Figure 1B ) Electrophoretic mobility shift assay ( EMSA ) to examine binding affinities of the GST - KNL2518 - 560 fragment for the CENP - A or H3 nucleosome . The molar ratio of KNL2518 - 560 against nucleosomes are indicated . D ) - F ) EMSA to examine binding affinities of the GST - KNL2 fragment for the CENP - A nucleosome : D , GST - KNL2347 - 560 ; E , GST - KNL2456 - 560 ; F , GST - KNL2456 - 516 ( CC - upstream ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B) Electrophoretic mobility shift assay (EMSA) to examine binding affinities of the GST-KNL2518-560 fragment for the CENP-A or H3 nucleosome. The molar ratio of KNL2518-560 against nucleosomes are indicated.D)-F) EMSA to examine binding affinities of the GST-KNL2 fragment for the CENP-A nucleosome: D, GST-KNL2347-560; E, GST-KNL2456-560; F, GST-KNL2456-516 (CC-upstream).


Figure 3B ) EMSA to examine contribution of the ggKNL2 R527 residue to the CENP - A nucleosome binding . D ) Expression of the WT or R527A mutant GFP - KNL2 in KNL2 conditional knock out chicken DT40 cells ( cKO - KNL2 ) . Expression of full - length KNL2 WT was conditionally turned off by tetracycline ( tet ) addition in cKO - KNL2 cells ( None ) . GFP - fused KNL2 WT or R527A mutant was stably expressed in the cKO - KNL2 cells . These cells were cultured in the presence or absence of tet ( + tet : KNL2 OFF or − tet : KNL2 ON ) for 48 h . α - Tubulin ( Tub ) was probed as a loading control . E ) Localization analyses of GFP - fused KNL2 WT or R527A mutant ( green ) . CENP - T was stained as a kinetochore marker ( red ) , and DNA was stained using DAPI ( blue ) . Magnified views are shown in insets . Scale bar indicates 10 μm . F ) Growth of the cKO - KNL2 cells expressing GFP - fused KNL2 WT or R527A mutant . The upper panel shows examined cell numbers at the indicated time after tet addition ( + tet : KNL2 OFF ) . The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number . The lower panel shows examined untreated cells ( - tet : KNL2 ON ) . Parental cKO - KNL2 chicken DT40 cells ( None ) were also examined .

O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B) EMSA to examine contribution of the ggKNL2 R527 residue to the CENP-A nucleosome binding.D) Expression of the WT or R527A mutant GFP-KNL2 in KNL2 conditional knock out chicken DT40 cells (cKO-KNL2). Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition in cKO-KNL2 cells (None). GFP-fused KNL2 WT or R527A mutant was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.E) Localization analyses of GFP-fused KNL2 WT or R527A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or R527A mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.


Figure 4B ) EMSA to examine contribution of the F535 and W536 residues in ggKNL2 to the CENP - A nucleosome binding . D ) Expression of GFP - KNL2 WT or CENP - A C - terminal tail binding mutants in cKO - KNL2 cells . Expression of full - length KNL2 WT was conditionally turned off by tetracycline ( tet ) addition to the cKO - KNL2 cells ( None ) . GFP - fused KNL2 WT , F535A , W536A or F535A / W536A was stably expressed in the cKO - KNL2 cells . These cells were cultured in the presence or absence of tet ( + tet : KNL2 OFF or − tet : KNL2 ON ) for 48 h . α - Tubulin ( Tub ) was probed as a loading control . E ) Localization analyses of GFP - fused KNL2 WT and F535A , W536A or F535A / W536A mutant ( green ) . CENP - T was stained as a kinetochore marker ( red ) , and DNA was stained using DAPI ( blue ) . Magnified views are shown in insets . Scale bar indicates 10 μm . F ) Growth of the cKO - KNL2 cells expressing GFP - fused KNL2 WT or each CENP - A C - terminal tail binding mutant . The upper panel shows examined cell numbers at the indicated time after tet addition ( + tet : KNL2 OFF ) . The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number . The lower panel shows examined untreated cells ( - tet : KNL2 ON ) . Parental cKO - KNL2 chicken DT40 cells ( None ) were also examined .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B) EMSA to examine contribution of the F535 and W536 residues in ggKNL2 to the CENP-A nucleosome binding.D) Expression of GFP-KNL2 WT or CENP-A C-terminal tail binding mutants in cKO-KNL2 cells. Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition to the cKO-KNL2 cells (None). GFP-fused KNL2 WT, F535A, W536A or F535A/W536A was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.E) Localization analyses of GFP-fused KNL2 WT and F535A, W536A or F535A/W536A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or each CENP-A C-terminal tail binding mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.


Figure 6A ) Immunostaining with anti - Mis18α antibody in AID - based KNL2 knockout cells expressing GFP fused KNL2 wild type or each KNL2 mutant in the presence of IAA for 15 h . Clear colocalization of KNL2 signals with Mis18α was observed ( Insets ) . DNA was stained with DAPI . Scale bar indicates 10 µm . B ) AID - based KNL2 knockout cells expressing GFP fused each KNL2 mutant and SNAP - tagged CENP - A were used . After addition of IAA , SNAP - CENP - A signals were visualized by TMR - Star staining . CENP - T was stained as a kinetochore marker . Representative images were presented . Mitotic centromere localization of each KNL2 mutant was shown as a merged image ( Insets ) : SNAP - CENP - A ( red ) and each KNL2 mutant ( green ) . DNA was stained with DAPI . Scale bar indicates 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Immunostaining with anti-Mis18α antibody in AID-based KNL2 knockout cells expressing GFP fused KNL2 wild type or each KNL2 mutant in the presence of IAA for 15 h. Clear colocalization of KNL2 signals with Mis18α was observed (Insets). DNA was stained with DAPI. Scale bar indicates 10 µm.B) AID-based KNL2 knockout cells expressing GFP fused each KNL2 mutant and SNAP-tagged CENP-A were used. After addition of IAA, SNAP-CENP-A signals were visualized by TMR-Star staining. CENP-T was stained as a kinetochore marker. Representative images were presented. Mitotic centromere localization of each KNL2 mutant was shown as a merged image (Insets): SNAP-CENP-A (red) and each KNL2 mutant (green). DNA was stained with DAPI. Scale bar indicates 10 µm.


Figure 7A ) KNL2 ( red ) localization in CENP - C conditional knockout chicken DT40 cells ( cKO - CENP - C ) with or without CENP - C expression . DNA was stained with DAPI ( blue ) . CENP - T was stained as a kinetochore marker ( light blue ) . Scale bar indicates 10 μm . B ) KNL2 intensities were evaluated in six cells before and after CENP - C knock out . KNL2 signal intensities on the mitotic centromeres ( 14 - 90 centromeres ) in each cell were plotted with the average and the standard deviation . The ratio of KNL2 intensities between cells before and after CENP - C knock out was estimated . KNL2 intensities were reduced by ~ 55 % , when CENP - C was knocked out . C ) Competitive pull - down assays for the CENP - A nucleosome binding of ggKNL2 and ggCENP - C . A complex of GST - KNL2456 - 560 with the CENP - A nucleosome ( CA - nuc ) was incubated in the absence or presence of MBP - CENP - C619 - 690 ( right panel ) or phosphorylated MBP - CENP - C619 - 690 ( middle panel ) and pulled down by GST affinity . The concentration of CENP - C619 - 690 was increased from 0 μM to 1 μM ( lanes 3 - 7 ) . The ratio of band intensities of pull - downed histones ( * ) to GST - KNL2456 - 560 in each lane ( lanes 3 - 7 ) was estimated and presented in the bar graph ( left panel ) . Data are mean with standard deviations calculated from the technical replicates , n = 3 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) KNL2 (red) localization in CENP-C conditional knockout chicken DT40 cells (cKO-CENP-C) with or without CENP-C expression. DNA was stained with DAPI (blue). CENP-T was stained as a kinetochore marker (light blue). Scale bar indicates 10 μm.B) KNL2 intensities were evaluated in six cells before and after CENP-C knock out. KNL2 signal intensities on the mitotic centromeres (14-90 centromeres) in each cell were plotted with the average and the standard deviation. The ratio of KNL2 intensities between cells before and after CENP-C knock out was estimated. KNL2 intensities were reduced by ~55%, when CENP-C was knocked out.C) Competitive pull-down assays for the CENP-A nucleosome binding of ggKNL2 and ggCENP-C. A complex of GST-KNL2456-560 with the CENP-A nucleosome (CA-nuc) was incubated in the absence or presence of MBP-CENP-C619-690 (right panel) or phosphorylated MBP-CENP-C619-690 (middle panel) and pulled down by GST affinity. The concentration of CENP-C619-690 was increased from 0 μM to 1 μM (lanes 3-7). The ratio of band intensities of pull-downed histones (*) to GST-KNL2456-560 in each lane (lanes 3-7) was estimated and presented in the bar graph (left panel). Data are mean with standard deviations calculated from the technical replicates, n=3.


Figure 1A , B Immunoblot analyses of the protein abundance of the METTL3 / METTL14 / WTAP heterotrimer in PBMCs ( A ) and BMDMs ( B ) upon challenge with VSV - eGFP , poly ( I : C ) , HSV - 1 or HSV60 at the indicated time points .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, B Immunoblot analyses of the protein abundance of the METTL3/METTL14/WTAP heterotrimer in PBMCs (A) and BMDMs (B) upon challenge with VSV-eGFP, poly(I:C), HSV-1 or HSV60 at the indicated time points.


