Journal: Cancers

Article Title: p66Shc Protein-Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer.

doi: 10.3390/cancers16193324

Figure Lengend Snippet: Figure 1. Characteristics of ShcA family protein. (A) Domain structure of ShcA isoforms. (B) Pro-oxidative signaling pathway of p66Shc isoform. PKCβ—protein kinase Cβ; S660-PKCβ— phosphorylated in S660 form of protein kinase Cβ; Pin1—prolyl isomerase; PP2A—serine/threonine protein phosphatase type 2; cyt. c—cytochrome c.

Article Snippet: pcDNA3.1his p66shc (#32574) and pcDNA3.1his p66shc S36A (#32575) plasmids were obtained from Addgene (Watertown, MA, USA).

Techniques:

Journal: Cancers

Article Title: p66Shc Protein-Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer.

doi: 10.3390/cancers16193324

Figure Lengend Snippet: Figure 2. The level of ShcA proteins (p66Shc, p52Shs, and p46Shc) in the MDA-MB-231 clones obtained through (A) lentiviral transfection or (B) genome editing using the CRISPR/Cas9 method by the Western Blot analysis. (C) The schematic of the U6gRNA-Cas9-2A-GFP plasmid, enabling genome editing in MDA-MB-231 cell lines. (D) Proliferation rates of the individual MDA-MB-231 clones.

Article Snippet: pcDNA3.1his p66shc (#32574) and pcDNA3.1his p66shc S36A (#32575) plasmids were obtained from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Transfection, CRISPR, Western Blot, Plasmid Preparation

Journal: Cancers

Article Title: p66Shc Protein-Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer.

doi: 10.3390/cancers16193324

Figure Lengend Snippet: Figure 3. The level of individual proteins involved in the pro-oxidative p66Shc signaling pathway. The levels of proteins were standardized to the GAPDH ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: pcDNA3.1his p66shc (#32574) and pcDNA3.1his p66shc S36A (#32575) plasmids were obtained from Addgene (Watertown, MA, USA).

Techniques:

Journal: Cancers

Article Title: p66Shc Protein-Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer.

doi: 10.3390/cancers16193324

Figure Lengend Snippet: Figure 9. Mitochondrial network morphology in the individual MDA-MB-231 clones treated (+DOX) and untreated (–DOX) with doxorubicin and mitochondrial membrane potential. The (A) Ctrl, (B) clone with elevated p66Shc protein levels, (C) clone with elevated levels of the S36A-mutated form of the p66Shc protein, and (D) clone with the knockout of p66Shc; nuclei were stained blue (DAPI) and the mitochondrial network was stained red (MitoRed); (E) mitochondrial membrane potential, * p < 0.05.

Article Snippet: pcDNA3.1his p66shc (#32574) and pcDNA3.1his p66shc S36A (#32575) plasmids were obtained from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Membrane, Knock-Out, Staining

Journal: Cancers

Article Title: p66Shc Protein-Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer.

doi: 10.3390/cancers16193324

Figure Lengend Snippet: Figure 1. Characteristics of ShcA family protein. (A) Domain structure of ShcA isoforms. (B) Pro-oxidative signaling pathway of p66Shc isoform. PKCβ—protein kinase Cβ; S660-PKCβ— phosphorylated in S660 form of protein kinase Cβ; Pin1—prolyl isomerase; PP2A—serine/threonine protein phosphatase type 2; cyt. c—cytochrome c.

Article Snippet: pcDNA3.1his p66shc (#32574) and pcDNA3.1his p66shc S36A (#32575) plasmids were obtained from Addgene (Watertown, MA, USA).

Techniques:

Journal: Cancers

Article Title: p66Shc Protein-Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer.

doi: 10.3390/cancers16193324

Figure Lengend Snippet: Figure 2. The level of ShcA proteins (p66Shc, p52Shs, and p46Shc) in the MDA-MB-231 clones obtained through (A) lentiviral transfection or (B) genome editing using the CRISPR/Cas9 method by the Western Blot analysis. (C) The schematic of the U6gRNA-Cas9-2A-GFP plasmid, enabling genome editing in MDA-MB-231 cell lines. (D) Proliferation rates of the individual MDA-MB-231 clones.

