkin expression levels in various brain regions are different from those of PINK1. (F) The ratios of PINK1 to vinculin and Parkin to vinculin in human tissue from E. BS: Brain stem; Cereb: cerebellum; Ctx: cortex; Hippo: hippocampus; SN: substantia nigra; Str: striatum.

Article Snippet: Table 1 | Main antibodies used in this study Antibody Host Dilution Cat# RRID Supplier Application PINK1 Mouse 1:50 E7B6 NA Homemade antibody by MerryBio, Nanjing China WB, IF, IHC PINK1 Rabbit 1:500 BC100-494 AB_10127658 Novus Biologicals , Centennial, CO, USA WB PINK1 Rat 1:500 MAB4357 AB_2164246 R&D Systems, Minneapolis, MN, USA IF PINK1 Sheep 1:500 S086D AB_2924297 MRC, Scotland, UK WB Parkin Mouse 1:1000 sc-32282 AB_628104 Santa Cruz Biotechnology, Santa Cruz, CA, USA WB, IF Parkin Mouse 1:1000 MAB5512 AB_2267915 Millipore, Temecula, CA, USA IF Vinculin Mouse 1:5000 MAB3574 AB_2304338 Millipore WB (Internal reference) NeuN Rabbit 1:5000 ab177487 AB_2532109 Abcam, Cambridge, MA, USA WB, IF (Neuron) SNAP25 Rabbit 1 μg/mL ab41455 AB_945552 Abcam WB (Synaptosomal) TOM20 Rabbit 1:500 #42406 AB_2687663 Cell Signaling Technolog, Danvers, MA, USA IF (outer mitochondrial membrane) PSD95 Rabbit 1:2000 ab238135 AB_2895158 Abcam WB (Synaptosomal) Calnexin Rabbit 1:1000 ab22595 AB_2069006 Abcam WB (Endoplasmic reticulum) MBP Rabbit 1:3000 ab218011 AB_2895537 Abcam WB (Myelin) Histone H3 Rabbit 1:3000 #9715 AB_331563 Cell Signaling Technology WB (Nuclei) GAPDH Mouse 1:5000 60004-1-Ig AB_2107436 Proteintech, Rosemont, IL, USA WB (Cytosol) VDAC1 Rabbit 1:3000 ab15895 AB_2214787 Abcam WB (Mitochondria) RSP6 Rabbit 1:3000 2217S AB_331355 Cell Signaling WB (Ribosome) GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IF: immunofluorescence; IHC: immunohistochemistry; MBP: myelin basic protein; NeuN: neuronal nuclei; PSD95: postsynaptic density protein-95; RPS6: ribosomal protein S6; SNAP25: 25 kDa synaptosome-associated protein; WB: western blotting.

Techniques: Expressing, Transfection, Western Blot, Mutagenesis

Journal: Neural Regeneration Research

Article Title: Differential distribution of PINK1 and Parkin in the primate brain implies distinct roles

doi: 10.4103/nrr.nrr-d-23-01140

Figure Lengend Snippet: Figure 2 ｜Postnatal expression of PINK1 and PINK1 knockdown in monkey brain. (A) Western blotting of the monkey cortex from WT and PINK1 mutant (M6) monkeys showing that both BC100-494 and S086D antibodies specifically recognize endogenous PINK1 that is knocked down in M6. (B) Western blot of cortical lysates from monkeys at ages postnatal day 1 (P1), P140, 2 years, and 10 years, and human cortex. Three different PINK1 antibodies (E7B6, BC100-494, and S086D) were used to probe the tissue. (C) E7B6 western blotting analysis of lysates from the cortex of WT monkeys (WT1, P1; WT2, 5 years) and PINK1 mutant monkeys (M7, aborted embryo day 139; M1, P7; M2, P7) and a 5-year-old monkey injected with AAV9-PINK1 gRNA/Cas9 to knock down PINK1 (KD). Knocking down PINK1 can cause neuronal loss in developing and adult monkey brains. (D) AAV9 viral expression of CRISPR/Cas9 to target the PINK1 gene in the prefrontal cortex in the 5-year-old WT adult monkey for 6 weeks resulted in a remarkable loss of neuronal cells, which was evident by the reduced number of NeuN-positive cells. E7B6 immunostaining showed a significant reduction of PINK1 protein in the AAV9- PINK1 gRNA/Cas9-injected region compared with that in the control gRNA/Cas9 injected-region. Arrows indicate AAV-infected cells. Green: PINK1 sgRNA-targeted cells; pink: PINK1 (E7B6) staining; red: NeuN staining; blue: nuclear (DAPI) staining. (E) Glial fibrillary acidic protein (GFAP) immunostaining of the cortex from the same brain sections in D showing that the remaining GFP-positive cells in the AAV9-PINK1 gRNA/Cas9-injected region are GFAP-positive astrocytes. Green: AAV-PINK1 gRNA expression; red: GFAP staining; blue: nuclear staining (DAPI). (F) Quantitative assessment of NeuN-positive neurons in D and GFAP-positive astrocytes in E (n = 5 sections for each group). Data shown as mean ± SEM (n = 5). *P < 0.05, ***P < 0.001 (two-tailed Student’s t-test). In D and E, scale bars: 20 μm. For immunocytochemical analysis, the numbers of cells labeled by specific antibodies per image (40×) from at least three independent experiments were quantified. DAPI: 4′,6-Diamidino-2-phenylindole; ns: not significant; PSD95: postsynaptic density protein-95.