Figure 2E A549 cells were stimulated with poly ( I : C ) for 16 hrs and then treated with PR - 171 ( 10 μM ) or PS - 341 ( 10 μM ) for additional 4 hrs and 8 hrs . The cell lysates were analyzed by immunoblotting . Representative images from 3 independent biological experiments are shown ( left ) , and the relative levels of WTAP are presented as the mean ± SD ( right ) . F A549 cells were stimulated with poly ( I : C ) for 16 hrs and then treated with CQ ( 50 mM ) or Baf A1 ( 0 . 2 mM ) for additional 4 hrs and 8 hrs . The cell lysates were analyzed by immunoblotting . LC3B served as a good marker of the autophagy process . Representative images from 3 independent biological experiments are shown ( left ) , and the relative levels of WTAP are presented as the mean ± SD ( right ) . H Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG - tagged WTAP , and HA - tagged K48 - linked , K63 - linked or wild - type ubiquitin and treated with DMSO or PR171 ( 10 μM ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2E A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with PR-171 (10 μM) or PS-341 (10 μM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right). F A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with CQ (50 mM) or Baf A1 (0.2 mM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. LC3B served as a good marker of the autophagy process. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right).H Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG-tagged WTAP, and HA-tagged K48-linked, K63-linked or wild-type ubiquitin and treated with DMSO or PR171 (10 μM).


Figure 3C qRT - PCR analyses to detect the mRNA abundances of IFNB1 , RANTES , DDX58 , and ISG15 in WT and WTAP - / - 293T cells followed by treatments with intracellular poly ( I : C ) , poly ( dA : dT ) , or VSV - eGFP . I , J Immunoblot analyses to detect the protein expression of ISGs in shCtrl and shWTAP A549 cells that were stimulated with poly ( I : C ) - LMW ( I ) or poly ( dA : dT ) ( J ) at different time points with the indicated antibodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C qRT-PCR analyses to detect the mRNA abundances of IFNB1, RANTES, DDX58, and ISG15 in WT and WTAP-/- 293T cells followed by treatments with intracellular poly(I:C), poly(dA:dT), or VSV-eGFP.I, J Immunoblot analyses to detect the protein expression of ISGs in shCtrl and shWTAP A549 cells that were stimulated with poly(I:C)-LMW (I) or poly(dA:dT) (J) at different time points with the indicated antibodies.


Figure 4A , B Immunoblot analyses of total and phosphorylated ( p - ) TBK1 and IRF3 in A549 cells transfected with Ctrl or WTAP - specific siRNA , followed by stimulation with poly ( I : C ) ( A ) or poly ( dA : dT ) ( B ) at different time points . E , F Immunoblot analyses of total and phosphorylated ( p - ) STAT1 in shCtrl and shWTAP A549 cells , followed by stimulation with IFN - α ( E ) or - β ( F ) at different time points .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, B Immunoblot analyses of total and phosphorylated (p-) TBK1 and IRF3 in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by stimulation with poly (I:C) (A) or poly (dA:dT) (B) at different time points.E, F Immunoblot analyses of total and phosphorylated (p-) STAT1 in shCtrl and shWTAP A549 cells, followed by stimulation with IFN-α (E) or -β (F) at different time points.


Figure 5A , B Immunoblot analyses to detect the protein expression of critical adaptors in the IFN - I and Jak - STAT signaling pathway in A549 cells after transfection with Ctrl or WTAP - specific siRNAs for 48 hrs . F , G Abundance of IRF3 ( F ) and IFNAR1 ( G ) transcripts ( detection site - 1 and - 2 ) among mRNA immunoprecipitated with anti - m6A antibody from shCtrl and shWTAP A549 cells . n = 3 independent biological replicates , and error bars represent standard deviations .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, B Immunoblot analyses to detect the protein expression of critical adaptors in the IFN-I and Jak-STAT signaling pathway in A549 cells after transfection with Ctrl or WTAP-specific siRNAs for 48 hrs.F, G Abundance of IRF3 (F) and IFNAR1 (G) transcripts (detection site-1 and -2) among mRNA immunoprecipitated with anti-m6A antibody from shCtrl and shWTAP A549 cells. n = 3 independent biological replicates, and error bars represent standard deviations.


Figure 6A Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 cells stimulated with poly ( I : C ) or poly ( dA : dT ) at the indicated time points . B Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 and L929 cells infected with VSV - eGFP at the indicated time points . H , Immunoblot analysis of IRF3 and IFNAR1 in A549 cells transfected with poly ( I : C ) - LMW or infected with VSV - GFP for 16 hrs and then treated with PR - 171 or DMSO for additional 4 and 8 hrs . Representative images from 3 independent biological experiments are shown ( H ) , protein abundance of IRF3 and IFNAR1 in A549 cells transfected with poly ( I : C ) - LMW or infected with VSV - GFP for 16 hrs and then treated with PR - 171 or DMSO for additional 4 and 8 hrs . the relative levels of IFNAR1 ( left ) and IRF3 ( right ) are presented as the mean ± SD ( I ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 cells stimulated with poly (I:C) or poly (dA:dT) at the indicated time points. B Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 and L929 cells infected with VSV-eGFP at the indicated time points.H, Immunoblot analysis of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. Representative images from 3 independent biological experiments are shown (H),protein abundance of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. the relative levels of IFNAR1 (left) and IRF3 (right) are presented as the mean ± SD (I).


Figure 1B . Significant increase of survival in Ppt1 - / - animals transplanted with HSCPs or BM isolated from Ppt1 + / + mice ; Log - rank : HSPCs / BM + / + tx vs BM - / - tx p < 0 . 001 ; BM - / - tx vs Ppt1 - / - UT p < 0 . 001 . D . Representative fluorescence microscope photomicrographs showing the distribution of donor - derived cells ( transduced with D . NGFR reporter gene , red ) and autofluorescent storage material ( AF , green ) in the brain of a Ppt1 - / - mouse euthanized at 240 days of age , after transplantation with HSPCs isolated from Ppt1 + / + mice . Insets in D show high magnification representative confocal scanning photomicrographs of DNGFR + AF + cells in different regions of the CNS . Arrows show DNGFR + cells engulfed with autofluorescent ( AF ) storage material . Scale bar ( main figure ) = 1mm ; scale bar ( insets ) = 25μm . G . Representative brightfield photomicrographs of Nissl - stained brain sections from wild type Ppt1 + / + mice and untreated or transplanted Ppt1 - / - mice . Black dashed box highlights a portion of the cortex , shown in the high magnification insets . Yellow lines represent the segments applied to measure cortical thickness ( see Materials and Methods for details on the quantification ) . Scale bar ( main figure ) = 1mm ; scale bar ( insets ) = 25μm . H . Histograms showing the quantification of cortical thickness . SM = somatomotor ; SS = somatosensory ; V = visual cortex . * * = p < 0 . 01 ; Kruskal Wallis followed by Dunn ' s post - hoc test ( for SM ) or ANOVA followed by Tukey ' s post - hoc test ( for SS and V ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Significant increase of survival in Ppt1-/- animals transplanted with HSCPs or BM isolated from Ppt1+/+ mice; Log-rank: HSPCs/BM+/+ tx vs BM-/- tx p<0.001; BM-/- tx vs Ppt1-/- UT p < 0.001.D. Representative fluorescence microscope photomicrographs showing the distribution of donor-derived cells (transduced with D.NGFR reporter gene, red) and autofluorescent storage material (AF, green) in the brain of a Ppt1-/- mouse euthanized at 240 days of age, after transplantation with HSPCs isolated from Ppt1+/+ mice. Insets in D show high magnification representative confocal scanning photomicrographs of DNGFR+AF+ cells in different regions of the CNS. Arrows show DNGFR+ cells engulfed with autofluorescent (AF) storage material. Scale bar (main figure) = 1mm; scale bar (insets) = 25μm.G. Representative brightfield photomicrographs of Nissl-stained brain sections from wild type Ppt1+/+ mice and untreated or transplanted Ppt1-/- mice. Black dashed box highlights a portion of the cortex, shown in the high magnification insets. Yellow lines represent the segments applied to measure cortical thickness (see Materials and Methods for details on the quantification). Scale bar (main figure) = 1mm; scale bar (insets) = 25μm. H. Histograms showing the quantification of cortical thickness. SM = somatomotor; SS = somatosensory; V = visual cortex. ** = p < 0.01; Kruskal Wallis followed by Dunn's post-hoc test (for SM) or ANOVA followed by Tukey's post-hoc test (for SS and V). D


Figure 2C . Significant increase of the survival in Ppt1 - / - mice receiving the administration of hPPT1 - LV transduced HSPCs . E , F . Significant prevention of pathological symptoms ( DSS higher than 4 and limbs stiffness ) in Ppt1 - / - mice transplanted with hPPT1 - LV transduced HSPCs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2C. Significant increase of the survival in Ppt1-/- mice receiving the administration of hPPT1-LV transduced HSPCs.E, F. Significant prevention of pathological symptoms (DSS higher than 4 and limbs stiffness) in Ppt1-/- mice transplanted with hPPT1-LV transduced HSPCs.


