hep27  (Santa Cruz Biotechnology)

Journal: American Journal of Cancer Research

Article Title: Gentian violet induces apoptosis and ferroptosis via modulating p53 and MDM2 in hepatocellular carcinoma

doi:

Figure Lengend Snippet: The high expression of MDM2 and p53 was dependent on Hep27. Western blot detection of MDM2, p53 and Hep27 expression in SK-HEP-1 and SMMC-7721 cells at the indicated treatment conditions for 48 h. From left to right for each cell line: DMSO, GV (100 nM for SK-HEP-1 and 300 nM for SMMC-7721), GV in cells transfected with Hep27 siRNA, GV co-treated with Fer-1 (2 μM).

Article Snippet: Western blotting Total protein was extracted using RIPA buffer (Solarbio, Beijing, China), resolved on SDS-PAGE, transferred to PVDF membrane, and probed with primary antibodies: Anti-p53 (Cell Signaling, 1:1000), Anti-MDM2 (Cell Signaling, 1:1000), Anti-Hep27 (Cell Signaling, 1:1000), and Anti-GAPDH (Cell Signaling, 1:1000).

Techniques: Expressing, Western Blot, Transfection

Journal: American Journal of Cancer Research

Article Title: Gentian violet induces apoptosis and ferroptosis via modulating p53 and MDM2 in hepatocellular carcinoma

doi:

Figure Lengend Snippet: The proposed molecular mechanisms of GV-induced killing of HCC. When NOX is inhibited by GV, Hep27 is increased, which leads to the blockade of the ubiquitination and degradation of p53 by MDM2. Consequently, the levels of p53 and MDM2 are increased. GV also directly increases p53 level. Increased p53 and MDM2 levels trigger ferroptosis and mitochondrial apoptosis (through the activation of caspase 8) by a coordinated ROS cross point and downstream signaling cascades. On the other hand, GV also triggers death receptor apoptosis (through the activation of caspase 9) by upregulating death receptors DR4/5 and ligands FAS-ligand and TRAIL.

Article Snippet: Western blotting Total protein was extracted using RIPA buffer (Solarbio, Beijing, China), resolved on SDS-PAGE, transferred to PVDF membrane, and probed with primary antibodies: Anti-p53 (Cell Signaling, 1:1000), Anti-MDM2 (Cell Signaling, 1:1000), Anti-Hep27 (Cell Signaling, 1:1000), and Anti-GAPDH (Cell Signaling, 1:1000).

Techniques: Activation Assay

Journal: American Journal of Cancer Research

Article Title: Gentian violet induces apoptosis and ferroptosis via modulating p53 and MDM2 in hepatocellular carcinoma

doi:

Figure Lengend Snippet: The high expression of MDM2 and p53 was dependent on Hep27. Western blot detection of MDM2, p53 and Hep27 expression in SK-HEP-1 and SMMC-7721 cells at the indicated treatment conditions for 48 h. From left to right for each cell line: DMSO, GV (100 nM for SK-HEP-1 and 300 nM for SMMC-7721), GV in cells transfected with Hep27 siRNA, GV co-treated with Fer-1 (2 μM).

Article Snippet: The relative gene expression level was calculated using the comparative Ct method and normalized to β-actin. siRNA transfection Knockdown of MDM2, p53 and Hep27 was performed using siRNA technology. siRNA oligos targeting MDM2 (sc-29394), p53 (sc-29435), Hep27 (sc-92153) or non-specific siRNAs were purchased from Santa Cruz Biotechnology (Dallas, TX). siRNAs were transfected into cells by electroporation using Nucleofector Device and the Nucleofector Kit L (Lonza, Basel, Switzerland).

