( B ) Average number of LGG - 1 : : GFP - containing puncta in daf - 16 ( mu86 ) ; daf - 2 ( e1370 ) double mutants compared to daf - 2 ( e1370 ) animals ; p = 0 . 50 , unpaired , two - tailed t - test . N2 wild - type animals ( WT ) are shown for comparison . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . The double mutant expressing the LGG - 1 reporter had a mean lifespan similar to non - transgenic daf - 16 ; daf - 2 double mutants ( data not shown , [ 65 ] ) . Please see Table S1 for quantification of all data .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(B) Average number of LGG-1::GFP-containing puncta in daf-16(mu86); daf-2(e1370) double mutants compared to daf-2(e1370) animals; p = 0.50, unpaired, two-tailed t-test. N2 wild-type animals (WT) are shown for comparison. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. The double mutant expressing the LGG-1 reporter had a mean lifespan similar to non-transgenic daf-16; daf-2 double mutants (data not shown, [65]). Please see Table S1 for quantification of all data.


C . Cell length profile as a function of the cell number position from the QC for three wild - type seedlings ( symbols , top ) at day 8 post germination and three simulated root files for the Sizer model ( symbols , bottom ) . Notice the use of a logarithmic scale in cell lengths , which visualizes the cell lengthlog10

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY

C. Cell length profile as a function of the cell number position from the QC for three wild-type seedlings (symbols, top) at day 8 post germination and three simulated root files for the Sizer model (symbols, bottom). Notice the use of a logarithmic scale in cell lengths, which visualizes the cell lengthlog10 rEZ


A . Cartoon of each cell terminal differentiation mechanism ( Ruler in gray , Timer in pink and Sizer in red ) and ( right ) juxtaposed confocal images of an 8 - day - old WT seedling with the zones being indicated . Colors of zones as in Fig . 1 . Note the differences in subindex nomenclature compared with Fig . 1 . B . Pseudocode of the algorithm used in each model .

O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O 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. Cartoon of each cell terminal differentiation mechanism (Ruler in gray, Timer in pink and Sizer in red) and (right) juxtaposed confocal images of an 8-day-old WT seedling with the zones being indicated. Colors of zones as in Fig.1. Note the differences in subindex nomenclature compared with Fig. 1. B. Pseudocode of the algorithm used in each model.


C - E . Relationships between pairs of phenotypic traits . C . Length of the elongation zone LEZ versus its number of cells , NEZ . Panels from left to right : the Ruler , Timer and Sizer models , and epidermal Col - 0 data . Symbols in the three left - most panels represent simulated data of individual simulated root files ( gray triangles for Ruler , pink squares for Timer and red diamonds for Sizer , n = 122 each ) . Data from individual epidermal files in Col - 0 is in green circles ( n = 122 from day 6 , 8 and 10 post germination ( Tables EV1 , EV2 ) ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient ( r ) for each pair of data are indicated . p stands for the p - value using standard Pearson correlation test . The epidermis exhibits of the number of cells in the EZ with its length , and also with 1 / ln ( rEZ ) . The epidermal mature cell length does not have correlation with the inverse of the elongation factor . These features are only reproduced in the Sizer model . For each model , the simulated roots and cells differ in the threshold value for cell elongation termination , the cell elongation rate , the meristematic activity and the initial cell length , according to a variability inferred from the epidermal Col - 0 data ( Appendix Fig . S4B - F ) . The parameter values are the same for the three models except for the threshold for cell elongation termination , which is specific of each model and has relative variability of 35 % ( Ruler ) , 7 % ( Timer ) and 26 % ( Sizer ) ( Table EV3 ) . Parameter values selected such that no statistical significance is found between model ( for each model ) and epidermal data in any of five phenotypic traits ( Wilcoxon - rank sum test , p - value > 0 . 01 , Table EV4 , Appendix Fig . S4B - F ) . Continuous lines are theoretical predictions for each model ( Appendix Text S1E ) . All models have the same dependence on the threshold value for differentiation ( blue line ) but each model has its own dependence on the the spatial profile of cells along the EZ ( rEZ ) ( lines in gray for Ruler , black and red for Timer and red for Sizer ) . The Timer model depends on the cell elongation dynamics and , in contrast with the Ruler and Sizer models , its relationships are not univocally defined by the spatial profile of cells along the EZ ( rEZ ) . For the Timer model , the continuous lines represent the theoretical prediction obtained when either relong ( black line ) or Rprod ( red line ) changes . For all the boxplots , boxes represent the interquartile range ( 25th - 75th percentiles , with the median indicated by the black horizontal line ) of the distribution , whiskers extend to the minimum and maximal values excluding outliers . Outliers are those values more ( less ) than 3 / 2 of upper ( lower ) quartile .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E. Relationships between pairs of phenotypic traits. C. Length of the elongation zone LEZ versus its number of cells, NEZ. Panels from left to right: the Ruler, Timer and Sizer models, and epidermal Col-0 data. Symbols in the three left-most panels represent simulated data of individual simulated root files (gray triangles for Ruler, pink squares for Timer and red diamonds for Sizer, n=122 each). Data from individual epidermal files in Col-0 is in green circles (n=122 from day 6, 8 and 10 post germination (Tables EV1, EV2)). Dashed lines are minimum square linear fits. Pearson correlation coefficient (r) for each pair of data are indicated. p stands for the p-value using standard Pearson correlation test. The epidermis exhibits of the number of cells in the EZ with its length, and also with 1/ln(rEZ). The epidermal mature cell length does not have correlation with the inverse of the elongation factor. These features are only reproduced in the Sizer model. For each model, the simulated roots and cells differ in the threshold value for cell elongation termination, the cell elongation rate, the meristematic activity and the initial cell length, according to a variability inferred from the epidermal Col-0 data (Appendix Fig. S4B-F). The parameter values are the same for the three models except for the threshold for cell elongation termination, which is specific of each model and has relative variability of 35% (Ruler), 7% (Timer) and 26% (Sizer) (Table EV3). Parameter values selected such that no statistical significance is found between model (for each model) and epidermal data in any of five phenotypic traits (Wilcoxon-rank sum test, p-value >0.01, Table EV4, Appendix Fig. S4B-F). Continuous lines are theoretical predictions for each model (Appendix Text S1E). All models have the same dependence on the threshold value for differentiation (blue line) but each model has its own dependence on the the spatial profile of cells along the EZ (rEZ) (lines in gray for Ruler, black and red for Timer and red for Sizer). The Timer model depends on the cell elongation dynamics and, in contrast with the Ruler and Sizer models, its relationships are not univocally defined by the spatial profile of cells along the EZ (rEZ). For the Timer model, the continuous lines represent the theoretical prediction obtained when either relong (black line) or Rprod (red line) changes. For all the boxplots, boxes represent the interquartile range (25th-75th percentiles, with the median indicated by the black horizontal line) of the distribution, whiskers extend to the minimum and maximal values excluding outliers. Outliers are those values more (less) than 3/2 of upper (lower) quartile.


