Figure S1.
Effect of patient variants on mRNA expression and of gnomAD variants on TLR signaling. (A)UNC93B1 mRNA level in primary fibroblasts of R525P P1 and three control primary fibroblast lines, assessed by qPCR and normalized to HPRT mRNA, and expressed as fold over one control fibroblast line dataset. Representative experiment. (B) NF-κB reporter luciferase activity following transfection of HEK293T cells with TLR7, TLR8, TLR3, or TLR9 plasmids and EV or WT UNC93B1 stimulated with indicated concentrations of R848, poly(I:C) or CpG-B. Data are expressed as the fold-induction of the RLU of the stimulated sample over the RLU of the respective NS sample for each UNC93B1 condition (“fold of respective NS”). Arrows indicate the dose of ligand chosen to study UNC93B1 variant gain of signaling in Fig. S1, C–F; and Fig. 2, C–F. Mean ± SEM of n = 2–4 experiments. (C–F) NF-κB reporter luciferase activity following transfection of HEK293T cells with (C) TLR7, (D) TLR8, (E) TLR3, (F) TLR9 plasmids and EV, WT, and gnomAD variant UNC93B1 stimulated respectively with (C) R848 0.01 µg/ml, (D) R848 0.1 µg/ml, (E) poly(I:C) 2.5 µg/ml, and (F) CpG-B 1 µM. Data are expressed as the fold-induction of the RLU of the stimulated sample over the RLU of the respective NS sample for each UNC93B1 condition (“fold stimulated”), normalized to the fold stimulated obtained for WT UNC93B1. Mean ± SEM of n = 3 experiments. One-way ANOVA with Dunnett’s post-hoc test. (G and H) NF-κB reporter luciferase activity following transfection of HEK293T cells with (G) TLR7 and (H) TLR8 plasmids and WT UNC93B1 together with the same amount of EV, WT and indicated variant UNC93B1 stimulated respectively with (G) R848 0.01 µg/ml, and (H) R848 0.1 µg/ml. Data are expressed as the fold-induction of the RLU of the stimulated sample over the RLU of NS sample for each UNC93B1 condition (“fold stimulated”), normalized to the fold stimulated obtained for WT UNC93B1. Mean ± SEM of n = 3 experiments. One-way ANOVA with Dunnett’s post-hoc test. (I) NF-κB reporter luciferase activity following transfection of HEK293T cells with TLR7, TLR8, TLR3, or TLR9 plasmids and EV, WT, or variant UNC93B1 without stimulation. Data are expressed as the RLU normalized to WT UNC93B1 RLU. Mean ± SEM of n = 2–4 experiments. One-way ANOVA with Dunnett’s post-hoc test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

Effect of patient variants on mRNA expression and of gnomAD variants on TLR signaling. (A) UNC93B1 mRNA level in primary fibroblasts of R525P P1 and three control primary fibroblast lines, assessed by qPCR and normalized to HPRT mRNA, and expressed as fold over one control fibroblast line dataset. Representative experiment. (B) NF-κB reporter luciferase activity following transfection of HEK293T cells with TLR7, TLR8, TLR3, or TLR9 plasmids and EV or WT UNC93B1 stimulated with indicated concentrations of R848, poly(I:C) or CpG-B. Data are expressed as the fold-induction of the RLU of the stimulated sample over the RLU of the respective NS sample for each UNC93B1 condition (“fold of respective NS”). Arrows indicate the dose of ligand chosen to study UNC93B1 variant gain of signaling in Fig. S1, C–F; and Fig. 2, C–F. Mean ± SEM of n = 2–4 experiments. (C–F) NF-κB reporter luciferase activity following transfection of HEK293T cells with (C) TLR7, (D) TLR8, (E) TLR3, (F) TLR9 plasmids and EV, WT, and gnomAD variant UNC93B1 stimulated respectively with (C) R848 0.01 µg/ml, (D) R848 0.1 µg/ml, (E) poly(I:C) 2.5 µg/ml, and (F) CpG-B 1 µM. Data are expressed as the fold-induction of the RLU of the stimulated sample over the RLU of the respective NS sample for each UNC93B1 condition (“fold stimulated”), normalized to the fold stimulated obtained for WT UNC93B1. Mean ± SEM of n = 3 experiments. One-way ANOVA with Dunnett’s post-hoc test. (G and H) NF-κB reporter luciferase activity following transfection of HEK293T cells with (G) TLR7 and (H) TLR8 plasmids and WT UNC93B1 together with the same amount of EV, WT and indicated variant UNC93B1 stimulated respectively with (G) R848 0.01 µg/ml, and (H) R848 0.1 µg/ml. Data are expressed as the fold-induction of the RLU of the stimulated sample over the RLU of NS sample for each UNC93B1 condition (“fold stimulated”), normalized to the fold stimulated obtained for WT UNC93B1. Mean ± SEM of n = 3 experiments. One-way ANOVA with Dunnett’s post-hoc test. (I) NF-κB reporter luciferase activity following transfection of HEK293T cells with TLR7, TLR8, TLR3, or TLR9 plasmids and EV, WT, or variant UNC93B1 without stimulation. Data are expressed as the RLU normalized to WT UNC93B1 RLU. Mean ± SEM of n = 2–4 experiments. One-way ANOVA with Dunnett’s post-hoc test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

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