Journal: Molecular Cancer Therapeutics

Article Title: Discovery and Characterization of Brigimadlin, a Novel and Highly Potent MDM2–p53 Antagonist Suitable for Intermittent Dose Schedules

doi: 10.1158/1535-7163.MCT-23-0783

Figure Lengend Snippet: MDM2 expression and brigimadlin dose-dependent regulation of p53 target genes and apoptosis in MDM2 -amplified and non-amplified solid tumor cell lines. A, MDM2 mRNA ( Ai ) and protein ( Aii ) expression in MDM2 -amplified and non-amplified cell lines. B, Brigimadlin dose-dependent upregulation of mRNA of seven genes in MDM2-amplified (SJSA-1) and non-amplified (U-2 OS) solid tumor cell lines (mean of technical replicates ± standard deviation). Bi , PUMA (BBC3); Bii , P21 (CDKN1A); Biii , MDM2 ; Biv , CD80 ; Bv , BAX ; Bvi , BIM ; Bvii , BCL-XL. One-step RT-PCR normalized to GAPDH: PUMA and P21 (24 hours) and BAX, BIM, and BCL-XL (48 hours). Two-step RT-PCR normalized to GAPDH: MDM2 and CD80 (48 hours). C, Western blot of biomarker proteins in SJSA-1 and U-2 OS cell lines after 24 and 72-hour treatment with brigimadlin. D, Brigimadlin dose and time dependence of cleaved PARP ( Di ) and cleaved caspase-3 ( Dii ) signal relative to vehicle (MSD apoptosis assay). amp, amplified; cl, cleaved; h, hour; rel, relative; TPM, transcripts per million.

Article Snippet: Cell or tumor lysates were prepared according to the Meso Scale Discovery (MSD) cell lysis protocol and analyzed in duplicate with MSD Apoptosis Panel Whole Cell Lysate Kit according to the manufacturer’s instructions.

Techniques: Expressing, Amplification, Standard Deviation, One Step RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Biomarker Discovery, Apoptosis Assay

Journal: Molecular Cancer Therapeutics

Article Title: Discovery and Characterization of Brigimadlin, a Novel and Highly Potent MDM2–p53 Antagonist Suitable for Intermittent Dose Schedules

doi: 10.1158/1535-7163.MCT-23-0783

Figure Lengend Snippet: Modulation of p53 target genes and markers of apoptosis in SJSA-1 tumor-bearing mice treated with brigimadlin. A, Mean (± SEM) change in GDF15 (MIC-1) levels in serum samples 24 hours after a single oral dose of brigimadlin. SJSA-1 tumor-bearing mice were treated with a single oral dose of either 0.25, 0.75, 2, or 10 mg/kg brigimadlin, respectively, or with vehicle. Absolute GDF15 levels are depicted in pg/mL serum. Error bars represent the SEM of three biological repeats. B, Mean (± SEM) change in mRNA levels of p53 target genes in SJSA-1 tumor samples 24 hours after a single oral dose of brigimadlin. SJSA-1 tumor-bearing mice were treated with a single oral dose of 0.25, 0.75, 2, or 10 mg/kg brigimadlin, respectively, or vehicle. Changes of mRNA levels are depicted as fold change relative to the vehicle control. Error bars represent the SEM of three biological repeats. C, Correlation of GDF15 protein levels from serum samples with fold change GDF15 mRNA in tumor samples. D, Brigimadlin concentration–time curve in plasma of SJSA-1 tumor-bearing mice treated with either brigimadlin 2 mg/kg single dose or 1 mg/kg BID × 1 day. Brigimadlin plasma concentration over time is shown. E, Dose-dependent mRNA modulation of p53 target genes in SJSA-1 tumors harvested at indicated posttreatment time points (two-step RT-PCR data normalized to GAPDH and relative to respective control): Ei , p21 (CDKN1A); Eii , MDM2 ; Eiii , GDF15 ; Eiv , PUMA (BBC3); Ev , CD80 ; Evi , BAX ; Evii , BIM ; Eviii , BCL-XL. F, Dose- and time-dependent modulation of apoptotic markers ( Fi , cleaved PARP and Fii , cleaved caspase-3) in SJSA-1 tumors harvested at indicated posttreatment time points (MSD apoptosis assay). BID, twice daily; Cl, cleaved; d, day; h, hours; rel, relative; SD, standard deviation; SEM, standard error of the mean.

