Journal: International journal of molecular sciences

Article Title: PHB2 Alleviates Neurotoxicity of Prion Peptide PrP 106-126 via PINK1/Parkin-Dependent Mitophagy.

doi: 10.3390/ijms242115919

Figure Lengend Snippet: Figure 4. PHB2 involves PrP106–126-induced mitophagy in primary neurons. (A) Cortical neurons were treated with PrP106–126 for the indicated periods, and then, the protein levels of PHB2 were analyzed using Western blotting. (B) Cortical neurons were transfected with shRNA-PHB2 (sh#1, sh#2) plasmids or shRNA-NC control plasmid before the incubation with PrP106–126 for 24 h. PHB2, TOMM20, and LC3B-II were analyzed using Western blotting. (C) Cortical neurons were transfected with different concentrations of flag-PHB2 vector or flag-PCMV control vector for 24 h and then incubated with PrP106–126 for another 24 h. PHB2, TOMM20, and LC3B-II proteins were analyzed using Western blotting, respectively. (D) Representative images of PrP106–126-induced mitophago- some (red) formation in PHB2-knockdown neurons or PHB2-overexpression neurons. Cortical cells were firstly transfected with shRNA-PHB2 (sh#1) and flag-PHB2, respectively, and 24 h later, these neurons were transfected with COX8-EGFP-mCherry. After 24 h, the neurons were subjected to the PrP106–126 treatment for another 24 h. Finally, the neurons were fixed for confocal microscopy.

Article Snippet: Rabbit polyclonal anti-prohibitin 2 (PHB2) (12295-1-AP) antibody, rabbit polyclonal antibeta actin (β-actin) antibody (20536-1-AP), and mouse monoclonal anti-GAPDH antibody (60004-1-Ig) were obtained from Proteintech Group (Chicago, IL, USA).

Techniques: Western Blot, Transfection, shRNA, Control, Plasmid Preparation, Incubation, Knockdown, Over Expression, Confocal Microscopy

Journal: International journal of molecular sciences

Article Title: PHB2 Alleviates Neurotoxicity of Prion Peptide PrP 106-126 via PINK1/Parkin-Dependent Mitophagy.

doi: 10.3390/ijms242115919

Figure Lengend Snippet: Figure 5. PHB2 is required for PINK1/Parkin-mediated mitophagy in PrP106–126-treated neu- rons. (A) Western blotting of PINK1 and Parkin proteins in mitochondria of PHB2-overexpression and PHB2-knockdown neurons with the PrP106–126 treatments (HSP60 served as loading control). (B) The colocalization between mitochondria (labeled with HSP60, red) and Parkin (green) in PHB2- knockdown and PHB2-overexpression neurons under the PrP106–126 treatment was observed using immunofluorescence imaging (nuclei, blue).

Article Snippet: Rabbit polyclonal anti-prohibitin 2 (PHB2) (12295-1-AP) antibody, rabbit polyclonal antibeta actin (β-actin) antibody (20536-1-AP), and mouse monoclonal anti-GAPDH antibody (60004-1-Ig) were obtained from Proteintech Group (Chicago, IL, USA).

Techniques: Western Blot, Over Expression, Knockdown, Control, Labeling, Imaging

Journal: International journal of molecular sciences

Article Title: PHB2 Alleviates Neurotoxicity of Prion Peptide PrP 106-126 via PINK1/Parkin-Dependent Mitophagy.

doi: 10.3390/ijms242115919

Figure Lengend Snippet: Figure 6. PHB2 attenuates PrP106–126-induced neuronal death. Primary neurons (A) or PHB2- knockdown neurons (B) or PHB2-overexpression neurons (C) were treated with PrP106–126, the cell viability were measured by using the CCK-8 assay. Bars represent means ± SD (n = 3). ** p < 0.01 indicate significant differences between the control groups. (D) Fluorescence images of cell apoptosis were detected using TUNEL assays (red) in control or treated cortical neurons (nuclei, blue).

