Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 1. TGF-β expression in osteosarcoma specimens and cell lines. (a) Immunohistochemical staining was used to analyze TGF-β expression in both osteosarcoma specimens and normal adjacent tissues. (b) Survival analysis was performed using the Kaplan-Meier method. Data are presented as mean ± SEM of three independent experiments. (c) TGF-β protein levels in osteosarcoma cell lines (*P < 0.05). (d) TGF-β mRNA expression in osteosarcoma cell lines (*P < 0.05).

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 2. Small interfering (si) RNA targeting of TGF-β (si-TGF-β) suppresses proliferation and promotes apoptosis in osteosarcoma. (a) RT-qPCR was employed to assess the knockdown efficiency of si-TGF-β, *P < 0.05 vs. the si-control group. (b) Western blotting was used to measure the knockdown efficiency of si-TGF-β, *P < 0.05 vs. the si-control group. (c)The MTT assay was used to assess the viability of U2OS and MG-63 cells transfected with si-control or si-TGF-β, *P < 0.05 vs. the si-control group. (d and e) The colony- forming ability of U2OS and MG-63 cells transfected with si-control or si-TGF -β, *P < 0.05 vs. the si-control group. (f and g) Flow cytometry analysis of the cell cycle phase distribution of U2OS and MG-63 cells transfected with si-control or si-TGF – β, *P < 0.05 vs. the si-control group. (h and i) The rate of apoptosis in U2OS and MG-63 cells transfected with si-TGF-β, *P < 0.05 vs. the si-control group. (j and k) TGF-β knockdown increases the expression of cleaved PARP, caspase-3, and BAX, *P < 0.05 vs. the si-control group.

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Quantitative RT-PCR, Knockdown, Control, Western Blot, MTT Assay, Transfection, Flow Cytometry, Expressing

Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 4. TGF-β enhances osteosarcoma cell stemness and increases the proportion of CD133+ cells. (a and b) Spheroid formation assay for U2OS and MG-63 cells transfected with si-TGF-β. Representative images (left panel) and statistical measurements (right panel). Representative micrographs of formed spheres were analyzed in cells treated with si-TGF-β or si-control. Scale bar, 100 μm. *P < 0.05 vs. the si-control group. (c and d) The percentage of CD133+ cells in U2OS- and MG-63-derived spheroids was analyzed by flow cytometry.*P < 0.05 vs. the si-control group. (e and f) Stem cell-associated protein expression was measured by western blot. *P < 0.05 vs. the si-control group.

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Tube Formation Assay, Transfection, Control, Derivative Assay, Flow Cytometry, Expressing, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: TGF-β is associated with poor prognosis and promotes osteosarcoma progression via PI3K/Akt pathway activation.

doi: 10.1080/15384101.2020.1805552

Figure Lengend Snippet: Figure 5. TGF-β regulates the PI3K/mTOR signaling pathway in osteosarcoma cells. (a) p-PI3K and p-Akt expression was measured by western blot, P < 0.05 vs. the si-control group. (b and c) The colony-forming ability of si-TGF-β-transfected U2OS and MG-63 cells with or without LY294002 treatment. P < 0.05 vs. the si-control group. (d and e) Transwell assays were used to assess the invasive ability of U2OS and MG-63 cells, P < 0.05 vs. the si-control group. (f and g) A wound-healing assay was performed to measure the migratory ability of U2OS and MG-63 cells, P < 0.05 vs. the si-control group. (h and i) Spheroid formation assay for U2OS/MG-63 cells transfected with si-TGF-β. Representative images (left panel) and statistical measurement (right panel). Representative micrographs of formed spheres were analyzed in cells treated with si-TGF-β or si-control with or without LY294002 treatment. Scale bar, 100 μm. *P < 0.05 vs. the si-control group. (j and k) The percentage of CD133+ cells in U2OS cell- and MG-63 cell-derived spheroids was analyzed by flow cytometry. *P < 0.05 vs. the si-control group.