Article Snippet: pcDNA3.1his p66shc (#32574) and pcDNA3.1his p66shc S36A (#32575) plasmids were obtained from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Transfection, CRISPR, Western Blot, Plasmid Preparation

Journal: Cancers

Article Title: p66Shc Protein-Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer.

doi: 10.3390/cancers16193324

Figure Lengend Snippet: Figure 3. The level of individual proteins involved in the pro-oxidative p66Shc signaling pathway. The levels of proteins were standardized to the GAPDH ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: pcDNA3.1his p66shc (#32574) and pcDNA3.1his p66shc S36A (#32575) plasmids were obtained from Addgene (Watertown, MA, USA).

Techniques:

Journal: Cancers

Article Title: p66Shc Protein-Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer.

doi: 10.3390/cancers16193324

Figure Lengend Snippet: Figure 9. Mitochondrial network morphology in the individual MDA-MB-231 clones treated (+DOX) and untreated (–DOX) with doxorubicin and mitochondrial membrane potential. The (A) Ctrl, (B) clone with elevated p66Shc protein levels, (C) clone with elevated levels of the S36A-mutated form of the p66Shc protein, and (D) clone with the knockout of p66Shc; nuclei were stained blue (DAPI) and the mitochondrial network was stained red (MitoRed); (E) mitochondrial membrane potential, * p < 0.05.

Article Snippet: pcDNA3.1his p66shc (#32574) and pcDNA3.1his p66shc S36A (#32575) plasmids were obtained from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Membrane, Knock-Out, Staining

Journal: Molecular Biology of the Cell

Article Title: Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene

doi: 10.1091/mbc.E13-11-0666

Figure Lengend Snippet: Nrf2 binds the human p66Shc promoter containing the ARE and AP1-like sequences. (A) K562 cells were treated with 20 μM hemin for 36 h, and nuclear (Nuc.) and cytoplasmic (Cyto.) fractions were used for Western blot with Nrf2, lamin B1, or LDH antibody. Lamin B1 and LDH are nuclear and cytoplasmic fraction markers, respectively. (B) Schematic of the 5′-promoter region of the human p66Shc gene, which contains an ARE and an AP1-like sequence. n = any base; M = A or C; R = A or G; Y = C or T. Asterisks indicate matched bases. (C) K562 cells treated with 0, 20, 40, and 75 μM hemin for 12 h were subjected to ChIP assays with control IgG or Nrf2 antibody, followed by real-time PCR using p66Shc primers spanning the ARE, AP1-like, a 2.3-kb upstream 5′-promoter region, and a β-globin promoter region. Data were normalized by input DNA and are shown as mean ± SD ( n = 3). (D) Nuclear extracts of K562 cells treated with 20 μM hemin for 36 h were used for pull-down assay. Nrf2 binding to a biotinylated double-strand p66Shc ARE (−107 to −82 base pairs) or a p66Shc AP1-like (−291 to −266 base pairs) probe was detected by Western blot using an Nrf2 antibody. A human ferritin H ARE (–4433 to –4412 base pairs) probe was used as a positive control and no DNA probe in the binding reaction as a negative control. Coomassie brilliant blue (CBB) staining is shown for verification of equal loading. (E) K562 cells were transfected with wild-type or mutant pGL3 p66Shc-450/+60 plasmid and incubated with 0, 20, and 40 μM hemin for 24 h. Firefly luciferase activity was normalized by Renilla luciferase activity and presented as relative to nontreated wild-type p66Shc-450/+60 plasmid. Top, asterisks indicate mutated nucleic acid. Bottom chart, asterisks indicate significantly different from nontreated cells transfected with wild-type p66Shc-450/+60 plasmid. * p < 0.005, ** p < 0.001 ( n > 3).

Article Snippet: The Nrf2 fragment was obtained from pOTB7Nrf2 plasmid (4548874; Invitrogen, Carlsbad, CA). pcDNA3.1His-p66Shc wild type and S36A mutant were provided by Toren Finkel, National Institutes of Health, Bethesda, MD , through Addgene (Cambridge, MA).