Techniques: Expressing, Western Blot, Transfection

Journal: American Journal of Cancer Research

Article Title: Gentian violet induces apoptosis and ferroptosis via modulating p53 and MDM2 in hepatocellular carcinoma

doi:

Figure Lengend Snippet: The proposed molecular mechanisms of GV-induced killing of HCC. When NOX is inhibited by GV, Hep27 is increased, which leads to the blockade of the ubiquitination and degradation of p53 by MDM2. Consequently, the levels of p53 and MDM2 are increased. GV also directly increases p53 level. Increased p53 and MDM2 levels trigger ferroptosis and mitochondrial apoptosis (through the activation of caspase 8) by a coordinated ROS cross point and downstream signaling cascades. On the other hand, GV also triggers death receptor apoptosis (through the activation of caspase 9) by upregulating death receptors DR4/5 and ligands FAS-ligand and TRAIL.

Article Snippet: The relative gene expression level was calculated using the comparative Ct method and normalized to β-actin. siRNA transfection Knockdown of MDM2, p53 and Hep27 was performed using siRNA technology. siRNA oligos targeting MDM2 (sc-29394), p53 (sc-29435), Hep27 (sc-92153) or non-specific siRNAs were purchased from Santa Cruz Biotechnology (Dallas, TX). siRNAs were transfected into cells by electroporation using Nucleofector Device and the Nucleofector Kit L (Lonza, Basel, Switzerland).

Techniques: Ubiquitin Proteomics, Activation Assay

Journal: ACS Central Science

Article Title: GlycoGrip : Cell Surface-Inspired Universal Sensor for Betacoronaviruses

doi: 10.1021/acscentsci.1c01080

Figure Lengend Snippet: (A(i)) Electrostatic potential map (ESP) of 1-up spike without glycans included for simple illustration. ESP is illustrated on a range from −4 to +4 kT/e. The RBD supersite is highlighted with an orange dashed line. (A(ii)) Rotated viewpoint of electrostatic potential map of 1-up spike. Again, ESP is illustrated on a range from −4 to +4 kT/e and the RBD HS supersite is highlighted with an orange dashed line. (A(iii)) Close up view of spike RBD (surface representation) and bound HEP octamer (hep8mer, licorice representation). Both spike RBD and hep8mer are colored according to their corresponding electrostatic potentials (ranging from −4 to +4 kT/e). (A(iv)) Close-ups of key interactions between hep8mer (licorice representation, orange carbon atoms) and spike RBD (gray cartoon representation) facilitated by R346, N354, K356, and R357. (B) Computational calculation of binding energy of hep8mer to spike RBD over a range of implicit solvent ionic strengths. (C) BLI results of HEP to spike in three different concentrations of NaCl (10, 75, and 150 mM). (D) Response of the lateral flow test in different ionic concentrations (10, 75, and 150 mM). (E) Screening results of HS15, HEP15, HEP27, CS25, DEX5, and DEX50 using LFSA. p values <0.05 (*), 0.01 (**) and 0.001 (***) determined using a one-way ANOVA with Tukey’s post hoc test.

Article Snippet: HEP27 (HP-207, porcine mucosa) was purchased from Creative PEGWorks.

Techniques: Binding Assay, Solvent

Journal: Molecular and Cellular Biology

Article Title: Mitochondrial HEP27 Is a c-Myb Target Gene That Inhibits Mdm2 and Stabilizes p53 ▿ †

doi: 10.1128/MCB.01284-09

Figure Lengend Snippet: Hep27 is a mitochondrial matrix protein. (A) U2OS cells infected by adenovirus (Ad) expressing control GFP or Mdm2 for 24 hours were immunoprecipitated with anti-Mdm2 2A10 antibody and resolved by SDS-PAGE. Hep27 was identified by mass spectrometry protein microsequencing analysis. First lane, Ad-GFP; second lane, protein ladder; third lane, Ad-Mdm2. (B) Immunofluorescence imaging of endogenous Hep27. U2OS cells were probed with anti-Hep27 antibody, Mitotracker CMXRos, and DAPI. Phase, phase-contrast image. (C) Expression constructs expressing full-length C-terminal Flag-tagged Hep27 or Hep27-DelN24 were transfected into U2OS cells. Anti-Flag M2 antibody was used to detect the flag-tagged protein. Mitotracker CMXRos was used to visualize mitochondria, and DAPI was used for nuclear staining. (D) Expression constructs expressing control GFP or HepMTS-GFP fusion protein were transfected into U2OS cells. Mitotracker CMXRos was used to visualize the mitochondria, and DAPI for nuclear staining. (E) Differential detergent fractionation was performed in triplicate in U2OS cells to fractionate endogenous Hep27. Hsp70 is the mitochondrial control, actin is the cytosolic control, and histone H3 is the nuclear control. Cyto, cytosolic compartment; Mito, mitochondrial compartment. (F) Transmission electron microscopy of endogenous Hep27 in U2OS cells. U2OS monolayers were fixed, probed with control IgG antibody (left) or anti-Hep27 primary antibody (right), and visualized by TEM.

