Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Structures and cytotoxic activities of magnolol, M2M, and MM1. ( a ) Chemical structures of magnolol, M2M, and MM1. ( b and c ) A375 and A431 skin cancer cell lines were treated with various concentrations of magnolol, M2M, or MM1 for 24 h. Their cytotoxic effects and the viabilities of the treated cells were detected with ( b ) SRB assays and ( c ) MTT assays. ( d ) Flow cytometric analysis of cell cycle. A375 and A431 cells were treated with 75 μ M of magnolol (Mag) or MM1 for 24 h. The treated cells were stained with propidium iodide and analyzed by flow cytometry. The percentages of cells in the sub-G1 region (M1) are indicated. Data shown in b and c are expressed as the mean±S.D. of two independent experiments. Symbols: * P <0.05; ** P <0.01; and *** P <0.001, as analyzed by unpaired t -tests. Data shown in d are from one of two similar results

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Effects of magnolol and MM1 on the clonogenic ability and anchorage-independent growth of skin cancer cells. ( a ) A375 (left panel) and A431 (right panel) cells were treated with various concentrations of magnolol or MM1 for 6 days and then cultured for an additional 8 days in the absence of drugs. The numbers of foci were scored, and the data are presented as the relative focus-forming ability (FFA). ( b ) A375 cells were treated with different concentrations of magnolol and MM1. The anchorage-independent growth was assessed as described in the Materials and Methods section. Data are expressed as the mean±S.D. of three independent experiments. Symbols: * P <0.05, as analyzed by unpaired t -tests

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Cell Culture

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Effect of magnolol and MM1 on the induction of apoptosis in A375 cells. ( a ) A375 cells were treated with magnolol or MM1 for 24 h, and western blotting was used to assess cell lysates for the cleavages of PARP and procaspase-7, -8, and -9. ( b and c ) Effect of caspase inhibition on the apoptosis of MM1-treated A375 cells. A375 cells were treated with 75 μ M MM1 in the presence or absence of the broad-spectrum caspase inhibitor, z-VAD-fmk (40 μ M), for 24 h. Phase-contrast images of MM1-treated cells treated with or without z-VAD-fmk are shown in b . Western blot analysis was used to assess cell lysates for the cleavages of procaspase-7, -8, and 9, with β -actin used as a loading control ( c )

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Inhibition, Control

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Effects of magnolol and MM1 on the growth of xenograft tumors in nude mice. A375 cells were injected subcutaneously into the flank of each mouse. When the tumor volumes reached about 50 mm 3 , the mice were i.p. injected with magnolol (1 mM in 100 μ l acetone), MM1 (1 mM in 100 μ l acetone), or DMSO (control) three times per week ( n =6 per group). The tumor volume ( a ) was determined twice weekly, while body weight ( b ) was measured daily. The xenograft tumors were excised from the mice at the end of experiment in 15 days. The sizes of representative tumors excised from the different groups are shown at the top of a , while hematoxylin–eosin staining (magnification, × 200, upper panel) and IHC staining for activated caspase-3 (magnification, × 200, lower panel) are shown in c . The results shown in a and b are presented as the means±S.D. of six mice. Symbols: * P <0.05 by unpaired t -tests

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Injection, Control, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: MM1 upregulates lncRNA GAS5. A375 cells were treated with or without 50 μ M MM1 for 24 h, and total RNA was subjected to microarray analysis as described in the Materials and Methods section. The heatmap shown in a presents a hierarchical clustering of the transcriptome profiles from duplicate cultures of untreated and MM1-treated A375 cells. The relative expression of each RNA transcript is indicated by color, and ranges from red (higher expression) to green (lower expression). ( b ) Functional classification of the differentially expressed genes in MM1-treated A375 cells, as assessed using the DAVID Database web server. The differentially expressed proteins are linked to a number of biological processes. ( c ) Upregulation of lncRNA GAS5 in magnolol- or MM1-treated A375 cells. A375 cells were treated with the indicated concentrations of magnolol or MM1 for 24 h, and the expression levels of lncRNA GAS5 were determined by real time RT-PCR, as described in the Materials and Methods section. The expressions of lncRNA GAS5 in the magnolol- and MM1-treated cells were normalized to that of the untreated cells, and are presented as relative expression levels. The data shown represent the mean±S.D. of three independent experiments. Symbol: ** P <0.01, as analyzed with the unpaired t -test

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Microarray, Expressing, Functional Assay, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: List of the 20 highest-scored lncRNA differentially expressed in MM1-treated A375 cells

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques:

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: lncRNA GAS5 plays a critical role in the MM1-mediated induction of apoptosis. ( a ) Effect of lncRNA GAS5 depletion on cell apoptosis. Left panel: A375 cells were transfected with siRNA against lncRNA GAS5 (si-lncRNA GAS5) at 50 nM or with nontargeting siRNA(−). After 48 h, the transfected cells were treated with 75 μ M of MM1 for 24 h. The levels of lncRNA GAS5 were assessed by real-time RT-PCR and expressed as relative to that of non-MM1-treated control (Left panel). The cleavage of PARP and caspase-9 was analyzed by western blotting (Right panel). β -actin served as an internal control. ( b and c ) Effect of lncRNA GAS5 overexpression on cell proliferation and apoptosis. A375 cells were transfected with pCDNA3.1-lncRNA GAS5 or the empty vector (control). After 48 h, the transfected cells were assayed for expression of lncRNA GAS5 ( b , left panel), for viability by MTT method ( b , right panel), and for the induction of apoptosis by phosphatidylserine exposure with Annexin V-FITC using flow cytometry ( c ). Data are expressed as the mean±S.D. of three independent experiments. Symbols: ** P <0.01; and *** P <0.001, as analyzed by unpaired t -tests

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Transfection, Quantitative RT-PCR, Control, Western Blot, Over Expression, Plasmid Preparation, Expressing, Flow Cytometry

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hypoxia-associated circRNA profiling and expression characteristics of Hsa_circ_0000566 in osteosarcoma (OS). (A) CircRNA microarray analysis reveals 35 upregulated and 23 downregulated circRNAs in OS cells under normoxic and hypoxic conditions. The black arrow represents Hsa_circ_0000566. (B) OS cells incubated under various oxygen concentrations. Total RNA extraction was performed for qRT-PCR assay. Western blotting was performed to determine the protein level of HIF-1α. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. Scale bars, 200 μm. (C) Hsa_circ_0000566 expression is much higher in primary OS tissue than in chondroma tissue. Results are representative images according to three different experiments. (D) Quantitative real-time polymerase chain reaction (qRT-PCR) results comparing Hsa_circ_0000566 mRNA expression in 12 OS and chondroma samples. Results are reported as mean ± SD, *p < 0.05, n = 12. (E) Hsa_circ_0000566 expression levels in hFOB1.19 and various OS cell lines. Results are reported as mean ± SD, *p < 0.05, n = 3. (F) Schematic diagram showing Hsa_circ_0000566 back-spliced by exons 2-11 of the VRK1 gene and the corresponding Sanger sequencing. (G) RT-PCR results validating the presence of Hsa_circ_0000566 in 143B and HOS cells. Various primers amplified the Hsa_circ_0000566 region in cDNA but not in genomic DNA. β-actin was used as the negative control. Divergent primers are presented as the opposite direction of the arrowhead, and the convergent primers were shown as the face-to-face direction of the arrowhead. (H) RT-PCR results indicating Hsa_circ_0000566 and VRK1 mRNA expression in untreated 143B and HOS cells and in the cells subjected to treatment with RNase-R. (I) RNA fluorescence in situ hybridization (FISH) results revealing Hsa_circ_0000566 localized mainly in the cytoplasm. Hsa_circ_0000566 probes were labeled with cy3 and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bars, 100 μm. (J) qRT-PCR determination of the main localization of Hsa_circ_0000566 in OS cells. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Microarray, Incubation, RNA Extraction, Quantitative RT-PCR, Western Blot, Standard Deviation, Real-time Polymerase Chain Reaction, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Fluorescence, In Situ Hybridization, Labeling, Staining

