Supplemetary Materials Materials and Methods Stable knockdown of HDACs in murine embryonic fibroblasts For establishing a transient knockdown of HDAC1, HDAC2 and HDAC3, immortalized Hdac3 fl/- Mef cells were transfected with mouse pLKO.1-puro, or pLKO.1 encoding shRNAs derived from the TRC1 library (http://www.broadinstitute.org/rnai/trc/lib): pLKO.1-puro-shHdac1 (cloneIDs TRCN0000039399, TRCN0000039400, TRCN0000039402, TRCN0000039403), pLKO.1-puro-shHdac2 (cloneIDs TRCN0000039395, TRCN0000039396, TRCN0000039397, TRCN0000039398), pLKO.1-puro-shHdac3 (cloneIDs TRCN0000039389, TRCN0000039390, TRCN0000039391, TRCN0000039392). For transient transfection 3.5x104 Mef cells were seeded in 48 well plates. 270ng plasmid DNA per well was transfected using Lipofectamine® LTX and PlusTM reagent from InvitrogenTM, following the manufacturer’s instructions. 24h post transfection, transfected cells were selected for 48h by adding 1µg/ml puromycin. Each shRNA-encoding vector was transfected in duplicate wells. One well was kept untreated, while the second well was stimulated with IL-1α (10ng/ml) for 3h. After harvesting the cells on ice in 1x PBS, the samples were prepared for RT-qPCR analysis using the TaqMan® PreAmp Cells-to-CT™ Kit from Ambion®, following the manufacture’s instructions. The expression of mCxcl2 (Mm00436450_m1) and mUbe2l3 (Mm00784559_s1) was determined by qPCR using the TaqMan® Fast universal PCR master mix and 7500 Fast real time PCR system from Applied Biosystems. Relative changes of mCxcl2 mRNA expression compared to the unstimulated pLKO.1 control were normalized to the expression of mUbe2l3 and quantified using the 2-ΔΔCt method. Chromatin immunopecipitation (ChIP) One 175-cm2 flask of confluent KB cells (corresponding to 2.5 - 3.0 x107 cells), treated as described in the figure legends, was used for each condition. Proteins bound to DNA 1 were cross-linked in vivo with 1% formaldehyde added directly to the medium. After 10 minutes incubation at room temperature, 0.1 M glycine was added for 5 minutes to stop the cross-linking. Then, cells were collected by scraping and centrifugation at 1,610 x g (5 minutes, 4°C), washed in cold PBS containing 1mM PMSF and centrifuged again at 1,610 x g (5 minutes, 4°C). Cells were lysed for 10 minutes on ice in 3ml ChIP lysis buffer (1% SDS, 10mM EDTA, 50mM Tris pH 8.1, 1mM PMSF, Roche protease inhibitor mix). The DNA was sheared by sonication (7 x 30s on / 30s off, 4 times; Bioruptor, Diagenode) and lysates cleared by centrifugation at 16,100 x g at 4°C for 15 minutes. Supernatants were collected and stored in aliquots at –80°C for subsequent ChIP. For determination of DNA concentration 20µl of sheared lysate was diluted with 100µl TE buffer including 10µg/ml RNAse A. After 30min at 37°C, 3.8µl proteinase K (20mg/ml) and 1% SDS was added and incubated for at least 2h at 37°C followed by overnight incubation at 65°C. Samples were resuspended in two volumes of buffer NTB (Macherey & Nagel) and DNA was purified using Nucleo Spin columns (Macherey & Nagel) according to the manufacturer’s instructions. DNA was eluted with 50µl 5mM Tris pH 8.5 and concentration was determined by Nano Drop. For CHIP, the following antibodies were used: anti-histone H3 (2µg, Abcam; ab1791), anti-acetyl-histone H3 (K9) (2µg, Millipore; 07-352), anti-NF-κB p65 (3µg, Santa Cruz; sc-372), anti-HDAC3 (4µg, Millipore; 17-10238), anti-phospho-Pol II (S5) (1.35µg, Abcam; ab5131), IgG (2µg, Cell Signaling; 2729). Antibodies were added to precleared lysate volumes equivalent to 25µg of chromatin. Then, 900µl of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 167mM NaCl, 16.7mM Tris/HCl pH 8.1) were added and the samples were rotated at 4°C overnight. Thereafter, 30µl of a protein A/G sepharose mixture, pre-equilibrated in ChIP dilution buffer was added to the lysates and incubation continued for 2h at 4°C. Beads were collected by centrifugation, washed once in 900µl ChIP low salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris pH 8.1, 150mM NaCl), once in 900µl ChIP high salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris pH 8.1, 500mM NaCl), once in 900µl ChIP LiCl buffer (0.25M LiCl, 1% NP40, 1% desoxycholate, 1mM EDTA, 10mM Tris pH 8.1) and twice in 900µl ChIP TE buffer (10mM Tris pH 8.1, 1mM EDTA) for 5 minutes at 4°C. Beads were finally resuspended in 100µl TE buffer including RNAse A (10mg/ml). In parallel, 1/10 volume 2 (2.5µg) of the initial lysate (input samples) were diluted with 100µl TE buffer including 10µg/ml RNAse A. After 30min at 37°C, 3.8µl proteinase K (20mg/ml) and 1% SDS were added and both, input and immunoprecipitates were incubated for at least 2h at 37°C followed by overnight incubation at 65°C. Samples were resuspended in two volumes of buffer NTB (Macherey & Nagel) and DNA purified using Nucleo Spin columns (Macherey & Nagel) according to the manufacturer’s instructions. DNA was eluted with 50µl 5mM Tris pH 8.5 and stored at –20°C until further use. Quantification of ChIP DNA by real-time PCR PCR products derived from ChIP were quantitated by real time PCR using the Fast ABI 7500 instrument (Applied Biosystems). The following primers were used as described in (37). Hhuman IL-8 promoter, sense 5’-aagaaaactttcgtcatactccg-3’, antisense 5’- tggctttttatatcatcaccctac-3’; human IL-8 upstream (negative control; gene free region 940bp upstream of IL-8 transcriptional start site): sense 5’-atcatgggtcctcagaggtcagac-3’, and antisense, 5’-ggtgggagggaggtgttatctaatg -3’. The reaction mixture contained 2µl of ChIP or input DNA (diluted 1:10 to represent 1% of input DNA), 