Journal: Biomolecules & Therapeutics

Article Title: AR-mTOR-SRF Axis Regulates HMMR Expression in Human Prostate Cancer Cells

doi: 10.4062/biomolther.2021.040

Figure Lengend Snippet: mTOR activity is critical for regulating the expression of HMMR protein. (A) Cells were exposed to a variety of signaling pathway inhibitors for 24 h and HMMR protein level was measured by immunoblotting analysis. (B-E) PC-3 and LNCaP cells were cultured for 24 h with various doses of LY294002 (0 to 10 nM), KU0063794 (0 to 800 nM), rapamycin (0, 200, and 500 nM), and Torin2 (0 to 500 nM). (F) PC-3 cells were incubated for the indicated time in the absence or presence of 200 nM Torin2. Twenty to 50 μg of protein from cell lysates was subjected to western blot analysis. Data shown are representative of two or three independent experiments (P9; PD98059, MEK inhibitor, SP; SP600125, JNK inhibitor, SB; SB202190, p38 MAPK inhibitor, LY; LY293002, PI3K inhibitor, P1; PD153035, EGFR inhibitor, TR; Torin2, mTOR inhibitor, KU; KU0063794, mTOR inhibitor, Rapa; Rapamycin, mTOR inhibitor).

Article Snippet: The sources for the following chemicals, reagents and antibodies were: Rapamycin (WAKO Chemical, Richmond, VA, USA); Torin 2 and Ku0063794 (TOCRIS, Bristol, UK); IGF-1 (BioVision, Milpitas, CA, USA); DHT, LY294002, SP600125, SB202190, PD153035, and PD98059 (Sigma Aldrich, St. Louis, MO, USA); characterized fetal bovine serum (FBS) (ATCC, Manassas, VA, USA); anti-HMMR (Bethyl, Montgomery, TX, USA); anti-p-Rb(S807/811), anti-CDK2, anti-E-cadherin (24E10), anti-vimentin (D21H3), anti-p-Erk1/2 (T202/Y204), and anti-Erk (Cell signaling, Danvers, MA, USA); anti-cyclin D2 (Santa Cruz, Dallas, TX, USA); anti-GAPDH (Bioworld Technology, St. Louis Park, MN, USA); anti-β-actin, anti-FlagM2, and dextran-charcoal stripped fetal bovine serum (DC-FBS) (Sigma Aldrich); characterized fetal bovine serum (FBS) (HyClone, Logan, UT, USA).

Techniques: Activity Assay, Expressing, Western Blot, Cell Culture, Incubation

Journal: Biomolecules & Therapeutics

Article Title: AR-mTOR-SRF Axis Regulates HMMR Expression in Human Prostate Cancer Cells

doi: 10.4062/biomolther.2021.040

Figure Lengend Snippet: Inactivation of mTOR suppresses mRNA levels both HMMR and HAS in dose dependent manner. (A, B) Total RNA was isolated from PC-3 (A) or LNCaP (B) cells stimulated with mTOR inhibitors (KU; KU0063794, Rapa; Rapamycin, TR2; Torin2) for 24 h. (C, D) Cells were exposed to various doses of Torin2 for 24 h. Quantitative real time PCR analysis was performed to determine message levels of HMMR and HAS isoforms. Data shown are the mean ± SD of triplicate determinations and representative of two or three independent experiments (A-D) ( # p <0.05 vs. control, * p <0.05, ** p <0.01, **** p <0.0001 vs. control).

Article Snippet: The sources for the following chemicals, reagents and antibodies were: Rapamycin (WAKO Chemical, Richmond, VA, USA); Torin 2 and Ku0063794 (TOCRIS, Bristol, UK); IGF-1 (BioVision, Milpitas, CA, USA); DHT, LY294002, SP600125, SB202190, PD153035, and PD98059 (Sigma Aldrich, St. Louis, MO, USA); characterized fetal bovine serum (FBS) (ATCC, Manassas, VA, USA); anti-HMMR (Bethyl, Montgomery, TX, USA); anti-p-Rb(S807/811), anti-CDK2, anti-E-cadherin (24E10), anti-vimentin (D21H3), anti-p-Erk1/2 (T202/Y204), and anti-Erk (Cell signaling, Danvers, MA, USA); anti-cyclin D2 (Santa Cruz, Dallas, TX, USA); anti-GAPDH (Bioworld Technology, St. Louis Park, MN, USA); anti-β-actin, anti-FlagM2, and dextran-charcoal stripped fetal bovine serum (DC-FBS) (Sigma Aldrich); characterized fetal bovine serum (FBS) (HyClone, Logan, UT, USA).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Control

