Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-199a-3p increases the anti-tumor activity of palbociclib in liver cancer models

doi: 10.1016/j.omtn.2022.07.015

Figure Lengend Snippet: miR-199a-3p increased palbociclib efficacy on human HCC cells in vitro and in vivo (A) Hep3B cells were transduced with an Adeno Associated Virus expressing miR-199a-3p (AAVV-199) or with a control AAVV (AAVV-CTRL) at MOI = 100. A group of cells received no treatment (NT). Palbociclib (PB) (20 μM) was added to cell culture 72 h before cell collection. The levels of miR-199a-3p were measured 120 h after transduction. (B) Viability and apoptosis were evaluated 120 h after transduction. Data are represented as mean +SD. (C) Western blot analysis and quantification of RB1, AKT, FOXM1, and their phosphorylated forms in Hep3B-treated cells. The values were normalized on the GAPDH expression. Because Hep3B cells exhibit a low level of full-length protein, digital images of RB1 and p-RB1 were acquired with an exposure time of 300 s instead of 30 s. (D) Tumor nodule volumes in DEN-treated TG221 mice of the following groups: (1) palbociclib (PB) (n = 11); (2) miR-199a-3p mimics (miR-199) (n = 9); (3) palbociclib + miR-199a-3p mimics (PB + miR-199) (n = 11); (4) sorafenib (SF) (n = 9); (5) vehicle/scramble oligonucleotides (CTRL) (n = 9). Experimental therapies started at 6 months, when all mice presented one or more tumor nodules in their livers. Single tumor nodules were monitored by ultrasound at the beginning and end of treatment. (E) Distribution of tumor nodule size measured at the time of euthanization. (F) Mice weight was monitored during treatment to check drug toxicity. Data are represented as mean ± SD. ∗p value ≤0.05; ∗∗p value ≤0.01; ∗∗∗p value ≤0.001; ∗∗∗∗p value ≤0.0001.

Article Snippet: After incubation with 5% Blocking agent, the membrane were incubated overnight at 4°C with the following antibodies and specific conditions: Rabbit antibodies against p-RB (Ser780, D59B7, #8180), p-AKT (Ser473, D9E XP, #4060), p-FOXM1 (Thr600, D9M6G, #14655), RB (D20, #9313), FOXM1 (D12D5, #5436) and PAK4 (#3242) were all from Cell Signaling Technology (Danvers, MA, USA) and were diluted in 5% w/v BSA (A4503, Sigma-Aldrich), 1X Tris-buffered saline (TBS; Bio-Rad Laboratories, Hercules, CA, USA), and 0.1% Tween 20 (Bio-Rad) and incubated at 4°C for 16 h. Rabbit antibody against AKT (C-20, sc-1618) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and was diluted in 1% w/v milk, 1X TBS, and 0.1% Tween 20 (Bio-Rad).

Techniques: In Vitro, In Vivo, Transduction, Virus, Expressing, Control, Cell Culture, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

doi: 10.1186/s13046-019-1504-5

Figure Lengend Snippet: Clinicopathological characteristics and TRIM6 expression ( n = 90)

Article Snippet: After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

doi: 10.1186/s13046-019-1504-5

Figure Lengend Snippet: Clinical significance of TRIM6 in CRC. a , The mRNA expression of TRIM6 was detected in 35 pairs of CRC samples and mucosa tissues (cohort 1) by qRT-PCR. The TRIM6 expression was normalized to GAPDH. b , The mRNA expression of TRIM6 in GSE GSE20842 dataset, which includes 65 paired samples of cancer and adjacent mucosa from patients with Stage II/III rectal adenocarcinomas. c , Representative images of immunohistochemical staining for TRIM6 in CRC samples and mucosa tissues from cohort 2. Scale bar: 100 μm. d , Survival analysis of patients with high (TRIM6 high ) or low expression of TRIM6 (TRIM6 low ). e , Multivariate regression analysis in cohort 2

Article Snippet: After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

doi: 10.1186/s13046-019-1504-5

Figure Lengend Snippet: Correlation of TRIM6 expression in colorectal cancer tissues with different clinicopathological features (n = 90)

Article Snippet: After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

doi: 10.1186/s13046-019-1504-5

Figure Lengend Snippet: TRIM6 knockdown inhibited CRC cell proliferation and induced G2/M arrest. a , The protein level of TRIM6 in human normal colorectal mucosa cell line (FHC) and CRC cell lines (LOVO, Sw620, Sw1116, HCT-8 and HCT116) was examined by western blotting. GAPDH was used as a loading control. b , Knocking down of TRIM6 in HCT-8 and HCT116 cells. Cells were infected with lentivirus expressing TRIM6 shRNA (shTRIM6–1, − 2 and − 3) or control shRNA (shNC). At 48 h post infection, protein was extracted and TRIM6 expression was examined by western blotting. C-F, The effects of TRIM6 on the proliferation, cell cycle distribution as well as expression of Cyclin B1 and c-Myc in HCT-8 and HCT116 cells were measured by CCK-8 ( c ), BrdU ( d ), flow cytometry analyses ( e ), and western blotting ( f ), respectively. * P < 0.05, ** P < 0.01, *** P < 0.001 vs shNC

Article Snippet: After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.

