( f ) Immunoblot analysis of supernatants and lysates of differentiated THP - 1 cells treated as in e ; supernatants were collected after culture for 6 h in serum - free medium . Scale bars ( b , c ) , 10 μm . Data are representative of at least two experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of supernatants and lysates of differentiated THP-1 cells treated as in e; supernatants were collected after culture for 6 h in serum-free medium. Scale bars (b,c), 10 μm. Data are representative of at least two experiments.


( a ) Immunoblot analysis of ASC immunoprecipitates of differentiated THP - 1 cells transfected for 2h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , probed for ubiquitin ( Ub ) , then stripped and reprobed for K63 - linked ubiquitin ( Ub - K63 ) , then stripped and reprobed for ASC .

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

(a) Immunoblot analysis of ASC immunoprecipitates of differentiated THP-1 cells transfected for 2h with poly(dA:dT) (1.5 μg/ml), probed for ubiquitin (Ub), then stripped and reprobed for K63-linked ubiquitin (Ub-K63), then stripped and reprobed for ASC.


( b ) Immunoassay of differentiated THP - 1 cells treated as in a with protein - dissociation buffer , followed by immunoprecipitation of ASC ( 24 kilodaltons ( kDa ) ) and immunoblot analysis .

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

(b) Immunoassay of differentiated THP-1 cells treated as in a with protein-dissociation buffer, followed by immunoprecipitation of ASC (24 kilodaltons (kDa)) and immunoblot analysis.


( c ) Confocal microscopy of differentiated THP - 1 cells transfected for 2 h with poly ( dA : dT ) , immunostained for ASC ( red ) and polyubiquitin ( green ) . PolyUb , polyubiquitination . Scale bars , 10 μm . Brackets ( right margins in a , b ) indicate smear of ubiquitinated proteins . Data are representative of three ( a , c ) or two ( b ) experiments .

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

(c) Confocal microscopy of differentiated THP-1 cells transfected for 2 h with poly(dA:dT), immunostained for ASC (red) and polyubiquitin (green). PolyUb, polyubiquitination. Scale bars, 10 μm. Brackets (right margins in a,b) indicate smear of ubiquitinated proteins. Data are representative of three (a,c) or two (b) experiments.


( a ) Immunoblot analysis of lysates of human monocytes - macrophages primed with LPS ( 10 ng ) and then transfected with poly ( dA : dT ) ( 1 . 5 μg / ml ) or treated with nigericin ( 4 μM ) for 6 h ( numbers below lanes as in Fig . 1d ) .

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

(a) Immunoblot analysis of lysates of human monocytes-macrophages primed with LPS (10 ng) and then transfected with poly(dA:dT) (1.5 μg/ml) or treated with nigericin (4 μM) for 6 h (numbers below lanes as in Fig. 1d).


( b ) Immunoblot analysis of processed caspase - 1 and IL - 1β ( top ) and enzyme - linked immunosorbent assay of IL - 1β ( middle ) in supernatants of monocytes - macrophages treated for 6 h with various combinations ( bottom ) of poly ( dA : dT ) ( b ) or nigericin ( c ) , plus 3 - MA and amino - acid starvation . * P 0 . 01 ( unpaired t test ) .

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

(b) Immunoblot analysis of processed caspase-1 and IL-1β (top) and enzyme-linked immunosorbent assay of IL-1β (middle) in supernatants of monocytes-macrophages treated for 6 h with various combinations (bottom) of poly(dA:dT) (b) or nigericin (c), plus 3-MA and amino-acid starvation. *P 0.01 (unpaired t test).


( d ) Confocal microscopy of LPS - primed human monocytes - macrophages transfected for 3 h with poly ( dA : dT ) ( top ) , treated for 3 h with nigericin ( middle ) or left unstimulated ( bottom ) , immunostained for p62 ( green ) and ASC ( red ) ; arrows indicate colocalized proteins . Second row , three - dimensional volume rendering reconstructed from confocal z - stack images . Scale bars , 10 μm . Data are representative of at least two experiments .