C - E . Relationships between pairs of phenotypic traits . D . Length of mature cell ( EZ cell closest to DZ ) lmax versus 1 / rEZ . Panels from left to right : the Ruler , Timer and Sizer models , and epidermal Col - 0 data . Symbols in the three left - most panels represent simulated data of individual simulated root files ( gray triangles for Ruler , pink squares for Timer and red diamonds for Sizer , n = 122 each ) . Data from individual epidermal files in Col - 0 is in green circles ( n = 122 from day 6 , 8 and 10 post germination ( Tables EV1 , EV2 ) ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient ( r ) for each pair of data are indicated . p stands for the p - value using standard Pearson correlation test . The epidermis exhibits of the number of cells in the EZ with its length , and also with 1 / ln ( rEZ ) . The epidermal mature cell length does not have correlation with the inverse of the elongation factor . These features are only reproduced in the Sizer model . For each model , the simulated roots and cells differ in the threshold value for cell elongation termination , the cell elongation rate , the meristematic activity and the initial cell length , according to a variability inferred from the epidermal Col - 0 data ( Appendix Fig . S4B - F ) . The parameter values are the same for the three models except for the threshold for cell elongation termination , which is specific of each model and has relative variability of 35 % ( Ruler ) , 7 % ( Timer ) and 26 % ( Sizer ) ( Table EV3 ) . Parameter values selected such that no statistical significance is found between model ( for each model ) and epidermal data in any of five phenotypic traits ( Wilcoxon - rank sum test , p - value > 0 . 01 , Table EV4 , Appendix Fig . S4B - F ) . Continuous lines are theoretical predictions for each model ( Appendix Text S1E ) . All models have the same dependence on the threshold value for differentiation ( blue line ) but each model has its own dependence on the the spatial profile of cells along the EZ ( rEZ ) ( lines in gray for Ruler , black and red for Timer and red for Sizer ) . The Timer model depends on the cell elongation dynamics and , in contrast with the Ruler and Sizer models , its relationships are not univocally defined by the spatial profile of cells along the EZ ( rEZ ) . For the Timer model , the continuous lines represent the theoretical prediction obtained when either relong ( black line ) or Rprod ( red line ) changes . For all the boxplots , boxes represent the interquartile range ( 25th - 75th percentiles , with the median indicated by the black horizontal line ) of the distribution , whiskers extend to the minimum and maximal values excluding outliers . Outliers are those values more ( less ) than 3 / 2 of upper ( lower ) quartile .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E. Relationships between pairs of phenotypic traits. D. Length of mature cell (EZ cell closest to DZ) lmax versus 1/rEZ. Panels from left to right: the Ruler, Timer and Sizer models, and epidermal Col-0 data. Symbols in the three left-most panels represent simulated data of individual simulated root files (gray triangles for Ruler, pink squares for Timer and red diamonds for Sizer, n=122 each). Data from individual epidermal files in Col-0 is in green circles (n=122 from day 6, 8 and 10 post germination (Tables EV1, EV2)). Dashed lines are minimum square linear fits. Pearson correlation coefficient (r) for each pair of data are indicated. p stands for the p-value using standard Pearson correlation test. The epidermis exhibits of the number of cells in the EZ with its length, and also with 1/ln(rEZ). The epidermal mature cell length does not have correlation with the inverse of the elongation factor. These features are only reproduced in the Sizer model. For each model, the simulated roots and cells differ in the threshold value for cell elongation termination, the cell elongation rate, the meristematic activity and the initial cell length, according to a variability inferred from the epidermal Col-0 data (Appendix Fig. S4B-F). The parameter values are the same for the three models except for the threshold for cell elongation termination, which is specific of each model and has relative variability of 35% (Ruler), 7% (Timer) and 26% (Sizer) (Table EV3). Parameter values selected such that no statistical significance is found between model (for each model) and epidermal data in any of five phenotypic traits (Wilcoxon-rank sum test, p-value >0.01, Table EV4, Appendix Fig. S4B-F). Continuous lines are theoretical predictions for each model (Appendix Text S1E). All models have the same dependence on the threshold value for differentiation (blue line) but each model has its own dependence on the the spatial profile of cells along the EZ (rEZ) (lines in gray for Ruler, black and red for Timer and red for Sizer). The Timer model depends on the cell elongation dynamics and, in contrast with the Ruler and Sizer models, its relationships are not univocally defined by the spatial profile of cells along the EZ (rEZ). For the Timer model, the continuous lines represent the theoretical prediction obtained when either relong (black line) or Rprod (red line) changes. For all the boxplots, boxes represent the interquartile range (25th-75th percentiles, with the median indicated by the black horizontal line) of the distribution, whiskers extend to the minimum and maximal values excluding outliers. Outliers are those values more (less) than 3/2 of upper (lower) quartile.


C - E . Relationships between pairs of phenotypic traits . E . NEZ versus 1 / ln ( rEZ ) , which can be interpreted as the ratio between the meristematic activity ( Rprod ) and the elongation rate ( relong ) . Panels from left to right : the Ruler , Timer and Sizer models , and epidermal Col - 0 data . Symbols in the three left - most panels represent simulated data of individual simulated root files ( gray triangles for Ruler , pink squares for Timer and red diamonds for Sizer , n = 122 each ) . Data from individual epidermal files in Col - 0 is in green circles ( n = 122 from day 6 , 8 and 10 post germination ( Tables EV1 , EV2 ) ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient ( r ) for each pair of data are indicated . p stands for the p - value using standard Pearson correlation test . The epidermis exhibits of the number of cells in the EZ with its length , and also with 1 / ln ( rEZ ) . The epidermal mature cell length does not have correlation with the inverse of the elongation factor . These features are only reproduced in the Sizer model . For each model , the simulated roots and cells differ in the threshold value for cell elongation termination , the cell elongation rate , the meristematic activity and the initial cell length , according to a variability inferred from the epidermal Col - 0 data ( Appendix Fig . S4B - F ) . The parameter values are the same for the three models except for the threshold for cell elongation termination , which is specific of each model and has relative variability of 35 % ( Ruler ) , 7 % ( Timer ) and 26 % ( Sizer ) ( Table EV3 ) . Parameter values selected such that no statistical significance is found between model ( for each model ) and epidermal data in any of five phenotypic traits ( Wilcoxon - rank sum test , p - value > 0 . 01 , Table EV4 , Appendix Fig . S4B - F ) . Continuous lines are theoretical predictions for each model ( Appendix Text S1E ) . All models have the same dependence on the threshold value for differentiation ( blue line ) but each model has its own dependence on the the spatial profile of cells along the EZ ( rEZ ) ( lines in gray for Ruler , black and red for Timer and red for Sizer ) . The Timer model depends on the cell elongation dynamics and , in contrast with the Ruler and Sizer models , its relationships are not univocally defined by the spatial profile of cells along the EZ ( rEZ ) . For the Timer model , the continuous lines represent the theoretical prediction obtained when either relong ( black line ) or Rprod ( red line ) changes . For all the boxplots , boxes represent the interquartile range ( 25th - 75th percentiles , with the median indicated by the black horizontal line ) of the distribution , whiskers extend to the minimum and maximal values excluding outliers . Outliers are those values more ( less ) than 3 / 2 of upper ( lower ) quartile .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E. Relationships between pairs of phenotypic traits. E. NEZ versus 1/ln(rEZ), which can be interpreted as the ratio between the meristematic activity (Rprod) and the elongation rate (relong). Panels from left to right: the Ruler, Timer and Sizer models, and epidermal Col-0 data. Symbols in the three left-most panels represent simulated data of individual simulated root files (gray triangles for Ruler, pink squares for Timer and red diamonds for Sizer, n=122 each). Data from individual epidermal files in Col-0 is in green circles (n=122 from day 6, 8 and 10 post germination (Tables EV1, EV2)). Dashed lines are minimum square linear fits. Pearson correlation coefficient (r) for each pair of data are indicated. p stands for the p-value using standard Pearson correlation test. The epidermis exhibits of the number of cells in the EZ with its length, and also with 1/ln(rEZ). The epidermal mature cell length does not have correlation with the inverse of the elongation factor. These features are only reproduced in the Sizer model. For each model, the simulated roots and cells differ in the threshold value for cell elongation termination, the cell elongation rate, the meristematic activity and the initial cell length, according to a variability inferred from the epidermal Col-0 data (Appendix Fig. S4B-F). The parameter values are the same for the three models except for the threshold for cell elongation termination, which is specific of each model and has relative variability of 35% (Ruler), 7% (Timer) and 26% (Sizer) (Table EV3). Parameter values selected such that no statistical significance is found between model (for each model) and epidermal data in any of five phenotypic traits (Wilcoxon-rank sum test, p-value >0.01, Table EV4, Appendix Fig. S4B-F). Continuous lines are theoretical predictions for each model (Appendix Text S1E). All models have the same dependence on the threshold value for differentiation (blue line) but each model has its own dependence on the the spatial profile of cells along the EZ (rEZ) (lines in gray for Ruler, black and red for Timer and red for Sizer). The Timer model depends on the cell elongation dynamics and, in contrast with the Ruler and Sizer models, its relationships are not univocally defined by the spatial profile of cells along the EZ (rEZ). For the Timer model, the continuous lines represent the theoretical prediction obtained when either relong (black line) or Rprod (red line) changes. For all the boxplots, boxes represent the interquartile range (25th-75th percentiles, with the median indicated by the black horizontal line) of the distribution, whiskers extend to the minimum and maximal values excluding outliers. Outliers are those values more (less) than 3/2 of upper (lower) quartile.