Article Snippet: Rabbit polyclonal anti-prohibitin 2 (PHB2) (12295-1-AP) antibody, rabbit polyclonal antibeta actin (β-actin) antibody (20536-1-AP), and mouse monoclonal anti-GAPDH antibody (60004-1-Ig) were obtained from Proteintech Group (Chicago, IL, USA).

Techniques: Knockdown, Over Expression, CCK-8 Assay, Control, Fluorescence, TUNEL Assay

Journal: International journal of molecular sciences

Article Title: PHB2 Alleviates Neurotoxicity of Prion Peptide PrP 106-126 via PINK1/Parkin-Dependent Mitophagy.

doi: 10.3390/ijms242115919

Figure Lengend Snippet: Figure 7. Schematic representation of PHB2-mediated mitophagy and neuronal death inhibition under PrP106–126 stimulation. PrP106–126 accumulates in mitochondria and leads to mitochondrial damage, which stabilizes PINK1 and recruits Parkin to mitochondria to mediate mitophagy. IMM component PHB2 acts as a mitophagy receptor and plays a vital role in PINK1/Parkin-dependent mitophagy. Endogenous and exogenous PHB2 functions as prosurvival proteins by mediating mi- tophagy and suppressing neuronal death. EX-PHB2, exogenous PHB2; EN-PHB2, endogenous PHB2.

Article Snippet: Rabbit polyclonal anti-prohibitin 2 (PHB2) (12295-1-AP) antibody, rabbit polyclonal antibeta actin (β-actin) antibody (20536-1-AP), and mouse monoclonal anti-GAPDH antibody (60004-1-Ig) were obtained from Proteintech Group (Chicago, IL, USA).

Techniques: Inhibition

Journal: Molecular Cancer Research

Article Title: Interaction of Delta-like 1 Homolog (Drosophila) with Prohibitins and Its Impact on Tumor Cell Clonogenicity

doi: 10.1158/1541-7786.mcr-13-0360

Figure Lengend Snippet: Figure 1. DLK1 interacts with the PHB1–PHB2 complex: A, WCLs were prepared by solubilizing BE(2)C cells in the modified RIPA buffer and incubated with monoclonal anti-DLK1 N-terminus antibody. Naïve mouse IgG2b was used as the isotype control. Immune complexes were separated by 10% SDS–PAGE and visualized by silver staining. PHB2 was identified by mass spectrometry. The asterisks represent heavy and light chains of IgG. B, reciprocal immunoprecipitation to confirm the interaction between DLK1 and PHB proteins using specific antibodies against DLK1 N-terminus, PHB2, and PHB1, respectively. Clean-Blot IP Detection Reagents (Thermo Scientific) were used to eliminate cross-reaction with IgG heavy and light chains. BE(2)C cells were either maintained under normal culture conditions (normoxia) or under hypoxia (1% O2; 16 hours) conditions. C, intracellular colocalization of DLK1 and PHBs as revealed by confocal microscopy. BE(2)C cells were fixed in cold methanol and then stained with anti-DLK1 þ anti-PHB1 or anti-DLK1 þ anti-PHB2 as described in Materials and Methods. Nuclei were counterstained with To-PRO3. Images were acquired using a Zeiss LSM 510 Meta confocal microscope.

Article Snippet: Immunoprecipitation was carried out by incubation of WCL (500 mg total protein) with 4 mg of monoclonal anti-N terminus DLK1 antibody (R&D Systems), rabbit anti-PHB2 antibody (Novus), or monoclonal anti-PHB1 antibody (Thermo Scientific; clone II-14-10) overnight at 4 C. Thirty mL of recombinant protein A agarose beads were then added and incubated for an hour at 4 C. Immune complexes were eluted in 1 SDS sample buffer and fractionated by SDS–PAGE under reducing conditions.