Journal: Biomolecules & Therapeutics

Article Title: AR-mTOR-SRF Axis Regulates HMMR Expression in Human Prostate Cancer Cells

doi: 10.4062/biomolther.2021.040

Figure Lengend Snippet: Androgen induces HMMR expression by activating mTOR, and silencing HMMR suppresses androgenic responses in LNCaP cells. (A) LNCaP cells were cultured for 24 h with the indicated pharmacological reagents before lysis (TR; Torin2, KU; KU0063794, RA; Rapamycin, DHT; Dihydrotestosterone). Protein samples and total RNA isolated were subjected to immunoblotting and quantitative real time PCR, respectively to measure HMMR expression. Data shown are the mean ± SD of triplicate determinations and representative of two or three independent experiments (**** p <0.0001 vs.Cont). (B) Cells were transiently transfected with 1 μg of total DNA including PSA-luciferase (PSA-luc) construct, cmv-renilla and either pCMV6 or pCMV6-flag-HMMR, followed by DHT (10 nM) treatment for 24 h (*** p <0.001, **** p <0.0001 vs. Cont, ## p <0.01, ### p <0.001 vs. DHT alone). (C) Separately, cells were transiently transfected with 1 μg of total DNA including either PSA-luciferase (PSA-luc) or MMTV-luc construct and cmv-renilla, and 200 nM of si-Cont or si-HMMR. Cells were then cultured for 24 h in the absence or presence of DHT (10 nM). Data shown are relative values of firefly luciferase activity normalized to renilla luciferase. Data shown are the mean ± SD of triplicate determinations and representative of two or three independent experiments (**** p <0.0001 vs.Cont, # p <0.05, &&&& p <0.0001 vs. DHT alone). (D) LNCaP was stimulated with 200 nM Rapamycin for 24 h, followed by DHT treatment for additional 24 h. The expression level of HMMR protein was measured by western blot analysis. Data shown are the mean ± SD of triplicate determinations and representative of two or three independent experiments (B-D).

Article Snippet: The sources for the following chemicals, reagents and antibodies were: Rapamycin (WAKO Chemical, Richmond, VA, USA); Torin 2 and Ku0063794 (TOCRIS, Bristol, UK); IGF-1 (BioVision, Milpitas, CA, USA); DHT, LY294002, SP600125, SB202190, PD153035, and PD98059 (Sigma Aldrich, St. Louis, MO, USA); characterized fetal bovine serum (FBS) (ATCC, Manassas, VA, USA); anti-HMMR (Bethyl, Montgomery, TX, USA); anti-p-Rb(S807/811), anti-CDK2, anti-E-cadherin (24E10), anti-vimentin (D21H3), anti-p-Erk1/2 (T202/Y204), and anti-Erk (Cell signaling, Danvers, MA, USA); anti-cyclin D2 (Santa Cruz, Dallas, TX, USA); anti-GAPDH (Bioworld Technology, St. Louis Park, MN, USA); anti-β-actin, anti-FlagM2, and dextran-charcoal stripped fetal bovine serum (DC-FBS) (Sigma Aldrich); characterized fetal bovine serum (FBS) (HyClone, Logan, UT, USA).

Techniques: Expressing, Cell Culture, Lysis, Isolation, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Construct, Activity Assay

Journal: BMC Cancer

Article Title: Identification of synthetic lethality of PRKDC in MYC-dependent human cancers by pooled shRNA screening

doi: 10.1186/1471-2407-14-944

Figure Lengend Snippet: MYC overexpression induces double-strand breaks in DNA. A) A SV40-transformed human lung cell line, WI-38 VA13, stably-expressing c-MYC, L-MYC, or N-MYC, were lyzed and immunoblots were analyzed for phosphorylated histone H2AX (anti-γH2AX) and GADPH (anti-GADPH). As a positive control for DNA damage detection, parental cells were treated with etoposide and analyzed via immunoblotting. Cell lysates were also analyzed for overexpression of MYC variants. B) The same cells lines as in A were treated with the PRKDC inhibitor, KU0060648, for 8 h, lyzed and immunoblotted for phosphorylated histone H2AX (anti-γH2AX) and γ-tubulin (anti-γ-tubulin). Signal intensities of immunoblots were quantified using ImageJ.