Techniques: Western Blot, Sequencing, Control, Real-time Polymerase Chain Reaction, Pull Down Assay, Binding Assay, Positive Control, Negative Control, Staining, Transfection, Mutagenesis, Plasmid Preparation, Incubation, Luciferase, Activity Assay

Journal: Molecular Biology of the Cell

Article Title: Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene

doi: 10.1091/mbc.E13-11-0666

Figure Lengend Snippet: p66Shc knockdown enhances ROS production and induces apoptosis through oxidative cell damage. (A) Whole-cell lysates isolated from K562 cells transfected with p66Shc-specific siRNA-1 or -2 (si p66Shc-1 or si p66Shc-2) and treated with 20 μM hemin for 24 h were subjected to Western blot with anti-Shc antibody, with GAPDH as a protein loading control. Short exposure (top) and long exposure (middle) of the same Shc Western blot. (B) Caspase 3 activity was measured using a luminogenic substrate containing the DEVD sequence as described in Materials and Methods . K562 cells were treated with 0 and 20 μM hemin for 2 d after p66Shc knockdown using si p66Shc-1 or si p66Shc-2. p values were calculated using Student's t -test. Asterisk indicates significant difference; * p < 0.001. Data are shown as mean ± SD ( n = 3). (C, D) si Control or p66Shc siRNA-1 (si p66Shc-1) was transfected into K562 cells; then cells were pretreated with 0 or 2 mM NAC before 0 or 20 μM hemin treatment for 2 d. (C) Caspase 3 assays were performed. p values were calculated using Student's t test. Asterisk indicates significant difference; * p < 0.001 (si p66Shc +He –NAC vs. all other conditions). Data are shown as mean ± SD ( n = 3). (D) Annexin V assays were performed. Representative flow cytometry plot. The number indicates the percentage of early apoptotic cell populations (annexin V positive and Fixable Viability Dye eFluor 780 negative). (E) K562 cells transfected with si p66Shc-1 were stimulated with 20 μM hemin for 0, 12, 24, 48, and 72 h, and ROS production was measured by flow cytometry using CM-H 2 DCFDA and MitoSox Red. Top, representative histogram; bottom, statistical charts (fold increase). Asterisk indicates significantly different from nontreated si Control sample. * p < 0.05. All error bars represent mean ± SD ( n > 3). (F) K562 cells transfected with si Control (si Ctrl) or si p66Shc-1 were pretreated with 0 or 2 mM NAC before 0 or 20 μM hemin treatment for 2 d. Total proteins were incubated with DNPH and analyzed by Western blot using anti-DNP antibody to detect carbonylated proteins. Coomassie brilliant blue (CBB) staining was used for verification of equal protein loading. (G, H) K562 cells were transfected with si Control or si p66Shc-1 and pcDNA3.1His empty vector (pcDNA), si p66Shc-1-resistant pcDNA3.1His-p66Shc wild type (WT), or -S36A mutant (S36A). G) Whole-cell lysates were used for Western blot with a Shc or GAPDH antibody. (H) K562 cells were treated with 0 and 20 μM hemin for 2 d after transfection, and caspase 3 assays were performed. Asterisk indicates significantly different from si p66Shc/pcDNA/+hemin sample. * p < 0.001, ** p = 0.003. All error bars represent mean ± SD ( n > 3). NS, no significant difference.

Article Snippet: The Nrf2 fragment was obtained from pOTB7Nrf2 plasmid (4548874; Invitrogen, Carlsbad, CA). pcDNA3.1His-p66Shc wild type and S36A mutant were provided by Toren Finkel, National Institutes of Health, Bethesda, MD , through Addgene (Cambridge, MA).

Techniques: Knockdown, Isolation, Transfection, Western Blot, Control, Activity Assay, Sequencing, Flow Cytometry, Incubation, Staining, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: P66Shc Mediates SUMO2-induced Endothelial Dysfunction

doi: 10.1101/2024.01.24.577109

Figure Lengend Snippet: (A) Immunoblot for Shc and SUMO2 expression in HUVECs following p66Shc knockdown (si-p66Shc) and SUMO2 overexpression (Ad-HA-SUMO2). (B and C) Quantification of ROS (MitoSOX fluorescence) in HUVECs from A. *P<0.05; n=24-32; student’s t-test. (D) Immunoblot for ICAM1 expression following p66Shc knockdown (si-p66Shc) in HUVECs with and without SUMO2 overexpression (Ad-HA-SUMO2). (E) Quantification of ICAM1 from D. *<0.05, **P<0.01; n=3; one-way ANOVA. Data represents mean ±SEM. Immunoblots are representative of at least three independent experiments. GC, scrambled siRNA used as control; HUVECs, human umbilical vein endothelial cells; ICAM1, intercellular adhesion molecule 1.