Simulation results are depicted in gray for Ruler , pink for Timer and red for Sizer models ( n = 30 simulated root files , each ) . Data from individual cortex tissue files in Col - 0 is in green ( n = 30 , day 6 post germination ( Tables EV1 , EV2 ) ) . Parameter values for the models ( Table EV3 ) selected such that no statistical significance is found between model ( for each model ) and cortex data in any of the five phenotypic traits depicted in A ( Wilcoxon - rank sum test , p - value > 0 . 01 , Table EV4 ) . For each model , the simulated roots and cells differ in the threshold value for cell elongation termination , the cell elongation rate , the meristematic activity and the initial cell length ( Table EV3 ) . The parameter values are the same for the three models except for the threshold for cell elongation termination , which is specific of each model and has relative variability of 40 % ( Ruler ) , 3 % ( Timer ) and 32 % ( Sizer ) ( Table EV3 ) . A . Boxplots for five phenotypic traits : elongation factor rEZ , mature cell length ( length of the EZ cell closest to the DZ ) lmax , length of the elongation zone LEZ , number of cells in the elongation zone NEZ and root growth rate . For the cortex Col - 0 data , the first four phenotypic traits are all measured in the same root files ( n = 30 , day 6 post germination ) , while the rootgrowth rate is measured on a different set of roots ( n = 22 , Methods ) . The Ruler and Timer models drive larger variability in the root growth rate .

O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O 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-TISSUE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O

Simulation results are depicted in gray for Ruler, pink for Timer and red for Sizer models (n=30 simulated root files, each). Data from individual cortex tissue files in Col-0 is in green (n=30, day 6 post germination (Tables EV1, EV2)). Parameter values for the models (Table EV3) selected such that no statistical significance is found between model (for each model) and cortex data in any of the five phenotypic traits depicted in A (Wilcoxon-rank sum test, p-value >0.01, Table EV4). For each model, the simulated roots and cells differ in the threshold value for cell elongation termination, the cell elongation rate, the meristematic activity and the initial cell length (Table EV3). The parameter values are the same for the three models except for the threshold for cell elongation termination, which is specific of each model and has relative variability of 40% (Ruler), 3% (Timer) and 32% (Sizer) (Table EV3). A. Boxplots for five phenotypic traits: elongation factor rEZ, mature cell length (length of the EZ cell closest to the DZ) lmax, length of the elongation zone LEZ , number of cells in the elongation zone NEZ and root growth rate. For the cortex Col-0 data, the first four phenotypic traits are all measured in the same root files (n=30, day 6 post germination), while the rootgrowth rate is measured on a different set of roots (n=22, Methods). The Ruler and Timer models drive larger variability in the root growth rate.


B - D . Relationships between pairs of the phenotypic traits depicted in A . Panels from left to right : the Ruler , Timer and Sizer models , and cortex Col - 0 data . Symbols in the three left - most panels represent simulated data of individual simulated root files ( gray triangles for Ruler , pink squares for Timer and red diamonds for Sizer , n = 30 each ) . Data from individual cortex files in Col - 0 is in green circles ( n = 30 ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient ( r ) for each pair of data are indicated . p stands for the p - value using standard Pearson correlation test . Continuos lines as in Fig . 2 C - E but for the corresponding parameter values ( Table EV3 ) .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. Relationships between pairs of the phenotypic traits depicted in A. Panels from left to right: the Ruler, Timer and Sizer models, and cortex Col-0 data. Symbols in the three left-most panels represent simulated data of individual simulated root files (gray triangles for Ruler, pink squares for Timer and red diamonds for Sizer, n=30 each). Data from individual cortex files in Col-0 is in green circles (n=30). Dashed lines are minimum square linear fits. Pearson correlation coefficient (r) for each pair of data are indicated. p stands for the p-value using standard Pearson correlation test. Continuos lines as in Fig. 2 C-E but for the corresponding parameter values (Table EV3).


C - D : NEZ versus 1 / lnrEZ in the presence or absence of the meristem - elongation correlation , when Rprod is changed two - fold .

O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O

C-D: NEZ versus 1/lnrEZ in the presence or absence of the meristem-elongation correlation, when Rprod is changed two-fold.


E - H : Theoretical distribution of mature cells length ( E , F ) and of root growth ( G , H ) in the presence ( E , G ) , or absence ( F , H ) of the correlation , for different values of the meristematic activity ( Rprod ) ( n = 35 ) . In all cases data was computed using the real experimental values for 1 / ln ( rEZ ) , 1 / ( 2ln ( rEZ ) ) and 2 / ( ln ( rEZ ) ) and hypothetical values for NEZ chosen to either preserve or not the correlation . p < 0 . 005 * * * , p < 0 . 01 * * , p < 0 . 05 * Boxplot representation as in Fig . 2 .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H: Theoretical distribution of mature cells length (E,F) and of root growth (G,H) in the presence (E,G), or absence (F,H) of the correlation, for different values of the meristematic activity (Rprod) (n=35). In all cases data was computed using the real experimental values for 1/ln(rEZ), 1/(2ln(rEZ)) and 2/(ln(rEZ)) and hypothetical values for NEZ chosen to either preserve or not the correlation. p<0.005 ***, p<0.01 **, p<0.05 * Boxplot representation as in Fig. 2.


A . Eight - day - old seedlings of WT , bri1 and pRPS5a : BRI1 : YFP ; bri1 . Scale bar 1 cm .

O O O O O O O B-ORGANISM O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O

A. Eight-day-old seedlings of WT, bri1 and pRPS5a:BRI1:YFP;bri1. Scale bar 1 cm.


B . Root - length measurements of 10 - day - old bri1 - 116 , bes1 - D and pRP5SA : BRI : YFP ; bri1 - 116 seedlings compared to the wild type . Symbols represent the mean of more than 20 plants , from 3 independent experiments . Bars show standard deviation . Straight lines represent the linear regression applied to each curve starting with day 4 post germination . In this way the root growth rate can be extracted for each phenotype ( See Table EV7 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O

B. Root-length measurements of 10-day-old bri1-116, bes1-D and pRP5SA:BRI:YFP;bri1-116 seedlings compared to the wild type. Symbols represent the mean of more than 20 plants, from 3 independent experiments. Bars show standard deviation. Straight lines represent the linear regression applied to each curve starting with day 4 post germination. In this way the root growth rate can be extracted for each phenotype (See Table EV7).


