Figure 6Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) staining of fetal liver cryosections of the indicated genotypes at E13 . 5 and quantification . Scale bar 100x = 200 µM , 400x = 50 µM . The percentage of TUNEL + area / field was assessed across three random fields from two control ( Chk1fl / + ) and two knock - out embryos ( Chk1fl / - Vav - Cre ) using ImageJ , as reported by us before ( 60 ) . Bars represent means ± S . D . Total fetal liver cell suspensions of Chk1fl / + ( + / + ) , Chk1fl / + Vav - Cre ( + / - ) and Chk1fl / - Vav - Cre ( - / - ) E13 . 5 embryos were processed for Western blot analysis using the indicated antibodies . Each lane represents samples isolated from an individual littermate embryo . DAPI - negative viable LK - and LSK - cells of the indicated genotypes ( littermates ) were sorted from E13 . 5 fetal livers and immediately fixed in 70 % EtOH . Fixed cell suspensions were stained for ɣH2A . X and DAPI intracellularly . Shown here are representative dot - plots and quantification of N = 4 embroys per genotype ; Bars represent means ± S . E . M . Total E13 . 5 fetal liver cells were processed for intracellular Ki67 staining in combination with cell surface antibody staining to discriminate LK ( top panel ) from LSK ( lower panel ) cells . Shown here are representative dot - plots of Ki67 and DAPI staining in LK and LSK cells , quantified in ( E ) . Bars represent means ± S . E . M . N = 4 for Chk1fl / + and N = 5 for Chk1fl / - Vav - Cre .

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

Figure 6Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of fetal liver cryosections of the indicated genotypes at E13.5 and quantification. Scale bar 100x = 200 µM, 400x = 50 µM. The percentage of TUNEL+ area/field was assessed across three random fields from two control (Chk1fl/+) and two knock-out embryos (Chk1fl/- Vav-Cre) using ImageJ, as reported by us before (60). Bars represent means ± S.D.Total fetal liver cell suspensions of Chk1fl/+ (+/+), Chk1fl/+ Vav-Cre (+/-) and Chk1fl/- Vav-Cre (-/-) E13.5 embryos were processed for Western blot analysis using the indicated antibodies. Each lane represents samples isolated from an individual littermate embryo.DAPI-negative viable LK- and LSK-cells of the indicated genotypes (littermates) were sorted from E13.5 fetal livers and immediately fixed in 70% EtOH. Fixed cell suspensions were stained for ɣH2A.X and DAPI intracellularly. Shown here are representative dot-plots and quantification of N=4 embroys per genotype; Bars represent means ± S.E.M.Total E13.5 fetal liver cells were processed for intracellular Ki67 staining in combination with cell surface antibody staining to discriminate LK (top panel) from LSK (lower panel) cells. Shown here are representative dot-plots of Ki67 and DAPI staining in LK and LSK cells, quantified in (E). Bars represent means ± S.E.M. N=4 for Chk1fl/+ and N=5 for Chk1fl/- Vav-Cre.


Figure 7Representative dot - plots and quantification of E13 . 5 fetal liver LSKhi / LSKlow cell phenotypes observed in embryos of the indicated genotypes . N = 21 for Chk1fl / + , N = 5 for Chk1fl / - Vav - Cre , N = 16 for Chk1fl / + Vav - BCL2 , N = 5 for Chk1fl / - Vav - Cre Vav - BCL2 , N = 2 for p53 - / - Chk1fl / + and N = 4 for p53 - / - Chk1fl / - Vav - Cre . Flow cytometry - based analysis of LSK cells stained with antibodies specific for CD34 and Flt3 , to discriminate LT - and ST - HSC from MPP within the LSK subset in fetal livers of embryos of the indicated genotypes . Left : N = 25 for Chk1fl / + , N = 7 for Chk1fl / + Vav - Cre and N = 7 for Chk1fl / - Vav - Cre . Right : N = 16 for Chk1fl / + Vav - BCL2 , N = 6 for Chk1fl / + Vav - Cre Vav - BCL2 and N = 5 for Chk1fl / - Vav - Cre Vav - BCL2 . Analysis of the colony formation potential of E14 . 5 total fetal liver cells of the indicated genotypes in methylcellulose . N = 3 for controls ( pooled ; 1 Chk1fl / + , 2 Chk1fl / + Vav - Cre ) , N = 3 for Chk1fl / - Vav - Cre and N = 3 for Chk1fl / - Vav - Cre Vav - BCL2 . Western blot analysis of fetal liver cells of the indicated genotypes ( each lane represents an embryo ) at E14 . 5 using 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 O O O O O O O O O O O O O O O O O O O O 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 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

Figure 7Representative dot-plots and quantification of E13.5 fetal liver LSKhi/LSKlow cell phenotypes observed in embryos of the indicated genotypes. N=21 for Chk1fl/+, N=5 for Chk1fl/- Vav-Cre, N=16 for Chk1fl/+ Vav-BCL2, N=5 for Chk1fl/- Vav-Cre Vav-BCL2, N=2 for p53-/- Chk1fl/+ and N=4 for p53-/- Chk1fl/- Vav-Cre.Flow cytometry-based analysis of LSK cells stained with antibodies specific for CD34 and Flt3, to discriminate LT- and ST-HSC from MPP within the LSK subset in fetal livers of embryos of the indicated genotypes. Left: N=25 for Chk1fl/+, N=7 for Chk1fl/+ Vav-Cre and N=7 for Chk1fl/- Vav-Cre. Right: N=16 for Chk1fl/+ Vav-BCL2, N=6 for Chk1fl/+ Vav-Cre Vav-BCL2 and N=5 for Chk1fl/- Vav-Cre Vav-BCL2.Analysis of the colony formation potential of E14.5 total fetal liver cells of the indicated genotypes in methylcellulose. N=3 for controls (pooled; 1 Chk1fl/+, 2 Chk1fl/+ Vav-Cre), N=3 for Chk1fl/- Vav-Cre and N=3 for Chk1fl/- Vav-Cre Vav-BCL2.Western blot analysis of fetal liver cells of the indicated genotypes (each lane represents an embryo) at E14.5 using the indicated antibodies.


Figure 8Three independent Chk1fl / fl mT / mG ( coloured ) and mT / mG ( grey ) Hoxb8 - FL progenitor cell lines were transduced with a retroviral construct carrying Cre ( pMIP - CRE - IRES - PURO ) and monitored over time for GFP - expression . Shown here are three technical replicates per genotype . Additionally , Chk1fl / fl mT / mG Hoxb8 - FL progenitor cells were FACS - sorted for GFP + and dsRED + cells 48h after transduction with the Cre - construct . Sorted cells were processed for western blotting using the indicated antibodies . Chk1fl / fl Hoxb8 - FL progenitor cells were transduced with a retroviral construct carrying Cre and GFP ( pMIG - CRE - IRES - GFP ) . Cells were FACS - sorted 72h after transduction with the retroviral construct and processed for western blotting using the indicated antibodies . Percentage of GFP + cells indicative for CRE expression and recombination in the peripheral blood after tamoxifen administration by gavage ( 5 x 2 mg , 200 μl / mouse ; daily ) . Bars represent means ± S . E . M . ( Day 0 N = 9 / 10 , WT / Chk1fl / fl , Day 7 N = 8 / 7 , Day 14 N = 3 / 3 ) . Quantification of Chk1 - del PCR - product enrichment in DNA from GFP + vs . Tomato + bone marrow cells sorted on day 8 after first TAM administration . Transgenic Cre levels were quantified in parallel for reference . Bars represent means ± S . E . M . from n = 5 mT / mG Vav - CreERT2 Chk1fl / fl mice . Percentage of living LK or LSK - cells and ( F ) percentage of GFP + LK or LSK - cells found in the bone marrow of mT / mG Vav - CreERT2 ( WT ) and mT / mG Vav - CreERT2 Chk1fl / fl ( Chk1fl / fl ) mice on day 8 or 15 post first TAM administration . Bars in ( E ) / ( F ) represent means ± S . E . M . ( Day 8 N = 8 / 8 , WT / Chk1fl / fl , Day 15 N = 7 / 8 ) . GFP + ( CRE ) and Tomato + ( CRE - ) total bone marrow cells were sorted on day 8 after first TAM - administration and processed for Western Blot using the indicated antibodies . Sorted cells from three animals - mT / mG Vav - CreERT2 ( WT ) and mT / mG Vav - CreERT2 Chk1fl / fl ( Chk1fl / fl ) - were pooled per lane . Thymocytes of the indicated genotypes were sorted based on GFP + and Tomato + on day 15 after the first TAM administration for western analysis using 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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

Figure 8Three independent Chk1fl/fl mT/mG (coloured) and mT/mG (grey) Hoxb8-FL progenitor cell lines were transduced with a retroviral construct carrying Cre (pMIP-CRE-IRES-PURO) and monitored over time for GFP-expression. Shown here are three technical replicates per genotype. Additionally, Chk1fl/fl mT/mG Hoxb8-FL progenitor cells were FACS-sorted for GFP+ and dsRED+ cells 48h after transduction with the Cre-construct. Sorted cells were processed for western blotting using the indicated antibodies.Chk1fl/fl Hoxb8-FL progenitor cells were transduced with a retroviral construct carrying Cre and GFP (pMIG-CRE-IRES-GFP). Cells were FACS-sorted 72h after transduction with the retroviral construct and processed for western blotting using the indicated antibodies.Percentage of GFP+ cells indicative for CRE expression and recombination in the peripheral blood after tamoxifen administration by gavage (5 x 2 mg, 200 μl/mouse; daily). Bars represent means ± S.E.M. (Day 0 N=9/10, WT/Chk1fl/fl, Day 7 N=8/7, Day 14 N=3/3).Quantification of Chk1-del PCR-product enrichment in DNA from GFP+ vs. Tomato+ bone marrow cells sorted on day 8 after first TAM administration. Transgenic Cre levels were quantified in parallel for reference. Bars represent means ± S.E.M. from n=5 mT/mG Vav-CreERT2 Chk1fl/fl mice.Percentage of living LK or LSK-cells and (F) percentage of GFP+ LK or LSK-cells found in the bone marrow of mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) mice on day 8 or 15 post first TAM administration. Bars in (E)/(F) represent means ± S.E.M. (Day 8 N=8/8, WT/Chk1fl/fl, Day 15 N=7/8).GFP+ (CRE) and Tomato+ (CRE-) total bone marrow cells were sorted on day 8 after first TAM-administration and processed for Western Blot using the indicated antibodies. Sorted cells from three animals - mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) - were pooled per lane.Thymocytes of the indicated genotypes were sorted based on GFP+ and Tomato+ on day 15 after the first TAM administration for western analysis using the indicated antibodies.


