Journal: The Journal of Biological Chemistry

Article Title: The Human COP9 Signalosome Protects Ubiquitin-conjugating Enzyme 3 (UBC3/Cdc34) from ?-Transducin Repeat-containing Protein (?TrCP)-mediated Degradation *

doi: 10.1074/jbc.M109.076661

Figure Lengend Snippet: UBC3 is a target of SCFβTrCP. A, UBC3 protein levels in CSN knockdown cells are rescued by CUL1 suppression. shRNA-transduced 293T cells were transfected with a control oligonucleotide or with two different siRNAs targeting CUL1. UBC3 content in total lysates was analyzed by Western blotting (upper panel). Cullin mRNA levels were analyzed by quantitative reverse transcription-PCR (lower panel). B, endogenous UBC3 and βTrCP interact. UBC3 (middle panel) and βTrCP (right panel) were immunoprecipitated (IP) from HEK293T cell lysates with specific antibodies. The control used was the irrelevant mouse (left panel) or rabbit (right panel) IgG antibody. WB, Western blot. C, the F-box is not required for βTrCP interaction with UBC3. 293T cells were transfected with the indicated vectors (full-length βTrCP (βTrCP-FL) and the F-box deletion mutant βTrCPΔFbox), and βTrCP was immunoprecipitated with anti-FLAG antibodies. UBC3 wt, wild-type UBC3. D, βTrCP overexpression promotes UBC3 ubiquitination. 293T cells were transfected as indicated. HA-UBC3 was immunoprecipitated with anti-HA antibody, and ubiquitinated forms of UBC3 were detected by anti-Myc Western blotting. E, down-regulation of βTrCP increases UBC3 protein levels. 293T cells were transfected with two different siRNA oligonucleotides that target both βTrCP1 and βTrCP2. F, down-regulation of SKP2 has no effect on UBC3 protein levels. 293T cells were transfected with two siRNA oligonucleotides targeting SKP2.

Article Snippet: Real-time fluorogenic reverse transcription-PCR (Applied Biosystems) was performed according to the manufacturer's protocol: 20 ng of cDNA was amplified with 4 μl of the “Assays-on-Demand” oligonucleotide in a final volume of 25 μl (Applied Biosystems TaqMan® gene expression assays COPS4, Hs00275045_m1; COPS5, Hs00272789_m1; CUL1, Hs00269187_m1; CUL4A, Hs00757716_m1; SKP2, Hs00180634_m1; BTRC, Hs00182707_m1; CDC34, Hs00362082_m1; UBE2D1, Hs00234280_m1; UBE2D2, Hs00366152_m1; UBE2D3, Hs00704312_m1; and CTNNB1, Hs00170025_m1).

Techniques: shRNA, Transfection, Western Blot, Immunoprecipitation, Mutagenesis, Over Expression

Journal: bioRxiv

Article Title: Targeted inhibition of SCF SKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma

doi: 10.1101/2023.05.13.540637

Figure Lengend Snippet: (A) mRNA levels of SKP2, E2f1, Aldh1a1, Aldh2, Aldh7a1, Kit and Prom1 in DKO cells relative to GAPDH were determined by qRT-PCR followed by SKP2 knockdown (SKO shSKP2). (B) Same genes were also compared among DKO and TKO tumor cells by qRT-PCR to determine mRNA levels relative to GAPDH. (C) Extreme limiting dilution analysis (ELDA) for assessing stem-like cell self-renewal ability. Representative figures were showing cell clusters but not sphere formation (Upper left); a cluster of differentiated and apoptotic cells showing no indication of sphere formation (Upper Middle), and a single sphere formation and scored as “positive” (Upper Right). Scale bars indicate 25 μm. The lower left figure showed sphere-forming capacity output using the previously described algorithm. Graphical representation in the lower right showed the reciprocal of stem cell frequency calculated. (D) ALDH activity staining of tumor cells of indicated genotypes. FACS (representative of three experiments) was used to detect and quantify the subpopulation that expresses a high level of ALDH (ALDH BR ). (E) Both DKO and TKO tumor cells were stained with CD117, (blue peak) or with the corresponding IgG control (red peak) antibodies. Flow cytometry was performed, and IgG staining was used for gating. Representative results of three experiments were shown on the left, and a quantification comparison of three experiments was on the right. Error bars are SEM. ∗P< 0.05, ∗∗P< 0.01, and ∗∗∗P< 0.001.

Article Snippet: Antibodies for Western blots included p27 (BD Bioscience, #610242), SKP2 (Proteintech, #15010-1-AP), p21 (Santa Cruz, #SC-397), and cleaved caspase 3 (#9661),α-tubulin (#2125), p53(#2524), SKP2 (#2652), Rb (#9309), GAPDH (#5174) were from Cell Signaling Technology.

Techniques: Quantitative RT-PCR, Knockdown, Activity Assay, Staining, Control, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Targeted inhibition of SCF SKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma

doi: 10.1101/2023.05.13.540637

Figure Lengend Snippet: A, Relative mRNA expression levels of TP53, RB1 (B) and SKP2 (C) as measured by RNA-seq in 24 PPTC osteosarcoma tumor samples. Relative mRNA expression levels were defined as counts. D. Western blot analysis for SKP2, TP53 and RB1 in protein lysates of the same OS samples as above. Three non-tumor mesenchymal cell lines, MSC (mesenchymal stem cells), NDHF (non-dermal human fibroblasts), NHOst (normal human osteoblasts), and 13 OS samples were included. GAPDH was used as a loading control. E. Heatmap of relative mRNA expression levels of TP53, RB1 and SKP2 as measured by RNA-seq in PPTC osteosarcoma tumor samples.

Article Snippet: Antibodies for Western blots included p27 (BD Bioscience, #610242), SKP2 (Proteintech, #15010-1-AP), p21 (Santa Cruz, #SC-397), and cleaved caspase 3 (#9661),α-tubulin (#2125), p53(#2524), SKP2 (#2652), Rb (#9309), GAPDH (#5174) were from Cell Signaling Technology.