0.25µM of primers and 10µl of Fast Sybr Green Mastermix (2x) (Applied Biosystems) in a total volume of 20µl. PCR cycles were as follows: 95°C (20s), 40x (95°C (3sec), 60°C (30sec)). Melting curve analysis revealed a single PCR product.. Calculation of enrichment by immunoprecipitation relative to the signals obtained for 1% input DNA was performed according to the following equation: percent of (input)=2-(Ct sample-Ct input). 3 Supplementary Figures Fig. S1. Apicidin inhibits IL-1-induced IL-8 expression in tumor cells. KB cells (upper graph) or A549 cells (lower graph) were pretreated for 24 h with increasing concentrations of the HDAC3 inhibitor apicidin, solvent control (DMSO, (1 %), or were left untreated. Thereafter cells were stimulated for 3 h with IL-1 as shown. RT-qPCR was used to determine IL-8 mRNA expression, bars show the mean IL-8 expression ±SEM relative to the unstimulated control (KB cells n=2, A549 cells n=3). Fig. S2. Tamoxifen has no impact on IL-1-inducible Cxcl2 expression. Two immortalized Mef lines were treated for 72h with 10µM tamoxifen followed by 30 min of IL-1 treatment as shown or were left untreated as indicated. Total RNA was isolated and Cxcl2 mRNA expression was quantified by RT-qPCR. Fig. S3. shRNA-mediated suppression of HDAC3 in murine fibroblasts impairs IL- 1-induced Cxcl2 expression. Hdac3 fl/- Mefs were transfected with empty pLKO.1 or with pLKO.1 containing the indicated shRNA-expression cassettes directed against HDAC3. Cells were selected for two days in puromycin (1µg/ml), stimulated for 3 h with IL-1 or were left untreated. Thereafter mRNA expression of Cxcl2 was analyzed by RT-qPCR and normalized to the expression of Ube2l3 as described in the Methods section. Shown are mean fold changes +/- SEM relative to the vector control from duplicate determinations. Fig. S4. TSA triggers histone H3 acetylation. The indicated HEK293IL-1R control and HDAC3 knockdown cells were treated with TSA (300ng/ml, 24 h) or IL-1 as shown. Nuclear extracts were analyzed by Western blotting for TSA-mediated hyperacetylation of histones by use of antibodies against acetylated histone H3 (Ac-(K9/14)-H3). Antibodies against beta actin were used as a loading control, a representative experiment is shown. 4 Fig. S5. HDAC3-knockdown in human cells suppresses expression of several IL-1- response genes. The indicated HEK293IL-1R control and HDAC3 knockdown cells were stimulated for various periods with IL-1 as indicated. RT-qPCR was used to quantify mRNA expression of the indicated human genes. Changes of mRNA expression are displayed relative to the untreated control. Error bars represent SEM from two independent experiments. Fig. S6. Microarray-based identification of HDAC3-dependent IL-1-response genes in murine cells. Hdac3 fl/+ or Hdac3 