Figure 1 ( D ) Immunofluorescence staining of the spinal cord at GW7 , GW8 , GW10 , GW11 , and GW23 for RBFOX3 ( red ) , OLIG2 ( green ) , and GFAP ( white ) . The appearance of astrocytes and oligodendrocytes was observed from GW8 onward . Scale bars : 100 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

Figure 1(D) Immunofluorescence staining of the spinal cord at GW7, GW8, GW10, GW11, and GW23 for RBFOX3 (red), OLIG2 (green), and GFAP (white). The appearance of astrocytes and oligodendrocytes was observed from GW8 onward. Scale bars: 100 μm.


Figure 2 ( C ) Immunofluorescence staining of the spinal cord from GW7 , GW8 , and GW10 for a dorsal marker ( PAX7 , green ) , neuronal precursor marker ( ASCL1 , red ) , and nuclei marker ( DAPI , blue ) . Images show the neuronal differentiation process on the dorsal side of the spinal cord . Scale bar : 100 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

Figure 2(C) Immunofluorescence staining of the spinal cord from GW7, GW8, and GW10 for a dorsal marker (PAX7, green), neuronal precursor marker (ASCL1, red), and nuclei marker (DAPI, blue). Images show the neuronal differentiation process on the dorsal side of the spinal cord. Scale bar: 100 μm.


Figure 3 ( C ) Violin plot showing the expression of selected genes . EGFR + Cluster 14 is circled with a red dotted line .

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

Figure 3(C) Violin plot showing the expression of selected genes. EGFR+ Cluster 14 is circled with a red dotted line.


Figure 4 ( D ) Violin plots produced by VlnPlot function of Seurat package representing changes in expression of EGFR , DLL1 , MIAT , and FAM189A2 in cluster 14 with development based on snRNA counts data .

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

Figure 4(D) Violin plots produced by VlnPlot function of Seurat package representing changes in expression of EGFR, DLL1, MIAT, and FAM189A2 in cluster 14 with development based on snRNA counts data.


Figure 5 ( B ) Immunofluorescence staining of spinal cord sections at GW7 , GW11 , GW17 , and GW23 for EGFR ( green ) and nuclei ( DAPI , blue ) . EGFR expression on the ventral side was observed in GW7 and GW11 spinal cord . EGFR expression on the dorsal side was observed in GW11 , GW17 , and GW23 spinal cord . Dorsal EGFR expression was mainly concentrated within the dorsal horn area . Scale bar : 100 μm . ( Top ) Higher - magnification view of boxes at bottom . White dashed lines indicate dorsal horn borders . ( D ) Representative immunofluorescence staining of a GW11 spinal cord section . Most dorsal EGFR - expressing cells at GW11 co - expressed GFAP and SOX9 , but not OLIG2 . Scale bars : 100 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 O O O O O O O O O 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

Figure 5(B) Immunofluorescence staining of spinal cord sections at GW7, GW11, GW17, and GW23 for EGFR (green) and nuclei (DAPI, blue). EGFR expression on the ventral side was observed in GW7 and GW11 spinal cord. EGFR expression on the dorsal side was observed in GW11, GW17, and GW23 spinal cord. Dorsal EGFR expression was mainly concentrated within the dorsal horn area. Scale bar: 100 μm. (Top) Higher-magnification view of boxes at bottom. White dashed lines indicate dorsal horn borders.(D) Representative immunofluorescence staining of a GW11 spinal cord section. Most dorsal EGFR-expressing cells at GW11 co-expressed GFAP and SOX9, but not OLIG2. Scale bars: 100 μm.


Figure 6GW11 EGFR - expressing cells exhibited radial glia morphology and most co - expressed SOX9 ( A ) but not OLIG2 ( B ) . However , EGFR co - expression with SOX9 or OLIG2 was found in GW17 EGFR - expressing cells ( C , D ) . White dashed lines marked dorsal horn borders . Higher - magnification view of boxes on left . Scale bars : 100 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

Figure 6GW11 EGFR-expressing cells exhibited radial glia morphology and most co-expressed SOX9 (A) but not OLIG2 (B). However, EGFR co-expression with SOX9 or OLIG2 was found in GW17 EGFR-expressing cells (C, D). White dashed lines marked dorsal horn borders. Higher-magnification view of boxes on left. Scale bars: 100 μm.


Figure 1 ( A ) Timelapse TIRF microscopy images showing an EL4 - MyD88 - GFP cell interacting with a IL - 1 functionalized SLB . MyD88 - GFP assembles into puncta that over time cluster and coalesce at the cell : SLB interface . Scale bar , 2 µm . ( C ) TIRF and brightfield microscopy images of EL4 - MyD88 - GFP cells incubated for 30 mins with IL - 1 functionalized SLBs formed off grid and on 1 and 2 . 5 µm grids . In the presence of a 1 and 2 . 5 µm grids , Myddosomes only coalesce within individual corrals and do not form multi - complex clusters . Scale bar , 5 µm . ( D ) Time series showing Myddosome formation in an EL4 cell interacting with both continuous and 2 . 5 µm gridded partitioned SLBs . t = 0s denotes the start of cellular observation . Scale bar , 5 µm . ( F ) Quantification of MyD88 - GFP puncta maximum fluorescence intensity normalized to GFP from cells stimulated off and on 2 . 5 or 1 µm grids , at a ligand density of 10 IL - 1 / µm2 . Violin plots show the distribution of average max puncta intensities from individual cells across replicates . Data points superimposed on the violin plots are the averages from independent experimental replicates . The average max MyD88 puncta intensity ( mean ± SEM ) : for off grid is 7 . 5 ± 1 . 8 GFPs , n = 8 biological replicates , with 88304 MyD88 - GFP puncta from 161 cells measured in total across all replicates ; for 2 . 5 µm grids is 5 . 2 ± 1 . 7 GFPs , n = 3 biological replicates , with 13164 MyD88 - GFP puncta from 31 cells measured in total across all replicates ; for 1 µm grids is 2 . 3 ± 0 . 1 GFPs , n = 8 biological replicates , with 126600 MyD88 - GFP puncta from 254 cells measured in total across all replicates . Bars represent mean ± SEM . ( G ) Widefield images showing RelA localization in unstimulated EL4 cells and EL4 cells stimulated by SLB formed on and off grids . EL4 were fixed 30 min after addition to IL - 1 - functionalized SLBs and stained for RelA ( magenta ) ; DAPI stained nuclei ( blue ) . Scale bar , 10 µm . ( H ) Quantification of RelA nucleus to cytoplasm ratio . Violin plots show the distribution of measurements from individual cells . Data points superimposed on the violin plots are the averages from independent experiments . The RelA nucleus to cytoplasm ratio of single cells marked with X in panel ( G ) are superimposed on the violin plot . RelA nucleus to cytoplasm ratio off grids , on 2 . 5 and 1 µm grids , and unstimulated conditions are 0 . 45 ± 0 . 01 , 0 . 34 ± 0 . 02 , 0 . 32 ± 0 . 02 , 0 . 21 ± 0 . 03 ( mean ± SEM ) respectively . The p value are * = 0 . 0133 and * * = 0 . 0027 . Bars represent mean ± SEM ( n = 3 to 5 biological replicates , with a total of 18370 , 3627 , 2988 and 965 cells measured off grids , on 2 . 5 µm and 1 µm grids , and unstimulated conditions respectively ) . Statistical significance is determined using unpaired two - tailed Student ' s t - 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Timelapse TIRF microscopy images showing an EL4-MyD88-GFP cell interacting with a IL-1 functionalized SLB. MyD88-GFP assembles into puncta that over time cluster and coalesce at the cell:SLB interface. Scale bar, 2 µm.(C) TIRF and brightfield microscopy images of EL4-MyD88-GFP cells incubated for 30 mins with IL-1 functionalized SLBs formed off grid and on 1 and 2.5 µm grids. In the presence of a 1 and 2.5 µm grids, Myddosomes only coalesce within individual corrals and do not form multi-complex clusters. Scale bar, 5 µm.(D) Time series showing Myddosome formation in an EL4 cell interacting with both continuous and 2.5 µm gridded partitioned SLBs. t = 0s denotes the start of cellular observation. Scale bar, 5 µm.(F) Quantification of MyD88-GFP puncta maximum fluorescence intensity normalized to GFP from cells stimulated off and on 2.5 or 1 µm grids, at a ligand density of 10 IL-1/µm2. Violin plots show the distribution of average max puncta intensities from individual cells across replicates. Data points superimposed on the violin plots are the averages from independent experimental replicates. The average max MyD88 puncta intensity (mean ± SEM): for off grid is 7.5 ± 1.8 GFPs, n = 8 biological replicates, with 88304 MyD88-GFP puncta from 161 cells measured in total across all replicates; for 2.5 µm grids is 5.2 ± 1.7 GFPs, n = 3 biological replicates, with 13164 MyD88-GFP puncta from 31 cells measured in total across all replicates; for 1 µm grids is 2.3 ± 0.1 GFPs, n = 8 biological replicates, with 126600 MyD88-GFP puncta from 254 cells measured in total across all replicates. Bars represent mean ± SEM.(G) Widefield images showing RelA localization in unstimulated EL4 cells and EL4 cells stimulated by SLB formed on and off grids. EL4 were fixed 30 min after addition to IL-1-functionalized SLBs and stained for RelA (magenta); DAPI stained nuclei (blue). Scale bar, 10 µm.(H) Quantification of RelA nucleus to cytoplasm ratio. Violin plots show the distribution of measurements from individual cells. Data points superimposed on the violin plots are the averages from independent experiments. The RelA nucleus to cytoplasm ratio of single cells marked with X in panel (G) are superimposed on the violin plot. RelA nucleus to cytoplasm ratio off grids, on 2.5 and 1 µm grids, and unstimulated conditions are 0.45 ± 0.01, 0.34 ± 0.02, 0.32 ± 0.02, 0.21 ± 0.03 (mean ± SEM) respectively. The p value are * = 0.0133 and ** = 0.0027. Bars represent mean ± SEM (n = 3 to 5 biological replicates, with a total of 18370, 3627, 2988 and 965 cells measured off grids, on 2.5 µm and 1 µm grids, and unstimulated conditions respectively). Statistical significance is determined using unpaired two-tailed Student's t-test.


