Supplementary Figure 1: Hsp60 / IEC mice are embryonically lethal (A) Light microscopic pictures show mouse embryos at developmental stage E12.
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- Amice Hodges
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1 Supplementary Figure 1: Hsp60 / IEC mice are embryonically lethal (A) Light microscopic pictures show mouse embryos at developmental stage E12.5 and E13.5 prepared from uteri of dams and subsequently genotyped. The visceral yolk sac is placed right next to the embryos. The table shows the frequency of offspring carrying the corresponding genotypes. (B) Representative agarose gels validating the Hsp60 knockout in embryonic abdominal samples of Hsp60 flox/flox X VillinCre Tg mice. 1
2 Supplementary Figure 2: Mild signs of mucosal inflammation in jejunum of Hsp60 / IEC mice (A) qrt-pcr analysis of cytokines and chemokines was performed on mrna isolated from whole jejunal tissue pieces (N=5 per genotype). Statistical analysis was performed via t-test comparing genotypes. (B) Representative pictures including detailed images show macrophage infiltrates into jejunal mucosa. Quantification of infiltrating F4/80-positive macrophage into jejunal mucosa (evaluation of 20 crypts of one representative mouse per genotype). Lines in the dot plots indicate mean numbers. Asterisk indicate significant differences *P<0.05; **P<0.01; ***P<
3 Supplementary Figure 3: HSP60 deficiency does not induce epithelial apoptosis or a loss of mitochondria 3
4 (A). The number and diameter of mitochondria in villus and crypt IEC was assessed by electron microscopy of jejunal sections. Arrows indicate differences in mitochondrial christae formation in in Hsp60 / IEC vs. CTRL mice. Right: Mitochondrial numbers and diameter in Hsp60 / IEC mice (N=4) and Hsp60 flox/flox control mice (N=3) (B) Ratio of mitochondrial to nuclear DNA measured by qpcr in IEC isolated from villi and crypt of Hsp60 / IEC mice and Hsp60 flox/flox control mice (N=6). (C) Representative images of jejunal sections from Hsp60 / IEC and Hsp60 flox/flox control mice stained via TUNEL assay to assess apoptotic DNA fragmentation including detailed images in higher magnification (DAPI stains nuclei in cyan). Quantification of TUNEL positive cells per crypt-villus unit (N=5). Positive controls from DNaseI treated tissue sections are indicated by white arrows. Lines in the dot plots indicate mean numbers. Statistical analyses were performed via t-tests comparing genotypes. 4
5 Supplementary Figure 4: Quantification of hyperproliferative, HSP60-positive crypt nodules induced by HSP60 loss in Hsp60 / IEC and in Chop -/- Hsp60 / IEC mice (A) Representative HSP60 IHC stainings of Hsp60 / IEC and Chop -/- Hsp60 / IEC mice along the small intestinal tract following proximal to distal compartments. (B) HSP60-positive crypt nodules were quantified along the intestinal tract using HSP60 IHC staining. Comparison of nodule perimeter of HSP60-positive crypt nodules of Hsp60 / IEC and Chop -/- Hsp60 / IEC mice (N=6) revealed no differences between genotypes. Lines in the dot plot indicate mean numbers. Statistical analyses were performed via t-tests comparing genotypes. 5
6 Supplementary Figure 5: Epithelial regeneration in Chop -/- Hsp60 / IEC mice 6