fl/- Mefs were treated with tamoxifen- or IL-1 as described in the legend for Fig. 2C. RNA was isolated and labeled cRNAs were hybridized to whole murine genome Agilent microarrays. Fluorescence intensity values were used to calculate ratios of gene expression. (A) Depicted are color-coded ratio values for 95 genes which were regulated by IL-1 by at least 1.5-fold in the same direction in both independent experiments. Green vertical bars indicate all genes with at least twofold regulation by IL- 1. Yellow bars indicate genes whose expression was reduced in the HDAC3 knockout by a least twofold in two independent experiments. (B) Depicted are color-coded ratios values for 490 genes which were regulated by at least twofold in response to tamoxifen. In this group, only 7 genes (1.4%) were also responsive to IL-1 as indicated by red bars. Fig. S7. Identification of p65 K4Q or p65 K5Q-suppressed or -induced genes. The entire data set which was retrieved by sequential filtering of the expression values of the microarray experiments as described in the legend of Fig. 9C and D is shown. Depicted are color-coded ratio values for IL-1-regulated (green vertical bars) and p65 NF-κB-dependent genes (A) and for genes regulated by p65 K4Q or by K5Q mutants (B). Numbers 1-24 indicate experimental conditions as described in the legend of Fig. 9C, D. For further details see the Material & Methods section. Fig. S8. DNA-binding of p65 is not required for HDAC3-mediated deacetylation. HEK293T cells were transfected with expression plasmids encoding His-tagged p65 wild type or a DNA-binding mutant (p65 E39I-His) along with YFP-CBP and increasing 5 concentrations of an expression vector for HDAC3-Flag (0.3µg and 1µg) as shown. One fraction of cells was purified under denaturing conditions by Ni-NTA columns and analyzed for p65 acetylation as displayed, another fraction was analyzed for adequate protein expression. Fig. S9. p65 NF-κB is deacetylated by HDAC1 or HDAC2 but not by HDAC5 and HDAC8. HEK293IL-1R cells were transiently transfected to express wild type His-tagged p65 along with untagged p300, HA-tagged HDAC3, FLAG-tagged HDAC2, 5, 8 or MYC- tagged HDAC1. Cells were lyzed under denaturing conditions, proteins were precipitated by TCA, redissolved and equal amounts were analyzed for general acetylation of p65 with a pan-acetyl-lysine specific antibody or with antibodies specific for acetylated p65 K310, K314 and K315 as shown. Expression of p65, HDACs and p300 was validated as indicated and equal loading of lanes was confirmed by anti-β-actin antibodies. The position of a molecular weight marker is indicated at the left. Fig. S10. shRNA-mediated suppression of HDAC2, or HDAC3 in murine fibroblasts impairs IL-1-induced Cxcl2 expression. Hdac3 fl/- Mefs were transfected with