Article Snippet: Osteosarcoma spheroids were cultured in a specialized growth medium (Celprogen Inc, Torrance, CA, USA) containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 20 ng/ml human platelet growth factor (Sigma-Aldrich), 100 ng/ml epidermal growth factor (Invitrogen), and 1% antimycotic (Invitrogen).

Techniques: Expressing, Western Blot, Control, Transfection, Wound Healing Assay, Tube Formation Assay, Derivative Assay, Flow Cytometry

Journal: Cell reports

Article Title: Uncovering genetic interactions in the DNA repair network in response to endogenous damage and ionizing radiation

doi: 10.1016/j.celrep.2025.116850

Figure Lengend Snippet: (A) Distribution of the observed phenotypes depleted in the basal screen results, indicating where the MRE11A-UBR5 gene pair falls relative to other depleted interactions. (B) Ranked observed phenotypes from the basal screen for both UBR5 and MRE11A . Blue/red regions highlight significantly enriched/depleted UBR5 -gene and MRE11A -gene interactions, respectively. Blue and red regions highlight gene combinations that are enriched above (blue) or depleted below (red) one standard deviation (+0.6981 and −0.7791) of the mean of all basal interactions and possess a Fisher-combined p < 0.05. (C) Time-lapse images of growth from Dox-induced dual-guide vectors with NT-1 x UBR5 -g2 or NT-1 x NT-2 in nuclear-EGFP-expressing cells co-cultured with nuclear-RFP-expressing cells grown in DMSO or 6.25 μM mirin (+mirin). Images are from one well per condition over the first 72 h of growth. Scale bar: 100 μM. (D) Measurements are calculated as the log 2 fold change (log 2 FC) of Dox induced over uninduced growth for the labeled condition, relative to the growth of co-cultured RFP+ cells, as seen in (C). Data represent mean ± SEM of 3 biological repliactes. The gray bar indicates the 72 h of growth shown in (C), though imaging continued. (E) Comparative growth differences at 96 h of two separate, inducible UBR5 guides from experiments performed similarly to those shown in (C) and (D). Bars are only shown for Dox-treated conditions but are normalized to uninduced, DMSO-treated control wells for each genotype and replicate. Mirin: 5 μM. Blue prediction bars are calculated from an additive model assuming no interaction, with the average effect of mirin on NT cells plus the average effect of UBR5 depletion for each guide and replicate. Data represent mean ± SEM of 2 biological replicates, and statistics represent a two-way ANOVA with Dunnett multiple comparisons test; p = 0.0277. (F) Western blot depicting robust UBR5 KO in two selected HT1080-6TG clones. (G) Dose curve of HT1080-6TG clones shown in (F) grown in increasing doses of mirin in a colony formation assay. IC50 was calculated from a sigmoidal interpolation of all datasets. Error bars: ± SEM from three biological replicates. Statistics match in color the clone they reference, relative to NT cells. Statistics: a two-way ANOVA with Dunnett’s multiple comparisons test between NT-treated cells and UBR5 -KO clones at each concentration; * p < 0.01 and **** p < 0.0001. (H) Western blot showing UBR5 depletion in U2-OS cells electroporated with either an sgNT-RNP or an sg UBR5 -RNP. (I) Log 2 of the relative confluence over time (relative to DMSO) for sgNT-RNP- or sg UBR5 -RNP-treated U2-OS cells from (H), grown with 25 μM mirin over 120 h. The shaded area around each solid line indicates the SEM from three biological replicates. Statistics: a two-way ANOVA at 120 h with Šídák’s multiple comparisons test; p = 0.0317. See also .

Article Snippet: U2-OS , ATCC , HTB-96.