7 (A) Schedule for oral tamoxifen administration to induce HSP60 deficiency and subsequent regeneration time. Representative H&E and corresponding HSP60 IHC stainings of Chop -/- Hsp60 / IEC mice along the intestinal tract following proximal to distal compartments. Detailed images of HSP60 IHC in higher magnification distinguish villus vs. crypt regions of the jejunum. Quantification of HSP60-positive and HSP60-negative crypt numbers of Chop -/- Hsp60 / IEC mice (N=4) at different time points after Hsp60 deletion indicate rapid tissue reconstitution. Bars represent mean +SEM. (B) IF Co-staining of HSP60 (green) and Ki67 (red) on jejunal sections including detailed images in higher magnification of Chop -/- Hsp60 / IEC mice and Chop -/- Hsp60 flox/flox controls (DAPI stains nuclei in cyan). Quantification of Ki67-positive cells in HSP60- positive and HSP60-negative crypts (N=5; 2 regions per mouse). (C) qpcr analysis of mtcoxi and Otc mrna expression in IEC isolated from the crypt compartment of Hsp60 / IEC and Chop -/- Hsp60 / IEC mice. Lines in the dot plots indicate mean numbers. Statistical analyses were performed via unpaired t-tests comparing genotypes. 7
8 Supplementary Figure 6: Expression of Hsp60 and Lgr5 following tamoxifen treatment of organoids (A) Schematic illustration of the experimental setup using small intestinal organoids. Organoids were isolated from Hsp60 flox/flox, VillinCreER T2-Tg mice and distributed to four protocols. (B) mrna expression levels of Hsp60 at d 1 and d 4, respectively, after tamoxifen treatment. (C) qrt-pcr analysis of Lgr5 mrna expression after tamoxifen treatment. Bars represent mean +SEM. Asterisk indicate significant differences ***P< One-Way ANOVA and appropriate post-hoc tests were used for all statistical analyses. Data from organoid experiments derive from at least 3 independent experiments. 8
9 Supplementary Figure 7: Epithelial expression of WNT-related factors and RSPO1 antibody validation 9
10 (A) mrna expression pattern of WNT-related factors found to be significantly altered in cryptderived IEC from Hsp60 / IEC mice in mucosal tissue (Mucosa), villus-derived IEC and cryptderived IEC from Hsp60 flox/flox and Hsp60 / IEC mice. Gene regulation is enriched in crypt-derived IEC fractions. (B) IF stainings of RSPO1 (red) and E-Cadherin (grey) at d 2 in jejunal sections from Hsp60 / IEC mice. A specific blocking peptide was used on consecutive sections (lower panel) with otherwise unchanged staining protocol (DAPI stains nuclei in cyan). (C) IF staining of RSPO1 (red) in splenic sections serving as positive tissue control. A specific blocking peptide was used on consecutive sections (lower panel) with otherwise unchanged staining protocol (DAPI stains nuclei in cyan). (D) mrna expression pattern of WNT-related factors found to be significantly altered in villus-derived IEC from Hsp60 / IEC mice in mucosal tissue (Mucosa), villus-derived IEC and crypt-derived IEC from Hsp60 flox/flox and Hsp60 / IEC mice. Gene regulation is enriched in villus-derived IEC fractions. Lines in the dot plots indicate mean numbers. Asterisk indicate significant differences *P<0.05; **P<0.01; ***P< All statistical analyses were performed via unpaired t-tests comparing genotypes. 10
11 Supplementary Figure 8: HSP60-deficient villus IEC express WNT factors Left: qrt-pcr analysis of known WNT ligands including the WNT enhancer Rspo1 in IEC isolated from villus tip epithelium of Hsp60 / IEC (N=6) vs. Hsp60 flox/flox mice (N=5). Statistical analysis was performed via t-test comparing genotypes at each time point. Asterisk indicate significant differences **P<0.01; ***P< Right: Correlation analysis (Pearson) of significantly regulated WNT factors compared to HSP60 levels in Hsp60 / IEC mice (the straight line indicates linear regression). P values indicate one-sided significance. 11
12 Supplementary Figure 9: WNT10A rescues intestinal organoid growth after HSP60 knockout 12