C . Confocal images of 8 - day - old wild type WT , bri1 - 116 and pRP5SA : BRI : YFP ; bri1 - 116 roots stained with PI . Green lines label the end of the transition zone and the yellow lines label the first root hair ( end of elongation zone ) . The inset shows pRP5SA expression domain of an 8 - day - old pRP5SA : BRI : YFP ; bri1 - 116 seedling . Green line labels the end of the meristematic zone , which coincides with the end of pRP5SA expression domain . Scale bars correspond to 100 microns .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O

C. Confocal images of 8-day-old wild type WT, bri1-116 and pRP5SA:BRI:YFP;bri1-116 roots stained with PI. Green lines label the end of the transition zone and the yellow lines label the first root hair (end of elongation zone). The inset shows pRP5SA expression domain of an 8-day-old pRP5SA:BRI:YFP;bri1-116 seedling. Green line labels the end of the meristematic zone, which coincides with the end of pRP5SA expression domain. Scale bars correspond to 100 microns.


C - H . Main relationships as in Fig . 2C - E between experimental values of the phenotypic traits used to assess the mechanism of differentiation in bri1 - 116 mutant ( C , E , G ) and pRP5SA : BRI : YFP ; bri1 - 116 line ( D , F , H ) for the epidermis ( left large plot ) and cortex ( right inset plot ) . Each circle denotes data extracted from a single root ( n = 126 ( epidermis ) and n = 25 ( cortex ) for bri1 - 116 and n = 90 ( epidermis ) and n = 17 ( cortex ) for pRP5SA : BRI : YFP ; bri1 - 116 , see Table EV1 , pooled from 6 , 8 and 10 day - old seedlings for epidermis and from day 6 for cortex ( data in Table EV5 ) ) . In G , H , the slope of the linear regression is 2 . 13 ± 0 . 04 for bri1 - 116 and 2 . 53 ± 0 . 04 for pRP5SA : BRI : YFP ; bri1 - 116 . Pearson correlation coefficient r is indicated and p - value ( p , using standard Person correlation test ) . In E , bri1 - 116 epidermal data do not conform to normal distributions .

O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-ORGANISM O B-TISSUE O O O O O B-TISSUE O O O O 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-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O 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-GENEPROD O O B-TISSUE O O O O O O O O

C-H. Main relationships as in Fig. 2C-E between experimental values of the phenotypic traits used to assess the mechanism of differentiation in bri1-116 mutant (C,E,G) and pRP5SA:BRI:YFP;bri1-116 line (D,F,H) for the epidermis (left large plot) and cortex (right inset plot). Each circle denotes data extracted from a single root (n=126 (epidermis) and n=25 (cortex) for bri1-116 and n=90 (epidermis) and n=17 (cortex) for pRP5SA:BRI:YFP;bri1-116, see Table EV1, pooled from 6, 8 and 10 day-old seedlings for epidermis and from day 6 for cortex (data in Table EV5)). In G, H, the slope of the linear regression is 2.13±0.04 for bri1-116 and 2.53±0.04 for pRP5SA:BRI:YFP;bri1-116. Pearson correlation coefficient r is indicated and p-value (p, using standard Person correlation test). In E, bri1-116 epidermal data do not conform to normal distributions.


Simulation results are depicted in gray for Ruler , pink for Timer and red for Sizer models ( n = 28 each ) . Data from cortex tissue in Col - 0 grown with 1µM PAC is in green ( n = 28 , day 6 post germination ) . Parameter values for the models ( Table EV3 ) selected such that no statistical significance is found between model ( for each model ) and cortex data in any of the five phenotypic traits depicted in A ( Wilcoxon - rank sum test , p - value > 0 . 01 , Table EV4 ) . For each model , the simulated roots and cells differ in the threshold value for cell elongation termination , the cell elongation rates , the meristematic activities and the initial cell length . The parameter values are the same for the three models except for the threshold for cell elongation termination , which has relative variability of 16 % ( Ruler ) , 2 % ( Timer ) and 14 % ( Sizer ) . A Boxplots for phenotypic traits : elongation factor rEZ , mature cell length ( length of the EZ cell closest to the DZ ) lmax , length of the elongation zone LEZ , number of cells in the elongation zone NEZ and root growth rate . For the cortex data , the first four phenotypic traits are all measured in the same root files ( n = 28 , day 6 post germination ) , while the root growth rate is measured on a different set of roots ( n = 40 , Methods ) .

O O 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-TISSUE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O 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-TISSUE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O O O O O O O O

Simulation results are depicted in gray for Ruler, pink for Timer and red for Sizer models (n=28 each). Data from cortex tissue in Col-0 grown with 1µM PAC is in green (n=28, day 6 post germination). Parameter values for the models (Table EV3) selected such that no statistical significance is found between model (for each model) and cortex data in any of the five phenotypic traits depicted in A (Wilcoxon-rank sum test, p-value >0.01, Table EV4). For each model, the simulated roots and cells differ in the threshold value for cell elongation termination, the cell elongation rates, the meristematic activities and the initial cell length. The parameter values are the same for the three models except for the threshold for cell elongation termination, which has relative variability of 16% (Ruler), 2% (Timer) and 14% (Sizer). A Boxplots for phenotypic traits: elongation factor rEZ , mature cell length (length of the EZ cell closest to the DZ) lmax, length of the elongation zone LEZ , number of cells in the elongation zone NEZ and root growth rate. For the cortex data, the first four phenotypic traits are all measured in the same root files (n=28, day 6 post germination), while the root growth rate is measured on a different set of roots (n=40, Methods).


B - D Relationships between pairs of the phenotypic traits depicted in A . Panels from left to right : the Ruler , Timer and Sizer models , and empirical cortex Col - 0 data . Symbols in the three left - most panels represent simulated data . Continuous lines are theoretical predictions for each model . In contrast with the Ruler and Sizer models , the theoretical Timer model relationships depend on which factor varies ( either the elongation rate ( black line ) or the meristematic activity ( red line ) ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient r for each pair of data .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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-D Relationships between pairs of the phenotypic traits depicted in A. Panels from left to right: the Ruler, Timer and Sizer models, and empirical cortex Col-0 data. Symbols in the three left-most panels represent simulated data. Continuous lines are theoretical predictions for each model. In contrast with the Ruler and Sizer models, the theoretical Timer model relationships depend on which factor varies (either the elongation rate (black line) or the meristematic activity (red line)). Dashed lines are minimum square linear fits. Pearson correlation coefficient r for each pair of data.


O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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-D Relationships between pairs of the phenotypic traits depicted in A. Panels from left to right: the Ruler, Timer and Sizer models, and empirical cortex Col-0 data. Symbols in the three left-most panels represent simulated data. Continuous lines are theoretical predictions for each model. In contrast with the Ruler and Sizer models, the theoretical Timer model relationships depend on which factor varies (either the elongation rate (black line) or the meristematic activity (red line)). Dashed lines are minimum square linear fits. Pearson correlation coefficient r for each pair of data.


( B ) Fold - change ± standard error in baseline IL - 6 plasma levels at week 12 from RA patients treated with placebo ( n = 47 ) , baricitinib 1 - mg ( n = 23 ) , 2 - mg ( n = 24 ) , 4 - mg ( n = 23 ) , and 8 - mg ( n = 23 ) in the phase 2b randomized , placebo - controlled study NCT01469013 . p values are for comparisons of baricitinib 1 - mg , 2 - mg , 4 - mg , and 8 - mg change from baseline compared to the placebo change from baseline . Treatment effects were estimated using a mixed effects repeated measures model ( SAS Proc Mixed ) using log10 transformed IL - 6 levels with an unstructured covariance matrix .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-TISSUE O O O O O B-DISEASE B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(B) Fold-change ± standard error in baseline IL-6 plasma levels at week 12 from RA patients treated with placebo (n=47), baricitinib 1-mg (n=23), 2-mg (n=24), 4-mg (n=23), and 8-mg (n=23) in the phase 2b randomized, placebo-controlled study NCT01469013. p values are for comparisons of baricitinib 1-mg, 2-mg, 4-mg, and 8-mg change from baseline compared to the placebo change from baseline. Treatment effects were estimated using a mixed effects repeated measures model (SAS Proc Mixed) using log10 transformed IL-6 levels with an unstructured covariance matrix.


