Journal: bioRxiv

Article Title: An interdependent Cbf1-CCAN interaction stabilizes the budding yeast kinetochore

doi: 10.64898/2026.03.25.714319

Figure Lengend Snippet: (A) CryoEM map of the Cbf1:CCAN complex bound to a fragment of C0N3 DNA containing the CDEI element (PDB: 8OVW). The interaction between Cbf1 and Okp1 is detailed in the inset. (B) Flag-tag immunoprecipitations of Cbf1-3Flag (SBY18421) and Cbf1-EW-3Flag (L283E, L287W; SBY22227) were immunoblotted against Okp1 to analyze co-purifying levels. (C) Immunoblots of DNA-bound proteins from de novo kinetochore assembly assays performed using extracts from asynchronously grown CBF1-3FLAG (SBY18421), cbf1Δ (SBY4958), and cbf1-EW-3FLAG (SBY22227) strains with the indicated DNA templates. (D) Tetrad dissection of a cross between cbf1-EW (SBY22227) and dsn1-3A (SBY14171) strains. The four spores from individual asci are aligned in horizontal rows. Orange circles represent spores with double mutant genotype. (E) Schematic of the TIRFM stability assay. Lysate is incubated on the TIRFM slide for 5 or 90 minutes before being washed off. Slides were then either imaged immediately or after 20 minutes. (F) Percentages of colocalization between CEN3 DNAs and Cbf1-GFP (SBY22129), Cbf1-EW-GFP (SBY22923) and Cbf1-GFP in ctf19Δ cells (SBY24889) as analyzed by TIRFM after 5 min or 90 min of incubation and imaged immediately post wash. Error bars represent the standard deviation over three biological repeats. At least 3000 DNA molecules were imaged for each biological replicate. (G) Same as in (E) but imaged 20 minutes post wash. (H) RT-qPCR analysis of cenRNA expression of CEN4, CEN5 , and CEN8 in wild type (SBY22452), cbf1Δ (SBY22454), and cbf1- EW (SBY22456) cells arrested in G1 with α-Factor. Expression levels were quantified relative to that of wild type (mean ± SD, n=3). Statistical significances were analyzed by unpaired t-tests (*, p<0.05; **, p<0.01).

Article Snippet: 1 μg of DNase-treated RNA was reverse transcribed using RevertAid Reverse Transcriptase (Thermo Fisher Scientific, #EP0442) in a 20 μL reaction using random hexamer and oligo(dT)18 primers and analyzed by qPCR using the Forget-Me-Not EvaGreen qPCR Master Mix (Biotium, #31045) with primers listed in Supplemental Table S3. qPCR was performed using a Quantstudio TM 5 Real-Time PCR System (Applied Biosystem).

Techniques: FLAG-tag, Western Blot, Dissection, Mutagenesis, Stability Assay, Incubation, Standard Deviation, Quantitative RT-PCR, Expressing

Journal: Oncogene

Article Title: Asparagine bioavailability regulates the translation of MYC oncogene

doi: 10.1038/s41388-022-02474-9

Figure Lengend Snippet: a Levels of MYC and ASNS protein in ALL cell lines were measured by western blot analysis. T-ALL lines: CCRF-CEM, P12 Ichikawa, HPB-ALL, KOPT-1, Jurkat, DND-41; B-ALL lines: Nalm-6, 697, RS4;11, Reh. b MYC and ASNS proteins were measured by western blot analysis in DND-41, Jurkat, RS4;11, Reh, HPB-ALL, and Nalm-6 cells following asparagine withdrawal from the culture media for 16 h. c qPCR analysis of ASNS mRNA in DND-41, RS4;11, HPB-ALL, Nalm-6, Jurkat and Reh cells following 16 h of asparagine starvation. d DND-41, RS;411, HPB-ALL, Nalm-6, Jurkat, and Reh cells were grown with or without exogenous asparagine for 3 days. Population doublings at day 3 were recorded. e GSEA of RS4;11 cells following asparagine depletion shows a significant downregulation of MYC target genes. f Western blot measurements of MYC and ASNS proteins in DND-41 and RS4;11 cells expressing control or ASNS cDNA. Cells were grown 16 h in asparagine-replete or -deficient media. g DND-41 and RS4;11 cells expressing control or ASNS cDNA were grown in asparagine-replete or -deficient media for 3 days. Population doublings at day 3 were recorded. h Western blot measurements of MYC and ASNS proteins in control and ASNS-deleted Jurkat cells grown for 16 h in asparagine-replete or -deficient media. i Control and ASNS-deleted Jurkat cells in panel (g) were grown in asparagine-replete or -deficient media for 6 days. Cell numbers were recorded every other day. p values were calculated at day 6. Results in panel (c), (d), (g) and (i) were presented as mean ± standard derivation (SD). p values were determined by using Student′s two-tailed unpaired t-test. (**p < 0.005; ****p < 0.0001).

Article Snippet: The synthesized cDNAs were then used for q-PCR using designated primers using BullsEye EvaGreen qPCR master mix (MIDSCI, Cat # BEQPCR) and run on QuantStudio 3 (Applied biosystems, Cat. # A28137).