empty pLKO.1 or with pLKO.1 containing the indicated shRNA-expression cassettes directed against HDAC1 (A), or HDAC2 (B). Cells were selected for two days in puromycin (1µg/ml), stimulated for 3 h with IL-1 (10ng/ml) or were left untreated. Thereafter mRNA expression of Cxcl2 was analyzed by RT-qPCR and normalized to the expression of Ube2l3 as described in the Methods section. Shown are mean fold changes +/- SEM relative to the vector control from duplicate determinations. Fig. S11: HDAC3 deletion does not affect TNF-induced genes. (A) Wild type (Hdac3 fl/+) or knockout (Hdac3 fl/-) Mefs were treated and analyzed exactly as described in the legend of Fig.2B except that cells were stimulated for 30 min with TNF (20ng/ml) instead of IL-1. RT-qPCR was used to quantify Cxcl2 mRNA expression. Shown are the mean –fold changes compared to the untreated Hdac3 fl/+ 6 control line, error bars represent SEM from three different experiments performed in duplicates. (B) Hdac3 fl/+ or Hdac3 fl/- Mefs were stimulated as described in (A) followed by RNA isolation and analysis of gene expression on whole murine genome Agilent microarrays. Fluorescence intensity values were used to calculate ratios of gene expression. Depicted are color-coded ratio values for 76 genes which were regulated by TNF by at least 1.5-fold in the same direction in both independent experiments. Purple vertical bars indicate all genes with at least twofold regulation by TNF. Blue bars indicate genes whose expression was reduced in the HDAC3 knockout by a least twofold in two independent experiments. Green bars indicate the overlap with IL-1-induced genes as shown in suppl. Fig.6B. 7 Supplementary Tables Table S1. Overlap between HDAC3-dependent IL-1 response genes and p65- dependent genes. The data set shown in Fig. S6 was used to extract 40 genes which are IL-1-dependent and whose expression is reduced by twofold in at least one of the two experiments. Using data described in Fig.9 and suppl. Fig. 7 the dependency of these genes on p65 NF-κB or its mutants is depicted. Table S2. HDAC3 is a global regulator of the transcriptional IL-1 response in human cells. Shown are gene identifiers, fluorescence intensity values and relative ratios of gene expression corresponding to the results depicted in Fig. 7B. Twofold regulated genes are indicated by yellow or green colors, respectively. Table S3. Microarray-based identification of HDAC3-dependent IL-1-response genes in murine cells.. Shown are gene identifiers, fluorescence intensity values and relative ratios of gene expression corresponding to the results depicted in Fig. S6A. Twofold regulated genes are indicated by yellow or green colors, respectively. 