Supplementary Figure 1: Co-localization of reconstituted L-PTC and dendritic cells
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1 a CD11c Na + K + ATPase Na + K + ATPase CD11c x-y CD11c Na + K + ATPase Na + K + ATPase CD11c x-z c b x-y view BoNT NAPs CD11c BoNT CD11c NAPs BoNT NAPs CD11c 90 x-z view Apical Basolateral Supplementary Figure 1: Co-localization of reconstituted and dendritic cells (DCs). (a) Alexa Fluor 488 labeled (green) was injected into ligated mouse intestinal loops and incubated for 3 h; FAE regions were stained with a anti-na + K + ATPase antibody (basolateral marker), Cy3 labeled anti-rabbit IgG antibody (red), and Pacific Blue labeled anti-cd11c antibody (DC marker, blue). X-Z images in lower panels correspond to the positions indicated by dotted lines in the X-Y images. Right panels show a higher-magnification image of the boxed region. were captured by DCs in the SED (arrows). (b and c) For reconstitution of the, Alexa Fluor 488 labeled BoNT (green) and Alexa Fluor 568 labeled NAPs (red) were mixed at a molar ratio of 5:1 (0 nm : 0 nm) in PBS (ph 6.0), and then incubated for 3 h at C. Reconstituted was confirmed by pull-down using lactose gel beads (EY Laboratories, Inc.), 1
2 which bound to HA, followed by immunoblotting with anti- antibody (data not shown). Reconstituted was injected into ligated intestinal loops and incubated for 3 h. FAE was stained with a Pacific Blue labeled anti-cd11c antibody (blue). (b) X-Y view. (c) Three-dimensional data, Top, X-Y view. Bottom, side (X-Z) view. X-Y images (b) correspond to the position indicated by dotted line in the X-Z view (c). For three-dimensional imaging, see Supplementary Movie 1. Scale bars: 10 µm (a, b, and higher-magnification images in a); 5 µm (c). Data are representative of two independent experiments. 2
3 a b stomach c UEA-1 UEA-1 a UEA-1 UEA-1 b UEA-1 UEA-1 c Supplementary Figure 2: Localization of orally administered in small intestine. Alexa Fluor 568 labeled (26.7 pmol: µg, red) was intragastrically administered to mice and incubated for 1 h. After incubation, the small intestine was removed, and PPs were excised from this tissue. PPs from duodenum (a), jejunum (b), or ileum (c) were stained with FITC labeled UEA-1 (green), and analyzed on a confocal laser microscope. Scale bar, 10 µm. Data are representative of two independent experiments. 3
4 NS Percent survival Per cent survival Cnt IgG Anti-RANKL Time (hour) Supplementary Figure 3: M-cell depletion does not influence lethality upon oral administration of M-PTC. Mice were treated intraperitoneally with anti-rankl antibody or control rat IgG. M-PTC were intragastrically (174.4 pmol: µg) administered to mice treated with anti-rankl antibody or rat IgG (n = 5 per group). Statistical analyses were performed with the log-rank test. NS, not significant. 4
5 mrna expression (Normalized to Gapdh) mrna expression (Normalized to Actb) Gp2 1 Prnp 2 Umod 3 Gp2 1 Prnp 2 Umod 3 Supplementary Figure 4: qpcr analysis of mouse GP2, PrP C, and uromodulin mrnas. Total RNA was prepared from FAE. Expression of each gene was analyzed by real-time qpcr. The expression levels of mrnas encoding GP2 (Gp2), PrP C (Prnp), and uromodulin (Umod) were normalized to the level of the mrna encoding glyceraldehyde-3-phosphate dehydrogenase gene (Gapdh, a) or β-actin (Actb, b). Error bars indicate s.d. (n =3). 5