Figure 3E . Representative fluorescence microscope photomicrographs showing autofluorescent storage material ( AF , green ) in different brain regions of Ppt1 - / - mice transplanted with hPPT1 - LV transduced HSPCs at 400 - 420 days of age , when the study was terminated . Mock transplanted and untreated Ppt1 - / - mice at about 250 days , i . e . humane end point , are shown as reference ; 450 days - old untreated wild type Ppt1 + / + are shown as control . Neurotrace fluorescent Nissl staining ( NT , red ) is used to highlight neurons in different brain regions . Arrows highlight the surviving Purkinje cells in the cerebellum of IV and ICV + IV transplanted mice ; arrowheads highlight the high number of AF + cells in the Purkinje cell layer of ICV transplanted or Ppt1 - / - untreated ( UT ) mice . Scale bar = 100μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3E. Representative fluorescence microscope photomicrographs showing autofluorescent storage material (AF, green) in different brain regions of Ppt1-/- mice transplanted with hPPT1-LV transduced HSPCs at 400-420 days of age, when the study was terminated. Mock transplanted and untreated Ppt1 -/- mice at about 250 days, i.e. humane end point, are shown as reference; 450 days-old untreated wild type Ppt1+/+ are shown as control. Neurotrace fluorescent Nissl staining (NT, red) is used to highlight neurons in different brain regions. Arrows highlight the surviving Purkinje cells in the cerebellum of IV and ICV+IV transplanted mice; arrowheads highlight the high number of AF+ cells in the Purkinje cell layer of ICV transplanted or Ppt1-/- untreated (UT) mice. Scale bar = 100μm.


Figure 4A . Quantification of cortical thickness in the somatosensory ( SS ) , somatomotor ( SM ) and visual ( V ) area , in untreated Ppt1 - / - or Ppt1 + / + mice and in mice transplanted ( tx ) with hPPT1 - LV transduced HSPCs IV , ICV or ICV + IV or mock - transplanted , analyzed at study termination . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; Kruskal Wallis followed by Dunn ' s post - hoc test . B . Quantification of CD68 immunoreactivity in the cortex , hippocampus , thalamus and cerebellum of untreated ( UT ) or gene therapy treated Ppt1 - / - mice . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 00001 ; Kruskal Wallis followed by Dunn ' s post - hoc test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Quantification of cortical thickness in the somatosensory (SS), somatomotor (SM) and visual (V) area, in untreated Ppt1-/- or Ppt1+/+ mice and in mice transplanted (tx) with hPPT1-LV transduced HSPCs IV, ICV or ICV+IV or mock-transplanted, analyzed at study termination. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; Kruskal Wallis followed by Dunn's post-hoc test.B. Quantification of CD68 immunoreactivity in the cortex, hippocampus, thalamus and cerebellum of untreated (UT) or gene therapy treated Ppt1-/- mice. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.00001; Kruskal Wallis followed by Dunn's post-hoc test.


Figure 5A . Significant increase of the survival in Ppt1 - / - mice transplanted at the symptomatic stage with hPPT1 - LV transduced HSPCs administered IV + ICV . E . Representative fluorescence microscope photomicrographs showing autofluorescent storage material ( AF , green ) in different brain regions of Ppt1 - / - mice transplanted with hPPT1 - LV transduced HSPCs at 18 - 20 weeks of age , analyzed at study termination . Ppt1 - / - mice mock transplanted at 18 - 20 weeks of age and analyzed at about 250 days , i . e . humane end point , are shown as reference . Neurotrace fluorescent Nissl staining ( NT , red ) is used to highlight neurons in different brain regions . Scale bar = 100 μm . F . Top panel : quantification of autofluorescent ( AF ) storage material in different brain regions of untreated or transplanted mice analyzed at study termination . * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; 2 - way ANOVA followed by Tukey ' s post - hoc test . Bottom panel : histograms showing the quantification of cortical thickness . SM = somatomotor ; SS = somatosensory ; V = visual cortex . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * * = p < 0 . 0001 ; Kruskal Wallis followed by Dunn ' s post - hoc test ( for SM and V area ) or ANOVA followed by Tukey ' s post - hoc test ( for SS area ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Significant increase of the survival in Ppt1-/- mice transplanted at the symptomatic stage with hPPT1-LV transduced HSPCs administered IV+ICV.E. Representative fluorescence microscope photomicrographs showing autofluorescent storage material (AF, green) in different brain regions of Ppt1-/- mice transplanted with hPPT1-LV transduced HSPCs at 18-20 weeks of age, analyzed at study termination. Ppt1 -/- mice mock transplanted at 18-20 weeks of age and analyzed at about 250 days, i.e. humane end point, are shown as reference. Neurotrace fluorescent Nissl staining (NT, red) is used to highlight neurons in different brain regions. Scale bar = 100 μm. F. Top panel: quantification of autofluorescent (AF) storage material in different brain regions of untreated or transplanted mice analyzed at study termination. ** = p<0.01; *** = p< 0.001; **** = p< 0.0001; 2-way ANOVA followed by Tukey's post-hoc test. Bottom panel: histograms showing the quantification of cortical thickness. SM = somatomotor; SS = somatosensory; V = visual cortex. * = p <0.05; ** = p<0.01; **** = p<0.0001; Kruskal Wallis followed by Dunn's post-hoc test (for SM and V area) or ANOVA followed by Tukey's post-hoc test (for SS area). S


Figure 1CD4 and CD8 T cells were stimulated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 beads as indicated . Cell lysates after 4 h of stimulation were analyzed by immunoblotting . Blots depict one representative donor out of three . CD4 T cells were stimulated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 ; and human IFNβ ( top ) and IP10 levels ( bottom ) in the supernatant were analyzed by ELISA at the indicated time points . Data panels depict mean ± SD of biological duplicates from three independent donors . CD4 T cells were pre - treated with cycloheximide ( 2 μg / ml ) for 2 h , then treated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 ( 3 / 28 ) for 4 h , then analyzed by quantitative PCR for IFNB1 and OASL expression . Data are depicted as mean + SEM of 3 independent donors ( for cGAMP and αCD3 / CD28 treated cells without CHX only 2 data points were obtained ) . Statistics were determined by a two - way ANOVA on log - transformed data using Šidák correction for multiple testing : * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ; ns , not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1CD4 and CD8 T cells were stimulated with cGAMP (20 μg/ml) and αCD3/CD28 beads as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. Blots depict one representative donor out of three.CD4 T cells were stimulated with cGAMP (20 μg/ml) and αCD3/CD28; and human IFNβ (top) and IP10 levels (bottom) in the supernatant were analyzed by ELISA at the indicated time points. Data panels depict mean ± SD of biological duplicates from three independent donors.CD4 T cells were pre-treated with cycloheximide (2 μg/ml) for 2 h, then treated with cGAMP (20 μg/ml) and αCD3/CD28 (3/28) for 4 h, then analyzed by quantitative PCR for IFNB1 and OASL expression. Data are depicted as mean + SEM of 3 independent donors (for cGAMP and αCD3/CD28 treated cells without CHX only 2 data points were obtained). Statistics were determined by a two-way ANOVA on log-transformed data using Šidák correction for multiple testing: ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant.


Figure 2CD4 T cells were stimulated with the indicated stimuli for 4 h . Lysates were analyzed by immunoblotting . One representative donor out of three is shown . CRISPR / Cas9 targeted CD4 T cells for the indicated genes were treated with the indicated stimuli for 48 h . Human IFNβ levels were analyzed by ELISA . Data are depicted as mean + SEM of 3 independent donors . Statistics indicate significance by two - way ANOVA with Dunnett correction for multiple testing : * * * * p < 0 . 0001 ; ns , not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2CD4 T cells were stimulated with the indicated stimuli for 4 h. Lysates were analyzed by immunoblotting. One representative donor out of three is shown.CRISPR/Cas9 targeted CD4 T cells for the indicated genes were treated with the indicated stimuli for 48 h. Human IFNβ levels were analyzed by ELISA. Data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: ****p < 0.0001; ns, not significant.


Figure 3CD4 T cells were pre - treated with Dasatinib ( 100 , 10 , 1 and 0 . 1 nM ) , Tacrolimus ( 1000 , 100 , 10 and 1 nM ) , TPCA - 1 ( 4 , 2 , 1 and 0 . 5 μM ) and Torin1 ( 50 , 5 , 0 . 5 and 0 . 05 nM ) for 30 min , then treated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 for 16 h . Ηuman IFNβ levels in the supernatant were analyzed by ELISA . Data panels depict mean of biological duplicates from three independent donors . CD4 T cells were treated with cGAMP and indicated stimuli together with increasing concentrations of TPCA - 1 for 4 h . Cell lysates were analyzed by immunoblotting . One representative donor out of two is shown . CD4 T cells were stimulated with cGAMP ( 20 μg / ml ) and additional stimuli as indicated . Cell lysates after 4 h of stimulation were analyzed by immunoblotting . One representative donor out of two is shown . CD4 T cells were stimulated with cGAMP ( 20 μg / ml ) and additional stimuli as indicated . Cell lysates after 4 h of stimulation were analyzed by immunoblotting . One representative donor out of two is shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3CD4 T cells were pre-treated with Dasatinib (100, 10, 1 and 0.1 nM), Tacrolimus (1000, 100, 10 and 1 nM), TPCA-1 (4, 2, 1 and 0.5 μM) and Torin1 (50, 5, 0.5 and 0.05 nM) for 30 min, then treated with cGAMP (20 μg/ml) and αCD3/CD28 for 16 h. Ηuman IFNβ levels in the supernatant were analyzed by ELISA. Data panels depict mean of biological duplicates from three independent donors.CD4 T cells were treated with cGAMP and indicated stimuli together with increasing concentrations of TPCA-1 for 4 h. Cell lysates were analyzed by immunoblotting. One representative donor out of two is shown. CD4 T cells were stimulated with cGAMP (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown. CD4 T cells were stimulated with cGAMP (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown.