Article Snippet: Table 1 | Main antibodies used in this study Antibody Host Dilution Cat# RRID Supplier Application PINK1 Mouse 1:50 E7B6 NA Homemade antibody by MerryBio, Nanjing China WB, IF, IHC PINK1 Rabbit 1:500 BC100-494 AB_10127658 Novus Biologicals , Centennial, CO, USA WB PINK1 Rat 1:500 MAB4357 AB_2164246 R&D Systems, Minneapolis, MN, USA IF PINK1 Sheep 1:500 S086D AB_2924297 MRC, Scotland, UK WB Parkin Mouse 1:1000 sc-32282 AB_628104 Santa Cruz Biotechnology, Santa Cruz, CA, USA WB, IF Parkin Mouse 1:1000 MAB5512 AB_2267915 Millipore, Temecula, CA, USA IF Vinculin Mouse 1:5000 MAB3574 AB_2304338 Millipore WB (Internal reference) NeuN Rabbit 1:5000 ab177487 AB_2532109 Abcam, Cambridge, MA, USA WB, IF (Neuron) SNAP25 Rabbit 1 μg/mL ab41455 AB_945552 Abcam WB (Synaptosomal) TOM20 Rabbit 1:500 #42406 AB_2687663 Cell Signaling Technolog, Danvers, MA, USA IF (outer mitochondrial membrane) PSD95 Rabbit 1:2000 ab238135 AB_2895158 Abcam WB (Synaptosomal) Calnexin Rabbit 1:1000 ab22595 AB_2069006 Abcam WB (Endoplasmic reticulum) MBP Rabbit 1:3000 ab218011 AB_2895537 Abcam WB (Myelin) Histone H3 Rabbit 1:3000 #9715 AB_331563 Cell Signaling Technology WB (Nuclei) GAPDH Mouse 1:5000 60004-1-Ig AB_2107436 Proteintech, Rosemont, IL, USA WB (Cytosol) VDAC1 Rabbit 1:3000 ab15895 AB_2214787 Abcam WB (Mitochondria) RSP6 Rabbit 1:3000 2217S AB_331355 Cell Signaling WB (Ribosome) GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IF: immunofluorescence; IHC: immunohistochemistry; MBP: myelin basic protein; NeuN: neuronal nuclei; PSD95: postsynaptic density protein-95; RPS6: ribosomal protein S6; SNAP25: 25 kDa synaptosome-associated protein; WB: western blotting.

Techniques: Expressing, Knockdown, Western Blot, Mutagenesis, Injection, CRISPR, Immunostaining, Control, Infection, Staining, Two Tailed Test, Labeling

Journal: Neural Regeneration Research

Article Title: Differential distribution of PINK1 and Parkin in the primate brain implies distinct roles

doi: 10.4103/nrr.nrr-d-23-01140

Figure Lengend Snippet: Figure 5 ｜Relative expression levels of PINK1 and Parkin in subcellular fractions isolated from the cortex of three monkeys aged 9, 24, and 26 years. The levels were calculated using ImageJ, and the values are ratios to “Total.” Data are presented as mean ± SEM (n = 3 for each group) and were analyzed by GraphPad Prism 8.0.2 Paired. LP: Layer pellet; LP1: myelin; LP2: other subcellular structures; LP3: synaptosome; LP4: mitochondria; P: pellets; S: supernatant fluid.