Figure 2 ( A ) TIRF images of fixed EL4 - MyD88 - GFP cells off grids and stained with antibodies against K63 - Ub , M1 - Ub , pIKK and pp65 . Cells were activated on IL - 1 functionalized SLBs for 30 min before fixation . Scale bar , 5 µm . ( B ) A magnified view of the large patch - like Myddosome clusters from the highlighted region of interest in panel A ( yellow box on merge images ) . Scale bar , 1 µm . ( C ) Structured illumination microscopy images of Myddosome clusters stained with anti - pIKK . Top row right , insets show detail of Myddosome staining with anti - pIKK . Inset taken from regions of interest overlaid the merge image ( yellow boxes 1 and 2 ) . Bottom row , x - z view slice taken from yellow line overlaid on the merge image ( top row ) . Myddosome and pIKK staining localize the cell - SLB interface . Blue dashed line defines the nucleus volume determined from the DAPI stain . Scale bar in main image and Z projection , 1 µm ; scale bar inset , 0 . 5 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 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

Figure 2(A) TIRF images of fixed EL4-MyD88-GFP cells off grids and stained with antibodies against K63-Ub, M1-Ub, pIKK and pp65. Cells were activated on IL-1 functionalized SLBs for 30 min before fixation. Scale bar, 5 µm. (B) A magnified view of the large patch-like Myddosome clusters from the highlighted region of interest in panel A (yellow box on merge images). Scale bar, 1 µm.(C) Structured illumination microscopy images of Myddosome clusters stained with anti-pIKK. Top row right, insets show detail of Myddosome staining with anti-pIKK. Inset taken from regions of interest overlaid the merge image (yellow boxes 1 and 2). Bottom row, x-z view slice taken from yellow line overlaid on the merge image (top row). Myddosome and pIKK staining localize the cell-SLB interface. Blue dashed line defines the nucleus volume determined from the DAPI stain. Scale bar in main image and Z projection, 1 µm; scale bar inset, 0.5 µm.


Figure 3 ( A and B ) Top , TIRF images of fixed EL4 - MyD88 - GFP cells incubated with IL - 1 functionalized SLBs for 30 minutes , and stained with antibodies against pp65 ( A ) or pIKK ( B ) . Scale bar , 5 µm . Region of interest ( red box , merge image ) shows an example of MyD88 - GFP puncta that colocalizes with pp65 ( A ) or pIKK ( B ) puncta . Bottom , 2D histograms of the distribution of MyD88 puncta intensity and associated pp65 ( A ) or pIKK ( B ) staining intensity . Linear fit is shown as a blue line superimposed on 2D histograms ( Pearson correlation coefficient , R , of linear fit labeled on 2D histograms ) . Blue shaded regions on scatter plot high MyD88 puncta classified as clustered Myddosomes . Bottom right , zoomed images of the region of interest ( red box overlaid merge image , top ) show MyD88 - GFP channel and associated pp65 ( A ) and pIKK ( B ) channel ( pp65 / pIKK images are displayed with Fire LUT ) . Red data points on the 2D histogram are from indicated puncta in the MyD88 - GFP image ( numbered red arrows ) . Scale bar , 1 µm . ( E and F ) 2D histogram of MyD88 - GFP puncta intensity and associated pp65 ( E ) or pIKK ( F ) staining intensity on 2 . 5 µm and 1 µm grids . Linear fit is shown as a blue line superimposed on 2D histograms ( Pearson correlation coefficient , R , of linear fit labeled on 2D histograms ) . ( G and H ) Quantification of mean pp65 ( G ) or pIKK ( H ) staining intensity for puncta classifieds as single or clustered Myddosomes , and MyD88 puncta formed on 2 . 5 and 1 µm grids . The normalized mean intensity for clusters , single Myddosomes MyD88 puncta on 2 . 5 and 1 µm grids are the following : for pp65 0 . 318 ± 0 . 044 , 0 . 163 ± 0 . 009 , 0 . 059 ± 0 . 005 and 0 . 057 ± 0 . 008 ; for pIKK 0 . 393 ± 0 . 051 , 0 . 130 ± 0 . 004 , 0 . 037 ± 0 . 006 and 0 . 035 ± 0 . 007 ( a . u . , mean ± SEM , mean value states in the order they appear on plot , left to right ) . Violin plots show the distribution of all segmented MyD88 puncta . Data points superimposed on the violin plots are the averages from independent experiments . p values are * < 0 . 05 , * * * < 0 . 001 , * * * * < 0 . 0001 . Bars represent mean ± SEM ( n = 3 - 4 biological replicates for pp65 , with 10273 , 14009 and 2657 puncta off grid , on 2 . 5 µm and 1 µm grid measured in total across all replicates ; n = 4 - 5 biological replicates for pIKK , with 2375 , 35496 and 59593 puncta off grid , on 2 . 5 µm and 1 µm grid measured in total across all replicates ) . Statistical significance is determined using unpaired two - tailed Student ' s t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 3(A and B) Top, TIRF images of fixed EL4-MyD88-GFP cells incubated with IL-1 functionalized SLBs for 30 minutes, and stained with antibodies against pp65 (A) or pIKK (B). Scale bar, 5 µm. Region of interest (red box, merge image) shows an example of MyD88-GFP puncta that colocalizes with pp65 (A) or pIKK (B) puncta. Bottom, 2D histograms of the distribution of MyD88 puncta intensity and associated pp65 (A) or pIKK (B) staining intensity. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R, of linear fit labeled on 2D histograms). Blue shaded regions on scatter plot high MyD88 puncta classified as clustered Myddosomes. Bottom right, zoomed images of the region of interest (red box overlaid merge image, top) show MyD88-GFP channel and associated pp65 (A) and pIKK (B) channel (pp65/pIKK images are displayed with Fire LUT). Red data points on the 2D histogram are from indicated puncta in the MyD88-GFP image (numbered red arrows). Scale bar, 1 µm.(E and F) 2D histogram of MyD88-GFP puncta intensity and associated pp65 (E) or pIKK (F) staining intensity on 2.5 µm and 1 µm grids. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R, of linear fit labeled on 2D histograms).(G and H) Quantification of mean pp65 (G) or pIKK (H) staining intensity for puncta classifieds as single or clustered Myddosomes, and MyD88 puncta formed on 2.5 and 1 µm grids. The normalized mean intensity for clusters, single Myddosomes MyD88 puncta on 2.5 and 1 µm grids are the following: for pp65 0.318 ± 0.044, 0.163 ± 0.009, 0.059 ± 0.005 and 0.057 ± 0.008; for pIKK 0.393 ± 0.051, 0.130 ± 0.004, 0.037 ± 0.006 and 0.035 ± 0.007 (a.u., mean ± SEM, mean value states in the order they appear on plot, left to right). Violin plots show the distribution of all segmented MyD88 puncta. Data points superimposed on the violin plots are the averages from independent experiments. p values are *<0.05, ***<0.001, ****<0.0001. Bars represent mean ± SEM (n = 3 - 4 biological replicates for pp65, with 10273, 14009 and 2657 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates; n = 4 - 5 biological replicates for pIKK, with 2375, 35496 and 59593 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates). Statistical significance is determined using unpaired two-tailed Student's t-test.


Figure 4 ( A and B ) Top , TIRF images of fixed EL4 - MyD88 - GFP cells incubated with IL - 1 functionalized SLBs for 30 minutes , and stained with anti - K63 - Ub ( A ) or anti - M1 - Ub ( B ) . Scale bar , 5 µm . Region of interest ( red box , merge image ) shows an example of MyD88 - GFP puncta that is colocalized with K63 - Ub ( A ) or M1 - Ub ( B ) puncta . Bottom , 2D histograms of the distribution of MyD88 puncta intensity and associated K63 - Ub ( A ) or M1 - Ub ( B ) staining intensity . Linear fit is shown as a blue line superimposed on 2D histograms ( Pearson correlation coefficient , R , of linear fit labeled on 2D histograms ) . Blue shaded region on scatter plot high MyD88 puncta classified as clustered Myddosomes . Bottom right , zoomed images of the region of interest ( red box overlaid merge image , top ) show MyD88 - GFP channel and associated K63 - Ub ( A ) and M1 - Ub ( B ) channel ( K63 / M1 - Ub images are displayed with Fire LUT ) . Red data points on the 2D histogram are from indicated puncta in the MyD88 - GFP image ( numbered red arrows ) . Scale bar , 1 µm . ( E and F ) 2D histograms of the distribution of MyD88 puncta intensity and associated K63 - Ub ( E ) or M1 - Ub ( F ) staining intensity on 2 . 5 µm and 1 µm grids . Linear fit is shown as a blue line superimposed on 2D histograms ( Pearson correlation coefficient of linear fit labeled on 2D histograms ) . ( G and H ) Quantification of mean K63 - Ub ( G ) or M1 - Ub ( H ) staining intensity for puncta classifieds as single or clustered Myddosomes , and MyD88 puncta formed on 2 . 5 and 1 µm grids . The normalized mean intensity for clustered , single Myddosomes , and MyD88 puncta on 2 . 5 and 1 µm grids are the following : for K63 - Ub 0 . 444 ± 0 . 030 , 0 . 257 ± 0 . 025 , 0 . 113 ± 0 . 015 and 0 . 104 ± 0 . 008 ; for M1 - Ub 0 . 520 ± 0 . 020 , 0 . 183 ± 0 . 008 , 0 . 174 ± 0 . 022 and 0 . 153 ± 0 . 016 ( a . u . , mean ± SEM , mean value states in the order they appear on plot , left to right ) . Violin plots show the distribution of all segmented MyD88 puncta . Data points superimposed on the violin plots are the averages from independent experiments . p values are * * < 0 . 01 , * * * < 0 . 001 . Bars represent mean ± SEM ( n = 3 - 4 biological replicates for K63 , with 14571 , 27494 and 24026 puncta off grid , on 2 . 5 µm and 1 µm grid measured in total across all replicates ; n = 3 - 4 biological replicates for M1 , with 3114 , 6091 and 1844 puncta off grid , on 2 . 5 µm and 1 µm grid measured in total across all replicates ) . Statistical significance is determined using unpaired two - tailed Student ' s t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 4(A and B) Top, TIRF images of fixed EL4-MyD88-GFP cells incubated with IL-1 functionalized SLBs for 30 minutes, and stained with anti-K63-Ub (A) or anti-M1-Ub (B). Scale bar, 5 µm. Region of interest (red box, merge image) shows an example of MyD88-GFP puncta that is colocalized with K63-Ub (A) or M1-Ub (B) puncta. Bottom, 2D histograms of the distribution of MyD88 puncta intensity and associated K63-Ub (A) or M1-Ub (B) staining intensity. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R, of linear fit labeled on 2D histograms). Blue shaded region on scatter plot high MyD88 puncta classified as clustered Myddosomes. Bottom right, zoomed images of the region of interest (red box overlaid merge image, top) show MyD88-GFP channel and associated K63-Ub (A) and M1-Ub (B) channel (K63/M1-Ub images are displayed with Fire LUT). Red data points on the 2D histogram are from indicated puncta in the MyD88-GFP image (numbered red arrows). Scale bar, 1 µm.(E and F) 2D histograms of the distribution of MyD88 puncta intensity and associated K63-Ub (E) or M1-Ub (F) staining intensity on 2.5 µm and 1 µm grids. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient of linear fit labeled on 2D histograms).(G and H) Quantification of mean K63-Ub (G) or M1-Ub (H) staining intensity for puncta classifieds as single or clustered Myddosomes, and MyD88 puncta formed on 2.5 and 1 µm grids. The normalized mean intensity for clustered, single Myddosomes, and MyD88 puncta on 2.5 and 1 µm grids are the following: for K63-Ub 0.444 ± 0.030, 0.257 ± 0.025, 0.113 ± 0.015 and 0.104 ± 0.008; for M1-Ub 0.520 ± 0.020, 0.183 ± 0.008, 0.174 ± 0.022 and 0.153 ± 0.016 (a.u., mean ± SEM, mean value states in the order they appear on plot, left to right). Violin plots show the distribution of all segmented MyD88 puncta. Data points superimposed on the violin plots are the averages from independent experiments. p values are **<0.01, ***<0.001. Bars represent mean ± SEM (n = 3 - 4 biological replicates for K63, with 14571, 27494 and 24026 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates; n = 3 - 4 biological replicates for M1, with 3114, 6091 and 1844 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates). Statistical significance is determined using unpaired two-tailed Student's t-test.