Techniques: Expressing, RNA Sequencing, Western Blot, Control

Journal: bioRxiv

Article Title: Targeted inhibition of SCF SKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma

doi: 10.1101/2023.05.13.540637

Figure Lengend Snippet: (A) Kaplan-Meier survival analysis comparing the DKO, DKOAA and TKO cohorts of mice undergoing tumorigenesis. P-value is by a log-rank test and indicated in the figure. (B) Representative H&E, immunostaining of p27 and PCNA, of mOS tissue from the indicated genotype and age. Scale bars = 200 μm (a,c,e,g,i,k) and 100 μm (b,d,f,h,j,l). (C) DKO mOS tumor tissue-derived organoid culture. Upper figures indicate the proliferation of one representative organoid in the process of 15 days (Scale bars = 200 μm). Lower figures showed representative H&E of mOS organoid, and osteocalcin, alkaline phosphatase (ALP), p27, SKP2, and PCNA by immunohistochemistry. (D) Three independent mOS derived tumor organoids from DKO (DKO-1, DKO-2, and DKO-3) and TKO (TKO-1, TKO-2, and TKO-3) were cultured for the indicated duration, and relative proliferation rates were determined by the CellTiter-Glo method ( P =0.035). (E) Graphs showing the proliferation of DKO, DKOAA and TKO tumor cells in monolayer cultures ( P =0.001 DKO vs. DKOAA, P <0.001 DKO vs. TKO, P =0.006 DKOAA vs. TKO). (F) Mice with limb tumors were randomly selected from each group (n=18 from DKO group and n=17 from TKO group) once the tumor reached a volume around 500mm 3 , and measured every three days. The tumor growth rate was plotted and compared among the two groups. (G) The age of mice was documented and compared among the two groups when palpable or visible limb tumors were first discovered ( P <0.001). Mice with measurable limb tumors at the same age (H) 28 weeks old, (I) 32 weeks old were randomly selected from each group, and tumor volume was measured and compared among two groups ( P <0.001 and P <0.001). Error bars are SEM. ∗P< 0.05, ∗∗P< 0.01, and ∗∗∗P< 0.001.

Article Snippet: Antibodies for Western blots included p27 (BD Bioscience, #610242), SKP2 (Proteintech, #15010-1-AP), p21 (Santa Cruz, #SC-397), and cleaved caspase 3 (#9661),α-tubulin (#2125), p53(#2524), SKP2 (#2652), Rb (#9309), GAPDH (#5174) were from Cell Signaling Technology.

Techniques: Immunostaining, Derivative Assay, Immunohistochemistry, Cell Culture

Journal: bioRxiv

Article Title: Targeted inhibition of SCF SKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma

doi: 10.1101/2023.05.13.540637

Figure Lengend Snippet: (A) Consecutive OS tumor sections of each genotype, as indicated, stained with H&E, TUNEL, and immunostaining of cleaved caspase-3. Scale bars = 200 μm (a, c, e, g, i, and k) and 100 μm (b, d, f, h, j, and l). (B) Quantification of apoptosis by TUNEL staining. Arrows demonstrate how total apoptosis was quantified. The bar graph was based on the average quantification of apoptosis from three mice. (C) SKP2 knockdown was achieved by shRNA and validated by qRT-PCR in DKO cells, followed by detection of mRNA levels of genes ( SKP2, E2f1, Bbc3, Bid, Bcl2l11, Casp3 ) relative to GAPDH. (D) DKO and TKO cells were subject to qRT-PCR to determine mRNA levels of the same genes relative to GAPDH. (E) 7-exceAAD and annexin V staining of tumor cells of the indicated genotypes. Flow cytometry was used to detect and quantify apoptosis in the sub-G1 population. Error bars indicate SEM of the means of three samples. (F) Propidium iodide-based DNA content FACS (representative of three experiments) to detect and quantify cell phases in the population, as marked and plotted on the right. (G) Protein levels were determined by western blotting of OS tissues from the indicated genotypes. (H) Kaplan-Meier plot comparing survival of NCI TARGET OS cohort stratified by dichotomized expression of the Reactome Apoptosis gene signature at the median. Red line indicates patients overexpressing genes associated with apoptosis. Error bars are SEM. ∗P< 0.05, ∗∗P< 0.01, and ∗∗∗P< 0.001.

Article Snippet: Antibodies for Western blots included p27 (BD Bioscience, #610242), SKP2 (Proteintech, #15010-1-AP), p21 (Santa Cruz, #SC-397), and cleaved caspase 3 (#9661),α-tubulin (#2125), p53(#2524), SKP2 (#2652), Rb (#9309), GAPDH (#5174) were from Cell Signaling Technology.

Techniques: Staining, TUNEL Assay, Immunostaining, Knockdown, shRNA, Quantitative RT-PCR, Flow Cytometry, Western Blot, Expressing

Journal: bioRxiv

Article Title: Targeted inhibition of SCF SKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma

doi: 10.1101/2023.05.13.540637

Figure Lengend Snippet: DKO and TKO cell viability following 72 hours of treatment with serial dilutions of SCF SKP2 inhibitors, C1 (A) or Penvenodistat (B) at indicated concentrations. Mice osteoblast (mOB) was used as the control. The cell viability was calculated relative to the treatment with DMSO (vehicle control) by the MTT method. Western blot of p21, p27 and cleaved caspase3 in DKO mOS, followed the treatment of C1 (C) or Penvenodistat (D) with indicated concentrations. (E-F) Three independent DKO tumor-derived organoids (DKO-1, DKO-2, and DKO-3) were treated with C1 (E) or Penvenodistat (F) for 96 hours with indicated concentrations. The organoid viability was determined by the CellTiter-Glo method. (G-H) Representative photographs of organoid death and degradation after C1 (G) or Penvenodistat (H) treatment at indicated concentrations and duration. Error bars are SEM.