Techniques: Standard Deviation, Expressing, Cell Culture, Labeling, Imaging, Control, Western Blot, Clone Assay, Colony Assay, Concentration Assay

Journal: BMC Genomics

Article Title: miR-486-5p expression is regulated by DNA methylation in osteosarcoma

doi: 10.1186/s12864-022-08346-6

Figure Lengend Snippet: miR-486-5p expression and methylation in osteosarcoma cell lines and patient samples. A . miR-486-5p expression level in normal bone ( n = 6), cell lines ( n = 17) and patient samples ( n = 9) using qRT-PCR. The expression level was quantified for the groups of samples. The values are shown relative to mean expression of bone. B . DNA methylation and miRNA expression levels in normal bone samples ( n = 4) and osteosarcoma cell lines ( n = 19) using arrays. The expression level of miRNAs (log2) and DNA methylation level (Beta, probe cg00176210) were obtained using Agilent miRNA array v2 and Illumina Infinium Methylation27 BeadChip technology, respectively. Values are given as mean (SD) with whiskers from min to max. P -values * < 0.01 and ** < 0.0001

Article Snippet: A panel of human osteosarcoma cell lines ( n = 21) composed of 143B, CAL-72, G-292, HAL, HOS, IOR/MOS, IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS18, IOR/SARG, KPD, MG-63, MHM, MNNG/HOS, OHS, OSA, Saos-2, U-2 OS and ZK-58 were obtained from ATCC ( www.lgcstandards-atcc.org ), DSMZ ( http://www.dsmz.de/ ) or research partners in the EU funded EuroBoNeT project [ ].

Techniques: Expressing, Methylation, Quantitative RT-PCR, DNA Methylation Assay

Journal: BMC Genomics

Article Title: miR-486-5p expression is regulated by DNA methylation in osteosarcoma

doi: 10.1186/s12864-022-08346-6

Figure Lengend Snippet: Expression and correlation of miR-486-5p and hsa-mir-486-1 (pri-mir486) in osteosarcoma cell lines upon 5-Aza-2′-deoxycytidine treatment. Relative expression level A. miR-485-5p and B. pri-mir486 after 72 h of 5-Aza treatment . The values are shown relative to treated cell lines (set to 1, horizontal line). Induction of > 30% shown as dotted line. Values are given as mean (SD). Correlation between expresion level of miR-485-5p and pri-mir486 in C. untreated and D. 5-Aza treated cell lines. The expression levels are quantified using qRT-PCR, and normalized against RNU44 for miR-486-5p and GAPDH for pri-mir486

Article Snippet: A panel of human osteosarcoma cell lines ( n = 21) composed of 143B, CAL-72, G-292, HAL, HOS, IOR/MOS, IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS18, IOR/SARG, KPD, MG-63, MHM, MNNG/HOS, OHS, OSA, Saos-2, U-2 OS and ZK-58 were obtained from ATCC ( www.lgcstandards-atcc.org ), DSMZ ( http://www.dsmz.de/ ) or research partners in the EU funded EuroBoNeT project [ ].

Techniques: Expressing, Quantitative RT-PCR

Journal: BMC Genomics

Article Title: miR-486-5p expression is regulated by DNA methylation in osteosarcoma

doi: 10.1186/s12864-022-08346-6

Figure Lengend Snippet: Expression and correlation of miR-486-5p and ANK1 before and after 5-Aza-2′-deoxycytidine treatment in osteosarcoma cell lines. A . Quantification of expression of miR-486-5p and ANK1 versions 1–4 in untreated cells. B . The induction of miR-486-5p and ANK1 versions 1–4 after 5-Aza treatment. The induction fold change is calculated as the ratio between expression of untreated and 5-Aza treated transcripts as quantified by qRT-PCR. The expression is normalized against RNU44 for miR-486-5p and GAPDH for ANK1 . Pearson’s Correlation r is calculated with and without the outlier sample MG-63

Article Snippet: A panel of human osteosarcoma cell lines ( n = 21) composed of 143B, CAL-72, G-292, HAL, HOS, IOR/MOS, IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS18, IOR/SARG, KPD, MG-63, MHM, MNNG/HOS, OHS, OSA, Saos-2, U-2 OS and ZK-58 were obtained from ATCC ( www.lgcstandards-atcc.org ), DSMZ ( http://www.dsmz.de/ ) or research partners in the EU funded EuroBoNeT project [ ].

