Journal: bioRxiv

Article Title: A FOXN1 mutation competitively displaces wild-type FOXN1 from higher-order nuclear condensates to cause immunodeficiency

doi: 10.1101/2021.03.28.437237

Figure Lengend Snippet: (a) Protein abundance of wild-type or Δ550 FOXN1 in anti-flag pull downs in lysates from 4D6 cells transfected to express wild-type (WT) or Δ550 FOXN1 tagged with a flag. The immunoprecipitates were analysed by Liquid Chromatography with tandem mass spectrometry (LC-MS-MS). Each point corresponds to an individual sample. The data is from a single experiment with three biological replicates per condition. Data shows the mean value and SD. (b) Normalised abundance of proteins identified by immunopreciptation and Liquid Chromatography with tandem mass spectrometry (LC-MS-MS). Comparison of 3 samples of 4D6 cells each transfected to express wild-type (WT) and Δ550 FOXN1 showing the normalized abundance of FOXN1 (red line), STAT6 (blue line), CBP (orange line), and YBX1 (green line). (c) Schematic representation of the generation of the 4D6-EYFP β5t FOXN1 CMV reporter cell line used for the validation of candidate proteins interacting with wild-type FOXN1. 4D6 cells were stably transduced to constitutively express FOXN1 under the CMV promoter that in turn controls the transcription of EYFP under the transcriptional control of the Psmb11 promoter which is a direct target of FOXN1 and drives β5t protein expression. (d) Flow cytometric gating strategy for the identification of EYFP positivity in live 4D6-EYFP β5t FOXN1 CMV cells that have been transfected with guide RNAs as identified by mruby2 positivity.

Article Snippet: Each pair of guides was cloned into the Cas9-2A-mRuby2 vector ( https://www.addgene.org/110164/ Addgene #110164) following the protocol of the Feng Zhang laboratory ( https://media.addgene.org/cms/filer_public/e6/5a/e65a9ef8-c8ac-4f88-98da-3b7d7960394c/zhang-lab-general-cloning-protocol.pdf ).

Techniques: Transfection, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: A FOXN1 mutation competitively displaces wild-type FOXN1 from higher-order nuclear condensates to cause immunodeficiency

doi: 10.1101/2021.03.28.437237

Figure Lengend Snippet: (a) STRING protein-protein interaction analysis of FLAG-tagged wild-type and Δ550 FOXN1 pulldowns from transfected 4D6 cells and subsequent analysis by LC-MS-MS. The links show previously verified interactions whereby the thickness of individual lines represents the strength of experimental evidence for such interactions. (b) FOXN1 and CBP co-localize in 4D6 cells. Indirect immunofluorescence microscopy combined with DAPI staining and confocal analysis of FLAG-tagged wild-type (WT) FOXN1 and endogenous CBP in 4D6 cells. The extent of CBP and FOXN1 colocalisation as assessed by Pearson’s correlation equals to 0.54 (where 0 equals to no colocalization and 1 to complete colocalization). (c) CBP and YBX1 are co-factors for FOXN1-mediated transcription. The frequency of cells positive for EYFP was measured using 4D6-EYFP β5t FOXN1 CMV cells that had a CrispR-mediated deletion of either CBP, YBX1, FOXN1 or CCR5. 4D6-EYFP β5t FOXN1 CMV cells transfected with only the cas9 vector served as an additional control. CrispR-targeted loss of FOXN1 and CCR5 served as a positive and negative control, respectively. The frequency of loss of EYFP expression was calculated relative to cells with loss of FOXN1 expression. The data is representative for at least 2(c) and 1 (b) independent experiments with three technical replicates each. Mean value and SD are shown and statistically compared by using a two-tailed unpaired t-test; p-values: ≥0.05 (ns), 0.0332(*), 0.0002 (***) <0.0001(****). See also and Table S3

Article Snippet: Each pair of guides was cloned into the Cas9-2A-mRuby2 vector ( https://www.addgene.org/110164/ Addgene #110164) following the protocol of the Feng Zhang laboratory ( https://media.addgene.org/cms/filer_public/e6/5a/e65a9ef8-c8ac-4f88-98da-3b7d7960394c/zhang-lab-general-cloning-protocol.pdf ).

Techniques: Transfection, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Microscopy, Staining, CRISPR, Plasmid Preparation, Negative Control, Expressing, Two Tailed Test