Figure 5 ( A and B ) Top : TIRF images of MyD88 - GFP and mScarlet - TRAF6 ( A ) or mScarlet - HOIL1 ( B ) . Region of interest ( yellow box , merge image ) shows an example of a MyD88 - GFP puncta colocalized with mScarlet - TRAF6 ( A ) or mScalet - HOIL1 ( B ) . Bottom : Time - series TIRF images from the region of interest ( left ) and fluorescence intensity times series ( right ) of MyD88 and TRAF6 ( A ) or HOIL1 ( B ) . ( C and D ) Quantifications of percentage of MyD88 - GFP puncta that colocalized with TRAF6 ( C ) or HOIL1 ( D ) grouped for all puncta , puncta containing ≤ 1x , > 1x and ≥ 2x Myddosome complexes . The percentages for TRAF6 ( C ) in these groups are 15 . 4 ± 2 . 4 % , 6 . 1 ± 1 . 8 % , 36 . 2 ± 5 . 2 % and 58 . 4 ± 5 . 9 % , respectively ( mean ± SEM ) . The percentages for HOIL1 ( D ) in these groups are 8 . 6 ± 0 . 5 % , 0 . 4 ± 0 . 2 % , 14 . 9 ± 1 . 9 % and 25 . 9 ± 5 . 0 % , respectively ( mean ± SEM ) . Violin plots indicate the distribution of individual cell measurements . Colored dots superimposed on the violin plots are the averages from independent experiments . Bars represent mean ± SEM ( n = 6 biological replicates for TRAF6 , with 191 cells measured in total across all replicates ,

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 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

Figure 5(A and B) Top: TIRF images of MyD88-GFP and mScarlet-TRAF6 (A) or mScarlet-HOIL1 (B). Region of interest (yellow box, merge image) shows an example of a MyD88-GFP puncta colocalized with mScarlet-TRAF6 (A) or mScalet-HOIL1 (B). Bottom: Time-series TIRF images from the region of interest (left) and fluorescence intensity times series (right) of MyD88 and TRAF6 (A) or HOIL1 (B).(C and D) Quantifications of percentage of MyD88-GFP puncta that colocalized with TRAF6 (C) or HOIL1 (D) grouped for all puncta, puncta containing ≤1x, >1x and ≥2x Myddosome complexes. The percentages for TRAF6 (C) in these groups are 15.4 ± 2.4%, 6.1 ± 1.8%, 36.2 ± 5.2% and 58.4 ± 5.9%, respectively (mean ± SEM). The percentages for HOIL1 (D) in these groups are 8.6 ± 0.5%, 0.4 ± 0.2%, 14.9 ± 1.9% and 25.9 ± 5.0%, respectively (mean ± SEM). Violin plots indicate the distribution of individual cell measurements. Colored dots superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 6 biological replicates for TRAF6, with 191 cells measured in total across all replicates,


Figure 6 ( A and B ) TIRF images of EL4 cells expressing MyD88 - GFP and mScarlet - TRAF6 stimulated on IL - 1 functionalized SLBs at a ligand density of 1 IL - 1 / µm2 off grids ( A ) or on 1 µm grids ( B ) . Kymographs derived from dashed lines overlaid TIRF images ( left panel ) . Scale bars , 5 µm . ( C ) Quantification of percentage of MyD88 - GFP puncta that colocalized with TRAF6 off grids and on 1 µm grids at a ligand density of 1 IL - 1 / µm2 , and the percentages are 16 . 2 ± 2 . 5 % and 6 . 1 ± 1 . 5 % , respectively ( mean ± SEM ) . Violin plots indicate the distribution of individual cell measurements . Colored dots superimposed on the violin plots are the averages from independent experiments . Bars represent mean ± SEM ( n = 4 experimental replicates off grids , with a total of 24315 MyD88 puncta from 91 cells ; n = 3 biological replicates on 1 µm grids , with a total of 23161 MyD88 puncta from 70 cells ) . Statistical significance is determined using unpaired two - tailed Student ' s t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(A and B) TIRF images of EL4 cells expressing MyD88-GFP and mScarlet-TRAF6 stimulated on IL-1 functionalized SLBs at a ligand density of 1 IL-1/µm2 off grids (A) or on 1 µm grids (B). Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bars, 5 µm. (C) Quantification of percentage of MyD88-GFP puncta that colocalized with TRAF6 off grids and on 1 µm grids at a ligand density of 1 IL-1/µm2, and the percentages are 16.2 ± 2.5% and 6.1 ± 1.5%, respectively (mean ± SEM). Violin plots indicate the distribution of individual cell measurements. Colored dots superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 4 experimental replicates off grids, with a total of 24315 MyD88 puncta from 91 cells; n = 3 biological replicates on 1 µm grids, with a total of 23161 MyD88 puncta from 70 cells). Statistical significance is determined using unpaired two-tailed Student's t-test.


Figure 7 ( A - B ) Histogram showing the lifetime of TRAF6 and HOIL1 recruitment to single Myddsomes , and clusters containing 2 - 4 and 5 - 7 Myddosome complexes , off ( blue ) and on ( red ) 1 µm grids .

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

Figure 7(A-B) Histogram showing the lifetime of TRAF6 and HOIL1 recruitment to single Myddsomes, and clusters containing 2-4 and 5-7 Myddosome complexes, off (blue) and on (red) 1 µm grids.


Figure 1A A heatmap showing the differential miRNAs expression as determined by miRNA - seq in progerin - expressing cells relative to control cells , P < 0 . 05 . Lowly expressed miRNAs were shown in blue , highly expressed ones in red . B Volcano Plot of top 10 miRNAs upregulated and 2 miRNAs downregulated in HGPS cells compared to control . C The level of top 10 upregulated miRNAs in HGPS cells was assessed by qPCR . D The level of novel - miR - 27 , miR - 3656 and miR - 59 in HGPS patient cells ( HGAFDFN003 p21 and HGAFDFN167 p19 ) versus control ( CRL - 1474 cells p28 ) was assessed by qPCR . HGAFDFN003 cells were transfected with anti - miR - ctrl , anti - miR - 3656 or anti - miR - 59 for 5 days , cells were subjected to SA - β - gal staining . HGAFDFN003 cells were transfected with anti - miR - ctrl , anti - miR - 3656 or anti - miR - 59 for 5 days , The percentage of the Ki67 positive cells was calculated by immunofluorescence ( F ) . HGAFDFN003 cells were transfected with anti - miR - ctrl , anti - miR - 3656 or anti - miR - 59 for 5 days The level of cyclin A2 and lamin B1 was detected by western blot ( G ) . Progerin - expressing CRL - 1474 cells were transfected with anti - miR - ctrl or anti - miR - 59 for 5 days . The percentage of SA - β - gal positive cells was calculated . Scale bars : 100 μm ( H ) . Progerin - expressing CRL - 1474 cells were transfected with anti - miR - ctrl or anti - miR - 59 for 5 days . The percentage of the Ki67 positive cells was calculated by immunofluorescence ( I ) . Progerin - expressing CRL - 1474 cells were transfected with anti - miR - ctrl or anti - miR - 59 for 5 days . The level of cyclin A2 and lamin B1 was detected by western blot ( J ) . K The level of miR - 59 was detected in 88 - and 92 - year - old cells versus 7 - year - old cells was assessed by qPCR . L 7 - year - old cells and 88 - year - old cells were transfected with anti - miR - ctrl or anti - miR - 59 , the level of cyclin A2 and lamin B1 was detected .

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 B-PANEL_START 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 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 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 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 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 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

Figure 1A A heatmap showing the differential miRNAs expression as determined by miRNA-seq in progerin-expressing cells relative to control cells, P< 0.05. Lowly expressed miRNAs were shown in blue, highly expressed ones in red.B Volcano Plot of top 10 miRNAs upregulated and 2 miRNAs downregulated in HGPS cells compared to control.C The level of top 10 upregulated miRNAs in HGPS cells was assessed by qPCR.D The level of novel-miR-27, miR-3656 and miR-59 in HGPS patient cells (HGAFDFN003 p21 and HGAFDFN167 p19) versus control (CRL-1474 cells p28) was assessed by qPCR.HGAFDFN003 cells were transfected with anti-miR-ctrl, anti-miR-3656 or anti-miR-59 for 5 days, cells were subjected to SA-β-gal staining. HGAFDFN003 cells were transfected with anti-miR-ctrl, anti-miR-3656 or anti-miR-59 for 5 days, The percentage of the Ki67 positive cells was calculated by immunofluorescence (F).HGAFDFN003 cells were transfected with anti-miR-ctrl, anti-miR-3656 or anti-miR-59 for 5 days The level of cyclin A2 and lamin B1 was detected by western blot (G).Progerin-expressing CRL-1474 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days. The percentage of SA-β-gal positive cells was calculated. Scale bars: 100 μm (H).Progerin-expressing CRL-1474 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days. The percentage of the Ki67 positive cells was calculated by immunofluorescence (I).Progerin-expressing CRL-1474 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days. The level of cyclin A2 and lamin B1 was detected by western blot (J).K The level of miR-59 was detected in 88- and 92-year-old cells versus 7-year-old cells was assessed by qPCR.L 7-year-old cells and 88-year-old cells were transfected with anti-miR-ctrl or anti-miR-59, the level of cyclin A2 and lamin B1 was detected.