Article Snippet: We also performed an in vitro sumoylation of p66Shc using recombinant His-tagged p66Shc protein and a commercially available kit (Enzo, Farmingdale, NY), which showed that recombinant p66Shc is modified by SUMO2 ( ).

Techniques: Western Blot, Expressing, Over Expression, Fluorescence

Journal: bioRxiv

Article Title: P66Shc Mediates SUMO2-induced Endothelial Dysfunction

doi: 10.1101/2024.01.24.577109

Figure Lengend Snippet: (A) Immunoblot for sumoylated p66Shc in HEK-293 cells overexpressing SUMO2 (HA-SUMO2) and p66Shc (Flag-p66Shc). (B) Immunoblot for p66Shc sumoylation in HEK-293 cells overexpressing SUMO2 (HA-SUMO2) and p66Shc (Flag-p66Shc) with or without overexpression of Ubc9 (HA-Ubc9) and SENP1 (Flag-SENP1). (C) Immunoblot for sumoylation of p66Shc in HUVECs overexpressing SUMO2 (Ad-HA-SUMO2) or p66Shc (Ad-Flag-p66Shc). (D) Immunoblot for p66Shc sumoylation in HUVEC cells overexpressing SUMO2 (Ad-HA-SUMO2) or p66Shc (Ad-Flag-p66Shc) in the presence or absence of anacardic acid. (E) Immunoblot for sumoylated recombinant p66Shc in the presence of absence of Ubc9. Immunoblots are representative of at least three independent experiments.

Article Snippet: We also performed an in vitro sumoylation of p66Shc using recombinant His-tagged p66Shc protein and a commercially available kit (Enzo, Farmingdale, NY), which showed that recombinant p66Shc is modified by SUMO2 ( ).

Techniques: Western Blot, Over Expression, Recombinant

Journal: bioRxiv

Article Title: P66Shc Mediates SUMO2-induced Endothelial Dysfunction

doi: 10.1101/2024.01.24.577109

Figure Lengend Snippet: (A) Modular structure of p66Shc showing conserved lysine-81 in the CH2 domain. (B) Tandem mass spectrometry of in vitro SUMO 2ylated recombinant p66Shc. (C) Immunoblot showing sumoylation of p66Shc in HEK-293 cells overexpressing p66ShcWT (Flag-p66ShcWT) or p66ShcK81R (Flag-p66ShcK81R) with and without SUMO2 (HA-SUMO2). (D) Immunoblot showing sumoylation of p66Shc in HUVECs overexpressing p66ShcWT (Ad-Flag-p66ShcWT) or p66ShcK81R (Ad-p66ShcK81R) with and without SUMO2 (Ad-HA-SUMO2). (E) Immunoblot for sumoylation of recombinant p66ShcWT or p66ShcK81R. Immunoblots are representative of at least three independent experiments. CH2, collagen homology 2; PTB, phosphotyrosine binding; CH1, Collagen homology 1; SH2, Src homology 2.

Article Snippet: We also performed an in vitro sumoylation of p66Shc using recombinant His-tagged p66Shc protein and a commercially available kit (Enzo, Farmingdale, NY), which showed that recombinant p66Shc is modified by SUMO2 ( ).

Techniques: Mass Spectrometry, In Vitro, Recombinant, Western Blot, Binding Assay

Journal: bioRxiv

Article Title: P66Shc Mediates SUMO2-induced Endothelial Dysfunction

doi: 10.1101/2024.01.24.577109

Figure Lengend Snippet: (A) Immunoblot for p66ShcWT in whole cell lysates or the mitochondrial fraction of HUVECs ectopically expressing SUMO2 (Ad-HA-SUMO2) and p66ShcWT (Ad-Flag-p66ShcWT). (B) Quantification of the relative abundance of p66Shc in the mitochondria from A. **P<0.01, ****P<0.0001; n=5; one-way ANOVA. (C) Immunoblot for p66Shc in whole cell lysates or the mitochondrial fraction of HUVECs ectopically expressing SUMO2 (Ad-HA-SUMO2) and p66ShcWT (Ad-Flag-p66ShcWT or p66ShcK81R (Ad-Flag-p66ShcK81R). (D) Quantification of relative abundance of p66Shc in the mitochondria from C. *P<0.05, **P<0.01; n=7; two-way ANOVA. (E) Immunoblot for SUMO2ylated p66Shc (SUMO2-p66Shc) in whole cell lysates and the mitochondrial fraction of HUVECs ectopically expressing SUMO2 (Ad-HA-SUMO2) and p66ShcWT (Ad-Flag-p66ShcWT). Cox IV was used as mitochondrial marker. Data represents mean ±SEM. Immunoblots are representative of at least three independent experiments. ▾-Non-specific band, △ -SUMO2-p66Shc.

