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1 DOI:.38/ncb1963 a wild 5.3kb 11.2kb targeting vector stop PCR primer KKpn NNhe targeted allele 5.7kb 6.8kb probe b d (g) genomic Southern blot Kpn I digest Nhe I digest / / / / / / 11.2 kb 5.7 kb 6.8 kb 5.3 kb 15 WT WT / 5 / (week) genomic PCR / / / e kbp.4 kbp OS/BS c primary osteoblasts kda OV/BV OAfull actin OAN O.Th (m) f 15 5 Ob.S/BS MS/BS MAR (m day 1 ) g CFUF CFUALP CFUO h ES/BS Oc.S/BS. i OPG (pg ml 1 ) OPG RANKL WT / Figure S1 Generation and phenotype of / mice. (a) Structure of the genomic locus, targeting vector, and targeted allele after homologous recombination (two sets of primers were used to distinguish mutant from WT loci; probes were used for genomic Southern blotting). (b) Southern blot analysis of tails digested with the indicated enzyme and PCR genotyping. Each band is detected in the expected manner. (c) Western blotting of using lysates from primary cultured osteoblasts. is expressed as fulllength (OAfull) and cleaved fragments (OAN) in primary osteoblasts, but is not detected in primary osteoblasts from / mice. (d) Representative body weight plotted against age in / (WT) and / littermates. / mice were born at the expected Mendelian ratios, viable, and had no histological evidence of internal organ dysfunction including the brain; however, both male and female / mice exhibit growth retardation. / mice displayed no obvious anatomical or histological abnormalities compared with WT. (e) Osteoid measurements reveal osteoid surface (OS/BS), osteoid volume (OV/BV), and osteoid thickness (O.Th) were significantly decreased in / (mean ± SD, N = 5, **p <.1, *p<.5; ttest). (f) Reductions in the mineral apposition rate (MAR) and mineralizing surface (MS/BS) with the decrease of the osteoblast surface (Ob.S/ BS) in / versus WT (mean ± SD, N = 5, **p <.1, *p<.5; ttest). (g) Total colony forming unitsfibroblasts (CFUF), colony forming unitsalkaline phosphatase (CFUALP), and colony forming unitsosteoblasts (CFUO) in bone marrow stromal cell cultures of WT and / mice (mean ± SD, N = 4, *p<.5; ttest). (h) Histomorphometric analysis reveals that a similarly eroded surface (ES/BS) and osteoclast surface (Oc.S/BS) are not significantly changed in / mice compared with WT (mean ± SD, N = 5). (i) OPG and RANKL levels in primary osteoblasts. Left; measurements of OPG in supernatants of WT and / osteoblast cultures. OPG are not significantly changed in / osteoblasts compared with WT (mean ± SD, N = 4). Right; RTPCR analysis of OPG and RANKL mrnas. There are no differences in the expressions of OPG and RANKL between WT and / osteoblasts Macmillan Publishers Limited. All rights reserved.
2 osteoclast chondrocyte osteocyte Figure S2 Electron microscopic analysis in tibiae of 4monthold WT and / mice. No structural changes are observed in osteocytes (scale Bar = 3 µm), osteoclasts (scale Bar = 6 µm), and chondrocytes (scale Bar = 6 µm) in / mice Macmillan Publishers Limited. All rights reserved.
3 a Col1a2 Ocn Bsp Alp b Ocn Osterix Osteonectin HSP47 c P3H1 actin Col1a2 Ocn Bsp WT / Alp Osterix actin Col1a2 O Bsp Alp Osterix actin Figure S3 Comparison of gene expressions in bone tissues between WT and / mice at postnatal 4 days (P4). (a) RTPCR analysis of P4 calvaria from WT and / mice. and Col1a2 mrnas are downregulated, and Osteocalcin (Ocn), Osteopontin (), Bone sialoprotein (Bsp), and Alkaline phosphatase (Alp) mrnas are upregulated in calvaria of / mice. (b) In situ hybridization analysis of Ocn and using P4 WT and / tibiae (scale Bar = 2 µm). (c) RTPCR analysis of various genes in cultured osteoblasts from WT and / mice. Right panel, quantitative analysis of RTPCR (mean ± SD, N = 3, **p<.1; ttest) Macmillan Publishers Limited. All rights reserved.