Figure 2HGAFDFN003 cells were transfected with anti - miR - ctrl or anti - miR - 59 for 5 days , the mRNA level of miR - 59 - targeting potential genes was assessed by qPCR ( B ) . HGAFDFN003 cells were transfected with anti - miR - ctrl or anti - miR - 59 for 5 days , The protein expression of HMGA1 and HMGA2 was detected by western blot ( C ) . D , After transfection with anti - miR - ctrl or anti - miR - 59 in progerin - expressing CRL - 1474 cells , the protein expression ( D ) of HMGAs were detected . After transfection with anti - miR - ctrl or anti - miR - 59 in progerin - expressing CRL - 1474 cells , mRNA level ( E ) of HMGAs were detected . Western blot analysis the expression of HMGAs ( F ) in miR - ctrl or miR - 59 mimics - transfected CRL - 1474 cells . qPCR analysis the mRNA levels ( G ) in miR - ctrl or miR - 59 mimics - transfected CRL - 1474 cells . Compared HGPS patient cells ( HGAFDFN003 p22 and HGAFDFN167 p20 ) with control ( CRL - 1474 p26 ) , the protein expression ( H ) of HMGAs were assessed . Compared HGPS patient cells ( HGAFDFN003 p22 and HGAFDFN167 p20 ) with control ( CRL - 1474 p26 ) , mRNA level ( I ) of HMGAs were assessed . The protein ( J ) level of HMGAs were detected in 7 , 88 and 92 - year - old cells . mRNA ( K ) level of HMGAs were detected in 7 , 88 and 92 - year - old cells . Transfected anti - miR - ctrl or anti - miR - 59 in 88 - year - old cells , the level of HMGAs was detected by Western blot ( L ) Transfected anti - miR - ctrl or anti - miR - 59 in 88 - year - old cells , the level of HMGAs was detected by qPCR ( M ) . N , O The binding sites of miR - 59 on HMGAs 3 ′ UTR were analyzed by TargetScan and miRanda . Luciferase experiments with the wild - type and the mutated 3 ′ UTR of HMGA1 ( N ) / HMGA2 ( 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 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 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 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 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 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 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 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 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 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 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

Figure 2HGAFDFN003 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days, the mRNA level of miR-59-targeting potential genes was assessed by qPCR (B).HGAFDFN003 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days, The protein expression of HMGA1 and HMGA2 was detected by western blot (C).D, After transfection with anti-miR-ctrl or anti-miR-59 in progerin-expressing CRL-1474 cells, the protein expression (D) of HMGAs were detected.After transfection with anti-miR-ctrl or anti-miR-59 in progerin-expressing CRL-1474 cells, mRNA level (E) of HMGAs were detected.Western blot analysis the expression of HMGAs (F) in miR-ctrl or miR-59 mimics-transfected CRL-1474 cells.qPCR analysis the mRNA levels (G) in miR-ctrl or miR-59 mimics-transfected CRL-1474 cells.Compared HGPS patient cells (HGAFDFN003 p22 and HGAFDFN167 p20) with control (CRL-1474 p26), the protein expression (H) of HMGAs were assessed.Compared HGPS patient cells (HGAFDFN003 p22 and HGAFDFN167 p20) with control (CRL-1474 p26), mRNA level (I) of HMGAs were assessed.The protein (J) level of HMGAs were detected in 7, 88 and 92-year-old cells.mRNA (K) level of HMGAs were detected in 7, 88 and 92-year-old cells.Transfected anti-miR-ctrl or anti-miR-59 in 88-year-old cells, the level of HMGAs was detected by Western blot (L)Transfected anti-miR-ctrl or anti-miR-59 in 88-year-old cells, the level of HMGAs was detected by qPCR (M).N, O The binding sites of miR-59 on HMGAs 3′ UTR were analyzed by TargetScan and miRanda. Luciferase experiments with the wild-type and the mutated 3′ UTR of HMGA1 (N) / HMGA2 (O).


Figure 3In Flag - HMGA1 or Flag - HMGA2 infected CRL - 1474 and HGPS cells ( HGAFDFN003 ) , the level of cyclin A2 , lamin B1 was detected by western blot ( A ) . In Flag - HMGA1 or Flag - HMGA2 infected CRL - 1474 and HGPS cells ( HGAFDFN003 ) , SA - β - gal staining were performed , the percentage of SA - β - gal positive cells was calculated . In Flag - HMGA1 or Flag - HMGA2 infected CRL - 1474 and HGPS cells ( HGAFDFN003 ) , Expression of Ki67 was analyzed by immunofluorescence . Scale bars : 50 μm ( C ) . Progerin - expressing CRL - 1474 cells were transfected with His - control or His - HMGA1 . SA - β - gal staining was performed . The percentage of SA - β - gal positive cells was calculated . Scale bars : 100 μm ( D ) . Progerin - expressing CRL - 1474 cells were transfected with His - control or His - HMGA1 . The level of cyclin A2 , lamin B1 was detected by western blot ( E ) . Infection with control shRNA ( shCtrl ) or HMGA1 / HMGA2 shRNA ( shHMGA1 and shHMGA2 ) in anti - miR - 59 - transfected HGPS cells ( HGAFDFN003 ) , cells were subjected to SA - β - gal staining . Scale bars : 100 μm ( F ) . Cells were subjected to immunofluorescence using anti - Ki67 ( red ) antibody ( left ) . The percentage of the Ki67 positive cells was calculated . Scale bars : 50 μm ( G ) . Infection with control shRNA ( shCtrl ) or HMGA1 / HMGA2 shRNA ( shHMGA1 and shHMGA2 ) in anti - miR - 59 - transfected HGPS cells ( HGAFDFN003 ) The level of cyclin A2 , lamin B1 , HMGA1 and HMGA2 was detected by western blot ( H ) .

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 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 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 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 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 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 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 3In Flag-HMGA1 or Flag-HMGA2 infected CRL-1474 and HGPS cells (HGAFDFN003), the level of cyclin A2, lamin B1 was detected by western blot (A).In Flag-HMGA1 or Flag-HMGA2 infected CRL-1474 and HGPS cells (HGAFDFN003), SA-β-gal staining were performed, the percentage of SA-β-gal positive cells was calculated.In Flag-HMGA1 or Flag-HMGA2 infected CRL-1474 and HGPS cells (HGAFDFN003), Expression of Ki67 was analyzed by immunofluorescence. Scale bars: 50 μm (C).Progerin-expressing CRL-1474 cells were transfected with His-control or His-HMGA1. SA-β-gal staining was performed. The percentage of SA-β-gal positive cells was calculated. Scale bars: 100 μm (D).Progerin-expressing CRL-1474 cells were transfected with His-control or His-HMGA1. The level of cyclin A2, lamin B1 was detected by western blot (E).Infection with control shRNA (shCtrl) or HMGA1/HMGA2 shRNA (shHMGA1 and shHMGA2) in anti-miR-59-transfected HGPS cells (HGAFDFN003), cells were subjected to SA-β-gal staining. Scale bars: 100 μm (F). Cells were subjected to immunofluorescence using anti-Ki67(red) antibody (left). The percentage of the Ki67 positive cells was calculated. Scale bars: 50 μm (G).Infection with control shRNA (shCtrl) or HMGA1/HMGA2 shRNA (shHMGA1 and shHMGA2) in anti-miR-59-transfected HGPS cells (HGAFDFN003) The level of cyclin A2, lamin B1, HMGA1 and HMGA2 was detected by western blot (H).


Figure 4A Interaction of endogenous HMGA1 with HMGA2 and TFIIH4 complex was analyzed by Co - IP assay using anti - HMGA1 antibody in H1299 cells . Pulled - down protein complexes were analyzed . Benzonase ( B ) was added in cell lysate . B Interaction of endogenous HMGA2 with HMGA1 and TFIIH4 complex was analyzed by Co - IP assay using anti - HMGA2 antibody in H1299 cells . Pulled - down protein complexes were analyzed . D Interaction of endogenous CDK7 with HMGAs , XPB and TFIIH4 was analyzed by Co - IP assay using anti - CDK7 antibody in H1299 cells . Pulled - down protein complexes were analyzed . HEK293T cells were transfected with Flag - HMGA1 ( E ) HMGA1 was IP with anti - Flag antibody and anti - CDK7 antibody . HEK293T cells were transfected with CDK7 ( F ) . HMGA1 or CDK7 was IP with anti - Flag antibody and anti - CDK7 antibody . G HEK293T cell lysates were incubated with GST or GST - HMGA1 sepharose beads . Pulled - down protein complexes were analyzed . I HEK293T cells were transfected with Flag - HMGA1 deletion mutants . IP of Flag was performed . Pulled - down protein complexes were analyzed . J HEK293T cell lysates were incubated with GST or GST - HMGA1 deletion mutants sepharose beads . Recombinant proteins from pull - down assays visualized by silver staining and western blot . K , L Progerin - expressing H1299 cells were transfected with His - control or His - HMGA1 . Immunoprecipitation of RNA Pol II ( K ) or CDK7 ( L ) with anti - RNAPII antibody or anti - CDK7 antibody was performed , respectively . Pulled - down protein complexes were analyzed . RNAPII , the RNAPII Ser2P and RNAPII Ser5P in HGAFDFN003 cells ( M ) compared to CRL - 1474 cells were detected by western blot . RNAPII , the RNAPII Ser2P and RNAPII Ser5P in 88 , 92 - year - old cells ( N ) compared to CRL - 1474 cells were detected by western blot . O Progerin - expressing CRL - 1474 cells were transfected with His - control or His - HMGA1 . Indicated proteins and modifications were detected by western blot . P Infection with siCtrl or siCDK7 in Flag - HMGA1 - transfected HGPS cells ( HGAFDFN003 ) , cyclin A1 , RNAPII , the RNAPII Ser2P and RNAPII Ser5P was detected . Q Infection with control shRNA ( shCtrl ) or HMGA1 shRNA ( shHMGA1 ) in anti - miR - 59 - transfected HGAFDFN003 cells . Indicated proteins and modifications were detected by western blot .