Article Snippet: We also performed an in vitro sumoylation of p66Shc using recombinant His-tagged p66Shc protein and a commercially available kit (Enzo, Farmingdale, NY), which showed that recombinant p66Shc is modified by SUMO2 ( ).

Techniques: Western Blot, Expressing, Marker

Journal: bioRxiv

Article Title: P66Shc Mediates SUMO2-induced Endothelial Dysfunction

doi: 10.1101/2024.01.24.577109

Figure Lengend Snippet: Schematic representation of key regulatory pathways impacted by SUMO2-p66Shc in endothelial cells, rescued by p66ShcK81R. Ingenuity Pathway Analysis (IPA) was performed on relative protein expression data from HUVECs expressing SUMO2 with p66ShcWT (top) or p66ShcK81R (bottom) compared to those expressing Ad-Lac Z. The figure illustrates the significant reversal observed in key regulatory pathways between p66ShcWT and p66ShcK81R conditions. Data are derived from a sample size of n=6.

Article Snippet: We also performed an in vitro sumoylation of p66Shc using recombinant His-tagged p66Shc protein and a commercially available kit (Enzo, Farmingdale, NY), which showed that recombinant p66Shc is modified by SUMO2 ( ).

Techniques: Expressing, Derivative Assay

Journal: Cell Death & Disease

Article Title: circ-CBFB upregulates p66Shc to perturb mitochondrial dynamics in APAP-induced liver injury

doi: 10.1038/s41419-020-03160-y

Figure Lengend Snippet: p66Shc silencing was induced via lentiviruses delivered to C57BL/6 mice exposed to APAP (300 mg/kg). a , b Liver p66Shc, p52Shc, and p46Shc protein, n = 3. ** p < 0.01 vs. the control group. c Serum ALT levels, n = 8. d Serum AST levels, n = 8. e H&E staining. Scale bar, 200 μm. f , g Mitochondrial dynamics perturbation was determined via TEM (×2000/×10,000, magnification, red arrow). h Liver H 2 O 2 levels, n = 8. i Liver GSH levels, n = 8. j Liver MDA levels, n = 8. k , l Liver p66Shc, p-p66Shc, OMA1, L-OPA1, S-OPA1, MFN2, DRP1, p-DRP1 protein, n = 3. m , n Liver OMA1 ubiquitination level, n = 3. * p < 0.05, ** p < 0.01 vs. the LV-control group; # p < 0.05, ## p < 0.01 vs. the APAP group.

Article Snippet: The primary antibodies included antibodies against the following: p66Shc (BD Biosciences, USA, 610878), p-p66Shc (Bioss, China, bs-3410R), CYP2E1 (Proteintech, China, 19937-1-AP), OMA1 (Santa, USA, sc-515788), OPA1 (Proteintech, China, 27733-1-AP), MFN2 (Bioss, China, bs-23685R), DRP1 (Wanleibio, China, WL03028), p-DRP1 (Abcam, USA, ab193216), and beta-actin (Proteintech, China, 60008-1-Ig).

Techniques: Staining

Journal: Cell Death & Disease

Article Title: circ-CBFB upregulates p66Shc to perturb mitochondrial dynamics in APAP-induced liver injury

