Skip to main content
. 2020 Oct 27;4(20):5269–5284. doi: 10.1182/bloodadvances.2020002013

Figure 4.

Figure 4.

The p.Trp547* CSF3R variant abrogates receptor surface expression, induces receptor secretion, and decreases CSF3R messenger RNA (mRNA) expression. (A) Flow cytometric analysis of CSF3R (CD114) surface expression on parental Ba/F3 cells, 3 Ba/F3 clones grown from single cells expressing p.Trp547* CSF3R (Trp547* #2, Trp547* #5, and Trp547* #6), and Ba/F3 cells expressing WT CSF3R. (B) Immunoblot analysis of whole-cell lysates and conditioned media harvested from cells expressing p.Trp547* CSF3R, WT CSF3R, or p.Pro784Thr CSF3R. Lanes indicate the relevant CSF3R forms, and M denotes molecular weight markers. Lanes 1 to 3 are whole-cell lysates, and lanes 5, 7, and 9 are conditioned media (CM). Lanes 4, 6, and 8 were intentionally left empty. (C) Quantitation of CSF3R mRNA transcripts by TaqMan quantitative reverse transcription polymerase chain reaction (qRT-PCR) in neutrophils (PMNs) isolated from 3 different healthy donors (blue) and the elderly patient with the heterozygous p.Trp547* CSF3R variant (red). CSF3R mRNA expression (expressed as 2^-ddCt) in PMNs isolated from the patient with the p.Trp547* allele was 0.207 ± 0.003, compared with 0.9 ± 0.1 for PMNs isolated from 3 healthy donors. A Q test to determine if the p.Trp547* expression value was outside the range of normal values yielded a P value of .045. Error bars show standard error. (D) Analysis of nonsense-mediated decay (NMD) of CSF3R mRNA transcripts in monocytes from the same patient in panel C with a heterozygous p.Trp547* CSF3R variant. Monocytes from the patient (red) and a normal donor (blue) were incubated in vitro in the absence (CHX0) or presence of 50 μg/mL of cycloheximide (CHX50). Total CSF3R mRNA transcript levels from triplicate samples were quantified using TaqMan qRT-PCR. Basal CSF3R mRNA expression was significantly decreased in the p.Trp547* monocytes compared with normal monocytes (P = .013). After CHX treatment, a significant change in CSF3R mRNA transcripts was detected in p.Trp547* monocytes (P < .001), but not in monocytes from the normal donor (P = .570). Error bars show the standard error.