Techniques: Western Blot, Infection, Expressing, shRNA, CCK-8 Assay, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

doi: 10.1186/s13046-019-1504-5

Figure Lengend Snippet: TRIM6 knockdown potentiated the anti-proliferative effects of 5-fluorouracil and oxaliplatin a , HCT-8 and HCT116 cells were infected with shTRIM6–1 or shNC, and treated with 40, 60, 80 or 100 μM L-OHP for 24 h. Cell proliferation was determined by CCK-8 assay, and IC50 was calculated. b , HCT-8 and HCT116 cells were infected with shTRIM6–1 or shNC, and treated with 300, 400, 500 or 600 μM 5-FU for 24 h. Cell proliferation was determined by CCK-8 assay, and IC50 was calculated. C-F, HCT-8 and HCT116 cells were infected with shTRIM6–1 or shNC, and treated with 64 μM L-OHP, 400 μM 5-FU or vehicle (DMSO) for 24 h. Cell apoptosis ( c , d ) and expression of cleaved-caspase3 (C-Casp3, e , f ) was determined. *** P < 0.001

Article Snippet: After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.

Techniques: Infection, CCK-8 Assay, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

doi: 10.1186/s13046-019-1504-5

Figure Lengend Snippet: TRIM6 interacted with TIS21 in CRC cells. a , pCMV-Tag2-TIRM6 or pCMV-Tag2 vector was transfected into 293 T cells, and 48 h later, cell lysates were prepared and subjected to immunoprecipitation (IP) experiments with anti-FLAG beads. After elusion with FLAG peptide, the immunoprecipitated protein complexes were resolved on SDS-PAGE, and stained with Coomassie Brilliant Blue. B, C, IP was carried out with TRIM6 antibody (TRIM6-Ab) /IgG ( b ) or TIS21 antibody (TIS21-Ab) /control IgG ( c ), and then western blotting was performed to analyze specific associations between TRIM6 and TIS21 in HCT-8 and HCT116 cells. D-E, GST pull-down assay. HCT-8 cells were lysed and incubated with GST, GST-tagged TRIM6 ( d ) and GST-tagged TIS21 ( e ) bound to glutathione beads, respectively. Proteins were detected as indicated. E, immunofluorescence staining of TRIM6 (Red) and TIS21 (Green) in HCT-8 and HCT116 cells. DAPI (blue) was used to label nuclei. Scale bar: 50 μm

Article Snippet: After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, SDS Page, Staining, Western Blot, Pull Down Assay, Incubation, Immunofluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

doi: 10.1186/s13046-019-1504-5

Figure Lengend Snippet: TRIM6 promoted TIS21 ubiquitination. a , b , Western blotting ( a ) and qRT-PCR ( b ) were used to detect TIS21 in HCT-8 and HCT116 cells infected with lentivirus expressing TRIM6 shRNA (shTRIM6–1, − 2) or control shRNA (shNC). c , HCT-8 cells were transfected with pcDNA3.1-myc-TIRM6 or pcDNA3.1-myc (Vector) for 24 h, and exposed to 20 mM cycloheximide (CHX, Sigma-Aldrich). Cell lysate was prepared at 0, 3 and 6 h after exposure and subjected to western blotting analysis. d , HCT-8 cells were transfected with pCMV-Tag2-TIRM6 or pCMV-Tag2 vector for 24 h and then treated with MG132 (10 μM) or DMSO for 20 h. Western blotting was used to detect TIS21. e , Cell lysates from HCT-8 cells infected with lentivirus expressing TRIM6 shRNA (shTRIM6–1) or control shRNA (shNC) were IP with TIS21-Ab/control IgG and then immunoblotted for ubiquitin (Ub). f , Ubiquitination assay. The 293 T cells were transfected with plasmids expressing myc-TRIM6, His-ubiquitin and FLAG-TIS21 (WT, K5R, K51R or K150R). Cell lysates were incubated with nickelnitrilotriacetic acid beads and subjected to western blotting with anti-FLAG

Article Snippet: After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.

