Immunostaining for CDH5 on heart sections shows a significantly increase of endothelial labeling in Npr3 - sCreER ; R26 - Confetti heart compared with Npr3 - CreER ; R26 - Confetti heart ( Tam ) . * P < 0 . 05 ; Data are mean ± s . e . m . ; n = 5 . Scale bars , 500 µm in c ; 100 µm in d . Each image is representative of 5 biological samples .

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Immunostaining for CDH5 on heart sections shows a significantly increase of endothelial cell labeling in Npr3-sCreER;R26-Confetti heart compared with Npr3-CreER;R26-Confetti heart (Tam). *P < 0.05; Data are mean ± s.e.m.; n = 5. Scale bars, 500 µm in c; 100 µm in d. Each image is representative of 5 individual biological samples.


Whole mount of E14 . 5 or E15 . 5 Npr3 - sCreER ; R26 - LZLT Npr3 - CreER ; R26 - LZLT mouse hearts . No tam is as control for detecting leakiness of sCreER or CreER . Representative Immunostaining for tdTomato , ESR CDH5 on heart sections .

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Whole mount fluorescent images of E14.5 or E15.5 Npr3-sCreER;R26-LZLT and Npr3-CreER;R26-LZLT mouse hearts. No tam is as control for detecting leakiness of sCreER or CreER. Representative Immunostaining images for tdTomato, ESR and CDH5 on heart sections.


of of endocardial expressing tdTomato in Npr3 - sCreER ; R26 - LZLT ( sCreER ) or Npr3 - CreER ; R26 - LZLT ( CreER ) hearts . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 . Scale bars , 500 µm . Each figure is a representative of 5 biological samples .

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Quantification of the percentage of endocardial cells expressing tdTomato in Npr3-sCreER;R26-LZLT (sCreER) or Npr3-CreER;R26-LZLT (CreER) hearts. Data are mean ± s.e.m.; n = 5; *P < 0.05. Scale bars, 500 µm. Each figure is a representative of 5 individual biological samples.


Immunostaining for VEGFR2 on Npr3 - sCreER ; Kdrflox / flox heart section from mice without tamoxifen treatment ( No Tam ) .

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Immunostaining for VEGFR2 on Npr3-sCreER;Kdrflox/flox heart section from mice without tamoxifen treatment (No Tam).


Immunostaining for VEGFR2 on mouse heart sections after tamoxifen treatment ( Tam ) . A , atrium ; V , ventricle ; TM , trabecular myocardium .

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Immunostaining for VEGFR2 on mouse heart sections after tamoxifen treatment (Tam). A, atrium; V, ventricle; TM, trabecular myocardium.


Immunostaining for tdTomato , VEGFR2 CDH5 on heart sections from mice of four different genotypes . data showed of VEGFR2 + endocardial ( ECs ) in tdTomato + ( tdT + ) in TM regions . Cartoon ( bottom ) indicate tdTomato + ( red ) VEGFR2 + ( green ) in TM or compact myocardium ( CM ) . Data are mean ± s . e . m . ; n = 5 . Scale bars , 100 µm . Each image is a representative of 5 biological samples .

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Immunostaining for tdTomato, VEGFR2 and CDH5 on heart sections from mice of four different genotypes. Quantification data showed the percentage of VEGFR2+ endocardial cells (ECs) in tdTomato+ (tdT+) cells in the TM regions. Cartoon images (bottom) indicate tdTomato+ cells (red) and VEGFR2+ cells (green) in the TM or compact myocardium (CM). Data are mean ± s.e.m.; n = 5. Scale bars, 100 µm. Each image is a representative of 5 individual biological samples.


Whole - mount of Col1a2 - sCreER ; R26 - tdTomato or Col1a2 - CreER ; R26 - tdTomato hearts . Inserts indicate bright - field .

Whole-mount fluorescence images of Col1a2-sCreER;R26-tdTomato or Col1a2-CreER;R26-tdTomato hearts. Inserts indicate bright-field images.


Immunostaining for tdTomato PDGFRa on heart sections shows similar patterning of tdTomato PDGFRa signals .

Immunostaining for tdTomato and PDGFRa on heart sections shows similar patterning of tdTomato and PDGFRa signals.


Immunostaining for tdTomato ESR on heart sections . Arrowheads indicate valve mesenchyme . of of PDGFRa + expressing tdTomato in Col1a2 - sCreER ; R26 - tdTomato ( sCreER ) or Col1a2 - CreER ; R26 - tdTomato ( CreER ) hearts . n . s . , non - significant ; Data are mean ± s . e . m . ; n = 6 ; n . s . , non - significant . of of tdTomato + expressing ESR . * P < 0 . 05 ; Data are mean ± s . e . m . ; n = 6 ; * P < 0 . 05 .

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Immunostaining for tdTomato and ESR on heart sections. Arrowheads indicate valve mesenchyme. Quantification of the percentage of PDGFRa+ cells expressing tdTomato in Col1a2-sCreER;R26-tdTomato (sCreER) or Col1a2-CreER;R26-tdTomato (CreER) hearts. n.s., non-significant; Data are mean ± s.e.m.; n = 6; n.s., non-significant. Quantification of the percentage of tdTomato+ cells expressing ESR. *P < 0.05; Data are mean ± s.e.m.; n = 6; *P < 0.05.


Immunostaining for PDGFRa in heart sections shows a significant increase of fibroblast labeling in valves of Col1a2 - sCreER ; R26 - Confetti heart compared with Col1a2 - CreER ; R26 - Confetti heart ( Tam ) . of of fibroblasts expressing reporters in cardiac valves . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 . Scale bars , 500 µm in c - e ; 100 µm in h . Each figure is a representative of 5 - 6 biological samples .

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Immunostaining for PDGFRa in heart sections shows a significant increase of fibroblast labeling in valves of Col1a2-sCreER;R26-Confetti heart compared with Col1a2-CreER;R26-Confetti heart (Tam). Quantification of the percentage of fibroblasts expressing fluorescent reporters in cardiac valves. Data are mean ± s.e.m.; n = 5; *P < 0.05. Scale bars, 500 µm in c-e; 100 µm in h. Each figure is a representative of 5-6 individual biological samples.


( A ) Comparative expression during murine pre - implantation development of Dppa2 ( red ) , Dppa4 ( blue ) , Dux ( black ) , Zscan4c ( ZGA marker , green ) . Each dot represents average value of single - -

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(A) Comparative expression during murine pre-implantation embryo development of Dppa2 (red), Dppa4 (blue), Dux (black), and Zscan4c (ZGA marker, green). Each dot represents the average value of single-cell RNA-seq


( C ) Sashimi plot representing coverage on Dppa4 gene of an sequencing of murine zygotes , early 2C , mid 2C late 2C Arcs depict splicing events , numbers their relative frequency ( i . e . reads across junctions ) .

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(C) Sashimi plot representing coverage on the Dppa4 gene of an RNA sequencing analysis of murine zygotes, early 2C, mid 2C and late 2C Arcs depict splicing events, and numbers their relative frequency (i.e. reads across junctions).


( A ) Average expression of Dppa2 Dppa4 in a single - - seq of mESCs sorted for expression of both Tomato GFP reporters driven by MERVL Zscan4 promoters , respectively , double - negative population .

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(A) Average expression of Dppa2 and Dppa4 in a single-cell RNA-seq analysis of mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population.


( B ) Fraction of GFP + in mESCs carrying a MERVL - GFP reporter depleted of Dppa2 or Dppa4 using shRNAs specific to transcripts or a control . Bars represent average , error bars SD ; n = 3 . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - .

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(B) Fraction of GFP+ cells in mESCs carrying a MERVL-GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control. Bars represent the average, error bars the SD; n = 3. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.


( C ) Dppa2 , Dppa4 , Dux Zscan4 gene expression in mESCs carrying a MERVL - GFP reporter depleted of Dppa2 or Dppa4 using shRNAs specific to transcripts or a control . Expression was normalized to Actb . Bars represent average , error bars SD ; n = 3 . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - .

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(C) Dppa2, Dppa4, Dux and Zscan4 gene expression in mESCs carrying a MERVL-GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.


- seq of WT Dppa2 ( D ) KO mESC clones . dot plot displays average gene expression of three independent clones from each type ; n = 3 .

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RNA-seq analysis of WT and Dppa2 (D) KO mESC clones. The dot plot displays the average gene expression of three independent clones from each cell type; n = 3.


- seq of WT Dppa4 ( E ) KO mESC clones . dot plot displays average gene expression of three independent clones from each type ; n = 3 .

RNA-seq analysis of WT and Dppa4 (E) KO mESC clones. The dot plot displays the average gene expression of three independent clones from each cell type; n = 3.