Article Snippet: The Hep27 sequences (Invitrogen) targeting the 3′ end of the coding sequence were RNAi 1, 5′-GGAACAUCAUCAGCUGCAGAGGAUU, and RNAi 2, 5′-CCUGGUCUCUUCCAUUGCAGCUUAU.

Techniques: Infection, Expressing, Immunoprecipitation, SDS Page, Mass Spectrometry, Immunofluorescence, Imaging, Construct, Transfection, Staining, Fractionation, Transmission Assay, Electron Microscopy

Journal: Molecular and Cellular Biology

Article Title: Mitochondrial HEP27 Is a c-Myb Target Gene That Inhibits Mdm2 and Stabilizes p53 ▿ †

doi: 10.1128/MCB.01284-09

Figure Lengend Snippet: Hep27 partially localizes to the nucleus. (A) U2OS cell monolayers were fixed and probed for endogenous Hep27 with anti-Hep27 antibody. Arrows denote gold bead-labeled Hep27 as detected by TEM. (B) Differential detergent fractionation was used to fractionate endogenous total (Tot) Hep27 from U2OS cells into the cytosolic (Cyto), mitochondrial (Mito), and nuclear (Nuc) compartments for detection by Western blot assay. Actin, Hsp70, and histone H3 were used as cytosolic, mitochondrial, and nuclear controls, respectively.

Article Snippet: The Hep27 sequences (Invitrogen) targeting the 3′ end of the coding sequence were RNAi 1, 5′-GGAACAUCAUCAGCUGCAGAGGAUU, and RNAi 2, 5′-CCUGGUCUCUUCCAUUGCAGCUUAU.

Techniques: Labeling, Fractionation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Mitochondrial HEP27 Is a c-Myb Target Gene That Inhibits Mdm2 and Stabilizes p53 ▿ †

doi: 10.1128/MCB.01284-09

Figure Lengend Snippet: Hep27 binds to Mdm2. (A) The TNT quick coupled transcription/translation system was used to synthesize [35S]methionine-labeled Mdm2 and Hep27-Flag. Reciprocal immunoprecipitations with anti-Mdm2 2A10 or anti-Flag M2 were detected by Western blot assay. Loading control shows 10% of the starting material. (B) Adenovirus (Ad) constructs expressing Mdm2 or Myc-tagged Hep27 were transduced into H1299 cells. Reciprocal immunoprecipitations with anti-Mdm2 2A10 or anti-Hep27 were detected by Western blot assay. Loading control shows 10% of the starting material. (C) Endogenous Mdm2 and Hep27 were immunoprecipitated from HepG2 cells using control (Con) anti-Myc 9E10 antibody or three different Mdm2 antibodies: 4B11, 2A10, and SMP14. Cells were incubated in the presence or absence of the proteasome inhibitor MG132 for six hours prior to IP. Loading control shows 10% of the starting material. (D) Expression constructs expressing Myc-tagged wild-type and C- or N-terminal Mdm2 truncation mutants were transfected into U2OS cells. Mdm2 constructs were immunoprecipitated with anti-Myc 9E10 antibody, and proteins detected by Western blot assay. Endogenous Rpl11 was used as an Mdm2 binding control. The loading control shows 10% of the starting material. The schematic (bottom) summarizes the experimental evidence that narrows Hep27 binding to Mdm2 amino acids 200 to 294.