Figure 4Freshly isolated CD4 T cells were treated with cGAMP , αCD3 / CD28 beads and ABT737 / S63845 ( A / S ) for 48 h and analyzed by flow cytometry . Treatment induced cell death was assessed by Annexin V and propidium iodide staining . Data are depicted as mean + SEM of 3 independent donors . Statistics indicate significance by two - way ANOVA with Dunnett correction for multiple testing : * * * p < 0 . 001 ; * p < 0 . 05 ; ns , not significant . CD4 T cells were treated with indicated concentrations of cGAMP for 18 h , and ABT737 / S63845 for 2 h . Lysates and supernatant precipitations were analyzed by immunoblotting . One representative donor out of two is shown . CRISPR / Cas9 targeted CD4 T cells for indicated genes were treated with the indicated stimuli for 48 h and analyzed by flow cytometry . Each panel shows propidium iodide and Annexin V intensity for one representative donor out of three . Digits indicate the percentage of the parental population ( single events without subcellular debris ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Freshly isolated CD4 T cells were treated with cGAMP, αCD3/CD28 beads and ABT737/S63845 (A/S) for 48 h and analyzed by flow cytometry. Treatment induced cell death was assessed by Annexin V and propidium iodide staining. Data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: ***p < 0.001; *p < 0.05; ns, not significant.CD4 T cells were treated with indicated concentrations of cGAMP for 18 h, and ABT737/S63845 for 2 h. Lysates and supernatant precipitations were analyzed by immunoblotting. One representative donor out of two is shown.CRISPR/Cas9 targeted CD4 T cells for indicated genes were treated with the indicated stimuli for 48 h and analyzed by flow cytometry. Each panel shows propidium iodide and Annexin V intensity for one representative donor out of three. Digits indicate the percentage of the parental population (single events without subcellular debris).


Figure 5CellTrace Violet ( CTV ) stained , freshly isolated CD4 and CD8 T cells were activated with αCD3 / CD28 beads , stimulated with cGAMP for 96 h and analyzed by flow cytometry . Each panel shows profiles from activated ( black ) , cGAMP - treated ( green ) and resting ( light gray ) single and live cells from one representative donor . Freshly isolated CD4 T cells were treated with cGAMP ( 40 μg / ml ) and αCD3 / CD28 ( 3 / 28 ) for 48 h and analyzed by Seahorse XF mito stress test . Data are depicted as mean ± SEM of 3 independent donors . Statistics indicate significance by two - way ANOVA with Šidák correction for multiple testing : * * p < 0 . 01 ; * p < 0 . 05 ; ns , not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5CellTrace Violet (CTV) stained, freshly isolated CD4 and CD8 T cells were activated with αCD3/CD28 beads, stimulated with cGAMP for 96 h and analyzed by flow cytometry. Each panel shows profiles from activated (black), cGAMP-treated (green) and resting (light gray) single and live cells from one representative donor.Freshly isolated CD4 T cells were treated with cGAMP (40 μg/ml) and αCD3/CD28 (3/28) for 48 h and analyzed by Seahorse XF mito stress test. Data are depicted as mean ± SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Šidák correction for multiple testing: **p < 0.01; *p < 0.05; ns, not significant.


Figure 1D : Analytical ultracentrifugation absorbance data analysis of N - NTD . The clear peak at ~ 15 . 4 kDa molecular weight ( MW ) corresponds to the N - NTD monomer . E : Analytical ultracentrifugation absorbance data analysis of N - CTD . The clear peak at ~ 28 . 7 kDa MW corresponds to the N - CTD dimer .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1D: Analytical ultracentrifugation absorbance data analysis of N-NTD. The clear peak at ~15.4 kDa molecular weight (MW) corresponds to the N-NTD monomer. E: Analytical ultracentrifugation absorbance data analysis of N-CTD. The clear peak at ~28.7 kDa MW corresponds to the N-CTD dimer.


Figure 1Immunofluorescence microscopy detection of Tmem117 in the mouse hypothalamus and pituitary : ( A , B ) Positive immunostaining was observed in the PVN , the SON and the posterior pituitary . ( C , D ) No immunostaining was detected in the hypothalamus and pituitary of mice with constitutive inactivation of Tmem117 in AVP neurons . Data information : 3V : 3rd ventricle , AP : Anterior pituitary , MZ : Medial zone , opt : optic tract , PP : Posterior pituitary , PVN : Paraventricular nucleus , SCh : Suprachiasmatic nucleus , SON : Supraoptic nucleus . Scale bar = 100μm for panelsImmunofluorescence microscopy detection of Tmem117 in the mouse pituitary : ( E ) Co - staining for Tmem117 and AVP in the PVN . ( F ) Co - staining for Tmem117 and AVP in the SON . ( G ) Co - staining for Tmem117 and AVP in the posterior pituitary . ( H , I ) Quantification of AVP + / Tmem117 + cells in the PVN and SON , respectively . Data information : 3V : 3rd ventricle , opt : optic tract , PP : Posterior pituitary , PVN : Paraventricular nucleus , SON : Supraoptic nucleus . Scale bar = 100μm for panels ( J ) Higher resolution depiction of an AVP + / Tmem117 + neuron in the PVN captured with structured illumination microscopy . The white arrows highlight representative positions of signal co - localization . Data information : 3V : 3rd ventricle , PVN : Paraventricular nucleus , Scale bar = 10μm for panel J .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Immunofluorescence microscopy detection of Tmem117 in the mouse hypothalamus and pituitary: (A, B) Positive immunostaining was observed in the PVN, the SON and the posterior pituitary. (C, D) No immunostaining was detected in the hypothalamus and pituitary of mice with constitutive inactivation of Tmem117 in AVP neurons. Data information: 3V: 3rd ventricle, AP: Anterior pituitary, MZ: Medial zone, opt: optic tract, PP: Posterior pituitary, PVN: Paraventricular nucleus, SCh: Suprachiasmatic nucleus, SON: Supraoptic nucleus. Scale bar=100μm for panelsImmunofluorescence microscopy detection of Tmem117 in the mouse pituitary: (E) Co-staining for Tmem117 and AVP in the PVN. (F) Co-staining for Tmem117 and AVP in the SON. (G) Co-staining for Tmem117 and AVP in the posterior pituitary. (H, I) Quantification of AVP+/Tmem117+ cells in the PVN and SON, respectively. Data information: 3V: 3rd ventricle, opt: optic tract, PP: Posterior pituitary, PVN: Paraventricular nucleus, SON: Supraoptic nucleus. Scale bar=100μm for panels(J) Higher resolution depiction of an AVP+/Tmem117+ neuron in the PVN captured with structured illumination microscopy. The white arrows highlight representative positions of signal co-localization. Data information: 3V: 3rd ventricle, PVN: Paraventricular nucleus, Scale bar= 10μm for panel J.


Figure 2 ( B ) Glycemia one hour after saline ( black ) or insulin ( red ) i . p . injection [ n = 15 - 16 mice per group ] . ( C ) CPP plasma levels one hour after saline or insulin injection [ n = 13 mice per group ; mean ± SEM for INS : WT 89 ± 5 vs KO 121 ± 13 pg / ml ] . ( D ) GCG plasma levels one hour after saline or insulin injection [ n = 15 - 16 mice per group ; mean ± SEM for INS : WT 124 ± 19 vs KO 186 ± 21 pg / ml ] . ( E ) Glycemia one hour after saline ( black ) or insulin ( red ) i . p . injection [ n = 18 - 22 mice per group ] . ( F ) CPP plasma levels one hour after saline or insulin injection [ n = 18 - 19 mice per group ; mean ± SEM for INS : WT 78 ± 6 vs KO 92 ± 8 pg / ml ] . ( G ) GCG plasma levels one hour after saline or insulin injection [ n = 17 - 20 mice per group ; mean ± SEM for INS : WT 242 ± 14 vs KO 277 ± 18 pg / ml ] . ( H - J ) Analysis of glycemic levels and of CPP and GCG secretion following insulin injection in female mice at each stage of the estrus cycle [ n = 3 - 7 mice per group ] . ( H ) Glycemic levels . ( I ) CPP plasma levels . ( J ) GCG plasma levels . Data information : d : day , di : diestrus , est : estrus , INS : insulin , met : metestrus , pro : proestrus , For panels B - G , bars correspond to the mean value per group . For panels H - J , lines correspond to the mean value per group and error bars represent ± SEM . B - G : 2 - way ANOVA RM with Bonferroni post - hoc test ; H - J : unpaired t - test ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Glycemia one hour after saline (black) or insulin (red) i.p. injection [n=15-16 mice per group]. (C) CPP plasma levels one hour after saline or insulin injection [n=13 mice per group; mean±SEM for INS: WT 89±5 vs KO 121±13 pg/ml]. (D) GCG plasma levels one hour after saline or insulin injection [n=15-16 mice per group; mean±SEM for INS: WT 124±19 vs KO 186±21 pg/ml]. (E) Glycemia one hour after saline (black) or insulin (red) i.p. injection [n=18-22 mice per group]. (F) CPP plasma levels one hour after saline or insulin injection [n=18-19 mice per group; mean±SEM for INS: WT 78±6 vs KO 92±8 pg/ml]. (G) GCG plasma levels one hour after saline or insulin injection [n=17-20 mice per group; mean±SEM for INS: WT 242±14 vs KO 277±18 pg/ml]. (H-J) Analysis of glycemic levels and of CPP and GCG secretion following insulin injection in female mice at each stage of the estrus cycle [n=3-7 mice per group]. (H) Glycemic levels. (I) CPP plasma levels. (J) GCG plasma levels. Data information: d: day, di: diestrus, est: estrus, INS: insulin, met: metestrus, pro: proestrus, For panels B-G, bars correspond to the mean value per group. For panels H-J, lines correspond to the mean value per group and error bars represent ± SEM. B-G: 2-way ANOVA RM with Bonferroni post-hoc test; H-J: unpaired t-test; *p<0.05, **p<0.01, ***p<0.001.