( F ) Comparative expression by qPCR of Dux , two downstream targets of DUX ( Zscan4 Tdpoz4 ) , two DUX - independent targets of DPPA2 DPPA4 ( Mael Tdrd1 ) in mESCs depleted of endogenous DPPA2 DPPA4 overexpressing ectopically DUX or GFP as a control . Expression was normalized to Actb . Bars represent average , error bars SD ; n =

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(F) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and two DUX-independent targets of DPPA2 and DPPA4 (Mael and Tdrd1) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DUX or GFP as a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3


( H ) Comparative expression of DUX - dependent , DPPA2 DPPA4 - dependent , of genes in mESCs sorted for expression of both Tomato GFP reporters driven by MERVL Zscan4 promoters , respectively , double - negative population ( unpaired t - ) .

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(H) Comparative expression of DUX-dependent, DPPA2 and DPPA4-dependent, and the rest of the genes in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population (unpaired t-test).


( A ) Comparative expression by qPCR of Dux , two downstream targets of DUX ( Zscan4 Tdpoz4 ) , three targets of DPPA2 DPPA4 independent of DUX ( Mael , Tdrd1 , Prex2 ) in mESCs depleted of endogenous DPPA2 DPPA4 overexpressing ectopically DPPA2 , DPPA4 or GFP as a control Expression was normalized to Actb . Bars represent average , error bars SD ; n = 3 . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - .

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(A) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and three targets of DPPA2 and DPPA4 independent of DUX (Mael, Tdrd1, Prex2) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DPPA2, DPPA4 or GFP as a control Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.


( B ) Average coverage normalized for sequencing depth of ChIP - seq signal of DPPA2 , DPPA4 or DPPA4 truncated of SAP domain overexpressed in mESCs in a window of 500bp around Dux gene ( n = 2 ) . Total input ( TI ) is shown in gray . Peaks over Dux gene were called in DPPA2 , DPPA4 DPPA4ΔSAP ChIP - seq .

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(B) Average coverage normalized for sequencing depth of the ChIP-seq signal of DPPA2, DPPA4 or DPPA4 truncated of the SAP domain overexpressed in mESCs in a window of 500bp around the Dux gene (n=2). Total input (TI) is shown in gray. Peaks over the Dux gene were called in DPPA2, DPPA4 and DPPA4ΔSAP ChIP-seq.


( C ) showing distribution of DUX , DPPA2 , DPPA4 DPPA4ΔSAP coverage in a ± 5kb window around TSS of DUX - dependent , DPPA2 DPPA4 - dependent random genes in mESCs .

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(C) Heatmap showing the distribution of DUX, DPPA2, DPPA4 and DPPA4ΔSAP coverage in a ± 5kb window around the TSS of DUX-dependent, DPPA2 and DPPA4-dependent and random genes in mESCs.


( D ) Boxplot / Jitterplot representing of GC nucleotide contents in peaks from DPPA2 , DPPA4 DUX ChIP , in a random shuffle of peaks .

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(D) Boxplot/Jitterplot representing the percentage of GC nucleotide contents in the peaks from DPPA2, DPPA4 and DUX ChIP, and in a random shuffle of peaks.


Total expression of transposable element subfamilies in ( A ) Dppa2 KO compared to WT mESC clones .

Total expression of transposable element subfamilies in (A) Dppa2 KO compared to WT mESC clones.


Total expression of transposable element subfamilies in ( B ) Dppa4 KO compared to WT mESC clones .

Total expression of transposable element subfamilies in (B) Dppa4 KO compared to WT mESC clones.


( C ) - seq of MERVL - int , L1Md _ A L1Md _ T in mESCs sorted for expression of both Tomato GFP reporters driven by MERVL Zscan4 promoters , respectively , double - negative population .

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(C) RNA-seq analysis of MERVL-int, L1Md_A and L1Md_T in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population.


( D ) displaying enrichment of DUX , DPPA2 , DPPA4 DPPA4ΔSAP binding at different transposable element families . Every subfamily enriched ( pval < 0 . 05 ) in at least one DPPA2 , DPPA4 or DPPA4ΔSAP replicate are shown .

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(D) Heatmap displaying the enrichment of DUX, DPPA2, DPPA4 and DPPA4ΔSAP binding at different transposable element families. Every subfamily enriched (pval < 0.05) in at least one DPPA2, DPPA4 or DPPA4ΔSAP replicate are shown.


( E ) MSA plot of DPPA2 , DPPA4 DPPA4ΔSAP ChIP - seq in mESCs showing enrichment over L1Md _ A , L1Md _ T MERVL - int sequences . Gaps are in grey sequences in with overlap of color - coded ChIP - seq signals . Upper plot shows average coverage of signal over aligned TEs .

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(E) MSA plot of DPPA2, DPPA4 and DPPA4ΔSAP ChIP-seq in mESCs showing enrichment over L1Md_A, L1Md_T and MERVL-int sequences. Gaps are in grey and sequences in white with overlap of color-coded ChIP-seq signals. Upper plot shows average coverage of the signal over the aligned TEs.


Proliferation curves of parental AGO2KO HeLaS3 are plotted ( n = 6 experimental replicates ) .

Proliferation curves of parental and AGO2KO HeLaS3 cells are plotted (n=6 experimental replicates).


Colony - forming activity of HeLaS3 AGO2KO was determined by colony - formation ( n = 3 experimental replicates ) .

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Colony-forming activity of HeLaS3 and AGO2KO cells was determined by colony-formation assay (n=3 experimental replicates).


Tumour growth curves of xenografts derived from HeLaS3 or AGO2KO are shown as tumour diameter at different time points ( n = 5 mice for each group ) .

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Tumour growth curves of xenografts derived from HeLaS3 or AGO2KO cells are shown as tumour diameter at different time points (n=5 mice for each group).


Xenograft tumour weights were measured 30 days post - injection ( n = 5 mice for each group ) .

Xenograft tumour weights were measured 30 days post- injection (n=5 mice for each group).


HeLaS3 were transduced with a lentiviral vector coding for human TERC ( HeLaS3 _ TERC ) or for GFP , as a control ( HeLaS3 _ GFP ) . relative expression of TERC was measured by RT - qPCR . HPRT1 was used as internal control ( n = 3 experimental replicates ) .

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HeLaS3 cells were transduced with a lentiviral vector coding for human TERC (HeLaS3_TERC) or for GFP, as a control (HeLaS3_GFP). The relative expression of TERC was measured by RT-qPCR. HPRT1 was used as internal control (n = 3 experimental replicates).


relative expression of terc - sRNA was measured in HeLaS3 _ TERC HeLaS3 _ GFP by RT - qPCR . RNU44 was used as internal control ( n = 3 experimental replicates ) .

The relative expression of terc-sRNA was measured in HeLaS3_TERC and HeLaS3_GFP by RT-qPCR. RNU44 was used as internal control (n = 3 experimental replicates).


Average telomere was measured from genomic of parental AGO2KO HeLaS3 , by qPCR amplification of telomere repeats ( T ) multicopy gene Alu ( R ) , as a reference . relative telomere ( T / R ) was plotted ( n = 3 experimental replicates )

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Average telomere length was measured from genomic DNA of parental and AGO2KO HeLaS3, by qPCR amplification of telomere repeats (T) and multicopy gene Alu (R), as a reference. The relative telomere length (T/R) was plotted (n=3 experimental replicates)


Representative of metaphase spreads obtained from HeLaS3 AGO2KO stained for telomeric sequences centromere 2 alphoid .

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Representative images of metaphase spreads obtained from HeLaS3 and AGO2KO cells stained for telomeric sequences and centromere 2 alphoid DNA.


Representative telomere distributions in parental AGO2KO HeLaS3 . Average telomere standard deviation , as well as total number of telomeres analyzed , are indicated . Notice marked shift towards short telomeres in AGO2KO compared with parental HeLa S3 .

Representative telomere length distributions in parental and AGO2KO HeLaS3. Average telomere length and the standard deviation, as well as the total number of telomeres analyzed, are indicated. Notice the marked shift towards short telomeres in AGO2KO cells compared with the parental HeLa S3 cell line.


Telomerase activity was detected by TRAP in parental HeLaS3 AGO2KO . 293T were used as a positive control . As negative controls , telomerase was heat - inactivated in extracts , in last lane no were added in lysis buffer . of telomerase products ( 6 bp - ladder ) was determined by ImageLab Software ( Biorad ) normalized with of Internal Control ( IC ) ( n = 3 experimental replicates ) .

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Telomerase activity was detected by TRAP in parental HeLaS3 and AGO2KO cells. 293T cells were used as a positive control. As negative controls, telomerase was heat- inactivated in cell extracts, in the last lane no cells were added in lysis buffer. Intensity of the telomerase products (6 bp-ladder) was determined by ImageLab Software (Biorad) and normalized with the intensity of the Internal Control (IC) (n=3 experimental replicates).


AGO2KO were transduced with a lentiviral vector coding for FLAG - HA - tagged AGO2 ( AGO2KO _ FH AGO2 ) or for GFP as a control ( AGO2KO _ GFP ) . of telomerase activity in AGO2KO _ FH AGO2 AGO2KO _ GFP , as assessed by TRAP , was plotted ( n = 3 experimental replicates ) .