( A and B ) Binding affinities in Kd are shown for baricitinib ( A and B ) , tofacitinib , upadacitinib , AZD7762 ( positive control ) , and broad - spectrum tyrosine kinase inhibitors sunitinib and erlotinib ( B ) . Kd values were estimated from an 11 - point concentration response curve with fixed top parameters and a four - parameter logistic model . Kd , equilibrium affinity constant ( n = 3 )

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY 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 and B) Binding affinities in Kd are shown for baricitinib (A and B), tofacitinib, upadacitinib, AZD7762 (positive control), and broad-spectrum tyrosine kinase inhibitors sunitinib and erlotinib (B). Kd values were estimated from an 11-point concentration response curve with fixed top parameters and a four-parameter logistic model. Kd, equilibrium affinity constant (n=3)


( C ) Protein and mRNA expression of relevant NAKs in primary human liver organoids ( n = 3 ) ( p < 0 . 05 ) . The immunofluorescence shows the juxtaposition of 1A9 ( signal from the SARS - CoV - 2 ) in red and ACE2 in green along with the spheroid nuclei in blue as a DAPI stain ( n = 1 ) . Colocalization of 1A9 and ACE2 is depicted with an arrow .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O

(C) Protein and mRNA expression of relevant NAKs in primary human liver organoids (n=3) (p<0.05). The immunofluorescence shows the juxtaposition of 1A9 (signal from the SARS-CoV-2) in red and ACE2 in green along with the spheroid nuclei in blue as a DAPI stain (n=1). Colocalization of 1A9 and ACE2 is depicted with an arrow.


( D , E ) Time course of days ( one , three , and six days ) post infection ( dpi ) using liver spheroids and SARS - CoV - 2 at 0 . 1 and 1 . 0 multiplicity of infection ( MOI ) ( n = 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 B-TISSUE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O

(D, E) Time course of days (one, three, and six days) post infection (dpi) using liver spheroids and SARS-CoV-2 at 0.1 and 1.0 multiplicity of infection (MOI) (n=1).


( F ) Viability of spheroids was quantified by measuring intracellular ATP concentrations . Note that no significant ( n . s . ; Student ' s t - test ) decrease in cell viability was observed for any concentration tested ( n = 10 ) .

O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O

(F) Viability of spheroids was quantified by measuring intracellular ATP concentrations. Note that no significant (n.s.; Student's t-test) decrease in cell viability was observed for any concentration tested (n=10).


( H ) Viral load in infected ( MOI 0 . 1 ) spheroids as measured by qPCR after treatment with 400 nM and 800 nM of baricitinib 48 hours after infection ( * p < 0 . 05 ; Student ' s t - test ) . Data are mean ± standard error of mean ( n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O 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) Viral load in infected (MOI 0.1) spheroids as measured by qPCR after treatment with 400 nM and 800 nM of baricitinib 48 hours after infection (*p<0.05; Student's t-test). Data are mean ± standard error of mean (n=3).


( A ) Detection of SARS - CoV - 2 in nasopharyngeal swabs and peripheral blood using the GeneFinderTM COVID - 19 Plus RealAmp Kit assay ( ELITechGroup S . p . A . , Turin , Italy ) . Three viral target genes , RdRp , N , and E , together with the housekeeping gene GAPDH were simultaneously amplified . Here the most sensitive target gene , N , is shown . Samples with Ct values > 40 were defined as negative . A dashed horizontal line indicates the cut - off . IL - 6 values are shown in the same graph . Time of baricitinib treatment is highlighted ( Patients A , B , and D for 10 days , Patient C for 12 days ) with total time of evaluation on the X - axis . Fever and cough symptoms are indicated . The bars in grey indicate that cough has improved but has not resolved .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-TISSUE O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O 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) Detection of SARS-CoV-2 in nasopharyngeal swabs and peripheral blood using the GeneFinderTM COVID-19 Plus RealAmp Kit assay (ELITechGroup S.p.A., Turin, Italy). Three viral target genes, RdRp, N, and E, together with the housekeeping gene GAPDH were simultaneously amplified. Here the most sensitive target gene, N, is shown. Samples with Ct values > 40 were defined as negative. A dashed horizontal line indicates the cut-off. IL-6 values are shown in the same graph. Time of baricitinib treatment is highlighted (Patients A, B, and D for 10 days, Patient C for 12 days) with total time of evaluation on the X-axis. Fever and cough symptoms are indicated. The bars in grey indicate that cough has improved but has not resolved.


( B ) Levels of CD3 + , CD4 + , CD8 + , B cells ( CD19 + ) and NK cells ( CD3 - , CD16 + , CD56 + ) were determined using the AQUIOS CL Flow Cytometry System ( Beckman Coulter ) .

O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(B) Levels of CD3+, CD4+, CD8+, B cells (CD19+) and NK cells (CD3-, CD16+, CD56+) were determined using the AQUIOS CL Flow Cytometry System (Beckman Coulter).


( C ) Total serum IgG , IgA , and IgM levels are shown for all patients .

O O O O B-TISSUE B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-ORGANISM O

(C) Total serum IgG, IgA, and IgM levels are shown for all patients.


Detection of IgG antibodies against the S1 / S2 antigens of SARS - CoV - 2 . Time of baricitinib treatment is indicated for all patients ( patients A , B , and D for 10 days , patient C for 12 days ) .

O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O

Detection of IgG antibodies against the S1/S2 antigens of SARS-CoV-2. Time of baricitinib treatment is indicated for all patients (patients A, B, and D for 10 days, patient C for 12 days).


( A ) Levels of D - dimer , CRP , ferritin , white blood cells , and neutrophils are shown for all patients .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE O O O O B-ORGANISM O

(A) Levels of D-dimer, CRP, ferritin, white blood cells, and neutrophils are shown for all patients.


( B ) Levels of aspartate aminotransferase , alanine aminotransferase , and bilirubin are shown for all patients . ALT , alanine aminotransferase ; AST , aspartate

O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD

(B) Levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin are shown for all patients. ALT, alanine aminotransferase; AST, aspartate aminotransferase


( C ) Chest CT scan for Patient C on day one and 19 from symptom onset showing clinical improvement over time . Day one CT scan shows ground glass opacity ( arrows ) sub - pleurally in the lower lobes bilaterally ( early stage one according to Pan et al ( Pan , Ye et al . , 2020 ) . Day 19 CT scan shows that consolidation was gradually absorbed with evident residual fibrosis and emphysema bubbles ( arrows ) in the site of the early lesions ( absorption stage four according to Pan et al ( Pan et al . , 2020 ) .

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-DISEASE O B-DISEASE O O O O O 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) Chest CT scan for Patient C on day one and 19 from symptom onset showing clinical improvement over time. Day one CT scan shows ground glass opacity (arrows) sub-pleurally in the lower lobes bilaterally (early stage one according to Pan et al (Pan, Ye et al., 2020). Day 19 CT scan shows that consolidation was gradually absorbed with evident residual fibrosis and emphysema bubbles (arrows) in the site of the early lesions (absorption stage four according to Pan et al (Pan et al., 2020).


