Supplementary Materials for

Size: px

Start display at page:

Download "Supplementary Materials for"

Silvester Knight
6 years ago
Views:

1 Supplementary Materials for Small heterodimer partner mediates liver X receptor (LXR) dependent suppression of inflammatory signaling by promoting LXR SUMOylation specifically in astrocytes Jee Hoon Lee, Hyunmi Kim, Soo Jung Park, Joo Hong Woo, Eun-hye Joe, Ilo Jou The PDF file includes: Corresponding author. jouilo@aumc.ac.kr Published 2 ugust 216, Sci. Signal. 9, ra78 (216) DOI: /scisignal.aaf485 Fig. S1. LXRs interact with and induce expression of in a receptordependent manner. Fig. S2. is involved in LXR-mediated signaling pathways in brain astrocytes. Fig. S3. contributes to LXR-mediated anti-inflammatory effects in brain astrocytes. Fig. S4. Interactions between and LXR are critical for LXR-mediated antiinflammatory effects. Fig. S5. SUMOylation mediates the LXR-dependent anti-inflammatory effect. Fig. S6. promotes LXR SUMOylation. Fig. S7. promotes protein stability. Fig. S8. promotes -mediated LXR SUMOylation by inhibiting SIH1 binding to. Table S1. List of sirn oligonucleotides. Legend for data file S1 Other Supplementary Material for this manuscript includes the following: (available at Data file S1 (Microsoft Excel format). Gene expression analysis of brain astrocytes upon knockdown.

con (kda) 13 95 72 55 43 STT1 ccession Protein protein coverage (%) PI (isoelectric point) Number of peak MW (kda) P39948 cyclin D1 76.4 5.88 1 22.71 2RZ48 ardet-iedl syndrome 5 42.8 5.18 72 36.

2 con (kda) STT1 ccession Protein protein coverage (%) PI (isoelectric point) Number of peak MW (kda) P39948 cyclin D RZ48 ardet-iedl syndrome P97947 Q9QXK nuclear receptor subfamily gro up number 2 (NR2) signal transducer and activator of transcription feno (h) C si- si- - (1 nm) (1 nm) (1 nm) GPDH α-tubulin α-tubulin TNF-α GPDH Figure S1. LXRs interact with and induce expression of in a receptor-dependent manner. () Primary astrocytes were stimulated with for 2 hours in the presence of. Cell lysates were immunoprecipitated with the antibody, loaded onto SDS-PGE, and the resultant gel was stained with rilliant lue G. Proteins in select bands were identified using MLDI-TOF-MS, as noted for STT1 and. The table displays the results of the interacting proteins. () strocytes were treated with or fenofibrate (feno) for the indicated times, and transcript and protein abundance were analyzed by RT-PCR (top) and Western blotting (bottom), respectively. (C) strocytes were transfected with an - or specific sirn duplex or control sirn (). fter 48 hours, cells were stimulated with for 2 or 6 hours in the absence or presence of LXR ligands. transcript and protein levels were examined using RT-PCR and Western blotting, respectively.

3 cytokine activity cytokine binding chemokine activiry chemokine receptor binding interleukin-1 receptor binding tumor necrosis factor receptor binding chemokine receptor activity chemokine binding cytokine glycoprotein disulfide bond inflammatory response GOTERM_MF FT (Gene Ontology) P value <.5(),.1(),.1() count SP_PIR_KEYWORDS (Functional categories) pyrogen macrophage lymphokine secreated lipoprotein P value <.5(),.1(),.1() immune response regulation of cytokine production positive regulation of cytokine production positive regulation of response to stimulus cytokine-mediated signaling pathway regulation of cytokine secretion inflammatory response protein kinase cascade cell migration regulation of intracellular signal transduction cytokine-cytokine receptor type I diabetes mellitus apoptosis chemokine signaling NOD-like receptor signaling Natural killer cell mediated cytotoxicity MPK signaing Jak-STT signaling GOTERM_P FT (Gene Ontology) P value <.5(),.1(),.1() count KEGG_Pathway (Pathway) P value <.5(),.1(),.1() count count Figure S2. is involved in LXR-mediated signaling pathways in brain astrocytes. Functional grouping and signaling pathways of differentially regulated genes in -specific or nonsilencing control sirn-transfected brain astrocytes treated with or without and. ll the genes identified were examined for biological function according to the Gene Ontology Consortium and grouped in the respective functional categories. Pathway analysis of differentially expressed genes was carried out based on the latest data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

si- STT1 IgG Input PCR: pstt1 binding site

medias (CMs) 2 si- si- 15 F 1 5 Figure S3.