Article Snippet: The Hep27 sequences (Invitrogen) targeting the 3′ end of the coding sequence were RNAi 1, 5′-GGAACAUCAUCAGCUGCAGAGGAUU, and RNAi 2, 5′-CCUGGUCUCUUCCAUUGCAGCUUAU.

Techniques: Labeling, Western Blot, Construct, Expressing, Immunoprecipitation, Incubation, Transfection, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Mitochondrial HEP27 Is a c-Myb Target Gene That Inhibits Mdm2 and Stabilizes p53 ▿ †

doi: 10.1128/MCB.01284-09

Figure Lengend Snippet: Hep27 binding to Mdm2 results in p53 stabilization. (A) Expression constructs expressing Mdm2, p53, Hep27, and Hep27-DelN24 were coexpressed in H1299 cells by transient transfection for 24 hours, and indicated proteins detected by Western blot assay. (B) Adenovirus (Ad) construct expressing Hep27-Flag was transduced into WI-38 primary fibroblasts for 24 hours, and indicated proteins detected by Western blot assay. (C) Hep27-DelN24 was transiently expressed in MCF7 cells for 24 hours, and indicated proteins detected by Western blot assay. (D) Two independent oligonucleotides were used to knock down Hep27 in MCF7 and U2OS cells. Endogenous Hep27 and p53 were assessed by Western blot assay. Con, control.

Article Snippet: The Hep27 sequences (Invitrogen) targeting the 3′ end of the coding sequence were RNAi 1, 5′-GGAACAUCAUCAGCUGCAGAGGAUU, and RNAi 2, 5′-CCUGGUCUCUUCCAUUGCAGCUUAU.

Techniques: Binding Assay, Expressing, Construct, Transfection, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Mitochondrial HEP27 Is a c-Myb Target Gene That Inhibits Mdm2 and Stabilizes p53 ▿ †

doi: 10.1128/MCB.01284-09

Figure Lengend Snippet: The proto-oncogene c-Myb regulates Hep27 expression. (A) WI-38 fibroblasts transfected with control vector or c-Myb expression construct were incubated for 36 hours. mRNA was harvested and reverse transcribed for qRT-PCR analysis targeting exons 1 and 2. Results are presented as expression levels of Hep27 relative to that of GAPDH control. (B) c-Myb expression construct was used to transfect WI-38 cells for 36 hours. Anti-Hep27 antibody was used to detect Hep27 expression by Western blot assay. (C) Short hairpin RNA constructs targeting GFP or c-Myb were used to establish stable MCF7 cell lines. Anti-c-Myb and anti-Hep27 antibodies were used to detect endogenous c-Myb and Hep27 levels by Western blot assay. (D) Schematic illustrating putative c-Myb response elements in the promoter of the Hep27 gene (DHRS2). The consensus Myb response element (MRE) is shown. Constructs for the minimal promoter mapping and site-directed mutagenesis are shown. (E and F) Luciferase reporter constructs were transfected into H1299 cells in the presence and absence of c-Myb coexpression for 48 hours. pGL3 Basic and c-Myb were transfected alone as controls, and β-galactosidase expressed with all samples as a transfection control. Relative light units (RLU) represent the relative expression of the luciferase/β-galactosidase ratio. Error bars represent standard errors of the means.

Article Snippet: The Hep27 sequences (Invitrogen) targeting the 3′ end of the coding sequence were RNAi 1, 5′-GGAACAUCAUCAGCUGCAGAGGAUU, and RNAi 2, 5′-CCUGGUCUCUUCCAUUGCAGCUUAU.