8 1400 KB 1200 1000 800 600 400 200 0 100 A549 80 60 40 20 0 apicidin (µg/ml, 24h) .1 1 10 .1 1 10 DMSO + + IL-1 + + + + + Figure S1 IL-8 mRNA (-fold) IL-8 mRNA (-fold) Mef lMiEnF eMK 12/5 wt mCxCL-2 MeMf ElFi nNCeor -D2AD wt mCxCL2 40 - - IL-1 3000 IL-1 30 2000 20 10 1000 0 0 - tamoxifen - tamoxifen Figure S2 Cxcl2 mRNA (-fold) Cxcl2 mRNA (-fold) 80 - 60 IL-1 40 20 0 Figure S3 Cxcl2 mRNA (-fold) pLKO.1 shHDAC3 (#1) shHDAC3 (#2) shHDAC3 (#3) shHDAC3 (#4) pS-Puro pS-HDAC3 IB: anti-(Ac)K9/14-H3 Ac-(K9/14)-H3 IB: anti--actin -actin 3h IL-1 + + + + TSA + + + + Figure S4 pS-Puro pS-HDAC3 250 200 75 150 50 100 25 50 0 0 125 25 100 20 75 15 50 10 25 5 0 0 - 0.5 1 3 8 16 24 IL-1(h) - 0.5 1 3 8 16 24 IL-1(h) Figure S5 mRNA (-fold) CXCL2 CXCL1 NFKBIA CXCL3 A log2 ratio -5 0 5 Cxcl2 Cxcl1 Ccl20 Cxcl10 Ccl7 Ccl2 Zfp36 Irf1 Nfkbiz Tnf Rnd1 Tnfaip3 Ch25h Cxcl5 Birc3 Serpine1 Il6 Ptgs2 1810011O10Rik Map3k8 Ier3 Mapk6 B2m Tlr2 Gadd45b Ier2 Btg2 Bcl10 Fos Junb Klf10 Zc3h12a 5830469G19Rik Atf3 Phlda1 Rgs16 Slc25a25 Csrnp1 Uncx Sdk2 Edn1 Gdf15 Nav3 Tnk1 Olfr1222 Tnip3 Egr2 Nfkbia Igf1 Pde4b Ammecr1l 2510017J16Rik Dusp1 Tslp Nfkbid Gdap5 6330407A03Rik Dusp8 Itgav Magix Rbbp8 Pim1 Vcam1 Ccrl2 Jun Slc43a2 8030431J09Rik Icam1 Neurl3 Dusp2 8430408G22Rik Elf3 Olfr661 Olfr391-ps Pax7 Ntsr1 Olfr971 Ttyh1 Srr Anks6 1700012D14Rik Ccl28 Galc Satb1 Wdr38 Tcstv1 9530013L04Rik Mxd4 Prickle2 Olfr975 2310035P21Rik 2610001A08Rik Pitx2 Hk3 Msh2 1 2 3 4 5 6 7 8 tamoxifen + + + + IL-1 + + + + Hdac3 fl/- 29 IL-1-regulated genes Cre-ER + + + + + + + + + suppressed by 2-fold in Hdac3 -/- cells in exp. 1 exp. 1 1 1 1 2 2 2 2 31 IL-1-regulated genes suppressed by 2-fold in Hdac3 -/- cells in exp. 2 35 IL-1-regulated genes by 2-fold in exp. 1 and exp. 2 Figure S6 B log2 ratio -5 0 5 Cxcl1 BC017612 Pkm2 Aldoart2 Slc5a3 Nrcam Angptl4 Cdhr1 Fam5c Ankrd37 Lgi2 Apln Gm129 Prss2 Pcdh9 Klhl1 Npy Prl2c5 Prl2c3 Ly6a Ly6f Podnl1 Casp4 Vwf Itgb2 Mmp10 Gsta3 Ampd3 4930452B06Rik Hmgcr Fdft1 Pmvk Ly6i Slc6a12 AI504432 Ereg Sox17 Fgf21 Tnfsf9 Prdm8 Il18rap Ptprb Lonrf3 Sgsm1 Mt2 Hsd17b7 Ccnd1 Fdps Prl7a2 Cyp51 Lrrc27 Mmp12 Fgl2 Hmgcs1 Eno3 Csta Kif1a Ccbe1 Pbp2 Jakmip2 Prkg2 Ldlr Kif21b Pdlim2 Crybb1 Pcyt2 Ctla2b Mvk Fabp4 Cd53 Ero1l Dach2 Klra12 Tmem171 Arap2 Vegfa Cldn15 Slc25a37 Gm1966 Dock5 Stard4 2010005H15Rik Adcy7 Unc93a Has2 Erdr1 1700016C15Rik March11 Ccdc88b Polr3g Gale Sh3bgrl2 Gramd1b Spn Stom 1600014C23Rik Elovl6 H2-DMb1 Dhcr7 Htr2b Mmab Syngr1 Fasn Serpinb6b Sqle Acsl4 Sc5d Trib3 Prelid2 Pappa Vcan 6330407J23Rik Dusp4 Ptpre Cd44 Fgf2 Fxyd5 Gvin1 H2-M3 Aacs Bnip3 Car5b Prl7a1 Mtap2 Adssl1 Ubash3b 1700027L20Rik Fgf7 Adcyap1r1 Fosl1 Ptgs2 Areg Phex Idi1 Vmn2r89 Prss46 Havcr2 Msln Gchfr Mvd Epas1 Klra22 Klra7 Klra15 Klra20 2310002L13Rik Gzme Gsta1 Gsta2 Rnf183 Scd1 Acss2 Angptl6 Aldh1l1 Arhgef16 Unc13a Ly6c2 Lss Acat2 Nsdhl Pcsk9 Ly6c1 Ctsw Cst6 Lrp8 Trim54 Apobec1 Cdca7l Slc2a6 Egln3 Tnni3 Insig1 Acat3 