Journal: Journal of Nanobiotechnology

Article Title: Matrix micro/nano-topography drives oncogenic signaling and drug response in a 3D osteosarcoma model

doi: 10.1186/s12951-026-04237-0

Figure Lengend Snippet: Potential metabolic detoxification mechanisms of drug resistance in the AlgHA model. ( A ) GSEA enrichment of pathways in ABC transporters, Metabolism of xenobiotics by cytochrome P450, and Drug metabolism-cytochrome P450. ( B ) PPI networks of drug resistance-related genes in the AlgHA model. ( C ) Fold change in genes expression related to ABC transporters, cytochrome P450, and glutathione S-transferase. The expression on TCPS was standardized to 1, n = 3. ( D ) CYP1B1 expression in OS patients with different Huvos pathological grades based on the GSE21257 dataset. ( E ) ABCB1 expression in metastatic OS patients with different Huvos pathological based on the GSE21257 dataset. ( F ) Cell viability assay of HOS cells treated with varying concentrations of WS-917 and TMS for 72 h, n ≥ 5. ( G ) Heat map of the IC 50 values under the three conditions, which were calculated using a variable-slope nonlinear regression model. Data are expressed as a percentage relative to the DMSO-treated control model. Error bars represent mean ± S.D. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: To investigate specific resistance mechanisms, WS-917 and TMS (both from MCE) were employed.

Techniques: Expressing, Viability Assay, Control

Journal: Molecular Medicine Reports

Article Title: MEK inhibitor enhanced the antitumor effect of oxaliplatin and 5-fluorouracil in MEK1 Q56P-mutant colorectal cancer cells

doi: 10.3892/mmr.2018.9730

Figure Lengend Snippet: Gene expression induced by U0126 combined with or without oxaliplatin/5-FU. (A) ERCC1 mRNA levels in SW48 cells were examined by RT-qPCR following treatment with U0126 and/or oxaliplatin. (B) TYMS mRNA levels in SW48 cells were examined by RT-qPCR following treatment with U0126 and/or 5-FU. The graph depicts the fold change in ERCC1/TYMS levels normalized to β-actin levels. Data are presented as means ± standard deviation. Western blot analysis results of (C) ERCC1 and (D) TYMS protein expression levels in cells treated with U0126 in combination with oxaliplatin or 5-FU, respectively. **P<0.01 vs. control group; ## P<0.01 vs. oxaliplatin/5-FU-treated group. 5-FU, 5-fluorouracil; ERCC1, excision repair cross-complementation group 1; TYMS, thymidylate synthase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Excision repair cross-complementation group 1 (ERCC1) rabbit polyclonal antibody (cat. no. 14586-1-AP) and TYMS rabbit polyclonal antibody (cat. no. 15047-1-AP) were purchased from ProteinTech Group, Inc. (Wuhan, China).

Techniques: Gene Expression, Quantitative RT-PCR, Standard Deviation, Western Blot, Expressing, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: American journal of human genetics

Article Title: Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

doi: 10.1016/j.ajhg.2022.06.014

Figure Lengend Snippet: Figure 1. TYMS deficiency in families affected by dyskeratosis congenita (A) Pedigrees of proband families are as shown and indicate the presence of the TYMS variant in the heterozygous (þ/) state. Black cir- cles and squares denote affected probands. (B–E) Photographs of affected probands show some of the clinical features: sparse hair, nail dystrophy, abnormal skin pigmentation, and abnormal dentition. (F) Lymphoblastoid cell lines from the probands and parents show reduced levels of TYMS expression compared to that in controls. All genes are normalized to MCM6 and TFRC. Data represent means 5 SD, n ¼ 3, p values determined by one-way ANNOVA. Samples include cell lines from probands (families 1, 2, and 3) and parents (families 1 and 3) and are compared with unrelated controls. (G and H) Reduced TYMS protein amounts in individual proband samples from three families are compared to those in parents and con- trols; b-actin is used as a loading control. (I and J) 5-fluorouracil (5-FU) sensitivity demonstrating increased toxicity in lymphoblastoid cells from the probands compared to par- ents and a control.

