1. Establishment of a ΔF508-CF promyelocytic cell line for cystic fibrosis research and drug screening 
Scott Jennings, Hang Pong Ng, Guoshun Wang 
Journal of Cystic Fibrosis 
Volume 18, Issue 1, Pages (January 2019)
DOI: /j.jcf
Copyright © 2018 European Cystic Fibrosis Society Terms and Conditions

2. Fig. 1
Schematic representation of the human CFTR Exon 11 region and ssOND homology-directed repair (HDR) template. A) The letters NIIFGVS indicate amino acid codons, with the highlighted F being lost in the case of ΔF508 mutation. The CRISPR/Cas9 sgRNA sequence is highlighted in blue, with the corresponding protospacer-adjacent motif (PAM) site highlighted in green. Red arrows indicate the site of the Cas9-induced double strand break (DSB). The HDR template is an ssODN with 36 bp of homology on the PAM-distal side of the DSB and 91 bp of homology on the PAM-proximal side of the DSB. B) The structure of the CFTR gene-editing vector, hCFTR-Cas9-eGFP. T2A: an adapted self-cleaving 2A peptide from picornaviruse to produce equimolar levels of multiple genes from the same mRNA. C) The regional sequence of WT-hCFTR-Exon-11 and the detail sequence of the designed ssODN.
Journal of Cystic Fibrosis  , 44-53DOI: ( /j.jcf )
Copyright © 2018 European Cystic Fibrosis Society Terms and Conditions

3. Fig. 2
Genotyping of single-cell clones. A) A representative gel image of PCR amplification of CFTR Exon 11 and SspI digestion for multiple single-cell clones. WT (+/+); homozygous ΔF508-CFTR (Δ/Δ); heterozygous ΔF508-CFTR (+/Δ). U: undigested PCR product; C: SspI-digested PCR product. B) Sequence comparison of CFTR Exon-11 region of the NCBI-published WT sequence, designed mutant sequence (ΔF508 + SspI), and an obtained homozygous clone (Δ/Δ) sequence.
Journal of Cystic Fibrosis  , 44-53DOI: ( /j.jcf )
Copyright © 2018 European Cystic Fibrosis Society Terms and Conditions

4. Fig. 3
Morphological and immunological determination of WT and ΔF508-CF neutrophils derived from the control and CFTR-gene-edited HL-60 cells. A-D) Morphological determination of WT and ΔF508-CF HL-60 cell differentiation. Giemsa staining of non-differentiated (A & C) or differentiated (B & D) HL-60 cells. WT (A & B) and ΔF508-CF (C & D). E-J) Immunological determination of WT and ΔF508-CF HL-60 cell differentiation and their CFTR expression. E & F) CD11b immunostaining. Differentiated (Diff.) or non-differentiated (Undiff.) WT or ΔF508-CF HL-60 cells were stained with an antibody against CD11b or an isotype control antibody (Isotype-Ctrl Ab). G & H) Surface CFTR immunostaining. Differentiated neutrophils from WT or ΔF508-CF HL-60 cells were stimulated with or without PMA, followed by staining with an antibody against an CFTR extracellular domain (CF3 Ab) or an isotype control antibody (Isotype-Ctrl Ab). I) Intracellular CFTR staining. Differentiated WT or ΔF508-CFTR (ΔF508) neutrophils were stained with an anti-CFTR antibody (Ab#450) or an isotype control antibody. J) Intracellular CFTR staining. Differentiated WT or ΔF508-CFTR (ΔF508) neutrophils were stained with an anti-CFTR antibody (Ab#596) or an isotype control antibody.
Journal of Cystic Fibrosis  , 44-53DOI: ( /j.jcf )
Copyright © 2018 European Cystic Fibrosis Society Terms and Conditions

5. Fig. 4
Oxidant production and microbial killing. A) Phagosomal HOCl production. Differentiated neutrophils from WT or ΔF508-CF HL-60 cells were allowed to phagocytose serum-opsonized P. aeruginosa. Chloride-dependent phagosomal HOCl production was assessed by R19-S oxidation assay. Fluorescence positivity and intensity were measured by flow cytometry, and the total fluorescence of all positive cells for each condition was plotted as percentage increase as related to the 0-mM chloride condition. B) Primitive oxidant production. Differentiated neutrophils from WT or ΔF508-CF HL-60 cells were pre-loaded with dihydrorhodamine 123 (DHR 123) and stimulated with 20 ng/ml PMA for 1 h. Total fluorescence were obtained by flow cytometry. C) Microbial killing. Differentiated neutrophils from WT or ΔF508-CF HL-60 cells were allowed to phagocytose serum-opsonized bacteria at a ratio of 1:5 (cell: bacteria). Chloride-dependent bacterial killing over 40 min at 37 °C was assessed by cell lysis, bacterial plating and colony numeration. Bacterial colony number under 122-mM chloride condition was related to that of 0-mM chloride condition.
Journal of Cystic Fibrosis  , 44-53DOI: ( /j.jcf )
Copyright © 2018 European Cystic Fibrosis Society Terms and Conditions

6. Fig. 5
Induced apoptosis and chemotaxis. A) Apoptosis. WT and ΔF508-CF neutrophils derived from their corresponding HL-60 cells were stimulated with PMA (20 ng/ml) for 1 h, followed by Annexin-V and propidium iodide stainings. Double asterisks (**) denote a significant difference between two compared groups by Student's t-test (p < .01, n = 4). B) Chemotaxis. WT and ΔF508-CF neutrophils derived from their corresponding HL-60 cells were set to migrate toward a control or an fMLF (100 nM) medium for 1 h. Double asterisks (**) denote a significant difference between two compared groups by Student's t-test (p < .01, n = 5).
Journal of Cystic Fibrosis  , 44-53DOI: ( /j.jcf )
Copyright © 2018 European Cystic Fibrosis Society Terms and Conditions