1700003F12Rik Aldoc Ak4 Speer3 Il6 Car6 Slc7a11 Acsbg1 Tm7sf2 Slc16a3 Mcpt8 4930583H14Rik A630095E13Rik Nfkbia Icam1 Neurl3 Gem Elf3 Pag1 Jazf1 Col5a3 Tgfa 4930444P10Rik Ms4a2 Ptplad2 Rbp4 Ccdc80 Klf15 Kcnj16 Entpd2 Cd1d2 Mfsd7c 5430407P10Rik Cpxm1 Enpep Cobl Sh3tc2 Tacc1 Efr3b Efna2 Il15 Lcorl Smagp Atl2 Synpo2 Wipf3 Plcb1 Ano1 Lgr6 IL-1-regulated genes: Mettl7a2 Rbm20 Galntl1 Ceacam1 Rasl11b Il6st 9030409G11Rik Hs3st3b1 Lama2 Mansc1 Mboat2 Lect2 Mfap5 Slc16a9 Vldlr Cldn2 Syt1 Lama3 Ccnjl 4930523C07Rik Bmp8a CNxeculr1l3 Tnfaip6 Tmtc1 Slc35f2 Pid1 Npr1 Ramp1 Camk4 D8Ertd82e Aplnr Lmod3 Col4a3 PIl6tgs2 Calml4 Tacstd2 Phyhip 4833424O15Rik Btbd3 Pfkfb3 Irf8 Klhl31 Samd5 Lce3c Dusp26 INl6fkbia Sp5 Tinag Ltbp2 Fam19a2 Pkhd1 Id4 Rgs6 Tnfrsf19 Sfrp2 Aard Itm2a NCxfkcbl1ia Sln Bean1 Nppa Cd79b Hist3h2ba Fam89a 2310043M15Rik Cxcr6 Slc16a7 Synm Tnnt3 EIclaf3m1 Wnt6 Lsp1 D3Bwg0562e Prr15 Ccdc88c Abca1 Pdzk1 Esr1 Hey1 Fibin P Rspo2 Ntegusr2l3 Ppp4r4 Abi3bp Spock2 Mylk 4933404M02Rik 2310015B20Rik 2900011O08Rik Zbtb8b 2310057J16Rik Tgfbi IEclaf3m1 D630039A03Rik Agt Lum LOC100503474 Keg1 Chrdl2 Adam5 Actr3b Sncaip Mcam Arhgap24 Cysltr1 Cftr Kcnn2 Popdc3 Omd Upk3b Slc1a6 Csf1r Pgr Hspa12a Ncald St6galnac2 Irx1 Nov Flrt3 Anpep Itga8 Scel Adam12 Aldh1a2 D630045M09Rik Col6a2 Tspan8 Adamts3 Gm106 Tmem86a Rai2 Padi2 Mme Gja1 Adra1b Amdhd1 Abcb11 Gbp3 Fry Ildr2 Bves Itgb6 Il1r1 Fst Cd55 Gpr160 Card10 Kctd12b Pde3a Gnai1 Chn2 Ramp2 Fgf5 Ncf2 Hey2 Col4a4 Dcn Fgf18 Icosl Nat8l Clstn2 Ggt6 Pcsk6 Cys1 Tspan33 Crb2 BC023202 Rassf10 Sdpr Dcdc2a Ppfia2 Kcnk4 Cdo1 Sox2 Krtap1-5 Kdr Kcng2 Ngfr Trdn Tmem56 Ceacam2 Slc35f1 Gap43 C77370 B3gnt8 Cldn10 Myh7 Bcam Krt7 Trpc6 Gabrb1 Kcnf1 1700024P16Rik Fam70a Cyp2s1 Kcne4 Gabra4 AI118078 C230098O21Rik Lrtm2 Cpne4 P4ha3 Gna14 Myh14 Zfp185 Dkk2 Tspan13 Thsd4 Dkk1 Dcaf12l1 E330013P04Rik Ttc22 Adamts16 Ptpn5 Mmp17 Sept4 Akr1c20 Fam19a1 Igfbp2 Gc 6330403K07Rik Igf2 Slc35d3 Rcsd1 Smtnl2 Tekt1 Prss12 Nkain4 C1qtnf3 Lrp2 Gm16485 Elmod1 4930449I04Rik Sh2d4a Greb1 Ptx3 Col2a1 Aldh1a3 Hist3h2bb-ps Cxcl17 1700011H14Rik Npy2r Oxtr Corin Wnt3 Clvs2 Cd200 Moxd1 Kit C4bp Gpr182 G6pc2 H19 Hpgd Lrrc55 Mro Grem2 Serpina1c Hpgds Chst8 Dio2 Pdzd2 Slc16a6 1700019N12Rik Lpl Pcp4l1 Akr1c14 Gjb2 2010300C02Rik C130060K24Rik Dhrs9 Dab1 Bmp3 Rasd1 Serpina1d 1 2 3 4 5 6 7 8 tamoxifen + + + + IL-1 + + + + Hdac3 fl/- Cre-ER + + + + + + + + + exp. 1 2 1 2 1 2 1 2 Figure S6 A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Figure S7 B p65 K4Q-induced genes p65 K4Q-repressed genes p65 K5Q-induced genes p65 K5Q-repressed genes IL-1-induced genes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Figure S7 Ni-NTA pulldown IB: anti-(Ac)K310-p65  Acetyl-K310 p65 IB: anti-p65  p65-His input IB: anti-FLAG  FLAG-HDAC3 IB: anti-p65  p65-His IB: anti-tubulin  tubulin p65-His + + + + p65 