Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer, pSi-shUSP22-1 or pSi-shUSP22-2 plasmid. ( A ) Western blot analysis of USP22 expression; β-actin was used as endogenous control. ( B ) Cell viability was measured using MTT assay at 1, 2, 3, and 4 d after transfection. ( C ) EdU staining used to examine the proliferation. Scale bar: 5 μm. ( D ) Percentage of EdU-positive staining in (C). ( E ) Effect of USP22 knockdown on cell proliferation evaluated by colony formation assay. After 21 d of transfection, cells were stained with Giemsa solution. ( F ) Histograms representing the colony formation number in (E). ( G ) Western blot conducted to analyze the expression of USP22 and proliferation-related proteins including Rb, p-Rb, Akt, p-Akt, and cyclin D2. β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pSilencer group (B, D, and F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, MTT Assay, Staining, Knockdown, Colony Assay

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer or pSi-shUSP22-1 plasmid. ( A and B ) After 48 h of transfection, the scratch wound-healing assay was performed to evaluate the effect of USP22 on cell migration. The representative images of cell migration were shown in (A), and the width of wounds at the indicated times was shown in (B). ( C ) Invasion of CAL-62 and 8505C cells analyzed by transwell assay. The invaded cells were stained with DAPI. Scale bar: 5 μm. ( D ) Number of DAPI-positive cells per field counted under a fluorescent microscopy. ( E ) Immunofluorescence staining was performed to analyze the effect of USP22 on the expression of E-cadherin and vimentin in 8505C cells. Scale bar: 5 μm. ( F and G ) Expression of invasion-related proteins containing BMI-1, E-cadherin, vimentin, and snail in ATC cells was measured by qPCR (F) and western blot (G). GAPDH and β-actin were used as endogenous controls, respectively. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with the group at 0 h after transfection (B). ## P < 0.01, as compared with mock or pSilencer group at 24 h after transfection (B). * P < 0.05, as compared with mock or pSilencer group (D and F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Wound Healing Assay, Migration, Transwell Assay, Staining, Microscopy, Immunofluorescence, Expressing, Western Blot

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer or pSi-shUSP22-1 plasmid. ( A ) Apoptosis of 8505C cells analyzed by flow cytometry. ( B ) Apoptosis rate in (A) was calculated. ( C ) Fluorescent TUNEL assay conducted to determine the apoptosis of 8505C cells. Scale: 10 μm. ( D ) Percentage of TUNEL-positive cells in (C). ( E ) CAL-62 and 8505C cell apoptosis evaluated by nucleosomal fragmentation assay. ( F ) Quantification of caspase-3 activity in CAL-62 and 8505C cells. ( G ) Western blot analyses of apoptosis-related protein (Bid, Bax, cl-caspase-3, caspase-3, and Bcl-2) expressions in CAL-62 and 8505C cells. β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pSilencer group (B and D–F).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, TUNEL Assay, Activity Assay, Western Blot, Control

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: Female six-week-old SCID mice were inoculated subcutaneously into right hind flanks or injected via tail vein with stably expressed pLKO.1 or pLKO.1-shUSP22 8505C-luc cells. Mock-treatment was used as control. ( A ) Tumor growth progression was measured by in vivo luciferase imaging of the xenografts at days 1, 5, 9, and 12 d after inoculation. ( B ) Representative gross photos of tumors 22 d after subcutaneous xenografting ( n = 6). ( C ) Tumor volumes of subcutaneous implantation models of ATC were monitored and calculated after 0, 7, 11, 13, 17, 20, and 22 d of inoculation. D–F. After 28 d of injection through tail vein, the lungs were removed and photographed ( D ), the number of metastatic nodules in lungs was counted ( E ), and the weight of lungs was measured ( F ). ( G ) Fluorescence TUNEL assay was carried out to determine cell apoptosis in the same tumor tissues as indicated above. The rate of TUNEL-positive cells was calculated. Scale bar: 10 μm. ( H ) qPCR assay was performed to detect the mRNA expression of USP22 in tumor tissues from (B). ( I ) Protein expression of USP22 in tumor tissues was analyzed by western blot. ( J ) Representative results of western blot analyses of cyclin D2, Akt, p-Akt, E-cadherin, vimentin, Bax, Bcl-2, cl-caspase-3, and caspase-3 in tumor tissues. (I and J) β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pLKO.1 group (C and E–H).

Article Snippet: Human ATC cell lines CAL-62 and 8505C were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), which certifies the origin and identity of the cells.