Article Snippet: Table 1 | Main antibodies used in this study Antibody Host Dilution Cat# RRID Supplier Application PINK1 Mouse 1:50 E7B6 NA Homemade antibody by MerryBio, Nanjing China WB, IF, IHC PINK1 Rabbit 1:500 BC100-494 AB_10127658 Novus Biologicals , Centennial, CO, USA WB PINK1 Rat 1:500 MAB4357 AB_2164246 R&D Systems, Minneapolis, MN, USA IF PINK1 Sheep 1:500 S086D AB_2924297 MRC, Scotland, UK WB Parkin Mouse 1:1000 sc-32282 AB_628104 Santa Cruz Biotechnology, Santa Cruz, CA, USA WB, IF Parkin Mouse 1:1000 MAB5512 AB_2267915 Millipore, Temecula, CA, USA IF Vinculin Mouse 1:5000 MAB3574 AB_2304338 Millipore WB (Internal reference) NeuN Rabbit 1:5000 ab177487 AB_2532109 Abcam, Cambridge, MA, USA WB, IF (Neuron) SNAP25 Rabbit 1 μg/mL ab41455 AB_945552 Abcam WB (Synaptosomal) TOM20 Rabbit 1:500 #42406 AB_2687663 Cell Signaling Technolog, Danvers, MA, USA IF (outer mitochondrial membrane) PSD95 Rabbit 1:2000 ab238135 AB_2895158 Abcam WB (Synaptosomal) Calnexin Rabbit 1:1000 ab22595 AB_2069006 Abcam WB (Endoplasmic reticulum) MBP Rabbit 1:3000 ab218011 AB_2895537 Abcam WB (Myelin) Histone H3 Rabbit 1:3000 #9715 AB_331563 Cell Signaling Technology WB (Nuclei) GAPDH Mouse 1:5000 60004-1-Ig AB_2107436 Proteintech, Rosemont, IL, USA WB (Cytosol) VDAC1 Rabbit 1:3000 ab15895 AB_2214787 Abcam WB (Mitochondria) RSP6 Rabbit 1:3000 2217S AB_331355 Cell Signaling WB (Ribosome) GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IF: immunofluorescence; IHC: immunohistochemistry; MBP: myelin basic protein; NeuN: neuronal nuclei; PSD95: postsynaptic density protein-95; RPS6: ribosomal protein S6; SNAP25: 25 kDa synaptosome-associated protein; WB: western blotting.

Techniques: Expressing, Isolation

Journal: The FASEB Journal

Article Title: Tmx2 Maintains Mitochondrial Function to Support Preimplantation Embryogenesis

doi: 10.1096/fj.202500640r

Figure Lengend Snippet: FIGURE 6 | Tmx2 knockdown inhibits mitophagy and autophagy in morula-stage embryos. (A-D) Immunostaining for PINK1 (A), PARKIN (B), MAP1LC3B (C), and LAMP1 (D) in control and Tmx2-knockdown embryos. Scale bars: 75 μm. (E-H) Quantification of immunofluorescence inten- sity for PINK1 (E), PARKIN (F), MAP1LC3B (G), and LAMP1 (H) in control and Tmx2-knockdown morula-stage embryos. Sample sizes: PINK1: Control = 16, siRNA2 = 14, siRNA3 = 19; PARKIN: Control = 21, siRNA2 = 20, siRNA3 = 23; MAP1LC3B: Control = 22, siRNA2 = 20, siRNA3 = 21; LAMP1: Control = 30, siRNA2 = 32, siRNA3 = 31. Statistical significance: **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Article Snippet: The primary antibodies used included: rabbit anti- TMX2 (Origene, TA341391, 1:50), goat anti- OCT4 (Abcam, ab27985, 1:200), mouse antiCDX2 (Biogenex, MU392A- UC, 1:100), goat anti- SOX17 (R&D Systems, AF1924, 1:100), rabbit anti- NANOG (Abcam, ab80892, 1:50), rabbit anti- PINK1 (Affinity, DF7742, 1:50), rabbit antiPARKIN (Abcepta, AP6402B, 1:50), rabbit anti- MAP1LC3B (CUSABIO, CSB- PA013403GA01HU, 1:50), and rabbit antiLAMP1 (Beyotime, AF7353, 1:100).

Techniques: Knockdown, Immunostaining, Control, Immunofluorescence