6 a P < 0.01 P < P < 0.01 Binding of (% of control) Gal Lac Glu Man Sialic acid Fuc GalNAc!" #" $" %" &" '" (" )" b Nontreated PNGase - PNGase + c Input Pull down 2 1 GST-Umod GST GST-Umod GST 2 1 P < GST-Umod Binding of (% of PNGase-) GST 0 PNGase - PNGase +!" #" Supplementary Figure 5: Binding of to GP2 and uromodulin is mediated by sugar chains. (a) ( nm) was pre-incubated with galactose (Gal), lactose (Lac), 6
7 glucose (Glu), mannose (Man), sialic acid, fucose (Fuc), or N-acetylgalactosamine (GalNAc) (10 mm), and then added to plates coated with mgp2-fc proteins. Bound was detected using anti-bont and HRP labeled anti-rabbit IgG antibodies. The data are expressed as a percentage of the binding of in the absence of added carbohydrates. Error bars indicate s.d. (n = 3). Statistical analyses were performed with the Student s t-test. (b) Deglycosylation of mgp2-fc was confirmed by SDS-PAGE. Binding of to deglycosylated mgp2-fc was analyzed by ELISA. Bound was detected using anti-bont and HRP labeled anti-rabbit IgG antibodies. Error bars indicate s.d. (n = 3). Statistical analyses were performed with the Student s t-test. (c) GST-mouse uromodulin or GST was incubated with Strep-Tactin Superflow agarose pre-bound to HA. HA-bound proteins were analyzed by immunoblotting with anti-gst antibody and HRP labeled anti-rabbit IgG antibody. 7
8 Input Pull down 2 1 mgp2 hgp2 MUC1 DPP4 PD1 TIM3 CD40 CD mgp2 hgp2 MUC1 DPP4 PD1 TIM3 CD40 CD24 Supplementary Figure 6: HA binds to various glycoproteins. Recombinant Fc proteins were incubated with Strep-Tactin Superflow agarose pre-bound to HA. HA-bound proteins were analyzed by immunoblotting using HRP labeled anti-human IgG antibody. Data are representative of two independent experiments. 8
9 MUC2 MUC2 phalloidin G Supplementary Figure 7: is trapped into the mucus layer. Alexa Fluor 488 labeled (green) were injected into ligated mouse intestinal loops and incubated for 1 h. Whole-mount specimens were fixed with Carnoy s solution and stained with anti-muc2 antibody and Cy3 labeled anti-mouse IgG antibody (red) and ifluor 405 labeled phalloidin (blue). was trapped in the mucus layer (arrows). G: goblet cell. Scale bar, 10 µm. Data are representative of two independent experiments. 9
10 D-10 UEA-1 D-10 UEA-1 Control (PBS) NAPs (NTNHA/HA) Supplementary Figure 8: NAPs (NTNHA/HA) disrupt the paracellular barrier around the M cells. NAPs (NTNHA/HA) and paracellular tracer (fixable FITC dextran 10K; D-10, green) or vehicle control (PBS) and the tracer were injected into ligated mouse intestinal loops and incubated for 2 h, and FAE regions were stained with rhodamine-labeled UEA-1 (red). Right panel shows a higher-magnification image of the boxed region. Paracellular tracer penetrated between M cells and enterocytes (arrows). Scale bars: 10 µm. Data are representative of two independent experiments. 10