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AGO2KO cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (AGO2KO_FH AGO2) or for GFP as a control (AGO2KO_GFP). Quantification of telomerase activity in AGO2KO_FH AGO2 and AGO2KO_GFP, as assessed by TRAP, was plotted (n=3 experimental replicates).


HeLaS3 were transduced with a lentiviral vector coding for FLAG - HA - tagged AGO2 ( HeLaS3 _ FH AGO2 ) or for GFP as a control ( HeLaS3 _ GFP ) . of telomerase activity in HeLaS3 _ FH AGO2 HeLaS3 _ GFP , as assessed by TRAP , was plotted ( n = 3 experimental replicates ) .

HeLaS3 cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (HeLaS3_FH AGO2) or for GFP as a control (HeLaS3_GFP). Quantification of telomerase activity in HeLaS3_FH AGO2 and HeLaS3_GFP, as assessed by TRAP, was plotted (n=3 experimental replicates).


relative expression of TERC was measured by RT - qPCR from total of HeLaS3 AGO2KO . HPRT1 was used as internal control ( n = 3 experimental replicates ) . qPCR of 3 ' - extended TERC transcript in cDNA from HeLaS3 AGO2KO generated using random hexamers . HPRT1 was used for normalization ( n = 3 experimental replicates ) . qPCR of TERC transcript in cDNA from HeLaS3 AGO2KO generated using oligo ( dT ) 10 priming . HPRT1 was used for normalization ( n = 3 experimental replicates ) .

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The relative expression of TERC was measured by RT-qPCR from total RNA of HeLaS3 and AGO2KO cells. HPRT1 was used as internal control (n = 3 experimental replicates). qPCR of 3'-extended TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using random hexamers. HPRT1 was used for normalization (n=3 experimental replicates). qPCR of TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using oligo(dT)10 priming. HPRT1 was used for normalization (n=3 experimental replicates).


Whole extract of HeLaS3 AGO2KO was analyzed by western blot for presence of TERT . GAPDH was used as loading control . Data are representative of 4 independent experiments . TERT level was quantified by ImageLab Software ( Biorad ) . GAPDH was use as internal control ( n = 4 experimental replicates )

Whole cell extract of HeLaS3 and AGO2KO was analyzed by western blot for the presence of TERT protein. GAPDH was used as loading control. Data are representative of 4 independent experiments. TERT protein level was quantified by ImageLab Software (Biorad). GAPDH was use as internal control (n=4 experimental replicates)


HeLaS3 AGO2KO extracts ( n = 3 ) , were immunoprecipitated using an anti - TERT antibody or IgG as mock IP . TERC HOTAIR ( as a negative control ) abundance was assessed by RT - qPCR . HPRT1 was used for normalization enrichment in TERT - RIP as compared to IgG RIP was plotted .

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HeLaS3 and AGO2KO cell extracts (n=3), were immunoprecipitated using an anti-TERT antibody or IgG as mock IP. TERC and HOTAIR (as a negative control) abundance was assessed by RT-qPCR. HPRT1 was used for normalization and enrichment in TERT-RIP as compared to IgG RIP was plotted.


AGO2KO _ FH AGO2 AGO2KO _ GFP extracts ( n = 3 experimental replicates ) were immunoprecipitated using an anti - TERT antibody or IgG as mock IP . TERC HOTAIR ( as a negative control ) abundance was assessed by RT - qPCR . HPRT1 was used for normalization enrichment in TERT - RIP as compared to IgG RIP was plotted .

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AGO2KO_FH AGO2 and AGO2KO_GFP cell extracts (n=3 experimental replicates) were immunoprecipitated using an anti-TERT antibody or IgG as mock IP. TERC and HOTAIR (as a negative control) abundance was assessed by RT-qPCR. HPRT1 was used for normalization and enrichment in TERT-RIP as compared to IgG RIP was plotted.


HeLaS3 whole - extract was immunoprecipitated using anti - AGO2 antibody or IgG , as mock IP . Whole lysates ( input ) immunoprecipitates were analyzed by western blot with anti - TERT antibody . As a positive control , presence in immunoprecipitates of GW182 , a known AGO2 - interacting , was assessed . Data are representative of three independent experiments .

HeLaS3 whole-cell extract was immunoprecipitated using anti- AGO2 antibody or IgG, as mock IP. Whole cell lysates (input) and immunoprecipitates were analyzed by western blot with anti-TERT antibody. As a positive control, the presence in immunoprecipitates of GW182, a known AGO2-interacting protein, was assessed. Data are representative of three independent experiments.


HeLaS3 whole - extract was immunoprecipitated using anti - AGO2 antibody or IgG , as mock IP . TERC enrichment in AGO2 RIP as compared to IgG RIP was assessed by RT - qPCR . 7SK was used for normalization ( n = 4 experimental replicates ) .

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HeLaS3 whole-cell extract was immunoprecipitated using anti- AGO2 antibody or IgG, as mock IP. TERC RNA enrichment in AGO2 RIP as compared to IgG RIP was assessed by RT-qPCR . 7SK RNA was used for normalization (n=4 experimental replicates).


RIP was performed from HeLaS3 _ FH AGO2 HeLaS3 _ GFP extract using anti - HA antibody or IgG , as negative control , followed by TERC detection by RT - qPCR . 7SK was used for normalization ( n = 3 experimental replicates ) .

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

RIP assay was performed from HeLaS3_FH AGO2 and HeLaS3_GFP cell extract using anti-HA antibody or IgG, as negative control, followed by TERC detection by RT-qPCR. 7SK RNA was used for normalization (n=3 experimental replicates).


HeLaS3 were transduced with a lentiviral vector coding for wild - type TERC ( HeLaS3 _ TERCwt ) , TERC mutated in position 313 - 340 ( HeLaS3 _ TERC ( 313 - 340mut ) ) or TERC delated in position 12 - 31 ( HeLaS3 _ TERC ( Δ 12 - 31 ) ) . Whole - lysates from HeLaS3 _ TERCwt , HeLaS3 _ TERC ( 313 - 340mut ) HeLaS3 _ TERC ( Δ 12 - 31 ) was immunoprecipitated using an anti - AGO2 antibody in order to assessed impact of TERC mutations on AGO2 binding . For each experiment enrichment of ectoTERC as compared to 7SK in RIP samples was normalized on amount of ectoTERC 7SK in input samples . Association of AGO2 with TERC ( 313 - 340mut TERC ( Δ 12 - 31 ) was compared to association with TERCwt ( n = 3 experimental replicates ) .

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

HeLaS3 cells were transduced with a lentiviral vector coding for wild-type TERC (HeLaS3_TERCwt), TERC mutated in position 313-340 (HeLaS3_TERC (313-340mut)) or TERC delated in position 12-31 (HeLaS3_TERC (Δ 12-31)). Whole-cell lysates from HeLaS3_TERCwt, HeLaS3_TERC(313-340mut) and HeLaS3_TERC (Δ 12-31) was immunoprecipitated using an anti-AGO2 antibody in order to assessed the impact of TERC mutations on AGO2 binding. For each experiment the enrichment of ectoTERC as compared to 7SK RNA in RIP samples was normalized on the amount of ectoTERC and 7SK RNA in input samples. Association of AGO2 with TERC(313-340mut and TERC (Δ 12-31) was compared to the association with TERCwt (n=3 experimental replicates).


HeLaS3 AGO2KO were transfected with a synthetic terc - sRNA or an siRNA with no target in humans ( siCtl ) , as a control . Four days after transfection , telomerase activity in 100 - 50 - lysate was assessed by TRAP . of telomerase products ( 6 bp - ladder ) was determined by ImageLab Software ( Biorad ) normalized with of Internal Control ( IC ) , ( n = 4 experimental replicates )

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

HeLaS3 and AGO2KO cells were transfected with a synthetic terc-sRNA or an siRNA with no target in humans (siCtl), as a control. Four days after transfection, telomerase activity in 100- and 50 cell-lysate was assessed by TRAP. Intensity of the telomerase products (6 bp-ladder) was determined by ImageLab Software (Biorad) and normalized with the intensity of the Internal Control (IC), (n=4 experimental replicates)


Comparison of colony forming unit ( CFU ) in feces from vehicle - ABX - treated mice ( n = 5 mice / group ) . n . d . , non - detectable .

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

Comparison of bacterial colony forming unit (CFU) in feces from vehicle- and ABX-treated mice (n = 5 mice/group). n.d., non-detectable.


Representative image measurements of lengths for CD31 + / LYVE - 1 + lacteal , CD31 + / LYVE - 1 - capillary plexus DAPI + villus .

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

Representative image and measurements of lengths for CD31+/LYVE-1+ lacteal, CD31+/LYVE-1- capillary plexus and DAPI+ villus.


comparisons of villi lengths absolute relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) ileum ( IL ) of vehicle - or ABX - treated mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) .

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

Images and comparisons of villi lengths and absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of vehicle- or ABX-treated mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group).


comparisons of number of Prox1 + LECs within initial 100 μm portion ( F ) presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit .

Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (F) presented as relative fluorescence intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit.


comparisons of VEGFR3 expression ( G ) , presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit .

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

Images and comparisons of the VEGFR3 expression (G), presented as relative fluorescence intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit.


comparison of VE - cadherin LEC junctions in lacteals of jejunum between vehicle - ABX - treated mice . Each boxed portion is highly magnified for visualization of detailed distributions of VE - cadherin to distinguish button - like ( red arrowheads ) , zipper - like ( open arrowheads ) mixed LEC junctions in lacteals ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 50 μm .

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

Images and comparison of VE-cadherin LEC junctions in lacteals of jejunum between vehicle- and ABX-treated mice. Each boxed portion is highly magnified for visualization of detailed distributions of VE-cadherin to distinguish button-like (red arrowheads), zipper-like (open arrowheads) and mixed LEC junctions in lacteals (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 50 μm.


Representative intravital imaging of lipid clearance from lamina propria via lacteals . Asterisks indicate site in lamina propria where ( FI ) was measured . Relative FI divided by mean FI of controls at 16 min ( left ) normalized FI divided by FI at 16 min in each villus ( right ) after initial loading of BODIPY - FA were quantified . For normalization of FI , villi that showed peak earlier or later than 16 min were excluded ( n = 5 - 7 mice / group , 5 - 10 villi / mouse ) . Scale bars , 100 μm .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative intravital imaging and quantification of lipid clearance from lamina propria via lacteals. Asterisks indicate the site in lamina propria where fluorescence intensity (FI) was measured. Relative FI divided by mean FI of controls at 16 min (left) and normalized FI divided by FI at 16 min in each villus (right) after initial loading of BODIPY-FA were quantified. For normalization of FI, villi that showed peak intensity earlier or later than 16 min were excluded (n = 5-7 mice/group, 5-10 villi/mouse). Scale bars, 100 μm.


Representative from transmission electron microscopic examination of lacteals of frequency of open junctions ( n = 4 mice / group , 4 - 6 / mouse ) . Jejunum of vehicle - ABX - treated mice were harvested 2 hours after oral lipid loading . Note that junction between LECs ( red colored bidirectional arrows ) is open in vehicle - treated mice , but not in ABX - treated mice . CM , chylomicron ; IS , interstitium of lamina propria ; LC , large caveola ; Lu , lacteal lumen ; Ve , vesicle containing lipoproteins . Scale bars , 1 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

Representative images from transmission electron microscopic examination of lacteals and quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Jejunum of vehicle- and ABX-treated mice were harvested 2 hours after oral lipid loading. Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice. CM, chylomicron; IS, interstitium of lamina propria; LC, large caveola; Lu, lacteal lumen; Ve, vesicle containing lipoproteins. Scale bars, 1 μm.


of frequency of open junctions ( n = 4 mice / group , 4 - 6 / mouse ) . Note that junction between LECs ( red colored bidirectional arrows ) is open in vehicle - treated mice , but not in ABX - treated mice .

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

quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice.


Comparisons of serum triglyceride ( TG ) free fatty ( FFA ) at indicated time points in vehicle - ABX - treated mice ( n = 5 - 7 mice / group ) .

O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Comparisons of serum triglyceride (TG) and free fatty acids (FFA) at indicated time points in vehicle- and ABX- treated mice (n = 5-7 mice/group).


comparisons of absolute relative lacteal lengths in jejunum between vehicle - or ABX - treated mice at P7 , P14 , P28 . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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

Images and comparisons of absolute and relative lacteal lengths in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of number of Prox1 + LECs within initial 100 μm portion in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between vehicle - or ABX - treated mice at P7 , P14 , P28 . Button - like ( red arrowheads ) zipper - like ( open arrowheads ) junctions are indicated in each magnified box in right ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparisons of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in right (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


comparisons of absolute relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) ileum ( IL ) of 8 - week old SPF , GF or CONV mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of 8-week old SPF, GF or CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of number of Prox1 + LECs within initial 100 μm portion ( C ) VEGFR3 expression ( D , presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from SPF , GF CONV mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (C) and VEGFR3 expression (D, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from SPF, GF and CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum among SPF , GF CONV mice . Button - like ( red arrowheads ) zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum among SPF, GF and CONV mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


comparison of VEGFR3 expression ( presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum between WT VR3iΔLEC mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparison of VEGFR3 expression (presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum between WT and VR3iΔLEC mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


comparisons of absolute relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) ileum ( IL ) of WT VR3iΔLEC mice . Note that , in addition to alterations in lacteal lengths in VR3iΔLEC mice , diameters of lacteal also shrank by VEGFR3 ablation . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and VR3iΔLEC mice. Note that, in addition to the alterations in the lacteal lengths in VR3iΔLEC mice, diameters of the lacteal also shrank by VEGFR3 ablation. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of number of Prox1 + LECs within initial 100 μm portion in CD31 + / LYVE - 1 + lacteals of jejunum from WT VR3iΔLEC mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from WT and VR3iΔLEC mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum from WT VR3iΔLEC mice . Button - like ( red arrowheads ) zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum from WT and VR3iΔLEC mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Comparisons of serum triglyceride ( TG ) at indicated time points in WT VR3iΔLEC mice ( n = 6 mice / group ) .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

Comparisons of serum triglyceride (TG) at indicated time points in WT and VR3iΔLEC mice (n = 6 mice/group).


Comparison of of VEGF - C in whole tissue of jejunum ileum from SPF , GF , CONV mice ( n = 3 - 5 mice / group ) .

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

Comparison of protein levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).


Comparison of mRNA of VEGF - C in whole tissue of jejunum ileum from SPF , GF , CONV mice ( n = 3 - 5 mice / group ) .

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

Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).


Comparison of mRNA of VEGF - C in sorted CD45 - stromal CD45 + MHCII + F4 / 80 + macrophages from jejunum ileum of vehicle - treated mice . mean of transcription of VEGF - C in CD45 - stromal was normalized to 1 , relative in macrophages were presented as fold change ( n = 4 mice / group ) .

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

Comparison of mRNA levels of VEGF-C in sorted CD45- stromal cells and CD45+ MHCII+ F4/80+ macrophages from jejunum and ileum of vehicle-treated mice. The mean of transcription levels of VEGF-C in CD45- stromal cells was normalized to 1, and the relative levels in macrophages were presented as fold change (n = 4 mice/group).


Comparisons of VEGF - C transcription level in CD45 - stromal ( left ) CD45 + MHCII + F4 / 80 + macrophages ( right ) in jejunum ileum of vehicle - ABX - treated mice . mean of transcription of VEGF - C in vehicle - treated mice was normalized to 1 , relative in ABX - treated mice were presented as fold change ( n = 5 - 6 mice / group ) .

Comparisons of VEGF-C transcription level in CD45- stromal cells (left) and CD45+ MHCII+ F4/80+ macrophages (right) in jejunum and ileum of vehicle- and ABX-treated mice. The mean of transcription levels of VEGF-C in vehicle-treated mice was normalized to 1, and the relative levels in ABX-treated mice were presented as fold change (n = 5 -6 mice/group).


comparison of F4 / 80 + macrophages in villi of duodenum ( DD ) , jejunum ( JJ ) , ileum ( IL ) from vehicle - or ABX - treated mice . Each dot indicates mean value of 5 sites in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparison of F4/80+ macrophages in the villi of duodenum (DD), jejunum (JJ), and ileum (IL) from vehicle- or ABX-treated mice. Each dot indicates mean value of 5 sites in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Representative flow cytometric comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + ( E ) macrophage gated on DAPI - CD45 + from jejunum ileum of vehicle - ABX - treated mice ( n = 5 mice / group ) .

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells (E) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).


Representative flow cytometric comparison of CX3CR1 + F4 / 80 + macrophage gated on DAPI - CD45 + MHCII + ( F ) macrophage gated on DAPI - CD45 + from jejunum ileum of vehicle - ABX - treated mice ( n = 5 mice / group ) .

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

Representative flow cytometric analysis and comparison of CX3CR1+ F4/80+ macrophage gated on DAPI- CD45+ MHCII+ cells (F) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).


Representative flow cytometric comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + from jejunum ileum of WT DTR mice ( n = 5 mice / group ) .

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

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and DTR mice (n = 5 mice/group).


Comparison of mRNA of VEGF - C in whole tissue of jejunum ileum from WT DTR mice ( n = 5 mice / group ) .

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

Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from WT and DTR mice (n = 5 mice/group).


comparisons of absolute relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) ileum ( IL ) of WT DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) VEGFR3 expression ( lower , presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT DTR mice . Button - like ( red arrowheads ) zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and DTR mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Representative flow cytometric comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + from jejunum ileum of WT MyD88ΔMP mice ( n = 5 mice / group ) .

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and MyD88ΔMP mice (n = 5 mice/group).