4 a BiP CHOP ATF4 XBP1 PDI fold induction BiP CHOP ATF4 XBP1 PDI ERdj4 EDEM WT / ERdj4 b EDEM c Tm (h) BiP BiP CHOP ATF4 XBP1 CHOP Perk ATF6 actin Figure S4 ER stress response in / osteoblasts. (a) RTPCR analysis of P4 calvaria in WT and / mice. The indicated UPRrelated genes in / calvaria are not changed compared with those of WT. A right panel shows the quantitative analysis of RTPCR (mean ± SD, N = 3). (b) In situ hybridization analysis of BiP and CHOP in WT and / tibiae at P4. There are no differences in the expressions of BiP and CHOP between WT and / tibia (scale Bar = 2 µm). (c) RTPCR analysis of UPRrelated genes in primary cultured osteoblasts in WT and / mice. Osteoblasts were exposed to 3 µg ml 1 tunicamycin (Tm) for the indicated time, and mrna was then extracted to examine the inductive level of each gene. The induction of each gene is almost the same in WT and / osteoblasts Macmillan Publishers Limited. All rights reserved.
5 a mock OAC2 OAC1 OAfull kda OAN actin b mock OAC1 OAC2 Ocn Bsp Alp Osterix ATF4 HSP47 BiP CHOP sec23a sec23b c non Tg WT BFA BMP2 / Hoechst Figure S5 Alterations of genes in MC3T3E1 cells infected with adenovirus expressing and immunostaining of in primary osteoblasts. (a) Western blotting of lysates from MC3T3E1 cells infected with each adenovirus using anti antibody. OAC1 and C2 are the adenovirus indicated as expressing clone1 and clone2, respectively. The expression levels of in infected cells are shown. (b) RTPCR analysis of bone formationrelated genes and ER stressrelated genes in MC3T3E1 cells infected with adenovirus expressing. The expression levels of these genes were not affected. (c) Indirect immunofluorescence with anti antibodies showing the subcellular localization of. Hoechst staining showing the nucleus. Cells were treated with 1 µm thapsigargin (TG) or 1 µg ml 1 brefeldin A (BFA) for 4 h. For BMP2 treatments, cells were cultured with ng ml 1 BMP2 for 5 days. Note that the immunoreactivities of are detected in the perinuclear area and also weak signals in nucleus under normal conditions. By contrast, signals accumulate in the nucleus after ER stress or treatments with BMP2 (scale Bar = µm). /; / osteoblasts Macmillan Publishers Limited. All rights reserved.
6 BMP2 fulllength mild ER stress ER mrna Nterminal nucleus secretion of bone matrix UPRE like sequence bone formation Figure S6 Putative mechanisms responsible for bone formation by in osteoblasts Macmillan Publishers Limited. All rights reserved.
7 Fig. 1c Fig. 2f Fig. 2g 3 bp actin TRAP 317 bp 153 bp Type I collagen control calvaria tibia control Type I collagen skin Fig. 3a Col1a2 actin actin actin 485 bp 3 bp 155 bp 153 bp calvaria tibia Fig. 3g Fig. 3h probe vector FLAGOAN competitor: wt competitor: mt antiflag wt mt IP: IP: IgG mock mock mock IP: acetylh3 mock input Fig. 4b 3 bp actin 153 bp Fig. 4a OCN actin 292 bp 155 bp actin Fig. S5a Fig. 4d actin actin actin Figure S7 Full scans of key figures Macmillan Publishers Limited. All rights reserved.