Figure 3C57BL6 / N male mice were injected i . p . with saline or insulin and c - Fos expression ( green ) in AVP ( magenta ) neurons was quantitated by immunofluorescence microscopy 2 hours later . ( A ) C - Fos expression in the PVN after saline injection . ( B ) C - Fos expression in the PVN after insulin injection ( C ) Quantitation of c - Fos positive cells in the PVN after saline and insulin injections [ n = 4 mice per group ] . Data information : 3V : 3rd ventricle , AVP : Vasopressin , INS : insulin , PVN : Paraventricular nucleus , SAL : saline , Scale bar = 100μm . Lines correspond to the mean value per group and error bars represent ± SEM . Unpaired t - test ; ns : p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 . C57BL6 / N male mice were injected i . p . with saline or insulin and c - Fos expression ( green ) in AVP ( magenta ) neurons was quantitated by immunofluorescence microscopy 2 hours later . ( D ) C - Fos expression in the SON after saline injection . ( E ) C - Fos expression in the SON after insulin injection ( F ) Quantitation of c - Fos positive cells in the SON after saline and insulin injections [ n = 4 mice per group ] . Data information : AVP : Vasopressin , INS : insulin , opt : optic tract , SAL : saline , SON : Supraoptic nucleus . Scale bar = 100μm . Lines correspond to the mean value per group and error bars represent ± SEM . Unpaired t - test ; ns : p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 . ( H ) Tmem117 mRNA levels in the PVN [ n = 4 mice per group ] . ( I ) Tmem117 mRNA levels in the SON [ n = 4 mice per group Data information : INS : insulin , PVN : Paraventricular nucleus , SAL : saline , SON : Supraoptic nucleus . Lines correspond to the mean value per group and error bars represent ± SEM . Unpaired t - test ; ns : p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C57BL6/N male mice were injected i.p. with saline or insulin and c-Fos expression (green) in AVP (magenta) neurons was quantitated by immunofluorescence microscopy 2 hours later. (A) C-Fos expression in the PVN after saline injection. (B) C-Fos expression in the PVN after insulin injection (C) Quantitation of c-Fos positive cells in the PVN after saline and insulin injections [n=4 mice per group]. Data information: 3V: 3rd ventricle, AVP: Vasopressin, INS: insulin, PVN: Paraventricular nucleus, SAL: saline, Scale bar=100μm. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.C57BL6/N male mice were injected i.p. with saline or insulin and c-Fos expression (green) in AVP (magenta) neurons was quantitated by immunofluorescence microscopy 2 hours later. (D) C-Fos expression in the SON after saline injection. (E) C-Fos expression in the SON after insulin injection (F) Quantitation of c-Fos positive cells in the SON after saline and insulin injections [n=4 mice per group]. Data information: AVP: Vasopressin, INS: insulin, opt: optic tract, SAL: saline, SON: Supraoptic nucleus. Scale bar=100μm. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.(H) Tmem117 mRNA levels in the PVN [n=4 mice per group]. (I)Tmem117 mRNA levels in the SON [n=4 mice per group Data information: INS: insulin, PVN: Paraventricular nucleus, SAL: saline, SON: Supraoptic nucleus. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.


Figure 4 ( B ) C - Fos expression in the SON Data information : AVP : Vasopressin INS : insulin opt : optic tract , SAL : saline , Scale bar = 100μm . ( G ) Membrane potential was increased comparably in response to 0 . 1 mM glucose in both groups [ n = 7 - 9 cells per group ] . ( H ) No change in membrane potential in NR neurons of both genotypes [ n = 9 cells per group ] . ( I ) Membrane resistance was decreased in 6 out of 9 AVPTM117WT and 6 out of 7 AVPTM117KO cells in response to 0 . 1 mM glucose [ n = 7 - 9 cells per group ] . ( J ) Membrane resistance in NR neurons was stable for both genotypes [ n = 9 cells per group ] . Data information : AVP : Vasopressin , d : day , GI : glucose inhibited , NR : non - responding , Lines correspond to the mean value per group and error bars represent ± SEM . G - J : paired t - test for all graphs except Δ membrane potential ( unpaired ) ; nsp > 0 . 05 , * p < 0 . 05 , * * * p > 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) C-Fos expression in the SON Data information: AVP: Vasopressin INS: insulin opt: optic tract, SAL: saline, Scale bar=100μm.(G) Membrane potential was increased comparably in response to 0.1 mM glucose in both groups [n=7-9 cells per group]. (H) No change in membrane potential in NR neurons of both genotypes [n=9 cells per group]. (I) Membrane resistance was decreased in 6 out of 9 AVPTM117WT and 6 out of 7 AVPTM117KO cells in response to 0.1 mM glucose [n=7-9 cells per group]. (J) Membrane resistance in NR neurons was stable for both genotypes [n=9 cells per group]. Data information: AVP: Vasopressin, d: day, GI: glucose inhibited, NR: non-responding, Lines correspond to the mean value per group and error bars represent ± SEM. G-J: paired t-test for all graphs except Δ membrane potential (unpaired); nsp>0.05, *p<0.05, ***p>0.001.


Figure 5 ( A ) Quantification of BiP mRNA per cell in AVP - positive cells of the SON [ n = 483 - 704 cells per group ; each group consisted of 4 mice ] . ( B ) Quantification of BiP mRNA per cell in AVP - negative cells of the SON [ n = 347 - 584 cells per group ; each group consisted of 4 mice ] . Data information : AVP : Vasopressin For the violin plots dashed lines correspond to the median value and dotted lines to the quartile values . unpaired t - test ; * p < 0 . 05 , * * * p > 0 . 001 . ( C ) Fluorescence microscopy detection of AVP mRNA ( green ) and BiP mRNA ( magenta ) in the SON . Data information : AVP : Vasopressin opt : optic tract Scale bar = 20μm . ( F ) Fluorescence microscopy detection of DHE - derived fluorescence ( red ) in the SON . ( G ) Quantification of DHE - derived fluorescence in the SON [ n = 8 SON per group ; each group consisted of 4 mice ] . Data information : AVP : Vasopressin , d : day , DHE : dihydroethidium , opt : optic tract , SON : Supraoptic nucleus , . Scale bar = 20μm . For panels G data are represented as mean ± SEM . G : unpaired t - test ; ; * p < 0 . 05 , * * * p > 0 . 001 . ( J ) Calcium recordings in AVP magnocellular terminals 10 minutes before and one hour after the i . p injection of insulin ( red and orange ) or saline ( black ) . The vertical arrow corresponds to the i . p . injection ( time = 0 ) . ( K ) Quantification of mean signal intensity in 10 - minute time bins . [ n = 6 - 8 mice per group ] . Data information : AUC : area under the curve , AVP : Vasopressin INS : insulin , SAL : saline For panels K , data are represented as mean ± SEM . K : 1 - way ANOVA with Tukey ' s post - hoc test ; * p < 0 . 05 , * * * p > 0 . 001 . ( M ) Fluorescence microscopy detection of GCaMP ( green ) in posterior pituitary . Immunofluorescent labelling was used to localize the AVP terminals ( magenta ) . Data information : AVP : Vasopressin , . Scale bar = 20μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Quantification of BiP mRNA per cell in AVP-positive cells of the SON [n=483-704 cells per group; each group consisted of 4 mice]. (B) Quantification of BiP mRNA per cell in AVP-negative cells of the SON [n=347-584 cells per group; each group consisted of 4 mice]. Data information: AVP: Vasopressin For the violin plots dashed lines correspond to the median value and dotted lines to the quartile values. unpaired t-test ; *p<0.05, ***p>0.001.(C) Fluorescence microscopy detection of AVP mRNA (green) and BiP mRNA (magenta) in the SON. Data information: AVP: Vasopressin opt: optic tract Scale bar=20μm.(F) Fluorescence microscopy detection of DHE-derived fluorescence (red) in the SON. (G) Quantification of DHE-derived fluorescence in the SON [n=8 SON per group; each group consisted of 4 mice]. Data information: AVP: Vasopressin, d: day, DHE: dihydroethidium , opt: optic tract, SON: Supraoptic nucleus,. Scale bar=20μm. For panels G data are represented as mean ± SEM. G: unpaired t-test; ; *p<0.05, ***p>0.001.(J) Calcium recordings in AVP magnocellular terminals 10 minutes before and one hour after the i.p injection of insulin (red and orange) or saline (black). The vertical arrow corresponds to the i.p. injection (time=0). (K) Quantification of mean signal intensity in 10-minute time bins. [n=6-8 mice per group]. Data information: AUC: area under the curve, AVP: Vasopressin INS: insulin, SAL: saline For panels K, data are represented as mean ± SEM. K: 1-way ANOVA with Tukey's post-hoc test; *p<0.05, ***p>0.001.(M) Fluorescence microscopy detection of GCaMP (green) in posterior pituitary. Immunofluorescent labelling was used to localize the AVP terminals (magenta). Data information: AVP: Vasopressin ,. Scale bar=20μm.