Techniques: Expressing, Transfection, Plasmid Preparation, Construct, Incubation, Quantitative RT-PCR, Western Blot, shRNA, Mutagenesis, Luciferase

Journal: Molecular and Cellular Biology

Article Title: Mitochondrial HEP27 Is a c-Myb Target Gene That Inhibits Mdm2 and Stabilizes p53 ▿ †

doi: 10.1128/MCB.01284-09

Figure Lengend Snippet: c-Myb induces p53 stabilization and activation in a Hep27-dependent manner. (A) Expression construct expressing c-Myb was transfected into MCF7 cells for 24 hours, and indicated proteins were detected by Western blot assay. Con, control. (B) Scrambled (Scr) RNAi and Hep27 oligonucleotides were transfected into MCF7 cells and incubated for 48 hours. Cells were washed, and an additional transfection performed with a c-Myb expression construct. Cells were incubated for an additional 24 hours and harvested for Western blot assay. (C) Short hairpin RNA constructs targeting GFP or c-Myb were used to establish stable MCF7 cell lines. Endogenous c-Myb, Hep27, and p53 levels were detected by Western blot assay. (D) Expression construct for c-Myb was transfected into MCF7 cells for 24 hours. Whole-cell lysate (Total) and the nuclear fraction (Nucleus) were assessed by Western blot assay. Actin is a cytosolic control, and histone H3 is a nuclear control. (E) Model for c-Myb-Hep27-Mdm2-p53 signaling. The proto-oncogene c-Myb induces the expression of full-length Hep27 preprotein. Hep27 is actively imported to the mitochondria by an N-terminal mitochondrial targeting signal (MTS). Once the protein is imported into the mitochondrial matrix, the MTS is cleaved off to produce mature Hep27 (Hep27m). A minor fraction of Hep27m translocates to the nucleus to bind to Mdm2, resulting in p53 stabilization and subsequent transactivation of downstream target genes.

Article Snippet: The Hep27 sequences (Invitrogen) targeting the 3′ end of the coding sequence were RNAi 1, 5′-GGAACAUCAUCAGCUGCAGAGGAUU, and RNAi 2, 5′-CCUGGUCUCUUCCAUUGCAGCUUAU.

Techniques: Activation Assay, Expressing, Construct, Transfection, Western Blot, Incubation, shRNA

Journal: Molecular and Cellular Biology

Article Title: Mitochondrial HEP27 Is a c-Myb Target Gene That Inhibits Mdm2 and Stabilizes p53 ▿ †

doi: 10.1128/MCB.01284-09

Figure Lengend Snippet: A potential c-Myb-Hep27-Mdm2-p53 pathway in breast cancer. The Miller et al. dataset (n = 236) (28) was classified into the breast tumor-intrinsic subtypes (basal-like, HER2-enriched, luminal [Lum] A, luminal B, and normal-like) using the PAM50 predictor (35). (A) Kaplan-Meier survival analysis of disease-specific survival stratified by subtype. P value was determined by log-rank test, testing the null hypothesis that the survival curves are identical across the subtypes. (B) Hep27 mRNA expression in ER− and ER+ tumors (n = 232). P values calculated by t test show different expression values across ER status or biologically defined breast tumor subtypes. (C) c-Myb and (D) Hep27 expression varies by intrinsic subtype. P values were determined by ANOVA, testing the null hypothesis that all group means are equal. (E) Hep27 expression in mutant-p53 (mut) and wild-type-p53 (wt) tumors. P values calculated by t test show different expression values across ER status or biologically defined breast tumor subtypes. (F) A p53 mutation signature (56) was applied to this dataset and correlated with breast tumor subtype. P values were determined by ANOVA, testing the null hypothesis that all group means are equal. For box and whisker plots, the horizontal bars represent the medians, the lower and upper hinges of the boxes represent the first and third quartiles, and the whiskers extend to ±1.58 × interquartile range/.

Article Snippet: The Hep27 sequences (Invitrogen) targeting the 3′ end of the coding sequence were RNAi 1, 5′-GGAACAUCAUCAGCUGCAGAGGAUU, and RNAi 2, 5′-CCUGGUCUCUUCCAUUGCAGCUUAU.

Techniques: Expressing, Mutagenesis, Whisker Assay