Journal: American Journal of Cancer Research

Article Title: METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m 6 A modifications and splicing factors

doi:

Figure Lengend Snippet: Establishment of stable METTL3-knockdown cell lines using osteosarcoma U2OS cells. A. Using the CRISPR-Cas9 system, METTL3-knockdown cells were established (2-9 and 2-14) along with control cells (NT2 and NT3). METTL3-knockdown efficiency was confirmed by the decreased METTL3 and METTL14 protein expression using western blot. B. Levels of m6A in total RNA isolated from each stable cell line was measured via ELISA.

Article Snippet: U2OS cells were purchased from the Korean Cell Line Bank.

Techniques: Knockdown, CRISPR, Control, Expressing, Western Blot, Isolation, Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m 6 A modifications and splicing factors

doi:

Figure Lengend Snippet: Enriched RBP motif in alternatively spliced genes. A. rMAPS captured a total of 19 RBPs that were enriched in alternatively spliced genes in METTL3-knockdown cells. Each RBP is represented with a different color based on the type of alternative splicing (alternative 3’ splice site: red, alternative 5’ splice site: blue, cassette exon: black, intron retention: green). B. The Pearson’s correlation coefficients between each RBP and METTL3 expression were analyzed in 12,839 TCGA pan-cancer patients. C. The m6A modification of the 3’UTR of SFPQ mRNA. The m6A sites in SFPQ mRNA was analyzed using SRAMP (https://www.cuilab.cn/sramp; left panel) and a red arrow indicates the predicted m6A site in the 3’UTR. MeRIP-qPCR analysis was performed to validate the predicted m6A site in U2OS cells (right panel). D. The mRNA expression levels of SFPQ and IGF2BP3 after knockdown of IGF2BP3. The mRNA expression levels were estimated by real-time PCR after transfecting 25 nM of IGF2BP3 siRNA for 48 h. E. Enrichment of IGF2BP3 in the 3’UTR of SFPQ mRNA. RIP-qPCR analysis was performed to detect binding of IGF2BP3 to the 3’UTR of SFPQ mRNA. F. The number of SFPQ peaks located within a distance of 1000 bp of alternatively spliced genes was counted.

Article Snippet: U2OS cells were purchased from the Korean Cell Line Bank.

Techniques: Knockdown, Alternative Splicing, Expressing, Modification, Real-time Polymerase Chain Reaction, Binding Assay

Journal: American Journal of Cancer Research

Article Title: METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m 6 A modifications and splicing factors

doi:

Figure Lengend Snippet: Alternatively spliced genes in METTL3-knockdown HULEC-5a and A375 cells. A. Alternative splicing was analyzed using JUM in METTL3-knockdown HULEC-5a and A375 cells using shRNA. The number of alternatively spliced genes is represented using a bar graph (p-value < 0.05 being significant). B. Venn diagram showing the number of common alternatively spliced genes between U2OS, HULEC-5a and A375 cells. C. Top 10 enriched GO terms of alternatively spliced genes in HULEC-5a and A375 cells.

Article Snippet: U2OS cells were purchased from the Korean Cell Line Bank.

Techniques: Knockdown, Alternative Splicing, shRNA

Journal: Materials

Article Title: The Biocompatibility and Self-Healing Effect of a Biopolymer’s Coating on Zn Alloy for Biomedical Applications

doi: 10.3390/ma16237486

Figure Lengend Snippet: Confocal images of U2OS cells with DAPI-stained nucleus after 7 days of incubation on casein-based layer (2cas1h) on tested materials: ZnMg3.2 alloy ( a ); casein coating (2cas1h) ( b ). Additional 3D visualization from Immaris software (Bitplane Scientific Software; version 10.0.0; Olympus, Zurich, Switzerland) for ZnMg3.2 alloy ( c ), casein coating (2cas1h) ( d ).

Article Snippet: Biological characterization was based on a human osteosarcoma cell line U2OS (cat. No. 92022711; Merck, Rahway, NJ, USA).

Techniques: Staining, Incubation, Software

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ALK2-R206H-mediated pSMAD1/5/8 formation is induced by ACVR2B more efficiently than by ACVR2A. U2OS cells were transfected with vectors encoding HA-ALK2-R206H (or HA-ALK2-WT) alone or with myc-tagged ACVR2A, ACVR2B, or ACVR2B-KD. After 24 h, cells were starved (2 h, 1% serum) and stimulated (or not; control) with ActA (4 nM, 60 min, 37 °C). Cells were lysed, subjected to SDS--PAGE, and immunoblotted for pSMAD1/5/8, tSMAD1/5/8, and β-actin. As shown in , the cell-surface levels of the tagged receptors were similar and were not affected by the co-expressed receptors. ( A , B ) Representative blots of ActA signaling to pSMAD1/5/8. ( C , D ) Quantification of ActA-mediated pSMAD1/5/8 formation. The bands were visualized by ECL and quantified by densitometry. Data are mean ± SEM of the pSMAD1/5/8 over tSMAD1/5/8 ratio of 5 ( C ) or 4 ( D ) independent experiments. The value obtained for ActA-stimulated cells co-transfected with HA-ALK2-R206H and myc-ACVR2B was taken as 1. Asterisks show significant differences between the pairs indicated by brackets, using one-way ANOVA and Bonferroni post hoc test (*, p < 0.02; **, p < 4 × 10 −3 ; ***, p < 8 × 10 −4 ; n.s. = not significant).

Article Snippet: The U2OS human cell line was authenticated by STR analysis at Microsynth AG (Balgach, Switzerland).

Techniques: Transfection, Control, SDS Page

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2B is superior to ACVR2A in eliciting ALK2-R206H-mediated transcriptional activation of the SMAD1/5/8 pathway. U2OS cells were co-transfected with BRE-Luc and pRL-TK, together with HA-ALK2-R206H or HA-ALK2-WT (alone or together with myc-ACVR2A, myc-ACVR2B, or myc-ACVR2B-KD). These constructs were replaced by empty vector for control samples. After 17 h, cells were starved without serum (5 h) and stimulated (or not; control) with ActA (2 nM, 19 h). Relative Luminescence Units (RLU) are expressed as mean fold induction ± SEM (n = 4 independent experiments). The results were normalized for transfection efficiency using Renilla luminescence by the DLR luminescence assay. The value in untreated, unstimulated cells was taken as 1. The cell-surface levels of the tagged receptors were not altered by the co-expressed receptors . Asterisks show significant differences between the pairs indicated by the brackets, using one-way ANOVA and Bonferroni post hoc test (**, p < 1 × 10 −3 ; ***, p < 5 × 10 −4 ; ****, p < 10 −4 ; n.s. = not significant).

Article Snippet: The U2OS human cell line was authenticated by STR analysis at Microsynth AG (Balgach, Switzerland).

Techniques: Activation Assay, Transfection, Construct, Plasmid Preparation, Control, Luminescence Assay