Figure 6 ( B ) Immunofluorescence microscopy detection of AVP in the PVN and the SON . Data information : 3V : 3rd ventricle , AVP : Vasopressin , KO : AVPTM117KO opt : optic tract , PVN : paraventricular nucleus , SON : Supraoptic nucleus , WT : AVPTM117WT . Scale bar = 100μm . ( C ) Quantitation of AVP positive cells in the PVN [ n = 3 - 4 mice per group ] . ( D ) Quantitation of AVP positive cells in the SON [ n = 3 - 4 mice per group ] . Data information : AVP : Vasopressin , KO : AVPTM117KO WT : AVPTM117WT . For panels C and D lines correspond to the mean value per group and error bars represent ± SEM . For panels C , D : unpaired t - test ; ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( E ) Glycemia one hour after saline ( black ) or insulin ( red ) i . p . injections in AVPTM117WT and AVPTM117KO male mice [ n = 8 - 9 mice per group ] . ( F ) CPP plasma levels one hour after saline or insulin injection in AVPTM117WT and AVPTM117KO male mice [ n = 8 mice per group ] . ( G ) GCG plasma levels one hour after saline or insulin injection in AVPTM117WT and AVPTM117KO male mice [ n = 8 - 9 mice per group ] . Data information : AVP : Vasopressin , d : day , INS : insulin , KO : AVPTM117KO , SAL : saline , WT : AVPTM117WT . For panels E - G bars correspond to the mean value per group and error bars represent ± SEM . E - G : 2 - way ANOVA for each timepoint in comparison to SAL baseline with Bonferroni post - hoc test ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) Immunofluorescence microscopy detection of AVP in the PVN and the SON. Data information: 3V: 3rd ventricle, AVP: Vasopressin, KO: AVPTM117KO opt: optic tract, PVN: paraventricular nucleus, SON: Supraoptic nucleus, WT: AVPTM117WT. Scale bar=100μm.(C) Quantitation of AVP positive cells in the PVN [n=3-4 mice per group]. (D) Quantitation of AVP positive cells in the SON [n=3-4 mice per group]. Data information: AVP: Vasopressin, KO: AVPTM117KO WT: AVPTM117WT. For panels C and D lines correspond to the mean value per group and error bars represent ± SEM. For panels C,D: unpaired t-test; ; *p<0.05, **p<0.01, ***p<0.001.(E) Glycemia one hour after saline (black) or insulin (red) i.p. injections in AVPTM117WT and AVPTM117KO male mice [n=8-9 mice per group]. (F) CPP plasma levels one hour after saline or insulin injection in AVPTM117WT and AVPTM117KO male mice [n=8 mice per group]. (G) GCG plasma levels one hour after saline or insulin injection in AVPTM117WT and AVPTM117KO male mice [n=8-9 mice per group]. Data information: AVP: Vasopressin, d: day, INS: insulin, KO: AVPTM117KO, SAL: saline, WT: AVPTM117WT. For panels E-G bars correspond to the mean value per group and error bars represent ± SEM. E-G: 2-way ANOVA for each timepoint in comparison to SAL baseline with Bonferroni post-hoc test; *p<0.05, **p<0.01, ***p<0.001.


Figure 1 A Whole mount FISH for piwi - 1 and immunostaining for BrdU at 1 - day , 1 - week and 5 - weeks post - BrdU administration . White boxes denote the magnified regions shown in the bottom panels . Scale bars , 30 µm . B Quantification of the percentage of BrdU label - retaining neoblasts . Data are presented as mean ± s . d . ( n = 4 - 6 animals per time point ) . C Double pulse experiment showing colocalization of BrdU after a 2 - week chase period with EdU after a 1 - day chase period . White boxes denote the magnified region shown on the left . Dashed lines mark the edges of the animal and white arrows mark colocalization . Scale bar , 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 A Whole mount FISH for piwi-1 and immunostaining for BrdU at 1-day, 1-week and 5-weeks post-BrdU administration. White boxes denote the magnified regions shown in the bottom panels. Scale bars, 30 µm.  B Quantification of the percentage of BrdU label-retaining neoblasts. Data are presented as mean ± s.d. (n = 4-6 animals per time point).  C Double pulse experiment showing colocalization of BrdU after a 2-week chase period with EdU after a 1-day chase period. White boxes denote the magnified region shown on the left. Dashed lines mark the edges of the animal and white arrows mark colocalization. Scale bar, 50 µm.


Figure 2 A Dissociated cells from unirradiated , 1 dpi or 7 dpi animals ( 60 Gy of irradiation ) were stained with Hoechst ( DNA ) and Pyronin Y ( RNA ) and sorted by FACS to reveal an RNAlow population . Percentages indicate the proportion of events within each gate . B Cell size and nucleus : cytoplasm ratio measured by CellMask ( cytoplasm ) and DAPI ( nucleus ) staining of FACS sorted cells at homeostasis ( n ≥ 47 per sample ) . Representative images are shown , with dashed lines to indicate the area measured . Scale bars , 5 µm . C EdU pulse - chase time course of label - retention in sorted RNAlow and S / G2 / M neoblasts ( n ≥ 3 individual experiments per time point ) . Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S / G2 / M or RNAlow neoblasts , respectively . Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S / G2 / M neoblasts . D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C . Red boxes denote examples of EdU - cells . Scale bars , 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 A Dissociated cells from unirradiated, 1 dpi or 7 dpi animals (60 Gy of irradiation) were stained with Hoechst (DNA) and Pyronin Y (RNA) and sorted by FACS to reveal an RNAlow population. Percentages indicate the proportion of events within each gate.  B Cell size and nucleus:cytoplasm ratio measured by CellMask (cytoplasm) and DAPI (nucleus) staining of FACS sorted cells at homeostasis (n ≥ 47 per sample). Representative images are shown, with dashed lines to indicate the area measured. Scale bars, 5 µm.  C EdU pulse-chase time course of label-retention in sorted RNAlow and S/G2/M neoblasts (n ≥ 3 individual experiments per time point). Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S/G2/M or RNAlow neoblasts, respectively. Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S/G2/M neoblasts.  D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C. Red boxes denote examples of EdU- cells. Scale bars, 5 µm.


Figure 3 A Area measurements of cells from the RNAlow , G1 and S / G2 / M gates in intact animals and at 6 and 48 hpa ( n ≥ 28 for each sample in the RNAlow plot , n ≥ 24 in the G1 plot , n ≥ 42 in the S / G2 / M plot ) . B RNAlow neoblast size in intact animals and at 48 , 72 and 96 hpa ( n ≥ 54 ) . C RNAlow neoblast size in intact animals and at 48 hours following amputation , poke or incision injuries ( n ≥ 61 ) . D RNAlow neoblast size in unirradiated animals and at 1 , 2 and 3 dpi ( 12 . 5 Gy . of irradiation , n ≥ 52 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 A Area measurements of cells from the RNAlow, G1 and S/G2/M gates in intact animals and at 6 and 48 hpa (n ≥ 28 for each sample in the RNAlow plot, n ≥ 24 in the G1 plot, n ≥ 42 in the S/G2/M plot).  B RNAlow neoblast size in intact animals and at 48, 72 and 96 hpa (n ≥ 54).  C RNAlow neoblast size in intact animals and at 48 hours following amputation, poke or incision injuries (n ≥ 61).  D RNAlow neoblast size in unirradiated animals and at 1, 2 and 3 dpi (12.5 Gy. of irradiation, n ≥ 52).


Figure 4 A Representative images of RNAlow , G1 and S / G2 / M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa . All images shown are from samples processed in the same experiment . Intact images are duplicated from Figure 2B . White arrows mark projections . Percentage of cells displaying projections in each condition is noted ( n > 100 cells counted ) . Scale bars , 5 µm . B Average projection length in RNAlow , G1 and S / G2 / M cells from intact animals and at 6 and 48 hpa ( n ≥ 13 ) . C - E Quantification of the number of projections per cell ( blue data ) and the sum total projection length per cell ( orange data ) in RNAlow ( C ) , G1 ( D ) or S / G2 / M ( E ) cells isolated from intact animals and at 6 and 48 hpa ( n ≥ 23 for projections / cell , n ≥ 10 for Σ projection length / cell ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 A Representative images of RNAlow, G1 and S/G2/M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa. All images shown are from samples processed in the same experiment. Intact images are duplicated from Figure 2B. White arrows mark projections. Percentage of cells displaying projections in each condition is noted (n > 100 cells counted). Scale bars, 5 µm. B Average projection length in RNAlow, G1 and S/G2/M cells from intact animals and at 6 and 48 hpa (n ≥ 13). C-E Quantification of the number of projections per cell (blue data) and the sum total projection length per cell (orange data) in RNAlow (C), G1 (D) or S/G2/M (E) cells isolated from intact animals and at 6 and 48 hpa (n ≥ 23 for projections/cell, n ≥ 10 for Σ projection length/cell).