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 contributes to in vitro osteosarcoma (OS) cell progression under hypoxic conditions. (A) Hsa_circ_0000566 overexpression and knockdown induced and repressed OS cell proliferation under hypoxia. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. Circ_0000566 represents Hsa_circ_0000566 overexpression, and si circ_0000566 represents Hsa_circ_0000566 knockdown. Vector and Si NC represents the negative control of Hsa_circ_0000566 overexpression and Hsa_circ_0000566 knockdown, respectively. (B) EdU exhibits the impact of Hsa_circ_0000566 on OS cell proliferation under hypoxia. Nuclei are stained with 4’,6-diamidino-2-phenylindole (DAPI). Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (C) Colony formation experiment verifies Hsa_circ_0000566 functions in OS cells under hypoxia. Results are reported as mean ± SD, *p < 0.05, n = 3. (D) Soft agar colony formation assay indicates the effects of Hsa_circ_0000566 on 143B and HOS cell colony forming capacity under hypoxia. Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (E) OS cell migration capacity as determined by Transwell™ migration assays. Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (F) Flow cytometry verifies Hsa_circ_0000566 functions in OS cell apoptosis. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vitro, Over Expression, Knockdown, Standard Deviation, Plasmid Preparation, Negative Control, Staining, Soft Agar Assay, Migration, Flow Cytometry

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 accelerates osteosarcoma (OS) glucose metabolism and regulates hypoxia-enhanced glycolysis. (A) Colors of the media indicate that Hsa_circ_0000566 silencing decreased lactate accumulation under hypoxia. (B-C) Quantitative real-time polymerase chain reaction (qRT-PCR) or western blots evaluating the expression levels of genes involved in glucose metabolism in 143B and HOS cells transfected with Hsa_circ_0000566-overexpressing, Hsa_circ_0000566 (shRNA), or vector plasmids. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. (D) Hsa_circ_0000566 knockdown in OS cells with decreased lactate accumulation, while Hsa_circ_0000566 overexpression has increased lactate accumulation. Results are reported as mean ± SD, *p < 0.05, n = 3. (E) Extracellular acidification rate (ECAR) indicates glycolysis rate. ECAR decreases in response to Hsa_circ_0000566 knockdown and increases in response to Hsa_circ_0000566 overexpression. Oxygen consumption rate (OCR) represented mitochondrial respiratory capacity. OCR is enhanced in response to Hsa_circ_0000566 silencing and reduced in response to Hsa_circ_0000566 overexpression in OS cells. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Transfection, shRNA, Plasmid Preparation, Standard Deviation, Knockdown, Over Expression

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 establishes interactions with HIF-1α and confers protection against ubiquitination-mediating degradation. (A) Effects of Hsa_circ_0000566 knockdown and Hsa_circ_0000566 overexpression on mRNA and protein expression in 143B and HOS cells under hypoxia. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. (B) Western blotting results revealing the impact of bortezomib treatment on the changes occurring at HIF-1α protein level mediated by Hsa_circ_0000566 silencing and vector transfection. (C) Western blotting assessment of the impact of CHX treatment on the variations in HIF-1α protein levels affected by Hsa_circ_0000566 silencing and vectors. Results are reported as mean ± SD, *p < 0.05, n = 3. (D) The western blot illustrates the effects of Hsa_circ_0000566 knockdown in the Hyp564 HIF-1α protein levels in the presence or absence of bortezomib treatment. (E) Immunoprecipitation assessing the HIF-1α ubiquitination levels in Hsa_circ_0000566 silencing and Hsa_circ_0000566 overexpressing osteosarcoma (OS) cells under hypoxia. Culture media were supplemented with bortezomib (250 nM) for 6 h. (F) The combination of Hsa_circ_0000566 with HIF-1α confirmed by radioimmunoprecipitation (RIP). Results are reported as mean ± SD, *p < 0.05, n = 3. (G) Pulldown assay validation of the interaction between Hsa_circ_0000566 and HIF-1α. (H) A RIP assay of HIF-1α regions interacting with Hsa_circ_0000566. Schematic diagram shows HIF-1α protein fragments. Results are reported as mean ± SD, *p < 0.05, n = 3. (I) Interaction profile between Hsa_circ_0000566 and HIF-1α obtained from catRAPID (left). (J) Schematic diagram showing Hsa_circ_0000566 RNA fragments. Combinative regions between Hsa_circ_0000566 and HIF-1α were identified by RIP assay. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Ubiquitin Proteomics, Knockdown, Over Expression, Expressing, Standard Deviation, Western Blot, Plasmid Preparation, Transfection, Immunoprecipitation, Biomarker Discovery

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 promotes osteosarcoma (OS) glucose metabolism and tumorigenesis progression in vivo. (A) 143B cells stably transfected with Hsa_circ_0000566 knockdown, HIF-1α overexpression, or empty vector plasmids. Nude mice were subcutaneously injected with 1 × 10 7 cells that were either stable negative controls or those with Hsa_circ_0000566 knockdown, HIF-1α overexpression, or Hsa_circ_0000566 knockdown. Thirty days after injection, the animals were euthanized, and their tumors dissected and photographed. (B) Tumor weight measurements on the same day the mice were euthanized. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 5. (C) Tumor volumes (ab2/2) were calculated every 6 d from the day after the mice were injected with stable OS cells. (D-E) Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) exhibit the expression levels of the genes involved in glycolysis metabolism. Results are reported as mean ± SD, *p < 0.05, n = 3. (F) Fluorescence in situ hybridization (FISH), hematoxylin and eosin (H&E) staining, and immunohistochemistry (IHC) analysis indicate the OS organization in mice and relative GLUT1, GLUT4, PDK1, PDK4, and LDHA protein levels in tumors from different groups. (G) In situ tumor formation experiment reveals that HIF-1α overexpression recovered Hsa_circ_0000566 knockdown-induced tumor attenuation. Results are reported as mean ± SD, *p < 0.05, n = 4. (H) Micro-computed tomography (CT) indicates the functions of HIF-1α and Hsa_circ_0000566 knockdown in bone loss. (I) H&E staining of lung metastasis. In mice injected in the tail vein with various stable 143B cells, lung metastasis was detected using an in vivo bioluminescence imaging system. Results are reported as mean ± SD, *p < 0.05, n = 5.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vivo, Stable Transfection, Transfection, Knockdown, Over Expression, Plasmid Preparation, Injection, Standard Deviation, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Fluorescence, In Situ Hybridization, Staining, Immunohistochemistry, In Situ, Micro-CT, Imaging

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited the proliferation and induced the apoptosis of PCa cells. The IC50 of mannose in ( a ) DU145 and ( b ) PC3 cells was determined using a CCK-8 assay. ( c ) Intracellular mannose concentration in PCa cells. Cell proliferation of ( d ) DU145 and ( e ) PC3 was assessed using growth curves, respectively. ( f ) Colony formation assays were performed and ( g ) colony numbers were counted in PCa cells. ( h ) Flow cytometric analysis was used to assess ( i ) the apoptosis rate of PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; IC50: the half-maximal inhibitory concentration; CCK-8: Cell Counting Kit-8; AAD: Aminoactinomycin D; APC: Allophycocyanin.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: CCK-8 Assay, Concentration Assay, Cell Culture, Cell Counting

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited tumor growth in a PCa xenograft model without affecting mice health. ( a ) Subcutaneous tumors from the xenograft model with DU145 cells. ( b ) Tumor growth was monitored for 30 days after mannose treatment. ( c ) The volume of tumor growth. ( d ) The weight of the tumors. ( e ) Intratumoral mannose concentration and ( f ) ATP content in subcutaneous tumors. The weights of ( g ) mice and ( h ) major metabolic organs. ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Concentration Assay, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted mitochondrial function, led to ROS overproduction, and activated Bax/Bak in PCa cells. ( a ) JC-1 staining and ( b ) rhodamine 123 staining were used to assess the MMP in DU145 and PC3 cells. ( c ) The ATP content in cells. ( d ) Mitochondrial ROS and ( e ) cellular ROS levels in cells. ( f and g ) The protein expression of Bax and Bak in cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine; Bax: BCL2-associated X; Bak: BCL2-antagonist/killer 1; ROS: reactive oxygen species; MMP: mitochondrial membrane potential; ATP: adenosine triphosphate; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Expressing, Cell Culture, Membrane