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

(d) Confocal microscopy of LPS-primed human monocytes-macrophages transfected for 3 h with poly(dA:dT) (top), treated for 3 h with nigericin (middle) or left unstimulated (bottom), immunostained for p62 (green) and ASC (red); arrows indicate colocalized proteins. Second row, three-dimensional volume rendering reconstructed from confocal z-stack images. Scale bars, 10 μm. Data are representative of at least two experiments.


( a ) Immunoblot analysis of lysates of LPS ( 20 ng / ml ) - primed mouse BMDMs exposed to LPS ( 500 ng / ml ) and ATP ( 3 μM ) , poly ( dA : dT ) ( 1 . 5 μg / ml ) or uric acid crystals ( 50 μg / ml ) , detecting GTP - bound RalB ( collected by RaBP1 - agarose affinity purification ) and RalB in total lysates .

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

(a) Immunoblot analysis of lysates of LPS (20 ng/ml)-primed mouse BMDMs exposed to LPS (500 ng/ml) and ATP (3 μM), poly(dA:dT) (1.5 μg/ml) or uric acid crystals (50 μg/ml), detecting GTP-bound RalB (collected by RaBP1-agarose affinity purification) and RalB in total lysates.


( b ) Immunoblot analysis of lysates and supernatants of mouse BMDMs transfected with control or RalB - specific siRNA on 2 sequential days , then primed with LPS and exposed for 6 h to poly ( dA : dT ) ( 1 . 5 μg / ml ) , probed for RalB , LC3 and IL - 1β ; supernatants were collected after overnight culture in serum - free medium ( numbers below lanes as in Fig . 1d ) . Nonsp , nonspecific band .

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

(b) Immunoblot analysis of lysates and supernatants of mouse BMDMs transfected with control or RalB-specific siRNA on 2 sequential days, then primed with LPS and exposed for 6 h to poly(dA:dT) (1.5 μg/ml), probed for RalB, LC3 and IL-1β; supernatants were collected after overnight culture in serum-free medium (numbers below lanes as in Fig. 1d). Nonsp, nonspecific band.


( c ) Immunoblot analysis of lysates of wild - type and Aim2 − / − mouse BMDMs transfected with control or AIM2 - specific siRNA on 2 sequential days , primed for 2 h with LPS and then exposed for 1 h to poly ( dA : dT ) ( numbers below lanes as in Fig . 1d ) .

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

(c) Immunoblot analysis of lysates of wild-type and Aim2−/− mouse BMDMs transfected with control or AIM2-specific siRNA on 2 sequential days, primed for 2 h with LPS and then exposed for 1 h to poly(dA:dT) (numbers below lanes as in Fig. 1d).


( d ) Immunoblot analysis of ASC immunoprecipitates from LPS - primed mouse BMDMs left untreated or transfected for 1 h with poly ( dA : dT ) and treated for 30 min with LPS and ATP . Mock , immunoprecipitation with control antibody .

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

(d) Immunoblot analysis of ASC immunoprecipitates from LPS-primed mouse BMDMs left untreated or transfected for 1 h with poly(dA:dT) and treated for 30 min with LPS and ATP. Mock, immunoprecipitation with control antibody.


( e ) Confocal microscopy of LPS - primed mouse BMDMs left untransfected or transfected for 30 min with poly ( dA : dT ) , immunostained for RalB and AIM2 ( left three columns ) ; RalB and ASC ( middle columns ) ; and beclin - 1 and AIM2 ( right two columns ) . Bottom row , 3 × electronic enlargement of areas outlined above . Scale bars , 10 μm . Data are representative of at least two experiments .