Article Snippet: Antibodies for Western blots included p27 (BD Bioscience, #610242), SKP2 (Proteintech, #15010-1-AP), p21 (Santa Cruz, #SC-397), and cleaved caspase 3 (#9661),α-tubulin (#2125), p53(#2524), SKP2 (#2652), Rb (#9309), GAPDH (#5174) were from Cell Signaling Technology.

Techniques: Control, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Targeted inhibition of SCF SKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma

doi: 10.1101/2023.05.13.540637

Figure Lengend Snippet: Tumors were initiated by the subcutaneous inoculation of DKO tumor-derived single-cell suspension. When tumors reached 100 mm 3 in size, mice were treated by subcutaneous administration of C1 (A; 40 mg/kg/day, P=0 . 009 ) or Penvenodistat (B; 90 mg/kg/day, P=0 . 022 ). The solvent for each drug was used as vehicle control. Tumor size was measured on the indicated days; data are means ± SEM of at least five mice per group. Mouse body weight during daily treatment with C1(C) ( P=0 . 664, 0 . 245 and 0 . 095 at day 0, 20 and 32, respectively ) or Pevonedistat (D) ( P=0 . 693, 0 . 467 and 0 . 220 at day 0, 20 and 32, respectively ), as indicated. (E & F) Western blot and quantitation of SKP2, p27 and cleaved caspase 3 in DKO tumor extracts from control and C1-or Pevonedistat-treated mice. Each lane in (E & F) is an individual mouse tumor. (G) Tumors isolated from drug-treated mice and control were analyzed for the expression of p27, PCNA, cleaved caspase 3 and TUNEL by immunohistochemistry. Statistical significance is indicated by *P < .05,**P < .01, ***P < .001. Column: mean; Error bars are SEM.

Article Snippet: Antibodies for Western blots included p27 (BD Bioscience, #610242), SKP2 (Proteintech, #15010-1-AP), p21 (Santa Cruz, #SC-397), and cleaved caspase 3 (#9661),α-tubulin (#2125), p53(#2524), SKP2 (#2652), Rb (#9309), GAPDH (#5174) were from Cell Signaling Technology.

Techniques: Derivative Assay, Suspension, Solvent, Control, Western Blot, Quantitation Assay, Isolation, Expressing, TUNEL Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) FBXW2 binds to SKP2 in vivo : Lysates from H1299 cells were pulled down with anti-FBXW2 (left panel) or anti-SKP2 (right panel), followed by IB. ( b ) FBXW2 failed to bind to a SKP2 mutant: H1299 cells were tranfected with indicated plasmids, followed by FLAG IP and HA IB or direct IB. ( c , d ) Ectopically expressed FBXW2 reduces the endogenous levels of SKP2 protein, but not mRNA: Cells were transfected with HA-FBXW2, followed by IB ( c ) or qRT-PCR for SKP2 ( d ). Error bars indicate mean +s.d. of three repeats. ( e , f ) FBXW2 overexpression decreases the levels of the exogenous and endogenous SKP2 proteins: Cells were co-transfected with indicated plasmids, followed by IB 48 h post transfection. ( g ) FBXW2 depletion increases the levels of SKP2 protein, but not mRNA: Cells were transfected with FBXW2 siRNA and scramble siRNA, followed by IB or qRT-PCR for SKP2. Error bars indicate mean+s.d. of three repeats. ( h ) FBXW2-induced SKP2 degradation is independent of CDH1: Cells were transfected with FLAG-FBXW2 and/or siRNA against CDH1, and then harvested for IB. ( i ) FBXW2 shortens SKP2 half-life: Cells were transfected with HA-FBXW2, and switched 48 h post transfection to fresh medium containing CHX for indicated periods with or without MG132 treatment for last 2 h, and then harvested for IB. The band density was quantified. ( j ) FBXW2 knockdown extends SKP2 half-life: Cells were transfected with siRNA targeting FBXW2. Cells were switched 48 h later to fresh medium containing CHX for indicated periods and harvested for IB. The band density was quantified. ( k , l ) FBXW2 promotes ubiquitylation of SKP2 ( k ), but not SKP2-3A mutant ( l ): Cells were transfected with indicated plasmids, followed by Ni-beads pull-down and IB for SKP2. ( m ) FBXW2 promotes SKP2 ubiquitylation by in vitro assay: H1299 cells were transfected with indicated plasmids. Pull-down purified FBXW2 and FBXW2ΔF (E3s), Pull-down purified SKP2 (substrate), were added into a reaction mixture containing ATP, ubiquitin, E1 and E2, followed by IB using anti-FLAG Ab. ( n ) FBXW2 promotes SKP2 ubiquitylation via K48 linkage: Cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down and IB for SKP2.

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: In Vivo, Mutagenesis, Transfection, Quantitative RT-PCR, Over Expression, Knockdown, In Vitro, Purification, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) Fluctuation of the levels of F-box proteins and VRK2 kinase during cell cycle progression: H358 cells were serum starved for 48 h, followed by serum addition. Cells were harvested at indicated time points and subjected to FACS and IB analyses using indicated Abs. ( b – f ) FBXW2-3A mutant rescues growth-promoting phenotype induced by β-TrCP1 overexpression ( b – d ), and wt FBXW2 rescues growth-promoting phenotype induced by SKP2 overexpression ( b , e , f ): H1299 cells were co-transfected with the indicated plasmids, followed by IB ( b ), ATP-lite proliferation assay ( n =3) ( c , e ) and clonogenic survival assay ( n =3) ( d , f ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01. ( g – i ) FBXW2 depletion stimulates cell growth, which is abrogated by simultaneous SKP2 depletion: H1299 cells were transfected with shRNAs targeting FBXW2 alone or in combination with shRNA targeting SKP2, along with scramble control, and then harvested for IB ( g ), ATP-lite proliferation assay ( n =3) ( h ) and clonogenic survival assay ( n =3) ( i ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001. ( j , k ) FBXW2 depletion stimulates tumour growth, which is abrogated by simultaneous SKP2 depletion: H1299 cells were transfected with shRNAs targeting FBXW2 alone or in combination with shRNA targeting SKP2, along with scramble control, followed by injection (1 × 10 6 cells) into nude mice. Tumour growth was observed for 33 days ( j ). Tumours were then harvested, photographed and weighted ( k ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: Mutagenesis, Over Expression, Transfection, Proliferation Assay, Clonogenic Cell Survival Assay, shRNA, Control, Injection

