Journal: International journal of oncology

Article Title: An asymmetrically dimethylarginated nuclear 90 kDa protein (p90aDMA) induced by interleukin (IL)-2, IL-4 or IL-6 in the tumor microenvironment is selectively degraded by autophagy.

doi: 10.3892/ijo.2016.3450

Figure Lengend Snippet: Figure 5. The p90aDMA and p70aDMA proteins accumulate in the nucleus of cancer cells and degrade in response to cellular stresses. (A) Nuclear and cytoplasmic extracts were prepared after MDA-MB‑231 cells were cultured with IL-2 or IL-6 as indicated. Fractions were analyzed by western blot analysis using antibody to ASYM24. TBP was probed as a loading control. (B and C) MDA-MB‑231 cells or HeLa cells were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 h. (D) HeLa cells were cultured in HBSS medium for 24 h to induce autophagy or left in the complete culture medium. The cells were harvested and nuclear and cytoplasmic extracts were prepared for western blot analysis using antibodies to LC3 and ASYM24, respectively. Fractionation efficiency was determined by localization of β-actin (cytoplasm) and TBP (nucleus).

Article Snippet: Target Source Host Dilution Catalog SYM11 Merck Millipore, MA, USA Rabbit 1:1,000 07-413 ASYM24 Merck Millipore, MA, USA Rabbit 1:500 07-414 LC3B Cell Signaling Technology, Inc. (CST), MA, USA Rabbit 1:1,000 #3868 ATG5 Cell Signaling Technology, Inc. (CST), MA, USA Rabbit 1:1,000 #12994 TBP Proteintech, Wu Han, China Rabbit 1:500 22006-1-AP β-actin Zen Bioscience, Cheng Du, China Mouse 1:20,000 70068 that were significantly reduced regardless of the interleukin stimulation (Fig. 2).

Techniques: Cell Culture, Western Blot, Control, Fractionation

Journal: Frontiers in Pharmacology

Article Title: Specnuezhenide Decreases Interleukin-1β-Induced Inflammation in Rat Chondrocytes and Reduces Joint Destruction in Osteoarthritic Rats

doi: 10.3389/fphar.2018.00700

Figure Lengend Snippet: Effect of SPN on IL-1β-induced nuclear translocation of NF-κB p65 and β-catenin. Nuclear and cytoplasmic extraction reagents were used to prepare nuclear (A) and cytoplasmic (B) extracts. Then, protein levels of β-catenin, NF-κB p65, GAPDH in the cytoplasm, and β-catenin, NF-κB p65, and TBP in the nucleus were assessed by Western blotting. GAPDH was used as an endogenous control in the cytoplasm, whereas TBP worked as an endogenous control in the nucleus ( n = 3 per group). (C) Chondrocytes were pretreated with SPN for 1 h, and then stimulated with IL-1β for 6 h for NF-κb-Luc activity analysis, or 12 h for TCF-LEF RE activity analysis ( n = 5 per group). Significance was calculated by one-way ANOVA with post hoc Tukey’s multiple comparisons test. ∗ p < 0.05, ∗∗∗ p < 0.001 versus the IL-1β+SPN (0 μM) group. # p < 0.05, ## p < 0.01, ### p < 0.001 versus the NC group.

Article Snippet: In the present study, Ab against MMP-3 (rabbit mAb, Abcam, ab52915), MMP-9 (rabbit mAb, Abcam, ab76003), IL-6 (10E5, mouse mAb, Santa Cruz, sc-57315), iNOS (rabbit mAb, Abcam, ab3523), COX2 (D5H5, rabbit mAb, Cell Signaling, #12282), collagen 2 (rabbit mAb, Abcam, ab188570), sox9 (rabbit mAb, Abcam, ab185966), β-catenin (D10A8, rabbit mAb, Cell Signaling, #8480p), non-phospho (active) β-catenin (Ser45) (D2U8Y, rabbit mAb, Cell Signaling, #19807S), NF-κB p65 (C22B4, rabbit mAb, Cell Signaling, #4764S), phosphor-NF-κB p65 (Ser536) (rabbit Ab, Cell Signaling, #3031), β-actin (mouse mAb, Abcam, ab8226), GAPDH (rabbit mAb, Cell Signaling, #5174), and TBP (rabbit Ab, Bosterbio, BA3586-2) were used.

