Volcano plots showing the differential expression of the genes after HNRNPC knock - down in the MCF7 ( A ) and T47D ( B ) cells . The ISGs with significant differential expression were marked as red dots . The cutoffs ( dashed line ) were set at the log2 fold - change > 1 . 5 or < - 1 . 5 and the P - value < 0 . 001 .

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

Volcano plots showing the differential expression of the genes after HNRNPC knock-down in the MCF7 (A) and T47D (B) cells. The ISGs with significant differential expression were marked as red dots. The cutoffs (dashed line) were set at the log2 fold-change > 1.5 or < -1.5 and the P-value < 0.001.


qPCR measurements of the expressions of ISGs upon knock - down of HNRNPC in MCF7 and T47D cells using

qPCR measurements of the expressions of ISGs upon knock-down of HNRNPC in MCF7 and T47D cells using siRNA


qPCR measurements of the expressions of ISGs upon knock - down of HNRNPC in MCF7 and T47D cells using CRISPR -

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qPCR measurements of the expressions of ISGs upon knock-down of HNRNPC in MCF7 and T47D cells using CRISPR-Cas9


11 TFs involved in the interferon response and signaling , which were identified by MARINa as master TFs upon HNRNPC knock - down . In each row , all genes were sorted ( from left to right ) by their differential expressions in siHNRNPC vs . control cells . The predicted target genes that are positive or negative regulated by the TF are marked as red or blue bars . All the TFs were sorted by the P - values ( FDR - corrected ) from the MARINa analysis . Ranks of these 11 TFs among all the master TFs identified by MARINa were provided to the right .

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

11 TFs involved in the interferon response and signaling, which were identified by MARINa as master TFs upon HNRNPC knock-down. In each row, all genes were sorted (from left to right) by their differential expressions in siHNRNPC vs. control cells. The predicted target genes that are positive or negative regulated by the TF are marked as red or blue bars. All the TFs were sorted by the P-values (FDR-corrected) from the MARINa analysis. Ranks of these 11 TFs among all the master TFs identified by MARINa were provided to the right.


Concentrations of IFNβ , measured by ELISA , in the culturing media of MCF7 and T47D cells 48 hours after siRNA transfections . siNC : non - targeting siRNA as a negative control , siHN - 1 : siRNA sequence 1 for HNRNPC , siHN - 2 : siRNA sequence 2 for HNRNPC , siLMNA : siRNA for LMNA as another negative control . Each sample has 3 replicates . Error bars represent mean ± SD .

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

Concentrations of IFNβ, measured by ELISA, in the culturing media of MCF7 and T47D cells 48 hours after siRNA transfections. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.


The normal MCF7 ( left ) and T47D ( right ) cells were cultured in the media collected from the corresponding cells 48 hours after siRNA - mediated gene knock - down . Expressions of the ISGs in these normal cells were measured with qPCR . Each sample has 3 replicates . Error bars represent mean ± SD .

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

The normal MCF7 (left) and T47D (right) cells were cultured in the media collected from the corresponding cells 48 hours after siRNA-mediated gene knock-down. Expressions of the ISGs in these normal cells were measured with qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.


Different doses of the IFNβ antibody were added to the media of the siRNA - transfected MCF7 ( left ) or T47D ( right ) cells right before the media were transferred to the wild type cells . The wild type MCF7 or T47D cells were then cultured in these media for 48 hours , and the expressions of ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD .

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

Different doses of the IFNβ antibody were added to the media of the siRNA-transfected MCF7 (left) or T47D (right) cells right before the media were transferred to the wild type cells. The wild type MCF7 or T47D cells were then cultured in these media for 48 hours, and the expressions of ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.


Growth curves of the MCF7 ( left ) and T47D ( right ) cells cultured in the media collected from the siRNA - transfected cells . Each sample has 3 replicates . Error bars represent mean ± SD .

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Growth curves of the MCF7 (left) and T47D (right) cells cultured in the media collected from the siRNA-transfected cells. Each sample has 3 replicates. Error bars represent mean ± SD.


In the siRNA - transfected MCF7 ( left ) or T47D cells ( right ) , the interferon signaling pathway was blocked at different stages by means of IFNβ neutralization The expressions of the ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD .

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In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of IFNβ neutralization The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.


In the siRNA - transfected MCF7 ( left ) or T47D cells ( right ) , the interferon signaling pathway was blocked at different stages by means of , IFNAR2 neutralization The expressions of the ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD .

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

In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of , IFNAR2 neutralization The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.


In the siRNA - transfected MCF7 ( left ) or T47D cells ( right ) , the interferon signaling pathway was blocked at different stages by means of JAK - STAT inhibition ( C ) . The expressions of the ISGs were measured by qPCR . Each sample has 3 replicates . Error bars represent mean ± SD .

In the siRNA-transfected MCF7 (left) or T47D cells (right), the interferon signaling pathway was blocked at different stages by means of JAK-STAT inhibition (C). The expressions of the ISGs were measured by qPCR. Each sample has 3 replicates. Error bars represent mean ± SD.


Growth curves of the MCF7 ( D ) or T47D cells ( E ) upon HNRNPC knock - down but with the interferon response blocked with the IFNβ antibody . Each sample has 3 replicates . Error bars represent mean ± SD .

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Growth curves of the MCF7 (D) or T47D cells (E) upon HNRNPC knock-down but with the interferon response blocked with the IFNβ antibody. Each sample has 3 replicates. Error bars represent mean ± SD.


Growth curves of the MCF7 ( left ) or T47D ( right ) cells upon HNRNPC knock - down and JAK - STAT inhibition with Ruxolitinib ( 5uM ) . Error bars represent mean ± SD . siNC : non - targeting siRNA as a negative control , siHN - 1 : siRNA sequence 1 for HNRNPC , siHN - 2 : siRNA sequence 2 for HNRNPC . Each sample has 3 replicates . Error bars represent mean ± SD .

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Growth curves of the MCF7 (left) or T47D (right) cells upon HNRNPC knock-down and JAK-STAT inhibition with Ruxolitinib (5uM). Error bars represent mean ± SD. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. Each sample has 3 replicates. Error bars represent mean ± SD.


Expression levels of ISGs ( A ) upon HNRNPC silencing in the cells with lentiviral shRNA - mediated stable knock - down of DDX58 or IFIH1 . Each sample has 3 replicates . Error bars represent mean ± SD .

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Expression levels of ISGs (A) upon HNRNPC silencing in the cells with lentiviral shRNA-mediated stable knock-down of DDX58 or IFIH1. Each sample has 3 replicates. Error bars represent mean ± SD.


Expression levels of IFNβ concentration in the media ( B ) upon HNRNPC silencing in the cells with lentiviral shRNA - mediated stable knock - down of DDX58 or IFIH1 . Each sample has 3 replicates . Error bars represent mean ± SD .

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

Expression levels of IFNβ concentration in the media (B) upon HNRNPC silencing in the cells with lentiviral shRNA-mediated stable knock-down of DDX58 or IFIH1. Each sample has 3 replicates. Error bars represent mean ± SD.


Growth curves of the DDX58 - and IFIH1 - deficient MCF7 ( left ) and T47D ( right ) cells upon HNRNPC knock - down . Each sample has 3 replicates . Error bars represent mean ± SD .

Growth curves of the DDX58- and IFIH1-deficient MCF7 (left) and T47D (right) cells upon HNRNPC knock-down. Each sample has 3 replicates. Error bars represent mean ± SD.


Xenograft tumor models with DDX58 - deficient MCF7 cells . Images , weights , and growth records of the xenograft tumor models in female NSG mice established from the DDX58 - deficient MCF7 cells upon lentivirus - mediated stable knock - down of LMNA or HNRNPC . Each group has 6 mice . The error bars represent the ± SEM .

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

Xenograft tumor models with DDX58-deficient MCF7 cells. Images, weights, and growth records of the xenograft tumor models in female NSG mice established from the DDX58-deficient MCF7 cells upon lentivirus-mediated stable knock-down of LMNA or HNRNPC. Each group has 6 mice. The error bars represent the ± SEM.


Xenograft tumor models with DDX58 - deficient MCF7 cells . Images , weights , and growth records of the xenograft tumors derived from DDX58 - deficient MCF7 cells , which were subjected to periodic siRNA injection , starting from 2 weeks after transplantation of the cells . Each group has 4 mice . The error bars represent the ± SEM .

Xenograft tumor models with DDX58-deficient MCF7 cells. Images, weights, and growth records of the xenograft tumors derived from DDX58-deficient MCF7 cells, which were subjected to periodic siRNA injection, starting from 2 weeks after transplantation of the cells. Each group has 4 mice. The error bars represent the ± SEM.


Immunofluorescence analysis of the dsRNA in MCF7 cells after knock - down of HNRNPC , with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) staining ( blue ) and anti - dsRNA antibody J2 ( green ) . Cells transfected with poly I : C was included as a positive control of dsRNA , and the cells treated with RNase III was used as a negative control . siNC : non - targeting siRNA as a negative control , siHN - 1 : siRNA sequence 1 for HNRNPC , siHN - 2 : siRNA sequence 2 for HNRNPC . The size of scale bar is 10um .

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

Immunofluorescence analysis of the dsRNA in MCF7 cells after knock-down of HNRNPC, with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) and anti-dsRNA antibody J2 (green). Cells transfected with poly I:C was included as a positive control of dsRNA, and the cells treated with RNase III was used as a negative control. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. The size of scale bar is 10um.


Counts of dsRNA regions in the siNC control cells or in the cells with siHNRNPC , identified in the dsRNA - enriched libraries with anti - dsRNA J2 pull - down or in the control libraries including the IgG control and the input control for dsRNA pull - down . Each bar represents the overlapping dsRNA species from two replicate experiments . Among each set of the dsRNA regions , the dsRNAs from Alus were marked in grey .

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Counts of dsRNA regions in the siNC control cells or in the cells with siHNRNPC, identified in the dsRNA-enriched libraries with anti-dsRNA J2 pull-down or in the control libraries including the IgG control and the input control for dsRNA pull-down. Each bar represents the overlapping dsRNA species from two replicate experiments. Among each set of the dsRNA regions, the dsRNAs from Alus were marked in grey.


MA plot showing all the dsRNA regions identified in the dsRNA - enriched libraries . For each dsRNA region , mean of the read counts ( log2 ) for two replicates of siHNRNPC and siNC cells was shown on the x - axis , and the average fold change ( log2 ) in siHNRNPC vs . siNC on the y - axis . The up - regulated dsRNA regions were marked in red and down - regulated regions in black .