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a – c ) Expression levels among three F-box proteins in lung cancer cell lines and tissues: Cell lysates from eight lung cancer cell lines and one immortalized line (BEAS-2B) were subjected to IB ( a ); SE: Short exposure; LE: Long exposure. Lung cancer tissue microarrays were stained with indicated Abs and photographed ( b , Scale bars, 100 μm), and data were then analysed using SPSS software to obtain coefficient ( c ; P <0.001, Pearson's test). ( d – f ) Protein expression of three F-box proteins in lung cancer and their relationship with patient survival: Continuous protein expression values were classified into the low and high groups with equal number of patients, and 5-year survival time was used for Kaplan-Meier survival analysis ( d – f ). Kaplan-Meier survival analysis indicated that patient with higher expression of FBXW2 was related to a better overall survival (log-rank test, P =0.032) ( e ); Higher expression of β-TrCP1 and SKP2 were related to a worse overall patient survival (log-rank test, P <0.001 and 0.001, respectively) ( d , f ).

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: Expressing, Staining, Software

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) FBXW2 mutants have reduced binding with SCF components: H1299 cells were transfected with indicated plasmids, followed by G418 selection. Resistant clones were pooled and subjected to FLAG-bead IP and IB with indicated Abs. ( b , c ) FBXW2 mutants MU-S84C ( b ) and MU-E269K ( c ) are unable to shorten SKP2 protein half-life: Stable H1299 cells were incubated with CHX for indicated time periods and harvested for IB. The band density was quantified. ( d , e ) Loss- or gain-of-function of FBXW2 mutants: H1299 cells stably expressing MU-S84C and MU-E269K mutants were subjected to ATP-lite proliferation assay ( n =3) ( d ), and clonogenic survival assay ( n =3) ( e ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001. ( f , g ) FBXW2 mutants either lose the tumour suppressor function or gain the oncogenic function in vivo : H1299 cells stably expressing MU-S84C and MU-E269K mutants (1 × 10 6 cells) were inoculated s.c. in both flanks of nude mice. The tumour growth was monitored twice a week for up to 28 days and growth curve plotted ( f ). Tumour tissues were harvested, photographed and weighed at 28 days ( g ). Student's t -test was used to compare each experimental group with the control group. Shown are mean±s.e.m., * P <0.05; ** P <0.01; *** P <0.001. ( h ) Immunohistochemical staining of xenograft tumour tissues. Tumour tissues from four groups of mice were fixed, sectioned and stained with indicated antibodies. Scale bars: 100 μm. Shown are mean±s.e.m., * P <0.05; ** P <0.01. ( i ) The oncogene-tumour suppressor-oncogene axis—a working model: During tumorigenesis, oncogenic β-TrCP1 is activated to promote ubiquitylation and degradation of FBXW2 tumour suppressor, resulting in abrogation of FBXW2-induced SKP2 degradation. Accumulated oncogenic SKP2 promotes ubiquitylation and degradation of tumour suppressors such as p21, p27, p130, FOXO1, and leading to activation of CDKs, E2F and mTOR, eventually to uncontrolled proliferation of cancer cells.

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: Binding Assay, Transfection, Selection, Clone Assay, Incubation, Stable Transfection, Expressing, Proliferation Assay, Clonogenic Cell Survival Assay, In Vivo, Control, Immunohistochemical staining, Staining, Activation Assay

Journal: Oncotarget

Article Title: Danshen improves survival of patients with advanced lung cancer and targeting the relationship between macrophages and lung cancer cells

doi: 10.18632/oncotarget.18767

Figure Lengend Snippet: ( A ) Total cell extracts of A549 cells or H460 cells were harvested from cells treated with DMSO or indicated concentrations of DT for 24 hours. The protein was immunoblotted with polyclonal antibodies specific for p -STAT3 or STAT3 or Skp2. β-actin was used as an internal loading control. ( B , C ) Total mRNA was extracted from the A549 cells (B) or H460 cells (C) after treat without or with indicated drugs for 24 hours. The coding regions of human Skp2 MMP2, and MMP9 were used as probes for real time polymerase chain reaction analysis. ( D ) ChIP assay for CCL2 promoter in A549 cells treated with indicated concentration of DT. ( E ) A549 cells or H460 cells were measured by XTT assay after 24–48 hours of culturing in the presence of different concentration DT. ( F ) For apoptosis assay, A549 cells or H460 cells were treated with 10–20 μM of DT for 24–48 hours. Cell apoptosis was detected by flow cytometry with annexin-V-FITC/PI dual staining. The representative histograms of flow cytometric analysis using double staining with annexin-V-FITC (FITC-A) and PI (PI-A). Q1 (annexin−V−/PI+) show necrosis cells; Q2 (annexin−V+/PI+) show the late apoptosis cells; Q3 (annexin−V−/PI−) show normal cells; Q4 (annexin−V+/PI−) show the early apoptosis cells. (Error bars = mean ± S.E.M. Asterisks (*) mark samples significantly different from DMSO group with p < 0.05).

Article Snippet: The primary antibodies used were: anti-phospho(tyr705)-STAT3 antibody ( p -STAT3)(Cell Signaling, ratio: 1:1000), anti-STAT3 antibody (Cell Signaling, ratio: 1:1000), anti-Skp2 antibody (Cell Signaling , ratio: 1:1000), anti-β-actin antibody (Santa Cruz, IB: 1:10000).