Techniques: Incubation, Control, SDS Page, Silver Staining, Mass Spectrometry, Immunoprecipitation, Confocal Microscopy, Staining, Microscopy

Journal: Molecular Cancer Research

Article Title: Interaction of Delta-like 1 Homolog (Drosophila) with Prohibitins and Its Impact on Tumor Cell Clonogenicity

doi: 10.1158/1541-7786.mcr-13-0360

Figure Lengend Snippet: Figure 2. DLK1-cyto domain interacts with the PHB1–PHB2 complex.WCLswerepreparedfrom ER cells overexpressing DLK1-FL, DLK1-EC domain, DLK1 Y339F/ S355A mutant (DLK1-DM), or DLK1 without cytoplasmic domain (DLK1- DIC). Expression of the different DLK1 constructs (indicated by arrows) was validated by Western blot analysis (A). Interactions between each DLK1 construct and PHBs were examined by immunoprecipitation with anti- DLK1 N-terminus antibody (B) and anti-PHB2 antibody (C). DLK1-cyto domain interacts with PHB2 (D). HEK293 cells were transiently transfected with Flag-DLK1-FL, Flag-tagged DLK1-cyto domain (Flag-DLK1-cyto), or the empty vector. WCL was subjected to immunoprecipitation using anti- Flag antibodies. DLK1 and PHB2 were then detected by Western blot analysis.

Article Snippet: Immunoprecipitation was carried out by incubation of WCL (500 mg total protein) with 4 mg of monoclonal anti-N terminus DLK1 antibody (R&D Systems), rabbit anti-PHB2 antibody (Novus), or monoclonal anti-PHB1 antibody (Thermo Scientific; clone II-14-10) overnight at 4 C. Thirty mL of recombinant protein A agarose beads were then added and incubated for an hour at 4 C. Immune complexes were eluted in 1 SDS sample buffer and fractionated by SDS–PAGE under reducing conditions.

Techniques: Mutagenesis, Expressing, Construct, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation

Journal: Molecular Cancer Research

Article Title: Interaction of Delta-like 1 Homolog (Drosophila) with Prohibitins and Its Impact on Tumor Cell Clonogenicity

doi: 10.1158/1541-7786.mcr-13-0360

Figure Lengend Snippet: Figure 3. Secreted DLK1-EC domain enhances DLK1–PHB interaction. A, ER cells expressing DLK1-EC domain (ER–EC), ER cells with empty vector (ER-V), or BE(2)C cells were cultured for 48 hours to approximately 80% confluence and maintained in the serum-free medium for 24 hours. Secreted proteins in conditioned media (CM) were precipitated by trichloroacetic acid and then subjected to Western blot analysis. DLK1-EC was detected by anti-DLK1 N-terminus antibodies, and the WCL from BE(2)C was used as a positive control for DLK1-FL. B, secreted DLK1-EC domain enhances DLK1–PHB interaction. BE(2)C or 3T3-L1 cells were incubated overnight with a 1:1 mixture of the ER cell–conditioned medium (ER–EC or ER-V) and the fresh culture medium containing 10% FBS. WCLs from these conditioned medium–treated cells were subjected to immunoprecipitation using anti-DLK1 N-terminus antibodies. The presence of PHB2 was examined by Western blot analysis. C, secreted DLK1-EC domain enhances clonogenicity. BE(2)C cells were plated at a clonal density (300 cells/well in 6-well plates) in a 1:1 mixture of the ER cell–conditioned medium (ER–EC or ER-V) and the fresh culture medium containing 10% FBS. After incubation for 10 days, tumor colonies were fixed and stained with Crystal Violet. D, colonies were counted and clonogenic efficiency or plating efficiency was calculated as number of colonies/number of input cells 100 (mean SEM; , P < 0.05).

Article Snippet: Immunoprecipitation was carried out by incubation of WCL (500 mg total protein) with 4 mg of monoclonal anti-N terminus DLK1 antibody (R&D Systems), rabbit anti-PHB2 antibody (Novus), or monoclonal anti-PHB1 antibody (Thermo Scientific; clone II-14-10) overnight at 4 C. Thirty mL of recombinant protein A agarose beads were then added and incubated for an hour at 4 C. Immune complexes were eluted in 1 SDS sample buffer and fractionated by SDS–PAGE under reducing conditions.