doi: 10.1038/s41419-020-03160-y

Figure Lengend Snippet: a AML12 cells were exposed to APAP (5 mM) and the p66Shc, p52Shc, and p46Shc proteins, n = 3. b – f AML12 cells were transfected with si-control or si-p66Shc and then exposed to APAP (5 mM). b H 2 O 2 levels, n = 8. c , d p66Shc, p-p66Shc, CYP2E1, OMA1, L-OPA1, S-OPA1, MFN2, DRP1, and p-DRP1 proteins, n = 3. e , f OMA1 ubiquitination level, n = 3. g , h AML12 cells were transfected with pcDNA 3.1 or pcDNA-p66Shc and then exposed to APAP and the p66Shc, OMA1, S-OPA1, MFN2, DRP1, and p-DRP1 proteins, n = 3. Mitochondrial ROS, cell apoptosis, and mitochondrial fragmentation were determined via representative fluorescence images of MitoSOX- ( i ), Tunel-( j ), and TOM20-stained ( k ) cells. Scale bar, 200 μm, 100 μm, or 12.5 μm. The colocalization of p66Shc and mitochondria (Mitotracker) ( l ), OMA1 and mitochondria (Mitotracker) ( m ) and p66Shc and OMA1 ( n ). Scale bar, 12.5 μm. ( o ) AML12 cells were transfected with si-control or si-p66Shc and then treated with APAP and MG132. Mitochondrial fragmentation was determined via representative fluorescence images of TOM20-stained cells. Scale bar, 12.5 μm. ** p < 0.01 vs. the si-control group; ## p < 0.01 vs. the APAP group; && p < 0.01 vs. the si-p66Shc group.

Article Snippet: The primary antibodies included antibodies against the following: p66Shc (BD Biosciences, USA, 610878), p-p66Shc (Bioss, China, bs-3410R), CYP2E1 (Proteintech, China, 19937-1-AP), OMA1 (Santa, USA, sc-515788), OPA1 (Proteintech, China, 27733-1-AP), MFN2 (Bioss, China, bs-23685R), DRP1 (Wanleibio, China, WL03028), p-DRP1 (Abcam, USA, ab193216), and beta-actin (Proteintech, China, 60008-1-Ig).

Techniques: Transfection, Fluorescence, TUNEL Assay, Staining

Journal: Cell Death & Disease

Article Title: circ-CBFB upregulates p66Shc to perturb mitochondrial dynamics in APAP-induced liver injury

doi: 10.1038/s41419-020-03160-y

Figure Lengend Snippet: AML12 cells were transected with pcDNA 3.1 or pcDNA-p66Shc and then incubated with mito-TEMPO under APAP treatment. a Mitochondrial ROS were determined via representative fluorescence images of MitoSOX-stained cells. Scale bar, 200 μm. b , c OMA1, S-OPA1, MFN2, DRP1, p-DRP1 protein, n = 3. d OMA1 ubiquitination level, n = 3. ** p < 0.01 vs. the pcDNA 3.1 group; ## p < 0.01 vs. the APAP group; && p < 0.01 vs. the pcDNA-p66Shc group.

Article Snippet: The primary antibodies included antibodies against the following: p66Shc (BD Biosciences, USA, 610878), p-p66Shc (Bioss, China, bs-3410R), CYP2E1 (Proteintech, China, 19937-1-AP), OMA1 (Santa, USA, sc-515788), OPA1 (Proteintech, China, 27733-1-AP), MFN2 (Bioss, China, bs-23685R), DRP1 (Wanleibio, China, WL03028), p-DRP1 (Abcam, USA, ab193216), and beta-actin (Proteintech, China, 60008-1-Ig).

Techniques: Incubation, Fluorescence, Staining

Journal: Cell Death & Disease

Article Title: circ-CBFB upregulates p66Shc to perturb mitochondrial dynamics in APAP-induced liver injury

doi: 10.1038/s41419-020-03160-y

Figure Lengend Snippet: a miR-185-5p has a predicted binding site in p66Shc from humans and mice. b Liver miR-185-5p expression, n = 6. c Hepatocytes miR-185-5p expression, n = 6. * p < 0.05, ** p < 0.01 vs. the control group. d , e AML12 cells were transfected with agomir-NC or miR-185-5p agomir. d miR-185-5p expression, n = 6. e p66Shc protein, n = 3. ** p < 0.01 vs. the ago-NC group. f , g AML12 cells were transfected with antagomir-NC or miR-185-5p antagomir. f miR-185-5p expression, n = 6. g p66Shc protein, n = 3. ** p < 0.01 vs. the ant-NC group. h AML12 cells were transfected with wild-type or mutant SHC1-3′-UTR luciferase constructs and with agomir-NC or miR-185-5p agomir. Luciferase activity was detected 48 h after transfection. ** p < 0.01 vs. the ago-NC group.