Article Snippet: Etoposide, NU-7441, and KU0060648 were obtained from Sigma (cat# E1383), Tocris Cookson Inc. (cat# 3712), and Axon Medchem BV (cat# Axon 1584), respectively.

Techniques: Over Expression, Transformation Assay, Stable Transfection, Expressing, Western Blot, Positive Control

Journal: Science signaling

Article Title: The injury response to DNA damage in live tumor cells promotes antitumor immunity.

doi: 10.1126/scisignal.abc4764

Figure Lengend Snippet: Fig. 8. Live injured chemotherapy-treated tumor cells require DNA-damage signaling for the DC-mediated T cell IFN- response. (A) B16-Ova cells were treated with doxorubicin at 0.5, 1, 2.5, or 5 M for 24 hours, washed, and incubated with primary bone marrow–derived dendritic cells (BMDCs) for another 24 hours. After this, OT-1 CD8+ T cells were added and evaluated for intracellular IFN- 15 hours later. Quantification of IFN-+CD8+ T cells from three independent experiments is shown. Error bars represent SEM. ****P < 0.0001 by ANOVA, followed by Dunnett’s multiple comparisons test. (B) B16-Ova cells were treated with doxorubicin, etoposide, or mitoxan- trone at the doses indicated for 24 hours and the live (attached) fractions were analyzed by fluorescence microscopy after staining with DAPI (blue channel) and anti-H2AX (red channel). Representative images are shown. Scale bar, 20 m. (C) Quantification of H2AX foci intensity from the experiment in (B) is shown. n ≥ 200 cells per condition. Width in violin plot indicates frequency for each observed value from maximum to minimum, with dotted line indicating median. ****P < 0.0001 by ANOVA followed by Dunnett’s multiple comparisons test compared to the DMSO control. (D) B16-Ova cells were treated with doxorubicin, etoposide, or mitoxantrone at the doses indicated for 24 hours, followed by separation of the live and dead fractions and analysis of respective lysates by Western blotting. Blots are representative of two independent experiments. (E) Quantification of IFN-+CD8+ T cells (from three independent experiments) induced by BMDCs after incubation with B16-Ova cells that were cotreated with 50 M etoposide and 10 M ATM kinase inhibitor KU-55933, ATR kinase inhibitor AZD6738, or DNA-PK inhibitor NU7441. First lane (−) defined as in Fig. 1C. Error bars represent SEM. **P < 0.01 and ****P < 0.0001 by ANOVA, followed by Dunnett’s multiple comparisons test versus the “Etop 50 M” group.

Article Snippet: KU-55933, AZD6738, and NU7441 were from SelleckChem.

Techniques: Incubation, Derivative Assay, Fluorescence, Microscopy, Staining, Control, Western Blot

Journal: Viruses

Article Title: Proteomic Analysis of Vero Cells Infected with Pseudorabies Virus

doi: 10.3390/v14040755

Figure Lengend Snippet: Primers used in this study.

Article Snippet: The primary antibodies used in this study were specific for XRCC5 (16389-1-AP, Proteintech, Rosemont, IL, USA), XRCC6 (10723-1-AP, Proteintech, Rosemont, IL, USA), β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA), VP5 (prepared in our lab), and gB (prepared in our lab).

Techniques: Sequencing

Journal: Viruses

Article Title: Proteomic Analysis of Vero Cells Infected with Pseudorabies Virus

doi: 10.3390/v14040755

Figure Lengend Snippet: Validation of proteomics data by western blot and RT-qPCR. ( A ) Vero cells infected with PRV for 24 h were collected and western blot was performed to detect the expression of XRCC5 and XRCC6 with corresponding antibodies. ( B ) The western blot and proteomics ratio of XRCC5 and XRCC6. ( C ) Relative XRCC6 transcription in Vero cells. ( D ) Relative XRCC5 transcription in Vero cells. ( E ) The expression of XRCC5 and XRCC6 in PK-15 infected with PRV. ( F ) The expression of XRCC5 and XRCC6 in CRL-2843 infected with PRV. ** indicates significance at a 99% confidence interval ( p < 0.01).

Article Snippet: The primary antibodies used in this study were specific for XRCC5 (16389-1-AP, Proteintech, Rosemont, IL, USA), XRCC6 (10723-1-AP, Proteintech, Rosemont, IL, USA), β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA), VP5 (prepared in our lab), and gB (prepared in our lab).

Techniques: Biomarker Discovery, Western Blot, Quantitative RT-PCR, Infection, Expressing