8 Supplement table fwd CCTTGTGCCTGTCAAGATGGAG rev GCAGCAGCCATGGCAGAGGAG osteocalcinfwd AAGCAGGAGGGCAATAAGGT osteocalcinrev AGCTGCTGTGACATCCATAC betaactinfwd TCCTCCCTGGAGAAGAGCTAC betaactinrev TCCTGCTTGCTGATCCACAT TRAPfwd AAATCACTCTTTAAGACCAG TRAPrev TTATTGAATAGCAGTGACAG procollagen 1a1fwd CCCCAACCCTGGAAACAGAC procollagen 1a1rev GGTCACGTTCAGTTGGTCAAAGG procollagen 1a2fwd GCAATCGGGATCAGTACGAA procollagen 1a2rev CTTTCACGCCTTTGAAGCCA OPNfwd TCACCATTCGGATGAGTCTG OPNrev ACTTGTGGCTCTGATGTTCC ALPfwd GCCCTCTCCAAGACATATA ALPrev CCATGATCACGTCGATATCC fwd CCGCACGACAACCGCACCAT rev CGCTCCGGCCCACAAATCTC Osterixfwd CTGGGGAAAGGAGGCACAAAGAAG Osterixrev GGGTTAAGGGGAGCAAAGTCAGAT osteonectinfwd ATTTGAGGACGGTGCAGAGG osteonectinrev TCTCGTCCAGCTCACACACCT HSP47fwd ACCACAGGATGGTGGACAACCGT HSP47rev ATCTCGCATCTTGTCTCCCTTGGG P3H1fwd GTGCAGGCAGATGACCTGGT P3H1rev TCACGCTGCTGACGGCAGCT BiPfwd GTTTGCTGAGGAAGACAAAAAGCTC BiPrev CACTTCCATAGAGTTTGCTGATAAT CHOPfwd GTCCAGCTGGGAGCTGGAAG CHOPrev CTGACTGGAATCTGGAGAG XBP1fwd ACACGCTTGGGAATGGACAC XBP1rev CCATGGGAAGATGTTCTGGG ATF4fwd GGACAGATTGGATGTTGGAGAAAATG ATF4rev GGAGATGGCCAATTGGGTTCAC PDIfwd CAAGATCAAGCCCCACCTGAT PDIrev AGTTCGCCCCAACCAGTACTT ERdj4fwd CCCCAGTGTCAAACTGTACCAG ERdj4rev AGCGTTTCCAATTTTCCATAAATT EDEMfwd TGGGTTGGAAAGCAGAGTGGC EDEMrev TCCATTCCTACATGGAGGTAGAAGGG PERKfwd TCTTGGTTGGGTCTGATGAAT PERKrev GATGTTCTTGCTGTAGTGGGGG ATF6fwd GGATTTGATGCCTTGGGAGTCAGAC ATF6rev ATTTTTTTCTTTGGAGTCAGTCCAT BSPfwd AACAATCCGTGCCACTCA BSPrev GGAGGGGGCTTCACTGAT OPGfwd AACCCCAGAGCGAAACACAGT OPGrev GGCTCTCCATCAAGGCAAGAA RANKLfwd CCAGCATCAAAATCCCAAGTT RANKLrev TCAAGGTTCTCAGTGGCACAT Sec23afwd GACCTACCACCCATCCAGTACGAG Sec23arev CTGCATGGACTCCTTCAGAGCCTG Sec23bfwd CAGGAGATGCTGGGCCTGACCAAGTC Sec23brev CCACAGATCTTCCACTGACTTGTG Macmillan Publishers Limited. All rights reserved.
(A) Dose response curves of HMLE_shGFP (blue circle), HMLE_shEcad (red square),
Supplementary Figures and Tables Figure S1. Validation of EMT-selective small molecules (A) Dose response curves of HMLE_shGFP (blue circle), HMLE_shEcad (red square), and HMLE_Twist (black diamond) cells
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DOI: 10.1038/ncb2607 Figure S1 Elf5 loss promotes EMT in mammary epithelium while Elf5 overexpression inhibits TGFβ induced EMT. (a, c) Different confocal slices through the Z stack image. (b, d) 3D rendering
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SUPPLEMENTARY INFORMATION Supplementary Figure 1. Generation of a conditional allele of the Kindlin-2 gene. (A) A restriction map of the relevant genomic region of Kindlin-2 (top), the targeting construct
SUPPLEMENTARY FIGURES AND TABLE
SUPPLEMENTARY FIGURES AND TABLE Supplementary Figure S1: Characterization of IRE1α mutants. A. U87-LUC cells were transduced with the lentiviral vector containing the GFP sequence (U87-LUC Tet-ON GFP).
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(A) PCR primers (arrows) designed to distinguish wild type (P1+P2), targeted (P1+P2) and excised (P1+P3)14-
1 Supplemental Figure Legends Figure S1. Mammary tumors of ErbB2 KI mice with 14-3-3σ ablation have elevated ErbB2 transcript levels and cell proliferation (A) PCR primers (arrows) designed to distinguish
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DOI:.38/ncb2822 a MTC02 FAO cells EEA1 b +/+ MEFs /DAPI -/- MEFs /DAPI -/- MEFs //DAPI c HEK 293 cells WCE N M C P AKT TBC1D7 Lamin A/C EEA1 VDAC d HeLa cells WCE N M C P AKT Lamin A/C EEA1 VDAC Figure
Supplementary Table 1. List of primers used in this study
Supplementary Table 1. List of primers used in this study Gene Forward primer Reverse primer Rat Met 5 -aggtcgcttcatgcaggt-3 5 -tccggagacacaggatgg-3 Rat Runx1 5 -cctccttgaaccactccact-3 5 -ctggatctgcctggcatc-3
Probe. Hind III Q,!?R'!! /0!!!!D1"?R'! vector. Homologous recombination
Supple-Zhang Page 1 Wild-type locus Targeting construct Targeted allele Exon Exon3 Exon Probe P1 P P3 FRT FRT loxp loxp neo vector amh I Homologous recombination neo P1 P P3 FLPe recombination Q,!?R'!!