Article Snippet: The primary antibodies included antibodies against the following: p66Shc (BD Biosciences, USA, 610878), p-p66Shc (Bioss, China, bs-3410R), CYP2E1 (Proteintech, China, 19937-1-AP), OMA1 (Santa, USA, sc-515788), OPA1 (Proteintech, China, 27733-1-AP), MFN2 (Bioss, China, bs-23685R), DRP1 (Wanleibio, China, WL03028), p-DRP1 (Abcam, USA, ab193216), and beta-actin (Proteintech, China, 60008-1-Ig).

Techniques: Binding Assay, Expressing, Transfection, Mutagenesis, Luciferase, Construct, Activity Assay

Journal: Cell Death & Disease

Article Title: circ-CBFB upregulates p66Shc to perturb mitochondrial dynamics in APAP-induced liver injury

doi: 10.1038/s41419-020-03160-y

Figure Lengend Snippet: AML12 cells were transfected with agomir-NC or miR-185-5p agomir and then exposed to APAP. a , b p66Shc, OMA1, S-OPA1, MFN2, DRP1, and p-DRP1 proteins, n = 3. c Mitochondrial fragmentation was determined via representative fluorescence images of TOM20-stained cells. Scale bar, 12.5 μm. * p < 0.05, ** p < 0.01 vs. the ago-NC group; # p < 0.05, ## p < 0.01 vs. the APAP group.

Article Snippet: The primary antibodies included antibodies against the following: p66Shc (BD Biosciences, USA, 610878), p-p66Shc (Bioss, China, bs-3410R), CYP2E1 (Proteintech, China, 19937-1-AP), OMA1 (Santa, USA, sc-515788), OPA1 (Proteintech, China, 27733-1-AP), MFN2 (Bioss, China, bs-23685R), DRP1 (Wanleibio, China, WL03028), p-DRP1 (Abcam, USA, ab193216), and beta-actin (Proteintech, China, 60008-1-Ig).

Techniques: Transfection, Fluorescence, Staining

Journal: Cell Death & Disease

Article Title: circ-CBFB upregulates p66Shc to perturb mitochondrial dynamics in APAP-induced liver injury

doi: 10.1038/s41419-020-03160-y

Figure Lengend Snippet: AML12 cells were transfected with si-control or si-CBFB and ant-NC or miR-185-5p antagomir and then exposed to APAP. a circ-CBFB expression, n = 6. b , c p66Shc, OMA1, S-OPA1, MFN2, DRP1, and p-DRP1 proteins, n = 3. d Mitochondrial fragmentation was determined via representative fluorescence images of TOM20-stained cells. Scale bar, 12.5 μm. e , f p66Shc, OMA1, S-OPA1, MFN2, DRP1, and p-DRP1 proteins, n = 3. g Mitochondrial fragmentation was determined via representative fluorescence images of TOM20-stained cells. Scale bar, 12.5 μm. ** p < 0.01 vs. the si-control group; ## p < 0.01 vs. the APAP group; && p < 0.01 vs. the si-CBFB group.

Article Snippet: The primary antibodies included antibodies against the following: p66Shc (BD Biosciences, USA, 610878), p-p66Shc (Bioss, China, bs-3410R), CYP2E1 (Proteintech, China, 19937-1-AP), OMA1 (Santa, USA, sc-515788), OPA1 (Proteintech, China, 27733-1-AP), MFN2 (Bioss, China, bs-23685R), DRP1 (Wanleibio, China, WL03028), p-DRP1 (Abcam, USA, ab193216), and beta-actin (Proteintech, China, 60008-1-Ig).

Techniques: Transfection, Expressing, Fluorescence, Staining

Journal: Cell Death & Disease

Article Title: circ-CBFB upregulates p66Shc to perturb mitochondrial dynamics in APAP-induced liver injury

doi: 10.1038/s41419-020-03160-y

Figure Lengend Snippet: circ-CBFB and miR-185-5p silencing was induced via lentiviruses delivered to C57BL/6 mice exposed to APAP. a H&E staining. Scale bar, 200 μm. b , c Mitochondrial dynamics perturbation was determined via TEM (×2000/×10,000, magnification, red arrow). d Serum ALT levels, n = 8. e Serum AST levels, n = 8. f Liver GSH levels, n = 8. g Liver MDA levels, n = 8. h , i Liver p66Shc, OMA1, S-OPA1, MFN2, DRP1, and p-DRP1 proteins, n = 3. ** p < 0.01 vs. the LV-control group; ## p < 0.01 vs. the APAP group; & p < 0.05, && p < 0.01 vs. the LV-CBFB group.