Article Snippet: The lentiviral induction was performed with cDNAs encoding GFP, GFP-tagged TYMS (Origene), and ENOSF1 shRNA (Santa Cruz Biotechnology), in control and probands’ cells by spinfection in the presence of polybrene (10 mg/mL).

Techniques: Variant Assay, Expressing, Control

Journal: American journal of human genetics

Article Title: Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

doi: 10.1016/j.ajhg.2022.06.014

Figure Lengend Snippet: Figure 2. TYMS deficiency impacts nucleotide metabolism, telomere maintenance, and genome instability in probands’ cells (A) Schematic diagram showing the proteins involved in different stages (indicated by arrows) of de novo and salvage pathways for dTTP synthesis. Abbreviations are as follows: RRM, ribonucleotide reductase catalytic subunits M1 and M2; NDPK, nucleoside diphosphate kinase; TYMS, thymidylate synthase; and TK1, thymidine kinase 1. (B–D) The effect of TYMS deficiency on dNTP pools. Cells from both probands and the control were harvested for analysis of dUMP, dTMP, and dTTP pools. The scattered dot plot represents 1 million cells per dot in each set. (E) Immunoblotting for key proteins in cell lysates of probands compared with unrelated controls. b-actin is used as a loading control. (F) Relative telomere lengths of probands are reduced in comparison with those of controls. Age-adjusted T/S ratios analyzed by the MMqPCR method show that probands with TYMS variants have shorter telomere lengths. T/S ratios from probands with either TERC or TINF2 variants are shown for comparison. (G) Telomere length measurement by flow-FISH in probands from families 1, 4, and 6. (H) Relative levels of telomerase activity in probands and age-matched control cells at passage 2 were determined by TRAP assay. ‘‘IS’’ indicates internal standard, and * refers to the position of loading dye across the lanes. (I) Oligo-dT(20)-primed mature TERC RNA transcripts are distinguished from random hexamer priming of cDNA acquired from RNA sam- ples from lymphoblastoid cell lines of probands (box represents mean and whiskers represent standard deviation). (J) Immunoblots showing levels of DNA-repair protein at steady state in cells from affected probands and controls. GAPDH is used to determine the loading control. (K and L) Cell viability in cells from probands, parents, and a control in the presence of hydroxyurea. (M) Immunoblots showing protein level after hydroxyurea treatment. a-tubulin is used as a loading control. (N) A representative image of gH2AX staining in control and index proband cells 24 h after release from hydroxyurea (HU) treatment. Images show DAPI-stained nuclei in blue and gH2AX in green. The scale bar represents 50 mm.

Article Snippet: The lentiviral induction was performed with cDNAs encoding GFP, GFP-tagged TYMS (Origene), and ENOSF1 shRNA (Santa Cruz Biotechnology), in control and probands’ cells by spinfection in the presence of polybrene (10 mg/mL).

Techniques: Control, Western Blot, Comparison, Activity Assay, TRAP Assay, Random Hexamer, Standard Deviation, Staining

Journal: American journal of human genetics

Article Title: Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

doi: 10.1016/j.ajhg.2022.06.014

Figure Lengend Snippet: Figure 3. Haplotype analysis and influence of ENOSF1 variants on TYMS expression (A) Inheritance of common polymorphisms and the variant of interest in the TYMS-ENOSF1 locus in families I–V. The ‘‘C-A-ins’’ haplo- type in red highlights the common inherited allele from the wild-type parent. The relative position of the TYMS-specific allele is high- lighted in green. na indicates that a sample was not available. A black-filled symbol indicates an affected individual; an open symbol indicates an unaffected individual; and a gray-filled symbol indicates an asymptomatic carrier of the TYMS exonic variant. The unique 28 bp polymorphic 50-UTR tandem-repeat sequence that is known as the TYMS enhancer region (TSER; rs45445694) and the 6 bp dele- tion in the 30 UTR (rs151264360) are shown. The TSER with three polymorphic repeats (3R) has greater TYMS expression levels when compared than the two-repeat sequence (2R), and this is further modulated by the presence of SNP G or C (rs2853542) within the 2R when 3R is present. (B) TYMS-ENOSF1 genomic locus depicting polymorphisms (red arrows) and intronic variants identified in ENOSF1 alleles (blue arrows) and the TYMSOS allele (black arrow) in individuals for whom parental samples were available. Exonic TYMS variants (green arrows) are from all probands in this study. An asterisk indicates a recurrent variant. (C) ENOSF1/TYMS transcript ratio in control and proband cells as well as an unaffected heterozygote parent TYMS carrier as analyzed by qPCR.