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted the balance of mitochondrial dynamics in PCa cells. ( a ) Mitochondria stained with MitoTracker Red were observed by confocal microscopy. ( b ) Mitochondrial structure under a transmission electron microscopy, and the mitochondrial cross-sectional area was quantified. ( c ) The protein expression of FIS1 in cells. Upregulated ( d ) FIS1 and ( e ) increased ATP content in PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. ATP: adenosine triphosphate; FIS1: fission, mitochondrial 1; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Expressing, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Downregulation of MPI expression enhances the anticancer effect of mannose. The expression of MPI in human prostate cancer tissues and its prognostic value. ( a and b ) The expression of MPI was silenced by siRNA-MPI and verified by western blotting. siRNA-MPI with different base sequences including si-1, si-2 and si-3 were used for downregulating the MPI expression in PCa cells. According to the degree of down-regulation of MPI protein, si-3 and si-2 with the best interference effect were applied to DU145 and PC3 cells, respectively. ( c ) Intracellular mannose concentration and ( d ) ATP content in cells. ( e ) Growth curves and ( f ) colony formation assays of cells. ( g ) The IHC scores for MPI expression in PCa tissues. ( h ) Kaplan–Meier curves of BCR-free survival and ( i ) overall survival for the low and high MPI expression groups of patients in the TCGA-PRAD dataset. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. MPI: mannose phosphate isomerase; si: siRNA, small interfering RNA; IHC: immunohistochemistry; PCa: prostate cancer; BCR: biochemical recurrence; TCGA-PRAD: The Cancer Genome Atlas-Prostate Adenocarcinoma; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TMA: tissue microarray; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Expressing, Western Blot, Concentration Assay, Cell Culture, Small Interfering RNA, Immunohistochemistry, Microarray

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited the proliferation and induced the apoptosis of PCa cells. The IC50 of mannose in ( a ) DU145 and ( b ) PC3 cells was determined using a CCK-8 assay. ( c ) Intracellular mannose concentration in PCa cells. Cell proliferation of ( d ) DU145 and ( e ) PC3 was assessed using growth curves, respectively. ( f ) Colony formation assays were performed and ( g ) colony numbers were counted in PCa cells. ( h ) Flow cytometric analysis was used to assess ( i ) the apoptosis rate of PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; IC50: the half-maximal inhibitory concentration; CCK-8: Cell Counting Kit-8; AAD: Aminoactinomycin D; APC: Allophycocyanin.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: CCK-8 Assay, Concentration Assay, Cell Culture, Cell Counting

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited tumor growth in a PCa xenograft model without affecting mice health. ( a ) Subcutaneous tumors from the xenograft model with DU145 cells. ( b ) Tumor growth was monitored for 30 days after mannose treatment. ( c ) The volume of tumor growth. ( d ) The weight of the tumors. ( e ) Intratumoral mannose concentration and ( f ) ATP content in subcutaneous tumors. The weights of ( g ) mice and ( h ) major metabolic organs. ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Concentration Assay, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted mitochondrial function, led to ROS overproduction, and activated Bax/Bak in PCa cells. ( a ) JC-1 staining and ( b ) rhodamine 123 staining were used to assess the MMP in DU145 and PC3 cells. ( c ) The ATP content in cells. ( d ) Mitochondrial ROS and ( e ) cellular ROS levels in cells. ( f and g ) The protein expression of Bax and Bak in cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine; Bax: BCL2-associated X; Bak: BCL2-antagonist/killer 1; ROS: reactive oxygen species; MMP: mitochondrial membrane potential; ATP: adenosine triphosphate; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Expressing, Cell Culture, Membrane

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted the balance of mitochondrial dynamics in PCa cells. ( a ) Mitochondria stained with MitoTracker Red were observed by confocal microscopy. ( b ) Mitochondrial structure under a transmission electron microscopy, and the mitochondrial cross-sectional area was quantified. ( c ) The protein expression of FIS1 in cells. Upregulated ( d ) FIS1 and ( e ) increased ATP content in PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. ATP: adenosine triphosphate; FIS1: fission, mitochondrial 1; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Expressing, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Downregulation of MPI expression enhances the anticancer effect of mannose. The expression of MPI in human prostate cancer tissues and its prognostic value. ( a and b ) The expression of MPI was silenced by siRNA-MPI and verified by western blotting. siRNA-MPI with different base sequences including si-1, si-2 and si-3 were used for downregulating the MPI expression in PCa cells. According to the degree of down-regulation of MPI protein, si-3 and si-2 with the best interference effect were applied to DU145 and PC3 cells, respectively. ( c ) Intracellular mannose concentration and ( d ) ATP content in cells. ( e ) Growth curves and ( f ) colony formation assays of cells. ( g ) The IHC scores for MPI expression in PCa tissues. ( h ) Kaplan–Meier curves of BCR-free survival and ( i ) overall survival for the low and high MPI expression groups of patients in the TCGA-PRAD dataset. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. MPI: mannose phosphate isomerase; si: siRNA, small interfering RNA; IHC: immunohistochemistry; PCa: prostate cancer; BCR: biochemical recurrence; TCGA-PRAD: The Cancer Genome Atlas-Prostate Adenocarcinoma; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TMA: tissue microarray; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Expressing, Western Blot, Concentration Assay, Cell Culture, Small Interfering RNA, Immunohistochemistry, Microarray

Journal: Neoplasia (New York, N.Y.)

Article Title: Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11

doi: 10.1016/j.neo.2021.04.001

Figure Lengend Snippet: LIN28A is overexpressed in human liver cancer. (A) Quantitative RT-PCR analysis of LIN28A mRNA levels in primary human hepatocytes (PHH) ( n = 10) as compared with human HCC cell lines (PLC ( n = 9), Hep3B ( n = 10), HepG2 ( n = 11)). (B,C) Quantitative RT-PCR analysis (A) and representative Western blot images depicting LIN28A mRNA and recombinant protein expression, respectively, after transfection of HepG2 cells (low endogenous LIN28A expression as compared with PLC and Hep33B) applying a LIN28A-overexpression (OE) plasmid vector (provided by G. Meister and described in detail elsewhere ( n = 4). (D) Immunofluorescence analysis of LIN28A expression and localization in two different HCC cell lines (PLC ( n = 2), Hep3B ( n = 2)) (representative images, 40-fold magnification). (E) LIN28A mRNA levels (qRT-PCR analysis) of paired human HCC and corresponding nontumorous liver tissue (CNTLT) samples ( n = 10pairs). (F-I) Immunohistochemical analysis of LIN28A protein expression and cellular localization patterns in patients applying tissue micro array derived paired HCC tissues and CNTLT. Staining intensity and percentage of positive cells were incorporated into a semiquantitative score describing "0" (very low/no expression), "1" (low/moderate expression) and "2" (strong expression). (F) depicts representative immunohistochemical images. (G) LIN28A protein expression score in human HCC tissues and CNTLT applying a tissue micro array (paired analyses). (H) LIN28A expression in HCC ( n = 103) as compared with CNTLT ( n = 115) (semiquantitative analysis applying Fisher's exact test). (I) Spearman correlation analysis of LIN28A expression levels in HCC patients with early (stage I & II) ( n = 71) compared with late (stage III and IV) tumor stages (UICC 2010). Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t-test (A,B), paired t-test (E,G), by two-sided Fisher's exact test (H), and Spearman correlation (I). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The human hepatocellular carcinoma (HCC) cell lines PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and HepG2 (ATCC HB-8065) were described in other studies [ , ].

Techniques: Quantitative RT-PCR, Western Blot, Recombinant, Expressing, Transfection, Over Expression, Plasmid Preparation, Immunofluorescence, Immunohistochemical staining, Microarray, Derivative Assay, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11

doi: 10.1016/j.neo.2021.04.001

Figure Lengend Snippet: LIN28A is a novel target gene of miR-622 in HCC. (A) MicroRNA recognition elements (MRE) in the LIN28A 3’UTR represent potential miR-622-binding sites and were identified applying the "TargetScan 7.2" database. (B) Quantitative RT-PCR analysis of LIN28A mRNA levels in control-transfected as compared to miR-622-transfected different HCC cell lines (PLC ( n = 3) ; Hep3B ( n = 3)). (C) Luciferase LIN28A 3′UTR-reporter (containing a conserved miR-622 MRE) activity in control-miR (CTR) as compared with miR-622-mimic (622)-transfected HCC cells (PLC) ( n = 3 ). (D) Schematic image depicting the pre-let-7-f-1 loop region (as predicted applying RNA-fold with the highlighted consensus motif for recognition and binding of LIN28 to pre-let-7-f-1 which was identified previously . (E) Schematic image depicting the pre-miR-622 stem loop (as predicted applying RNA-fold and miRNAMap ) and magnification of the pre-miR-622 loop region containing the identic sequence motif as identified previously for recognition and binding of LIN28 to pre-let-7. (F) Hypothesis image: According to pre-let-7, LIN28A might also bind to the terminal loop of pre-miR-622 with subsequent ZCCHC11-mediated oligouridylation followed by degradation of pre-miR-622. (G) RNA-pulldown ( n = 2) depicting binding of LIN28A to the let-7-g hairpin (positive control). No binding was found applying a negative control miR without the potential consensus motif (miR-18b) as well as a miR-622 and a miR-622-mutated (mutated consensus motif) hairpin. Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t-test (A,B). * P < 0.05, *** P < 0.001.