Article Snippet: The primary antibodies included antibodies against the following: p66Shc (BD Biosciences, USA, 610878), p-p66Shc (Bioss, China, bs-3410R), CYP2E1 (Proteintech, China, 19937-1-AP), OMA1 (Santa, USA, sc-515788), OPA1 (Proteintech, China, 27733-1-AP), MFN2 (Bioss, China, bs-23685R), DRP1 (Wanleibio, China, WL03028), p-DRP1 (Abcam, USA, ab193216), and beta-actin (Proteintech, China, 60008-1-Ig).

Techniques: Staining

Journal: Cancers

Article Title: p66Shc Protein—Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer

doi: 10.3390/cancers16193324

Figure Lengend Snippet: Characteristics of ShcA family protein. ( A ) Domain structure of ShcA isoforms. ( B ) Pro-oxidative signaling pathway of p66Shc isoform. PKCβ—protein kinase Cβ; S660-PKCβ—phosphorylated in S660 form of protein kinase Cβ; Pin1—prolyl isomerase; PP2A—serine/threonine protein phosphatase type 2; cyt. c—cytochrome c.

Article Snippet: p66Shc-S36A , Mouse , Merck KGaA, Darmstadt, Germany.

Techniques:

Journal: Cancers

Article Title: p66Shc Protein—Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer

doi: 10.3390/cancers16193324

Figure Lengend Snippet: Characteristics of individual genetic modifications performed in clones of MDA-MB-231 and MCF-7 cell lines.

Article Snippet: p66Shc-S36A , Mouse , Merck KGaA, Darmstadt, Germany.

Techniques: Clone Assay, Modification, Transfection, Plasmid Preparation, Over Expression, Residue, Mutagenesis, Knock-Out

Journal: Cancers

Article Title: p66Shc Protein—Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer

doi: 10.3390/cancers16193324

Figure Lengend Snippet: List of used primary and secondary antibodies.

Article Snippet: p66Shc-S36A , Mouse , Merck KGaA, Darmstadt, Germany.

Techniques: Transduction

Journal: Cancers

Article Title: p66Shc Protein—Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer

doi: 10.3390/cancers16193324

Figure Lengend Snippet: The level of ShcA proteins (p66Shc, p52Shs, and p46Shc) in the MDA-MB-231 clones obtained through ( A ) lentiviral transfection or ( B ) genome editing using the CRISPR/Cas9 method by the Western Blot analysis. ( C ) The schematic of the U6gRNA-Cas9-2A-GFP plasmid, enabling genome editing in MDA-MB-231 cell lines. ( D ) Proliferation rates of the individual MDA-MB-231 clones.

Article Snippet: p66Shc-S36A , Mouse , Merck KGaA, Darmstadt, Germany.

Techniques: Clone Assay, Transfection, CRISPR, Western Blot, Plasmid Preparation

Journal: Cancers

Article Title: p66Shc Protein—Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer

doi: 10.3390/cancers16193324

Figure Lengend Snippet: The level of individual proteins involved in the pro-oxidative p66Shc signaling pathway. The levels of proteins were standardized to the GAPDH ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: p66Shc-S36A , Mouse , Merck KGaA, Darmstadt, Germany.

Techniques:

Journal: Cancers

Article Title: p66Shc Protein—Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer

doi: 10.3390/cancers16193324

Figure Lengend Snippet: Mitochondrial network morphology in the individual MDA-MB-231 clones treated (+DOX) and untreated (–DOX) with doxorubicin and mitochondrial membrane potential. The ( A ) Ctrl, ( B ) clone with elevated p66Shc protein levels, ( C ) clone with elevated levels of the S36A-mutated form of the p66Shc protein, and ( D ) clone with the knockout of p66Shc; nuclei were stained blue (DAPI) and the mitochondrial network was stained red (MitoRed); ( E ) mitochondrial membrane potential, * p < 0.05.

Article Snippet: p66Shc-S36A , Mouse , Merck KGaA, Darmstadt, Germany.

Techniques: Clone Assay, Membrane, Knock-Out, Staining