Journal: Scientific Reports

Article Title: Down-regulation of Skp2 expression inhibits invasion and lung metastasis in osteosarcoma

doi: 10.1038/s41598-018-32428-9

Figure Lengend Snippet: Skp2 is overexpressed in osteosarcoma cell lines and high Skp2 levels are correlated with a worse prognosis. ( A ) Quantitative RT-PCR. Skp2 mRNA expression in 5 standard and 8 patient-derived osteosarcoma cell lines was significantly increased compared to normal human osteoblasts (NHOst). (B , C) Skp2 protein levels were elevated in standard (B) and patient-derived (C) osteosarcoma cell lines compared to NHOsts. (D) Kaplan-Meier analysis. Raw Skp2 expression data was retrieved from NCBI GEO and correlated with survival data from the R2 platform. The median Skp2 mRNA expression was used as a cutoff to distinguish low vs. high expression. High Skp2 expression correlated significantly with a worse metastasis-free survival. (E) Tissue microarrays. Overall survival was compared in osteosarcoma patients whose tumors expressed low (- and +) vs. high (++ and +++) Skp2 (negative =<1% stained cells; (+) = 1–10%; (++) = 10–50%; (+++) =>50%). By log-rank test, the high Skp2 expression group sustained a worse overall survival than the low expression group. (F) Representative pictures of IHC scoring for Skp2. Statistical significance is indicated by: *p < 0.05, **p < 0.01, ***p < 0.001. Column: mean; Error bars: SD.

Article Snippet: After deparaffinization, rehydration, and blocking, sections were incubated with an anti-Skp2 antibody (2652, Cell Signaling Technology, Beverly, MA) at 4 °C overnight, followed by incubation with a secondary antibody for 30 minutes at room temperature.

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Staining

Journal: Scientific Reports

Article Title: Down-regulation of Skp2 expression inhibits invasion and lung metastasis in osteosarcoma

doi: 10.1038/s41598-018-32428-9

Figure Lengend Snippet: Proliferation, invasion, and mechanistic studies following knockdown of Skp2 in osteosarcoma cells. ( A , B ) MTT proliferation assays. Compared to control transfected cells, the proliferation of (A) 143B and (B) SaOS-2 Skp2-knockdown cells were significantly reduced. ( C – F ) Matrigel invasion assay. Compared to controls, invasion through Matrigel is significantly reduced in Skp2-knockdown 143B (C) and SaOS-2 (E) cell lines. (D , F) Representative pictures of the invasion chambers. Skp2 knockdown was confirmed by qRT-PCR in 143B ( G ) and SaOS-2 ( H ) cell lines. Western blots showed that p21 and cleaved caspase-3 levels were elevated, while RhoA and MMP-9 were reduced after Skp2 knockdown ( I ). Statistical significance is indicated by: *p < 0.05, **p < 0.01, ***p < 0.001. Column: mean; Error bars: SD.

Article Snippet: After deparaffinization, rehydration, and blocking, sections were incubated with an anti-Skp2 antibody (2652, Cell Signaling Technology, Beverly, MA) at 4 °C overnight, followed by incubation with a secondary antibody for 30 minutes at room temperature.

Techniques: Knockdown, Control, Transfection, Invasion Assay, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Down-regulation of Skp2 expression inhibits invasion and lung metastasis in osteosarcoma

doi: 10.1038/s41598-018-32428-9

Figure Lengend Snippet: Skp2 knockdown inhibits tumor growth and lung metastasis in an orthotopic osteosarcoma model. ( A) Experimental design. 5 × 10 5 143B/shctrl or 143B/shSkp2 cells were implanted into the proximal tibias of mice. Legs were amputated once any tumor reached 1.5 cm in diameter. Animals were kept for 2 weeks after amputation and then sacrificed. (B) Representative pictures of tibia tumors. (C) Tumor volumes of the Skp2-knockdown group were significantly than the control group. (D) Representative pictures of lungs harvested from mice injected with transfected 143B/shctrl and 143B/shSkp2 cell lines. (E) The mean lung weight (LW) normalized to body weight (BW) was significantly greater in the control group compared to the Skp2-knockdown group. (F) Skp2 knockdown significantly reduced the number of metastatic lung nodules. (G) Expression of Skp2 from two different lungs was examined by Western blot. No Skp2 expression was observed in143B/shSkp2 lung tissue. (H) Representative pictures of hematoxylin-eosin (HE) and IHC staining of lung tissues. Lower levels of Skp2 immunostaining were detected in metastatic lung nodules from the 143B/shSkp2 group. Statistical significance is indicated by: *p < 0.05, **p < 0.01, ***p < 0.001. Column: mean; Error bars: SD.