11 a 1 Marker M-PTC BoNT NAPs 1 Marker Strep-HA1 FLAG-HA2 Strep-HA3 HA1-FLAG HA1-FLAG N285A Strep-HA3 R528A NTNHA BoNT HC HA3 BoNT LC HA3b HA1 HA HA3a 15 HA2 15 HA2 b c Marker HA1 HA3 HA2/HA3 HA1/HA2/HA3 HA3 Marker WT N285A R528A N285A/R528A HA3 HA1 HA HA2 15 HA2 Supplementary Figure 9: Toxins, NAPs, and recombinant HA proteins used in this 11
12 study. (a) SDS-PAGE of M-PTC,, BoNT, NAPs (2.0 µg), and recombinant HA protein (1.0 µg). HA3 protein is cleaved to yield two fragments, designated HA3a and HA3b (alternatively termed HA and HA52, respectively), in and NAPs 8. (b and c) Recombinant HA1, HA2, and HA3 proteins were mixed at a molar ratio of 4:4:1 ( µm: µm:5 µm) in PBS (ph 7.4), and then incubated for 3 h at C. Reconstituted HA (complex of Strep-HA1, FLAG-HA2, and Strep-HA3, b) and reconstituted HA harboring mutant HA1 or HA3 (complex of HA1-FLAG [N285A], FLAG-HA2, and Strep-HA3 [R528A], c) were confirmed by pull-down using Strep-Tactin Superflow agarose and SDS-PAGE. 12
13 Fig. 3a Input Pull down Fig. 5c 2 1 Fig. 5e Fig. 5f
14 Supplementary Fig. 5b Supplementary Fig. 5c Supplementary Fig. 6 Input Pull down Supplementary Fig. 9a Supplementary Fig. 9b Supplementary Fig. 9c Supplementary Figure 10: Uncropped scan images of immunoblots and SDS-PAGE gels (CBB staining). The red boxed regions in the uncropped scan images were used in the manuscript. The right side of the uncropped chemiluminescence image is a bright-field image of the blot membrane, showing molecular markers (kda). 14
15 Supplementary Table 1: Primers sequences used in this study. Primer sequence (5-3 ) Strep-HA1-F GGAACGGTACCCAGTAATCCAAAATTCATTAAATG Strep-HA1-R ATAGTCGACTTATGGGTTACGAATATTCCA FLAG-HA2-F TGAATAAGCTTTCAGTTGAAAGAACTTTTCTAC FLAG-HA2-R CTTTGGTACCTTATATTTTTTCAAGTTTGAAC Strep-HA3-F AAAGTTAGGTACCCTAGTGATACTATTGATTTAG Strep-HA3-R CGTGTCGACTTAATTAGTAATATCTATATGC HA1-FLAG-F CATGCCATGGTAATCCAAAATTCATTAAA HA1-FLAG-R CGGGATCCTTACTTGTCATCGTCATCCTTGTAGTCTGGGTTACGAATATTCCATTTC HA1-FLAG N285A-F GATGATGCTCAGAAATGGAATATTCGT HA1-FLAG N285A-R TTTCTGAGCATCATCTCCATGATAATT Strep-HA3 R528A-F TATACAGCTCAAAGCCCTGATGTCCATG Strep-HA3 R528A-R GCTTTGAGCTGTATAGTAATGTGCACC mgp2-fc-f GAAGATCTACCATGAAAAGGATGGTGGGTT mgp2-fc-r CCGCTCGAGTGTAGTGTGGGGAGTCCCC hgp2-fc-f CGCGGATCCACCATGGAAAGGATGGTGGGC hgp2-fc-r CCGCTCGAGTCCATTCATGACACCGGGAGA Umod-Fc-F CGCAGATCTACCATGGGGATCCCTTTGACC Umod-Fc-R CGCGTCGACCTTGGACACTGAGGCCTGG PrP C -Fc-F CGGGATCCACCATGGCGAACCTTGGCTACT 15
16 Supplementary Table 1: Primers sequences used in this study (continued). Primer sequence (5-3 ) PrP C -Fc-R CGCTCGAGGGATCTTCTCCCGTCGTAATAG GST-mGP2-F TCCAGGGGCCCCTGGGATCTACAATACATCAAGGTTATGGGAG GST-mGP2-R ATGCGGCCGCTCGAGTCATGTAGTGTGGGGAGTCCCCTTC GST-Umod-F TCCAGGGGCCCCTGGGAAGTAACTCAACAGAAGCGAGAC GST-Umod-R ATGCGGCCGCTCGAGTCACTTGGACACTGAGGCCTGGAC Used for qpcr Actb-F GAGCGCAAGTACTCTGTGTG Actb-R AACGCAGCTCAGTAACAGTCC Gapdh-F TGTGTGCGTCGTGGATCTGA Gapdh-R TTGCTGTTGAAGTCGCAGGAG Gp2-F AACTGCTATGCCACCCCTTC Gp2-R TCGGCTTTCTGAGGACACAC Umod-F CAGGTGTTTCCAGGACAGAGG Umod-R CATTCAGAACACCGTCTCGC Prnp-F GGTCCCTTTGATGGAGTCTGTC Prnp-R TGTGGATGCTCTAGCTATCCCA Used for mgp2 transfection mgp2-f GAAGATCTACCATGAAAAGGATGGTGGGTT mgp2-r CCGCTCGAGGAACAGTAGAGCCAGGAAGAC 16
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