Techniques: Western Blot, Quantitative RT-PCR, Infection, Expressing, shRNA, Transfection, Plasmid Preparation, Ubiquitin Assay, Incubation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

doi: 10.1186/s13046-019-1504-5

Figure Lengend Snippet: TIS21/FoxM1 was essential for TRIM6-inhibited CRC cell proliferation and cell cycle progression. a , Sw620 cells were transfected with plasmids expressing TRIM6, TIS21 or vector. Overexpression of TRIM6 or TIS21 was confirmed by western blotting. B-F, Sw620 cells were divided into four groups: Vector (cells transfected with vector), TRIM6 (cells transfected with plasmid expressing TRIM6), TIS21 (cells transfected with plasmid expressing TIS21) and TRIM6 + TIS21 (cells transfected with plasmid expressing TRIM6 and plasmid expressing TIS21). CCK-8 ( b ), BrdU ( c ), flow cytometry analyses ( d ) and western blotting ( e , f ) were performed to determine the effects of TRIM6 and TIS21 on the proliferation, cell cycle distribution and relative protein expression, respectively. F, HCT-8 cells were transfected with plasmids expressing FoxM1 or vector. Overexpression of TRIM6 or TIS21 was confirmed by western blotting. G-J, HCT-8 cells were divided into four groups: Vector+shNC (cells treated with vector and control shRNA), Vector+shTRIM6 (cells treated with vector and TRIM6 shRNA), FoxM1 + shNC (cells treated with plamid expressing FoxM1 and control shRNA) and FoxM1 + shTRIM6 (cells treated with plamid expressing FoxM1 and TRIM6 shRNA). CCK-8 ( g ), BrdU ( h ), flow cytometry analyses ( i ) and western blotting ( j ) were performed to determine the effects of FoxM1 overexpression and TRIM6 knockdown on the proliferation, cell cycle distribution and relative protein expression, respectively. * P < 0.05, **P < 0.05, *** P < 0.001

Article Snippet: After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.

Techniques: Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot, CCK-8 Assay, Flow Cytometry, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

doi: 10.1186/s13046-019-1504-5

Figure Lengend Snippet: TRIM6 knockdown inhibited tumorigenesis of CRC cells. HCT-8 cells stably expressed TRIM6 shRNA (shTRIM6) or control shRNA (shNC) were injected into nude mice. Xenograft growth curve ( a ), photograph of the xenografts ( b ), tumor weight ( c ), representative images of Ki67 staining ( d ), and representative western blot ( e ) are shown. Scale bar: 50 μm. *P < 0.05, **P < 0.05, ***P < 0.001

Article Snippet: After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.

Techniques: Stable Transfection, shRNA, Injection, Staining, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

doi: 10.1186/s13046-019-1504-5

Figure Lengend Snippet: Correlation analyses in colorectal tissues. a , Western blotting analysis of TRIM6, TIS21, FoxM1, p-FoxM1 (Thr 600) and p-FoxM1 (Ser 35) in 6 normal mucosa samples and 10 CRC samples. b , Quantification of the western blotting data. c , Pearson correlation scatter plots in colorectal tissues. d , Representative images of IHC staining of TRIM6, TIS21, p-FoxM1 (Thr 600) and p-FoxM1 (Ser 35) in CRC samples (Case 1 and Case 2). Scale bar: 100 μm

Article Snippet: After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.

Techniques: Western Blot, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

doi: 10.1186/s13046-019-1504-5

Figure Lengend Snippet: TRIM6 expression level influenced the anti-proliferative effect of FoxM1 inhibitor TST. A-C, qRT-PCR (A) was performed to detect TRIM6 mRNA expression in primary CRC cells. Cells L1-L6, had a relative lower level of TRIM6 expression than cells H1-H8. Primary CRC cells were treated with 400 μM 5-FU, 64 μM L-OHP, 2 μM TST or vehicle (DMSO) for 48 h. CCK-8 assay was carried out to determine the inhibition rate of cell proliferation (%). D-G, a xenograft mouse model was established by inoculation of HCT116 or SW620 cells to nude mice. On the 12th day after inoculation, the mice were treated with TST or Vehicle (DMSO). Tumor volume (D), tumor images (E), tumor weight (F) and overall survival (G) were shown. *P < 0.05, ** P < 0.01, ***P < 0.001 vs. Vehicle. H, Schematic representation of the regulation of CRC cell proliferation by TRIM6/TIS21/FoxM1

Article Snippet: After incubation with anti-TRIM6 (Bioss Inc., Woburn, Massachusetts, USA), anti-TIS21 (Abcam, Cambridge, MA, USA), anti-p-FoxM1(Thr600) (Affinity, Cincinnati, OH, USA), anti-p-FoxM1(Ser35) (Affinity) overnight at 4 °C, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Longislandbio, Shanghai, China) for 1 h at RT, developed with a DAB staining kit (Longislandbio) and counterstained with hematoxylin.

Techniques: Expressing, Quantitative RT-PCR, CCK-8 Assay, Inhibition