Techniques: Expressing, Plasmid Preparation, Cell Culture, Western Blot, Positive Control, Incubation, Immunoprecipitation, Staining

Journal: Molecular Cancer Research

Article Title: Interaction of Delta-like 1 Homolog (Drosophila) with Prohibitins and Its Impact on Tumor Cell Clonogenicity

doi: 10.1158/1541-7786.mcr-13-0360

Figure Lengend Snippet: Figure 5. The DLK1–PHB pathway regulates cancer cell stemness. BE(2)C cells were treated with pooled siRNA oligos specific for DLK1, PHB1, PHB2, and control (SMARTpool; Dharmacon), respectively. Efficiency of knockdown was examined by Western blot analysis (A) and quantitative RT-PCR (B), respectively. The quantitative RT-PCR data were analyzed by one-way ANOVA (P < 0.001). For pairwise comparison, , P < 0.01 versus their respective siCtrl. C, the DLK1–PHB pathway is required for maintaining self-renewal. BE(2)C cells were treated with siRNAs discussed in (A). Tumor sphere formation assay was performed by plating 5,000 cells per well in polyHEMA-coated 12-well plates. Tumor cell spheres were allowed to grow for 3 days in the serum-free sphere medium. Data shown were mean numbers of spheres per well SEM. Data in the left were first analyzed by one-way ANOVA (P < 0.0001; n ¼ 6), followed by pairwise comparison (, P < 0.0001 vs. siCtrl and #, P < 0.01 vs. siPHB1 or siDLK1). Data on the right were analyzed by an unpaired, two-tailed Student t test of siDLK1 versus siCtrl in each PHB group; , P < 0.003; n ¼ 3. D, the DLK1–PHB pathway is required for clonogenicity. siRNA-treated BE(2)C cells were plated at 300 cells per well in 6-well plats and were cultured for 10 days. Colonies were fixed and stained with Crystal Violet and counted. Clonogenic efficiency ¼ percentage of colonies per input SEM (n ¼ 6). Data on the left were first analyzed by one-way ANOVA (P < 0.0001), followed by pairwise comparison (, P < 0.0001 vs. siCtrl and #, P < 0.01 vs. siPHB1 or siDLK1). Data on the right were also analyzed by one-way ANOVA (P < 0.03 for the siDLK1 group). For comparison with vector/siCtrl, , P < 0.04 (Student t test).

Article Snippet: Immunoprecipitation was carried out by incubation of WCL (500 mg total protein) with 4 mg of monoclonal anti-N terminus DLK1 antibody (R&D Systems), rabbit anti-PHB2 antibody (Novus), or monoclonal anti-PHB1 antibody (Thermo Scientific; clone II-14-10) overnight at 4 C. Thirty mL of recombinant protein A agarose beads were then added and incubated for an hour at 4 C. Immune complexes were eluted in 1 SDS sample buffer and fractionated by SDS–PAGE under reducing conditions.

Techniques: Control, Knockdown, Western Blot, Quantitative RT-PCR, Comparison, Tube Formation Assay, Two Tailed Test, Cell Culture, Staining, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: Nuclear distribution of eIF3g and its interacting nuclear proteins in breast cancer cells

doi: 10.3892/mmr.2016.4935

Figure Lengend Snippet: Nuclear localization of eIF3g. (A) Results of fractionation of the-Bcap37/Tet-on-eIF3g cells. GAPDH was used as the cytoplasmic marker and Histone H4 was used as the nuclear marker. The proteins samples were subjected to 12% SDS-PAGE, transferred onto a polyvinylidene fluo -ride membrane and probed by specific antibodies. (B) Immunofluorescent staining was performed. Confocal microscope images for eIF3g are shown (magnification, ×100). eIF3g, eukaryotic translation initiation factor 3 subunit g; W, whole cell lysate; C, cytoplasmic fraction; N, nuclear fraction.

Article Snippet: Briefly, the human breast cancer Bcap37 cells were cultured in RPMI 1640 medium (Boster Biological Technology) supplemented with 10% fetal bovine serum (FBS; Gbico; Thermo Fisher Scientific, Inc., Waltham, MA, USA) or 10% Tet-approved FBS (Clontech Laboratories, Mountain View, CA, USA) in a humidified 37°C incubator.