Article Snippet: Cell or tumor lysates were prepared according to the Meso Scale Discovery (MSD) cell lysis protocol and analyzed in duplicate with MSD Apoptosis Panel Whole Cell Lysate Kit according to the manufacturer’s instructions.

Techniques: Control, Concentration Assay, Clinical Proteomics, Reverse Transcription Polymerase Chain Reaction, Apoptosis Assay, Standard Deviation

Journal:

Article Title: Suppression of breast cancer metastasis through the inhibition of VEGF-mediated tumor angiogenesis

doi:

Figure Lengend Snippet: Adenoviral-mediated p16 expression as determined by Western blot analysis. MDA-MB-231 cells were grown in culture dishes and transduced by recombinant adenovirus expressing p16 (AdRSVp16) at moi of 200. Seventy-two h later the cells were harvested and cell lysate was subjected to a Western blot analysis using anti-p16 antibody as the primary antibody. The same blot was immunoblotted with anti-actin antibody as an internal control.

Article Snippet: A recombinant adenovirus (Ad-HIF-1α/HT2) expressing fusion protein of hypoxia inducible factor-1α (HIF-1α) and a reporter gene HaloTag (HT2) (Promega) was used in this assay to trace the cellular localization of HIF-1α protein.

Techniques: Expressing, Western Blot, Recombinant, Control

Journal: American Journal of Translational Research

Article Title: Deferoxamine enhances the migration of dental pulp cells via hypoxia-inducible factor 1α

doi:

Figure Lengend Snippet: DPCs were transfected with pAd-GFP-shRNA-HIF-1α for 48 h. (A1: Fluorescence microscopy. A2: Inverted phase-contrast microscopy). DPCs were transfected with pAd-GFP for 48 h (B1: Fluorescence microscopy. B2: Inverted phase-contrast microscopy). The control group can be observed in (C1 and C2) (100 ×).

Article Snippet: A recombinant adenovirus vector carrying the GFP, the shRNA-HIF-1α targeting the human HIF-1α gene (pAd-GFP-shRNA-HIF-1α), or the control adenovirus (pAd-GFP) were constructed by Novobio Biotechnology (Shanghai, China).

Techniques: Transfection, shRNA, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Chitosan Particles Complexed with CA5-HIF-1α Plasmids Increase Angiogenesis and Improve Wound Healing

doi: 10.3390/ijms241814095

Figure Lengend Snippet: Luciferase expression over time when using chitosan particles for plasmid transfection. Verification of transfection efficiency of luciferase plasmids (Luc) using chitosan particles (CPs) in an in vivo reporter assay wound model. Statistical significance was calculated using two-way ANOVA with Holm–Sidak’s multiple comparison test (* p < 0.05) ( n = 3).

Article Snippet: Plasmid DNA encoding the human CA5-HIF-1α gene and firefly luciferase gene was provided by Nature Technology Corporation (NTC) (Lincoln, NE, USA). pCMV-GFP was a gift from Connie Cepko (Addgene plasmid #11153; http://n2t.net/addgene:11153 ; RRID:Addgene_11153 (accessed on 12 July 2021).

Techniques: Luciferase, Expressing, Plasmid Preparation, Transfection, In Vivo, Reporter Assay, Comparison