Techniques: Western Blot, Expressing, Control, Two Tailed Test

Journal: Oncogene

Article Title: Asparagine bioavailability regulates the translation of MYC oncogene

doi: 10.1038/s41388-022-02474-9

Figure Lengend Snippet: a Levels of MYC mRNA were measured by qPCR in DND-41 cells cultured in asparagine-replete or -deficient media for 6 h. Result was presented as mean ± standard derivation (SD). Differences in expression was calculated using Student′s two-tailed unpaired t-test. (ns not significant; **p < 0.01). b Levels of MYC and HSP90 were measured in DND-41 cells cultured in asparagine-deficient media for 6 h. Protein extraction were collected at indicated time points and analyzed by western blot. c Schematic model detailing the three possible routes asparagine may regulate the expression of MYC protein post-transcriptionally. (1) Asparagine suppresses MYC protein degradation by proteasome; (2) Asparagine activates mTORC1 to stimulate MYC mRNA translation; (3) Asparagine depletion activates GCN2 and induces eIF2α phosphorylation-dependent inhibition of MYC mRNA translation. d DND-41 and RS4;11 cells were cultured in asparagine-replete or -deficient media for 6 h with or without a proteasomal inhibitor MG-132 (1 μM). MYC protein was measured by western blot analysis. e DND-41 cells cultured in asparagine-replete or -deficient media, followed by treatment with cycloheximide (2 μg/mL) starting at 0 min for the indicated time points. Equal amounts of protein lysates were analyzed by western blot to measure c-MYC protein, with HSP90 included as a loading control. f DND-41 and RS4;11 cells expressing control or ASNS cDNA were cultured in asparagine-replete or -deficient media for 6 h. MYC, ASNS, ATF4 and p-S6K(T389) were measured by western blot analysis. g DND-41 cells were cultured at the indicated conditions for 6 h. MYC and p-S6K(T389) were measured by western blot analysis. h DND-41 cells were cultured in asparagine-deficient media for 16 h followed by asparagine re-supplementation in the presence of mTOR inhibitors rapamycin (100 nM) and Torin1 (100 nM) for 6 h. MYC, p-mTOR(S2448), p-S6K(T389) and p-4EBP1(S65) were measured by western blot analysis. i DND-41 and RS4;11 cells were cultured in asparagine-deficient media for 6 h in the presence of ISRIB (250 nM) or GCN2iB (1.5 μM). Puromycin (90 μM) was added 10 min before protein harvest. MYC, ATF4, p-S6K(T389) and puromycin-incorporated polypeptides were detected by western blot analysis. Global protein synthesis rates were analyzed by quantifying the intensity of individual lanes in the puromycin blot relative to the loading control using the ImageJ software.

Article Snippet: The synthesized cDNAs were then used for q-PCR using designated primers using BullsEye EvaGreen qPCR master mix (MIDSCI, Cat # BEQPCR) and run on QuantStudio 3 (Applied biosystems, Cat. # A28137).

Techniques: Cell Culture, Expressing, Two Tailed Test, Protein Extraction, Western Blot, Phospho-proteomics, Inhibition, Control, Software

Journal: Oncogene

Article Title: Asparagine bioavailability regulates the translation of MYC oncogene

doi: 10.1038/s41388-022-02474-9

Figure Lengend Snippet: a DND-41 cells were cultured for 6 h in asparagine-replete (blue), -deficient medium (orange) or -deficient medium with GCN2iB (1.5 μM, green), and lysates were prepared and separated by sucrose gradient centrifugation. Gradient fractions were monitored by absorbance at 245 nm and monosomes and polysomes are indicated. The ratio between polysome and monosome was listed in the table. The result is from one of the representative profiles of each condition. b Levels of c-MYC mRNAs in each individual sucrose gradient fraction were measured by qPCR. For each fraction, results were normalized to a spiked luciferase mRNA control and plotted as a percentage of the total mRNA. Statistical significance was estimated using two-way ANOVA using the recommended Sidak method for multiple comparisons. (**p < 0.01; and ****p < 0.0001). c Levels of ATF4 mRNAs in each individual sucrose gradient fraction were quantified in a similar way as panel (b) (**p < 0.01; ***p < 0.001; ****p < 0.0001). d Lentiviral vector expressing control or MYC cDNA with HA tag on the C-terminus without endogenous UTRs were introduced into DND-41 cells. Cells were cultured in asparagine-replete or -deficient media for 6 h. MYC protein was measured by western blot analyses using c-MYC antibody and anti-HA antibody. ASNS protein was undetectable and Jurkat cells were used as a positive control.

Article Snippet: The synthesized cDNAs were then used for q-PCR using designated primers using BullsEye EvaGreen qPCR master mix (MIDSCI, Cat # BEQPCR) and run on QuantStudio 3 (Applied biosystems, Cat. # A28137).

Techniques: Cell Culture, Gradient Centrifugation, Luciferase, Control, Plasmid Preparation, Expressing, Western Blot, Positive Control

Journal: BMC Plant Biology

Article Title: Comparative transcriptome analysis revealed that tomato response to salt stress induced by exogenous GABA and functional verification of SlGAD1

doi: 10.1186/s12870-025-07986-6

Figure Lengend Snippet: qPCR verification of differential gene expression in GN and N treatments. A qPCR verification of GNs. B qPCR verification of Ns. C Correlation analysis between transcriptome data and qPCR validation of GNs. D Correlation analysis between transcriptome data and qPCR validation of Ns

Article Snippet: The gene-specific primers used are shown in Table S1. qRT‒PCR was performed according to the instructions for the Fast Super EvaGreen qPCR Master Mix Kit (US Everbright ® , Inc.).

Techniques: Gene Expression, Biomarker Discovery