E39I-His + + + + YFP-CBP + + ++ + + ++ FLAG-HDAC3 + + + + Figure S8 kD TCA lysates IB: anti‐(Ac)K310‐p65 70  acetyl‐K310 p65 IB: anti‐(Ac)K314‐p65 70   acetyl‐K314 p65 70   acetyl‐K315 p65 IB: anti‐(Ac)K315‐p65 IB: anti‐(Ac)‐lysine 70   pan acetyl‐lysine p65 IB: anti‐p65 70   p65 55 IB: anti‐HDAC3  HDAC3‐HA  HDAC5‐Flag 130 95 IB: anti‐FLAG 70  HDAC2‐Flag 55  HDAC8‐Flag 70  unspecific IB: anti‐MYC  HDAC1‐Myc  p300 IB: anti‐p300 170 IB: anti‐‐actin 40  ‐actin p65-His + + + + + + + p300 + + + + + + HDAC1-MYC + HDAC2-FLAG + HDAC3-HA + HDAC5-FLAG + HDAC8-FLAG + Figure S9 2500 A - 2000 IL-1 1500 1000 500 0 1400 1200 - B 1000 IL-1 800 600 400 200 0 Figure S10 Cxcl2 mRNA (-fold) Cxcl2 mRNA (-fold) pLKO.1 pLKO.1 shHDAC2 (#1) shHDAC1 (#1) shHDAC2 (#2) shHDAC1 (#2) shHDAC2 (#3) shHDAC1 (#3) shHDAC2 (#4) shHDAC1 (#4) A 800 600 400 200 0 tamoxifen + + + + TNF + + + + Hdac3 fl/+Cre-ER + + + + + Hdac3 fl/- Cre-ER + + + + + Figure S11 Cxcl2 mRNA (-fold) B log2 ratio -5 0 5 1 2 3 4 5 6 7 8 tamoxifen + + + + TNF + + + + 10 TNFα-regulated genes suppressed by 2-fold in Hdac3 fl/- + + + + + + + + + Hdac3 -/- cells in exp. 1 Cre-ER exp. 1 1 1 1 2 2 2 2 12 TNFα-regulated genes suppressed by 2-fold in Hdac3 -/- cells in exp. 2 29 TNFα-regulated genes 2-fold in exp. 1 and exp. 2 60 IL-1-regulated genes 1.5-fold in exp. 1 and exp. 2 Figure S11 IL-1-induced IL-1-induced basal basal expression expression expression expression expression expression reduced in suppressed suppressed suppressed suppressed suppressed by EntrezGeneID GenbankAccession GeneSymbol HDAC3 -/- in p65 -/- by p65 K4Q by K5Q by K4Q K5Q 1 320897 AK033113 8030431J09Rik x 2 213393 NM_145980 8430408G22Rik x x 3 225339 AK030009 Ammecr1l x 4 12010 AK086184 B2m x 5 11796 BC011338 Birc3 x x 6 20296 NM_011333 Ccl2 x x 7 20297 NM_016960 Ccl20 x 8 20306 NM_013654 Ccl7 x x 9 54199 NM_017466 Ccrl2 x x x x 10 12642 NM_009890 Ch25h x x 11 14825 NM_008176 Cxcl1 x 12 15945 NM_021274 Cxcl10 x x 13 20310 NM_009140 Cxcl2 x x x x 14 20311 NM_009141 Cxcl5 x x 15 13537 NM_010090 Dusp2 x 16 18218 NM_008748 Dusp8 x 17 13654 NM_010118 Egr2 x x x 18 13710 NM_007921 Elf3 x 19 14281 NM_010234 Fos x 20 15894 NM_010493 Icam1 x x x 21 16000 AK050118 Igf1 x 22 16362 NM_008390 Irf1 x x x x 23 16410 AK171739 Itgav x 24 16476 NM_010591 Jun x 25 50772 AK011505 Mapk6 x 26 214854 NM_153408 Neurl3 x x 27 18035 NM_010907 Nfkbia x x 28 80859 NM_030612 Nfkbiz x x x 29 18578 AK171700 Pde4b x x x 30 18712 NM_008842 Pim1 x x x 31 225182 AK087007 Rbbp8 x 32 223881 NM_172612 Rnd1 x x x x 33 18787 AK040662 Serpine1 x x x 34 215113 AK090207 Slc43a2 x x 35 24088 NM_011905 Tlr2 x x x x 36 21926 NM_013693 Tnf x x x 37 21929 NM_009397 Tnfaip3 x x x x x x 38 53603 NM_021367 Tslp x x x 39 22329 NM_011693 Vcam1 x x x x x x 40 22695 NM_011756 Zfp36 x x 40 19 15 14 2 2 Table S1