Comparisons of mRNA of VEGF - C in sorted MHCII + F4 / 80 + macrophages from jejunum ileum of WT MyD88ΔMP mice . mean of transcription of VEGF - C in WT mice was normalized to 1 , relative level in MyD88ΔMP mice were presented as fold change ( n = 5 mice / group ) .

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

Comparisons of mRNA levels of VEGF-C in sorted MHCII+ F4/80+ macrophages from jejunum and ileum of WT and MyD88ΔMP mice. The mean of transcription levels of VEGF-C in WT mice was normalized to 1, and the relative level in MyD88ΔMP mice were presented as fold change (n = 5 mice/group).


comparisons of absolute relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) ileum ( IL ) of WT MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) VEGFR3 expression ( lower , presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT MyD88ΔMP mice . Button - like ( red arrowheads ) zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and MyD88ΔMP mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Representative confocal of - type - specific promoter : : GFP : AGO1 translational fusions .

Representative confocal images of cell-type-specific promoter::GFP:AGO1 translational fusions.


Immunoblot of FLAG : AGO1 IPed in each root - tip layer . Scale bars : 50μM , NT : Non - transformed . CB : Coomassie blue .

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Immunoblot analysis of FLAG:AGO1 IPed in each root-tip layer. Scale bars: 50μM, NT: Non-transformed. CB: Coomassie blue.


Left : input IPed AGO1 following post - lysis addition of a radiolabeled siRNA duplex ( siGFP ) . Right : northern of AGO1 - bound sRNAs .

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Left: input and IPed AGO1 following post-lysis addition of a radiolabeled siRNA duplex (siGFP). Right: northern analysis of AGO1-bound sRNAs.


Layer - specific distribution of AGO1 - loaded miRNAs .

Layer-specific distribution of all AGO1-loaded miRNAs.


Northern of layer - specific AGO1 - loaded miRNAs using sRNAs IPed independently from that used for deep - sequencing . Bar - plots underneath each panel display deep - sequencing results as normalized reads .

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Northern analysis of layer-specific AGO1-loaded miRNAs using sRNAs IPed independently from that used for deep-sequencing. Bar-plots underneath each panel display the deep-sequencing results as normalized reads counts.


Representative confocal of - type - specific promoter : : GFP transcriptional fusions in hyl1 vs WT roots .

Representative confocal images of cell-type-specific promoter::GFP transcriptional fusions in hyl1 vs WT roots.


Hierarchical clustering of validated miRNA targets between different layers in either WT or hyl1 roots .

Hierarchical clustering of validated miRNA targets between different cell layers in either WT or hyl1 roots.


qRT - PCR validation of three distinct classes of direct miRNA target transcripts from total root - , total root polysome - or epidermis - specific polysome - associated . AT2G37050 kinase is positively regulated by LAC17 . Scale bars : 50μM . qPCR values were normalized to internal control ACTIN2 .

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qRT-PCR validation of three distinct classes of direct miRNA target transcripts from total root-, total root polysome- or epidermis-specific polysome-associated RNA. The AT2G37050 kinase is positively regulated by LAC17. Scale bars: 50μM. All qPCR values were normalized to the internal control ACTIN2.


miRoot snapshots of whole - root ( left ) layer - specific ( right ) LAC17 mRNA accumulation in WT versus hyl1 , as assessed by polysome profiling .

miRoot snapshots of whole-root (left) and layer-specific (right) LAC17 mRNA accumulation in WT versus hyl1, as assessed by polysome profiling.


miRoot snapshot of - type specific miRNAs showing , in this case , a sum group of miR156 family . right panel shows gradient of expression revealed upon removal of stele ( blue box ) .

miRoot snapshot of cell-type specific miRNAs showing, in this case, a sum group of the miR156 family. The right panel shows the gradient of expression revealed upon removal of the stele (blue box).


Snapshot of SPL6 miR156 translatome in WT hyl1 backgrounds . Removal of stele ( blue box ) reveals subtleties of miR156 - mediated gene regulation in other layers , resulting in a near - mirror image of ( A ) .

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Snapshot of the SPL6 miR156 translatome in WT and hyl1 backgrounds. Removal of the stele (blue box) reveals subtleties of miR156-mediated gene regulation in the other layers, resulting in a near-mirror image of (A).


Confocal image of pmiR396a : : H2B : GFP . Normalized read from AGO1 - IP - Seq independent northern of AGO1 - loaded miR396 .

Confocal image of pmiR396a::H2B:GFP. Normalized read counts from AGO1-IP-Seq and independent northern analysis of AGO1-loaded miR396.


Representative confocal image of pGRF2 : : H2B : GFP translational fusion .

Representative confocal image of the pGRF2::H2B:GFP translational fusion.


Focused miRoot of GRF2 translatome signal in WT versus hyl1 columella . Graph : GRF2 normalized read .

Focused miRoot analysis of the GRF2 translatome signal in WT versus hyl1 columella. Graph: GRF2 normalized read counts.


Representative confocal of pGRF2 : : GRF2 : GFP translational fusion in WT or hyl1 backgrounds .

Representative confocal images of the pGRF2::GRF2:GFP translational fusion in WT or hyl1 backgrounds.


Focused miRoot of PLT1 translatome signal in WT versus hyl1 columella .

Focused miRoot analysis of the PLT1 translatome signal in WT versus hyl1 columella.


Layer - specific normalized read of miR169 paralogs ( separated into 4 different groups on identity : a , b - c , d - g , h - n ) from AGO1 - IP - deep - Seq independent northern , as in Fig . 1F .

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Layer-specific normalized read counts of miR169 paralogs (separated into 4 different groups based on sequence identity: a, b-c, d-g, h-n) from AGO1-IP-deep-Seq and independent northern analysis, as in Fig.1F.


- type specific AGO1 - IP signals for each miR169 - group with ( upper ) or without ( lower panel ) miRoot scale normalization option .

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Cell-type specific AGO1-IP signals for each miR169 sequence-group with (upper) or without (lower panel) the miRoot scale normalization option.


Predicted base - pairing of indicated miR169 - group to four NF - YA target transcripts . Pairing with expectation value greater than 3 was not considered . color gradient ( pale : low , : strong ) qualitatively approximates pairing contribution of each miR169 - group according to upper panel of ( B ) . miRoot snapshots of layer - specific translatome signals of four out - of - seven miR169 NF - YA targets in WT versus hyl1 backgrounds .

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Predicted base-pairing of indicated miR169 sequence-group to four NF-YA target transcripts. Pairing with expectation value greater than 3 was not considered. The color gradient (pale: low, dark: strong) qualitatively approximates the pairing contribution of each miR169 sequence-group according to the upper panel of (B). miRoot snapshots of layer-specific translatome signals of four out-of-seven miR169 NF-YA targets in WT versus hyl1 backgrounds.


Browser representation independent northern AGO1 - loaded miR165 / 166 .

Browser representation and independent northern analysis and AGO1-loaded miR165/166.


miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds .

miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds.


Representative confocal of pmiR395 ( a / c / e ) : : H2B : GFP transcriptional fusions under SO4 - - deficient or SO4 - - proficient growth conditions . Northern of AGO1 - loaded miR395 under conditions in ( C ) .

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Representative confocal images of pmiR395(a/c/e)::H2B:GFP transcriptional fusions under SO4--deficient or SO4--proficient growth conditions. Northern analysis of AGO1-loaded miR395 under the conditions in (C).


Representative confocal of pAPS4 : : APS4 : GFP translational fusion under conditions in ( C ) .

Representative confocal images of the pAPS4::APS4:GFP translational fusion under the conditions in (C).


Representative confocal image of pSHR : : GFP : P19 translational fusion ( B ) principle of P19 - miRNA duplex sequestration .

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Representative confocal image of the pSHR::GFP:P19 translational fusion and (B) principle of P19-based miRNA duplex sequestration.


Binding of miR395 by SHR : : HA : P19 under SO4 - - sufficient ( + ) - deficient ( - ) growth conditions .

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Binding of miR395 by SHR::HA:P19 under SO4--sufficient (+) and -deficient (-) growth conditions.


qRT - PCR of polysome - associated APS4 in cortex or epidermis in presence ( + ) or absence ( - ) of SHR : : HA : P19 . Signals are relative to SHR : : HA : P19 ( - ) lines given a value of 1 .

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qRT-PCR analysis of polysome-associated APS4 in the cortex or epidermis in the presence (+) or absence (-) of SHR::HA:P19. Signals are relative to SHR::HA:P19 (-) lines given a value of 1.


Representative confocal image of pmiR397b : : H2B : GFP transcriptional fusion ( left ) browser AGO1 - loaded signal for miR397b ( right ) .

Representative confocal image of the pmiR397b::H2B:GFP transcriptional fusion (left) and the browser AGO1-loaded signal for miR397b (right).


Cross section of Arabidopsis leaf showing - types isolated via Meselect .

Cross section of Arabidopsis leaf showing cell-types isolated via Meselect.


miR160 northern in whole - leaf , vasculature epidermis . U6 : loading control .

miR160 northern analysis in whole-leaf, vasculature and epidermis. U6: loading control.