Article Snippet: The human hepatocellular carcinoma (HCC) cell lines PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and HepG2 (ATCC HB-8065) were described in other studies [ , ].

Techniques: Binding Assay, Quantitative RT-PCR, Control, Transfection, Luciferase, Activity Assay, Sequencing, Positive Control, Negative Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11

doi: 10.1016/j.neo.2021.04.001

Figure Lengend Snippet: ZCCHC11 is overexpressed in HCC. (A) Quantitative RT-PCR analysis of ZCCHC11 mRNA levels in primary human hepatocytes (PHH) ( n = 9) as compared with human HCC cell lines (PLC ( n= 10), Hep3B ( n= 6), HepG2 ( n= 15)). (B) Representative Western blot image and (densitometric) quantification of ZCCHC11 protein levels in PHH ( n = 3) as compared with human HCC cell lines (PLC ( n = 4), Hep3B ( n = 4), HepG2 ( n = 4)). (C) ZCCHC11 mRNA levels (qRT-PCR analysis) in paired human HCC tissue samples and corresponding nontumorous liver tissues (CNTLT) ( n = 32pairs). (D) Transcriptomics data of ZCCHC11 RNA expression levels in HCC ( n = 369) and nontumorous liver tissues ( n = 50) derived from TCGA-data applying the Gene Expression Profiling Interactive Analysis (GEPIA) database. (E) ZCCHC11 overexpression in HCC tissues ( n = 2) as compared with nontumorous liver tissues ( n = 2) applying the human proteinatlas database (representative images). (E-H) Confirmation of ZCCHC11 protein expression and cellular localization patterns in patients applying tissue micro array derived paired HCC tissues and CNTLT. Staining intensity and percentage of positive cells were incorporated into a semi-quantitative score describing "0" (very low/no expression), "1" (low/moderate expression) and "2" (strong expression). (H) Representative immunohistochemistry images and paired quantification of ZCCHC11 expression score (G) as well as semiquantitative analysis applying Fischer's exact test (H) in HCC as compared with CNTLT. Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t-test (A,B,D), paired t-test (G), and two-sided Fisher's exact test (H). * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: The human hepatocellular carcinoma (HCC) cell lines PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and HepG2 (ATCC HB-8065) were described in other studies [ , ].

Techniques: Quantitative RT-PCR, Western Blot, RNA Expression, Derivative Assay, Gene Expression, Over Expression, Expressing, Microarray, Staining, Immunohistochemistry

Journal: Neoplasia (New York, N.Y.)

Article Title: Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11

doi: 10.1016/j.neo.2021.04.001

Figure Lengend Snippet: ZCCHC11 reveals oncogenic functions in HCC. (A-C) HCC cell lines (Hep3B ( n = 2), PLC ( n = 2)) were transfected with a control si-RNA-pool or with a specific si-RNA-pool targeting ZCCHC11 for 48 h. (A) ZCCHC11 mRNA levels as determined by qRT-PCR analysis. (B) Migration as determined applying Boyden chamber cell migration assays. (C) Colony numbers as determined applying clonogenic assays. (D) Real-time cell proliferation analysis of HCC cells transfected with an si-RNA-Pool directed against ZCCHC11 or a control si-RNA-Pool ( n = 4). (E) Correlated ZCCHC11 and CDK2, CDK4, CDK12, CDK14, CDK17, CDK20 RNA expression levels (log 2 (TPM)), respectively, in HCC tissues. TCGA-derived data were used applying the Gene Expression Profiling Interactive Analysis (GEPIA) database. The data represent RNA expression levels (log 2 (TPM)) in HCC tissues. (F,G) TCGA-derived datasets deposited on the Gene Expression Profiling Interactive Analysis (GEPIA) database (F) or the ProteinAtlas database (G), respectively. "HR": Hazard ratio. Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t-test (A,B,C,D) and Pearson correlation analysis (E). Survival analysis was performed computationally applying log-rank testing and hazard ratio estimates (F) or applying log-rank testing (Mantel-Cox) (G). * P < 0.05, ** P < 0.01, ns: non-significant.

Article Snippet: The human hepatocellular carcinoma (HCC) cell lines PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and HepG2 (ATCC HB-8065) were described in other studies [ , ].

Techniques: Transfection, Control, Quantitative RT-PCR, Migration, RNA Expression, Derivative Assay, Gene Expression

Journal: Neoplasia (New York, N.Y.)

Article Title: Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11

doi: 10.1016/j.neo.2021.04.001

Figure Lengend Snippet: ZCCHC11 is regulated by miR-622 in HCC. (A) Immunohistochemical analysis and correlation of ZCCHC11 and LIN28A expression levels applying tissue micro arrays (staining intensity and percentage of positive cells for both ZCCHC11 and LIN28A were incorporated into a semi-quantitative score describing "0" (very low/no expression), "1" (low/moderate expression) and "2" (strong expression)) ( n = 102). (B) MicroRNA recognition elements (MRE) in the ZCCHC11 3’UTR represent potential miR-622-binding sites and were identified applying the "TargetScan 7.2" database. (C) Quantitative RT-PCR analysis of ZCCHC11 mRNA levels in control-transfected as compared to miR-622-transfected different HCC cell lines (PLC ( n = 2) ; Hep3B ( n = 3)). (D) Representative Western blot images and (densitometric) quantification of ZCCHC11 protein levels in control-transfected as compared to miR-622-transfected different HCC cell lines (PLC ( n = 2) ; Hep3B ( n = 2)). (E) Luciferase ZCCHC11 3′UTR-reporter (containing a conserved miR-622 MRE) activity in control-miR (CTR) as compared with miR-622-mimic (622)-transfected HCC cells (PLC) ( n = 3 ). (F,G) TCGA-derived datasets and the Gene Expression Profiling Interactive Analysis (GEPIA) database were used for overall survival analysis comparing high and low ZCCHC11/miR-622-ratios (F) or high and low miR-622/ZCCHC11-ratios (G), respectively in HCC patients. "HR": Hazard ratio. Data are presented as the mean ± SEM. Statistical significance was determined by two-sided Fisher's exact test together with Spearman correlation analysis (A), and 2-tailed, unpaired t-test (C,D,E). Survival analysis was performed computationally applying log-rank testing and hazard ratio estimates (F,G). * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: The human hepatocellular carcinoma (HCC) cell lines PLC (ATCC CRL-8024), Hep3B (ATCC HB-8064) and HepG2 (ATCC HB-8065) were described in other studies [ , ].

Techniques: Immunohistochemical staining, Expressing, Staining, Binding Assay, Quantitative RT-PCR, Control, Transfection, Western Blot, Luciferase, Activity Assay, Derivative Assay, Gene Expression

Journal: Therapeutic Advances in Musculoskeletal Disease

Article Title: Cyclin-dependent kinase 7 (CDK7) is an emerging prognostic biomarker and therapeutic target in osteosarcoma

doi: 10.1177/1759720X21995069

Figure Lengend Snippet: Expression of CDK7 in osteosarcoma cell lines, osteosarcoma patient fresh tissues, and osteosarcoma tissue microarray (TMA). (a) Expression levels of CDK7 protein in osteosarcoma cell lines (U2OS, KHOS, 143B, Saos-2, MG63, and MNNG/HOS) were higher than the expression of CDK7 in the normal osteoblast cell line (HOB-c, NHOst) as measured by Western blot. (b) Densitometry quantification of the Western blots of CDK7 from (a), presented as relative to tubulin expression. The data represent the mean ± SE of the experiment carried out in triplicate. (c) CDK7 expression in seven human osteosarcoma fresh tissues measured by Western blot. (d) Densitometry quantification of the Western blots of CDK7 from (c), presented as relative to tubulin expression. The data represent the mean ± SE of the experiment carried out in triplicate. (e) Representative images of different immunohistochemistry staining intensities of CDK7 and HE staining in an osteosarcoma TMA are shown in osteosarcoma tissues. Based on the CDK7 staining intensity within the tumor samples, the staining patterns were divided into six groups: no positive staining (0); <10% positive cells (1+); 10–25% positive cells (2+); 26–50% positive cells (3+); 51–75% positive cells (4+); >75% positive cells (5+). (Original magnification, 400×; scale bar, 50 µm). (f) Tumors with the staining score of ⩽2+ were defined as the low CDK7 expression group, ⩾3+ were defined as the high CDK7 expression group. Pie chart representing relative frequency of different CDK7 expression levels in osteosarcoma TMA. CKD7, cyclin-dependent kinase 7.

