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

Journal: Nucleic Acids Research

Article Title: FOXN3 integrates the KU70/KU80/SREBP-1 complex to regulate lipid metabolism in non-alcoholic fatty liver disease

doi: 10.1093/nar/gkag171

Figure Lengend Snippet: Depletion of FOXN3 or KU70/KU80 suppresses SREBP-1 transcriptional activity by disrupting the recruitment of SREBP-1 to its target gene elements. ( A ) Anti-SREBP-1 CUT&Tag analysis in HepG2 cells treated with FFA (400 μM, 24 h) was performed to examine the impact of FOXN3 knockdown on the binding profiles of SREBP-1 to lipogenic response genes. The graphs represent the proportion of reads enriched in the peak region relative to every one million total reads. ( B ) ChIP analysis was conducted in HepG2 cells treated with FFA (400 μM, 24 h) to assess the effect of FOXN3 knockout on the distribution of SREBP-1 at lipogenic response gene promoters. ( C ) ChIP analysis was conducted in HepG2 cells treated with FFA (400 μM, 24 h) to assess the effect of KU70 and KU80 knockdown on the distribution of SREBP-1 at lipogenic response gene promoters. ( D ) ChIP analysis was conducted in HepG2 cells treated with FFA (400 μM, 24 h) to assess the effect of KU70 and KU80 knockdown on the distribution of FOXN3 at lipogenic response gene promoters. ( E ) qPCR analysis was conducted in HepG2 cells treated with FFA (400 μM, 24 h) to assess the effect of KU70 and KU80 knockdown on the levels of lipogenic response genes. ( F ) A anti-Flag Co-IP assay was performed in FFA-treated (400 μM, 24 h) HepG2 cells stably expressing Flag-tagged FOXN3 to assess the effect of KU70 or KU80 knockdown on the interaction between FOXN3 and nuclear SREBP-1. The cells were treated with MG-132 (20 μM, 4 h) prior to collection. ( G ) Anti-KU70 Co-IP analysis was conducted in WT and FOXN3-knockout HepG2 cells treated with FFA (400 μM, 24 h) to assess the impact of FOXN3 deficiency on the association between KU70 and nuclear SREBP-1. ( H ) A anti-Flag Co-IP assay was performed in FFA-treated (400 μM, 24 h) HepG2 cells transfected with Flag-tagged KU80 to assess the effect of FOXN3 knockout on the interaction between KU80 and nuclear SREBP-1. ( I ) A anti-Flag Co-IP assay was performed in HepG2 cells transfected with the indicated plasmids to assess the effect of His-tagged KU70 overexpression on the interaction between FOXN3 and nuclear SREBP-1. The cells were treated with MG-132 (20 μM, 4 h) prior to collection. ( J ) A anti-Flag Co-IP assay was performed in HepG2 cells transfected with the indicated plasmids to assess the effect of HA-tagged FOXN3 overexpression on the interaction between KU80 and nuclear SREBP-1. The data B–E were assessed via two-tailed Student’s t -tests and are shown as means ± S.D. The blotting data F–J were quantified as the mean fold change and subjected to two-tailed Student’s t -tests; * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Quantitative real-time PCR was performed using the EvaGreen qPCR Master Mix (G891, Applied Biological Materials).

Techniques: Activity Assay, Knockdown, Binding Assay, Knock-Out, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Transfection, Over Expression, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: FOXN3 integrates the KU70/KU80/SREBP-1 complex to regulate lipid metabolism in non-alcoholic fatty liver disease