Techniques: Control, Real-time Polymerase Chain Reaction, Concentration Assay, XTT Assay, Apoptosis Assay, Flow Cytometry, Staining, Double Staining

Journal: Science signaling

Article Title: Skp2-dependent reactivation of AKT drives resistance to PI3K inhibitors

doi: 10.1126/scisignal.aao3810

Figure Lengend Snippet: (A) Representative images of MDA-MB-231 spheroids, from parental or BKM-120–resistant cells, that were infected with SKP2 shRNA lentiviral vector or control pLKO, and grown for 14 days in a 3-dimensional Matrigel/growth media mixture and treated with BKM-120 and/or MK-2206 (each 1 μM). Scale bar, 500 μm (4x magnification).

Article Snippet: After mixing the resulting template with Skp2 (Mm00449925_m1) or glyceraldehyde-3-phospahte dehydrogenase (GAPDH; Mm99999915_g1) primers and ABI Taqman Fast Universal PCR Master Mix, the RT-PCR reaction was performed with the ABI-7500 Fast Real-time PCR system.

Techniques: Infection, shRNA, Plasmid Preparation, Control

Journal: Science signaling

Article Title: Skp2-dependent reactivation of AKT drives resistance to PI3K inhibitors

doi: 10.1126/scisignal.aao3810

Figure Lengend Snippet: (A) Immunoblotting of lysates from MDA-MB-231, parental and BKM-120–resistant, cells that were infected with SKP2 shRNA lentiviral vector or control pLKO. Cells were serum-starved overnight (−) or stimulated with IGF-1 for 20 min (+).

Article Snippet: After mixing the resulting template with Skp2 (Mm00449925_m1) or glyceraldehyde-3-phospahte dehydrogenase (GAPDH; Mm99999915_g1) primers and ABI Taqman Fast Universal PCR Master Mix, the RT-PCR reaction was performed with the ABI-7500 Fast Real-time PCR system.

Techniques: Western Blot, Infection, shRNA, Plasmid Preparation, Control

Journal: Aging (Albany NY)

Article Title: Chikusetsu saponin IVa protects pancreatic β cell against intermittent high glucose-induced injury by activating Wnt/β-catenin/TCF7L2 pathway

doi: 10.18632/aging.102702

Figure Lengend Snippet: CHS protected against cell cycle arrest of islet cells from IHG. ( A ) The βTC3 cell proliferation was measured by a BrdU labeling and detection kit. Inverted microscope (200×) images. ( B ) The cell cycle related proteins (Cyclin D1, Skp2, p21, andp27) were measured by western bolting. Data are representative of three independent experiments. ## P <0.01 vs. NG treatment group, ** P <0.01 vs. SHG treatment group, ΔΔ P <0.01 vs. IHG treatment group.

Article Snippet: Bax (No. 2772), Bcl-2 (No. 3498), Bad (No. 9292), Cleaved-caspase 3 (No. 9661), Cleaved-PARP (No. 94885), Cytochrome c (No. 11940), β-actin (No. 4970), VDAC (No. 12454), Cyclin D1 (No. 2978), Skp2 (No. 4313), p21 (No. 2947), p27 (No. 3688), p53 (No. 2524), Wnt3a (No. 2721), β-Catenin (No. 8480), c-Myc (No. 9402) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Labeling, Inverted Microscopy, Western Blot

Journal: Aging (Albany NY)

Article Title: Chikusetsu saponin IVa protects pancreatic β cell against intermittent high glucose-induced injury by activating Wnt/β-catenin/TCF7L2 pathway

doi: 10.18632/aging.102702

Figure Lengend Snippet: Involvement of Wnt/TCF7L2 signaling in the protective effect of CHS on islets β cell. βTC3 cells were treated with CHS and IHG or SHG, then the RNA was extracted by TRIZOL. The mRNA expression levels of Wnt2 ( A ), Wnt3a ( B ), Wnt4 ( C ), Wnt5a ( D ), and Wnt10b ( E ) were measured by RT-PCR. ( F ) The protein expression levels of Wnt3a was measured by western blotting after different treatments. The effects of CHS on β-catenin expression levels in the cytoplasm ( G ) and nuclear ( H ) were measured by western blotting. ( I ) The effects of CHS on protein expression levels of Axin-2, c-Myc, Naked-1 and P-GSK-3β. βTC3 cells were treated with CHS and XAV-939 (a Wnt/β-catenin antagonist, 10μM), and then subjected to IHG. ( J ) The protein expression levels of TCF7L2, cyclin D1, skp2, p53, p21 and GIPR were measured by western blotting. ( K ) Cell variability was measured by CCK8 assay. ( L ) Insulin secretion levels was measured by an insulin RIA kit. ( M ) BrdU positive ratio was calculated from the BrdU positive cell numbers vs total cell numbers. Data are representative of three independent experiments. ## P <0.01 vs. NG treatment group, ** P <0.01 vs. IHG treatment group, ΔΔ P <0.01 vs. scrb treatment group, && P <0.01 vs.CHS treatment group.

Article Snippet: Bax (No. 2772), Bcl-2 (No. 3498), Bad (No. 9292), Cleaved-caspase 3 (No. 9661), Cleaved-PARP (No. 94885), Cytochrome c (No. 11940), β-actin (No. 4970), VDAC (No. 12454), Cyclin D1 (No. 2978), Skp2 (No. 4313), p21 (No. 2947), p27 (No. 3688), p53 (No. 2524), Wnt3a (No. 2721), β-Catenin (No. 8480), c-Myc (No. 9402) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, CCK-8 Assay

Journal: Aging (Albany NY)