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

MA plot showing all the dsRNA regions identified in the dsRNA-enriched libraries. For each dsRNA region, mean of the read counts (log2) for two replicates of siHNRNPC and siNC cells was shown on the x-axis, and the average fold change (log2) in siHNRNPC vs. siNC on the y-axis. The up-regulated dsRNA regions were marked in red and down-regulated regions in black.


( A ) Immunofluorescence labeling of FTSJ1 - Flag ( red ) in HEK293T cells . The nucleus was stained by DAPI ( blue ) . Scale bar , 20 μm .

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(A) Immunofluorescence labeling of FTSJ1-Flag (red) in HEK293T cells. The nucleus was stained by DAPI (blue). Scale bar, 20 μm.


( B ) Subcellular localization of FTSJ1 . Cytoplasmic ( C ) and nuclear ( N ) fractions were separated from HEK293T cells expressing FTSJ1 - Flag . α - Tubulin and Lamin A / C were used as indicators of the cytoplasmic and nuclear fractions , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Subcellular localization of FTSJ1. Cytoplasmic (C) and nuclear (N) fractions were separated from HEK293T cells expressing FTSJ1-Flag. α-Tubulin and Lamin A/C were used as indicators of the cytoplasmic and nuclear fractions, respectively.


( C ) Co - IP assays of the interaction between FTSJ1 and WDR6 in co - transfected HEK293T cells . Anti - HA IP ( top ) and anti - Flag IP ( middle ) were immunoblotted by anti - HA and anti - Flag antibodies , respectively . Bottom : cell lysates immunoblotted by anti - HA or anti - Flag antibodies , with GAPDH serving as a loading control .

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(C) Co-IP assays of the interaction between FTSJ1 and WDR6 in co-transfected HEK293T cells. Anti-HA IP (top) and anti-Flag IP (middle) were immunoblotted by anti-HA and anti-Flag antibodies, respectively. Bottom: cell lysates immunoblotted by anti-HA or anti-Flag antibodies, with GAPDH serving as a loading control.


( D ) Co - IP assays of the interaction between FTSJ1 and WDR6 with RNase A addition .

(D) Co-IP assays of the interaction between FTSJ1 and WDR6 with RNase A addition.


B , C ) Co - IP assays of the interaction between FTSJ1 - Flag or truncated FTSJ1 mutants and WDR6 - HA in co - transfected HEK293T cells . Anti - Flag IP ( top ) were respectively immunoblotted by anti - HA and anti - Flag antibodies . Bottom : cell lysates immunoblotted by anti - HA or anti - Flag antibodies , with GAPDH serving as a loading control .

B, C) Co-IP assays of the interaction between FTSJ1-Flag or truncated FTSJ1 mutants and WDR6-HA in co-transfected HEK293T cells. Anti-Flag IP (top) were respectively immunoblotted by anti-HA and anti-Flag antibodies. Bottom: cell lysates immunoblotted by anti-HA or anti-Flag antibodies, with GAPDH serving as a loading control.


E ) IP assays of FTSJ1 - Asn221DIZPK . The empty vector was transfected as a negative control . ( F ) A list of top FTSJ1 - Asn221DIZPK putative interacting proteins identified by IP - MS in HEK293T cells .

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E) IP assays of FTSJ1-Asn221DIZPK. The empty vector was transfected as a negative control. (F) A list of top FTSJ1-Asn221DIZPK putative interacting proteins identified by IP-MS in HEK293T cells.


Mass chromatograms of the nucleosides , Cm ( Q1 / Q3 = 258 . 1 / 112 . 1 ) , Gm ( Q1 / Q3 = 298 . 1 / 152 . 1 ) and A ( Q1 / Q3 = 268 . 1 / 136 . 2 ) of tRNAPhe ( GAA ) isolated from WT , ftsj1 KO and wdr6 KO cells . Target peaks are indicated by black triangles . Cm , 2 ' - O - methylcytidine ; Gm , 2 ' - O - methylguanosine ; Q1 / Q3 : the mass of the precursor ion and the mass of the product ion .

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

Mass chromatograms of the nucleosides, Cm (Q1/Q3=258.1/112.1), Gm (Q1/Q3=298.1/152.1) and A (Q1/Q3=268.1/136.2) of tRNAPhe(GAA) isolated from WT, ftsj1 KO and wdr6 KO cells. Target peaks are indicated by black triangles. Cm, 2'-O-methylcytidine; Gm, 2'-O-methylguanosine; Q1/Q3: the mass of the precursor ion and the mass of the product ion.


Mass chromatograms of the nucleosides m1G ( Q1 / Q3 = 298 . 1 / 166 . 1 ) , m7G ( Q1 / Q3 = 298 . 1 / 166 . 1 ) and m2G ( Q1 / Q3 = 298 . 1 / 166 . 1 ) , o2yW ( Q1 / Q3 = 541 . 2 / 409 . 0 ) , m5C ( Q1 / Q3 = 258 . 1 / 126 . 1 ) and A ( Q1 / Q3 = 268 . 1 / 136 . 2 ) of tRNAPhe ( GAA ) isolated from WT , ftsj1 KO and wdr6 KO cells . Target peaks are indicated by black triangles . m2G , 2 - methylguanosine ; D , dihydrouridine ; m22G , N2 , N2 - dimethylguanosine ; m5C , 5 - methylcytidine ; m7G , 7 - methylguanosine ; m5U , 5 - methyluridine ; m1A : 1 - methyladenosine ; ψ : pseudouridine ; o2yW , peroxywybutosine . Q1 / Q3 : the mass of the precursor ion and the mass of the product ion . Combined the retention time of standard product , we marked the o2yW in Figure 3G . Considering the change of this peak area was consistent with that of o2yW , we speculated the other peak was generated by the intermediate product of o2yW with nature isotope labelled .

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

Mass chromatograms of the nucleosides m1G (Q1/Q3=298.1/166.1), m7G (Q1/Q3=298.1/166.1) and m2G (Q1/Q3=298.1/166.1), o2yW (Q1/Q3=541.2/409.0), m5C (Q1/Q3=258.1/126.1) and A (Q1/Q3=268.1/136.2) of tRNAPhe(GAA) isolated from WT, ftsj1 KO and wdr6 KO cells. Target peaks are indicated by black triangles. m2G, 2-methylguanosine; D, dihydrouridine; m22G, N2,N2-dimethylguanosine; m5C, 5-methylcytidine; m7G, 7-methylguanosine; m5U, 5-methyluridine; m1A: 1-methyladenosine; ψ: pseudouridine; o2yW, peroxywybutosine. Q1/Q3: the mass of the precursor ion and the mass of the product ion. Combined the retention time of standard product, we marked the o2yW in Figure 3G. Considering the change of this peak area was consistent with that of o2yW, we speculated the other peak was generated by the intermediate product of o2yW with nature isotope labelled.


( B , C ) Quantification of the Cm / A and Gm / A of tRNAPhe ( GAA ) , △ ftsj1 _ tRNAPhe ( GAA ) , and m1G37 _ tRNAPhe ( GAA ) after incubation with FTSJ1 , WDR6 , and FTSJ1 - WDR6 by UPLC - MS / MS analysis , respectively . Error bars represent the standard deviation of three independent experiments . n . d . , not detected .

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(B, C) Quantification of the Cm/A and Gm/A of tRNAPhe(GAA), △ftsj1_tRNAPhe(GAA), and m1G37_tRNAPhe(GAA) after incubation with FTSJ1, WDR6, and FTSJ1-WDR6 by UPLC-MS/MS analysis, respectively. Error bars represent the standard deviation of three independent experiments. n.d., not detected.


( D ) UPLC - MS / MS analysis of 2 ' - O - methylation of m1G37 _ tRNAPhe ( GAA ) , - G34A , - G34C and G34U after incubation with FTSJ1 - WDR6 . 2 μL of Gm ( 1 ng / mL ) , Am ( 1 ng / mL ) , Cm ( 1 ng / mL ) and Um ( 5 ng / mL ) standards were injected to UPLC - MS / MS as control . cps , counts per second .

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(D) UPLC-MS/MS analysis of 2'-O-methylation of m1G37_tRNAPhe(GAA), -G34A, -G34C and G34U after incubation with FTSJ1-WDR6. 2 μL of Gm (1 ng/mL), Am (1 ng/mL), Cm (1 ng/mL) and Um (5 ng/mL) standards were injected to UPLC-MS/MS as control. cps, counts per second.


A ) The SAM binding affinity of FTSJ1 as measured by ITC .

A) The SAM binding affinity of FTSJ1 as measured by ITC.


( B The binding affinity of FTSJ1 alone or FTSJ1 - WDR6 for tRNA analyzed by the gel mobility shift assay .

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(B The binding affinity of FTSJ1 alone or FTSJ1-WDR6 for tRNA analyzed by the gel mobility shift assay.


C ) For the reaction in figure C , 0 . 2 μΜ GST , 0 . 2 μΜ GST - WDR6 , 1 μM FTSJ1 , the mixture of 1 μM FTSJ1 with 0 . 2 μΜ GST - WDR6 or 0 . 2 μΜ GST , 0 . 5 mM SAM and 250 nM tRNAPhe ( GAA ) transcript were incubated .

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C) For the reaction in figure C, 0.2 μΜ GST, 0.2 μΜ GST-WDR6, 1 μM FTSJ1, the mixture of 1 μM FTSJ1 with 0.2 μΜ GST-WDR6 or 0.2 μΜ GST, 0.5 mM SAM and 250 nM tRNAPhe(GAA) transcript were incubated.


The representative graphs for quantification of the expected Nm32 / A % and Nm34 / A % ratios of G37 - tRNAPhe ( GAA ) , tRNALeu ( UAA ) , tRNATrp ( CCA ) , tRNAArg ( ACG ) , tRNAArg ( UCG ) , and A37 - tRNAMet ( CAU ) , which were isolated from WT , ftsj1 KO , and wdr6 KO cells by UPLC - MS / MS analysis . Error bars represent the standard deviation of three independent experiments . p values were determined using two - tailed Student ' s t - test for paired samples . * * * p < 0 . 001 . n . s . , no significance . n . d . , not detected .