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

(e) Confocal microscopy of LPS-primed mouse BMDMs left untransfected or transfected for 30 min with poly(dA:dT), immunostained for RalB and AIM2 (left three columns); RalB and ASC (middle columns); and beclin-1 and AIM2 (right two columns). Bottom row, 3× electronic enlargement of areas outlined above. Scale bars, 10 μm. Data are representative of at least two experiments.


( a ) Species relative abundance as a function of time for all pairwise communities . Experimental measurements and model fits based on T3 are represented as data points and lines , respectively . In each subplot , time and species relative abundance are displayed on the x and y - axis , respectively . Stars denote datasets with a sum of mean squared errors greater than 0 . 15 . Error bars represent 1 s . d . from the mean of at least three biological replicates . ( b ) Temporal changes in species relative abundance of a selected set of pairwise assemblages inoculated at 5 % species A , 95 % species B or 95 % species A , 5 % species B based on OD600 values . Time and relative abundance are represented on the x and y - axis , respectively . Data points and lines represent experimental measurements and model fits to T3 , respectively . Error bars represent 1 s . d . from the mean of at least three biological replicates . Stars denote datasets with a sum of mean squared errors greater than 0 . 15 . ( c ) Inferred inter - species interaction coefficients for the gLV model trained on T3 . Gray and green edges denote negative ( αij < 0 ) and positive ( αij > 0 ) interaction coefficients . The edge width and node size represent the magnitude of the inter - species interaction coefficient and steady - state monospecies abundance ( xe = - μiαii - 1 ) , respectively . To highlight significant interactions , inter - species interaction coefficients with a magnitude less than 1e - 5 were not displayed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Species relative abundance as a function of time for all pairwise communities. Experimental measurements and model fits based on T3 are represented as data points and lines, respectively. In each subplot, time and species relative abundance are displayed on the x and y-axis, respectively. Stars denote datasets with a sum of mean squared errors greater than 0.15. Error bars represent 1 s.d. from the mean of at least three biological replicates. (b) Temporal changes in species relative abundance of a selected set of pairwise assemblages inoculated at 5% species A, 95% species B or 95% species A, 5% species B based on OD600 values. Time and relative abundance are represented on the x and y-axis, respectively. Data points and lines represent experimental measurements and model fits to T3, respectively. Error bars represent 1 s.d. from the mean of at least three biological replicates. Stars denote datasets with a sum of mean squared errors greater than 0.15. (c) Inferred inter-species interaction coefficients for the gLV model trained on T3. Gray and green edges denote negative (αij < 0) and positive (αij > 0) interaction coefficients. The edge width and node size represent the magnitude of the inter-species interaction coefficient and steady-state monospecies abundance (xe = -μiαii-1), respectively. To highlight significant interactions, inter-species interaction coefficients with a magnitude less than 1e-5 were not displayed.