Article Title: Chikusetsu saponin IVa protects pancreatic β cell against intermittent high glucose-induced injury by activating Wnt/β-catenin/TCF7L2 pathway

doi: 10.18632/aging.102702

Figure Lengend Snippet: Effects of CHS on HBP1 expression in islets β cell. ( A ) Time dependent effects of CHS on the expression HBP1. ( B ) Dose dependent effects of CHS on the expression HBP1. ** P <0.01 vs. 0h or 0mM treatment group. ( C ) Effects of CHS on HBP1 expression after IHG or SHG treatment. βTC3 cells were transfected with the empty vector pcDNA3 (scrb) or vectors encoding HA-tagged wild-type HBP1 (HBP1) at 30nM, and then cells were exposed to different treatments as indicated. ( D ) The protein expression levels of TCF7L2, GIPR, cyclinD1, Skp2, p53 and p21 were measured by western blotting. ( E ) Insulin secretion levels was measured by an insulin RIA kit. ( F ) Cell variability was measured by CCK8 assay. ( G ) BrdU positive ratio was calculated from the BrdU positive cell numbers vs total cell numbers. βTC3 cells were transfected with scrb and siHBP1 (30nM) for 24h, and then cells were exposed to different treatments as indicated. ( H ) The protein expression levels of GIPR, cyclinD1, and p53 were measured by western blotting. ( I ) Cell variability was measured by CCK8 assay. ( J ) Insulin secretion levels was measured by an insulin RIA kit. ( K ) BrdU positive ratio was calculated from the BrdU positive cell numbers vs total cell numbers. Data are representative of three independent experiments. ## P <0.01 vs. NG treatment group, ΔΔ P <0.01 vs. IHG treatment group, && P <0.01 vs.scrb treatment group.

Article Snippet: Bax (No. 2772), Bcl-2 (No. 3498), Bad (No. 9292), Cleaved-caspase 3 (No. 9661), Cleaved-PARP (No. 94885), Cytochrome c (No. 11940), β-actin (No. 4970), VDAC (No. 12454), Cyclin D1 (No. 2978), Skp2 (No. 4313), p21 (No. 2947), p27 (No. 3688), p53 (No. 2524), Wnt3a (No. 2721), β-Catenin (No. 8480), c-Myc (No. 9402) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, CCK-8 Assay

Journal: Aging (Albany NY)

Article Title: Chikusetsu saponin IVa protects pancreatic β cell against intermittent high glucose-induced injury by activating Wnt/β-catenin/TCF7L2 pathway

doi: 10.18632/aging.102702

Figure Lengend Snippet: Effect of CHS on the HBP1/Wnt/ TCF7L2 pathway in vivo. DM model was induced by HFD and STZ, and the CHS (120 mg/kg) or positive control rosiglitazone (Ros, 2 mg/kg) was given through intragastric administration one time every day for 30days. FBG ( A ), FINS ( B ), GIP ( C ) and GLP-1 ( D ) levels in serum were measured using relative kits. ( E ) Effects of CHS on the expression levels of β-catenin in nuclear. ( F ) Effects of CHS on the expression levels of apoptosis related proteins (cleaved-caspase 3, bax, bad, cytochrome C) in WT and β-catenin -/- DM mice. ( G ) Effects of CHS on the expression levels of Wnt3a and HBP1 in DM mice. ( H ) Effects of CHS on the expression levels of TCF7L2 related proteins (TCF7L2, c-Myc, cyclinD1, Skp2, p53, p21, GLP-1R, GIPR and p27) in WT and β-catenin -/- DM mice. Data are representative of three independent experiments. ## P <0.01 vs. ND mice group, ** P <0.01 vs. DM mice group, && P <0.01 vs. WT DM mice group. ( I ) Potential mechanism underlying the protective effects of CHS on IHG induced cell injuries in DM mice and pancreas islet cells.

Article Snippet: Bax (No. 2772), Bcl-2 (No. 3498), Bad (No. 9292), Cleaved-caspase 3 (No. 9661), Cleaved-PARP (No. 94885), Cytochrome c (No. 11940), β-actin (No. 4970), VDAC (No. 12454), Cyclin D1 (No. 2978), Skp2 (No. 4313), p21 (No. 2947), p27 (No. 3688), p53 (No. 2524), Wnt3a (No. 2721), β-Catenin (No. 8480), c-Myc (No. 9402) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: In Vivo, Positive Control, Expressing

Journal: Pharmaceuticals

Article Title: Tyrphostin AG1024 Suppresses Coronaviral Replication by Downregulating JAK1 via an IR/IGF-1R Independent Proteolysis Mediated by Ndfip1/2_NEDD4-like E3 Ligase Itch

doi: 10.3390/ph15020241

Figure Lengend Snippet: NEDD4-like Itch E3 inhibition restored the AG1024 downregulated JAK1. ( A ) Neither the NEDD4 inhibitor heclin ( a ) nor the skp2 inhibitors SKPin C1 and CC220 ( b ) restored AG1024 downregulated JAK1, whereas the NEDD4 inhibitor heclin restored ouabain diminished JAK1 protein level. ( B ) Itch E3 inhibitors clomipramine and CPZ significantly restored the AG1024 downregulated JAK1. TGEV infected (MOI: 7) ST cells were treated with ouabain, AG1024, heclin, MLN4924, SKPin C1, CC220, clomipramine, or CPZ as indicated and harvested at 5 h.p.i. MLN4924, a NEDD8 inhibitor, served as a non-specific reference control, and the concentrations were used as reported . The concentrations of SKPin C1, CC220, clomipramine, or CPZ used herein were based on previous reports [ , , , , ]. The resulting lysates were subjected to western analyses. *, p < 0.05; **, p < 0.01. Results shown are averages ± SD from three independent experiments.

Article Snippet: DMSO (≧99.5%), human insulin (I2643, ≧98%, HPLC), ouabain (O3125, ≧95%, HPLC), and clomipramine hydrochloride (C7291, ≧98%, HPLC) were purchased from Sigma-Aldrich (St. Louis, MO, USA); MG132 (474790, ≧98%, HPLC) from Merck Millipore Calbiochem (Merck, La Jolla, CA, USA); AG1024 (S1234, ≧99.7%, HPLC), picropodophyllin (PPP, S7668, ≧99.5%, HPLC), MLN4924 (S7109, ≧99%, HPLC), chlorpromazine (CPZ, S5749, ≧97%, HPLC), Skp2 inhibitor C1 (SKPin C1, S8652, ≧99%, HPLC) and iberdomide (CC-220, S8760, ≧98%, HPLC) from Selleckchem (Houston, TX, USA); heclin (5433, ≧98%, HPLC) from Tocris Bioscience (Minneapolis, MN, USA); and human IGF-1 (CYT-216, ≧98%, SDS-PAGE) from ProSpec (Rehovot, Israel).