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

The representative graphs for quantification of the expected Nm32/A% and Nm34/A% ratios of G37-tRNAPhe(GAA), tRNALeu(UAA), tRNATrp(CCA), tRNAArg(ACG), tRNAArg(UCG), and A37-tRNAMet(CAU), which were isolated from WT, ftsj1 KO, and wdr6 KO cells by UPLC-MS/MS analysis. Error bars represent the standard deviation of three independent experiments. p values were determined using two-tailed Student's t-test for paired samples. ***p < 0.001. n.s., no significance. n.d., not detected.


( A , B ) The growth curve of WT and two ftsj1 KO HEK293T cell lines under normal culture or in the culture condition with 0 . 2 mg / mL paromomycin assayed by cell counting kit - 8 proliferation analysis .

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(A, B) The growth curve of WT and two ftsj1 KO HEK293T cell lines under normal culture or in the culture condition with 0.2 mg/mL paromomycin assayed by cell counting kit-8 proliferation analysis.


( C , D ) The growth curve of WT with or without overexpressing tRNAPhe ( GAA ) under normal culture or with 0 . 2 mg / mL paromomycin .

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(C, D) The growth curve of WT with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin.


( E , F ) The growth curve of ftsj1 KO cells with or without overexpressing tRNAPhe ( GAA ) under normal culture or with 0 . 2 mg / mL paromomycin .

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(E, F) The growth curve of ftsj1 KO cells with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin.


( H , I ) The translation efficiency of F - luc with the 6 × UUC codons or 6 × UUU codons .

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

(H, I) The translation efficiency of F-luc with the 6×UUC codons or 6×UUU codons.


( J , K ) The translation efficiency of F - luc with all UUC codons or all UUU codons .

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

(J, K) The translation efficiency of F-luc with all UUC codons or all UUU codons.


( C ) Co - immunoprecipitation using BmN4 cells transfected with the Flag - tagged DEAD - box helicases identified by shotgun proteomics . The Flag - tagged proteins and Ago3 were detected by western blotting . Full - length Flag - tagged proteins are indicated with red asterisks . h . c . : heavy chain .

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

(C) Co-immunoprecipitation using BmN4 cells transfected with the Flag-tagged DEAD-box helicases identified by shotgun proteomics. The Flag-tagged proteins and Ago3 were detected by western blotting. Full-length Flag-tagged proteins are indicated with red asterisks. h.c.: heavy chain.


( F , G ) Analysis of Siwi - piRNA and Ago3 - piRNA levels in Siwi and Ago3 complexes with DDX43 , respectively . The WB ( western blotting ) and NB ( northern blotting ) signals represent the PIWI protein and piRNA levels , respectively ( see also Figs EV1A and EV1B ) . The relative ratios of piRISC were calculated based on the signal intensities of the PIWI proteins and piRNAs .

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

(F, G) Analysis of Siwi-piRNA and Ago3-piRNA levels in Siwi and Ago3 complexes with DDX43, respectively. The WB (western blotting) and NB (northern blotting) signals represent the PIWI protein and piRNA levels, respectively (see also Figs EV1A and EV1B). The relative ratios of piRISC were calculated based on the signal intensities of the PIWI proteins and piRNAs.


( B ) Immunoprecipitation and western blotting showing that DDX43 associated with Vret and Ago3 .

(B) Immunoprecipitation and western blotting showing that DDX43 associated with Vret and Ago3.


( C , D ) Immunoprecipitation of endogenous Siwi , DDX43 , and Vret bound with Ago3 from DDX43 - depleted ( C ) or Vret - depleted ( D ) cells . The factors were detected by western blotting . Ago3 interacts with Vret and DDX43 independently of the other .

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

(C, D) Immunoprecipitation of endogenous Siwi, DDX43, and Vret bound with Ago3 from DDX43-depleted (C) or Vret-depleted (D) cells. The factors were detected by western blotting. Ago3 interacts with Vret and DDX43 independently of the other.


( D ) RNA unwinding assays upon Ago3 - piRISC - dependent target RNA cleavage Recombinant Vasa or DDX43 was added and incubated in the reaction mixture upon cleavage and the materials were separated using a magnet to supernatant ( Sup . ) and bead ( Beads ) fractions . RNAs cleaved by Ago3 are shown as " Cleaved RNA " . DDX43 but not Vasa released the cleaved target RNA from Ago3 - piRISC .

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

(D) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage Recombinant Vasa or DDX43 was added and incubated in the reaction mixture upon cleavage and the materials were separated using a magnet to supernatant (Sup.) and bead (Beads) fractions. RNAs cleaved by Ago3 are shown as "Cleaved RNA". DDX43 but not Vasa released the cleaved target RNA from Ago3-piRISC.


( F ) RNA unwinding assays upon Ago3 - piRISC - dependent target RNA cleavage as in ( D ) . Recombinant DDX43 was added and incubated in the reaction mixture upon cleavage for 0 min ( i . e . , no cleavage ) , 15 min , 30 min , and 60 min , and the materials were separated using a magnet to supernatant ( Sup . ) and bead ( Beads ) fractions . RNAs cleaved by Ago3 are shown as " Cleaved RNA " . DDX43 fails to unwind cleaved RNAs from Siwi - piRISC . − DDX43 means no DDX43 added in the reaction mixture .

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

(F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage as in (D). Recombinant DDX43 was added and incubated in the reaction mixture upon cleavage for 0 min (i.e., no cleavage), 15 min, 30 min, and 60 min, and the materials were separated using a magnet to supernatant (Sup.) and bead (Beads) fractions. RNAs cleaved by Ago3 are shown as "Cleaved RNA". DDX43 fails to unwind cleaved RNAs from Siwi-piRISC. − DDX43 means no DDX43 added in the reaction mixture.


( C ) Immunoprecipitation of Ago3 with Flag - tagged DDX43 WT and its D399A and E400Q mutants . Ago3 and DDX43 were detected by western blotting using anti - Ago3 and anti - Flag - antibodies , respectively . Flag - EGFP was used as a negative control . The E400Q mutation in DDX43 impairs the interaction between Ago3 and DDX43 . IgG h . c . : IgG heavy chain .

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

(C) Immunoprecipitation of Ago3 with Flag-tagged DDX43 WT and its D399A and E400Q mutants. Ago3 and DDX43 were detected by western blotting using anti-Ago3 and anti-Flag-antibodies, respectively. Flag-EGFP was used as a negative control. The E400Q mutation in DDX43 impairs the interaction between Ago3 and DDX43. IgG h.c.: IgG heavy chain.


( F ) RNA unwinding assays upon Ago3 - piRISC - dependent target RNA cleavage . Unwinding activities of DDX43 WT and its D399A , E400Q , and ∆ KH mutants are shown . RNAs cleaved by Ago3 are shown as " Cleaved RNA " .

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

(F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage. Unwinding activities of DDX43 WT and its D399A, E400Q, and ∆KH mutants are shown. RNAs cleaved by Ago3 are shown as "Cleaved RNA".


C ) ELISA binding results of mAbs specific for hRSV F ( left panels ) or hMPV F ( right panels ) with the proteins indicated at left . mAbs specific of hRSV F bind epitopes of antigenic site II ( Mota and 47F ) or antigenic site IV ( 101F ) . mAs specific of hMPV F bind epitopes of antigenic site II ( MF9 , MF12 , MF14 and MF15 ) or antigenic site IV ( MF16 ) .

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

C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind epitopes of antigenic site II (Mota and 47F) or antigenic site IV (101F). mAs specific of hMPV F bind epitopes of antigenic site II (MF9, MF12, MF14 and MF15) or antigenic site IV (MF16).


Binding of Mz and 47F Fabs to the immobilized proteins indicated at left . Sensorgrams of eight different concentrations are shown in each panel . The calculated binding parameters are shown in the Table . RUs , response units .

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

Binding of Mz and 47F Fabs to the immobilized proteins indicated at left. Sensorgrams of eight different concentrations are shown in each panel. The calculated binding parameters are shown in the Table. RUs, response units.


Groups of BALB / c female mice ( n = 5 ) were inoculated twice ( four weeks apart ) i . m . with 10 µg / dose of the proteins indicated at the top of each panel , folded in their respective postfusion conformations . One week after the last dose , mice were sacrificed and their blood collected . A ) Each mouse serum ( identified by a number ) was tested in ELISA for antibody binding to the postfusion F proteins indicated at the bottom . The results represented in the y axis for each individual mouse serum correspond to the inverse dilution that gave 50 % of maximal binding , expressed in log10 units ( EC50 ( log10 ) ) . Each number represents an individual mouse . Short horizontal bars indicate mean values for each group . P values were calculated as indicated under Statistical Analysis . Only relevant P values are shown . * P = 0 . 00045 . Differences were considered significant when P < 0 . 05 .

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Groups of BALB/c female mice (n = 5) were inoculated twice (four weeks apart) i.m. with 10 µg/dose of the proteins indicated at the top of each panel, folded in their respective postfusion conformations. One week after the last dose, mice were sacrificed and their blood collected. A) Each mouse serum (identified by a number) was tested in ELISA for antibody binding to the postfusion F proteins indicated at the bottom. The results represented in the y axis for each individual mouse serum correspond to the inverse dilution that gave 50% of maximal binding, expressed in log10 units (EC50 (log10)). Each number represents an individual mouse. Short horizontal bars indicate mean values for each group. P values were calculated as indicated under Statistical Analysis. Only relevant P values are shown. *P = 0.00045. Differences were considered significant when P < 0.05.


Groups of BALB / c female mice ( n = 5 ) were inoculated twice ( four weeks apart ) i . m . with 10 µg / dose of the proteins indicated at the top of each panel , folded in their respective postfusion conformations . One week after the last dose , mice were sacrificed and their blood collected . B ) Mouse sera were also tested in microneutralization assays with hRSV and hMPV , as indicated at the bottom . The results represented in the y axis correspond to the inverse dilution that inhibited 50 % of the viral infectivity ( IC50 ( log10 ) ) . P values : * P = 0 . 01001 and * * P = 0 . 00271 . Long horizontal lines in A and B indicate detection limits .

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

Groups of BALB/c female mice (n = 5) were inoculated twice (four weeks apart) i.m. with 10 µg/dose of the proteins indicated at the top of each panel, folded in their respective postfusion conformations. One week after the last dose, mice were sacrificed and their blood collected. B) Mouse sera were also tested in microneutralization assays with hRSV and hMPV, as indicated at the bottom. The results represented in the y axis correspond to the inverse dilution that inhibited 50% of the viral infectivity (IC50 (log10)). P values: *P = 0.01001 and **P = 0.00271. Long horizontal lines in A and B indicate detection limits.