13 Organoids were isolated from Hsp60 flox/flox, VillinCreER T2-Tg mice and distributed to four protocols. (A) Experimental scheme to show the effects of d WNT10A supplementation (100ng/mL) on Hsp60 deficient small intestinal organoids. Lower panel: representative pictures of the indicated treatments and time points. (B) Measurement of organoid area (left) and life cell protease activity measured by fluorescence (right) following indicated treatments. (C) Quantification of de novo crypt formation (left); a, b, c, significantly different from each other, Kruskal Wallis test on ranks followed by Dunn s test. Right: qrt-pcr analysis of Lgr5 mrna expression in organoids in response to WNT10A treatment. (D) qpcr analysis of Hsp60 mrna expression. Bars represent mean +SEM. Asterisk indicate significant differences *P<0.05; **P<0.01; ***P< Unless otherwise indicated, One-Way ANOVA and appropriate post-hoc tests were used for all statistical analyses. Data from organoid experiments derive from at least 3 independent experiments. 13
14 Supplementary Figure 10: Oxidative stress associated with HSP60 deficiency-induced mitochondrial dysfunction seems to play a minor role in IEC (A) Representative 8-OHdG IHC stainings of Hsp60 / IEC and Hsp60 flox/flox control mice. Detailed images of 8-OHdG IHC in higher magnification distinguish villus vs. hyperproliferative nodules/ non-nodular crypt regions of the jejunum. Positive control: tissue showing neoplastic changes associated with inflammation. (B) qrt-pcr analysis of oxidative stress-associated genes in IEC isolated from Hsp60 / IEC and Hsp60 flox/flox control mice (N=5). Asterisk indicate significant differences *P<0.05; **P<0.01; ***P< Statistical analyses were performed using unpaired t-test. 14
15 Supplementary Figure 11: Uncropped versions of agarose gels from the main figures 15
16 Uncropped versions of agarose gels shown in (A) Figure 1B, (B) Figure 1D, (C) Figure 2, (D) Figure 4C, (E) Figure 6A, (F) Supplementary Figure 1B. 16
17 Supplementary Table 1. Primer sequences for genotyping Allele Primers (5 3 ) Allele Primers (5 3 ) Hsp60 KO Hsp60 flox Chop KO Chop WT tctgcctgcttcttctgccttca VillinCre Tg caagcctggctcgacggcc accagaacaacctcaggcctcaat cgcgaacatcttcaggttct accaagaccctgtactcttaacc VillinCreER T2-Tg accatatccaccgagtc aacttgacctagatgttgtgtgg aggaatgcgatgaagtag atgcccttacctatcgtg Lgr5CreER T2 - cactgcattctagttgtgg aacgccagggttttcccagtca Egfp Tg cggtgcccgcagcgag atgcccttacctatcgtg gagactctggctactcatcc gcagggtcaagagtagtg DNA control ccttcagcaagagctggggac 17
18 Supplementary Table 2. Antibodies and dilutions used for IF, IHC and WB Primary antibodies: Companies: Dilutions: anti-hsp60 (goat /rabbit (WB)) Santa Cruz Biotechnology, Santa Cruz, CA 1:200/ 1:1000 (WB) anti-ki67 (rabbit) abcam, Cambridge, UK 1:400 anti-olfm4 (rabbit) Biorbyt, Cambridge, UK 1:200 anti-rspo1 (rabbit) Sigma-Aldrich 1:100 anti-gfp (rabbit) Cell Signalling Technology, Danvers, MA 1:100 anti-e-cadherin (mouse) abcam, Cambridge, UK 1:300 anti-αsma (mouse) abcam, Cambridge, UK 1:100 anti-wnt10a (rabbit) abcam, Cambridge, UK 1:100 anti-lysozyme (goat) Santa Cruz Biotechnology, Santa Cruz, CA 1:25 anti-citrate synthase (rabbit) abcam, Cambridge, UK 1:500 (WB) anti-β-actin Cell Signalling Technology, Danvers, MA 1:1000 anti-8-ohdg (mouse) abcam, Cambridge, UK 1:1000 Alexa Flour 488 anti-mouse Biolegend, San Diego, CA 1:1000 F4/80 Secondary antibodies: Companies: Dilutions: HRP-conjugated donkey dianova, Hamburg, Germany 1:300 anti-rabbit IgG HRP-conjugated donkey Sigma-Aldrich, St. Louis, MO 1:300 anti-goat IgG Biotin-conjugated donkey abcam, Cambridge, UK 1:500 anti-rabbit IgG Alexa Fluor donkey anti-goat Life Technologies, Carlsbad, CA 1: / 546 Alexa Fluor donkey antirabbit Life Technologies, Carlsbad, CA 1: Alexa Fluor donkey antimouse 647 Life Technologies, Carlsbad, CA 1:200 18