Figure 5 B FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment . C RNAlow neoblast size in intact animals and at 48 hpa in control or TOR KD animals ( n ≥ 55 ) . D Quantification of H3P immunostaining in intact animals and at 48 hpa in control or TOR KD animals ( n = 5 - 10 ) . E RNAlow neoblast size in intact animals and at 48 hpa in control , raptor or rictor KD animals ( n ≥ 54 ) . F FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals . Percentages indicate the proportion of events within each gate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 B FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment.  C RNAlow neoblast size in intact animals and at 48 hpa in control or TOR KD animals (n ≥ 55).  D Quantification of H3P immunostaining in intact animals and at 48 hpa in control or TOR KD animals (n = 5-10).  E RNAlow neoblast size in intact animals and at 48 hpa in control, raptor or rictor KD animals (n ≥ 54).  F FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals. Percentages indicate the proportion of events within each gate.


Figure 6A Representative WISH images for Lrig - 1 . B RNAlow neoblast size in intact animals following control or Lrig - 1 knockdown ( n ≥ 53 technical replicates ) . C Quantification of H3P immunostaining in intact animals following control or Lrig - 1 knockdown ( n = 9 - 10 technical replicates ) . D Quantification of H3P immunostaining at homeostasis ( fixed at 7 days post - RNAi , white region ) and at 48 hpa ( amputated at 5 days post - RNAi and fixed at 7 days post - RNAi , yellow region ) during the first round of regeneration in control or Lrig - 1 KD animals ( n = 10 - 17 ) . E Quantification of unpigmented blastema area ( as % total tissue area ) in regenerating control or Lrig - 1 KD animals from 2 dpa to 7 dpa during the first round of regeneration ( n = 10 animals per condition per time point ) . The same animals were used for all timepoints . Representative images from 7 dpa are shown . Scale bars , 500 µm . F Quantification of H3P immunostaining at homeostasis ( fixed at 14 days post - RNAi , white region ) and at 48 hpa ( amputated at 12 days post - RNAi and fixed at 14 days post - RNAi , yellow region ) during a second round of regeneration in control or Lrig - 1 KD animals ( n = 10 - 18 ) . G Quantification of unpigmented blastema area ( as % total tissue area ) in regenerating control or Lrig - 1 KD animals from 2 dpa to 7 dpa during the second round of regeneration ( n = 9 - 10 animals per condition per time point ) . The same animals were used for all timepoints . Representative images from 7 dpa are shown . Scale bars , 500 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Representative WISH images for Lrig-1. B RNAlow neoblast size in intact animals following control or Lrig-1 knockdown (n ≥ 53 technical replicates).  C Quantification of H3P immunostaining in intact animals following control or Lrig-1 knockdown (n = 9-10 technical replicates).  D Quantification of H3P immunostaining at homeostasis (fixed at 7 days post-RNAi, white region) and at 48 hpa (amputated at 5 days post-RNAi and fixed at 7 days post-RNAi, yellow region) during the first round of regeneration in control or Lrig-1 KD animals (n = 10-17).  E Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the first round of regeneration (n = 10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm.  F Quantification of H3P immunostaining at homeostasis (fixed at 14 days post-RNAi, white region) and at 48 hpa (amputated at 12 days post-RNAi and fixed at 14 days post-RNAi, yellow region) during a second round of regeneration in control or Lrig-1 KD animals (n = 10-18).  G Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the second round of regeneration (n = 9-10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm.


Figure 1 ( A - D ) Images of a representative wild - type adult eye ( A ) or adult eyes containing mtm mutant clones ( B and C ) . Quantification of relative eye sizes ( n = 10 ) ( D ) . ( F - G ) A pupal eye disc containing mtm mutant clones ( marked by the absence of GFP ) was stained for Dlg . Note the increased interommatidial cells in the mtm mutant clone . Quantification of numbers of interommatidial cells per ommatidium ( n = 10 ) ( G ) . ( K ) RNAi of Mtm reduces Yki phosphorylation . Kc167 cells were incubated with dsRNA of gfp or mtm for three days before western blot analysis . Total cell lysates were probed with anti - phospho - Yki ( p - Yki ) antibody . Note that RNAi of mtm decreased Yki phosphorylation .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A-D) Images of a representative wild-type adult eye (A) or adult eyes containing mtm mutant clones (B and C). Quantification of relative eye sizes (n=10) (D).(F-G) A pupal eye disc containing mtm mutant clones (marked by the absence of GFP) was stained for Dlg. Note the increased interommatidial cells in the mtm mutant clone. Quantification of numbers of interommatidial cells per ommatidium (n=10) (G).(K) RNAi of Mtm reduces Yki phosphorylation. Kc167 cells were incubated with dsRNA of gfp or mtm for three days before western blot analysis. Total cell lysates were probed with anti-phospho-Yki (p-Yki) antibody. Note that RNAi of mtm decreased Yki phosphorylation.


Figure 4PI ( 3 ) P expression levels detected by a 2xFYVE - GFP reporter in a third instar larval wing disc ( A ) and eye disc ( B ) containing mtm mutant clones ( marked by the absence of RFP ) . Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis . Note the strong increases of PI ( 3 ) P levels in the mtm mutant clones . Quantification of the mean FYVE - GFP fluorescence intensity of mtm mutant clones and surrounding wild - type ( WT ) cells are shown ( C ) . Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis . Note the strong increases of PI ( 3 ) P levels in the mtm mutant clones .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4PI(3)P expression levels detected by a 2xFYVE-GFP reporter in a third instar larval wing disc (A) and eye disc (B) containing mtm mutant clones (marked by the absence of RFP). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.Quantification of the mean FYVE-GFP fluorescence intensity of mtm mutant clones and surrounding wild-type (WT) cells are shown (C). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.


Figure 1Display of differentially expressed genes in NeuNT tumors ( WT , TNCKO ) as volcano plot representing all associated genes in WT tumors . N = 3 tumors per genotype . ( A ) Log2 Fold Change ( X - axis ) and the negative log10 ( P . Value ) ( Y - axis ) is displayed , the blue line indicating p = 0 . 05 ( Y - axis ) , points above the line , p < 0 . 05 . The dashed red lines indicate the log 2 Fold Change cutoff of - 0 . 25 and 0 . 25 , respectively ( X - axis ) . Green dots represent genes that display both statistical significance ( p < 0 . 05 ) and fold changes - 0 . 25 and 0 . 25 , respectively . Selected genes regulated by TNC are marked in red ( type I interferon response ) or pink ( Cxcl12 ) dot .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Display of differentially expressed genes in NeuNT tumors (WT, TNCKO) as volcano plot representing all associated genes in WT tumors. N = 3 tumors per genotype. (A) Log2 Fold Change (X-axis) and the negative log10 (P.Value) (Y-axis) is displayed, the blue line indicating p = 0.05 (Y-axis), points above the line, p < 0.05. The dashed red lines indicate the log 2 Fold Change cutoff of -0.25 and 0.25, respectively (X-axis). Green dots represent genes that display both statistical significance (p < 0.05) and fold changes -0.25 and 0.25, respectively. Selected genes regulated by TNC are marked in red (type I interferon response) or pink (Cxcl12) dot.


Figure 2 ( A ) Representative staining images ( N = 5 ) of tissue from TNC high ( WT / shC ) and TNC low ( KO / shTNC ) tumors ( N = 5 per condition ) with the indicated antibodies . Arrows point at the respective immune subtype . Scale bar , 50 µm . ( D , E ) Apoptosis measurement upon tumor staining for cl . casp3 ( D ) and signal quantification per field ( E ) in TNC high and TNC low tumors ( N = 7 per condition ) , 2 sections per tumor , 5 random fields . * * * p = 0 . 0006 , Mann - Whitney test , Mean ± SEM . Scale bar , 50 µm . ( I , J ) Detection ( I ) and quantification ( J ) of CD8 TIL in lung metastasis of NeuNT WT and TNCKO mice ( N = 3 mice , n = 7 and 12 lung metastases , respectively ) . Scale bar , 100 µm . * * p = 0 . 0097 , Mann Whitney test . Mean ± SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative staining images (N = 5) of tissue from TNC high (WT/shC) and TNC low (KO/shTNC) tumors (N = 5 per condition) with the indicated antibodies. Arrows point at the respective immune subtype. Scale bar, 50 µm.(D, E) Apoptosis measurement upon tumor staining for cl. casp3 (D) and signal quantification per field (E) in TNC high and TNC low tumors (N = 7 per condition), 2 sections per tumor, 5 random fields. *** p = 0.0006, Mann-Whitney test, Mean ± SEM. Scale bar, 50 µm.(I, J) Detection (I) and quantification (J) of CD8 TIL in lung metastasis of NeuNT WT and TNCKO mice (N = 3 mice, n = 7 and 12 lung metastases, respectively). Scale bar, 100 µm. ** p = 0.0097, Mann Whitney test. Mean ± SEM.