Techniques: Expressing, Quantitative RT-PCR

Journal: BMC Genomics

Article Title: miR-486-5p expression is regulated by DNA methylation in osteosarcoma

doi: 10.1186/s12864-022-08346-6

Figure Lengend Snippet: Genome-wide methylation profile of the mir-486/ANK1 locus and miR-486-5p expression levels in osteosarcoma patient samples. A. Representation of average methylation profile across the mir-486 / ANK1 locus in osteosarcoma patient samples ( n = 10) and bone ( n = 4). Methylation level (Beta) is shown for the individual CpG sites (ticks on horizontal axis) from the HumanMethylation450 BeadChips. The probes are ordered along the locus and intervals are not in scale. CGIs are shown as grey boxes with number refering to CpG count (from UCSC Genome Browser NCBI, GRCh37/hg19 assembly). Horizontal lines below plot show representative transcript variants of the respective mRNA genes (RefSeq) with exons as vertical bars, not in scale. Shaded vertical box: CGI shown in detail in B. Black, osteosarcomas; Grey, bone. B. Probe level methylation for CGI CpG79 in osteosarcoma patient samples and bone. Methylation levels as determined by Infinium 450 k arrays are given in Beta values for the individual CpG sites (cg). The selected CpGs within CGI CpG79 are highlighted with a shaded vertical box in A. Methylated: Beta 0.7–1.0; partially methylated: Beta 0.3–0.7; unmethylated: Beta< 0.3. C. Expression level of miR-486-5p in osteosarcoma patient samples and bone based on qRT-PCR. The osteosarcoma samples are grouped based on methylation status for probe cg08194989 on Infinium 450 k arrays, while the bone samples are shown as one group. Horizontal line: mean value

Article Snippet: A panel of human osteosarcoma cell lines ( n = 21) composed of 143B, CAL-72, G-292, HAL, HOS, IOR/MOS, IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS18, IOR/SARG, KPD, MG-63, MHM, MNNG/HOS, OHS, OSA, Saos-2, U-2 OS and ZK-58 were obtained from ATCC ( www.lgcstandards-atcc.org ), DSMZ ( http://www.dsmz.de/ ) or research partners in the EU funded EuroBoNeT project [ ].

Techniques: Genome Wide, Methylation, Expressing, Quantitative RT-PCR

Journal: BMC Genomics

Article Title: miR-486-5p expression is regulated by DNA methylation in osteosarcoma

doi: 10.1186/s12864-022-08346-6

Figure Lengend Snippet: Methylation distributions in osteosarcoma assessed by quantitative methylation-specific PCR. The methylation was measured for the groups of normal bones and osteosarcoma cell lines, xenografts and patient samples. PMR, percent of methylated reference. Horizontal black line: mean values

Article Snippet: A panel of human osteosarcoma cell lines ( n = 21) composed of 143B, CAL-72, G-292, HAL, HOS, IOR/MOS, IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS18, IOR/SARG, KPD, MG-63, MHM, MNNG/HOS, OHS, OSA, Saos-2, U-2 OS and ZK-58 were obtained from ATCC ( www.lgcstandards-atcc.org ), DSMZ ( http://www.dsmz.de/ ) or research partners in the EU funded EuroBoNeT project [ ].