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 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 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 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 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 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 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 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 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 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 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

Figure 4A Interaction of endogenous HMGA1 with HMGA2 and TFIIH4 complex was analyzed by Co-IP assay using anti-HMGA1 antibody in H1299 cells. Pulled-down protein complexes were analyzed. Benzonase (B) was added in cell lysate. B Interaction of endogenous HMGA2 with HMGA1 and TFIIH4 complex was analyzed by Co-IP assay using anti-HMGA2 antibody in H1299 cells. Pulled-down protein complexes were analyzed.D Interaction of endogenous CDK7 with HMGAs, XPB and TFIIH4 was analyzed by Co-IP assay using anti-CDK7 antibody in H1299 cells. Pulled-down protein complexes were analyzed.HEK293T cells were transfected with Flag-HMGA1 (E) HMGA1 was IP with anti-Flag antibody and anti-CDK7 antibody.HEK293T cells were transfected with CDK7 (F). HMGA1 or CDK7 was IP with anti-Flag antibody and anti-CDK7 antibody.G HEK293T cell lysates were incubated with GST or GST-HMGA1 sepharose beads. Pulled-down protein complexes were analyzed.I HEK293T cells were transfected with Flag-HMGA1 deletion mutants. IP of Flag was performed. Pulled-down protein complexes were analyzed.J HEK293T cell lysates were incubated with GST or GST-HMGA1 deletion mutants sepharose beads. Recombinant proteins from pull-down assays visualized by silver staining and western blot.K, L Progerin-expressing H1299 cells were transfected with His-control or His-HMGA1. Immunoprecipitation of RNA Pol II (K) or CDK7 (L) with anti-RNAPII antibody or anti-CDK7 antibody was performed, respectively. Pulled-down protein complexes were analyzed.RNAPII, the RNAPII Ser2P and RNAPII Ser5P in HGAFDFN003 cells (M) compared to CRL-1474 cells were detected by western blot.RNAPII, the RNAPII Ser2P and RNAPII Ser5P in 88, 92-year-old cells (N) compared to CRL-1474 cells were detected by western blot.O Progerin-expressing CRL-1474 cells were transfected with His-control or His-HMGA1. Indicated proteins and modifications were detected by western blot.P Infection with siCtrl or siCDK7 in Flag-HMGA1-transfected HGPS cells (HGAFDFN003), cyclin A1, RNAPII, the RNAPII Ser2P and RNAPII Ser5P was detected.Q Infection with control shRNA (shCtrl) or HMGA1 shRNA (shHMGA1) in anti-miR-59-transfected HGAFDFN003 cells. Indicated proteins and modifications were detected by western blot.


Figure 5The mRNA level of cell cycle genes in HGPS patient cells ( HGAFDFN003 and HGAFDFN167 ) ( D ) was assessed by qPCR . The mRNA level of cell cycle genes in HGPS and 88 - and 92 - year - old cells ( E ) was assessed by qPCR . F HGAFDFN003 cells were infected with His - HMGA1 and the mRNA level of cell cycle genes was assessed by qPCR . G Infection with control shRNA ( shCtrl ) or HMGA1 shRNA ( shHMGA1 ) in anti - miR - 59 - transfected HGAFDFN003 cells . The mRNA level of cell cycle genes was assessed by qPCR .

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 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 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

Figure 5The mRNA level of cell cycle genes in HGPS patient cells (HGAFDFN003 and HGAFDFN167) (D) was assessed by qPCR.The mRNA level of cell cycle genes in HGPS and 88- and 92-year-old cells (E) was assessed by qPCR.F HGAFDFN003 cells were infected with His-HMGA1 and the mRNA level of cell cycle genes was assessed by qPCR.G Infection with control shRNA (shCtrl) or HMGA1 shRNA (shHMGA1) in anti-miR-59-transfected HGAFDFN003 cells. The mRNA level of cell cycle genes was assessed by qPCR.


Figure 6A miR - 59 was detected in heart , liver , lung , kidney , skin , muscle , and brain of LmnaG609G / G609G mice by qPCR at 12 weeks of life ( n = 5 for each group ) . B , C The mRNA level of Hmga1 ( B ) and Hmga2 ( C ) was detected in heart , liver , lung , kidney , skin , muscle and brain of LmnaG609G / G609G mice by qPCR at 12 weeks of life ( n = 5 for each group ) . D Transfected anti - miR - Ctrl or anti - miR - 59 in progerin - expressing NIH3T3 cells . Western blot analysis of HMGAs . G , H Luciferase experiments with the wild - type and the mutated 3 ′ UTR of Hmga1 and Hmga2 .

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

Figure 6A miR-59 was detected in heart, liver, lung, kidney, skin, muscle, and brain of LmnaG609G/G609G mice by qPCR at 12 weeks of life (n=5 for each group).B, C The mRNA level of Hmga1 (B) and Hmga2 (C) was detected in heart, liver, lung, kidney, skin, muscle and brain of LmnaG609G/G609G mice by qPCR at 12 weeks of life (n=5 for each group).D Transfected anti-miR-Ctrl or anti-miR-59 in progerin-expressing NIH3T3 cells. Western blot analysis of HMGAs.G, H Luciferase experiments with the wild-type and the mutated 3′ UTR of Hmga1 and Hmga2.


Figure 7A The in vivo therapy scheme . Eight - day - old mice were injected intraperitoneally with AAV9 - mCherry - anti - miR - 59 ( 5x1011 viral particles ) . The mCherry signals were detected 30 days post - injection ( DPI ) in LmnaG609G / G609G mice versus PBS - injected control ( dorsal ) . B Expression of the mCherry reporter in different organs at 30DPI in LmnaG609G / G609G mice versus PBS - injected control . Hrt , Mscl , Ver , Kid , Liv : heart , Muscle , vertebrae , kidney , liver . AAV9 - mCherry - anti - miR - 59 was injected into 8 - day - old mice and detected at different points . C , D The gross morphology of the wild - type and LmnaG609G / G609G mice which injected with anti - ctrl or anti - miR - 59 for 14 weeks . E Progression of body weight of male mice transduced with anti - Ctrl versus anti - miR - 59 was detected from 4 weeks of life . The sample size ( n ) indicates the initial number of mice at week 4 , initial n = 10 WT ; n = 9 anti - Ctrl - transduced mice ; n = 7 anti - miR - 59 - transduced mice . Shown are mean values ± standard deviations , two - sided t - test was used when comparing anti - Ctrl - transduced mice and anti - miR - 59 - transduced mice from week 6 - 16 . F Body weight comparison at 7 - and 11 - week - old male mice ( n = 9 anti - Ctrl - transduced mice ; n = 7 anti - miR - 59 - transduced mice ) . Data are presented as the mean ± SD ( * P < 0 . 05 , * * P < 0 . 01 ) . P - values were calculated by unpaired Student ' s t - test . G Progression of body weight of female mice transduced with anti - Ctrl versus anti - miR - 59 was detected from four weeks of life . The sample size ( n ) indicates the initial number of mice at week 4 , initial n = 5 WT ; n = 6 anti - Ctrl - transduced mice ; n = 6 anti - miR - 59 - transduced mice . Shown are mean values ± standard deviations , two - sided t - test was used when comparing anti - Ctrl - transduced mice and anti - miR - 59 - transduced mice from week 6 - 16 . H Body weight comparison at 7 - and 11 - week - old female mice ( n = 6 for each group ) . Data are presented as the mean ± SD ( * P < 0 . 05 ) . P - values were calculated by unpaired Student ' s t - test . I Kaplan - Meier survival plot of the wild - type and LmnaG609G / G609G mice which injected with anti - Ctrl or anti - miR - 59 ( n = 12 WT ; n = 13 anti - Ctrl - transduced mice ; n = 13 anti - miR - 59 - transduced mice , two - sided log - rank 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 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 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 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 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 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 7A The in vivo therapy scheme. Eight-day-old mice were injected intraperitoneally with AAV9-mCherry-anti-miR-59 (5x1011 viral particles). The mCherry signals were detected 30 days post-injection (DPI) in LmnaG609G/G609G mice versus PBS-injected control (dorsal).B Expression of the mCherry reporter in different organs at 30DPI in LmnaG609G/G609G mice versus PBS-injected control. Hrt, Mscl, Ver, Kid, Liv: heart, Muscle, vertebrae, kidney, liver. AAV9-mCherry-anti-miR-59 was injected into 8-day-old mice and detected at different points.C, D The gross morphology of the wild-type and LmnaG609G/G609G mice which injected with anti-ctrl or anti-miR-59 for 14 weeks.E Progression of body weight of male mice transduced with anti-Ctrl versus anti-miR-59 was detected from 4 weeks of life. The sample size (n) indicates the initial number of mice at week 4, initial n=10 WT; n=9 anti-Ctrl-transduced mice; n=7 anti-miR-59-transduced mice. Shown are mean values ± standard deviations, two-sided t-test was used when comparing anti-Ctrl-transduced mice and anti-miR-59-transduced mice from week 6-16.F Body weight comparison at 7- and 11-week-old male mice (n=9 anti-Ctrl-transduced mice; n=7 anti-miR-59-transduced mice). Data are presented as the mean ± SD (*P<0.05, **P<0.01). P-values were calculated by unpaired Student's t-test.G Progression of body weight of female mice transduced with anti-Ctrl versus anti-miR-59 was detected from four weeks of life. The sample size (n) indicates the initial number of mice at week 4, initial n=5 WT; n=6 anti-Ctrl-transduced mice; n=6 anti-miR-59-transduced mice. Shown are mean values ± standard deviations, two-sided t-test was used when comparing anti-Ctrl-transduced mice and anti-miR-59-transduced mice from week 6-16.H Body weight comparison at 7- and 11-week-old female mice (n=6 for each group). Data are presented as the mean ± SD (*P<0.05). P-values were calculated by unpaired Student's t-test.I Kaplan-Meier survival plot of the wild-type and LmnaG609G/G609G mice which injected with anti-Ctrl or anti-miR-59 (n=12 WT; n=13 anti-Ctrl-transduced mice; n=13 anti-miR-59-transduced mice, two-sided log-rank test).