doi: 10.1093/nar/gkag171

Figure Lengend Snippet: The disruption of FOXN3 significantly reduces the biosynthesis of fatty acid and cholesterol. ( A ) Western blot analysis of liver tissues from mice administered a HFD for 0, 12, and 20 weeks. ( B–D ) Western blot analysis was conducted to assess FOXN3 levels in primary hepatocytes (B), as well as in HepG2 (C) and Huh7 (D) cells, following treatment with FFA at the specified time points. ( E ) RNA sequencing analysis presenting a volcano plot that illustrates the differential gene expression in HepG2 cells treated with FFA (400 μM, 24 h) following FOXN3 knockout. ( F ) The 1845 downregulated genes identified from the RNA sequencing data following FOXN3 knockout in HepG2 cells were subjected to KEGG analysis. ( G ) A heatmap was generated from the mRNA sequencing analysis to examine the expression changes of lipogenic response genes following FOXN3 knockout. ( H ) A quantitative PCR (qPCR) assay was conducted in HepG2 cells treated with FFA (400 μM, 24 h), both with and without FOXN3 knockout, to assess the RNA levels of representative lipogenic response genes. ( I ) Western blot analysis was performed in WT and FOXN3-knockout HepG2 cells to examine the protein levels of representative lipogenic response genes. The cells were treated with FFA (400 μM, 24 h) prior to collection. ( J ) Bodipy staining analysis of lipid accumulation in WT and FOXN3-knockout HepG2 cells, treated with or without FFA (400 μM, 24 h). ( K and L ) Cellular TG (K) and TC (L) levels in WT and FOXN3-knockout HepG2 cells, with and without FFA (400 μM, 24 h) treatment, were detected. ( M ) Bodipy staining analysis of lipid accumulation in Huh7 cells with or without siRNA-mediated FOXN3 knockdown, in the presence or absence of FFA (400 μM, 24 h). ( N and O ) Cellular TG (N) and TC (O) levels in Huh7 cells with or without siRNA-mediated FOXN3 knockdown, in the presence or absence of FFA (400 μM, 24 h), were detected. The data A, B, C, D, H, and I were assessed via a two-tailed Student’s t -test, and the data K, L, N, and O were assessed via one-way ANOVA. The blotting data A was quantified as means using ImageJ software ( n = 3), the data B, C, D, and I was quantified as the mean fold change from two independent experiments, also using ImageJ software, and the data H, K, L, N, and O are shown as the mean ± S.D.; * P < 0.05, ** P < 0.01.

Article Snippet: Quantitative real-time PCR was performed using the EvaGreen qPCR Master Mix (G891, Applied Biological Materials).

Techniques: Disruption, Western Blot, RNA Sequencing, Gene Expression, Knock-Out, Generated, Sequencing, Expressing, Real-time Polymerase Chain Reaction, Staining, Knockdown, Two Tailed Test, Software

Journal: Nucleic Acids Research

Article Title: FOXN3 integrates the KU70/KU80/SREBP-1 complex to regulate lipid metabolism in non-alcoholic fatty liver disease

doi: 10.1093/nar/gkag171

Figure Lengend Snippet: Hepatocyte-specific FOXN3 knockout impedes hepatic lipid production in mice. ( A–C ) Analysis of fasting body weight (A), food intake (B), liver weight, and liver-to-body weight ratio (C) in FOXN3 Flox and HepKO mice following 20 weeks of NCD or HFD administration ( n = 8 for each group). ( D ) Serum transaminase levels in FOXN3 Flox and HepKO mice were assessed after 20 weeks of administration of a NCD or a HFD ( n = 8 for each group). ( E ) Fasting blood glucose levels in FOXN3 Flox and HepKO mice were assessed after 20 weeks of administration of a NCD or a HFD ( n = 8 for each group). ( F ) Blood glucose levels of Flox and HepKO mice were assessed through intraperitoneal GTT and ITT following 20 weeks of HFD administration. ( G and H ) Serum (G) and hepatic (H) TG and TC levels were detected in FOXN3 Flox and HepKO mice were assessed after 20 weeks of administration of a NCD or a HFD ( n = 8 for each group). ( I ) H&E staining and Oil Red O staining were performed on Flox control and HepKO mice after 20 weeks of administration of a NCD or a HFD; scale bar: 100 μm. ( J ) Western blot analysis was conducted to assess the protein expression of lipogenic response genes in the livers of Flox control and HepKO mice after 20 weeks of administration of a NCD or a HFD ( n = 3 for each group). ( K ) qPCR analysis was conducted to evaluate the RNA expression of lipogenic response genes in the livers of Flox control and HepKO mice after 20 weeks of administration of a NCD or a HFD ( n = 3 for each group). The data A–E, G, H, and K were assessed via one-way ANOVA, the data F was assessed via two-way ANOVA, and the data are shown as the mean ± S.D., with “ns” indicating not significant. The blotting data J was quantified as the mean fold change and analyzed using a two-tailed Student’s t -test with ImageJ software ( n = 3); * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Quantitative real-time PCR was performed using the EvaGreen qPCR Master Mix (G891, Applied Biological Materials).