19 Supplementary Table 3. Primer sequences for quantitative real-time PCR and UPL probe number Gene Primers (5 3 ) Probe Gene Primers (5 3 ) Probe Hsp60 tcttcaggttgtggcagtca cgctgggagagcgtactg 1 Wnt7a cccctcttctccaaacactg cgataatcgcataggtgaagg 12 Hsp10 ggcccgagttcagagtcc tctgtccagcggcagttac 77 Wnt7b tgtcaaagagcggaagaaactt tcttgttgcagatgatgttgg 49 ClpP gcaacaagaagcccattcat actgcggctgtgacgagt 26 Wnt8a gtactgcattgtgtcgtagatgg cccgaactccacgttgtc 75 Chop gcgacagagccagaataaca gcctcggagactttgacaac 91 Wnt8b gatgcacttccttctggaaca ctccccagagccaacctt 76 mtcoxi cagaccgcaacctaaacaca cgagtggacttccacaacaa 25 Wnt9a ttctgggtgcccaaagaat ggcatttgcaagtggtttc 19 Pgc1 gagcgaaccttaagtgtggaa ccaagagaggaagcaaggac 52 Wnt9b tcttggttggctttatgagga tctcaggccgctcttcac 105 Otc gctgtcatggtatccctgct ggcgctcctgttcttccta 99 Wnt10a tttccttttgacaggcatca gtcgttgggtgctgacct 71 Olfm4 gaaattcgagagagagttttctaagg aatgcggatccacaacaac 92 Wnt10b gacctctactcggaccgtca ctccaacaggtcttgaattgg 27 Lgr5 cttcactcggtgcagtgct caggatcccaagccaataaa 60 Wnt11 cagccagctaccaaataggtg tccagggaggcacgtaga 94 Axin2 gagagtgagcggcagagc ccctctttggctatgagctg 96 Wnt16 cggctgactcgttctcct actgcatgacctggtgacag 94 Wnt1 tactggcactgaccgctct cgacatgaacaaatgcatca 25 Rspo1 cttggaatccgtcaacaggt ctcctgacacttggtgcaga 5 Wnt2 cagagatcacagcctctttgg tgcctatgtctcagcctcttc 101 Tnf gcgtaaacaaaggccgatt gaggccatttgggaacttct 49 Wnt2b ccgggaccacactgtcttt cctttggaccctctgacttg 16 gctgacgagatagcatagacga Inf agcgttcattgtctcagagcta 63 Wnt3 ctcgctggctacccaattt gatggatgctaccaaactggat 81 Il-6 gaggccagagatgtgtactgc ccaggtagctatggtactccaga 6 Wnt3a cttagtgctctgcagcctga agactccagccacactccaa 76 Kc gagtgctcagagaggagtactgg tgacagcgcagctcattg 83 Wnt4 actggactccctccctgtct catccacgtgttggctca 62 Mcp-1 tgcccttgtcactgcaaa gatcatcttgctggtgaatgagt 62 Wnt5a acgcttcgcttgaattcct gctgccgtcattttctgc 55 Ip-10 cccgggcttaatattccaa tctcactggcccgtcatc 3 Wnt5b agcaccgtggacaacacat tccactcatggcaaattcaa 53 Gapdh aaggcagtctctcggctacc tttgatgttagtggggtctcg 9 Wnt6 gtgcaactgcacaacaacg ggaacggaggcagcttct 62 19
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SUPPLEMENTARY INFORMATION
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Figure S1. Reduction in glomerular mir-146a levels correlate with progression to higher albuminuria in diabetic patients.
Supplementary Materials Supplementary Figures Figure S1. Reduction in glomerular mir-146a levels correlate with progression to higher albuminuria in diabetic patients. Figure S2. Expression level of podocyte
Supplementary Materials and Methods
Supplementary Materials and Methods Hepatocyte toxicity assay. Freshly isolated hepatocytes were incubated for overnight with varying concentrations (-25 µm) of sodium glycochenodeoxycholate (GCDC) or
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Bezzi et al., Supplementary Figure 1 *** Nature Medicine: doi: /nm Pten pc-/- ;Zbtb7a pc-/- Pten pc-/- ;Pml pc-/- Pten pc-/- ;Trp53 pc-/-
Gr-1 Gr-1 Gr-1 Bezzi et al., Supplementary Figure 1 a Gr1-CD11b 3 months Spleen T cells 3 months Spleen B cells 3 months Spleen Macrophages 3 months Spleen 15 4 8 6 c CD11b+/Gr1+ cells [%] 1 5 b T cells
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SUPPLEMENTARY INFORMATION
doi:10.1038/nature12652 Supplementary Figure 1. PRDM16 interacts with endogenous EHMT1 in brown adipocytes. Immunoprecipitation of PRDM16 complex by flag antibody (M2) followed by Western blot analysis
c Ischemia (30 min) Reperfusion (8 w) Supplementary Figure bp 300 bp Ischemia (30 min) Reperfusion (4 h) Dox 20 mg/kg i.p.