Groups of BALB / c mice ( n = 5 ) were inoculated three times , four weeks apart , with 20 µg of the postfusion F proteins indicated above each panel . One group ( n = 5 ) was inoculated only with the CpG adjuvant , as indicated . Two other groups of mice ( n = 8 ) were similarly inoculated with either F - 415 or F - 416 , as indicated at top of the panels . A ) Mouse sera , collected just before challenging with hRSV ( A2 strain ) , were used in microneutralization assays against the viruses indicated at bottom . Statistical results are shown as in Fig . 3B . * P = 0 . 00085 and * * P = 0 . 01986 .

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

Groups of BALB/c mice (n = 5) were inoculated three times, four weeks apart, with 20 µg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. A) Mouse sera, collected just before challenging with hRSV (A2 strain), were used in microneutralization assays against the viruses indicated at bottom. Statistical results are shown as in Fig. 3B. *P = 0.00085 and **P = 0.01986.


Groups of BALB / c mice ( n = 5 ) were inoculated three times , four weeks apart , with 20 µg of the postfusion F proteins indicated above each panel . One group ( n = 5 ) was inoculated only with the CpG adjuvant , as indicated . Two other groups of mice ( n = 8 ) were similarly inoculated with either F - 415 or F - 416 , as indicated at top of the panels . B ) Five days after virus inoculation , mice were sacrificed and the amount of virus in lung extracts was measured in a plaque assay . Mean virus titer for each group is indicated by short horizontal bar .

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

Groups of BALB/c mice (n = 5) were inoculated three times, four weeks apart, with 20 µg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. B) Five days after virus inoculation, mice were sacrificed and the amount of virus in lung extracts was measured in a plaque assay. Mean virus titer for each group is indicated by short horizontal bar.


C ) ELISA binding results of mAbs specific for hRSV F ( left panels ) or hMPV F ( right panels ) with the proteins indicated at left . mAbs specific of hRSV F bind to epitopes of antigenic site Ø ( D25 ) , antigenic site II ( Mz and 47F ) or antigenic site IV ( 101F and 107F ) . mAs specific of hMPV F bind to epitopes of antigenic site II ( MF14 ) , antigenic site IV ( MF11 , MF16 and MF29 ) or an epitope shared by hRSV F and hMPV F ( MPE8 ) .

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

C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind to epitopes of antigenic site Ø (D25), antigenic site II (Mz and 47F) or antigenic site IV (101F and 107F). mAs specific of hMPV F bind to epitopes of antigenic site II (MF14), antigenic site IV (MF11, MF16 and MF29) or an epitope shared by hRSV F and hMPV F (MPE8).


F . Groups of BALB / c female mice ( n = 5 ) were inoculated twice with 10 µg of the proteins indicated at the top of each panel , folded in either the prefusion conformation for hRSV F , F - 410 and F - 411 or in the postfusion conformation for hMPV F . One week after the last dose , mice were sacrificed and their blood collected . A ) Each mouse serum was tested in ELISA for antibody binding to the F proteins indicated at the bottom of each panel . * P = 0 . 00046 and * * P = 0 . 02459 .

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

F. Groups of BALB/c female mice (n = 5) were inoculated twice with 10 µg of the proteins indicated at the top of each panel, folded in either the prefusion conformation for hRSV F, F-410 and F-411 or in the postfusion conformation for hMPV F. One week after the last dose, mice were sacrificed and their blood collected. A) Each mouse serum was tested in ELISA for antibody binding to the F proteins indicated at the bottom of each panel. *P = 0.00046 and **P = 0.02459.


Groups of BALB / c female mice ( n = 5 ) were inoculated twice with 10 µg of the proteins indicated at the top of each panel , folded in either the prefusion conformation for hRSV F , F - 410 and F - 411 or in the postfusion conformation for hMPV F . One week after the last dose , mice were sacrificed and their blood collected . B ) Mouse sera were also tested in microneutralization assays with hRSV and hMPV , indicated at the bottom . * P = 0 . 04138 , * * P = 0 . 04831 and * * * P = 0 . 00339 . The results in A and B are presented as in Fig . 3 .

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

Groups of BALB/c female mice (n = 5) were inoculated twice with 10 µg of the proteins indicated at the top of each panel, folded in either the prefusion conformation for hRSV F, F-410 and F-411 or in the postfusion conformation for hMPV F. One week after the last dose, mice were sacrificed and their blood collected. B) Mouse sera were also tested in microneutralization assays with hRSV and hMPV, indicated at the bottom. *P = 0.04138, **P = 0.04831 and ***P = 0.00339. The results in A and B are presented as in Fig. 3.


( B and C ) IC50 values for MPA - response curves using cell viability assay were shown . Human AML cell lines ( with MLL - fusions : MV4 ; 11 , MOLM13 , NOMO1 and THP1 , without MLL - fusions : HL60 , Kasumi - 1 , OCI - AML3 and U937 ) , human cord blood ( CB ) CD34 + cells , and those expressing MLL - AF9 ( CB - MLL - AF9 # 1 , 2 , 3 ) or MLL - ENL ( CB # MLL - ENL - 1 , 2 ) , and patient cells with or without MLL - fusions ( AML # 1 , 2 , 3 or AML # 4 , 5 , 6 ) were treated with titrating doses of MPA ( 1 - 100 μM ) for 72 hours in three technical replicates . Data information : All data are shown as mean ± SEM .

O O O O O O O O B-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 O O O O O O O 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-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

(B and C) IC50 values for MPA-response curves using cell viability assay were shown. Human AML cell lines (with MLL-fusions: MV4;11, MOLM13, NOMO1 and THP1, without MLL-fusions: HL60, Kasumi-1, OCI-AML3 and U937), human cord blood (CB) CD34+ cells, and those expressing MLL-AF9 (CB-MLL-AF9#1, 2, 3) or MLL-ENL (CB#MLL-ENL-1, 2), and patient cells with or without MLL-fusions (AML#1, 2, 3 or AML#4, 5, 6) were treated with titrating doses of MPA (1-100 μM) for 72 hours in three technical replicates. Data information: All data are shown as mean ± SEM.


( D ) Response curves of FF - 10501 - 01 ( 1 - 100 μM ) for 72 hours in three technical replicates .

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(D) Response curves of FF-10501-01 (1-100 μM) for 72 hours in three technical replicates.


( E ) FCM plots of the cell cycle and apoptosis analyses in MLL - AF9 expressing CB cells and MV4 ; 11 cells . Cells were incubated with / without 1 μM MPA and 100 mM Guanosine for 36 hours . Numbers indicate the frequency of S / G2 / M - phase cells ( left ) and Annexin V + cells ( right ) .

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(E) FCM plots of the cell cycle and apoptosis analyses in MLL-AF9 expressing CB cells and MV4;11 cells. Cells were incubated with/without 1 μM MPA and 100 mM Guanosine for 36 hours. Numbers indicate the frequency of S/G2/M-phase cells (left) and Annexin V+ cells (right).


( F and G ) Immunophenotypes and Wright - Giemsa staining of MLL - AF9 expressing CB cells after four days of culture with / without 3 . 3 μM MPA and 100 μM Guanosine ( Scale bar : 20 mm ) .

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(F and G) Immunophenotypes and Wright-Giemsa staining of MLL-AF9 expressing CB cells after four days of culture with/without 3.3 μM MPA and 100 μM Guanosine (Scale bar: 20 mm).


( H ) MLL - ENL - expressing CB cells and MOLM13 cells were transduced with Cas9 together with non - targeting ( NT ) sgRNA or IMPDH2 - targeting sgRNAs ( IMPDH2 - KO1 and KO2 ) co - expressing tRFP657 . Changes in the frequency of tRFP657 + cells ( sgRNA - transduced cells ) in cell cultures are shown . Results are normalized to the frequency of tRFP657 + cells at day 4 , set to 1 .

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

(H) MLL-ENL-expressing CB cells and MOLM13 cells were transduced with Cas9 together with non-targeting (NT) sgRNA or IMPDH2-targeting sgRNAs (IMPDH2-KO1 and KO2) co-expressing tRFP657. Changes in the frequency of tRFP657+ cells (sgRNA-transduced cells) in cell cultures are shown. Results are normalized to the frequency of tRFP657+ cells at day 4, set to 1.


( I ) The tRFP657 + cells in ( H ) were sorted and subjected to western blotting . The depletion of IMPDH2 protein in MLL - ENL cells and MOLM13 cells was confirmed by western blotting .

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

(I) The tRFP657+ cells in (H) were sorted and subjected to western blotting. The depletion of IMPDH2 protein in MLL-ENL cells and MOLM13 cells was confirmed by western blotting.


( A ) Immunoblotting of the cord blood cells and those expressing RUNX1 - ETO or MLL - AF9 . The cells were incubated with / without 1 μM MPA ( M ) and 100 μM Guanosine ( G ) for 24 h .

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(A) Immunoblotting of the cord blood cells and those expressing RUNX1-ETO or MLL-AF9. The cells were incubated with/without 1 μM MPA (M) and 100 μM Guanosine (G) for 24 h.


( B ) CB - MLL - AF9 # 2 cells were transduced with a Non - targeting ( NT ) control and an shRNA targeting p21 ( sh - p21 ) . Cells were incubated with / without 1 μM MPA ( M ) and 100 μM guanosine ( G ) for 24 hours . Total cell lysates were analyzed by western blotting using the antibodies for p21 and Tubulin .

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(B) CB-MLL-AF9#2 cells were transduced with a Non-targeting (NT) control and an shRNA targeting p21 (sh-p21). Cells were incubated with/without 1 μM MPA (M) and 100 μM guanosine (G) for 24 hours. Total cell lysates were analyzed by western blotting using the antibodies for p21 and Tubulin.


( C ) CB - MLL - AF9 # 2 cells transduced with NT or sh - p21 shRNAs were incubated with MPA at the indicated concentration for 72 hours . Cell viability assays were performed using WST - 1 in three technical replicates .

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(C) CB-MLL-AF9#2 cells transduced with NT or sh-p21 shRNAs were incubated with MPA at the indicated concentration for 72 hours. Cell viability assays were performed using WST-1 in three technical replicates.


( D ) CB - MLL - AF9 # 2 cells were transduced by vector control or p53DD ( dominant - negative ) and were incubated with / without 1 μM MPA ( M ) and 100 μM guanosine ( G ) for 24 h . Total cell lysates were analyzed by western blotting using the antibodies for p53 and Tubulin . Note the elevated levels of endogenous p53 protein ( upper arrow ) in p53DD ( lower arrow ) - expressing cells .