Western of promoter : : H2B : GFP translational fusions shown in ( 5C ) in types isolated in ( G ) .

Western analysis of the promoter::H2B:GFP translational fusions shown in (5C) in the cell types isolated in (G).


qRT - PCR of ARF17 in whole - leaves or in separated types in WT versus hyl1 backgrounds .

qRT-PCR analysis of ARF17 in whole-leaves or in separated cell types in WT versus hyl1 backgrounds.


( A ) exclusion chromatography profile of NS1 pull - down experiments showing a clear shift after incubation in complete or heat - inactivated human serum ( solid dotted black lines , respectively ) from same healthy donor compared to NS1 alone ( blue ) . NS1 interaction partners were identified by SDS - PAGE N - terminal sequencing as Apolipoprotein B scaffold of low - density lipoproteins ( LDL ) in first SEC elution peak , ApoA - I scaffold of high - density lipoproteins ( HDL ) in second elution peak .

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(A) Size exclusion chromatography profile of NS1 pull-down experiments showing a clear shift after incubation in complete or heat-inactivated human serum (solid and dotted black lines, respectively) from the same healthy donor compared to the NS1 protein alone (blue line). NS1 protein interaction partners were identified by SDS-PAGE and N-terminal sequencing as the Apolipoprotein B scaffold of the low-density lipoproteins (LDL) in the first SEC elution peak, and the ApoA-I protein scaffold of the high-density lipoproteins (HDL) in the second elution peak.


( B ) Biolayer interferometry ( BLI ) profiles corresponding to binding of NS1 at various concentrations respective to human HDL ( left panel ) human LDL ( central panel ) particles . - dependence of steady - state signal corresponding to binding of NS1 to HDL ( black dots ) LDL ( dots ) is shown on right - side panel . measurements were replicated at least three times using novel biosensors samples . Data points error bars correspond to mean ± SD .

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(B) Biolayer interferometry (BLI) profiles corresponding to the binding of NS1 at various concentrations respective to human HDL (left panel) and human LDL (central panel) particles. The concentration-dependence of the steady-state signal corresponding to the binding of NS1 to HDL (black dots) and LDL (white dots) is shown on the right-hand side panel. The measurements were replicated at least three times using novel biosensors and samples. Data points and error bars correspond to the mean ± SD.


( C ) Typical sedimentation coefficient distribution of NS1 or human HDL alone or pre - incubated together ( mixture of NS1 HDL at a 1 : 1 or 5 : 1 mass ) monitored using an interferometric detector . Peaks were integrated for detectors ( interference absorbance at 280nm ) . calculated stoichiometries are indicated for each peak . Solutions were equilibrated at 20 ° C for 2h before sedimentation velocity .

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(C) Typical sedimentation coefficient distribution of NS1 or human HDL alone or pre-incubated together (mixture of NS1 and HDL at a 1:1 or 5:1 mass ratio) monitored using an interferometric detector. Peaks were integrated for all the detectors (interference and absorbance at 280nm). The calculated stoichiometries are indicated for each peak. Solutions were equilibrated at 20°C for 2h before sedimentation velocity analysis.


( A , B ) Electron microscopy observations from left to right : a representative image , followed by three most representative classes of ( A ) purified HDL particles ( B ) NS1 - HDL complexes . bar : 50 nm , Black bar : 20 nm .

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(A, B) Electron microscopy observations from left to right: a representative image, followed by the three most representative classes of (A) purified HDL particles and (B) NS1-HDL complexes. White bar: 50 nm, Black bar: 20 nm.


( D ) Differential scanning calorimetry ( DSC ) of NS1 alone ( blue ) or of an NS1 - HDL mixture at a 2 : 1 molar ( orange ) . Of note , HDL particles alone did not generate any signal in range tested .

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(D) Differential scanning calorimetry (DSC) of NS1 alone (blue line) or of an NS1-HDL mixture at a 2:1 molar ratio (orange). Of note, the HDL particles alone did not generate any signal in the temperature range tested.


( E ) Binding inhibition of hexameric NS1 to HDL with anti - ApoA - I polyclonal antibodies ( anti - ApoA - I Ab ) measured by BLI .

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(E) Binding inhibition of hexameric NS1 to HDL with anti-ApoA-I polyclonal antibodies (anti-ApoA-I Ab) measured by BLI.


( A - D ) Human macrophages were incubated for 24h with different potential effectors ( NS1 , HDL , mix NS1 - HDL ) or with control suspensions ( PBS buffer , LPS , mix LPS - HDL ) . LPS stimulation was used as a positive control in presence or absence of HDL provided values consistent between experiments . Phosphate buffer used in SEC purification step was used as a negative control . culture supernatants were clarified tested with a Luminex to quantify amount of ( A ) TNF - α , ( B ) Il - 6 , ( C ) Il - 1ß ( D ) Il - 10 released in extracellular medium . Data reported on graphs correspond to biological replicates of macrophages isolated from four blood donors ( n = 9 for TNF - α , Il - 6 Il - 1ß , n = 5 for Il - 10 ) . Data represent mean + / - . A Mann - Whitney was used to assess statistical significance of differences observed between mean cytokine in different culture supernatants . Not significant : ns , * p < 0 . 05 , * * * p < 0 . 001 .

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(A-D) Human primary macrophages were incubated for 24h with the different potential effectors (NS1, HDL, mix NS1-HDL) or with control suspensions (PBS buffer, LPS, mix LPS-HDL). LPS stimulation was used as a positive control in the presence or absence of HDL and provided values consistent between experiments. Phosphate buffer used in the SEC purification step was used as a negative control. Cell culture supernatants were clarified and tested with a Luminex assay to quantify the amount of (A) TNF-α, (B) Il-6, (C) Il-1ß and (D) Il-10 released in the extracellular medium. Data reported on the graphs correspond to biological replicates of macrophages isolated from four blood donors (n=9 for TNF-α, Il-6 and Il-1ß, n=5 for Il-10). Data represent mean +/- SEM. A Mann-Whitney test was used to assess the statistical significance of differences observed between mean cytokine levels in different cell culture supernatants. Not significant: ns, * p < 0.05, *** p < 0.001.


of ( B ) NS1 , ( C ) NS1 - ApoA - I , ( D ) NS1 - ApoE ( E ) NS1 - ApoB NS1 - ApoA - I NS1 - ApoB complexes are representative of NS1 - HDL NS1 - LDL species , respectively , while NS1 - ApoE - positive complexes remain to be fully characterized . Errors bars indicate .

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levels of (B) NS1, (C) NS1-ApoA-I, (D) NS1-ApoE and (E) NS1-ApoB NS1-ApoA-I and NS1-ApoB complexes are representative of NS1-HDL and NS1-LDL complex species, respectively, while the NS1-ApoE-positive complexes remain to be fully characterized. Errors bars indicate SEM.


of ( F ) total cholesterol , ( G ) HDL - cholesterol ( H ) triglycerides . Errors bars indicate .

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levels of (F) total cholesterol, (G) HDL-cholesterol and (H) triglycerides. Errors bars indicate SEM.


( I ) Purified NS1 from different flaviviruses ( yellow fever , YF ; ZIKA ; West Nile , WN ; Japanese encephalitis , JE ; Native Antigen Company ) were spiked in normal human plasma NS1 - ApoA - I complexes further detected by ELISA . Data represent mean values of two technical replicates .

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(I) Purified NS1 from different flaviviruses (yellow fever, YF; ZIKA; West Nile, WN; Japanese encephalitis, JE; The Native Antigen Company) were spiked in normal human plasma and NS1-ApoA-I complexes further detected by ELISA. Data represent the mean values of two technical replicates.


Spearman correlations between expression sensitivity to Plk1 inhibitors ( BI2536 , GSK461364 , BRD - K70511574 , GW - 843682X ) , on data from Cancer Therapeutics Response Portal v2 database expression data derived from MD Anderson Project database ( Li et al , 2017 ) . with expression that correlate with under curve ( AUC ) values for at least two drugs are included in graph . color of data point represent direction significance ( p value ) of correlation . Red indicates a positive correlation with drug resistance green a positive correlation with drug sensitivity ( i . e . , in red are more highly expressed in resistant lines ) .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Spearman correlations between protein expression and sensitivity to Plk1 inhibitors (BI2536, GSK461364, BRD-K70511574, and GW-843682X), based on data from the Cancer Therapeutics Response Portal v2 database and protein expression data derived from the MD Anderson Cell Line Project database (Li et al, 2017). Proteins with expression levels that correlate with area under the curve (AUC) values for at least two drugs are included in the graph. The color and size of the data point represent the direction and significance (p value) of the correlation. Red indicates a positive correlation with drug resistance and green a positive correlation with drug sensitivity (i.e., proteins in red are more highly expressed in resistant cell lines).