Figure 3 ( E ) Analysis of Cxcl12 ( qRT - PCR ) in tumor cells upon pretreatment with the indicated inhibitors for 45 minutes ( GW , BOP , DMSO and PBS as control respectively ) or 60 minutes ( Sunitinib ) and 6 hours ( Cli95 ) ( 6 hours DMSO as control ) before incubation with TNC for 24 hours . N = 6 independent experiments , * * p = 0 . 0022 , Mann Whitney test . Mean ± SEM . CXCL12 binding to TNC as determined by negative EM imaging ( G ) , followed by quantification ( H ) . ( G ) scale bars , 100 nm ( left ) , 50 nm ( right ) , the arrowheads point at gold labeled CXCL12 and the asterisk points at the fibrinogen globe of the murine TNC monomer . ( H ) Representation of TNC monomer , oligomerization domain ( triangle ) to form hexamers as seen in ( G ) , FNIII repeats ( grey boxes , contant domains , white boxes , alternative domains ) , fibrinogen like domain ( circle ) . Representative result ( 3 independent experiments ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(E) Analysis of Cxcl12 (qRT-PCR) in tumor cells upon pretreatment with the indicated inhibitors for 45 minutes (GW, BOP, DMSO and PBS as control respectively) or 60 minutes (Sunitinib) and 6 hours (Cli95) (6 hours DMSO as control) before incubation with TNC for 24 hours. N = 6 independent experiments, ** p = 0.0022, Mann Whitney test. Mean ± SEM.CXCL12 binding to TNC as determined by negative EM imaging (G), followed by quantification (H). (G) scale bars, 100 nm (left), 50 nm (right), the arrowheads point at gold labeled CXCL12 and the asterisk points at the fibrinogen globe of the murine TNC monomer. (H) Representation of TNC monomer, oligomerization domain (triangle) to form hexamers as seen in (G), FNIII repeats (grey boxes, contant domains, white boxes, alternative domains), fibrinogen like domain (circle). Representative result (3 independent experiments).


Figure 4Attraction and retention were determined towards coatings with collagen I ( COL ) , fibronectin ( FN ) or TNC in the presence of CXCL10 , CCL21 , CXCL12 ( B , C ) 3 independent experiments in duplicates , Mann - Whitney test , ns > 0 . 05 ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 005 . Mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Attraction and retention were determined towards coatings with collagen I (COL), fibronectin (FN) or TNC in the presence of CXCL10, CCL21, CXCL12 (B, C) 3 independent experiments in duplicates, Mann-Whitney test, ns > 0.05; * p <0.05; **p < 0.01; ***p < 0.005. Mean ± SEM.


Figure 5 ( B , C ) Imaging and signal quantification upon staining for cl . casp3 and DAPI in PBS and AMD treated tumors , N = 5 tumors per condition , 2 sections per tumor , 5 random fields , * * p = 0 . 0079 , Mann - Whitney test . Mean ± SEM . Scale bar , 50µm , arrows point at cl . Casp3 . ( D , E ) CD8 TIL abundance and localization in PBS and AMD treated tumors , arrow points at CD8 TIL , scale bar , 50µm ( D ) . ( E ) Quantification of CD8 T cells per randomly chosen fields . N = 5 tumors per condition , 2 sections per tumor , 5 random fields , * * p = 0 . 0079 , Mann - Whitney test . Mean ± SEM . ( F ) Gzmb levels in PBS and AMD treated tumors . N = 10 tumors , * * * * p < 0 . 0001 , unpaired t - test . Mean ± SEM . ( ( I , J ) Abundance and quantification of F4 / 80 + macrophages in PBS and AMD treated tumors , ( I ) staining for the indicated molecules , ( arrow , macrophages in the stroma ; asterisk , macrophages in the tumor nest ) , ( J ) quantification of F4 / 80 + cells inside the tumor nest . N = 5 tumors per condition , 2 sections per tumor , 5 random fields , * * p = 0 . 0079 , Mann - Whitney test . Mean ± SEM . Scale bar , 50 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B, C) Imaging and signal quantification upon staining for cl. casp3 and DAPI in PBS and AMD treated tumors, N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. Scale bar, 50µm, arrows point at cl. Casp3.(D, E) CD8 TIL abundance and localization in PBS and AMD treated tumors, arrow points at CD8 TIL, scale bar, 50µm (D). (E) Quantification of CD8 T cells per randomly chosen fields. N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. (F) Gzmb levels in PBS and AMD treated tumors. N = 10 tumors, **** p < 0.0001, unpaired t-test. Mean ± SEM. ( (I, J) Abundance and quantification of F4/80+ macrophages in PBS and AMD treated tumors, (I) staining for the indicated molecules, (arrow, macrophages in the stroma; asterisk, macrophages in the tumor nest), (J) quantification of F4/80+ cells inside the tumor nest. N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. Scale bar, 50 µm.


Figure 6 ( I - L ) qRT - PCR assessment of Neu ( I ) , Cd8a ( J ) , Ifnγ ( K ) and Gzmb ( L ) by qRT - PCR . PBS ( N = 16 ) and AMD ( N = 19 ) treated mice , * , p = 0 . 0364 , Mann whitney test ( I ) , * * , p = 0 . 0086 unpaired t - test ( J ) , * * , p = 0 . 0055 , unpaired t - test ( K ) , * * * , p = 0 . 0003 unpaired t - test ( L ) , respectively . The central band represents the median , the ends of the box the first and the third quartiles , and the whiskers reach from each quartile to the minimum or maximum .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(I-L) qRT-PCR assessment of Neu (I), Cd8a (J), Ifnγ (K) and Gzmb (L) by qRT-PCR. PBS (N = 16) and AMD (N = 19) treated mice, *, p = 0.0364, Mann whitney test (I), **, p = 0.0086 unpaired t-test (J), **, p = 0.0055, unpaired t-test (K), ***, p = 0.0003 unpaired t-test (L), respectively. The central band represents the median, the ends of the box the first and the third quartiles, and the whiskers reach from each quartile to the minimum or maximum.


Figure 7 ( A - C ) Adjacent human breast cancer tissue microarray ( TMA ) sections were stained with the indicated antibodies , scale bar , 50 μm , arrows point at CD8 TIL ( A ) and , CD8 TIL were counted per field ( B ) , or in the stroma and nest , respectively ( C ) , N = 103 TNC + tumors , N = 116 TNC - tumors . . ( B ) * * p = 0 . 0055 , Fisher ' s exact test . The central band represents the median , the ends of the box the first and the third quartiles , and the whiskers reach from each quartile to the minimum or maximum . ( C ) * * p = 0 , 0055 , Mann - Whitney test . ( D - G ) Kaplan Meier analysis to address correlation of TNC expression ( D ) , total numbers of CD8 TIL ( E ) , combined high ( above median ) CD8 TIL and high or low ( above / below median ) TNC ( F ) and stromal CD8 cells ( G ) with metastasis - free survival ( MFS ) of breast cancer patients . Stromal CD8 - or CD8 + is defined as below or above median stromal CD8 TIL per area . Log - rank test . ( H , I ) Correlation of TNC and CD8a levels with overall survival ( OS ) using survival analysis on breast cancer patient cohort GSE 19783 - GPL6580 . Hazard ratio ( HR ) and p values are indicated in Appendix Fig . S5N . Log - rank test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A-C) Adjacent human breast cancer tissue microarray (TMA) sections were stained with the indicated antibodies, scale bar, 50 μm, arrows point at CD8 TIL (A) and, CD8 TIL were counted per field (B), or in the stroma and nest, respectively (C), N = 103 TNC+ tumors, N = 116 TNC- tumors.. (B) ** p = 0.0055, Fisher's exact test. The central band represents the median, the ends of the box the first and the third quartiles, and the whiskers reach from each quartile to the minimum or maximum. (C) ** p = 0,0055, Mann-Whitney test.(D-G) Kaplan Meier analysis to address correlation of TNC expression (D), total numbers of CD8 TIL (E), combined high (above median) CD8 TIL and high or low (above/below median) TNC (F) and stromal CD8 cells (G) with metastasis-free survival (MFS) of breast cancer patients. Stromal CD8- or CD8+ is defined as below or above median stromal CD8 TIL per area. Log-rank test.(H, I) Correlation of TNC and CD8a levels with overall survival (OS) using survival analysis on breast cancer patient cohort GSE 19783-GPL6580. Hazard ratio (HR) and p values are indicated in Appendix Fig. S5N. Log-rank test.


Figure 8 ( A ) Representative IHC images of CXCL12 intensity and staining pattern in the TMA . N = 219 tumors . Upper panel , scale bar , 400 μm ; lower panel , scale bar , 100 μm . Arrows point at punctate staining in the stroma . ( D ) Kaplan Meier survival analysis of breast cancer patients upon stratification into tumors with a CXCL12 Hscore below or above the median , p = 0 . 002 . Mann - Whitney test . ( E ) Survival analysis of breast cancer patients in the GSE 19783 - GPL6580 cohort upon stratification into tumors with high or low expression of TNC and CXCL12 . Log - rank test . HR and p values are indicated ,

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Representative IHC images of CXCL12 intensity and staining pattern in the TMA. N = 219 tumors. Upper panel, scale bar, 400 μm; lower panel, scale bar, 100 μm. Arrows point at punctate staining in the stroma.(D) Kaplan Meier survival analysis of breast cancer patients upon stratification into tumors with a CXCL12 Hscore below or above the median, p = 0.002. Mann-Whitney test. (E) Survival analysis of breast cancer patients in the GSE 19783-GPL6580 cohort upon stratification into tumors with high or low expression of TNC and CXCL12. Log-rank test. HR and p values are indicated,