Figure 6.
Figure 6. LUBAC components regulate TLR3 signaling induced by poly(I:C) ex vivo and in vivo. (A) SHARPIN protein expression of PMKs from WT mice (n = 2) and cpdm (n = 4) littermates was analyzed by Western blot. Actin served as loading control. (B) PMKs isolated from newborn WT (n = 2) and cpdm (n = 4) littermates were stimulated by poly(I:C) as indicated. Concentrations of murine CCL5 (mCCL5) and murine CCL20 (mCCL20) in the supernatants were determined by ELISA after 24 h of stimulation. One representative of two independent experiments performed in triplicates is shown. (C) PMKs from WT mice (n = 2) and cpdm littermates (n = 2) were isolated at the age of 4 wk and cells were stimulated with 50 µg/ml poly(I:C) in absence versus presence of 250 ng/ml CHX. Loss of cell viability was determined after 48 h of stimulation. One representative of two independent experiments performed in triplicates is shown. (D) PMKs from cpdm+/− (n = 3), Tlr3−/−.cpdm+/− (n = 3), and Tlr3−/−.cpdm−/− (n = 3) littermates isolated at the age of 4 wk were stimulated with 100 µg/ml poly(I:C) and concentration of mCCL5 was determined in the supernatant after 24 h by ELISA. (E) HOIP protein expression of PMKs isolated from one litter of newborn Cre+ Hoipfl/fl mice (n = 4) cultured in absence versus presence of 1 µM 4-OHT for at least 72 h was analyzed by Western blot. Actin served as loading control. (F) PMKs with 4-OHT–inducible deletion of HOIP from Cre+ Hoipfl/fl newborn littermates (n = 4) were stimulated with poly(I:C) as indicated. Concentrations of mCCL5 and mCCL20 were determined by ELISA after 24 h of stimulation. One representative of two independent experiments performed in triplicates is shown. (G) Loss of cell viability was determined in parallel. (H) Naked poly(I:C) was injected into the ear pinnae of WT and cpdm littermates (n = 5) and induction of il6 and ifnb mRNA was assessed by RT-PCR. (I) Naked poly(I:C) was injected into the ear pinnae of WT and Tlr3−/− littermates (n = 5) and ifnb mRNA level was assessed by RT-PCR. Values are plotted as means ± SD for the indicated number of mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001, unpaired Student’s t test. Images are representative of at least two independent experiments.

LUBAC components regulate TLR3 signaling induced by poly(I:C) ex vivo and in vivo. (A) SHARPIN protein expression of PMKs from WT mice (n = 2) and cpdm (n = 4) littermates was analyzed by Western blot. Actin served as loading control. (B) PMKs isolated from newborn WT (n = 2) and cpdm (n = 4) littermates were stimulated by poly(I:C) as indicated. Concentrations of murine CCL5 (mCCL5) and murine CCL20 (mCCL20) in the supernatants were determined by ELISA after 24 h of stimulation. One representative of two independent experiments performed in triplicates is shown. (C) PMKs from WT mice (n = 2) and cpdm littermates (n = 2) were isolated at the age of 4 wk and cells were stimulated with 50 µg/ml poly(I:C) in absence versus presence of 250 ng/ml CHX. Loss of cell viability was determined after 48 h of stimulation. One representative of two independent experiments performed in triplicates is shown. (D) PMKs from cpdm+/− (n = 3), Tlr3−/−.cpdm+/− (n = 3), and Tlr3−/−.cpdm−/− (n = 3) littermates isolated at the age of 4 wk were stimulated with 100 µg/ml poly(I:C) and concentration of mCCL5 was determined in the supernatant after 24 h by ELISA. (E) HOIP protein expression of PMKs isolated from one litter of newborn Cre+ Hoipfl/fl mice (n = 4) cultured in absence versus presence of 1 µM 4-OHT for at least 72 h was analyzed by Western blot. Actin served as loading control. (F) PMKs with 4-OHT–inducible deletion of HOIP from Cre+ Hoipfl/fl newborn littermates (n = 4) were stimulated with poly(I:C) as indicated. Concentrations of mCCL5 and mCCL20 were determined by ELISA after 24 h of stimulation. One representative of two independent experiments performed in triplicates is shown. (G) Loss of cell viability was determined in parallel. (H) Naked poly(I:C) was injected into the ear pinnae of WT and cpdm littermates (n = 5) and induction of il6 and ifnb mRNA was assessed by RT-PCR. (I) Naked poly(I:C) was injected into the ear pinnae of WT and Tlr3−/− littermates (n = 5) and ifnb mRNA level was assessed by RT-PCR. Values are plotted as means ± SD for the indicated number of mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001, unpaired Student’s t test. Images are representative of at least two independent experiments.

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