Techniques: Inhibition, Infection, Control, Western Blot

Journal: Molecular medicine reports

Article Title: Interference of Skp2 effectively inhibits the development and metastasis of colon carcinoma.

doi: 10.3892/mmr.2014.2308

Figure Lengend Snippet: Figure 1. Identification of the most effective siRNA of the Skp2 gene in SW620 cells. The cells were plated in 48-well plates and three pairs of Skp2‑siRNA were transfected into the SW620 cells. The expression levels of the Skp2 gene were detected 48 h later by (A) quantitative polymerase chain reaction and (B) western blot analysis. β-actin was used an internal reference. Results are presented as the mean ± standard error of the mean. **P<0.01, compared with the control group. Skp2, S-phase kinase-associated protein 2; si, small interfering.

Article Snippet: Skp2 p45 shRNA (h) Lentiviral Particles were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Molecular medicine reports

Article Title: Interference of Skp2 effectively inhibits the development and metastasis of colon carcinoma.

doi: 10.3892/mmr.2014.2308

Figure Lengend Snippet: Figure 2. Distribution of the different groups of cells in the cell cycle phases. Cell cycle analysis of the SW620 cells was performed 48 h after transfection with Skp2-siRNA or scrambled siRNA. (A) The proportion of the cells in each phase was determined by flow cytometry of propidium iodide-stained cells and (B) the data were quantified. Results are presented as the mean ± standard error of the mean. *P<0.05 and **P<0.01, compared with the respective control group. si, small interfering; Skp2, S-phase kinase-associated protein 2.

Article Snippet: Skp2 p45 shRNA (h) Lentiviral Particles were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Cell Cycle Assay, Transfection, Flow Cytometry, Staining, Control

Journal: Molecular medicine reports

Article Title: Interference of Skp2 effectively inhibits the development and metastasis of colon carcinoma.

doi: 10.3892/mmr.2014.2308

Figure Lengend Snippet: Figure 4. Induction of apoptosis was determined by PI-Annexin V staining. The cells were transfected with Skp2-siRNA and scrambled siRNA for 48 h. PI-Annexin V‑positive cells were analyzed by (A) fluorescence-activated cell sorting and (B) the percentage of the PI-Annexin V-positive cells was quantified and shown in the histograms. Results are presented as the mean ± standard error of the mean. **P<0.01, compared with the control group or the group treated with scrambled siRNA. Skp2, S-phase kinase-associated protein 2; si, small interfering; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Article Snippet: Skp2 p45 shRNA (h) Lentiviral Particles were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Staining, Transfection, Fluorescence, FACS, Control

Journal: Molecular medicine reports

Article Title: Interference of Skp2 effectively inhibits the development and metastasis of colon carcinoma.

doi: 10.3892/mmr.2014.2308

Figure Lengend Snippet: Figure 3. In vitro scratch assay. (A) Untreated group, (B) SW620 colon cancer cells treated with scrambled siRNA for 12 h after the scratch was performed and (C) SW620 cells transfected with Skp2-siRNA for 12 h after the scratch was performed. si, small interfering; Skp2, S-phase kinase-associated protein 2.

Article Snippet: Skp2 p45 shRNA (h) Lentiviral Particles were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: In Vitro, Wound Healing Assay, Transfection

Journal: Molecular medicine reports

Article Title: Interference of Skp2 effectively inhibits the development and metastasis of colon carcinoma.

doi: 10.3892/mmr.2014.2308

Figure Lengend Snippet: Figure 5. Inteference of Skp2 inhibited proliferation of SW620 cells, as detected by an MTT assay. SW620 cells (1x105) were planted in a 96-well plate and treated for different time periods in the presence of Skp2 siRNA or scrambled siRNA. The number of SW620 cells was measured. Untreated cells were used as the negative control. Data are shown as the mean ± stan dard error of the mean of at least three independent experiments on different individuals. **P<0.01 compared with the control group or the group treated with scrambled siRNA. OD, optical density; si, small interfering; Skp2, S-phase kinase‑associated protein 2.

Article Snippet: Skp2 p45 shRNA (h) Lentiviral Particles were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: MTT Assay, Negative Control, Control

Journal: Molecular medicine reports

Article Title: Interference of Skp2 effectively inhibits the development and metastasis of colon carcinoma.

doi: 10.3892/mmr.2014.2308

Figure Lengend Snippet: Figure 8. Survival rate of nude mice following lethal SW620 cell challenge. The nude mice were challenged subcutaneously with 5x105 SW620 colon cancer cells in the flank area. The survival rate at 29 days was determined as follows: 100 x (number of survivors)/(number of challenged mice). Each group contained ten mice. **P=0.003 vs. scrambled group, P=0.006 vs. con trol group. si, small interfering; Skp2, S-phase kinase‑associated protein 2.

Article Snippet: Skp2 p45 shRNA (h) Lentiviral Particles were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques:

Journal: Molecular medicine reports

Article Title: Interference of Skp2 effectively inhibits the development and metastasis of colon carcinoma.

doi: 10.3892/mmr.2014.2308

Figure Lengend Snippet: Figure 7. Paraffin-embedded samples were analyzed by immunohisto chemical staining for Skp2. Skp2 protein was mainly located and expressed in the nucleus of the tumor cells. Skp2 expression in tumor tissues of the (A) control, (B) scrambled siRNA and (C) Skp2 siRNA groups (images of Skp2 staining were captured at x400 original magnification). Skp2, S-phase kinase‑associated protein 2; si, small interfering.