Article Snippet: The human osteosarcoma cell lines U2OS, Saos-2, MG63, MNNG/HOS, and 143B were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Expressing, Microarray, Western Blot, Immunohistochemistry, Staining

Journal: Therapeutic Advances in Musculoskeletal Disease

Article Title: Cyclin-dependent kinase 7 (CDK7) is an emerging prognostic biomarker and therapeutic target in osteosarcoma

doi: 10.1177/1759720X21995069

Figure Lengend Snippet: Effects of the CDK7 inhibitor BS-181 on the activity of CDK7 and cell growth in osteosarcoma cell lines. BS-181, at the indicated concentrations, inhibited osteosarcoma cell proliferation in (a) cell growth and proliferation of KHOS, and (b) U2OS cell lines, which was determined by MTT assays after treatment with BS-181 for 6 days. The data represent the mean ± SE of two experiments carried out in triplicate. (c) Microscopy images of morphologic changes and a reduction in cell number after 72 h of BS-181 treatment (scale bar, 100 µm). (d) The expression of proteins involved in the CDK7-signaling pathway in the KHOS osteosarcoma cell line was examined by Western blot after 48 h of BS-181 treatment. (e) Semiquantitative analysis of (d) densitometry relative to tubulin. The data represent the mean ± SE of the experiment carried out in triplicate. (f) The expression of proteins involved in the CDK7-signaling pathway in the U2OS osteosarcoma cell line was examined by Western blot after 48 h of BS-181 treatment. (g) Semiquantitative analysis of (f) densitometry relative to tubulin. The data are mean ± SE of the experiment carried out in triplicate. CKD7, cyclin-dependent kinase 7; RNAPII, RNA polymerase II.

Article Snippet: The human osteosarcoma cell lines U2OS, Saos-2, MG63, MNNG/HOS, and 143B were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Activity Assay, Microscopy, Expressing, Western Blot

Journal: Therapeutic Advances in Musculoskeletal Disease

Article Title: Cyclin-dependent kinase 7 (CDK7) is an emerging prognostic biomarker and therapeutic target in osteosarcoma

doi: 10.1177/1759720X21995069

Figure Lengend Snippet: CDK7 inhibition reduced osteosarcoma cell clonogenicity in vitro and decreased the spheroid diameter of osteosarcoma cell lines in a three-dimensional (3D) cell culture. (a) Representative results of colony formation in KHOS and U2OS. The numbers of colonies and their sizes were markedly decreased in cells treated with BS-181. (b, c) Representative images of KHOS and U2OS were measured after 10 µM of BS-181 treatment in a 3D cell culture. Spheroid formation of (b) KHOS and (c) U2OS were significantly smaller than untreated cells at all observation points. Cell fluorescence images of spheroid formation were taken after 12 days of cultivation (scale bar, 100 µm). (d, e) The average relative spheroid diameter of (b) KHOS and (c) U2OS treated with CDK7 inhibitor compared with untreated cells at an observation point of 12 days. The data are mean ± SE of the two experiments carried out in triplicate (** indicates p < 0.001). CKD7, cyclin-dependent kinase 7.

Article Snippet: The human osteosarcoma cell lines U2OS, Saos-2, MG63, MNNG/HOS, and 143B were purchased from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Inhibition, In Vitro, Cell Culture, Fluorescence

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) The Cancer Genome Atlas (TCGA) database analyses demonstrate RBM3 mRNA levels are significantly higher in colon cancer (T) (n=286) than in normal colon (N) (n=41) (p<0.001). (B) RBM3 mRNA expression from a cDNA array of colon tumor (T) samples (n=24) and matched adjacent normal colon tissue (N) (n=24) normalized to β-actin, shows a significant increase of RBM3 expression in tumor tissue (p=0.023). (C) Immunohistochemistry (IHC) of a colon cancer tumor microarray shows that RBM3 is upregulated in colon adenocarcinoma along with lymph node metastasis and liver metastasis as compared to the normal colon, lymph node, and liver. (D) Composite score of colon cancer TMA shows significantly higher expression of RBM3 in tumor (n=28) (p=0.002) and metastasis (n=30) (p=0.036) as compared to normal tissue. RBM3 expression is also increased in the different stages of colon cancer (Stage I (n = 3), Stage II (n = 12) (p=0.015), Stage III (n= 11) (p=0.07), Stage IV (n = 2) and metastasis (n=30) (p=0.036)) as compared to normal colon, liver and lymph node (n=3 each). (E) Western blot analysis of RBM3 protein expression in established colon cancer cell lines HCT116, DLD1, SW480, SW620, HT29, RKO and LST17T as compared to normal colon epithelial cells (FHC cell line). Data in are represented as ± SEM. Also, see Supplementary figure 1

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Expressing, Immunohistochemistry, Microarray, Western Blot

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) Volcano plots for RNA-sequencing (RNA-seq) showing differentially expressed lncRNA in both HCT116 and DLD1 RBM3 overexpressing (RBM3 O/E) cell lines compared to empty vector (EV) cell lines. (B) Venn diagram depicting the number of co-expressed and uniquely expressed lncRNA found in RNA-seq. (C) The volcano plots for RNA-immunoprecipitation coupled sequencing (RNA-IP seq) showing differentially expressed lncRNA in both HCT116 and DLD1, RBM3 overexpressing cell lines compared to empty vector cell lines. (D) Venn diagram depicting the number of co-expressed and uniquely expressed lncRNA found in RNA-IP seq. (E-F) Plot from REVIGO software showing the Gene Ontology (GO) terms enriched in lncRNA (RNA-seq) for HCT116 RBM3 (E). The graph has been modified to highlight the GO terms and color key. The box plot drawn highlighting enriched GO terms(F). (G) RT-PCR validation of lncRNA identified through RNA-seq in colon cancer cell lines compared to normal FHC cells. (H) RT-PCR validation of lncRNA identified through RNA-seq, in the RBM3 overexpressing (RKO, HCT116, and DLD1) cells, show increased expression of lnc-HOTAIR, lnc-TUG1, lnc-Flii- 1, lnc-LSAMP-3. (I) Western blot analysis for the expression demonstrating levels of RBM3 in the distal colon in representative wild type littermates and RBM3 transgenic mice (RBM3 O/E). (J) RT-PCR analysis demonstrating increased expression of lncRNA lnc-Flii-1 and lnc-LSAMP- 3 compared to GAPDH in representative wild type (WT) littermates and RBM3 overexpressing transgenic mice (RBM3 O/E). Data in are represented as ± SEM. Also, see Supplementary figure 2.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: RNA Sequencing, Plasmid Preparation, RNA Immunoprecipitation, Sequencing, Software, Modification, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Expressing, Western Blot, Transgenic Assay