Techniques: Fractionation, Marker, SDS Page, Membrane, Staining, Microscopy

Journal: Molecular Medicine Reports

Article Title: Nuclear distribution of eIF3g and its interacting nuclear proteins in breast cancer cells

doi: 10.3892/mmr.2016.4935

Figure Lengend Snippet: co-IP of proteins with anti-eIF3g antibody in the nuclear fraction of Bcap37/Tet-on-eIF3g cells. The nuclear protein (200 µ g) was incubated with the 5 µ g anti-eIF3g antibody and 50 µ l protein A/G PLUS-Agarose. Bands that were clearly present and with clear edges for excision in the co-IP sample but absent in the control sample (bands 1, 2, 3 and 4) were selected for protein characterization by mass spectrometry analysis. co-IP, co-immunoprecipitation; eIF3g, eukaryotic translation initiation factor 3 subunit g.

Article Snippet: Briefly, the human breast cancer Bcap37 cells were cultured in RPMI 1640 medium (Boster Biological Technology) supplemented with 10% fetal bovine serum (FBS; Gbico; Thermo Fisher Scientific, Inc., Waltham, MA, USA) or 10% Tet-approved FBS (Clontech Laboratories, Mountain View, CA, USA) in a humidified 37°C incubator.

Techniques: Co-Immunoprecipitation Assay, Incubation, Control, Mass Spectrometry, Immunoprecipitation

Journal: Molecular Medicine Reports

Article Title: Nuclear distribution of eIF3g and its interacting nuclear proteins in breast cancer cells

doi: 10.3892/mmr.2016.4935

Figure Lengend Snippet: eIF3g interacts with hnRNP U, HSZFP36 and β-actin. (A) Nuclear extracts of Bcap37/Tet-on-eIF3g cells were subjected to immunoprecipitation with anti-eIF3g (5 µ g), anti-hnRNP U (5 µ g), anti-HSZFP36 (5 µ g) and anti-β-actin (5 µ g) antibodies, respectively. Precipitated proteins were resolved by 10% SDS-PAGE and analyzed by western blotting with anti-eIF3g antibody. Arrowheads indicate eIF3g. (B) Anti-eIF3g antibody-immunoprecipitated proteins were analyzed by western blotting with anti-hnRNP U antibody. Arrowheads indicate hnRNP U. (C) Anti-eIF3g (5 µ g) and anti-hnRNP U (5 µ g) antibody-immunoprecipitated proteins were analyzed by western blotting with anti-HSZFP36 antibody. Arrowheads indicate HSZFP36. (D) Anti-eIF3g (5 µ g) and anti-β-actin (5 µ g) antibody-immunoprecipitated proteins were analyzed by western blotting with anti-β-actin antibody. Arrowheads indicate β-actin. (E) Results of western blotting from the in vitro crosslinking. The nuclear extract from Bcap37/Tet-on-eIF3g cells was crosslinked by DSS, subject ed to 8% SDS-PAGE and electrotransferred onto a polyvinylidene fluoride membrane, followed by incubation with antibodies specific for eIF3g, hnRNP U, HSZFP36 and β-actin. Arrowheads indicate larger complex bands; arrows indicate smaller complex bands. (F) GST-pulldown results. GST or GST-eIF3g proteins were incubated with nuclear lysates of Bcap37 cells and glutathione beads; bound proteins were resolved by 10% SDS-PAGE and detected by western blotting. hnRNP U, HSZFP36 and β-actin were pulled down by GST-eIF3g, with GST protein and GSH beads alone used as negative controls. Arrowheads indicate eIF3g in the Bcap37 cells; arrows indicate the GST-eIF3g protein band. eIF3g, eukaryotic translation initiation factor 3 subunit g; hnRNP U, heterogeneous nuclear ribonucleoprotein U; GST, glutathione S-transferase; GSH, glutathione.