B . The 5 ' UTR of rne is bound by RNase E and non - genomically encoded A - tails are maximally recovered - 9nt from the rne start codon . Known stem loop strucutres ( HP1 - 3 ) and the ribosomal binding site ( RBS ) are shaded grey .

O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD 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. The 5'UTR of rne is bound by RNase E and non-genomically encoded A-tails are maximally recovered -9nt from the rne start codon. Known stem loop strucutres (HP1-3) and the ribosomal binding site (RBS) are shaded grey.


C . RNase E binding and oligoadenylation of the pdlB - yigL dicistronic transcript . The reported RNase E cleavage site ( red dashed line ) and SgrS binding site ( grey shading ) are indicated .

O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O

C. RNase E binding and oligoadenylation of the pdlB-yigL dicistronic transcript. The reported RNase E cleavage site (red dashed line) and SgrS binding site (grey shading) are indicated.


D . Length of non - genomically encoded oligo ( A ) tails recovered from RNase E bound reads .

O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O

D. Length of non-genomically encoded oligo(A) tails recovered from RNase E bound reads.


E . Position of Hfq binding and oliogadenylation relative to RNase E binding peaks . The cumulative position of Hfq and oligoadenylation peaks were determined relative to RNase E binding peaks for 672 RNase E binding sites that were within 1kb of an Hfq binding peak . A detailed description of the data processing is presented in the Appended Supplementary Methods .

O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

E. Position of Hfq binding and oliogadenylation relative to RNase E binding peaks. The cumulative position of Hfq and oligoadenylation peaks were determined relative to RNase E binding peaks for 672 RNase E binding sites that were within 1kb of an Hfq binding peak. A detailed description of the data processing is presented in the Appended Supplementary Methods.


B . Hfq binding ( blue ) , RNase E binding ( grey ) , and oligoadenylation of the MicA binding site in the ompA mRNA . The MicA seed sequence is shaded blue .

O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O

B. Hfq binding (blue), RNase E binding (grey), and oligoadenylation of the MicA binding site in the ompA mRNA. The MicA seed sequence is shaded blue.


C . Interaction strength ( Gibbs free energy ) of all RNA - RNA interactions recovered by RNase E - CLASH ( solid line ) and for randomly paired hybrid read halves ( dashed line ) .

O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O

C. Interaction strength (Gibbs free energy) of all RNA-RNA interactions recovered by RNase E-CLASH (solid line) and for randomly paired hybrid read halves (dashed line).


D . Interaction strength of sRNA - mRNA hybrid read halves ( solid line ) and randomly paired sRNA and mRNA read halves ( dashed line ) .

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

D. Interaction strength of sRNA-mRNA hybrid read halves (solid line) and randomly paired sRNA and mRNA read halves (dashed line).


E . Position of sRNA - mRNA interactions relative to the mRNA start codon .

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O

E. Position of sRNA-mRNA interactions relative to the mRNA start codon.


RNA classes are labeled on the outer ring ( rRNA has been omitted to clarify interactions between other RNA classes ) . RNA - RNA interactions with more than two unique hybrid sequences are presented . The thickness of the link represents the number of unique hybrid sequences recovered ( up to a maximum of 50 sequences ) . RNA - RNA interactions where both hybrid halves overlap an Hfq binding site are colored blue and non - overlapping interactions are colored green .

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

RNA classes are labeled on the outer ring (rRNA has been omitted to clarify interactions between other RNA classes). RNA-RNA interactions with more than two unique hybrid sequences are presented. The thickness of the link represents the number of unique hybrid sequences recovered (up to a maximum of 50 sequences). RNA-RNA interactions where both hybrid halves overlap an Hfq binding site are colored blue and non-overlapping interactions are colored green.


A B ( top ) . Sequences interacting with ChiX ( A ) or ompA ( B ) were analyzed for conserved motifs using MEME . Motifs that were enriched in the target RNAs and are shown above the heatmap with proportion of target RNAs carrying the motif ( N ) and the expected value for the motif ( e - val ) . The complementary sequence motifs within ChiX or ompA are shown below the logo . ( Middle ) Heatmaps showing the position of predicted basepairing for each interaction within ChiX or ompA . Grey bars boxed below indicate the position of basepairing with experimentally verified mRNAs ( known interactions ) . ( Bottom ) Hfq ( blue ) and RNase E ( grey ) binding sites within ChiX and ompA . Hfq and RNase E - bound sequence , and non - genomically encoded oligo ( A ) tails within ChiX and ompA are shown as line plots where the x - axis position correlates with heatmaps above .

O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

A B (top). Sequences interacting with ChiX (A) or ompA (B) were analyzed for conserved motifs using MEME. Motifs that were enriched in the target RNAs and are shown above the heatmap with proportion of target RNAs carrying the motif (N) and the expected value for the motif (e-val). The complementary sequence motifs within ChiX or ompA are shown below the logo. (Middle) Heatmaps showing the position of predicted basepairing for each interaction within ChiX or ompA. Grey bars boxed below indicate the position of basepairing with experimentally verified mRNAs (known interactions). (Bottom) Hfq (blue) and RNase E (grey) binding sites within ChiX and ompA. Hfq and RNase E-bound sequence, and non-genomically encoded oligo(A) tails within ChiX and ompA are shown as line plots where the x-axis position correlates with heatmaps above.


C , D E . Cumulative plots of oligoadenylation ( C ) , Hfq ( D ) and RNase E ( E ) binding at predicted seed motifs .

O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O

C, D E. Cumulative plots of oligoadenylation (C), Hfq (D) and RNase E (E) binding at predicted seed motifs.


A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for frdA - RyhB ( A ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for frdA-RyhB (A). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for zapB - RyhB ( B ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for zapB-RyhB (B). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for hdeA - RyhB ( C ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for hdeA-RyhB (C). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) hdeA - GadY ( D ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) hdeA-GadY (D). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for rssA - RyeB ( E ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for rssA-RyeB (E). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for chuA - Esr41 ( F ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for chuA-Esr41 (F). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for cirA - Esr41 ( G ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for cirA-Esr41 (G). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for the sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) and are shown ( left ) for bfr - Esr41 ( H ) . Point mutations and predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median fluorescence intensity ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - tailed t - test was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for bfr-Esr41 (H). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - C . FACS analysis of constitutively expressed sfGFP fusions to chuA ( A ) , cirA ( B ) , or bfr ( C ) in the presence of Esr41 ( orange ) , RyhB ( blue ) , or a scrambled RNA control ( red ) . The histogram shows GFP fluorescence for each sRNA - mRNA fusion .

O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O

A-C. FACS analysis of constitutively expressed sfGFP fusions to chuA (A), cirA (B), or bfr (C) in the presence of Esr41 (orange), RyhB (blue), or a scrambled RNA control (red). The histogram shows GFP fluorescence for each sRNA-mRNA fusion.


D . Esr41 confers resistance to Colicin 1A and 1B in the sensitive background , E . coli DH5alpha . Top agar lawns of DH5alpha expressing a control scrambled RNA ( pJV300 ) , Esr41 ( pZE12 : : esr41 ) , or RyhB ( pZE12 : : ryhB ) were spotted with colicins indicated ( top ) . Zones of clearing indicate sensitivity to the tested colicin .

O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

D. Esr41 confers resistance to Colicin 1A and 1B in the sensitive background, E. coli DH5alpha. Top agar lawns of DH5alpha expressing a control scrambled RNA (pJV300), Esr41 (pZE12::esr41), or RyhB (pZE12::ryhB) were spotted with colicins indicated (top). Zones of clearing indicate sensitivity to the tested colicin.