Techniques: Injection, Stable Transfection, Control, In Vivo, Luciferase, Imaging, Fluorescence, TUNEL Assay, Expressing, Western Blot

Journal: American Journal of Cancer Research

Article Title: Anaplastic thyroid cancer cell-secreted TGFβ1 plays a key role in inducing macrophage polarization of human monocytes

doi: 10.62347/BHFA4606

Figure Lengend Snippet: The effect of transforming growth factor β1 (TGFβ1) on the phenotype of THP-1 cells. (A-I-A-III) Soluble factors derived from anaplastic thyroid cancer (ATC) cells induced morphological changes in THP-1 cells. Monocytes were treated for 18 hours (h) with 8505C-derived conditioned media (CM) and TGFβ1 20 ng/ml. Representative bright-field imaging of THP-1 cells control (A-I) or treated with 8505C-derived CM (A-II) and TGFβ1 20 ng/ml (A-III). (B) Proliferation of THP-1 cells incubated with 8505C-derived CM and TGFβ1 12.5 ng/ml for 24 h and 48 h, estimated by cell counting. (C) Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay to detect human C-C motif chemokine ligand 13 (hCCL13), hDectin1 and CD163 in THP-1 cells grown under normal conditions (control) and treated with 8505C-derived CM and TGFβ1 20 ng/ml for 24 h. (D, E) Flow cytometry analysis showed significant induction of hDectin1 in THP-1 cells treated with 8505C-derived CM and TGFβ1 20 ng/ml for 24 h compared to THP-1 control (E). Representative histograms and quantification (D) are shown. (F) Concentrations of TGFβ1 in Dulbecco’s modified Eagle’s medium (DMEM) alone, DMEM FBS 5% (control) and in the CM of 8505C and C643 cells, determined by enzyme-linked immunosorbent assay (ELISA) as described in Methods and Materials. Data are expressed as mean ± SEM. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001.

Article Snippet: Human ATC cell line 8505C was obtained from the European Collection of Authenticated Cell Cultures while the ATC-derived C643 cell line was obtained from the University of Colorado Cancer Center Cell Bank.

Techniques: Derivative Assay, Imaging, Control, Incubation, Cell Counting, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Flow Cytometry, Modification, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: Anaplastic thyroid cancer cell-secreted TGFβ1 plays a key role in inducing macrophage polarization of human monocytes

doi: 10.62347/BHFA4606

Figure Lengend Snippet: SNAIL and SLUG are up-regulated in THP-1 cell activation induced by cancer cell-secreted TGFβ1. (A, B) Immunoblot analysis of SNAIL in THP-1 cells grown under normal conditions (control) or exposed to CM derived from 8505C cells and TGFβ1 20 ng/ml for 18 h and 24 h (A). The figure shows a representative Western blot of 4 independent experiments. Quantification of relative expression of SNAIL protein in human monocytes after using βActin as loading control (B). (C) Expression levels of SNAIL messenger RNA (mRNA) levels by RT-qPCR in control and THP-1 cells treated for 12 h and 24 h with 8505C-derived CM and TGFβ1 20 ng/ml. (D, E) Immunoblot for SLUG from THP-1 cells treated with CM derived from 8505C cells and TGFβ1 20 ng/ml for 18 h and 24 h. The same blot was stripped and re-blotted using anti-βActin antibody. The figure shows a representative Western blot of 4 independent experiments (D). Expression levels of SLUG mRNA levels by RT-qPCR in control and THP-1 cells treated for 18 h and 24 h with 8505C-derived CM and TGFβ1 20 ng/ml. Data are expressed as mean ± SEM. **P<0.005, ***P<0.0005, ****P<0.0001. ns, not significant.

Article Snippet: Human ATC cell line 8505C was obtained from the European Collection of Authenticated Cell Cultures while the ATC-derived C643 cell line was obtained from the University of Colorado Cancer Center Cell Bank.

Techniques: Activation Assay, Western Blot, Control, Derivative Assay, Expressing, Quantitative RT-PCR

Journal: American Journal of Cancer Research

Article Title: Anaplastic thyroid cancer cell-secreted TGFβ1 plays a key role in inducing macrophage polarization of human monocytes

doi: 10.62347/BHFA4606

Figure Lengend Snippet: TGFβ receptor inhibition affects 8505C-derived CM induced macrophage polarization as well as expression of SNAIL and SLUG. (A-C) THP-1 cells pre-incubated with SB431542 (20 µM) or dimethyl sulfoxide (DMSO) as control for 1 h, were treated with 8505C-derived CM for 18 h and 24 h. The expression of hCCL13 (A), hDectin1 (B) and hCD163 (C) were analyzed by RT-qPCR. (D, E) RT-qPCR of SNAIL (D) and SLUG (E) expression in THP-1 cells pre-treated with SB431542 (20 µM) before 8505C-derived CM treatment for 18 h and 24 h. Data are expressed as mean ± SEM. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. ns, not significant.