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A-D) Quantitative PCR shows increased mRNA levels of VEGFA, ZEB1, TWIST, and SNAI2 in RBM3 overexpressing cells compared to empty vector cell lines. (E) Plot for tube length of the endothelial tubular network formed by HUVEC cell in (G). Increased tube formation in HUVEC cells treated by condition media from HCT116 (p=0.038) and DLD1 (p=0.004) RBM3 overexpressing compared to empty vector cells (F) Plot showing increase in migration of HCT116 (p=0.001), DLD1 (p=0.02) and RKO (p=0.04) RBM3 overexpressing cells compared to empty vector performed by wound healing assay. (G) Plot showing increase in migration of HCT116 (p=0.006), DLD1 (p=0.002) and RKO (p=0.006) RBM3 overexpressing cells compared to empty vector performed by transwell migration assay. (H) Plot showing increase in invasion of HCT116 (p<0.001), DLD1 (p<0.01) and RKO (p<0.01) RBM3 overexpressing cells compared to empty vector performed by transwell invasion assay. Data in are represented as ± SEM. Also, see Supplementary figure 3.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Migration, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A-D) The plot of tumor weights (A, C) and tumor volumes (B, D) from HCT116 and DLD1 empty vector and RBM3 overexpressing xenografts. The tumor volume and weight were significantly increased in RBM3 overexpression compared to control (p<0.05) (E) RT-PCR validation of lncRNA in the tumor xenograft tissues, in both HCT116 and DLD1 xenografts. RBM3 overexpressing xenografts show an increase in lnc-Flii-1 and lnc-LSAMP-3 expression compared to empty vector. (F) RT-PCR validation for increased expression of TWIST1, SNA2 and VEGFA in HCT116 and DLD1 empty vector and RBM3 overexpressing xenograft tumors tissues. (G) Western blot analysis of tumors from HCT116 and DLD1 xenograft tissues for VEGF expression. (H) Increassed RBM3 and PCNA levels in HCT116 and DLD1 RBM3 overexpressing xenograft tissues as compared to empty vector as seen by immunohistochemistry analysis. (I) Increase in percentage of PCNA positive nuclei in RBM3 overexpressing HCT116 (p=0.022) and DLD1 (p=0.025) xenograft tissues immunohistochemistry. The data for are presented as the means ± SEM, (n=10). Also, see Supplementary figure S5.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Plasmid Preparation, Over Expression, Control, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Expressing, Western Blot, Immunohistochemistry

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) Protein expression of RBM3 decreases in shRNA knockdown clones (2,3,5) compared to scramble (scr1,scr2) in the HCT116 cell line by Western blot analysis. (B) RT-PCR validation for the decreased expression of lnc-LSAMP-3 and lnc-Flii-1 in HCT116 RBM3 shRNA knocked down clone compared to scramble. (C) Plot for decrease in percentage proliferation of the RBM3 shRNA knockdown clones over empty vector for sh2 (proliferation= 55.06%, p=0.04), sh3 (proliferation= 53.8%, p=0.0023), sh5 (proliferation= 47.45%, p=0.0001). (D) The plot showing decrease in percent migration in HCT116 shRNA knocked down clones (sh2 p=0.044 and sh5 p=0.048) compared to scramble performed by Transwell migration. (E) The plot showing decrease in percent invasion in HCT116 shRNA knocked down clones (sh2 p=<0.001 and sh5 p=0.001) compared to scramble performed by Transwell invasion assay. (F and G) The plot of tumor weights (F) and tumor volumes (G) from HCT116 scramble (scr2) and RBM3 shRNA knockdown (shRNA-2) xenografts. Both tumor volume and weight were significantly reduced in RBM3 knockdown compared to scramble control (p<0.05). (H) Kaplan-Meier analysis showing increased percent survival after the AOM/DSS induced carcinogenesis in cre inducible RBM3 knockout (KO) compared to wild type (WT) mice. (I) Plot showing decreased intestinal permeability in RBM3 knockout mice as compared to the wild type littermates (p=0.04). (J) The plot showing decreased number of tumors in the distal colon observed after the AOM/DSS induced carcinogenesis in cre inducible RBM3 knockout compared to wild type mice (p=0.007). (K) Representative image of H and E staining of the distal colon with tumors after the AOM/DSS induced carcinogenesis in RBM3 knockout and wild type mice. (L) Decrease in expression of lnc-LSAMP-3 and lnc-Flii-1 seen by PCR analysis in RBM3 knockout mice as compared to the wild type mice. The data for are presented as the means ± SEM, (mice n=10). Also, see Supplementary figure 6.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Expressing, shRNA, Knockdown, Clone Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Plasmid Preparation, Migration, Transwell Invasion Assay, Control, Knock-Out, Permeability, Staining

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A and B) RT-PCR validation of lnc-LSAMP-3 and lnc-Flii-1 knocked down in HCT116 (A) and DLD1 (B) RBM3 O/E and empty vector cells. (C and D) RT-PCR validation of VEGFA, ZEB1 and TWIST in HCT116 (C) and DLD1 (D) RBM3 O/E and empty vector cells, after lncRNA (lnc-Flii-1 and lnc-LSAMP-3) knockdown. (E) Plot showing decreased percent migration of HCT116 empty vector and RBM3 overexpressing cells having knockdown of lncRNA lnc-LSAMP-3 (Empty vector p=0.017, RBM3 O/E p=0.031) and lnc-Flii-1(Empty vector p=ns, RBM3 O/E p=0.011) performed by scratch plate assay. (F) Plot showing decreased tube length of the endothelial tubular network formed by HUVEC cell treated by condition media from knockdown of lncRNA lnc-LSAMP-3 and lnc-Flii-1(Empty vector p=0.012, RBM3 O/E p=0.022) in HCT116 cells. (G-J)The plot showing decrease in tumor weight (G, I) and tumor volumes (H, J) from HCT116 and DLD1 xenografts treated with intratumoral injection of si+ LNA gapmer for lnc-Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control (p< 0.05). (K and L) RT-PCR validation for the decreased expression of lncRNA (lnc-LSAMP-3 and lnc- Flii-1) in the tumor xenograft of HCT116 (K) and DLD1(L) treated with intratumoral injection of si+ LNA gapmer for lnc-Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control. (M and N) Western blot showing decreased protein expression of SNAI2 and VEGF in HCT116 (M) and DLD1 (N) tumor xenograft treated with intratumoral injection of si+ LNA gapmer for lnc- Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control. The data for are presented as the means ± SEM. Also, see figure Supplementary figure

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Plasmid Preparation, Knockdown, Migration, Injection, Control, Expressing, Western Blot

Journal: Nucleic Acids Research

Article Title: A transcriptomic and proteomic map of primary human cell types

doi: 10.1093/nar/gkaf1498

Figure Lengend Snippet: Cell type specific genes demonstrated at mRNA level. (A) Heat map of genes with cell type specific expression. (B) Heat map of selected genes with endothelial or epithelial cell type specific expression. (C) Immunohistochemistry staining of protein C1orf116 across human tissues showing its epithelial cell type specific expression. The panel shows a magnified view (20×) from tissue microarrays.

Article Snippet: The Human Protein Atlas (HPA) project provided a tissue-based map of the human proteome through transcriptomics and tissue microarray-based immunohistochemistry analysis [ , ].

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Nature Communications

Article Title: The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer

doi: 10.1038/s41467-021-22101-7

Figure Lengend Snippet: a Analysis of somatic alterations of AAMDC using cancer genomic data sets and tools available from cBioPortal (see “Methods”). The frequency of amplification is shown as a percentage and the sample numbers are shown in brackets. METABRIC Molecular Taxonomy of Breast Cancer International Consortium, TCGA The Cancer Genome Atlas, BRCA Breast Cancer, INSERM Institut national de la santé et de la recherche médicale, MBC Metastatic Breast Cancer, NSCLC non-small-cell lung carcinoma, FHCRC Fred Hutchinson Cancer Research Center, NEPC National Environment Protection Council, PanCan Pan-Cancer. b Kaplan–Meier survival plots for patients with tumors expressing high (red) or low (green) levels of AAMDC mRNA. The lower left plots correspond to luminal B tumors treated with tamoxifen (see “Methods”). The p value shown for each plot is determined by the log-rank test. GEO Gene Expression Omnibus, GSE genomic spatial event, NSCLC non-small-cell lung carcinoma. c Localization of the AAMDC protein in tumors from a breast tissue microarray (TMA) assessed by immunohistochemistry (IHC). Representative IHC sections of normal breast tissue, estrogen receptor-negative (ER − ) tumor tissue, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) are shown. 0, 1+, 2+, 3+ indicate the staining intensity score. d Associations between AAMDC expression (IHC) and lymph node metastasis (LN + ) as well as tumor grade, tumor size (T3-4), and ER positivity (ER + ) by AAMDC localization from the same TMA. Statistical significance is indicated by Chi-square analysis with a one-tailed p -value relative to ER − tissue. For T3-4: * p = 0.03; for LN + : * p = 0.03; for ER + , from left to right: * p = 0.003, * p = 0.005, * p = 0.005. n = 60 biologically independent samples. Full details of the TMA are provided in Supplementary Table . e Frequency of AAMDC amplification/polysomy in a cohort of 119 luminal B breast cancer specimens. Representative fluorescence in situ hybridization (FISH) images are indicated, with specific probes for AAMDC (red) and Centromere enumeration 11 probe for chromosome 11 ( C11 , green). The full clinical and pathological features of these tumors are shown in Supplementary Data . f Real-time expression analyses (qRT-PCR) of AAMDC in luminal, non-luminal, and normal-like breast cells. Significance levels are determined relative to MCF-12A by Ordinary one-way ANOVA with Dunnett multiple comparison test. Data are presented as mean values ± SEM (* p = 0.0217, ** p = 0.0018, **** p < 0.0001). n = 3 biologically independent RNA extractions. Representative images of immunocytochemistry (ICC) and FISH of selected luminal cell lines are presented. HuMECs non-transformed human mammary epithelial cells.