Figure 8A Running ability of male mice transduced with anti - Ctrl versus anti - miR - 59 at 12 weeks of life ( n = 5 for each group ) . Masson staining showed the perivascular interstitial fibrosis in the heart and quadriceps muscle ( B ) The boxes and arrows denoted the aortic adventitia . Masson staining showed the adventitial fibrosis ( blue areas ) and the adventitial width of aorta ( C ) . The boxes and arrows denoted the aortic adventitia . D H & E staining in skin showed the epidermis and fat layer of skin . Scale bars , 150 μm . The boxes and arrows denoted epidermis and vertical bars indicated fat layer . E The level of miR - 59 was detected in heart , liver , lung , kidney , skin , and muscle of LmnaG609G / G609G mice ( n = 5 for each group ) . F , G The mRNA level of Hmga1 ( F ) and Hmga2 ( G ) was detected in heart , liver , lung , kidney , skin , and muscle of LmnaG609G / G609G mice ( n = 5 for each group ) . H Hmgas immunohistochemistry in skin of WT mice and LmnaG609G / G609G mice . Scale bars , 50 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 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 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 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 8A Running ability of male mice transduced with anti-Ctrl versus anti-miR-59 at 12 weeks of life (n=5 for each group).Masson staining showed the perivascular interstitial fibrosis in the heart and quadriceps muscle (B) The boxes and arrows denoted the aortic adventitia.Masson staining showed the adventitial fibrosis (blue areas) and the adventitial width of aorta (C). The boxes and arrows denoted the aortic adventitia.D H&E staining in skin showed the epidermis and fat layer of skin. Scale bars, 150 μm. The boxes and arrows denoted epidermis and vertical bars indicated fat layer.E The level of miR-59 was detected in heart, liver, lung, kidney, skin, and muscle of LmnaG609G/G609G mice (n=5 for each group). F, G The mRNA level of Hmga1 (F) and Hmga2 (G) was detected in heart, liver, lung, kidney, skin, and muscle of LmnaG609G/G609G mice (n=5 for each group). H Hmgas immunohistochemistry in skin of WT mice and LmnaG609G/G609G mice. Scale bars, 50 μm. D


Figure 1Percentage of the total signal intensity attributed to phosphopeptides ( blue bars ; 11 unique peptide ions ) and non - phosphorylated peptides ( grey bars ; 25 unique peptide ions ) following SAX fractionation at ( A ) pH 6 . 0 , ( B ) pH 6 . 8 and ( C ) pH 8 . 0 , with later fractions consisting of up to 100 % phosphopeptide ion signal . Representative pHis - containing myoglobin peptide ( LFTGHPETLEK ; solid line ) , and its non - phosphorylated counterpart ( LFTGHPETLEK ; dashed line ) , quantified across all 16 SAX fractions ; SAX was performed at pH 6 . 0 ( red ) , pH 6 . 8 ( blue ) or pH 8 . 0 ( green ) . % of the total peak area of each individual peptide across the gradient is plotted for each fraction . In order to assess pHis stability and effects of SAX separation in a complex mixture ( black ) , SAX was also repeated at pH 6 . 8 with phosphorylated myoglobin spiked into a human cell lysate prior to digestion . Data for the five pHis - containing myoglobin peptides is shown in Fig . EV3 .

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

Figure 1Percentage of the total signal intensity attributed to phosphopeptides (blue bars; 11 unique peptide ions) and non-phosphorylated peptides (grey bars; 25 unique peptide ions) following SAX fractionation at (A) pH 6.0, (B) pH 6.8 and (C) pH 8.0, with later fractions consisting of up to 100% phosphopeptide ion signal. Representative pHis-containing myoglobin peptide (LFTGHPETLEK; solid line), and its non-phosphorylated counterpart (LFTGHPETLEK; dashed line), quantified across all 16 SAX fractions; SAX was performed at pH 6.0 (red), pH 6.8 (blue) or pH 8.0 (green). % of the total peak area of each individual peptide across the gradient is plotted for each fraction. In order to assess pHis stability and effects of SAX separation in a complex mixture (black), SAX was also repeated at pH 6.8 with phosphorylated myoglobin spiked into a human cell lysate prior to digestion. Data for the five pHis-containing myoglobin peptides is shown in Fig. EV3.


Figure 2siRNA - mediated knockdown of PHPT1 in HeLa cells ( 24 h ) with reference to GAPDH loading control . NT - non - targeting siRNA control . Representative SAX profile of trypsin digested HeLa lysate ( Abs280 nm ) . Representative SAX profile of trypsin digested HeLa lysate ( Abs280 nm ) . Base peak chromatograms are shown for select SAX fractions following high - resolution LC - MS / MS using an Orbitrap Fusion mass spectrometer : ( D ) fraction 3 ( green ) ; ( E ) fraction 6 ( red ) ; ( F ) fraction 10 ( grey ) . Peptides were fragmented by HCD , with neutral loss of 98 amu from the precursor ions triggering EThCD ( Ferries et al , 2017 ) . Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot . The ptmRS node was used for phosphosite localisation . Analysis was performed on three independent biological replicates for each condition ( NT or PHPT1 siRNA ) .

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 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

Figure 2siRNA-mediated knockdown of PHPT1 in HeLa cells (24 h) with reference to GAPDH loading control. NT - non-targeting siRNA control.Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm).Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm). Base peak chromatograms are shown for select SAX fractions following high-resolution LC-MS/MS using an Orbitrap Fusion mass spectrometer: (D) fraction 3 (green); (E) fraction 6 (red); (F) fraction 10 (grey). Peptides were fragmented by HCD, with neutral loss of 98 amu from the precursor ions triggering EThCD (Ferries et al, 2017). Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot. The ptmRS node was used for phosphosite localisation. Analysis was performed on three independent biological replicates for each condition (NT or PHPT1 siRNA).


Figure 5Comparison of triplet score ( ≥ 5 % S / N ) following HCD of unique phosphopeptides ( 5 % FDR ) identified from UPAX separated tryptic digests of HeLa cell derived phosphopeptides . Site of phosphorylation ( ptmRS score ≥ 0 . 90 ) is indicated . % of phosphopeptides exhibiting either no neutral loss ( Triplet = 0 ) , or neutral loss of any 1 ( Triplet = 1 ) , 2 ( Triplet = 2 ) , or 3 ions from the precursor ( ∆ 80 , ∆ 98 and / or ∆ 116 amu ) . Distribution of triplet score following HCD fragmentation as a function of site localisation confidence ( ptmRS score ≥ 0 . 75 , ≥ 0 . 90 , ≥ 0 . 95 , ≥ 0 . 99 ) for unique singly phosphorylated peptide as a function of the residue phosphorylated . Numbers of phosphopeptides ( ptmRS ≥ 0 . 90 ) exhibiting different combinations of the three neutral loss species ( ∆ 80 , ∆ 98 and / or ∆ 116 amu from precursor ) for each of the phosphorylated residues .

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 5Comparison of triplet score (≥5% S/N) following HCD of unique phosphopeptides (5% FDR) identified from UPAX separated tryptic digests of HeLa cell derived phosphopeptides. Site of phosphorylation (ptmRS score ≥ 0.90) is indicated. % of phosphopeptides exhibiting either no neutral loss (Triplet =0), or neutral loss of any 1 (Triplet = 1), 2 (Triplet =2), or 3 ions from the precursor (∆80, ∆98 and/or ∆116 amu). Distribution of triplet score following HCD fragmentation as a function of site localisation confidence (ptmRS score ≥ 0.75, ≥ 0.90, ≥ 0.95, ≥ 0.99) for unique singly phosphorylated peptide as a function of the residue phosphorylated. Numbers of phosphopeptides (ptmRS ≥ 0.90) exhibiting different combinations of the three neutral loss species (∆80, ∆98 and/or ∆116 amu from precursor) for each of the phosphorylated residues.


Figure 1A Immunohistochemical analysis of LONP1 expression in CKD children with mild ( n = 11 ) , moderate ( n = 10 ) , or severe fibrosis ( n = 9 ) . N = 4 in Normal group . D Western blot analysis for the expression of LONP1 in UUO models at different time points . F Deposition of total fibrosis in kidney tissues was determined by Masson ' s trichrome staining . Scale bar : 50μm . G Sirius red staining in WT and cKI mice after UUO . Scale bar : 50μm . J , K Western blot and densitometric analysis for the expression of FN1 , Collagen I , Collagen III and TGF - β1 ( monomer ) in WT and cKI mice after UUO ( n = 3 or 4 , biological replicates ) . M Succinate dehydrogenase ( SDH ) staining in WT and cKI mice after UUO . Scale bar : 50μm . N Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKI mice after UUO . Scale bar : 500nm .

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 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 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 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

Figure 1A Immunohistochemical analysis of LONP1 expression in CKD children with mild (n=11), moderate (n=10), or severe fibrosis (n=9). N=4 in Normal group.D Western blot analysis for the expression of LONP1 in UUO models at different time points.F Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm. G Sirius red staining in WT and cKI mice after UUO. Scale bar: 50μm.J, K Western blot and densitometric analysis for the expression of FN1, Collagen I, Collagen III and TGF-β1 (monomer) in WT and cKI mice after UUO (n=3 or 4, biological replicates).M Succinate dehydrogenase (SDH) staining in WT and cKI mice after UUO. Scale bar: 50μm.N Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKI mice after UUO. Scale bar: 500nm.


Figure 2A Deposition of total fibrosis in kidney tissues was determined by Masson ' s trichrome staining . Scale bar : 50μm . B Sirius red staining in WT and cKO mice after UUO . Scale bar : 50μD , E Western blot and densitometric analysis for the expression of FN1 , Collagen I , Collagen III and α - SMA in WT and cKO mice after UUO ( n = 3 , biological replicates ) . I Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKO mice after UUO . Scale bar : 500nm .

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 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

Figure 2A Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm. B Sirius red staining in WT and cKO mice after UUO. Scale bar: 50μD, E Western blot and densitometric analysis for the expression of FN1, Collagen I, Collagen III and α-SMA in WT and cKO mice after UUO (n=3, biological replicates).I Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKO mice after UUO. Scale bar: 500nm.


Figure 3C Masson ' s trichrome staining of WT and cKI mice after 5 / 6Nx . Scale bar : 100μm . D qRT - PCR analysis of FN1 , Collagen I and Collagen III in WT and cKI mice after 5 / 6Nx ( n = 6 in cKI + Sham group , n = 5 in the other three groups , biological replicates ) G Masson ' s trichrome staining of WT and cKO mice after 5 / 6Nx . Scale bar : 100μm . H qRT - PCR analysis of FN1 , Collagen I , Collagen III and α - SMA in WT and cKO mice after 5 / 6Nx ( n = 5 in WT + Sham group , n = 7 in cKO + Sham group , n = 6 in 5 / 6Nx groups , biological replicates ) . I , J Western blot and densitometric analysis for the expression of Collagen I and α - SMA in WT and cKO mice after 5 / 6Nx ( n = 4 , biological replicates ) . L Transmission electron microscopy images of the mitochondria in tubular cells of WT and cKO mice after 5 / 6Nx . Scale bar : 500nm .

