Journal: PLOS One

Article Title: Poldip2 deficiency attenuates lung disease severity in a mouse model of COVID-19

doi: 10.1371/journal.pone.0348065

Figure Lengend Snippet: Male and female hACE2/Poldip2 mice were infected with SARS-CoV-2 by nasal inoculation. Lungs were collected 7 days later for RNA preparation. Viral loads were measured by RT-qPCR using primer pairs and TaqMan probes specific for the viral gene N (A) or subgenomic E (sgE) RNA (B). Bars represent means ± SEM of data from n = 5-7 animals. Groups were compared using two-tailed Mann-Whitney tests: **P < 0.01.

Article Snippet: Reverse transcription was performed using Protoscript II reverse transcriptase (M0368, New England Biolabs) with random primers. cDNA was amplified with 2X Forget-Me-Not EvaGreen qPCR Master Mix with Low ROX (31045, Biotium) and primers against mouse TNFα, IL-1β, IL-6, MCP1, CXCL1, IFN-γ, Poldip2 and human ACE2 ( ).

Techniques: Infection, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: PLOS One

Article Title: Poldip2 deficiency attenuates lung disease severity in a mouse model of COVID-19

doi: 10.1371/journal.pone.0348065

Figure Lengend Snippet: Male and female hACE2/Poldip2 mice were euthanized 7 days after intranasal administration of PBS or SARS-CoV-2. Bronchoalveloar lavage (BAL) fluid was collected before harvesting lungs for RNA extraction as in Figure 3. mRNAs were measured by RT-qPCR in lung tissue (A) and protein ELISAs were carried out in BAL (B). Bars represent means ± SEM of data from n = 4-17 animals. Basal protein levels in +/ + PBS were 40 ± 20 pg/ml (MCP1) and 111 ± 31 pg/ml (CXCL1). Data were analyzed using 2-way ANOVA: ns, not significant; ** P < 0.01; *** P < 0.001.

Article Snippet: Reverse transcription was performed using Protoscript II reverse transcriptase (M0368, New England Biolabs) with random primers. cDNA was amplified with 2X Forget-Me-Not EvaGreen qPCR Master Mix with Low ROX (31045, Biotium) and primers against mouse TNFα, IL-1β, IL-6, MCP1, CXCL1, IFN-γ, Poldip2 and human ACE2 ( ).

Techniques: RNA Extraction, Quantitative RT-PCR

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