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(D) CB-MLL-AF9#2 cells were transduced by vector control or p53DD (dominant-negative) and were incubated with/without 1 μM MPA (M) and 100 μM guanosine (G) for 24 h. Total cell lysates were analyzed by western blotting using the antibodies for p53 and Tubulin. Note the elevated levels of endogenous p53 protein (upper arrow) in p53DD (lower arrow)-expressing cells.


( E ) Cells were incubated with MPA at the indicated concentration with / without 100 μM guanosine for 72 hours then measured with WST - 1 in three technical replicates .

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(E) Cells were incubated with MPA at the indicated concentration with/without 100 μM guanosine for 72 hours then measured with WST-1 in three technical replicates.


( F ) Mouse bone marrow c - Kit + cells derived from Trp53 - / - mice were transduced with MLL - AF9 - GFP and were transplanted into mice to generate p53 - deficient ( p53 - / - ) leukemia cells . GFP + MLL - AF9 leukemia cells were collected from bone marrows of vehicle - or FF - 10501 - 01 - treated wild - type and p53 - / - leukemic mice . Expression levels of p53 and Tubulin were assessed by western blotting 24 hours after the treatment .

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(F) Mouse bone marrow c-Kit+ cells derived from Trp53-/- mice were transduced with MLL-AF9-GFP and were transplanted into mice to generate p53-deficient (p53-/-) leukemia cells. GFP+ MLL-AF9 leukemia cells were collected from bone marrows of vehicle- or FF-10501-01-treated wild-type and p53-/- leukemic mice. Expression levels of p53 and Tubulin were assessed by western blotting 24 hours after the treatment.


( H ) Kaplan - Meier survival curves of p53 - / - MLL - AF9 leukemia mice treated with vehicle or FF - 10501 - 01or DS - 5272 ( n = 6 per group ) . Statistical significance was evaluated by the log - rank test ( left ) . Frequency of GFP + leukemia cells in peripheral blood at day 19 ( right , n = 6 per group ) . A two - tailed unpaired t - test was used for the comparison . Data information : All data are shown as mean ± SEM .

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(H) Kaplan-Meier survival curves of p53-/- MLL-AF9 leukemia mice treated with vehicle or FF-10501-01or DS-5272 (n = 6 per group). Statistical significance was evaluated by the log-rank test (left). Frequency of GFP+ leukemia cells in peripheral blood at day 19 (right, n = 6 per group). A two-tailed unpaired t-test was used for the comparison. Data information: All data are shown as mean ± SEM.


( B ) The TLR - expressing BaκB cells were stimulated with the corresponding TLR ligands ( 100 ng / mL Pam3CSK4 for TLR2 , 100 ng / mL LPS for TLR4 , 10 ng / mL Flagellin for TLR5 , 100 ng / ml R848 for TLR7 ) with titrating doses of MPA ( 1 - 10 μM ) . GFP expression was assessed 24 hours after the stimulation .

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(B) The TLR-expressing BaκB cells were stimulated with the corresponding TLR ligands (100 ng/mL Pam3CSK4 for TLR2, 100 ng/mL LPS for TLR4, 10 ng/mL Flagellin for TLR5, 100 ng/ml R848 for TLR7) with titrating doses of MPA (1 - 10 μM). GFP expression was assessed 24 hours after the stimulation.


( C ) 293T cells were transfected with FLAG - tagged TRAF6 and HA - tagged K63 - ubiquitin . After 24 hours , these cells were treated with 3 or 10 μM MPA ( M - 3 μM or M - 10 μM ) with / without 100 μM guanosine ( G ) . IRAK1 / 4 inhibitor ( IRAK1 / 4 i ) was also used as a control . Whole - cell extracts were immunoprecipitated with anti - FLAG antibody , and ubiquitinated TRAF6 was detected with anti - HA antibody .

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(C) 293T cells were transfected with FLAG-tagged TRAF6 and HA-tagged K63-ubiquitin. After 24 hours, these cells were treated with 3 or 10 μM MPA (M-3 μM or M-10 μM) with/without 100 μM guanosine (G). IRAK1/4 inhibitor (IRAK1/4 i) was also used as a control. Whole-cell extracts were immunoprecipitated with anti-FLAG antibody, and ubiquitinated TRAF6 was detected with anti-HA antibody.


( D , Murine bone marrow - derived macrophages ( BMDMs ) were treated with vehicle , 10 μM MPA alone ( D ) N = 3 ( technical replicates ) per group . mRNA levels of TNF - α and IL - 1β were measured by qPCR . Two - tailed unpaired t - tests were used for the comparison in ( D ) . Data information : All data are shown as mean ± SEM .

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(D, Murine bone marrow-derived macrophages (BMDMs) were treated with vehicle, 10 μM MPA alone (D) N=3 (technical replicates) per group. mRNA levels of TNF-α and IL-1β were measured by qPCR. Two-tailed unpaired t-tests were used for the comparison in (D). Data information: All data are shown as mean ± SEM.


E ) Murine bone marrow - derived macrophages ( BMDMs ) were treated with vehicle , 10 μM MPA alone or co - treated with vehicle , 10 μM MPA ( M ) , 100 ng / mL Pam3CSK4 and 100 μM Guanosine ( G ) for 4 hours ( E ) , as indicated . N = 3 ( technical replicates ) per group . mRNA levels of TNF - α and IL - 1β were measured by qPCR . Ordinary one - way ANOVA was used for the comparison in ( E ) . Data information : All data are shown as mean ± SEM .

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E) Murine bone marrow-derived macrophages (BMDMs) were treated with vehicle, 10 μM MPA alone or co-treated with vehicle, 10 μM MPA (M), 100 ng/mL Pam3CSK4 and 100 μM Guanosine (G) for 4 hours (E), as indicated. N=3 (technical replicates) per group. mRNA levels of TNF-α and IL-1β were measured by qPCR. Ordinary one-way ANOVA was used for the comparison in (E). Data information: All data are shown as mean ± SEM.


( F ) THP1 cells were pretreated with 10 μM MPA for 2 hours and then treated with 1000 ng / mL Pam3CSK4 for 0 , 30 , 60 , and ( 120 ) minutes . Cells were lysed and subjected to SDS - PAGE . Levels of total IκBα , phosphate - p38 , total p38 , and GAPDH were evaluated by western blotting .

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(F) THP1 cells were pretreated with 10 μM MPA for 2 hours and then treated with 1000 ng/mL Pam3CSK4 for 0, 30, 60, and (120) minutes. Cells were lysed and subjected to SDS-PAGE. Levels of total IκBα, phosphate-p38, total p38, and GAPDH were evaluated by western blotting.


( G ) MLL - AF9 - expressing CB cells ( CB - MLL - AF9 # 1 ) were pretreated with 10 μM MPA for 2 hours and then treated with 1000 ng / mL Pam3CSK4 for 0 , 30 , 60 Cells were lysed and subjected to SDS - PAGE . Levels of total IκBα , phosphate - p38 , total p38 were evaluated by western blotting .

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(G) MLL-AF9-expressing CB cells (CB-MLL-AF9#1) were pretreated with 10 μM MPA for 2 hours and then treated with 1000 ng/mL Pam3CSK4 for 0, 30, 60 Cells were lysed and subjected to SDS-PAGE. Levels of total IκBα, phosphate-p38, total p38 were evaluated by western blotting.


( A ) RT - qPCR quantification of relative miR - 21 - 3p and miR - 21 - 5p levels in mouse brain , heart , kidney and epidermis . N = 4 animals per group , one representative experiment is shown out of three independent replicates .

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(A) RT-qPCR quantification of relative miR-21-3p and miR-21-5p levels in mouse brain, heart, kidney and epidermis. N=4 animals per group, one representative experiment is shown out of three independent replicates.


( B ) Fluorescent miR - 21 - 3p in situ hybridization ( pink ) in dorsal skin of acutely irradiated ( Ac - UV ) and non - irradiated ( no UV ) Ppard + / + and - / - mice . E : epidermis ; D : dermis ; HF : hair follicle . Magnification bar :

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(B) Fluorescent miR-21-3p in situ hybridization (pink) in dorsal skin of acutely irradiated (Ac-UV) and non-irradiated (no UV) Ppard +/+ and -/- mice. E: epidermis; D: dermis; HF: hair follicle. Magnification bar: 100m


( C ) RT - qPCR quantification of relative pri - miR - 21 and miR - 21 - 3p levels in the epidermis ( left and middle ) , and of relative miR - 21 - 3p in the dermis ( right ) of acutely irradiated ( Ac - UV ; + ) and non - irradiated ( - ) Ppard + / + and - / - mice . Pri - miR - 21 : Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 022 ; miR - 21 - 3p : Ppard + / + no UV vs Ppard + / + Ac - UV P = 0 . 017 , Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 008 . N = 3 - 4 animals per group , one representative experiment is shown out of three independent 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 O O O O O O O O O O

(C) RT-qPCR quantification of relative pri-miR-21 and miR-21-3p levels in the epidermis (left and middle), and of relative miR-21-3p in the dermis (right) of acutely irradiated (Ac-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. Pri-miR-21: Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.022; miR-21-3p: Ppard +/+ no UV vs Ppard +/+ Ac-UV P = 0.017, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.008. N=3-4 animals per group, one representative experiment is shown out of three independent replicates.


( D ) RT - qPCR quantification of relative miR - 21 - 3p levels in total skin of chronically irradiated ( Chr - UV ; + ) and non - irradiated ( - ) Ppard + / + and - / - mice . miR - 21 - 3p : Ppard + / + no UV vs Ppard + / + Chr - UV P = 0 . 008 , Ppard + / + Chr - UV vs Ppard - / - Chr - UV P = 0 . 002 , ns : non significant . N = 4 animals per groups , one representative experiment is shown out of two independent replicates .

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(D) RT-qPCR quantification of relative miR-21-3p levels in total skin of chronically irradiated (Chr-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. miR-21-3p: Ppard +/+ no UV vs Ppard +/+ Chr-UV P = 0.008, Ppard +/+ Chr-UV vs Ppard -/- Chr-UV P = 0.002, ns: non significant. N=4 animals per groups, one representative experiment is shown out of two independent replicates.