Article Snippet: Briefly, the human breast cancer Bcap37 cells were cultured in RPMI 1640 medium (Boster Biological Technology) supplemented with 10% fetal bovine serum (FBS; Gbico; Thermo Fisher Scientific, Inc., Waltham, MA, USA) or 10% Tet-approved FBS (Clontech Laboratories, Mountain View, CA, USA) in a humidified 37°C incubator.

Techniques: Immunoprecipitation, SDS Page, Western Blot, In Vitro, Membrane, Incubation

Journal: Molecular Medicine Reports

Article Title: Nuclear distribution of eIF3g and its interacting nuclear proteins in breast cancer cells

doi: 10.3892/mmr.2016.4935

Figure Lengend Snippet: Co-localization of eIF3g, hnRNP U, HSZFP36 and β-actin. (A) Bcap37/Tet-on-eIF3g cells were incubated with anti-eIF3g, anti-hnRNP U, anti-HSZFP36 and anti-β-actin antibodies, respectively (magnification, ×100). (B) Results of western blot analysis. Extracts from Bcap37/Tet-on-eIF3g cells were probed with anti-eIF3g, anti-hnRNP U, anti-HSZFP36 and anti-β-actin antibodies, respectively. (C) Co-localization of eIF3g, HSZFP36 and β-actin in the nuclei (magnification, ×100). eIF3g, eukaryotic translation initiation factor 3 subunit g; hnRNP U, heterogeneous nuclear ribonucleoprotein U; W, whole cell lysate; C, cytoplasmic fraction; N, nuclear fraction.

Article Snippet: Briefly, the human breast cancer Bcap37 cells were cultured in RPMI 1640 medium (Boster Biological Technology) supplemented with 10% fetal bovine serum (FBS; Gbico; Thermo Fisher Scientific, Inc., Waltham, MA, USA) or 10% Tet-approved FBS (Clontech Laboratories, Mountain View, CA, USA) in a humidified 37°C incubator.

Techniques: Incubation, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Pgam5 aggravates hyperglycemia-induced myocardial dysfunction through disrupting Phb2-dependent mitochondrial dynamics

doi: 10.7150/ijms.92872

Figure Lengend Snippet: Pgam5 controlled mitophagy and mitochondrial fission through Phb2. A-C. RNA was isolated from cardiomyocytes and then the transcription of Drp1, Mff, and Fis1 was measured via qPCR. Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. D-F. RNA was isolated from cardiomyocytes and then the transcription of Parkin, Fundc1, and Bnip3 was measured via qPCR. Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. #p<0.05.

Article Snippet: The adenovirus Phb2 (Catalog No. sc-419296-LAC) was obtained from Santa Cruz Biotechnology, Inc. and subjected to conventional methods for purification and infectious virus titer determination, as per reference .

Techniques: Isolation, Transfection

Journal: International Journal of Medical Sciences

Article Title: Pgam5 aggravates hyperglycemia-induced myocardial dysfunction through disrupting Phb2-dependent mitochondrial dynamics

doi: 10.7150/ijms.92872

Figure Lengend Snippet: Phb2 overexpression reduced mitochondrial dysfunction upon hyperglycemia stress. A. JC-1 probe was used to stain mitochondrial membrane potential. B. ROS production was measured in cardiomyocytes. Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. C-F. The activities of catalase, glutaredoxin, thioredoxin reductase (TrxR), and peroxiredoxin were determined by ELISA. Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. #p<0.05.

Article Snippet: The adenovirus Phb2 (Catalog No. sc-419296-LAC) was obtained from Santa Cruz Biotechnology, Inc. and subjected to conventional methods for purification and infectious virus titer determination, as per reference .

Techniques: Over Expression, Staining, Membrane, Transfection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Medical Sciences

Article Title: Pgam5 aggravates hyperglycemia-induced myocardial dysfunction through disrupting Phb2-dependent mitochondrial dynamics

doi: 10.7150/ijms.92872

Figure Lengend Snippet: Phb2 overexpression maintained cardiomyocyte viability and function upon hyperglycemia stress. A. Cell viability was determined by MTT assay. Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. B-G. Pgam5 siRNA was transfected into cardiomyocytes and then the contractile was measured, including resting length remained, peak shortening (PK), maximal velocity of shortening (dL/dt), and time-to-peak shortening, maximal velocity of relengthening (-dL/dt), and time-to-90% relengthening (TR90). Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. #p<0.05.