( a ) Stacked bar plots of all 11 and 12 - member ( full ) multi - species communities . The text above each subplot denotes the absent organism in the community . Time and relative abundance are represented on the x and y - axis of each subplot , respectively . Colors represent different organisms in the community . ( b ) Difference in community assembly for single - species dropout communities . Difference in community assembly was computed as $ \ sum _ { i = 1 , \ } ^ { 11 } { \ frac { 1 } { 7 } \ sum _ { j = 1 } ^ { 7 } { ( { \ widehat { v } } _ { ji , FULL } - v _ { ji , X } ) } } ^ { 2 } $ where i and j represent species and time points , respectively . X represents a single - species dropout community lacking the organism X . v ̂ denotes the renormalized relative abundance of the shared set of species in the full ( 12 - member ) and single - species dropout community . Here $ { \ widehat { v } } _ { i } = \ \ frac { v _ { i } } { \ sum _ { i \ \ in C } ^ { } v _ { i } } $ where C denotes the set of 11 shared organisms in the full and single - species dropout community . Data points represent biological replicates ( n = 6 , circles ) and the mean of biological replicates ( blue squares ) , respectively . ( c ) Box plot of the Pearson correlation coefficient for three model training sets ( left ) including T1 : monospecies ( M ) ; T2 : M and pairwise 1 ( PW1 ) ; and T3 : M , PW1 and pairwise 2 ( PW2 ) . On each box , the red line represents the median , the edges of the box are the 25th and 75th percentiles , the whiskers extend to the most extreme data points and the outliers are plotted as red crosses . ( d ) Box plot of the sum of mean squared errors for the model predictions of the time - resolved measurements of multi - species communities trained on T1 , T2 or T3 training sets . On each box , the red line represents the median , the edges of the box are the 25th and 75th percentiles , the whiskers extend to the most extreme data points and the outliers are plotted as red crosses . ( e ) Comparison between model prediction based on T1 - T4 and experimental data for each organism in the full community as a function of time . On each subplot , the x and y - axis represent time and relative abundance of each species , respectively . Data points ( red ) and lines denote experimental data and model predictions based on T1 - T4 . Species names are displayed in the upper left corner of each subplot . Error bars represent 1 s . d . from the mean of six biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stacked bar plots of all 11 and 12-member (full) multi-species communities. The text above each subplot denotes the absent organism in the community. Time and relative abundance are represented on the x and y-axis of each subplot, respectively. Colors represent different organisms in the community. (b) Difference in community assembly for single-species dropout communities. Difference in community assembly was computed as $\sum_{i = 1,\ }^{11}{\frac{1}{7}\sum_{j = 1}^{7}{({\widehat{v}}_{ji,FULL} - v_{ji,X})}}^{2}$ where i and j represent species and time points, respectively. X represents a single-species dropout community lacking the organism X. v̂ denotes the renormalized relative abundance of the shared set of species in the full (12-member) and single-species dropout community. Here ${\widehat{v}}_{i} = \ \frac{v_{i}}{\sum_{i\ \in C}^{}v_{i}}$ where C denotes the set of 11 shared organisms in the full and single-species dropout community. Data points represent biological replicates (n = 6, circles) and the mean of biological replicates (blue squares), respectively. (c) Box plot of the Pearson correlation coefficient for three model training sets (left) including T1: monospecies (M); T2: M and pairwise 1 (PW1); and T3: M, PW1 and pairwise 2 (PW2). On each box, the red line represents the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points and the outliers are plotted as red crosses. (d) Box plot of the sum of mean squared errors for the model predictions of the time-resolved measurements of multi-species communities trained on T1, T2 or T3 training sets. On each box, the red line represents the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points and the outliers are plotted as red crosses. (e) Comparison between model prediction based on T1-T4 and experimental data for each organism in the full community as a function of time. On each subplot, the x and y-axis represent time and relative abundance of each species, respectively. Data points (red) and lines denote experimental data and model predictions based on T1-T4. Species names are displayed in the upper left corner of each subplot. Error bars represent 1 s.d. from the mean of six biological replicates.


( a ) Bipartite network of species ( left ) and metabolites ( right ) for metabolites that decreased by at least two - fold compared to the abundance of each metabolite at the beginning of the experiment . Colors represent modules containing many overlapping interactions in the network . Network partitioning into modules was performed using BiMat ( Flores Metabolites in bold were depleted and secreted by distinct organisms . ( b ) Bipartite network of species ( left ) and metabolites ( right ) for metabolites that increased in abundance by at least two - fold compared to the beginning of the experiment . Metabolites highlighted in bold were depleted or secreted by different species . Colors represent modules containing many overlapping interactions in the network . Network partitioning into modules was performed using BiMat (

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(a) Bipartite network of species (left) and metabolites (right) for metabolites that decreased by at least two-fold compared to the abundance of each metabolite at the beginning of the experiment. Colors represent modules containing many overlapping interactions in the network. Network partitioning into modules was performed using BiMat(Flores Metabolites in bold were depleted and secreted by distinct organisms. (b) Bipartite network of species (left) and metabolites (right) for metabolites that increased in abundance by at least two-fold compared to the beginning of the experiment. Metabolites highlighted in bold were depleted or secreted by different species. Colors represent modules containing many overlapping interactions in the network. Network partitioning into modules was performed using BiMat(Flores


( c ) Scatter plot of the number of consumed metabolites that decreased by at least two - fold compared to the beginning of the experiment vs . the OD600 value of the monospecies culture at the corresponding time point . Error bars represent 1 s . d . from the mean of three biological replicates .