Techniques: Knock-Out, Staining, Control, Western Blot, Expressing, RNA Expression, Two Tailed Test, Software

Journal: Nucleic Acids Research

Article Title: FOXN3 integrates the KU70/KU80/SREBP-1 complex to regulate lipid metabolism in non-alcoholic fatty liver disease

doi: 10.1093/nar/gkag171

Figure Lengend Snippet: Hepatocyte-specific FOXN3 overexpression accelerates lipid and cholesterol biosynthesis in mice. ( A-C ) Analysis of fasting body weight ( A ), food intake ( B ), liver weight, and liver-to-body weight ratio ( C ) was conducted in mice with and without hepatic FOXN3 overexpression following 20 weeks of administration of a NCD or a HFD (n = 6 for each group). ( D ) Serum transaminase levels in mice with and without hepatic FOXN3 overexpression were assessed after 20 weeks of administration of a NCD or a HFD. ( E ) Fasting blood glucose levels in mice with and without hepatic FOXN3 overexpression were assessed after 20 weeks of administration of a NCD or a HFD (n = 6 for each group). ( F ) Blood glucose levels in mice with and without hepatic FOXN3 overexpression were assessed through intraperitoneal GTT and ITT following 20 weeks of HFD administration. ( G ) H&E staining and Oil Red O staining were performed on mice with and without hepatic FOXN3 overexpression after 20 weeks of administration of a NCD or a HFD. Scale bar, 100 μm. ( H, I ) Serum ( H ) and hepatic ( I ) TG and TC levels in mice with and without hepatic FOXN3 overexpression were detected after 20 weeks of administration of a NCD or a HFD (n = 6 for each group). ( J ) Western blot analysis was conducted to assess the protein expression of lipogenic response genes in the livers of mice with and without hepatic FOXN3 overexpression after 20 weeks of administration of a NCD or a HFD (n = 3 for each group). ( K ) qPCR analysis was conducted to evaluate the RNA expression of lipogenic response genes in the livers of mice with and without hepatic FOXN3 overexpression after 20 weeks of administration of a NCD or a HFD (n = 3 for each group). The data A-E, H, I, and k were assessed via one-way ANOVA, and the data F was assessed via two-way ANOVA. All the data are shown as the mean ± S.D., with “ns” indicating not significant. The blotting data J was quantified as the mean fold change and analyzed using a one-way ANOVA with ImageJ software (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Quantitative real-time PCR was performed using the EvaGreen qPCR Master Mix (G891, Applied Biological Materials).

Techniques: Over Expression, Staining, Western Blot, Expressing, RNA Expression, Software

Journal: Nucleic Acids Research

Article Title: FOXN3 integrates the KU70/KU80/SREBP-1 complex to regulate lipid metabolism in non-alcoholic fatty liver disease

doi: 10.1093/nar/gkag171

Figure Lengend Snippet: Inhibition of FOXN3 phosphorylation at S83 and S85 enhances SREBP-1 transcriptional activity. ( A ) Western blot analysis was performed to assess the levels of total FOXN3 and its phosphorylation at S83 and S85 in the livers of mice administered a HFD for 0, 12, and 20 weeks. ( B–D ) Western blot analysis was performed to assess the levels of total FOXN3 and its phosphorylation at S83 and S85 in HepG2 (B) and Huh7 (C) cells, as well as in MPHs (D) exposed to FFA treatment at the indicated time points. ( E ) A luciferase assay was conducted in FOXN3-knockout HepG2 cells to assess the effect of WT FOXN3 and its S83,85A mutant on the transcriptional activity of SREBP-1. A plasmid containing an SREBP element was co-transfected with a plasmid expressing either Flag-tagged WT FOXN3 or its S83,85A mutant. The cells were treated with FFA (400 μM, 24 h) prior to collection. ( F ) qPCR analysis was conducted to measure the RNA levels of lipogenic response genes in FOXN3-knockout HepG2 cells infected with lentivirus expressing either Flag-tagged WT FOXN3 or its S83,85A mutant. The cells were treated with FFA (400 μM, 24 h) prior to collection. ( G ) Western blot analysis was conducted to measure the protein levels of lipogenic response genes in FOXN3-knockout HepG2 cells infected with lentivirus expressing either Flag-tagged WT FOXN3 or its S83,85A mutant. The cells were treated with FFA (400 μM, 24 h) prior to collection. ( H ) Anti-SREBP-1 and anti-Flag ChIP analyses were conducted to measure the binding capacity of SREBP-1 to the promoters of lipogenic response genes in FOXN3-knockout HepG2 cells infected with lentivirus expressing either Flag-tagged WT FOXN3 or its S83,85A mutant. The cells were treated with FFA (400 μM, 24 h) prior to collection. ( I ) A Co-IP assay was performed in FOXN3-knockout HepG2 cells transfected with Flag-tagged WT FOXN3 or its S83,85A mutant to assess their interaction with the KU70/KU80/SREBP-1 complex. The cells were treated with MG-132 (20 μM, 4 h) prior to collection. The data in E, F, and H were assessed via two-tailed Student’s t -tests and are shown as means ± S.D. The blotting data A–D, J, and I were quantified as the mean fold change and subjected to two-tailed Student’s t -tests; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Quantitative real-time PCR was performed using the EvaGreen qPCR Master Mix (G891, Applied Biological Materials).