( E ) RT - qPCR quantification of relative miR - 21 - 3p level in the epidermis of Ppard + / + and - / - mice , acutely irradiated ( Ac - UV ; + ) or non - irradiated ( - ) , treated with the PPAR / antagonist GSK0660 ( + ) or vehicle ( - ) , as indicated . miR - 21 - 3p : Ppard + / + no UV vs Ppard + / + Ac - UV P = 0 . 005 , Ppard + / + Ac - UV vs Ppard + / + Ac - UV / GSK0660 P = 0 . 04 , N = 5 ( Ppard + / + ) to 3 ( Ppard - / - ) animals per group . One representative experiment is shown out of two independent

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(E) RT-qPCR quantification of relative miR-21-3p level in the epidermis of Ppard +/+ and -/-mice, acutely irradiated (Ac-UV; +) or non-irradiated (-), treated with the PPAR/ antagonist GSK0660 (+) or vehicle (-), as indicated. miR-21-3p: Ppard +/+ no UV vs Ppard +/+ Ac-UV P = 0.005, Ppard +/+ Ac-UV vs Ppard +/+ Ac-UV/GSK0660 P = 0.04, N=5 (Ppard +/+) to 3 (Ppard -/-) animals per group. One representative experiment is shown out of two independent replicates


( F ) RT - qPCR quantification of relative miR - 21 - 3p levels in HaCat cells treated with the PPAR / agonists GW501516 , GW0742 ( + ) or vehicle ( - ) as indicated . miR - 21 - 3p : Veh vs GW501516 P = 4E - 04 , Veh vs GW0742 P = 2E - 04 . N = 2 - 3 biological replicates , one representative experiment is shown out of two independent replicates .

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(F) RT-qPCR quantification of relative miR-21-3p levels in HaCat cells treated with the PPAR/ agonists GW501516, GW0742 (+) or vehicle (-) as indicated. miR-21-3p: Veh vs GW501516 P = 4E-04, Veh vs GW0742 P = 2E-04. N=2-3 biological replicates, one representative experiment is shown out of two independent replicates.


( A ) RT - qPCR quantification of relative pri - miR - 21 level in HaCaT human keratinocytes treated with the PPAR / agonist GW0742 ( + ) or vehicle ( - ) , with ( + ) or without ( - ) cycloheximide ( Cyclo ) as indicated . Pri - miR - 21 : Veh vs GW0742 P = 0 . 009 . N = 3 biological replicates , one representative experiment is shown out of two independent replicates .

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(A) RT-qPCR quantification of relative pri-miR-21 level in HaCaT human keratinocytes treated with the PPAR/ agonist GW0742 (+) or vehicle (-), with (+) or without (-) cycloheximide (Cyclo) as indicated. Pri-miR-21: Veh vs GW0742 P = 0.009. N=3 biological replicates, one representative experiment is shown out of two independent replicates.


( B ) RT - qPCR quantification of relative pri - miR - 21 , pre - miR - 21 , miR - 21 - 5p and miR - 21 - 3p levels in HaCaT cells treated for 24h with 2 or 5ng / ml of recombinant human TGFβ - 1 ( + ) or vehicle ( - ) as indicated . Pri - miR - 21 : Veh vs TGF - 1 5ng / ml P = 0 . 029 ; Pre - miR - 21 : Veh vs TGF - 1 5ng / ml P = 0 . 001 ; miR - 21 - 5p : Veh vs TGF - 1 5ng / ml P = 0 . 002 ; miR - 21 - 3p : Veh vs TGF - 1 5ng / ml P = 1 . 7E - 05 . N = 3 biological replicates , one representative experiment is shown out of two independent 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 O O O O O O

(B) RT-qPCR quantification of relative pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in HaCaT cells treated for 24h with 2 or 5ng/ml of recombinant human TGFβ-1 (+) or vehicle (-) as indicated. Pri-miR-21: Veh vs TGF-1 5ng/ml P = 0.029; Pre-miR-21: Veh vs TGF-1 5ng/ml P = 0.001; miR-21-5p: Veh vs TGF-1 5ng/ml P = 0.002; miR-21-3p: Veh vs TGF-1 5ng/ml P = 1.7E-05. N=3 biological replicates, one representative experiment is shown out of two independent replicates .


( C ) RT - qPCR quantification of pri - miR - 21 , pre - miR - 21 , miR - 21 - 5p and miR - 21 - 3p levels in HaCat cells treated for 24h with the PPAR / agonist GW0742 ( + ) , TGFβ receptor inhibitor SB431542 ( + ) , or vehicle ( - ) as indicated . Pri - miR - 21 : GW0742 vs SB431542 P = 0 . 004 , GW0742 vs GW0742 / SB431542 P = 0 . 015 ; Pre - miR - 21 : Veh vs GW0742 P = 0 . 022 , GW0742 vs SB431542 P = 0 . 036 , GW0742 vs GW0742 / SB431542 P = 0 . 024 ; miR - 21 - 5p : GW0742 vs SB431542 P = 0 . 034 , GW0742 vs GW0742 / SB431542 P = 0 . 024 ; miR - 21 - 3p : Veh vs GW0742 P = 0 . 036 , GW0742 vs SB431542 P = 0 . 011 , GW0742 vs GW0742 / SB431542 P = 0 . 008 . N = 3 biological replicates , one representative experiment is shown out of two independent replicates .

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(C) RT-qPCR quantification of pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in HaCat cells treated for 24h with the PPAR/ agonist GW0742 (+), TGFβ receptor inhibitor SB431542 (+), or vehicle (-) as indicated. Pri-miR-21: GW0742 vs SB431542 P = 0.004, GW0742 vs GW0742/SB431542 P = 0.015; Pre-miR-21: Veh vs GW0742 P = 0.022, GW0742 vs SB431542 P = 0.036, GW0742 vs GW0742/SB431542 P = 0.024; miR-21-5p: GW0742 vs SB431542 P = 0.034, GW0742 vs GW0742/SB431542 P = 0.024; miR-21-3p: Veh vs GW0742 P = 0.036, GW0742 vs SB431542 P = 0.011, GW0742 vs GW0742/SB431542 P = 0.008. N=3 biological replicates, one representative experiment is shown out of two independent replicates.


( D ) RT - qPCR quantification of relative Tgfb1 level in the epidermis of acutely irradiated ( Ac - UV ) and non - irradiated Ppard + / + and - / - mice . Tgfb1 : Ppard + / + no UV vs Ac - UV P = 0 . 034 , Ppard + / + Ac - UV vs Ppard Ac - UV P = 0 . 030 . N = 2 - 3 animals per group , one representative experiment is shown out of three independent replicates .

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(D) RT-qPCR quantification of relative Tgfb1 level in the epidermis of acutely irradiated (Ac-UV) and non-irradiated Ppard +/+ and -/- mice. Tgfb1: Ppard +/+ no UV vs Ac-UV P= 0.034, Ppard +/+ Ac-UV vs Ppard Ac-UV P= 0.030. N=2-3 animals per group, one representative experiment is shown out of three independent replicates.


( E ) RT - qPCR quantification of relative miR - 21 - 5p and miR - 21 - 3p levels in the skin of Ppard + / + mice treated with the TGFβ receptor inhibitor SB431542 ( + ) or vehicle ( - ) , with ( + ) or without ( - ) acute UV exposure ( Ac - UV ) . miR - 21 - 5p : no UV vs Ac - UV P = 0 . 038 , Ac UV vs Ac UV / SB431542 P = 0 . 028 ; miR - 21 - 3p : no UV vs Ac - UV P = 0 . 017 ; Ac - UV vs Ac - UV / SB431542 P = 0 . 035 . N = 3 animals per group , one representative experiment is shown out of three independent replicates .

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(E) RT-qPCR quantification of relative miR-21-5p and miR-21-3p levels in the skin of Ppard +/+ mice treated with the TGFβ receptor inhibitor SB431542 (+) or vehicle (-), with (+) or without (-) acute UV exposure (Ac-UV). miR-21-5p: no UV vs Ac-UV P = 0.038, Ac UV vs Ac UV/SB431542 P = 0.028; miR-21-3p: no UV vs Ac-UV P = 0.017; Ac-UV vs Ac-UV/SB431542 P = 0.035. N=3 animals per group, one representative experiment is shown out of three independent replicates.


( A ) RT - qPCR quantification of relative SMAD7 expression level in the epidermis of acutely irradiated ( Ac - UV ; + ) ex vivo biopsies of human normal skin . SMAD7 : no UV vs Ac - UV P = 0 . 032 . N = 4 biological replicates , one representative experiment is shown out of three independent experiments performed with the skin of three different donors .

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

(A) RT-qPCR quantification of relative SMAD7 expression level in the epidermis of acutely irradiated (Ac-UV; +) ex vivo biopsies of human normal skin. SMAD7: no UV vs Ac-UV P = 0.032. N=4 biological replicates, one representative experiment is shown out of three independent experiments performed with the skin of three different donors.


( B ) Left panel : wild - type ( WT SMAD7 3 ' UTR ) and mutated ( MUT SMAD7 3 ' UTR ; * : mutated nucleotides ) miR - 21 - 3p binding sequences in the humanSMAD7 3 ' UTR . Right panel : Luciferase reporter assay with wild - type ( WT SMAD7 3 ' UTR ) or mutated ( MUT SMAD7 3 ' UTR ) SMAD7 3 ' UTR in HEK293 cells overexpressing miR - 21 - 3p ( miR - 21 - 3p mimic ) or a scrambled sequence ( Control ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: wild-type (WT SMAD7 3'UTR) and mutated (MUT SMAD7 3'UTR; *: mutated nucleotides) miR-21-3p binding sequences in the humanSMAD7 3'UTR. Right panel: Luciferase reporter assay with wild-type (WT SMAD7 3'UTR) or mutated (MUT SMAD7 3'UTR) SMAD7 3'UTR in HEK293 cells overexpressing miR-21-3p (miR-21-3p mimic) or a scrambled sequence (Control).