T H E J O U R N A L O F C E L L B I O L O G Y
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Supplementary methods:
Supplementary methods: Primers sequences used in real-time PCR analyses: β-actin F: GACCTCTATGCCAACACAGT β-actin [11] R: AGTACTTGCGCTCAGGAGGA MMP13 F: TTCTGGTCTTCTGGCACACGCTTT MMP13 R: CCAAGCTCATGGGCAGCAACAATA
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Supplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein content relative to GAPDH in two independent experiments.
Figure S1. Generation of inducible PTEN deficient mice and the BMMCs (A) B6.129 Pten loxp/loxp mice were mated with B6.
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supplementary information
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DOI:.38/ncb3399 a b c d FSP DAPI 5mm mm 5mm 5mm e Correspond to melanoma in-situ Figure a DCT FSP- f MITF mm mm MlanaA melanoma in-situ DCT 5mm FSP- mm mm mm mm mm g melanoma in-situ MITF MlanaA mm mm
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DOI: 10.1038/ncb2566 Figure S1 CDKL5 protein expression pattern and localization in mouse brain. (a) Multiple-tissue western blot from a postnatal day (P) 21 mouse probed with an antibody against CDKL5.
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Supplemental Figure 1. Furin is efficiently deleted in CD4 + and CD8 + T cells. a, Western blot for furin and actin proteins in CD4cre-fur f/f and fur f/f Th1 cells. Wild-type and furin-deficient CD4 +
Supplemental Figure 1
1 Supplemental Figure 1 Effects of DATE shortening on HGF promoter activity. The HGF promoter region (-1037 to +56) containing wild-type (30As) or truncated DATE (26As, 27As, 28A, 29As) from breast cancer
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1. Supplementary Figures and Legends Supplementary Fig. 1. S1P-mediated transcriptional regulation of integrins expressed in OP/monocytoid cells. Real-time quantitative PCR analyses of mrna for two integrins,
Supplementary data Supplementary Figure 1 Supplementary Figure 2
Supplementary data Supplementary Figure 1 SPHK1 sirna increases RANKL-induced osteoclastogenesis in RAW264.7 cell culture. (A) RAW264.7 cells were transfected with oligocassettes containing SPHK1 sirna
Predictive PP1Ca binding region in BIG3 : 1,228 1,232aa (-KAVSF-) HEK293T cells *** *** *** KPL-3C cells - E E2 treatment time (h)
Relative expression ERE-luciferase activity activity (pmole/min) activity (pmole/min) activity (pmole/min) activity (pmole/min) MCF-7 KPL-3C ZR--1 BT-474 T47D HCC15 KPL-1 HBC4 activity (pmole/min) a d
A. One to three months of age. Anterior Lens (Mean ± SEM) Posterior Lens (Mean ± SEM) Mid Lens (Mean ± SEM) Cornea (Mean ± SEM) Genotype
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Kidney. Heart. Lung. Sirt1. Gapdh. Mouse IgG DAPI. Rabbit IgG DAPI
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Downstream Col1A locus S P P P EP Genotyping with P1, P2 frt PGKneopA + frt hygro-pa Targeting vector Genotyping with P3, P4 P1 pcag-flpe P2 P3 P4 frt SApA CAG LSL PGKATG frt hygro-pa C. D. E. ormal KRAS
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Supplementary Figures
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p = formed with HCI-001 p = Relative # of blood vessels that formed with HCI-002 Control Bevacizumab + 17AAG Bevacizumab 17AAG
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control kda ATGL ATGLi HSL 82 GAPDH * ** *** WT/cTg WT/cTg ATGLi AKO/cTg AKO/cTg ATGLi WT/cTg WT/cTg ATGLi AKO/cTg AKO/cTg ATGLi iwat gwat ibat
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