E . Esr41 confers a competitive disadvantage on EHEC under iron - limiting conditions . Wild type EHEC and isogenic ∆ esr41 strain ( black ) , or EHEC ∆ esr41and the chromosomally repaired strain EHEC ∆ esr41 : : esr41 were inoculated at equal densities and cultured in MEM - HEPES media for 3 days . The proportion of each strain was determined at each time point ( days ) and is expressed as a ratio relative to the starting inoculum where 1 is an equal fitness ( see Appended Supplementary Methods ) .

O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Esr41 confers a competitive disadvantage on EHEC under iron-limiting conditions. Wild type EHEC and isogenic ∆esr41 strain (black), or EHEC ∆esr41and the chromosomally repaired strain EHEC ∆esr41::esr41 were inoculated at equal densities and cultured in MEM-HEPES media for 3 days. The proportion of each strain was determined at each time point (days) and is expressed as a ratio relative to the starting inoculum where 1 is an equal fitness (see Appended Supplementary Methods).


A - D The morphology of uninjected , FLT3 / WT mRNA and FLT3 / ITD mRNA injected ( 150 ng per embryo ) embryos on day 2 post fertilization ( dpf ) . Data information : ov : otic vesicles Scale bar = 500 μm .

O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O

A-D The morphology of uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected (150 ng per embryo) embryos on day 2 post fertilization (dpf). Data information: ov: otic vesicles Scale bar = 500 μm.


E - H Whole mount in situ hybridization ( WISH ) of notochord - specific marker col9a2 in uninjected , FLT3 / WT mRNA and FLT3 / ITD mRNA injected embryos on 2 dpf . Data information : ov : otic vesicles ; cm : cephalic mesoderm ; nc : notochord . Scale bar = 500 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-TISSUE O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O O O O O O O

E-H Whole mount in situ hybridization (WISH) of notochord-specific marker col9a2 in uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected embryos on 2 dpf. Data information: ov: otic vesicles; cm: cephalic mesoderm; nc: notochord. Scale bar = 500 μm.


FLT3 signaling was detected by Western blotting in 293FT cells - transfected with FLT3 / ITD mRNA ( I )

B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O O O O O O

FLT3 signaling was detected by Western blotting in 293FT cells-transfected with FLT3/ITD mRNA (I)


FLT3 signaling was detected by Western blotting in zebrafish embryos - injected with FLT3 / ITD mRNA ( J ) .

B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-TISSUE O O O B-GENEPROD O O O O O O O

FLT3 signaling was detected by Western blotting in zebrafish embryos-injected with FLT3/ITD mRNA (J).


K The effect of FLT3 inhibitor quizartinib ( Qui ) on the dorsalization and axis - duplication phenotype - induced by FLT3 / ITD mRNA injection in zebrafish . Data information : the experiments were performed in triplicates and the data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . NS , not significant .

O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K The effect of FLT3 inhibitor quizartinib (Qui) on the dorsalization and axis-duplication phenotype-induced by FLT3/ITD mRNA injection in zebrafish. Data information: the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant.


Quantification of fst expression by RT - qPCR ( L ) after FLT3 / ITD overexpression in zebrafish embryos at 6 hpf . Data information : the experiments were performed in triplicates and the data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) . NS , not significant .

O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of fst expression by RT-qPCR (L) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf. Data information: the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant.


Quantification of fst expression by Western Blotting ( M ) , after FLT3 / ITD overexpression in zebrafish embryos at 6 hpf .

O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-ORGANISM B-TISSUE O O O O

Quantification of fst expression by Western Blotting (M), after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.


Quantification of fst expression by WISH ( N ) after FLT3 / ITD overexpression in zebrafish embryos at 6 hpf .

O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-ORGANISM B-TISSUE O O O O

Quantification of fst expression by WISH (N) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.


A - C WISH of fst in FLT3 / WT - ( A ) , FLT3 / ITD - plasmid DNA injected zebrafish embryos without ( B ) or with ( C ) quizartinib treatment ( 2 . 5 μM ) from 6 to 36 hpf . fst expression was expanded by FLT3 / ITD DNA in 86 % of embryos ( B , arrow , 32 / 37 ) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83 % of embryos ( C , 29 / 35 ) . Data information : Scale bar = 500 μm .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-ORGANISM B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O O O O O O O O O O O O O

A-C WISH of fst in FLT3/WT- (A), FLT3/ITD- plasmid DNA injected zebrafish embryos without (B) or with (C) quizartinib treatment (2.5 μM) from 6 to 36 hpf. fst expression was expanded by FLT3/ITD DNA in 86% of embryos (B, arrow, 32/37) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83% of embryos (C, 29/35). Data information: Scale bar = 500 μm.


Generation and characterization of FLT3 / ITD - transgenic zebrafish . GFP expression was detected by florescent microscopy ( F - H ) and in blood circulation and thymus by WISH ( I and J , blue arrow ) in WT sibling and Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) embryos at 4 dpf . FLT3 / ITD - positive zebrafish ( F1 ) were confirmed by PCR genotyping of GFP and FLT3 / ITD using genomic DNA from fin clip of WT siblings and Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at two months old . Fish 4 , 5 and 6 showed germline transmission of FLT3 / ITD transgene ( K ) . Data information : Scale bar = 500 μm .

O O O O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-TISSUE O O B-TISSUE O B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-TISSUE O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

Generation and characterization of FLT3/ITD-transgenic zebrafish. GFP expression was detected by florescent microscopy (F-H) and in blood circulation and thymus by WISH (I and J, blue arrow) in WT sibling and Runx1-FLT3/ITD transgenic zebrafish (F1) embryos at 4 dpf. FLT3/ITD-positive zebrafish (F1) were confirmed by PCR genotyping of GFP and FLT3/ITD using genomic DNA from fin clip of WT siblings and Runx1-FLT3/ITD transgenic zebrafish (F1) at two months old. Fish 4, 5 and 6 showed germline transmission of FLT3/ITD transgene (K). Data information: Scale bar = 500 μm.


Kidney marrow ( KM ) was collected from Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at 18 months old . The morphology and hematopoietic composition of KM from WT siblings ( n = 6 ) and Runx1 - FLT3 / ITD transgenic ( n = 6 ) zebrafish were examined by Giemsa staining ( L ) and flow cytometry ( abbreviation for panel M : M , Myeloid cells ; P , Progenitor cells ; L , Lymphoid cells ; E , Erythroid cells ) . Data are presented in box plot . * * P < 0 . 01 ( Student ' s t - test ) .

B-TISSUE I-TISSUE O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O

Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by Giemsa staining (L) and flow cytometry (abbreviation for panel M: M, Myeloid cells; P, Progenitor cells; L, Lymphoid cells; E, Erythroid cells). Data are presented in box plot. **P<0.01 (Student's t-test).


Kidney marrow ( KM ) was collected from Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at 18 months old . The morphology and hematopoietic composition of KM from WT siblings ( n = 6 ) and Runx1 - FLT3 / ITD transgenic ( n = 6 ) zebrafish were examined by flow cytometry Data are presented in box plot . The whiskers , boxes , and central lines in panel N represented the minimum - to - maximum values , 25th - to - 75th percentile , and the 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - test ) .

B-TISSUE I-TISSUE O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by flow cytometry Data are presented in box plot. The whiskers, boxes, and central lines in panel N represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).


O Expression of fst was detected by RT - qPCR in KM from WT sibling and Runx1 - FLT3 / ITD transgenic zebrafish at 18 months old . The RT - qPCR experiments were performed in triplicates and data were presented as mean ± SEM . * * P < 0 . 01 .

