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. 2018 Dec 10;129(1):388–405. doi: 10.1172/JCI98869

Figure 7. Correlation of reduced C1INH secretion in skin-derived fibroblasts derived from a patient carrying the c.551_685del mutation with C1INH accumulation in ER.

Figure 7

(A) C1INH in medium from control (NHDF-03, NHDF-05, NHDF-15) and patient-derived fibroblasts. (B) Evaluation of SERPING1 mRNA expression levels in control and patient-derived fibroblasts by qPCR. Equal expression of normal and mutant SERPING1 alleles in fibroblasts derived from the patient carrying the c.551_685del mutation. Inset: Agarose gel showing PCR products from PCR amplification (using primers spanning the deletion) on cDNA synthesized from RNA purified from either control NHDF-03 fibroblasts or patient-derived fibroblasts. (C) Restricted C1INH secretion induced by lentiviral delivery of SERPING1[c.551_685del]. Control NHDF-03 fibroblasts were transduced with lentiviral vectors encoding C1INHGly162_Pro206del (or eGFP as control) at an estimated MOI of 1. (D) Structure of the ER in control fibroblasts and fibroblasts derived from the c.551_685del patient. The fibroblasts were transfected with 900 ng plasmid encoding the Tomato fluorescence gene fused to an ER-targeting sequence to visualize ER (red). Cells were fixated 48 hours after transfections, and nuclei visualized with Hoechst (blue). Scale bar: 10 μm. (E) Accumulation of endogenous C1INH within the ER of patient-derived fibroblasts. The fibroblasts were transfected as in D, fixated, and incubated with Hoechst to visualize the nuclei (blue) and C1INH antibody to visualize both normal and mutated C1INH (green). Scale bars: 10 μm. (A–C) The experiment was carried out in triplicate (n = 3), and data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, patient-derived fibroblasts compared with NHDF-05 (A) or NHDF-15 (B) or transduced compared with untransduced NHDF-03 (C). Statistical analyses were performed by 1-way ANOVA with Dunnett’s multiple comparison test. (D–E) Data are representative of findings from more than 3 biological replicates. (A–E) Similar results were seen in at least 2 independent experiments.