( to D) Primary astrocytes were transfected

fter transfection, - or -treated astrocytes

(), immunocytochemistry (C), or ChIP assay

4 Relative migration (fold) TNF-α (pg/ml) Merge TNF-α si- C si- ctin E 1 5 si- D IP si- STT1 IgG Input PCR: pstt1 binding site of IRF-1 (-158 ~ -8) astrocyte-conditioned medias (CMs) 2 si- si- 15 F 1 5 Figure S3. contributes to LXR-mediated anti-inflammatory effects in brain astrocytes. ( to D) Primary astrocytes were transfected with -specific (si-) or a control sirn (). Western blot analysis confirmed gene knockdown in cells (). fter transfection, - or -treated astrocytes were stimulated with and prepared for ELIS (), immunocytochemistry (C), or ChIP assay (D). (E and F) Representative images (E) and analysis (F) of a microglia migration transwell assay, in which the bottom well contained conditioned media from astrocytes transfected with si- or. Scale bar, 4 μm. p<.5 (n=3; mean±s.d.).

Merge TNF-α TNF-α (pg/ml) mock WT -WT -MT M1 M2 M3 C 18 27 PTILYTLLSP 118 129

-WT -MT F IP -WT -MT STT1 IgG Input PCR: pstt1 binding site of IRF-1 (-158 ~ -8)

Interactions between and LXR are critical for LXR-mediated anti-inflammatory effects.

() HEK293T cells were transfected with -tagged wild-type (WT) or mutant [M1-M3; ()].

[-MT; as in Fig. 1H, a.k.a. the M1 mutant in ()].

5 Merge TNF-α TNF-α (pg/ml) mock WT -WT -MT M1 M2 M3 C PTILYTLLSP PSILKKILLEEP GRLRILLM M1 M2 M3 IP D ctin actin input -WT -MT E -WT -MT F IP -WT -MT STT1 IgG Input PCR: pstt1 binding site of IRF-1 (-158 ~ -8) Figure S4. Interactions between and LXR are critical for LXR-mediated anti-inflammatory effects. () Schematic of the protein, showing the sequences and locations of the three putative nuclear receptor binding motifs. rrows show the amino acids changed to alanine in the three mutants, M1-M3. () HEK293T cells were transfected with -tagged wild-type (WT) or mutant [M1-M3; ()]. Subsequently, cell lysates were immunoprecipitated with the indicated antibodies, followed by immunoblot analysis. (C to F) Primary astrocytes were transfected with -tagged wild-type (-WT) or mutant [-MT; as in Fig. 1H, a.k.a. the M1 mutant in ()]. Western blot analysis confirmed overexpression in cells (C). fter transfection, or -treated astrocytes were stimulated with and prepared for ELIS (D), immunocytochemistry (E), or ChIP assay (F). Input indicates control PCR and shows the amount of IRF-1 promoter DN present in each sample before ChIP. p<.5 (n=3; mean ±S.D.). Scale bars, 5 μm.

6 IL-1β (fold) IL-1β (fold) TNF-α (fold) TNF-α (fold) si-sumo1 si-sumo2 -WT -K3R Figure S5. SUMOylation mediates the LXR-dependent anti-inflammatory effect. () qrt-pcr for the indicated mrns in astrocytes transfected with SUMO1- or SUMO2-specific sirn and stimulated with in the presence of individual LXR ligands. () qrt-pcr for the indicated mrns in astrocytes transfected with -tagged wild-type (-WT) or a SUMOylation site-mutant (-K3R) and subsequently treated with in the absence or presence of LXR ligands. p<.5 (n=3; mean ±S.D.).