Spearman correlations between expression sensitivity to Plk1 inhibitors ( BI2536 GSK461364 AUC values for two Plk1 inhibitors are compared with expression of selected ( cMet , E - cadherin , β - catenin , Snail ) ; each data point represents an non - small lung cancer . E - cadherin , β catenin , cMet expression correlated with drug resistance .

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Spearman correlations between protein expression and sensitivity to Plk1 inhibitors (BI2536 GSK461364 AUC values for two Plk1 inhibitors are compared with expression of selected proteins (cMet, E-cadherin, β-catenin, and Snail); each data point represents an individual non-small cell lung cancer cell line. E-cadherin, β catenin, and cMet protein expression correlated with drug resistance.


Three epithelial / resistant non - small lung cancer lines were treated with 5 ng / ml TGF - β for 14 days to induce a mesenchymal phenotype , which was confirmed with Western blot ( left ) qPCR ( right ) of epithelial - to - mesenchymal transition ( EMT ) markers ( A ) .

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Three epithelial/resistant non-small cell lung cancer cell lines were treated with 5 ng/ml TGF-β for 14 days to induce a mesenchymal phenotype, which was confirmed with Western blot (left) and qPCR (right) analysis of epithelial-to-mesenchymal transition (EMT) markers (A).


Parental TGF - β isogenic lines were treated with 50nM volasertib for 72 hours . were then harvested , lysates were immunoblotted for PARP γH2AX were subsequently quantitated normalized with β - actin ( B ) .

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Parental and TGF-β isogenic cell lines were treated with 50nM volasertib for 72 hours. Cells were then harvested, and lysates were immunoblotted for cleaved PARP and γH2AX proteins were subsequently quantitated and normalized with β-actin (B).


Apoptosis was measured by Apo - BrdU ( C )

Apoptosis was measured by the Apo-BrdU assay (C)


DNA damage was measured by the Comet assay (D).


Thirty - three were differentially regulated between epithelial mesenchymal NSCLC lines after treatment with volasertib , including those involved in cMet / FAK / Src signaling axis ( blue text ) those involved in PI3K / Akt signaling axis ( orange text ) .

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Thirty-three proteins were differentially regulated between epithelial and mesenchymal NSCLC cell lines after treatment with volasertib, including those involved in the cMet/FAK/Src signaling axis (blue text) and those involved in the PI3K/Akt signaling axis (orange text).


same lines treated identically were subjected to immunoblotting for indicated ( upper ) with densitometric normalized with β - actin ( lower ) .

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The same cell lines treated identically were subjected to immunoblotting for the indicated proteins (upper) with densitometric quantification normalized with β-actin (lower).


Epithelial ( red text ) mesenchymal ( blue text ) NSCLC lines transfected with 10nM siRNA as indicated for 48 hours . NT , non - targeting control subjected to Western blotting with indicated antibodies . Data are means ± standard error of mean from three independent experiments . Significant differences using two - sided Student t are indicated by * p < 0 . 05 .

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Epithelial (red text) and mesenchymal (blue text) NSCLC cell lines transfected with 10nM siRNA as indicated for 48 hours. NT, non-targeting control and subjected to Western blotting with the indicated antibodies. Data are means ± standard error of the mean from three independent experiments. Significant differences using two-sided Student t test are indicated by *p < 0.05.


H1975 Calu6 were treated with indicated inhibitors for 4 hours or 24 hours . were then harvested , lysates were immunoblotted for indicated .

H1975 and Calu6 cells were treated with indicated inhibitors for 4 hours or 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


viability was measured using CellTiter - Glo in NSCLC lines treated with volasertib , tepotinib , or a 1 : 2 of both for 72 hours . Left : representative viability graph of treated with indicated drugs . Right : depicting calculated combination indices at Fa = 0 . 25 Fa = 0 . 5 .

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Cell viability was measured using CellTiter-Glo in NSCLC cell lines treated with volasertib, tepotinib, or a 1:2 ratio of both for 72 hours. Left: representative cell viability graph of cells treated with the indicated drugs. Right: heatmap depicting the calculated combination indices at Fa = 0.25 and Fa = 0.5.


Apoptosis was measured using Apo - BrdU in indicated lines treated with indicated drugs for 24 hours .

Apoptosis was measured using the Apo-BrdU assay in the indicated cell lines treated with the indicated drugs for 24 hours.


Immunoblots from treated with indicated drugs for 24 hours ( left ) with densitometric normalized with β - actin ( right ) .

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Immunoblots from cells treated with indicated drugs for 24 hours (left) with densitometric quantification normalized with β-actin (right).


tested lines were treated as indicated for 24 hours allowed to grow in drug - free medium for 15 - 20 days to form colonies , which were counted using ImageJ .

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All tested cell lines were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ.


Apoptosis was measured using Apo - BrdU in indicated lines transfected with 10nM siRNA as indicated for 48 hours . NT , non - targeting control .

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Apoptosis was measured using the Apo-BrdU assay in the indicated cell lines transfected with 10nM siRNA as indicated for 48 hours. NT, non-targeting control.


Immunoblots from transfected with siRNA as indicated for 48 hours ( left ) with densitometric normalized with β - actin ( right ) . Data are means ± standard error of mean from three independent experiments . Significant differences using two - way of variance with Bonferroni or Benjamini - Hochberg ( BH ) correction for multiple comparison are indicated ( * p < 0 . 01 ) . Mesenchymal epithelial NSCLC lines are indicated in blue red text , respectively .

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Immunoblots from cells transfected with siRNA as indicated for 48 hours (left) with densitometric quantification normalized with β-actin (right). Data are means ± standard error of the mean from three independent experiments. Significant differences using two-way analysis of variance with Bonferroni or Benjamini-Hochberg (BH) correction for multiple comparison are indicated (*p < 0.01). Mesenchymal and epithelial NSCLC cell lines are indicated in blue and red text, respectively.


Mice bearing epithelial ( red text ) mesenchymal ( blue text ) non - small lung cancer tumors were treated with vehicle control , volasertib ( 30 mg / kg per week intravenously ) , tepotinib ( 25 mg / kg per day orally ) , or combination for 5 to generate growth curves of - derived xenografts ( PDX ; A ) xenografts change in volume at end of treatment ( normalized to day zero ) was calculated with each data point representing an mouse , bar is mean value ± standard error of 10 mice for each

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Mice bearing epithelial (red text) and mesenchymal (blue text) non-small cell lung cancer tumors were treated with vehicle control, volasertib (30 mg/kg per week intravenously), tepotinib (25 mg/kg per day orally), or the combination for 5 weeks to generate tumor growth curves of patient-derived xenografts (PDX; A) and cell line xenografts The percent change in tumor volume at the end of treatment (normalized to day zero) was calculated with each data point representing an individual mouse, and the bar is the mean value ± standard error of 10 mice for each group


growth curve of mesenchymal PDX TC424 ( top ) xenograft Calu6 ( bottom ) upon treatment for 5 after cessation of treatment ( recovery phase ) . Data are means ± standard error of mean from five mice for each group .

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Tumor growth curve of mesenchymal PDX TC424 (top) and cell line xenograft Calu6 (bottom) upon treatment for 5 weeks and after cessation of treatment (recovery phase). Data are means ± standard error of the mean from five mice for each group.


Kaplan - Meier survival curves for mesenchymal PDX TC424 ( top ) xenograft Calu6 ( bottom ) with due to excessive burden as endpoint .

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Kaplan-Meier survival curves for mesenchymal PDX TC424 (top) and cell line xenograft Calu6 (bottom) with death due to excessive tumor burden as the endpoint.


of TUNEL - positive in paraffin - embedded xenograft tissue sections .

Quantification of TUNEL-positive cells in paraffin-embedded xenograft tissue sections.


Calu6 , H157 , H1355 TPR - Met - expressing or vector control ( pBABE ) - expressing were treated as indicated for 24 hours apoptosis was measured using Apo - BrdU .

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Calu6, H157, and H1355 TPR-Met-expressing or vector control (pBABE)-expressing cells were treated as indicated for 24 hours and apoptosis was measured using the Apo-BrdU assay.


Parental TPR - Met - expressing lines were treated with indicated drugs for 24 hours . were then harvested , lysates were immunoblotted for indicated . β - actin was used as a loading control .

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Parental and TPR-Met-expressing cell lines were treated with the indicated drugs for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. β-actin was used as a loading control.


Parental TPR - Met - expressing lines were treated as indicated for 24 hours allowed to grow in drug - free medium for 15 - 20 days to form colonies , which were counted using ImageJ . Data are means ± standard error of mean from three independent experiments . Significant differences using two - way of variance with Bonferroni or Benjamini - Hochberg correction for multiple comparisons are indicated .

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Parental and TPR-Met-expressing cell lines were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ. Data are means ± standard error of the mean from three independent experiments. Significant differences using two-way analysis of variance with Bonferroni or Benjamini-Hochberg correction for multiple comparisons are indicated.


Mesenchymal NSCLC lines with noted MET alterations were incubated with 50nM volasertib for four hours subjected to immunoblotting with indicated antibodies . * p < 0 . 05 . * * p < 0 . 01 .