Article Snippet: The lentiviral induction was performed with cDNAs encoding GFP, GFP-tagged TYMS (Origene), and ENOSF1 shRNA (Santa Cruz Biotechnology), in control and probands’ cells by spinfection in the presence of polybrene (10 mg/mL).

Techniques: Expressing, Variant Assay, Sequencing, Control

Journal: American journal of human genetics

Article Title: Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

doi: 10.1016/j.ajhg.2022.06.014

Figure Lengend Snippet: Figure 4. Post-transcriptional epistatic silencing of TYMS by elevated ENOSF1 in cells of the affected probands (A) RNA expression of TYMS and ENOSF1 after rescue by a GFP-TYMS lentiviral particle. Expression is relative to the control-GFP in each proband. (B) Immunoblotting of TYMS protein in control and proband cells transduced with lentivirus particles encoding GFP alone and GFP- tagged TYMS cDNA. (C) The RNA secondary structures of both TYMS and ENOSF1 in this RactIP predicted region is modelled with the RNAfold webserver under default parameters. The purple-colored dashed lines indicate base pairing of RNA residues between TYMS and ENOSF1. (D) RNA expression of TYMS and ENOSF1 after transduction with lentiviral particles encoding ENOSF1 shRNA. Expression is relative to the control-GFP shRNA in each proband. (E) Immunoblotting of TYMS protein in control and proband cells transduced with lentivirus particles encoding ENOSF1 shRNA. (F) Cellular sensitivity to 5-flurouracil (5-FU) in control and proband cells after transduction of lentivirus particles encoding ENOSF1 RNAi. For (A), (D), and (F), each experiment was performed in duplicate and analyzed in triplicate.

Article Snippet: The lentiviral induction was performed with cDNAs encoding GFP, GFP-tagged TYMS (Origene), and ENOSF1 shRNA (Santa Cruz Biotechnology), in control and probands’ cells by spinfection in the presence of polybrene (10 mg/mL).

Techniques: RNA Expression, Expressing, Control, Western Blot, Transduction, shRNA

Journal: Journal of Biological Chemistry

Article Title: Cryopyrin-induced Interleukin 1β Secretion in Monocytic Cells

doi: 10.1074/jbc.m401178200

Figure Lengend Snippet: FIG. 4. Cryopyrin requires oligomerization for ASC binding and IL-1 secretion. A, schematic representation of the cryopyrinPD-Fpk3 or Fpk3 vector. B, 1 108 THP-1 cryopyrinPD-Fpk3 and Fpk3 stable cells were immunoprecipitated (IP) with polyclonal myc Ab (top panel), and total lysates (bottom panel) from stable cells were immunoblotted with monoclonal ASC Ab. C, 0.5 106 THP-1 cells stably expressing with cryopyrinPD-Fpk3 or Fpk3 vector in the presence () or absence () of Fpk3 ligand AP1510 for 0, 4, and 8 h followed by detection of IL-1 secretion. The results are given as the mean S.D. of triplicate cultures.

Article Snippet: The antibodies used for immunoprecipitation and immunodetection were anti-FLAG monoclonal (Santa Cruz), anti-Myc polyclonal (Santa Cruz), anti-ASC polyclonal ( ProSci), anti-ASC monoclonal (21), and anti-tubulin monoclonal (Sigma).

Techniques: Binding Assay, Plasmid Preparation, Immunoprecipitation, Stable Transfection, Expressing