Article Snippet: Human ATC cell line 8505C was obtained from the European Collection of Authenticated Cell Cultures while the ATC-derived C643 cell line was obtained from the University of Colorado Cancer Center Cell Bank.

Techniques: Inhibition, Derivative Assay, Expressing, Incubation, Control, Quantitative RT-PCR

Journal: Journal of the Endocrine Society

Article Title: Reevaluation of the Effect of Iodine on Thyroid Cell Survival and Function Using PCCL3 and Nthy-ori 3-1 Cells

doi: 10.1210/jendso/bvaa146

Figure Lengend Snippet: 131 Iodine ( 131 I) uptake in PCCL3, Nthy-ori 3-1, 8505C, and HepG2 cells, and thyrotropin receptor (TSHR) and sodium/iodine symporter (NIS) messenger RNA (mRNA) expression in PCCL3 and Nthy-ori 3-1 cells. A, Cells were cultured in the presence or absence of thyrotropin (TSH) and/or perchlorate and then incubated with 10 μCi/mL 131 I for 30 minutes. 131 I taken up into the cells was measured as described in “Materials and Methods.” Data are means ± SD (n = 3). B, Total RNA was extracted from PCCL3 and Nthy-ori 3-1 cells and subjected to RT-PCR for TSHR, NIS, and β-actin mRNAs as described in “Materials and Methods.” Data are means ± ranges (n = 2).

Article Snippet: Human anaplastic thyroid cancer cell line 8505C and human hepatocellular cancer cell line HepG2 were obtained from RIKEN Bioresource Center, cultured in RPMI 1640 with 10% FBS and antibiotics, and used as controls.

Techniques: Expressing, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of the Endocrine Society

Article Title: Reevaluation of the Effect of Iodine on Thyroid Cell Survival and Function Using PCCL3 and Nthy-ori 3-1 Cells

doi: 10.1210/jendso/bvaa146

Figure Lengend Snippet: Cytotoxicity of iodine in PCCL3, Nthy-ori 3-1, 8505C, and HepG2 cells. The cells, cultured in the presence or absence of perchlorate, were incubated with up to 5 × 10 –2 M iodine for 24 hours and cell viability was determined as described in “Materials and Methods.” Data are means ± SD (n = 3).

Article Snippet: Human anaplastic thyroid cancer cell line 8505C and human hepatocellular cancer cell line HepG2 were obtained from RIKEN Bioresource Center, cultured in RPMI 1640 with 10% FBS and antibiotics, and used as controls.

Techniques: Cell Culture, Incubation

Journal: Journal of experimental & clinical cancer research : CR

Article Title: miRNA-106a directly targeting RARB associates with the expression of Na(+)/I(-) symporter in thyroid cancer by regulating MAPK signaling pathway.

doi: 10.1186/s13046-016-0377-0

Figure Lengend Snippet: Fig. 3 a, MTT Cell viability assays: relative cell viability in parental 8505C and transfected sublines [*, P < 0.01, miR106a(−) and U0126 vs. control; #, P < 0.01, miR106a(−) + RARB(−) and miR106a(−) + MAP3K2 vs. miR106a(−)]; b, MTT Cell viability assays: relative cell viability in parental CGTH-W3 and transfected sublines [*, P < 0.01, miR106a(+) and RARB(−) vs. control; #, P < 0.01, miR106a(+) + U0126 vs. miR106a(+)]; c and d, flow cytometry analysis of cell cycle in 8505C and 8505C-miR106a(−)

Article Snippet: 8505C-miR106a(−) + MAP3K2(+) 8505C-miR106a(−) cells were transiently transfected with MAP3K2 (NM_006609) Human cDNA ORF Clone (OriGene, Cat. # RC223988) using 100 μl Opti-MEM I (Gibco, Cat. # 51985091) and 3 μL Turbofectin 8.0 (OriGene, Cat. # TF81001).

Techniques: Transfection, Control, Flow Cytometry