O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

O Expression of fst was detected by RT-qPCR in KM from WT sibling and Runx1-FLT3/ITD transgenic zebrafish at 18 months old. The RT-qPCR experiments were performed in triplicates and data were presented as mean ± SEM. **P<0.01.


P Detection of FST expression , p - ERK1 / 2 , and p - CREB in mononuclear cells from normal peripheral blood stem cell ( PBSC ) and FLT3 / ITD AML patients ( diagnostic samples with leukemia blasts > 80 % ) by Western blotting . ^ : non - specific staining of p - ATF1 protein due to the conserved motif .

O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-GENEPROD O O B-DISEASE B-ORGANISM O O O O B-DISEASE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O

P Detection of FST expression, p-ERK1/2, and p-CREB in mononuclear cells from normal peripheral blood stem cell (PBSC) and FLT3/ITD AML patients (diagnostic samples with leukemia blasts > 80%) by Western blotting. ^: non-specific staining of p-ATF1 protein due to the conserved motif.


B - C The direct binding of p - CREB to human FST promoter was detected by ChIP - PCR ( B ) and ChIP - qPCR ( C ) . c - Fos was used as positive control of p - CREB target gene . Normal IgG was used as negative control of ChIP . Data information the experiments were performed in triplicates , and the data were presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) .

O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-ORGANISM B-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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-C The direct binding of p-CREB to human FST promoter was detected by ChIP-PCR (B) and ChIP-qPCR (C). c-Fos was used as positive control of p-CREB target gene. Normal IgG was used as negative control of ChIP. Data information the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


D Dual luciferase assay demonstrating the direct binding of pCREB on human FST promoter . pRL - CMV , Renilla luciferase vector ; pGL - CRE - and pGL - CRE + , firefly luciferase expression driven by human FST promoter with deleted CRE site ( CRE - ) or wildtype ( CRE + ) ; p - GFPSpark , GFP expressing vector ; p - CREBY134F , CREBY134F - GFP expressing vector . Data information : the experiments were performed in triplicates , and the data were presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O B-ORGANISM B-GENEPROD O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O B-ORGANISM B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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 Dual luciferase assay demonstrating the direct binding of pCREB on human FST promoter. pRL-CMV, Renilla luciferase vector; pGL-CRE- and pGL-CRE+, firefly luciferase expression driven by human FST promoter with deleted CRE site (CRE-) or wildtype (CRE+); p-GFPSpark, GFP expressing vector; p-CREBY134F, CREBY134F-GFP expressing vector. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


E FST expression and FLT3 / ITD signaling were detected by Western blotting in Ba / F3 - parental ( P in short ) and Ba / F3 - FLT3 / ITD ( ITD in short ) cells .

O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O O O O

E FST expression and FLT3/ITD signaling were detected by Western blotting in Ba/F3-parental (P in short) and Ba/F3-FLT3/ITD (ITD in short) cells.


F - H Phosphor - flow analysis of p - CREB in Ba / F3 - parental , Ba / F3 - FLT3 / ITD , and Ba / F3 - FLT3 / ITD cells - treated with FLT3 inhibitor Quizartinib ( Qui in short ) . Isotype antibody were used as control to calculate the mean fluorescence intensity ( MFI ) ratio ( F - G ) . The transcription and expression of Fst was detected by RT - qPCR after Quizartinib treatment ( 10 nM ) in Ba / F3 - FLT3 / ITD cells for one day ( H ) . Data information : the experiments were performed in triplicates , and the data were presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H Phosphor-flow analysis of p-CREB in Ba/F3-parental, Ba/F3-FLT3/ITD, and Ba/F3-FLT3/ITD cells-treated with FLT3 inhibitor Quizartinib (Qui in short). Isotype antibody were used as control to calculate the mean fluorescence intensity (MFI) ratio (F-G). The transcription and expression of Fst was detected by RT-qPCR after Quizartinib treatment (10 nM) in Ba/F3-FLT3/ITD cells for one day (H). Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


I - K The expression of FST and phosphorylation of CREB were detected by Western Blotting ( I and K ) and phospho - flow analysis ( J ) in MOLM - 13 ( I ) and Ba / F3 - FLT3 / ITD ( K ) cells treated with Quizartinib and BRD7389 for one day , respectively . Data information : the experiments were performed in triplicates , and the data were presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) .

O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K The expression of FST and phosphorylation of CREB were detected by Western Blotting (I and K) and phospho-flow analysis (J) in MOLM-13 (I) and Ba/F3-FLT3/ITD (K) cells treated with Quizartinib and BRD7389 for one day, respectively. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


L RSK and FST expression were detected by Western Blotting after p90RSK knockout by CRISPR / Cas9 in MOLM - 13 cells .

O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

L RSK and FST expression were detected by Western Blotting after p90RSK knockout by CRISPR/Cas9 in MOLM-13 cells.


M The phosphorylation of CREB and FST expression were detected by Western Blotting in Ba / F3 - FLT3 / ITD cells treated with CREB inhibitor 666 - 15 for one day . ^ : non - specific staining of p - ATF1 protein due to the conserved motif .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O

M The phosphorylation of CREB and FST expression were detected by Western Blotting in Ba/F3-FLT3/ITD cells treated with CREB inhibitor 666-15 for one day. ^: non-specific staining of p-ATF1 protein due to the conserved motif.


N CREB and FST expression were detected by Western Blotting after CREB knockout by CRISPR / Cas9 in MOLM - 13 cells .

N CREB and FST expression were detected by Western Blotting after CREB knockout by CRISPR/Cas9 in MOLM-13 cells.


O The growth of Ba / F3 - parental ( with IL - 3 ) , Ba / F3 - FLT3 / ITD ( without IL - 3 ) and Ba / F3 - FLT3 / ITD ( with IL - 3 ) cells was measured after three days treatment of CREB inhibitor 666 - 15 in vitro . Data information : the experiments were performed in triplicates , and the data were presented as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) .

O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O 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 growth of Ba/F3-parental (with IL-3), Ba/F3-FLT3/ITD (without IL-3) and Ba/F3-FLT3/ITD (with IL-3) cells was measured after three days treatment of CREB inhibitor 666-15 in vitro. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


P The rescue effect of CREB inhibitor 666 - 15 on FLT3 / ITD - induced dorsalization and axis duplication in zebrafish embryos at 1 dpf .

O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O

P The rescue effect of CREB inhibitor 666-15 on FLT3/ITD-induced dorsalization and axis duplication in zebrafish embryos at 1 dpf.


A FST expression in different AML cell lines was detected by Western Blotting .

O B-GENEPROD B-EXP_ASSAY O O B-DISEASE O O O O O B-EXP_ASSAY I-EXP_ASSAY O

A FST expression in different AML cell lines was detected by Western Blotting.


FST317 and FST344 overexpression resulted in significant increases of FST transcription by RT - qPCR and protein by Western Blot ( B ) Green , ML - 2 - GFP ; Blue , ML - 2 - FST317 ; Red , ML - 2 - FST344 . The RT - qPCR experiments were performed in triplicates ( B ) . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . ns : Not significant .

B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FST317 and FST344 overexpression resulted in significant increases of FST transcription by RT-qPCR and protein by Western Blot (B) Green, ML-2-GFP; Blue, ML-2-FST317; Red, ML-2-FST344. The RT-qPCR experiments were performed in triplicates (B). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.


FST317 and FST344 overexpression promoted ML - 2 cell growth in vitro ( C ) . Green , ML - 2 - GFP ; Blue , ML - 2 - FST317 ; Red , ML - 2 - FST344 . Data information : data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . ns : Not significant .

B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FST317 and FST344 overexpression promoted ML-2 cell growth in vitro (C). Green, ML-2-GFP; Blue, ML-2-FST317; Red, ML-2-FST344. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.