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Mesenchymal NSCLC cell lines with the noted MET alterations were incubated with 50nM volasertib for four hours and subjected to immunoblotting with the indicated antibodies. *p < 0.05. **p < 0.01.


viability measured by CellTiter - Glo in Calu6 parental Calu6 - VAR treated with different concentrations of volasertib for 72 hours .

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Cell viability measured by CellTiter-Glo in Calu6 parental and Calu6-VAR cells treated with different concentrations of volasertib for 72 hours.


Micrograph shows acquisition of a mesenchymal - to - epithelial transition phenotype in VAR .

Micrograph shows acquisition of a mesenchymal-to-epithelial transition phenotype in VAR cells.


Basal mRNA expression in parental VAR for indicated was measured using immunoblotting ( upper ) reverse - transcriptase PCR ( lower ) , with GAPDH expression used for normalization .

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Basal protein and mRNA expression in parental and VAR cells for the indicated proteins was measured using immunoblotting (upper) and reverse-transcriptase PCR (lower), with GAPDH expression used for normalization.


Parental VAR were treated as indicated for 48 hours apoptosis was measured using Apo - BrdU .

Parental and VAR cells were treated as indicated for 48 hours and apoptosis was measured using the Apo-BrdU assay.


Parental VAR were treated with indicated drug concentrations for 48 hours . were then harvested , lysates were immunoblotted for indicated .

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Parental and VAR cells were treated with the indicated drug concentrations for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Parental VAR were treated as indicated for 24 hours allowed to grow in drug - free medium for 15 - 20 days to form colonies , which were counted using ImageJ .

Parental and VAR cells were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ.


Epithelial mesenchymal non - small lung cancer ( NSCLC ) lines after treatment with 50nM volasertib for 24 hours . were then harvested , lysates were immunoblotted for indicated .

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Epithelial and mesenchymal non-small cell lung cancer (NSCLC) cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Epithelial mesenchymal NSCLC lines after Plk1 silencing with siRNA for 48 hours . were then harvested , lysates were immunoblotted for indicated .

Epithelial and mesenchymal NSCLC cell lines after Plk1 silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Calu6 H1792 mesenchymal NSCLC lines after VIM silencing with siRNA for 48 hours . were then harvested , lysates were immunoblotted for indicated .

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Calu6 and H1792 mesenchymal NSCLC cell lines after VIM silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Calu6 ( mesenchymal ) H1975 ( epithelial ) NSCLC lines after Plk1 INTB1 silencing with siRNA for 48 hours . were then harvested , lysates were immunoblotted for indicated .

Calu6 (mesenchymal) and H1975 (epithelial) NSCLC cell lines after Plk1 and INTB1 silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Calu6 ( mesenchymal ) H1975 ( epithelial ) NSCLC lines after treatment with fibronectin for 24 hours . were then harvested , lysates were immunoblotted for indicated .

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Calu6 (mesenchymal) and H1975 (epithelial) NSCLC cell lines after treatment with fibronectin for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Calu6 H1975 NSCLC lines after treatment with 50nM volasertib for 24 hours . were then harvested , lysates were immunoprecipitated ( IP ) using β1 - integrin , vimentin , Plk1 antibodies , then immunoblotted for indicated .

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Calu6 and H1975 NSCLC cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoprecipitated (IP) using β1-integrin, vimentin, and Plk1 antibodies, then immunoblotted for the indicated proteins.


Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from CK - p25 mice . Colors indicate fold change relative to control on a log2 - scale . Row colors ( left ) indicate peptides from predicted - type specific . Green = Astrocyte , Orange = Neuron , Blue = Microglia , Purple = Oligodendrocyte .

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Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from CK-p25 animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte.


Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from 5XFAD mice . Colors indicate fold change relative to control on a log2 - scale . Row colors ( left ) indicate peptides from predicted - type specific . Green = Astrocyte , Orange = Neuron , Blue = Microglia , Purple = Oligodendrocyte .

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Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from 5XFAD animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte.


Colors indicate fold change relative to control on a log2 - scale . Row colors ( left ) indicate peptides from predicted - type specific . Green = Astrocyte , Orange = Neuron , Blue = Microglia , Purple = Oligodendrocyte . ( E ) showing enriched hippocampus phosphotyrosine peptides from 6 month old Tau P301S .

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Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte. (E) Heatmap showing enriched hippocampus phosphotyrosine peptides from 6 month old Tau P301S animals.


( F - G ) Relative abundances across tissues for phosphotyrosine peptides : ( F ) SVyTEIK ( Siglec - F pY561 ) ; ( G ) LyDFVK ( Inpp5d pY868 ) . Bars indicate mean ± 95 % confidence interval ( CI ) ; n = 3 - 8 samples ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided .

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(F-G) Relative abundances across all tissues for phosphotyrosine peptides: (F) SVyTEIK (Siglec-F pY561); (G) LyDFVK (Inpp5d pY868). Bars indicate mean ± 95% confidence interval (CI); n = 3-8 samples; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


( A ) Immunofluorescence ( IF ) showing Siglec - F Iba1 localization in hippocampus CA3 region of 3wk induced CK ( top ) or CK - p25 ( bottom ) mice . Scale bars = 50 μm . Colors are : Magenta = Iba1 , Cyan = Siglec - F . are 60x super - resolution max z - stack projections taken from coronal slices . ( B - C ) coverage of ( B ) Iba1 ( C ) Iba1 - proximal Siglec - F between CK CK - p25 mice across hippocampal regions . Bars indicate mean ± 95 % CI ; n = 3 - 4 ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided .

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(A) Immunofluorescence (IF) staining showing Siglec-F and Iba1 localization in hippocampus CA3 region of 3wk induced CK (top) or CK-p25 (bottom) mice. Scale bars = 50 μm. Colors are: Magenta = Iba1, Cyan = Siglec-F. Images are 60x super-resolution max z-stack projections taken from coronal slices. (B-C) Percent area coverage of (B) Iba1 and (C) Iba1-proximal Siglec-F between CK and CK-p25 mice across hippocampal regions. Bars indicate mean ± 95% CI; n = 3-4 animals; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


( D - E ) IF of Siglec - F Iba1 in ( D ) 5XFAD 2 month hypothalamus ( HY ) ( E ) 5XFAD 6 month dentate gyrus ( DG ) . Yellow = Aβ from methoxy X04 ( D ) or D54D2 ( E ) . Scale bars = 50 μm .

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(D-E) IF staining of Siglec-F and Iba1 in (D) 5XFAD 2 month hypothalamus (HY) and (E) 5XFAD 6 month dentate gyrus (DG). Yellow = Aβ from methoxy X04 (D) or D54D2 (E). Scale bars = 50 μm.


( F ) coverage of Iba1 - proximal Siglec - F relative to total Iba1 across analyzed thalamic , cortical , hippocampal regions of 6 month WT ( non - Tg ) 5XFAD mice . Box indicates quartiles whiskers indicate last datum within 1 . 5 inter - quartile range ; n = 15 - 40 ( 3 - 5 ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . ( G ) coverage of Siglec - F localized to Iba1 compared to total Aβ plaque volume in each field of view across analyzed from 5XFAD non - Tg control mice . Linear regression R2 value is shown for with ≥ 10 μm2 total amyloid volume .

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(F) Percent area coverage of Iba1-proximal Siglec-F relative to total Iba1 across all analyzed thalamic, cortical, and hippocampal regions of 6 month WT (non-Tg) and 5XFAD mice. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 15-40 images (3-5 animals); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. (G) Percent area coverage of Siglec-F localized to Iba1 compared to total Aβ plaque volume in each field of view across all analyzed images from 5XFAD and non-Tg control mice. Linear regression R2 value is shown for images with ≥10 μm2 total amyloid volume.


( F ) coverage of Iba1 - proximal Siglec - F relative to total Iba1 across analyzed thalamic , cortical , hippocampal regions of 6 month WT ( non - Tg ) 5XFAD mice . Box indicates quartiles whiskers indicate last datum within 1 . 5 inter - quartile range ; n = 15 - 40 ( 3 - 5 ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided .

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(F) Percent area coverage of Iba1-proximal Siglec-F relative to total Iba1 across all analyzed thalamic, cortical, and hippocampal regions of 6 month WT (non-Tg) and 5XFAD mice. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 15-40 images (3-5 animals); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


( H ) Cumulative distribution function ( CDF ) of between Siglec - F + Iba1 + mask voxels nearest Aβ plaques in 6 month 5XFAD mice . Each value in CDF represents mean calculated from a single z - stack of a ROI containing ≥ 1 plaque of ≥ 10 μm2 . n = 55 ( 4 ) .

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(H) Cumulative distribution function (CDF) of the distance between Siglec-F+ and Iba1+ mask voxels and nearest Aβ plaques in 6 month 5XFAD mice. Each value in the CDF represents the mean distance calculated from a single z-stack of a ROI containing ≥ 1 plaque of size ≥10 μm2. n = 55 images (4 animals).