Techniques: Translocation Assay, Extraction, Western Blot, Control, Activity Assay

Journal: Nature Communications

Article Title: Increased global transcription activity as a mechanism of replication stress in cancer

doi: 10.1038/ncomms13087

Figure Lengend Snippet: ( a ) TBP mRNA quantification by quantitative reverse transcriptase–PCR in BJ-HRAS V12 cells 72 h after RAS induction. TBP mRNA levels were normalized to GAPDH and control. ( b ) Protein levels of TBP and β-ACTIN after RAS induction for the times indicated. ( c ) Densitometry quantification of TBP levels based on western blotting as in b after RAS induction for the times indicated. Values were normalized to 72 h control. N =3 (24 and 48 h), N =6 (72 h). ( d ) Twenty-four hours after RAS induction, cells were transfected with TBP siRNA (TBPsi #1) or control siRNA (nonTsi). Cells were processed for DNA fibre analysis or western blotting 48 h later and for 53BP1 staining 24 h later. ( e ) Protein levels of TBP, HRAS and GAPDH (loading control) 72 h after RAS induction and 48 h after siRNA transfection. ( f ) Quantification of nascent RNA synthesis by EU incorporation ±TBPsi #1 72 h after RAS induction. N =3. ( g ) Distribution of replication fork speeds ±TBPsi #1 72 h after RAS induction. N =3. ( h ) Median replication fork speeds ±TBPsi #1 72 h after RAS induction. N =3. ( i ) Percentages of cells containing more than eight 53BP1 foci, ±TBPsi #1 96 h after RAS induction. N =3. ( j ) Median replication fork speeds in cells treated with TBPsi #1 and DRB 72 h after RAS induction, compared with TBPsi #1 or DRB alone. N =3. ( k ) Percentages of cells treated with TBPsi #1 and DRB containing more than eight 53BP1 foci after 96 h after RAS induction, compared with TBPsi #1 or DRB alone. N =3. ( l ) Model for the role of TBP in HRAS V12 -induced replication stress. Means ±s.e.m. (bars) are shown. Student's t -test, * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: For BJ-TBPind cells, human TBP complementary DNA (Origene, SC118124) was inserted into a pInducer20 lentivirus construct to generate TBP-pInducer20 and infected BJ-hTert cells were selected with 500 μg ml −1 G418 (Gibco).

Techniques: Western Blot, Transfection, Staining

Journal: Nature Communications

Article Title: Increased global transcription activity as a mechanism of replication stress in cancer

doi: 10.1038/ncomms13087

Figure Lengend Snippet: ( a ) Protein levels of TBP, p53 and TUBULIN (loading control) in BJ-TBPind cells treated with doxycycline for 1–6 days, to induce TBP overexpression. No doxycycline was used as a control. ( b ) Nascent RNA synthesis as measured by EU incorporation after TBP induction for 3 days. Scale bars, 10 μm. ( c ). Quantification of nuclear EU intensity after TBP induction for 1–4 days. N =4 (con, day 1, 2 and 4), N =5 (day 3). ( d ) Median replication fork speeds in after TBP induction. N =2 (day 1, 2 and 4), N =4 (con, day 3). Asterisks compare with control. ( e ) Nascent RNA synthesis as measured by EU incorporation in cells treated with DRB or dimethylsulfoxide (DMSO) (control) for 100 min, 72 h after TBP induction. N =3. ( f ) Median replication fork speeds in BJ-TBPind cells treated with DRB 72 h after TBP induction. N =3. ( g ). Percentage of cells displaying more than eight 53BP1 foci or micronuclei after TBP induction. Right panel: representative images of cells with 53BP1 foci and micronuclei. Asterisks compare with control. N =2–7. ( h ) Cell cycle distribution of 53BP1-positive cells 96 h after TBP induction as determined by co-staining with Cyclin A. N =2. ( i ) Representative images and percentages of β-galactosidase staining after TBP induction for 1–6 days. Scale bars, 100 μm. ( j ) Model of how HRASV 12 and other growth factor oncogenes such as epidermal growth factor receptor (EGFR) induce replication stress by increasing transcription through TBP and other transcription factors. Additional mechanisms, such as reactive oxygen species, may also contribute to HRASV 12 -induced DNA damage. Means ±s.e.m. (bars) are shown. Student's t -test, * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: For BJ-TBPind cells, human TBP complementary DNA (Origene, SC118124) was inserted into a pInducer20 lentivirus construct to generate TBP-pInducer20 and infected BJ-hTert cells were selected with 500 μg ml −1 G418 (Gibco).

Techniques: Over Expression, Staining