Techniques: Inhibition, Phospho-proteomics, Activity Assay, Western Blot, Luciferase, Knock-Out, Mutagenesis, Plasmid Preparation, Transfection, Expressing, Infection, Binding Assay, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: FOXN3 integrates the KU70/KU80/SREBP-1 complex to regulate lipid metabolism in non-alcoholic fatty liver disease

doi: 10.1093/nar/gkag171

Figure Lengend Snippet: Ablation of FOXN3 phosphorylation at S83 and S85 promotes fatty acid and cholesterol biosynthesis both in vivo and in vitro . ( A–C ) Analysis of fasting body weight (A), food intake (B), liver weight, and liver-to-body weight ratio (C) in WT and Foxn3 KI/KI mice following 20 weeks of HFD administration ( n = 6 for each group). ( D ) Fasting blood glucose levels in WT and Foxn3 KI/KI mice were assessed after 20 weeks of administration of a NCD or a HFD ( n = 6 for each group). ( E ) Blood glucose levels in WT and Foxn3 KI/KI mice were assessed through intraperitoneal GTT and ITT following 20 weeks of HFD administration. ( F ) Serum transaminase levels in WT and Foxn3 KI/KI mice were assessed after 20 weeks of administration of a NCD or a HFD ( n = 6 for each group). ( G ) Serum TG and TC levels in WT and Foxn3 KI/KI mice were detected after 20 weeks of administration of a NCD or a HFD ( n = 6 for each group). ( H ) Hepatic TG and TC levels in WT and Foxn3 KI/KI mice were detected after 20 weeks of administration of a NCD or a HFD ( n = 6 for each group). ( I ) H&E staining and Oil Red O staining were performed on WT and Foxn3 KI/KI mice after 20 weeks of administration of a NCD or a HFD; scale bar: 100 μm. ( J ) Western blot analysis was conducted to assess the protein expression of lipogenic response genes in the livers of WT and Foxn3 KI/KI mice after 20 weeks of administration of a NCD or a HFD. ( K ) qPCR analysis was conducted to evaluate the RNA expression of lipogenic response genes in the livers of WT and Foxn3 KI/KI mice after 20 weeks of administration of a NCD or a HFD. ( L ) Bodipy staining analysis of lipid accumulation in MPHs isolated from WT or Foxn3 KI/KI mice. The cells were treated with or without FFA (400 μM, 24 h) prior to collection. ( M and N ) Cellular TG (M) and TC (N) levels in MPHs isolated from WT or Foxn3 KI/KI mice. The cells were treated with or without FFA (400 μM, 24 h) prior to collection. The data A–D, F, G, I, and K were assessed via two-tailed Student’s t -tests, the data M and N were assessed via one-way ANOVA, the data E was assessed via two-way ANOVA. All these data are shown as means ± S.D. The blotting data J was quantified as the mean fold change and subjected to two-tailed Student’s t -tests. * P < 0.05, ** P < 0.01, *** P < 0.001; “ns” indicating not significant.

Article Snippet: Quantitative real-time PCR was performed using the EvaGreen qPCR Master Mix (G891, Applied Biological Materials).

Techniques: Phospho-proteomics, In Vivo, In Vitro, Staining, Western Blot, Expressing, RNA Expression, Isolation, Two Tailed Test