Journal: Oncogenesis

Article Title: The HIF/PHF8/AR axis promotes prostate cancer progression

doi: 10.1038/oncsis.2016.74

Figure Lengend Snippet: PHF8 is upregulated by HIFs during hypoxia and regulates histone methylation in response to hypoxia. ( a – c ) LNCaP ( a ), 22RV1 ( b ) and DU145 ( c ) cells were cultured first at normoxic conditions and then shifted to hypoxic conditions for 0, 12, 24 h. Cells were collected and whole-cell lysates were prepared. Proteins were separated on 8% SDS–polyacrylamide gel electrophoresis and western blotting was performed with antibodies against HIF1α, HIF2α, PHF8, GLUT-1 and β-actin as indicated. ( d – f ) LNCaP ( d ), 22RV1 ( e ) and DU145 ( f ) cells were cultured first at normoxic conditions and then shifted to hypoxic conditions for 0, 12, 24 h. The total RNAs were then prepared and quantitative RT–PCR for PHF8 was performed. The results are presented as the mean±s.e.m. of three independent experiments. ( g ) Knockdown HIF2α or HIF1α abrogated hypoxia-induced upregulation of PHF8 proteins. LNCaP cells stably infected with control shRNA (shCtrl) or shHIF2α (left panel) /shHIF1α (right panel) were cultured under normoxic (N) or hypoxic (H) conditions for 12 h and then collected for western blot analysis using antibodies as indicated. ( h ) Hypoxia-induced PHF8 expression correlates with reduced levels of H3K9me1/2 and H4K20me1. On the left panel, LNCaP cells were subjected to normoxic and hypoxic conditions for 12 h. Whole-cell lysates were prepared and proteins were separated on SDS–polyacrylamide gel electrophoresis. Then western blots were conducted with antibodies against H3K9me1, H3K9me2, H3K9me3, H4K20me1, H3, H4, PHF8 and β-actin as indicated. On the right panel, LNCaP cells were stably infected with control shRNA or shPHF8 and the histone methylation status under normoxic conditions were analyzed by western blot. Note that PHF8 knockdown resulted in increased levels of H3K9me1/2 and H4K20me1 but not H3K9me3, indicating that PHF8 controls global levels of H3K9me1/2 and H4K20me1 in LNCaP cells. The quantitative analysis of western blot results was based on Image J software version 1.44p (NIH, USA).

Article Snippet: Control shRNA or shRNAs specific for human HIF1α (SH001530), HIF2α (SH001430) and PHF8 (SH184896) mRNAs were purchased from Yingrun Biotechnology Inc. (Changsha, China) ( ).

Techniques: Methylation, Cell Culture, Polyacrylamide Gel Electrophoresis, Western Blot, Quantitative RT-PCR, Stable Transfection, Infection, shRNA, Expressing, Software

Journal: Oncogenesis

Article Title: The HIF/PHF8/AR axis promotes prostate cancer progression

doi: 10.1038/oncsis.2016.74

Figure Lengend Snippet: Castration-induced hypoxia upregulates HIFs and PHF8 in prostate cancer tissues ( a ) Western blotting was conducted on samples from pre-and post-castration treatment in the same patients, using antibodies against HIF1α, HIF2α and PHF8 and β-actin. The intensities of the bands were estimated by Image J and normalized by comparing to that of the β-actin. Results from three representative patients were shown. ( b ) The representative results of immunohistochemistry analysis on pre- and post-castration prostate cancer tissues. The pre- and post-castration prostate cancer samples from 14 prostate cancer patients were stained with antibodies against HIF1α, HIF2α and PHF8. Original magnification was × 400. ( c ) The staining intensities were semi-quantified and the averages of the optical densities are shown in the bar graphs. Student's t -tests were used to compare differences between two groups, **P <0.01.

Article Snippet: Control shRNA or shRNAs specific for human HIF1α (SH001530), HIF2α (SH001430) and PHF8 (SH184896) mRNAs were purchased from Yingrun Biotechnology Inc. (Changsha, China) ( ).

Techniques: Western Blot, Immunohistochemistry, Staining

Journal: Oncogenesis

Article Title: The HIF/PHF8/AR axis promotes prostate cancer progression

doi: 10.1038/oncsis.2016.74

Figure Lengend Snippet: A proposed working model of the HIF/PHF8/AR axis in CRPC progression. Prostate cancer- and castration-induced hypoxia upregulate both HIF1α and HIF2α in prostate cancer cells, which subsequently upregulates PHF8 expression. Under both normoxic and hypoxic conditions, PHF8 interacts with and facilitates AR transcriptional activation. Mechanistically, PHF8 specifically modifies the histone tails through its demethylase activity in the promoter regions of AR target genes and enhances the expression of AR targets. HIFs, PHF8 and AR thus act together to favor the development and/or progression of CRPC.

Article Snippet: Control shRNA or shRNAs specific for human HIF1α (SH001530), HIF2α (SH001430) and PHF8 (SH184896) mRNAs were purchased from Yingrun Biotechnology Inc. (Changsha, China) ( ).

Techniques: Expressing, Activation Assay, Activity Assay