NF-κB transcriptional activity, but not IκB degradation, is decreased in Lrrk2-KD cells.

<p>(<b>A</b>) Parental (−), control (Con) and Lrrk2-KD (KD) cells were transfected with a 5× NF-κB-luciferase reporter plasmid (pDNA), and luciferase activity was measured 3 h after LPS stimulation. (<b>B</b>) Cells were treated with LPS for the indicated times, and IκB levels were analyzed by Western blotting. Relative IκB levels were quantified using α-tubulin as an internal control. Values in (A) and (B) are means ± SEMs of three independent experiments (*<i>p</i><0.05).</p

LPS-induced p38 phosphorylation is specifically inhibited in Lrrk2-KD cells.

<p>(<b>A, C, D</b>) Cells were incubated with LPS (100 ng/mL) for the indicated times (A, C) or 30 min (D), and the levels of phosphorylated p38 (p-p38), JNK (p-JNK), ERK (p-ERK), and total p38 (A) or total and phosphorylated MKK3/6 (C, D) were determined by Western blotting. α-tubulin was used as an internal control. (<b>B, E</b>) Band intensities of p-p38 (B) and p-MKK3/6 (D) were quantified using a densitometer. Values are means ± SEMs of three independent experiments (*<i>p</i><0.05, **, <i>p</i><0.01 vs. control). Data are representative of three independent experiments.</p

List of primer sequences used for qRT-PCR.¹

1<p><b>Fwd</b>, Forward primer; <b>Rev</b>, Reverse primer.</p

Effect of hLRRK2 overexpression on the NF-κB signaling pathway and NF-κB activity in HEK293T cells.

<p>(<b>A</b>) HEK293T cells (parental or TCM-HEK) were transfected with a 5× NF-κB–luciferase reporter construct, empty vector (mock), or hLRRK2 expression vector. One unit is the basal NF-κB activity detected in untreated parental HEK293T cells. <i>Left:</i> NF-κB activity was measured by luciferase assay. <i>Right:</i> LRRK2 expression was confirmed by Western blotting for c-myc. β-actin was used as a loading control. Values are means ± SEMs of three independent experiments. (**<i>p</i><0.01 vs. mock). (<b>B</b>) LPS induced a dose-dependent increase in NF-κB activity in TCM-HEK cells with or without LRRK2. Cells were treated with the indicated amount of LPS for 6 h. The values shown are fold-induction relative to the values of NF-κB activity in unstimulated TCM-HEK cells, expressed as means ± SEMs of three independent experiments (*<i>p</i><0.05, **<i>p</i><0.01 vs. mock). (<b>C</b>) <i>Upper panel:</i> TCM-HEK cells were transfected with LRRK2-WT and each LRRK2 mutant, and NF-κB activity was analyzed after treating with LPS (10 ng/mL) for 6 h. <i>Lower panel:</i> Expression of LRRK2 mutants was detected by Western blotting. Values are means ± SEMs of three independent experiments. (<b>D</b>) TCM-HEK cells were transfected with mock, G2019S (100, 250 and 750 ng), and G2385R (100 and 500 ng) hLRRK2 expression vector. Values are means ± SEMs of three independent experiments. <i>Upper panel:</i> NF-κB activity measured by luciferase assay. <i>Lower panel:</i> LRRK2 levels determined by Western blotting (*p<0.05; <i>ns</i>, not significant). (<b>E</b>) NF-κB DNA-binding activity was analyzed by EMSA. Nuclear extracts were obtained from mock and LRRK2-WT-overexpressing TCM-HEK cells stimulated with LPS (10 ng/mL) for the indicated times. (<b>F</b>) A supershift assay was performed using nuclear extracts obtained 1 h after LPS treatments and preincubated with NF-κB p50 and p65 antibodies. Arrowheads indicate p50/p65 complex. Specific binding (arrowhead) was analyzed using an excess (20×) of unlabeled (Cold) DNA.</p

Attenuation of inflammatory responses in Lrrk2-KD microglia.