a Marker Ripk3 +/ 5 bp 3 bp b Ischemia (3 min) Reperfusion (4 h) d 2 mg/kg i.p. 1 w 5 w Sacrifice for IF size A subset for echocardiography and morphological analysis c Ischemia (3 min) Reperfusion (8
Supplementary Figure 1.
Supplementary Figure 1. Increased β cell mass and islet diameter in βtsc2 -/- mice up to 35 weeks A: Reconstruction of multiple anti-insulin immunofluorescence images showing differences in β cell mass
Trim29 gene-targeting strategy. (a) Genotyping of wildtype mice (+/+), Trim29 heterozygous mice (+/ ) and homozygous mice ( / ).
Supplementary Figure 1 Trim29 gene-targeting strategy. (a) Genotyping of wildtype mice (+/+), Trim29 heterozygous mice (+/ ) and homozygous mice ( / ). (b) Immunoblot analysis of TRIM29 in lung primary
Genotype analysis by Southern blots of nine independent recombinated ES cell clones by
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Islet viability assay and Glucose Stimulated Insulin Secretion assay RT-PCR and Western Blot
Islet viability assay and Glucose Stimulated Insulin Secretion assay Islet cell viability was determined by colorimetric (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay using CellTiter
Supplementary Figure 1
Supplementary Figure 1 The average sigmoid parametric curves of capillary dilation time courses and average time to 50% peak capillary diameter dilation computed from individual capillary responses averaged
Nature Biotechnology: doi: /nbt Supplementary Figure 1
Supplementary Figure 1 Overview of the transplant procedure and supplementary data to Figure 1. a. Under isofluorane anesthesia, the lumen of the colon is washed by a gentle PBS enema. b. Using a p200
Supplementary Figure 1. Spatial distribution of LRP5 and β-catenin in intact cardiomyocytes. (a) and (b) Immunofluorescence staining of endogenous
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Tcf21 MCM ; R26 mtmg Sham GFP Col 1/3 TAC 8W TAC 2W. Postn MCM ; R26 mtmg Sham GFP Col 1/3 TAC 8W TAC 2W
A Tcf21 MCM ; R26 mtmg Sham GFP Col 1/3 Tcf21 MCM ; R26 mtmg TAC 2W Tcf21 MCM ; R26 mtmg TAC 8W B Postn MCM ; R26 mtmg Sham GFP Col 1/3 Postn MCM ; R26 mtmg TAC 2W Postn MCM ; R26 mtmg TAC 8W Supplementary
Mitosis. Single Nano Micro Milli Macro. Primary. PCNA expression
a b c DAPI YFP CC3 DAPI YFP PCNA DAPI YFP ph3 DAPI YFP KI67 e 6 Mitosis f 1 PCNA expression %ph3 + /YFP + n= 63 87 61 3 13 8 n= 15 3 9 1 5 %PCNA+/YFP+ 8 6 Supplementary Figure 1. Proliferation/apoptosis
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Supplemental Table 1: Demographics and characteristics of study participants. Male, n (%) 3 (20%) 6 (50%) Age, years [mean ± SD] 33.3 ± ± 9.
![Supplemental Table 1: Demographics and characteristics of study participants. Male, n (%) 3 (20%) 6 (50%) Age, years [mean ± SD] 33.3 ± ± 9.](/thumbs/90/103696086.jpg "Supplemental Table 1: Demographics and characteristics of study participants. Male, n (%) 3 (20%) 6 (50%) Age, years [mean ± SD] 33.3 ± ± 9.")