Article Snippet: The adenovirus Phb2 (Catalog No. sc-419296-LAC) was obtained from Santa Cruz Biotechnology, Inc. and subjected to conventional methods for purification and infectious virus titer determination, as per reference .

Techniques: Over Expression, MTT Assay, Transfection

Journal: iScience

Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

doi: 10.1016/j.isci.2023.106293

Figure Lengend Snippet: RX-375 induces phosphorylation of AMPKα and directly binds to PHBs, related to Figures S1 and (A) The structure of RX-375. (B) HepG2 cells were incubated in the presence of [ 14 C]acetate and with the indicated concentrations of RX-375 or metformin (Met) for 3 h, after which fatty acid synthesis was measured. (C) HepG2 cells were incubated with the indicated concentrations of RX-375 or metformin for 3 h, lysed, and subjected to immunoblot analysis with antibodies to Thr 172 -phosphorylated (pT172) or total forms of AMPKα or to Ser 79 -phosphorylated (pS79) or total forms of ACC. Representative blots as well as the p -AMPKα/AMPKα and p-ACC/ACC ratios determined by densitometry and expressed relative to the corresponding value for control (untreated) cells are shown. (D and E) Primary human hepatocytes were incubated with 1 μM RX-375, 10 mM metformin, or dimethyl sulfoxide (DMSO) vehicle as well as in the presence (D) or absence (E) of [ 14 C]acetate for 3 h, and were then assayed for fatty acid synthesis (D) or subjected to immunoblot analysis of AMPKα and ACC phosphorylation (E). The p-AMPKα/AMPKα and p-ACC/ACC ratios were determined by densitometry. (F) Schematic illustration of target identification with RX-375-conjugated beads. MALDI-TOF MS, matrix-assisted laser desorption/ionization–time of flight mass spectrometry. (G and H) HepG2 cell lysates were incubated with control or RX-375-conjugated beads for 24 hat 4°C, after which bound proteins were eluted from the beads and subjected to SDS–polyacrylamide gel electrophoresis (PAGE) followed by silver staining (G) or by immunoblot analysis with antibodies to PHB1, to PHB2, to AMPKα, and to AMPKβ1/2 (H). All quantitative data are means ± s.e.m. from three independent experiments (n = 3). ∗p < 0.05 versus DMSO, Dunnett’s multiple comparison test (D and E).

Article Snippet: PHB2 (Mm00476104_m1) , Life Technologies , Cat#4331182.

Techniques: Phospho-proteomics, Incubation, Western Blot, Control, Drug discovery, Mass Spectrometry, Polyacrylamide Gel Electrophoresis, Silver Staining, Comparison