Article Snippet: Skp2 p45 shRNA (h) Lentiviral Particles were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Staining, Expressing, Control

Journal: Molecular medicine reports

Article Title: Interference of Skp2 effectively inhibits the development and metastasis of colon carcinoma.

doi: 10.3892/mmr.2014.2308

Figure Lengend Snippet: Figure 6. Expression levels of p27kip1 were increased in SW620 cells. SW620 cells (2x105) were planted in a 24-well plate and transfected for 24 h in the presence of Skp2 siRNA or scrambled siRNA. Untreated cells were used as the negative control. Total cell lysates were analyzed for Skp2, p27 and β-actin by western blot analysis. β-actin was used as an internal refer ence. Skp2, S-phase kinase‑associated protein 2; si, small interfering.

Article Snippet: Skp2 p45 shRNA (h) Lentiviral Particles were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Negative Control, Western Blot

Journal: Toxicology Reports

Article Title: Upregulation of cyclin-dependent kinase inhibitors CDKN1B and CDKN1C in hepatocellular carcinoma-derived cells via goniothalamin-mediated protein stabilization and epigenetic modifications

doi: 10.1016/j.toxrep.2015.01.010

Figure Lengend Snippet: In Huh-7 cells, goniothalamin stabilized nuclear CDKN1B via downregulation of its E3 ligase SKP2 level. (A) GTN treatment for 24 h upregulated CDKN1B while downregulated SKP2 protein level [DMSO 24 h/Cycloheximide (CHX) 0 h vs. GTN 24 h/CHX, 0 h]. CHX-chase (0, 2, 4, 8 h) along with immunoblotting assays estimated that the half-life of CDKN1B and SKP2 proteins were approximately 6 h. Compared to the DMSO group, GTN treatments prolonged the stabilities of high CDKN1B and low SKP2 protein levels. (B) GTN treatments did not alter CDKN1B and SKP2 mRNA levels in a time course experiment, compared to their corresponding controls (DMSO). (C) MG132 (5 μM) increased SKP2 protein abundance and further restored GTN-inhibited SKP2 protein level. DMSO (control), GTN and MG132 were added at time 0, 0 and 16 h, respectively; the total reaction time was 20 h. (D) GTN treatments for 8, 16 and 24 h downregulated SKP2, nevertheless, did not alter FZR1 (an E3 ligase of SKP2) protein abundance. (E) Fractionation of nuclear and cytosolic proteins and immunoblotting analysis indicated that GTN notably induced nuclear CDKN1B, however, downregulated nuclear SKP2, CCNE1, CDK2, pCDKN1B(T187) protein levels. PARP1 and GAPDH were served as nuclear and cytosolic control, respectively. All experiments were triplicated and results are expressed as mean ± SEM. Representative immunoblotting images are shown. Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: TaqMan ® chemistry with primers and probes (Life Technologies): CDKN1B (#4331182; 151 bp, Hs01597588_m1, NM_004064.3 ), CDKN1C (#4331182; 91 bp, Hs00175938_m1, NM_000076.2 , NM_001122630.1 , NM_001122631.1 ), SKP2 (#4331182; 59 bp, Hs01021864_m1, NM_001243120.1 , NM_005983.3 , NM_032637.3 ) and GAPDH (#4453320; 93 bp, Hs02758991_g1, NM_001256799.1 , NM_002046.4 ) were used for quantitative RT-PCR.

Techniques: Western Blot, Quantitative Proteomics, Control, Fractionation

Journal: Toxicology Reports

Article Title: Upregulation of cyclin-dependent kinase inhibitors CDKN1B and CDKN1C in hepatocellular carcinoma-derived cells via goniothalamin-mediated protein stabilization and epigenetic modifications

doi: 10.1016/j.toxrep.2015.01.010

Figure Lengend Snippet: In Hep-3B cells, goniothalamin induced CDKN1C mRNA and subsequent protein levels via upregulation of acetyl-H3 and H3K9/14ac levels. Immunoblotting identified that SKP2 (an CDKN1C-specific E3 ligase) protein level was almost unchanged after GTN treatments for 8, 16 and 24 h. (B) Quantitative RT-PCR showed that GTN upregulated CDKN1C mRNA levels in a time-dependent manner. (C) Treatments with trichostatin A (TSA; 100 ng/mL) and GTN (15 μM) for 24 h but not 5-Aza-2′-deoxycytidine (5-Aza-dC; 10 μM) for 72 h, upregulated CDKN1C mRNA level, compared to the control group (DMSO). Combined treatments of TSA and GTN further upregulated CDKN1C mRNA level, compared to either treatment with TSA or GTN alone. (D) CDKN1C translation was delayed to 28 h when combined treatments with TSA and GTN, compared to its transcription level at 24 h. (E, F) ITSA1 (50 μM, 24 h), a TSA-specific inhibitor, downregulated endogenous and GTN-induced CDKN1C mRNA and protein levels, compared to the controls (DMSO). Similarly, CDKN1C translation was delayed to 28 h when combined treatments with GTN and ITSA1. ITSA1 further downregulated GTN-induced CDKN1C mRNA and protein levels. (G) Immunoblotting analysis demonstrated that TSA and GTN notably upregulated acetyl-H3 but not acetyl-H4 protein level, with an additive effect. (H) Histone extraction and immunoblotting analysis further demonstrated that both TSA and GTN increased H3K9/14-acetylated proteins in Hep-3B cells, with a slightly additive effect. All experiments were triplicated and results are expressed as mean ± SEM. For immunoblotting analysis, representative images are shown. GAPDH, ACTB and H3 were served as loading controls. Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: TaqMan ® chemistry with primers and probes (Life Technologies): CDKN1B (#4331182; 151 bp, Hs01597588_m1, NM_004064.3 ), CDKN1C (#4331182; 91 bp, Hs00175938_m1, NM_000076.2 , NM_001122630.1 , NM_001122631.1 ), SKP2 (#4331182; 59 bp, Hs01021864_m1, NM_001243120.1 , NM_005983.3 , NM_032637.3 ) and GAPDH (#4453320; 93 bp, Hs02758991_g1, NM_001256799.1 , NM_002046.4 ) were used for quantitative RT-PCR.

Techniques: Western Blot, Quantitative RT-PCR, Control, Extraction