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(c) Scatter plot of the number of consumed metabolites that decreased by at least two-fold compared to the beginning of the experiment vs. the OD600 value of the monospecies culture at the corresponding time point. Error bars represent 1 s.d. from the mean of three biological replicates.


Example time trace when no binding event occurs ( green laser excitation ) . EmCCD image shows signal from green and red channels before the addition of Cy3 - labelled DNA when illuminated with green laser . Size of EmCCD image is 25µm x 50 µm . Example time trace showing a binding event ( green laser excitation ) . EmCCD image shows signal from green and red channels after the addition of Cy3 - labeled DNA when illuminated with green laser . Size of EmCCD image is 25µm x 50 µm .

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Example time trace when no binding event occurs (green laser excitation). EmCCD image shows signal from green and red channels before the addition of Cy3-labelled DNA when illuminated with green laser. Size of EmCCD image is 25µm x 50 µm.   Example time trace showing a binding event (green laser excitation). EmCCD image shows signal from green and red channels after the addition of Cy3-labeled DNA when illuminated with green laser. Size of EmCCD image is 25µm x 50 µm.


Example time traces when DNA containing 3 PAM sequences is added ( top ) and when DNA containing a single PAM is added showing both short and long binding events ( bottom ) .

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Example time traces when DNA containing 3 PAM sequences is added (top) and when DNA containing a single PAM is added showing both short and long binding events (bottom).


FRET histograms of all binding events for each construct . Y axis represents total number of counts , divided by the number of immobilized molecules in the field of view .

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FRET histograms of all binding events for each construct. Y axis represents total number of counts, divided by the number of immobilized molecules in the field of view.


Bar plot of all dwelltime values for all constructs showing short binding events for all DNA and long events only for the constructs containing PAM sequences . The times shown are mean values of dwelltimes obtained during four different experiments on four different days . Error bars represent standard error of the mean .

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Bar plot of all dwelltime values for all constructs showing short binding events for all DNA and long events only for the constructs containing PAM sequences. The times shown are mean values of dwelltimes obtained during four different experiments on four different days. Error bars represent standard error of the mean.


Example dwelltime histograms from negative control and a construct containing a single PAM . Negative control dwelltime distribution is characterized by a single exponential decay ( top ) and dwelltime distribution for PAM - containing DNA is fitted by a double - exponential decay ( blue line ) ( bottom ) . Equation for the double exponential decay fit : y = A1e - t / τ1 + A2e - t / τ2 ; ΔA1 = 796 . 4 ΔA2 = 28 . 3 . Red line shows what a single - exponential decay fit for such distribution would be . Errors represent standard error of the mean .

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

Example dwelltime histograms from negative control and a construct containing a single PAM. Negative control dwelltime distribution is characterized by a single exponential decay (top) and dwelltime distribution for PAM-containing DNA is fitted by a double-exponential decay (blue line) (bottom). Equation for the double exponential decay fit: y=A1e-t/τ1 + A2e-t/τ2; ΔA1= 796.4 ΔA2= 28.3. Red line shows what a single-exponential decay fit for such distribution would be. Errors represent standard error of the mean.