Article Snippet: For experiments involving non-genetically manipulated human mammary epithelial cells (HuMECs) (Fig. ), human primary mammary epithelial cells were purchased from ATCC (HMEC, PCS-600-010).

Techniques: Amplification, Expressing, Gene Expression, Microarray, Immunohistochemistry, In Situ, Staining, One-tailed Test, Fluorescence, In Situ Hybridization, Quantitative RT-PCR, Comparison, Immunocytochemistry, Transformation Assay

Journal: Frontiers in Oncology

Article Title: lncRNA MIR22HG-Derived miR-22-5p Enhances the Radiosensitivity of Hepatocellular Carcinoma by Increasing Histone Acetylation Through the Inhibition of HDAC2 Activity

doi: 10.3389/fonc.2021.572585

Figure Lengend Snippet: (A) Microarray results indicating that MIR22HG expression in MHCC97H cells increased significantly after irradiation. (B) Kaplan-Meier curves of the overall survival. The P -value was computed using the log-rank test. (C) mRNA expression of MIR22HG and miR-22-5p in tumor (n = 16) and non-tumor (n = 16) tissue. (D) miR-22-5p expression in blood samples of healthy individuals and patients with HCC before and after radiotherapy. (E, F) mRNA expression of MIR22HG and miR-22-5p in HCC cells Huh7, HepG2, Hep3B, BEL-7402, MHCC97H, and MHCC97L and in the human normal liver cell L02. *Compared with the L02 group. Mean ± SD (n = 3 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The hepatoma cell lines, Huh7, HepG2, Hep3B, BEL-7402, MHCC97H, and MHCC97L as well as human normal liver cell line L02 were purchased from the American Type Culture Collection (ATCC).

Techniques: Microarray, Expressing, Irradiation

Journal: Frontiers in Oncology

Article Title: lncRNA MIR22HG-Derived miR-22-5p Enhances the Radiosensitivity of Hepatocellular Carcinoma by Increasing Histone Acetylation Through the Inhibition of HDAC2 Activity

doi: 10.3389/fonc.2021.572585

Figure Lengend Snippet: (A) Microarray results indicating that HDAC2 expression in MHCC97H cells decreased significantly after irradiation. (B) HDAC1, HDAC2, and HDAC3 activity in tumor (n = 16) and non-tumor (n = 16) tissues. (C) The activity of HDAC2 in the HCC cells Huh7, HepG2, Hep3B, BEL-7402, MHCC97H, and MHCC97L and the human normal liver cells L02. *Compared with the L02 group. (D) The activity of HDAC2 in HepG2 and MHCC97H cells treated with different doses of irradiation. * Compared with the HepG2 and MHCC97H groups. (E, F) The mRNA expression of MIR22HG and miR-22-5p in HepG2 and MHCC97H cells treated with different doses of irradiation (1, 2, 4, and 6 Gy). *Compared with HepG2 and MHCC97H groups. (G) Western blot analysis of histone H3K4ac, H3K9ac, H3K23ac, H3k27ac, and H3 in HepG2 and MHCC97H cells. The treatments were irradiation of 4 Gy alone, irradiation of 4 Gy + histone acetyltransferase inhibitor C646, irradiation of 4 Gy + histone deacetylation inhibitor Santacruzamate A. (H, I) Expression of MIR22HG and miR-22-5p in HepG2 and MHCC97H cells. The treatments were irradiation 4 Gy alone, irradiation of 4 Gy + histone acetyltransferase inhibitor C646, irradiation of 4Gy + histone deacetylation inhibitor Santacruzamate A. * Compared with the NC group. (J) ChIP-PCR showing the amplified signal of the MIR22HG promoter region. The treatments were irradiation of 4 Gy alone, irradiation of 4 Gy + histone acetyltransferase inhibitor C646, and irradiation of 4 Gy + histone deacetylation inhibitor Santacruzamate A. Results showed that both irradiation and drug intervention in HDAC2 can alter amplified signals of the MIR22HG promoter region. Mean ± SD (n = 3 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. San A, Santacruzamate A.

Article Snippet: The hepatoma cell lines, Huh7, HepG2, Hep3B, BEL-7402, MHCC97H, and MHCC97L as well as human normal liver cell line L02 were purchased from the American Type Culture Collection (ATCC).

Techniques: Microarray, Expressing, Irradiation, Activity Assay, Western Blot, Amplification

Journal: Frontiers in Oncology

Article Title: lncRNA MIR22HG-Derived miR-22-5p Enhances the Radiosensitivity of Hepatocellular Carcinoma by Increasing Histone Acetylation Through the Inhibition of HDAC2 Activity

doi: 10.3389/fonc.2021.572585

Figure Lengend Snippet: (A) Human miR-22 is located in exon 2 of MIR22HG . miR-22-5p is conserved between humans, mice, rats, rabbits, dogs, and elephants. (B) The miR-22-5p expression level was detected in MIR22HG-overexpressing HepG2 and MHCC97H cells using qRT-PCR. (C) Correlation of MIR22HG and miR-22-5p expression in TCGA cohorts. (D) Wound healing assay showing the migration of HepG2 and MHCC97H cells with different treatments. *Compared with the HepG2 and MHCC97H groups. (E) Colony formation of HepG2 and MHCC97H cells with different treatments. *Compared with the HepG2 and MHCC97H groups. (F) MTT assay showing the cell proliferation of HepG2 and MHCC97H cells with different treatments. Mean ± SD (n = 3 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The hepatoma cell lines, Huh7, HepG2, Hep3B, BEL-7402, MHCC97H, and MHCC97L as well as human normal liver cell line L02 were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Quantitative RT-PCR, Wound Healing Assay, Migration, MTT Assay

Journal: Frontiers in Oncology

Article Title: lncRNA MIR22HG-Derived miR-22-5p Enhances the Radiosensitivity of Hepatocellular Carcinoma by Increasing Histone Acetylation Through the Inhibition of HDAC2 Activity

doi: 10.3389/fonc.2021.572585

Figure Lengend Snippet: (A) MTT assay showing the cell proliferation of HepG2 and MHCC97H cells with different treatments. *Compared with HepG2 + 4 Gy and MHCC97H + 4 Gy groups. (B) Wound healing assay showing the migration of HepG2 and MHCC97H cells with different treatments. *Compared with HepG2 + 4 Gy and MHCC97H + 4 Gy groups. (C) Colony formation of HepG2 and MHCC97H cells with different treatments. *Compared with HepG2 + 4 Gy and MHCC97H + 4 Gy groups. (D) EdU assay showing the cell proliferation of HepG2 and MHCC97H cells with different treatments. Mean ± SD (n = 3 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. San A, Santacruzamate A; KO MIR22HG, knockout MIR22HG.

Article Snippet: The hepatoma cell lines, Huh7, HepG2, Hep3B, BEL-7402, MHCC97H, and MHCC97L as well as human normal liver cell line L02 were purchased from the American Type Culture Collection (ATCC).

Techniques: MTT Assay, Wound Healing Assay, Migration, EdU Assay, Knock-Out

Journal: Frontiers in Oncology

Article Title: lncRNA MIR22HG-Derived miR-22-5p Enhances the Radiosensitivity of Hepatocellular Carcinoma by Increasing Histone Acetylation Through the Inhibition of HDAC2 Activity

doi: 10.3389/fonc.2021.572585

Figure Lengend Snippet: (A, B) Tumor tissues isolated from the indicated mice on day 25 post-transplant. Tumor growth curve in nude mice. After tumor cells were injected subcutaneously into the right flank of the abdomen of the nude mice, the short and long diameters of the tumors were measured every 3 days and tumor volumes (mm 3 ) were calculated. (C, D) qPCR of MIR22HG and miR-22-5p mRNA expression in HepG2 and MHCC97H xenografts. (E) Immunohistochemistry showing the Ki67 expression of HepG2 and MHCC97H xenografts. Mean ± SD (n = 3 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The hepatoma cell lines, Huh7, HepG2, Hep3B, BEL-7402, MHCC97H, and MHCC97L as well as human normal liver cell line L02 were purchased from the American Type Culture Collection (ATCC).