Article Snippet: After deparaffinization, rehydration, and blocking, sections were incubated with an anti-Skp2 antibody (2652, Cell Signaling Technology, Beverly, MA) at 4 °C overnight, followed by incubation with a secondary antibody for 30 minutes at room temperature.

Techniques: Knockdown, Control, Injection, Transfection, Expressing, Western Blot, Immunohistochemistry, Immunostaining

Journal: Scientific Reports

Article Title: Down-regulation of Skp2 expression inhibits invasion and lung metastasis in osteosarcoma

doi: 10.1038/s41598-018-32428-9

Figure Lengend Snippet: FKA exerts inhibitory effects by targeting Skp2 in osteosarcoma cells. Quantitative RT-PCR. Treatment with FKA markedly decreased SKP2 mRNA levels in 143B (A) and SaOS-2 (B) cell lines. (C) Western blot showing FKA-mediated suppression of Skp2 and Cul1-Nedd8 in a dose-dependent manner. Levels of the cell cycle regulator p21 (a known Skp2 target) were also elevated, along with the apoptosis markers cleaved Caspase-3 and cleaved PARP. Other Skp2 targets such as RhoA and MMP-9, were also inhibited by FKA. Independent t-tests were used to evaluate differences in qRT-PCR data. Statistical significance is indicated by: ****p < 0.0001. Column: mean value; Error bars: SD.

Article Snippet: After deparaffinization, rehydration, and blocking, sections were incubated with an anti-Skp2 antibody (2652, Cell Signaling Technology, Beverly, MA) at 4 °C overnight, followed by incubation with a secondary antibody for 30 minutes at room temperature.

Techniques: Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Down-regulation of Skp2 expression inhibits invasion and lung metastasis in osteosarcoma

doi: 10.1038/s41598-018-32428-9

Figure Lengend Snippet: Oral treatment with FKA blocks osteosarcoma lung metastasis in vivo . ( A ) Experimental design. (B , D) FKA treatment significantly reduced the number of metastatic surface lung nodules. (C) Mean normalized lung weight (LW) to body weight (BW) was significantly lower in the FKA-treated group compared to the FKA-treated group. (E) Western blot showing Skp2 protein expression in two representative lung specimens harvested from control and FKA-treated mice. Skp2 expression in lung tissue lysates was markedly suppressed after FKA treatment (F) Representative pictures of HE and IHC staining of lung tissues. Independent t-tests were used to compare differences in the LW/BW ratio and lung nodules between control and FKA-treated groups. Statistical significance is indicated by: *p < 0.05, **p < 0.01, ***p < 0.001. Column: mean; Error bars: SD.

Article Snippet: After deparaffinization, rehydration, and blocking, sections were incubated with an anti-Skp2 antibody (2652, Cell Signaling Technology, Beverly, MA) at 4 °C overnight, followed by incubation with a secondary antibody for 30 minutes at room temperature.

Techniques: In Vivo, Western Blot, Expressing, Control, Immunohistochemistry

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 3 CLOCK interference promotes GCs proliferation. A The interference efficiency of CLOCK was measured using RT-qPCR. Data are expressed as mean ± SEM (n = 5), **P < 0.01. B Western blotting reveals the expression levels of CLOCK. C Quantification of the western blot analysis. Data are expressed as mean ± SEM (n = 3), *P < 0.05. D EdU staining was used to detect the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 5), **P < 0.01. E CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 5), *P < 0.05. F RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 5), *P < 0.05, **P < 0.01. G Western blot analysis of proliferation-related gene protein level (CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A). GAPDH as a housekeeping protein. H Quantifying the Western blot analysis of CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), *P < 0.05

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Transfection

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 6 ASB9 interference promotes GCs proliferation. A RT-qPCR detected the interference efficiency of ASB9. Data are expressed as mean ± SEM (n = 6), **P < 0.01. B Western blotting reveals the expression levels of ASB9. C Quantification of the western blot analysis. Data are expressed as mean ± SEM (n = 3), *P < 0.05. D EdU staining was used to detect the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 4), **P < 0.01. E CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 16), ****P < 0.0001. F RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 5), *P < 0.05, **P < 0.01. G Western blot analysis of proliferation-related gene protein level (ASB9, CCNB1, CCNE1, CDK4, and CDKN1A). H Quantifying the western blot analysis of CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), *P < 0.05, **P < 0.01. I RNA expression of ASB9 in GCs. ZT: zone time

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Transfection, RNA Expression