-WT si-hdc4 HDC4 py-stt1 HDC4 SUMO2/3 HDC4 actin IP WCL E1 E2 SUMO2/3 GST-HDC4 His- SUMO2/3 72 55 72 55 M r (K) WT

() Coimmunoprecipitation in astrocytes transfected with -tagged -WT or with HDC4 sirn and treated with in the

7 -WT si-hdc4 HDC4 py-stt1 HDC4 SUMO2/3 HDC4 actin IP WCL E1 E2 SUMO2/3 GST-HDC4 His- SUMO2/ M r (K) WT MT SUMO2/3- SUMO2/3- Figure S6. promotes LXR SUMOylation. () Coimmunoprecipitation in astrocytes transfected with -tagged -WT or with HDC4 sirn and treated with in the absence or presence of. () was immunoprecipitated from the cells and an in vitro SUMOylation assay was performed in a reaction with the elements as indicated atop the Western blot.

Relative density si- actin 1.2 1.8.6.4.2 1.5 fold change1.5 n.s n.

s D si- Myc- E - - Myc-WT DPI Myc- SUMO1 IP: - Myc-D1 - Myc-D2 - Myc-D3 Myc input SUMO1

() strocytes transfected with si- or were treated and, followed by immunoblotting with

and intensities were quantified via scanning densitometry (lower panel). p<.

() Primary astrocytes were transfected with -specific sirn or with -WT for 48 hours.

(C) was immunoprecipitated from the cells and an in vitro ubiquitination assay was

(ub) and. n.s, non-specific. (D) Primary astrocytes were transfected with si- or Myc-.

8 Relative density si- actin fold change1.5 n.s n.s con si- -WT C E1/TP E2 ub E3 GST-SIH1 His- ub -Ub -Ub n.s D si- Myc- E - - Myc-WT DPI Myc- SUMO1 IP: - Myc-D1 - Myc-D2 - Myc-D3 Myc input SUMO1 Myc actin WCL Figure S7. promotes protein stability. () strocytes transfected with si- or were treated and, followed by immunoblotting with anti- antibody (upper panel). and intensities were quantified via scanning densitometry (lower panel). p<.5 (n=3; mean ±S.D.). () Primary astrocytes were transfected with -specific sirn or with -WT for 48 hours. transcript abundance was determined with qrt-pcr. n.s, non-significant. (C) was immunoprecipitated from the cells and an in vitro ubiquitination assay was performed in a reaction with elements as indicated atop the Western blots for ubiquitin (ub) and. n.s, non-specific. (D) Primary astrocytes were transfected with si- or Myc-. t 48 hours after transfection, cells were stimulated with with Protein interactions were measured via immunoprecipitation using the antibody. (E) HEK293T cells overexpressing - and either Myc--WT or one of three Myc- deletion mutants (D1 D3; Fig. 4F) were analyzed using PL to detect interactions between and variants. Scale bars, 1 μm.

9 Figure S8. promotes -mediated SUMOylation by inhibiting SIH1 binding to. () Primary astrocytes were transfected with - or SIH1-specific sirn. t 48 hours after transfection, cells were stimulated with with Protein interactions were measured via immunoprecipitation using the antibody. ( and C) Primary astrocytes were transfected with si- or. SIH transcript and protein amounts were determined using qrt-pcr () and Western blotting (C), respectively. Transcripts Target sequences 5'- UGGGGCGUUCGGG-3' 5'- GGCGUCCGCUUG-3' 5 -CUCGCUCUCCUGU-3 SIH1 5 -CGGUUUGUGUUCGU-3 SIH2 5 -CUGCCCGUUGGUCUCU-3 5'-CUUCGGGUUCGGC-3' HDC4 5'-CCGUGGGCUUCGGUUU-3' SUMO1 5'-GGGGCGUGUUG-3' SUMO2 5'-CGCGGCCCGG-3' Table S1. List of sirn oligonucleotides. The 5-3 sequence of the sirns used in this study, corresponding to the encoded protein (left), are listed. Data file S1. Gene expression analysis of brain astrocytes upon knockdown. Microarray data pertaining to the heatmap in Fig. 1C, provided in an Excel sheet in the online supplemental materials. The data are also available in NCI GEO, accession number GSE fc, fold change between the conditions noted (coded 1-4, legend at top).

RAW264.7 cells stably expressing control shrna (Con) or GSK3b-specific shrna (sh-

1 a b Supplementary Figure 1. Effects of GSK3b knockdown on poly I:C-induced cytokine production. RAW264.7 cells stably expressing control shrna (Con) or GSK3b-specific shrna (sh- GSK3b) were stimulated