Journal: iScience

Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

doi: 10.1016/j.isci.2023.106293

Figure Lengend Snippet: PHBs are negative regulators of AMPK, related to Figures S3 and (A) HepG2 cells transfected with control (siCtrl) or PHB1 and PHB2 (siPHB1/2) siRNAs were subjected to immunoblot analysis with antibodies to the indicated proteins. The p-AMPKα/AMPKα, p-ACC/ACC, PHB1/β-actin, and PHB2/β-actin ratios were determined by densitometry and are expressed relative to the corresponding mean value for siCtrl-transfected cells. (B) HepG2 cells transfected with siCtrl or siPHB1/2 were incubated with [ 14 C]acetate and then assayed for fatty acid synthesis. (C) Immunoblot analysis of a whole cell extract (WCE) as well as of isolated mitochondrial (Mito) and cytosolic fractions of HepG2 cells with antibodies to AMPKα, to PHB1, to PHB2, to HSP90 (cytosolic marker), and to COX IV (mitochondrial marker). (D) Mitochondrial lysates prepared from HepG2 cells were subjected to immunoprecipitation (IP) with antibodies to AMPKα or with control immunoglobulin G (IgG), and the resulting precipitates as well as the original lysates (Input) were subjected to immunoblot analysis with antibodies to the indicated proteins. (E) Recombinant GST-PHB1 or GST alone, each bound to glutathione-agarose beads, was incubated with recombinant His 6 -tagged AMPK (α2β1γ1) in the absence or presence of RX-375 (2 μM), after which bead-bound proteins were subjected to immunoblot analysis with antibodies to the indicated proteins. (F) HepG2 cells expressing 3×Flag-tagged PHB1 and Myc epitope-tagged AMPKα2 were incubated with RX-375 (1 μM) for 3 h, after which cell lysates were subjected to immunoprecipitation with antibodies to Flag and the resulting precipitates were subjected to immunoblot analysis with antibodies to Myc and to Flag. All quantitative data are means ± s.e.m. from five (B) or six (A) independent experiments (n = 5 or 6, respectively). ∗p < 0.05 versus siCtrl, unpaired two-tailed Student’s t test (A and B).

Article Snippet: PHB2 (Mm00476104_m1) , Life Technologies , Cat#4331182.

Techniques: Transfection, Control, Western Blot, Incubation, Isolation, Marker, Immunoprecipitation, Recombinant, Expressing, Two Tailed Test

Journal: iScience

Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

doi: 10.1016/j.isci.2023.106293

Figure Lengend Snippet: Treatment with RX-375 or knockdown of hepatic PHBs reduces the triglyceride content and inhibits fatty acid synthesis in the liver of db/db mice, related to Figures S5 and (A) RX-375 (30 mg/kg) or vehicle (Veh) was administered by oral gavage to male db/db mice (6 weeks of age) that had been deprived of food for 3 h. At 1 h after dosing, the mice were killed for examination of AMPKα phosphorylation in the liver by immunoblot analysis. The p-AMPKα/AMPKα ratio was determined by densitometry and expressed relative to the mean value for vehicle-treated mice. (B–E) RX-375 (30 mg/kg) or vehicle was orally administered to 6-week-old male db/db or db/m+ mice twice a day for 21 days (B). The plasma triglyceride concentration was determined at 14 days after treatment onset (C), and the triglyceride content (D), and fatty acid synthesis (E) in the liver were measured at 21 days. (F–J) Male db/db mice at 7 weeks of age were injected with an adenovirus encoding a PHB1 shRNA (shPHB1) or the corresponding empty virus (shCtrl) and were killed for analysis of the liver 2 weeks later (F). The relative abundance of PHB1 and PHB2 mRNAs was determined by reverse transcription (RT) and real-time PCR (PCR) analysis (G), that of PHB1 and PHB2 proteins was determined by immunoblot analysis (H), and triglyceride content (I) and fatty acid synthesis (J) were also measured. All quantitative data are means ± s.e.m. for 3 (A), 6 (C–E), or 10 (G–J) mice per group (n = 3, 6, and 10, respectively). ∗p < 0.05 versus db/db mice treated with vehicle or shCtrl, †p < 0.05 versus db/m+ mice with the unpaired two-tailed Student’s t test (A, C–E, G–J). (K) Proposed mechanism for regulation of AMPKα phosphorylation by PHBs and for the amelioration of dyslipidemia by RX-375. TG, triglyceride.

Article Snippet: PHB2 (Mm00476104_m1) , Life Technologies , Cat#4331182.

Techniques: Knockdown, Phospho-proteomics, Western Blot, Clinical Proteomics, Concentration Assay, Injection, shRNA, Virus, Reverse Transcription, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: iScience

Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

doi: 10.1016/j.isci.2023.106293

Figure Lengend Snippet:

Article Snippet: PHB2 (Mm00476104_m1) , Life Technologies , Cat#4331182.

Techniques: Produced, FLAG-tag, Control, Recombinant, Membrane, Transfection, Reverse Transcription, Silver Staining, Isolation, Cell Culture, Expressing, Plasmid Preparation, Luciferase, shRNA, Software