Techniques: Isolation, Injection, Expressing, Immunohistochemistry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: SULF2 secreted by HCC cells induced the transformation from HSCs to CAFs. (A) Both RT-PCR and western immunoblotting assays showed that there was more expression of α-SMA, FAP, and POSTN in LX2 cells co-cultured with Hep3B SULF2 cells (LX2 SULF2 cells) than those co-cultured with Hep3B Vector cells (the protein was extracted from total cells). (B) LX2 cells co-cultured with Huh7 Scr shRNA cells expressed more α-SMA, FAP, and POSTN than those with Huh7 SULF2 shRNA cells. Cell fractions: LX2 cells (the protein was extracted from total cells). SULF2, sulfatase 2; HCC, hepatocellular carcinoma; CAF, carcinoma-associated fibroblast.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: Transformation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Cell Culture, Plasmid Preparation, shRNA

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: SULF2 activated HSCs to CAFs via mediating TGFβ1/SMAD3 signaling. (A) LX2 cells co-cultured with Hep3B SULF2 cells (LX2 SULF2 cells) were found to express more expression of both TGFβ1 and p-SMAD3 than those with Hep3B Vector cells (LX2 Vector cells) by western immunoblotting and RT-PCR. Cell fractions: LX2 cells (the protein was extracted from total cells). (B) Microarray profiling of mRNA expression showed that there was a significantly higher mRNA expression of TGFβ1 signaling downstream genes including BMP7, MAPK9, JUN, PMEPA1, MAPK12, SERPINE1, MAPK13, RUNX2, and RUNX3 in LX2 SULF2 cells than LX2 Vector cells. (C) Immunofluorescence staining displayed that more TGFβ1 protein was bound in the cell membrane of LX2 SULF2 cells than LX2 Vector cells. (D) Both luciferase reporter and ChIP assays confirmed that p-SMAD3 was bound with the α-SMA promoter directly in LX2 cells. (E) ChIP assay showed that p-SMAD3 protein was able to bind with the promoter of POSTN in LX2 cells treated with TGFβ1. SULF2, sulfatase 2; HCC, hepatocellular carcinoma; CAF, carcinoma-associated fibroblast; ChIP, chromatin immunoprecipitation.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: Cell Culture, Expressing, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Microarray, Immunofluorescence, Staining, Membrane, Luciferase, Chromatin Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: CAFs activated SDF-1/CXCR4/PI3K/AKT pathway and consequently inhibited HCC cell apoptosis. (A) Both RT-PCR and western immunoblotting assays showed that LX2 SULF2 cells expressed significantly more SDF-1 than LX2 Vector cells. Cell fractions: LX2 cells. (B) It was found by ELISA assay that there was more SDF-1 protein in conditioned medium from LX2 SULF2 cells than that from LX2 Vector cells (the protein was extracted from total cells). (C) Microarray profiling of mRNA expression showed that most downstream genes of SDF-1/CXCR4 signaling were remarkably upregulated in Hep3B CAFs cells in contrast to Hep3B Vector cells. (D) Co-culture with CAFs was found by western immunoblotting to increase expression of SDF-1, CXCR4, p-PI3K, and p-AKT in Hep3B cells, while treatment of CXCR4 inhibitor (AMD3100) did not alter the expression of phosphorylation of PI3K, AKT, BAD, caspase 9, and FKHRL 1 in Hep3B cells. Cell fractions: Hep 3B cells (the protein was extracted from total cells). (E) By both DAPI staining and caspase 3/7 activity assay, it was found that co-culture with CAFs suppressed cell apoptosis, whereas AMD3100 treatment did not influence Hep3B cell apoptosis apparently. CAF, carcinoma-associated fibroblast; HCC, hepatocellular carcinoma; SULF2, sulfatase 2.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Microarray, Expressing, Co-Culture Assay, Phospho-proteomics, Staining, Activity Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: CAFs driven by SULF2 induced HCC EMT phenotype via upregulating SNAI1 by activating SDF-1/CXCR4 signaling. (A) By western immunoblotting assay, it was found that Hep3B LX2 SULF2 cells had significantly more expression of SNAI1, N-cadherin, and Vimentin and less E-cadherin expression than Hep3B LX2 Vector cells, while co-culture with LX2 SULF2 cells did not alter the expression of E-cadherin, SNAI1, N-cadherin, and Vimentin after treatment of AMD3100. Cell fractions: Hep 3B cells (the protein was extracted from total cells). (B) As assessed by wound healing assay, the migration ability of Hep3B cells was found to be accelerated by co-culture with LX2 SULF2 cells, which was weakened by treatment of AMD3100. (C) Transwell invasion assay showed that co-culture with LX2 SULF2 cells consolidated the invasion capacity of Hep3B cells and AMD3100 treatment attenuated the regulatory effect of co-culture with LX2 SULF2 cells on the Hep3B cell invasion ability. CAF, carcinoma-associated fibroblast; SULF2, sulfatase 2; HCC, hepatocellular carcinoma; EMT, epithelial-to-mesenchymal transition.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: Western Blot, Expressing, Plasmid Preparation, Co-Culture Assay, Wound Healing Assay, Migration, Transwell Invasion Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: SDF-1/CXCR4 axis inhibited miR-153-3p expression and then induced EMT of HCC cells via upregulating SNAI1. (A) The data from TargetScan 7.2 database showed the positions 440–447 of SNAI1 3′-UTR had the complementary sequence of miR-153-3p. (B) As assessed by qRT-PCR, it was found that Hep3B LX2 SULF2 cells had significantly less miR-153-3p in contrast to Hep3B LX2 Vector cells. (C) Western immunoblotting showed that enhanced expression of miR-153-3p in SNU398 cells decreased expression of SNAI1, N-cadherin, and Vimentin and upregulated E-cadherin magnificently. Cell fractions: SNU398 cells (the protein was extracted from total cells). (D) Knockdown of miR-153-3p with miRNA inhibitors in Hep3B cells resulted in upregulation of SNAI1, N-cadherin, and Vimentin, while it led to loss of E-cadherin. Cell fractions: Hep 3B cells (the protein was extracted from total cells). (E) Luciferase reporter assay demonstrated that miR-153-3p overexpression by mimics treatment markedly decreased the luciferase activity of plasmid containing wt 3′-UTR of SNAI1 rather than mt 3′-UTR of SNAI1 in SNU398 cells. EMT, epithelial-to-mesenchymal transition; HCC, hepatocellular carcinoma; SULF2, sulfatase 2.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: Expressing, Sequencing, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Knockdown, Luciferase, Reporter Assay, Over Expression, Activity Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

doi: 10.3389/fcell.2021.631931

Figure Lengend Snippet: OIP5-AS1 accelerated the growth of HCC in vivo and induced EMT phenotype of HCC cells via upregulating SNAI1 expression. (A) By measuring tumor growth curves and tumor weight, it was found that knockdown of OIP5-AS1 attenuated the growth of HCC xenografts. (B) qRT-PCR analysis showed that SNU398 OIP5-AS1 cells had significantly less OIP5-AS1 expression than SNU398 Scr cells. (C) SNU398 OIP5-AS1 cells were found by qRT-PCR assay to express more miR-153-3p expression than SNU398 Scr cells. (D) IHC staining was carried out in HCC xenograft tissues, and it was found that there was magnificently lower level of both SNAI1 and Vimentin, and more E-cadherin is detected in SNU398 OIP5-AS1 cells than SNU398 Scr cells. HCC, hepatocellular carcinoma; EMT, epithelial-to-mesenchymal transition; IHC, immunohistochemical.

Article Snippet: The human HCC cell lines (Hep3B, PLC/PRF/5, and SNU398), non-HCC cell line (LO2), and the HSC cell line (LX2 cells) were purchased from American Type Culture Collection (Manassas, VA, United States).

Techniques: In Vivo, Expressing, Knockdown, Quantitative RT-PCR, Immunohistochemistry, Immunohistochemical staining