<p>(<b>A</b>) BV-2 microglia were infected with lentivirus expressing non-targeted (Con) or Lrrk2-targeted shRNA (KD). Two stable clones of each group were selected. Expression levels of Lrrk2 mRNA and protein were analyzed by qRT-PCR (left) and Western blotting (right), respectively. Parental (−), con, and KD cells were treated with or without 100 ng/mL LPS (B–E), 10 µg/mL LTA (F), 500 ng/mL CL097 (G), or 500 ng/ml ODN1668 (H) for indicated times (B, D, E), 12 h (C), 24 h (F), or 48 h (G, H). (<b>B</b>) TNF-α and IL-6 secretion into the culture medium were analyzed by ELISA. (<b>C, D, F–H</b>) iNOS protein expression was assayed by Western blotting (C), and NO release was measured using the Griess reagent, as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034693#s2" target="_blank">Methods</a> (D, F–H). (<b>E</b>) TNF-α, IL-1β, IL-6 and iNOS mRNA levels were analyzed by qRT-PCR. Gapdh mRNA and α-tubulin protein levels were analyzed as internal controls for qRT-PCR and Western blotting, respectively. Values are means ± SEMs (*p<0.05, **<i>p</i><0.01 vs. control). Data are representative of at least three independent experiments unless indicated otherwise.</p

Effect of hLRRK2 overexpression on p38 and JNK phosphorylation in HEK293T cells.

<p>(<b>A</b>) LPS-responsive HEK293T cells (TCM-HEK), prepared as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034693#s2" target="_blank">Methods</a>, were co-transfected with empty pcDNA3.1 for the following control experiments. LPS (100 ng/mL) induced phosphorylation of p38, and JNK was as analyzed by Western blotting. (<b>B, D</b>) TCM-HEK cells co-transfected with c-myc-tagged hLRRK2 (WT, G2019S [2019], D1994A [1994], and G2385R [2385]) were treated with LPS (100 ng/mL) for the indicated times. Empty vector (mock) was used as a control. c-myc and α-tubulin were used as markers of LRRK2 expression and loading controls, respectively. Phosphorylation levels of p38, JNK, and MKK3/6 were analyzed by Western blotting. Phosphorylation of JNK was indicated with arrowhead. (<b>C</b>) Band intensities in (B) were quantified using a densitometer. Values are means ± SEMs of three independent experiments (<i>+p</i> = 0.054, <i>*p</i><0.05, **<i>p</i><0.01 vs. mock in LPS-untreated group [−LPS]; <i>#p</i><0.05; ##<i>p<0</i>.01 vs. mock in LPS-treated group [+LPS]; <i>ns</i>, not significant). Data are representative of three independent experiments.</p

The DNA-binding ability of p50 is increased in Lrrk2-KD microglia in response to LPS stimulation.

<p>(<b>A</b>) The DNA-binding activity of NF-κB was analyzed by EMSA. Nuclear extracts were prepared from control and Lrrk2-KD cells at the indicated times after LPS treatment. Specific binding was analyzed using an excess (20×) of unlabeled (Cold) consensus NF-κB sequence. Two specific NF-κB-DNA complex bands (arrowhead and arrow) were detected. (<b>B</b>) A supershift assays were performed using NF-κB p50 and p65 antibodies. Nuclear extracts obtained 30 min after LPS treatment were preincubated with antibodies. Arrows and arrowheads in (A, B) indicate p50/p50 complex and p50/p65 complex, respectively. (<b>C</b>) DNA affinity precipitation assays were performed using nuclear extracts prepared from untreated and LPS-treated control and Lrrk2-KD microglia. Nuclear extracts (100 µg protein) were incubated with biotin-labeled NF-κB consensus sequence, and then precipitated with streptavidin-conjugated agarose beads. The amount of p50 or p65 in the nuclear extracts (nuclear extract), and bound to DNA (DNA-binding) were analyzed by Western blotting. TBP was used as a nuclear marker. The arrowhead in (C) indicates p50. Data are representative of three independent experiments.</p