Techniques: Methylation

Journal: BMC Genomics

Article Title: miR-486-5p expression is regulated by DNA methylation in osteosarcoma

doi: 10.1186/s12864-022-08346-6

Figure Lengend Snippet: Introduction of miR-486-5p in osteosarcoma cell lines. Osteosarcoma cells (OSA, OHS and U-2 OS) were transiently transfected with synthetic miR-486-5p mimics or a negative control. Cellular proliferation rates were determined by live cell imaging for 48 h using the IncuCyte, measuring cell confluence over time. One representative experiment of three is shown ( n = 3). Error bars represent the standard error of means of values for replicate wells ( n ≥ =5)

Article Snippet: A panel of human osteosarcoma cell lines ( n = 21) composed of 143B, CAL-72, G-292, HAL, HOS, IOR/MOS, IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS18, IOR/SARG, KPD, MG-63, MHM, MNNG/HOS, OHS, OSA, Saos-2, U-2 OS and ZK-58 were obtained from ATCC ( www.lgcstandards-atcc.org ), DSMZ ( http://www.dsmz.de/ ) or research partners in the EU funded EuroBoNeT project [ ].

Techniques: Transfection, Negative Control, Live Cell Imaging

Journal: PLoS ONE

Article Title: A p53 Drug Response Signature Identifies Prognostic Genes in High-Risk Neuroblastoma

doi: 10.1371/journal.pone.0079843

Figure Lengend Snippet: (A) Consistent with our previously data , Western blotting showed an increase in p53 levels within 4 hours exposure to Nutlin-3a in p202, p218, and IMR32 cells, confirming that the p53 signaling pathway is active in our system. (B) Apoptosis induction was tested in five established neuroblastoma lines (JF, IMR32), early passage neuroblastoma tumor cultures (p202, p218, H), and four non-neuroblastoma human solid tumors including colorectal (HCT116), breast (MCF7) and osteosarcoma (SJSA-1). SJSA-1 line differs by the level of MDM2 expression, being amplified 25 fold. A p53 mutant neuroblastoma line (SJ3-12), which lacks part of the DNA binding domain, was used as control. Proliferating cells were treated with Nutlin-3a for 24 hours and TdT-positive fraction was measured by flow cytometry. (C) Pathways enriched upon Nutlin-3a treatment: p53 signaling (GSEA ID: M6370), DNA damage (GSEA ID: M8378), and chemotherapy response (bleo_human_lymph) genes. Vertical columns separate Nutlin-3a versus Nutlin-3b at 3, 8, and 16 hours, horizontal rows indicate genes.

Article Snippet: JF (ATCC), IMR32 (ATCC), LAN5 (LS Metelitsa, Houston TX), and LAN1 (ATCC) human NB lines were maintained in RPMI 1640; human colorectal cancer cell line, HCT 116 (ATCC), and human breast cancer cell line, MCF7 (ATCC), in McCoy’s 5A and DMEM plus 1% insulin respectively; human osteosarcoma line, SJSA-1 (ATCC), in RPMI 1640 medium with 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate; primary neuroblastoma lines (p202, p218, pH) (Texas Children’s Cancer Center, Houston, TX) and CHLA255 line (LS Metelitsa) in IMDM with 20% FBS and 0.1% ITS.

Techniques: Western Blot, Expressing, Amplification, Mutagenesis, Binding Assay, Control, Flow Cytometry

Journal: Cells

Article Title: Ischemic Stroke Risk Associated with Mitochondrial Haplogroup F in the Asian Population

doi: 10.3390/cells9081885

Figure Lengend Snippet: HIF-1α expressions in different cybrids under hypoxia-ischemia. Comparison of HIF-1α expression under hypoxic-ischemic conditions in cybrids harboring common mitochondrial haplogroups found in the ethnic Chinese population (B4, B5, D4, D5, F1, F2, N9). The 143B cybrid was used to represent the Caucasian population. Actin was used as a loading control. Data represent the mean ± SD of at least three independent experiments ( # p < 0.005). N was used to represent normoxic and H to represent the hypoxic condition for 24 h.

Article Snippet: MtDNA-depleted ρ0 cells were established by the treatment of human osteosarcoma 143B cells (BCRC 60439) (Bioresource Collection and Research Center, Taipei, Taiwan) with ethidium bromide for three months, and illustration of their mtDNA-less cellular status by RT-PCR has been previously described [ , ].

Techniques: Comparison, Expressing, Control