( C ) Left panel : normalized expression data of SMAD7 mRNA obtained from genomic microarray analysis of human HaCaT cells treated with a miR - 21 - 3p mimic ( miR - 21 - 3p mimic ) or a scrambled sequence ( control ) . N = 3 biological replicates . Middle panel : western blot quantification of SMAD7 protein level ( normalized to GAPDH protein level ) in human HaCaT cells treated with a miR - 21 - 3p mimic ( miR - 21 - 3p mimic ) or a scrambled sequence ( control ) . SMAD7 : control vs miR - 21 - 3p mimic P = 0 . 031 . N = 3 biological replicates , Right panel : western blot of SMAD7 and GAPDH proteins in human HaCaT cells treated with a miR - 21 - 3p mimic ( miR - 21 - 3p mimic ) or a scrambled sequence ( control ) , N = 3 biological replicates ; one representative experiment is shown out of two independent 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 O O O O O O O O O O O O O O O O O O O O O

(C) Left panel: normalized expression data of SMAD7 mRNA obtained from genomic microarray analysis of human HaCaT cells treated with a miR-21-3p mimic (miR-21-3p mimic) or a scrambled sequence (control). N=3 biological replicates. Middle panel: western blot quantification of SMAD7 protein level (normalized to GAPDH protein level) in human HaCaT cells treated with a miR-21-3p mimic (miR-21-3p mimic) or a scrambled sequence (control). SMAD7: control vs miR-21-3p mimic P = 0.031. N=3 biological replicates, Right panel: western blot of SMAD7 and GAPDH proteins in human HaCaT cells treated with a miR-21-3p mimic (miR-21-3p mimic) or a scrambled sequence (control), N=3 biological replicates; one representative experiment is shown out of two independent replicates.


( D ) Western blot of Smad7 from epidermis of acutely irradiated ( Ac - UV ; + ) Ppard + / + and - / - mice . Loading control : GAPDH .

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

(D) Western blot of Smad7 from epidermis of acutely irradiated (Ac-UV; +) Ppard +/+ and -/- mice. Loading control: GAPDH.


( E ) RT - qPCR quantification of relative TGFB1 , SERPINE1 , p21 , RUNX and SNAI2 levels in human HaCaT cells treated with a miR - 21 - 3p mimic ( miR - 21 - 3p mimic ) or a scrambled sequence ( control ) with ( + ) or without ( - ) treatment with 2ng / ml recombinant TGFβ - 1 . SERPINE1 : TGF - 1 2ng / ml control vs miR - 21 - 3p mimic P = 0 . 004 ; p21 : TGF - 1 2ng / ml control vs miR - 21 - 3p mimic P = 0 . 03 ; RUNX : TGF - 1 2ng / ml control vs miR - 21 - 3p mimic P = 0 . 04 . N = 3 biological 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

(E) RT-qPCR quantification of relative TGFB1, SERPINE1, p21, RUNX and SNAI2 levels in human HaCaT cells treated with a miR-21-3p mimic (miR-21-3p mimic) or a scrambled sequence (control) with (+) or without (-) treatment with 2ng/ml recombinant TGFβ-1. SERPINE1: TGF-1 2ng/ml control vs miR-21-3p mimic P = 0.004; p21: TGF-1 2ng/ml control vs miR-21-3p mimic P =0.03; RUNX: TGF-1 2ng/ml control vs miR-21-3p mimic P = 0.04. N=3 biological replicates.


( C ) RT - qPCR analysis of IL6 , IL1B , IL1RAP , PTGS2 , CCL5 , CXCL10 and CASP14 levels in HaCat cells transfected with a miR - 21 - 3p mimic or a scrambled sequence ( control ) . IL6 : control vs miR - 21 - 3p mimic P = 0 . 05 ; IL1B : control vs miR - 21 - 3p mimic P = 0 . 005 ; IL1RAP : control vs miR - 21 - 3p mimic P = 0 . 008 ; PTGS2 : control vs miR - 21 - 3p mimic P = 0 . 050 ; CCL5 : control vs miR - 21 - 3p mimic P = 0 . 001 ; CXCL10 : control vs miR - 21 - 3p mimic P = 0 . 012 ; CASP14 : control vs miR - 21 - 3p mimic P = 9E - 05 . N = 3 biological replicates , one representative experiment is shown out of three independent 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 O O O O O O O O O O O O O O O O

(C) RT-qPCR analysis of IL6, IL1B, IL1RAP, PTGS2, CCL5, CXCL10 and CASP14 levels in HaCat cells transfected with a miR-21-3p mimic or a scrambled sequence (control). IL6: control vs miR-21-3p mimic P = 0.05; IL1B: control vs miR-21-3p mimic P = 0.005; IL1RAP: control vs miR-21-3p mimic P = 0.008; PTGS2: control vs miR-21-3p mimic P = 0.050; CCL5: control vs miR-21-3p mimic P = 0.001; CXCL10: control vs miR-21-3p mimic P = 0.012; CASP14: control vs miR-21-3p mimic P = 9E-05. N=3 biological replicates, one representative experiment is shown out of three independent replicates.


( A ) RT - qPCR quantification of Il6 , Il1b , Ptgs2 and Ccl5 levels in the epidermis of acutely irradiated ( Ac - UV ; + ) and non - irradiated ( - ) Ppard + / + and - / - mice . Il6 : Ppard + / + no UV vs Ac - UV P = 7E - 04 , Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 008 ; Il1b : Ppard + / + no UV vs Ac - UV P = 0 . 010 , Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 016 , Ppard - / - no UV vs Ac - UV P = 0 . 015 ; Ptgs2 : Ppard + / + no UV vs Ac - UV P = 0 . 028 , Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 028 , Ppard - / - no UV vs Ac - UV P = 8E - 04 ; Ccl5 : Ppard + / + no UV vs Ac - UV P = 0 . 002 , Ppard + / + Ac - UV vs Ppard - / - Ac - UV P = 0 . 014 , Ppard - / - no UV vs Ac - UV P = 0 . 010 ; ns : non significant . N = 4 animals per group , one representative experiment is shown out of three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) RT-qPCR quantification of Il6, Il1b, Ptgs2 and Ccl5 levels in the epidermis of acutely irradiated (Ac-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. Il6: Ppard +/+ no UV vs Ac-UV P = 7E-04, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.008; Il1b: Ppard +/+ no UV vs Ac-UV P = 0.010, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.016, Ppard -/- no UV vs Ac-UV P = 0.015; Ptgs2: Ppard +/+ no UV vs Ac-UV P = 0.028, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.028, Ppard -/- no UV vs Ac-UV P = 8E-04; Ccl5: Ppard +/+ no UV vs Ac-UV P = 0.002, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.014, Ppard -/- no UV vs Ac-UV P = 0.010; ns: non significant. N=4 animals per group, one representative experiment is shown out of three independent replicates.


( B ) Left panel : Immunostaining for infiltrating F4 / 80 - positive macrophages in acutely irradiated ( Ac - UV ) and non - irradiated ( no UV ) Ppard + / + and - / - miceskin sections . Magnification bar : 200m . Right panel : RT - qPCR quantification of relative levels of the macrophage marker Emr1 mRNA in total skin of acutely irradiated ( Ac - UV ; + ) and non - irradiated ( - ) Ppard + / + and - / - mice . Emr : Ppard + / + no UV vs Ac UV P = 0 . 034 ; Ppard + / + Ac UV vs Ppard - / - Ac UV P = 0 . 033 . N = 4 animals per group , one representative experiment is shown out of three independent 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

(B) Left panel: Immunostaining for infiltrating F4/80-positive macrophages in acutely irradiated (Ac-UV) and non-irradiated (no UV) Ppard +/+ and -/- miceskin sections. Magnification bar: 200m. Right panel: RT-qPCR quantification of relative levels of the macrophage marker Emr1 mRNA in total skin of acutely irradiated (Ac-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. Emr: Ppard +/+ no UV vs Ac UV P = 0.034; Ppard +/+ Ac UV vs Ppard -/- Ac UV P = 0.033. N=4 animals per group, one representative experiment is shown out of three independent replicates.


( C ) RT - qPCR quantification of relative levels of Il6 , Il1b , Ptgs2 and Tnfa in mouseepidermis acutely irradiated ( Ac - UV ; + ) and non - irradiated ( - ) , treated with miR - 21 - 3p inhibitor ( + ) or mismatched control ( - ) as indicated .

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

(C) RT-qPCR quantification of relative levels of Il6, Il1b, Ptgs2 and Tnfa in mouseepidermis acutely irradiated (Ac-UV; +) and non-irradiated (-), treated with miR-21-3p inhibitor (+) or mismatched control (-) as indicated.


( A ) RT - qPCR quantification of relative levels of pri - miR - 21 , pre - miR - 21 , miR - 21 - 5p and miR - 21 - 3p levels in healthy human skin and human squamous cell carcinomas ( SCC ) . pri - miR - 21 Healthy skin vs SCC P = 0 . 043 ; pre - miR - 21 Healthy skin vs SCC P = 3E - 05 ; miR - 21 - 5p Healthy skin vs SCC P = 7E - 05 ; miR - 21 - 3p Healthy skin vs SCC P = 1 . 4E - 06 . ≥ 5 independent biopsies per condition .

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

(A) RT-qPCR quantification of relative levels of pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in healthy human skin and human squamous cell carcinomas (SCC). pri-miR-21 Healthy skin vs SCC P = 0.043; pre-miR-21 Healthy skin vs SCC P = 3E-05; miR-21-5p Healthy skin vs SCC P = 7E-05; miR-21-3p Healthy skin vs SCC P = 1.4E-06. ≥ 5 independent biopsies per condition.


( B ) RT - qPCR quantification of relative levels of pri - miR - 21 , pre - miR - 21 , miR - 21 - 5p and miR - 21 - 3p levels in healthy human skin and humanpsoriasis lesions . pri - miR - 21 Healthy skin vs Psoriasis P = 0 . 034 ; miR - 21 - 5p Healthy skin vs Psoriasis P = 0 . 034 ; miR - 21 - 3p Healthy skin vs Psoriasis P = 0 . 036 ; ns : non significant , ≥ 4 independent biopsies per condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR quantification of relative levels of pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in healthy human skin and humanpsoriasis lesions. pri-miR-21 Healthy skin vs Psoriasis P = 0.034; miR-21-5p Healthy skin vs Psoriasis P = 0.034; miR-21-3p Healthy skin vs Psoriasis P = 0.036; ns: non significant, ≥ 4 independent biopsies per condition.


( C ) RT - qPCR quantification of relative levels of IL6 , IL1B , PTGS2 and TNFA levels in epidermis of abdominal healthy humanskin biopsies exposed to acute UV ex vivo , with or without topical treatment with miR - 21 - 3p inhibitor ( + ) or mismatched control ( - ) as indicated . ns : not significant . IL6 : no UV vs Ac - UV P = 0 . 036 , Ac - UV vs Ac - UV / miR - 21 - 3p inhibitor P = 0 . 019 ; IL1B : no UV vs Ac - UV P = 0 . 002 , Ac - UV vs Ac - UV / miR - 21 - 3p inhibitor P = 0 . 023 ; PTGS2 : no UV vs Ac - UV P = 0 . 008 , Ac - UV vs Ac - UV / miR - 21 - 3p inhibitor P = 0 . 022 ; TNFA : no UV vs Ac - UV P = 0 . 032 , ns = non significant . ≥ 7 replicates , a pool of three independent experiments performed with the skin of three different donors is shown .