O B-PANEL_START 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 B-PANEL_START 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 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 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

Figure 3C Masson's trichrome staining of WT and cKI mice after 5/6Nx. Scale bar: 100μm.D qRT-PCR analysis of FN1, Collagen I and Collagen III in WT and cKI mice after 5/6Nx (n=6 in cKI+Sham group, n=5 in the other three groups, biological replicates)G Masson's trichrome staining of WT and cKO mice after 5/6Nx. Scale bar: 100μm.H qRT-PCR analysis of FN1, Collagen I, Collagen III and α-SMA in WT and cKO mice after 5/6Nx (n=5 in WT+Sham group, n=7 in cKO+Sham group, n=6 in 5/6Nx groups, biological replicates).I, J Western blot and densitometric analysis for the expression of Collagen I and α-SMA in WT and cKO mice after 5/6Nx (n=4, biological replicates).L Transmission electron microscopy images of the mitochondria in tubular cells of WT and cKO mice after 5/6Nx. Scale bar: 500nm.


Figure 4A Quantification of mitochondrial ROS production ( n = 4 , biological replicates ) . D qRT - PCR analysis of FN1 , Collagen I , Collagen III , Collagen IV , α - SMA and Vimentin ( n = 3 , biological replicates ) . Measurement of OCR using an XF96 Extracellular Flux Analyzer ( n = 8 or 10 in each group , biological replicates ) . Western blot analysis for the expression of Collagen I , α - SMA , and Vimentin ( n = 3 , two independent experiments were carried out ) .

O B-PANEL_START 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 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

Figure 4A Quantification of mitochondrial ROS production (n=4, biological replicates).D qRT-PCR analysis of FN1, Collagen I, Collagen III, Collagen IV, α-SMA and Vimentin (n=3, biological replicates).Measurement of OCR using an XF96 Extracellular Flux Analyzer (n=8 or 10 in each group, biological replicates).Western blot analysis for the expression of Collagen I, α-SMA, and Vimentin (n=3, two independent experiments were carried out).


Figure 5C Western blot validation of the up - regulated proteins in mitochondria ( n = 2 , biological replicates ) . E GST pull down analysis of interaction between LONP1 and HMGCS2 . G Immunofluorescence co - localization analysis of LONP1 , HMGCS2 and mitochondria in mPTC ( up , scale bar : 20μm ) and human kidney tissue ( down , scale bar : 200μm ) .

O B-PANEL_START 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 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

Figure 5C Western blot validation of the up-regulated proteins in mitochondria (n=2, biological replicates).E GST pull down analysis of interaction between LONP1 and HMGCS2.G Immunofluorescence co-localization analysis of LONP1, HMGCS2 and mitochondria in mPTC (up, scale bar: 20μm) and human kidney tissue (down, scale bar: 200μm).


Figure 6A Immunohistochemical analysis of HMGCS2 expression in CKD children . Scale bar : 50μm . B Immunohistochemical semi - quantitative IOD analysis of HMGCS2 ( n = 8 in Control group , n = 15 in CKD group ) . Western blot analysis of HMGCS2 in WT and cKI mice of UUO model ( n = 3 or 4 , biological replicates ) . western blotting analysis of Hmgcs2 expression in WT and Hmgcs2 + / - mice ( n = 3 , biological replicates ) .

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 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

Figure 6A Immunohistochemical analysis of HMGCS2 expression in CKD children. Scale bar: 50μm. B Immunohistochemical semi-quantitative IOD analysis of HMGCS2 (n=8 in Control group, n=15 in CKD group).Western blot analysis of HMGCS2 in WT and cKI mice of UUO model (n=3 or 4, biological replicates).western blotting analysis of Hmgcs2 expression in WT and Hmgcs2+/- mice (n=3, biological replicates).


Figure 7G , H Representative immunoblot and densitometric analysis of HMGCS2 , FN1 , Collagen III , α - SMA and Vimentin in mPTCs treated as indicated . Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel ( n = 3 in each group ) . J - N Representative Western blot and densitometric analysis of LONP1 , FN1 , α - SMA and Vimentin of each group was shown . Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel ( n = 3 in each group ) . mPTCs were transfected with siNC or siLONP1 , and then pre - treated with 84 - B10 ( 10 μM ) 2h before TGF - β1 ( 10ng / mL ) treatment for another 24h .

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

Figure 7G, H Representative immunoblot and densitometric analysis of HMGCS2, FN1, Collagen III, α-SMA and Vimentin in mPTCs treated as indicated. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n=3 in each group).J-N Representative Western blot and densitometric analysis of LONP1, FN1, α-SMA and Vimentin of each group was shown. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n=3 in each group). mPTCs were transfected with siNC or siLONP1, and then pre-treated with 84-B10 (10 μM) 2h before TGF-β1 (10ng/mL) treatment for another 24h.


Figure 8A Fibrosis deposition of kidney tissues was determined by Sirius red staining in UUO model . Scale bar : 50μm . B Quantification of fibrotic area in UUO model ( n = 10 in each group , biological replicates ) . G , H Fibrosis deposition of kidney tissues was determined by Sirius red staining and quantified in UIRI model ( n = 10 in each group , biological replicates ) . Scale bar : 50μm .

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 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

Figure 8A Fibrosis deposition of kidney tissues was determined by Sirius red staining in UUO model. Scale bar: 50μm. B Quantification of fibrotic area in UUO model (n=10 in each group, biological replicates).G, H Fibrosis deposition of kidney tissues was determined by Sirius red staining and quantified in UIRI model (n=10 in each group, biological replicates). Scale bar: 50μm.


Figure 1A Activity of an NF - κB reporter relative to vehicle control in human embryonic kidney cells that express TLR4 ( hTLR4 ) or an isogenic control cell line that does not express TLR4 ( null2 ) and were stimulated with vehicle ( veh ) , 1 ng / mL LPS , 400 μM nickel chloride , or 25 , 50 or 100 μM platinum ( II ) chloride and platinum ( IV ) chloride ( n = 3 independent biological replicates ) . B As per panel ( A ) but secreted IL - 8 was monitored as a metric of TLR4 activation upon stimulation with 50 pg / mL LPS , 200 μM nickel chloride , 100 μM platinum ( II ) chloride or 100 μM platinum ( IV ) chloride ( n = 4 independent biological replicates ) . Data Information : For all panels actual individual data from each experiment are plotted as box ( 25th and 75th percentile borders ; median central band ) with Tukey whiskers . Statistical analyses were assessed by 2 - way ANOVA : in A ) hTLR4 compared to null2 cells ; in B ) agonist treatment compared to non - treated ( nil ) ; ns , not significant ; * * , P < . 01 ; ; * * * * , P < . 0001 ( Dunnett ' s test , A ,

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

Figure 1A Activity of an NF-κB reporter relative to vehicle control in human embryonic kidney cells that express TLR4 (hTLR4) or an isogenic control cell line that does not express TLR4 (null2) and were stimulated with vehicle (veh), 1 ng/mL LPS, 400 μM nickel chloride, or 25, 50 or 100 μM platinum(II) chloride and platinum(IV) chloride (n=3 independent biological replicates). B As per panel (A) but secreted IL-8 was monitored as a metric of TLR4 activation upon stimulation with 50 pg/mL LPS, 200 μM nickel chloride, 100 μM platinum(II) chloride or 100 μM platinum(IV) chloride (n=4 independent biological replicates). Data Information: For all panels actual individual data from each experiment are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers. Statistical analyses were assessed by 2-way ANOVA: in A) hTLR4 compared to null2 cells; in B) agonist treatment compared to non-treated (nil); ns, not significant ; **, P<.01; ; ****, P<.0001 (Dunnett's test, A, B


Figure 2A IL - 8 secretion in HEK cells stably expressing hTLR4 but not MD - 2 ( HEK - isoTLR4 ) , transfected with empty vector ( EV ) or MD - 2 and left untreated ( nil ) or treated with 1 ng / mL LPS , 200 μM nickel chloride , 2 μg / ml HMGB1 , 100 μM platinum ( II ) chloride , 100 μM platinum ( IV ) chloride or 25 μM cisplatin ( n = 3 or 4 independent biological replicates ) . C IL - 8 secretion in HeLa cells transfected with non - targeting ( siNT ) or TLR4 - targeting ( siTLR4 ) siRNA and left untreated ( nil ) , or treated with 30 μM cisplatin ( n = 3 independent biological replicates ) . Mock cells were not subject to siRNA treatment prior . Data Information : Actual individual data are plotted as box ( 25th and 75th percentile borders ; median central band ) with Tukey whiskers ( A , C ) Statistical analyses were determined in comparison to nil treatments using 2 - way ( A , C ) ns , not significant ; * , P < . 05 ; * * * * , P < . 0001 ( Dunnett ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2A IL-8 secretion in HEK cells stably expressing hTLR4 but not MD-2 (HEK-isoTLR4), transfected with empty vector (EV) or MD-2 and left untreated (nil) or treated with 1 ng/mL LPS, 200 μM nickel chloride, 2 μg/ml HMGB1, 100 μM platinum(II) chloride, 100 μM platinum(IV) chloride or 25 μM cisplatin (n=3 or 4 independent biological replicates). C IL-8 secretion in HeLa cells transfected with non-targeting (siNT) or TLR4-targeting (siTLR4) siRNA and left untreated (nil), or treated with 30 μM cisplatin (n=3 independent biological replicates). Mock cells were not subject to siRNA treatment prior. Data Information: Actual individual data are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers (A, C) Statistical analyses were determined in comparison to nil treatments using 2-way (A, C) ns, not significant; *, P<.05; ****, P<.0001 (Dunnett's test).


Figure 3A - HEI - OC1 cells containing a Tlr4 deletion ( Tlr4 - / - ) were compared to HEI - OC1 non - targeting ( NT ) control cells and assessed for cell viability ( A ) , AnnexinV / propidium iodide staining ( B ) , following cisplatin treatment at the indicated concentrations ( n = 3 independent biological replicates ) . Data Information : In all panels data are presented as mean and standard deviation . Statistical comparisons to NT at the same cisplatin concentration were assessed by 2 - way ( A , B ) * , P < . 05 ; * * , P < . 0001 ( Bonferroni 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 O O

Figure 3A- HEI-OC1 cells containing a Tlr4 deletion (Tlr4-/-) were compared to HEI-OC1 non-targeting (NT) control cells and assessed for cell viability (A), AnnexinV/propidium iodide staining (B), following cisplatin treatment at the indicated concentrations (n=3 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to NT at the same cisplatin concentration were assessed by 2-way (A, B) *, P<.05; **, P<.0001(Bonferroni test).