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: 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: 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