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: Journal of Cellular Physiology

Article Title: Integrin-linked kinase regulates migration and proliferation of human intestinal cells under a fibronectin-dependent mechanism

doi: 10.1002/jcp.21963

Figure Lengend Snippet: Knockdown of ILK expression inhibits HIEC cell proliferation. HIEC and Caco-2/15 cells were transfected with 40 µM of siCNS or siILK. A: Proliferation indexes were determined by BrdU incorporation on transfected cells that were seeded on plastic or on exogenous fibronectin, and stained using an anti-BrdU antibody. BrdU-positive cells were counted and expressed as a percentage of total cells determined by DAPI staining. Results are expressed as mean ± standard error of the mean (SEM) and are from three independent experiments. * P < 0.05 in a paired t -test. B: HIEC-transfected cells were analyzed by Western blot for protein expression levels of ILK, pRB (upper band, hyperphosphorylated form; lower band, hypophosphorylated form), cyclin D, p21, and p27. β-actin was used as a loading control. Representative Western analysis is from at least three independent experiments. n.s., not significant.

Article Snippet: Rabbit primary antibodies used were directed against: laminin (WB: 1/1,000, IF: 1/200) (EHS; Sigma–Aldrich, Oakville, ON), α5 integrin (WB: 1/1,000) (AB1928, Millipore, Temecula, CA), αV integrin (WB: 1/1,000) (AB1930, Millipore), p21 (WB: 1/2,000) (Santa Cruz Biotechnology, Inc.), p27 (WB: 1/2,000) (Santa Cruz Biotechnology, Inc.), and cyclin D1 (WB: 1/5,000) (Santa Cruz Biotechnology, Inc.).

Techniques: Knockdown, Expressing, Transfection, BrdU Incorporation Assay, Staining, Western Blot, Control

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti‐ eIF3d (WB 1:2,000) , Bethyl , Cat # A301‐758A; RRID: AB_1210970.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Magnetic Beads, Software, Microscopy, Microarray, Imaging, Flow Cytometry, Mutagenesis, Gel Extraction, cDNA Synthesis, Sample Prep, Clear Native PAGE, Bicinchoninic Acid Protein Assay