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

(C) RT-qPCR quantification of relative levels of IL6, IL1B, PTGS2 and TNFA levels in epidermis of abdominal healthy humanskin biopsies exposed to acute UV ex vivo, with or without topical treatment with miR-21-3p inhibitor (+) or mismatched control (-) as indicated. ns: not significant. IL6: no UV vs Ac-UV P = 0.036, Ac-UV vs Ac-UV/miR-21-3p inhibitor P = 0.019; IL1B: no UV vs Ac-UV P = 0.002, Ac-UV vs Ac-UV/miR-21-3p inhibitor P = 0.023; PTGS2: no UV vs Ac-UV P = 0.008, Ac-UV vs Ac-UV/miR-21-3p inhibitor P = 0.022; TNFA: no UV vs Ac-UV P = 0.032, ns = non significant. ≥ 7 replicates, a pool of three independent experiments performed with the skin of three different donors is shown.


( B ) Representative eTLCs ( left ) and corresponding elongation kinetics ( right ) for the CGA - CGA inhibitory pair ( red ) and the CGC optimal pair ( green ) . Product formation is normalized to the fraction of Met ICs that form Met - Puro when reacted with puromycin ( Fig EV1A ) .

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

(B) Representative eTLCs (left) and corresponding elongation kinetics (right) for the CGA-CGA inhibitory pair (red) and the CGC optimal pair (green). Product formation is normalized to the fraction of Met ICs that form Met-Puro when reacted with puromycin (Fig EV1A).


( C ) Representative eTLCs ( left ) and corresponding elongation kinetics ( right ) for the CGA - CCG inhibitory pair ( red ) and the CGC - CCA optimal pair ( green ) . Product formation is normalized to the fraction of Met ICs that form Met - Puro when reacted with puromycin ( Fig EV1A ) .

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

(C) Representative eTLCs (left) and corresponding elongation kinetics (right) for the CGA-CCG inhibitory pair (red) and the CGC-CCA optimal pair (green). Product formation is normalized to the fraction of Met ICs that form Met-Puro when reacted with puromycin (Fig EV1A).


( A ) Comparison of observed rates of elongation for inhibitory pairs ( red ) and their optimal controls ( green ) at limiting tRNA concentrations ( hatched bars , 15 to 25 nM aa - tRNA ) and saturating tRNA concentrations ( solid bars , 150 to 250 nM aa - tRNA ) .

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

(A) Comparison of observed rates of elongation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA).


( B ) Comparison of total peptide formation for inhibitory pairs ( red ) and their optimal controls ( green ) at limiting tRNA concentrations ( hatched bars , 15 to 25 nM aa - tRNA ) and saturating tRNA concentrations ( solid bars , 150 to 250 nM aa - tRNA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of total peptide formation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA).


( C ) Elongation kinetics for the CGA - CGA inhibitory codon pair with the native arginine ICGtRNA ( red ) or the non - native arginine UCGtRNA ( pink ) and for the CGC - CGC optimal control pair with the native arginine ICGtRNA ( green ) .

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

(C) Elongation kinetics for the CGA-CGA inhibitory codon pair with the native arginine ICGtRNA (red) or the non-native arginine UCGtRNA (pink) and for the CGC-CGC optimal control pair with the native arginine ICGtRNA (green).


( A ) Meta - analysis of footprint size of all 17 inhibitory pairs identified aligning the first codon of the pair in the ribosomal P site .

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

(A) Meta-analysis of footprint size of all 17 inhibitory pairs identified aligning the first codon of the pair in the ribosomal P site.


( B ) Metacodon analysis of 21 nt RPFs centered at the first codon of each inhibitory pair ( red ) compared to their corresponding optimal pair ( green ) . P - values were determined by Student ' s t - test between each inhibitory and the corresponding optimal pair ( * * p < 0 . 01 , n = 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Metacodon analysis of 21 nt RPFs centered at the first codon of each inhibitory pair (red) compared to their corresponding optimal pair (green). P-values were determined by Student's t-test between each inhibitory and the corresponding optimal pair (** p < 0.01, n=2).


( C ) Comparison of 21 nt RPFs aligned at all 17 inhibitory codons from libraries made with CHX / ANS ( blue ) and CHX / TIG ( black ) ( left ) to their corresponding optimal pairs with the same antibiotic combination ( right ) . No significant difference found ( n . s . : p = 0 . 9961 , n = 2 ) between CHX - TIG and CHX - ANS .

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

(C) Comparison of 21 nt RPFs aligned at all 17 inhibitory codons from libraries made with CHX/ANS (blue) and CHX/TIG (black) (left) to their corresponding optimal pairs with the same antibiotic combination (right). No significant difference found (n.s.: p=0.9961, n=2) between CHX-TIG and CHX-ANS.


( A ) Relative mRNA levels of FX in CD45 + leukocytes in the peripheral blood of E0771 ( abbreviated to E ) or LLC tumour - bearing mice . The mean sizes of E0771 and LLC tumours were 9 . 8 mm and 9 . 5 mm , respectively . Shown are averages ( N = 8 mice / group ) with SEM and one - way ANOVA

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

(A) Relative mRNA levels of FX in CD45+ leukocytes in the peripheral blood of E0771 (abbreviated to E) or LLC tumour-bearing mice. The mean sizes of E0771 and LLC tumours were 9.8 mm and 9.5 mm, respectively. Shown are averages (N = 8 mice/group) with SEM and one-way ANOVA


( B ) Relative mRNA levels of FX in CD45 + leukocytes in various organs such as lung ( Lu ) , Liver ( Li ) , spleen ( Sp ) , bone marrow ( BM ) and lymph node ( Lymph ) derived from no tumour - bearing or E0771 - bearing mice . " Tumour " stands for mRNA levels of FX in E0771 tumours . Shown are averages ( N = 6 : organs from no tumour - bearing mice , N = 6 : organs from E0771 - bearing mice ) with SEM and one - way

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative mRNA levels of FX in CD45+ leukocytes in various organs such as lung (Lu), Liver (Li), spleen (Sp), bone marrow (BM) and lymph node (Lymph) derived from no tumour-bearing or E0771-bearing mice. "Tumour" stands for mRNA levels of FX in E0771 tumours. Shown are averages (N = 6: organs from no tumour-bearing mice, N = 6: organs from E0771-bearing mice) with SEM and one-way ANOVA


( C ) Immunohistochemical quantifications of FX expression in CD45 + leukocytes in the liver and lungs of no tumour - bearing or E0771 - bearing mice . Shown are averages ( N = 6 ) with SEM and Welch " s t - test

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

(C) Immunohistochemical quantifications of FX expression in CD45+ leukocytes in the liver and lungs of no tumour-bearing or E0771-bearing mice. Shown are averages (N = 6) with SEM and Welch"s t-test


( D ) Immunohistochemical quantifications of fibrinogen deposition in no tumour - bearing - and tumour - bearing - mouse lungs . Shown are averages ( N = 7 / group , 14 random fields ) with SEM . Welch " s t - test

(D) Immunohistochemical quantifications of fibrinogen deposition in no tumour-bearing- and tumour-bearing- mouse lungs. Shown are averages (N = 7/ group, 14 random fields) with SEM. Welch"s t-test


( E ) Representative photos of immunohistochemical co - localisation of FX + CD45 + cells and fibrinogen deposition in tumour - bearing mouse lungs . Circles show high fibrinogen deposition areas ( scale bar , 50 μm )

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

(E) Representative photos of immunohistochemical co-localisation of FX+CD45+ cells and fibrinogen deposition in tumour-bearing mouse lungs. Circles show high fibrinogen deposition areas (scale bar, 50 μm)


( B ) KikGR red cells were detected in the TCM - stimulated - liver and lungs after liver exposure to violet light ( arrow ) . Images taken from animals with no light exposure were also shown ( scale bar , 100 μm )

(B) KikGR red cells were detected in the TCM-stimulated- liver and lungs after liver exposure to violet light (arrow). Images taken from animals with no light exposure were also shown (scale bar, 100 μm)


( C ) Flow cytometric quantifications of photoconverted HepELs in TCM ( 3 times ) - stimulated KikGR mouseliver and lungs . Cells were isolated 72 h after photoconversion . Ratio was calculated as the number of photoconverted cells ( KikGR redin comparison with the number of liver or lung cells pre - sorted with CD45 - beads . Shown are averages ( N = 5 ) with SEM and Welch " s t -

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

(C) Flow cytometric quantifications of photoconverted HepELs in TCM (3 times)-stimulated KikGR mouseliver and lungs. Cells were isolated 72 h after photoconversion. Ratio was calculated as the number of photoconverted cells (KikGR redin comparison with the number of liver or lung cells pre-sorted with CD45-beads. Shown are averages (N = 5) with SEM and Welch"s t-test


( D ) Surface marker analyses of photoconverted KikGR cells . Vertical axes represent ratio of each marker + cell / photoconverted KikGR cell . Shown are averages ( conCM , control : N = 4 , TCM : N = 9 ) with SEM and Welch " s t - test

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(D) Surface marker analyses of photoconverted KikGR cells. Vertical axes represent ratio of each marker+ cell/photoconverted KikGR cell. Shown are averages (conCM, control: N = 4, TCM: N = 9) with SEM and Welch"s t-test


( E ) Representative immunostaining images of FX expression in NK1 . 1 + , CD11c + , B220 + and CD4 + cells in tumour - bearing lungs ( scale bar , 10 μm )

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(E) Representative immunostaining images of FX expression in NK1.1+, CD11c+, B220+ and CD4+ cells in tumour-bearing lungs (scale bar, 10 μm)


( A ) Flow cytometric analysis of B220 + CD11c + NK1 . 1 + cells in the lung of TCM - stimulated mice . Values in the dot plots present ratios for total cells gated KikGR red and KikGR green cell

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(A) Flow cytometric analysis of B220+CD11c+NK1.1+ cells in the lung of TCM-stimulated mice. Values in the dot plots present ratios for total cells gated KikGR red and KikGR green cell