SUPPLEMENTAL DATA Supplemental Table 1: Demographics and characteristics of study participants Lean (n=15) Obese (n=12) Male, n (%) 3 (20%) 6 (50%) Age, years [mean ± SD] 33.3 ± 9.5 44.8 ± 9.1 White, n
GPR120 *** * * Liver BAT iwat ewat mwat Ileum Colon. UCP1 mrna ***
a GPR120 GPR120 mrna/ppia mrna Arbitrary Units 150 100 50 Liver BAT iwat ewat mwat Ileum Colon b UCP1 mrna Fold induction 20 15 10 5 - camp camp SB202190 - - - H89 - - - - - GW7647 Supplementary Figure
Nature Medicine: doi: /nm.4322
1 2 3 4 5 6 7 8 9 10 11 Supplementary Figure 1. Predicted RNA structure of 3 UTR and sequence alignment of deleted nucleotides. (a) Predicted RNA secondary structure of ZIKV 3 UTR. The stem-loop structure
well for 2 h at rt. Each dot represents an individual mouse and bar is the mean ±
Supplementary data: Control DC Blimp-1 ko DC 8 6 4 2-2 IL-1β p=.5 medium 8 6 4 2 IL-2 Medium p=.16 8 6 4 2 IL-6 medium p=.3 5 4 3 2 1-1 medium IL-1 n.s. 25 2 15 1 5 IL-12(p7) p=.15 5 IFNγ p=.65 4 3 2 1
p = formed with HCI-001 p = Relative # of blood vessels that formed with HCI-002 Control Bevacizumab + 17AAG Bevacizumab 17AAG
A.. Relative # of ECs associated with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 ol b p < 0.001 Relative # of blood vessels that formed with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 l b p = 0.002 Control IHC:
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Supplementary Material
Supplementary Material Fig. S1. Gpa33 -/- mice do not express Gpa33 mrna or GPA33 protein. (A) The Gpa33 null allele contains a premature translational stop codon (STOP) in exon 2, and almost all of the
X P. Supplementary Figure 1. Nature Medicine: doi: /nm Nilotinib LSK LT-HSC. Cytoplasm. Cytoplasm. Nucleus. Nucleus
a b c Supplementary Figure 1 c-kit-apc-eflu780 Lin-FITC Flt3-Linc-Kit-APC-eflu780 LSK Sca-1-PE-Cy7 d e f CD48-APC LT-HSC CD150-PerCP-cy5.5 g h i j Cytoplasm RCC1 X Exp 5 mir 126 SPRED1 SPRED1 RAN P SPRED1
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Supporting Information
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A263 A352 A204. Pan CK. pstat STAT3 pstat3 STAT3 pstat3. Columns Columns 1-6 Positive control. Omentum. Rectosigmoid A195.
pstat3 75 Pan CK A A263 A352 A24 B Columns 1-6 Positive control A195 A22 A24 A183 Rectal Nodule STAT3 pstat3 STAT3 pstat3 Columns 7-12 Omentum Rectosigmoid Left Ovary Right Ovary Omentum Uterus Uterus
Fig 1 CD163. CD11b S100A9. Sirius Red. 100μm ** ** CD163. CD11b S100A9 ** Sirius Red (PL) Sirius Red SUM Mo.
T47D T47D + o SU-59 Fig SU-59 + o IHC score (-3) IHC score (-2) CD63 3 2 IHC score (-3) CD63 3 ** 2 CDb CDb * * SA9 SA9 ** * 2 IHC score (-4) αsa αsa 4 ** ** 2 Sirius Red μm IHC score (%) Sirius Red 8
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Supplementary Figure 1 Lymphocytes can be tracked for at least 4 weeks after
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Supplementary Figure 1 IL-27 IL
Tim-3 Supplementary Figure 1 Tc0 49.5 0.6 Tc1 63.5 0.84 Un 49.8 0.16 35.5 0.16 10 4 61.2 5.53 10 3 64.5 5.66 10 2 10 1 10 0 31 2.22 10 0 10 1 10 2 10 3 10 4 IL-10 28.2 1.69 IL-27 Supplementary Figure 1.
Supplementary Figure 1 IMQ-Induced Mouse Model of Psoriasis. IMQ cream was
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Supplementary Information
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