Scatter plot showing the average dwelltime ( Δτav ) values for the cases of 2 ( d2 ) and 4 ( d4 ) nucleotide separation between the PAM sites . The values are averages of four measurements over four different days for each case . Error bars represent the standard error of the mean . The average dwelltime was obtained using the equation τav = ( A1Δτ12 + A2τ22 ) / ( A1Δτ1 + A2Δτ2 )

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

Scatter plot showing the average dwelltime (Δτav) values for the cases of 2 (d2) and 4 (d4) nucleotide separation between the PAM sites. The values are averages of four measurements over four different days for each case. Error bars represent the standard error of the mean. The average dwelltime was obtained using the equation τav=(A1Δτ12+A2τ22)/(A1Δτ1+A2Δτ2)


Example trace showing direct FRET transitions for a 5 - PAM containing DNA construct with separation between PAM sites being 4 nucleotides .

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Example trace showing direct FRET transitions for a 5-PAM containing DNA construct with separation between PAM sites being 4 nucleotides.


Histogram showing the percentage of events that are longer than τ1 showing FRET fluctuations for multiple PAM constructs with PAM separation of 4 nucleotides . The values are averages of four measurements over four different days . The error bars represent standard error of the mean .

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

Histogram showing the percentage of events that are longer than τ1 showing FRET fluctuations for multiple PAM constructs with PAM separation of 4 nucleotides. The values are averages of four measurements over four different days. The error bars represent standard error of the mean.


FRET histogram of all binding events from the construct containing a single PAM next to the partial target . Y axis represents total number of counts .

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

FRET histogram of all binding events from the construct containing a single PAM next to the partial target. Y axis represents total number of counts.


Example FRET traces from single - target multiple - PAM experiments . Top trace shows an example of a single - step binding event and a binding event that starts at a lower FRET state before transitioning to productive binding high - FRET state . Bottom trace shows an example of FRET fluctuations between the high - FRET state and lower FRET states .

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

Example FRET traces from single-target multiple-PAM experiments. Top trace shows an example of a single-step binding event and a binding event that starts at a lower FRET state before transitioning to productive binding high-FRET state. Bottom trace shows an example of FRET fluctuations between the high-FRET state and lower FRET states.


A histogram showing the percentage of dynamic . The percentage values were obtained from five experiments on five different days . Error bars represent standard error of the mean . The percentage of events showing dynamic binding behavior was calculated by dividing the number of binding events that showed such behavior by the total number of binding events .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 histogram showing the percentage of dynamic. The percentage values were obtained from five experiments on five different days. Error bars represent standard error of the mean. The percentage of events showing dynamic binding behavior was calculated by dividing the number of binding events that showed such behavior by the total number of binding events.


A scatter plot showing the time Cas9 spends in a lower FRET state before transitioning to a high - FRET state . The dwelltimes increase with the number of PAMs adjacent to the target . Such dwelltime could not be determined for a construct containing a single PAM . The values were obtained from five experiments on five different days . Error bars represent standard error of the mean .

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

A scatter plot showing the time Cas9 spends in a lower FRET state before transitioning to a high-FRET state. The dwelltimes increase with the number of PAMs adjacent to the target. Such dwelltime could not be determined for a construct containing a single PAM. The values were obtained from five experiments on five different days. Error bars represent standard error of the mean.


Example fluorescence traces showing Cas9 transitioning between H and L targets with increasing target distances ( right ) . FRET histograms constructed from events that show fluctuations showing two FRET peaks corresponding to binding to either target . High FRET peak stays the same in each histogram , but lower FRET peak moves to lower FRET values as the distance between targets increases .

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Example fluorescence traces showing Cas9 transitioning between H and L targets with increasing target distances (right). FRET histograms constructed from events that show fluctuations showing two FRET peaks corresponding to binding to either target. High FRET peak stays the same in each histogram, but lower FRET peak moves to lower FRET values as the distance between targets increases.


A scatter plot showing the probability of Cas9 transitioning between targets before dissociation . The values are averages from five measurements and error bars represent standard error of the mean . The transition probability ( p = T / ( T + D ) ) was calculated by summing up all the number of FRET transitions ( T ) and dividing T by the sum of T and the total number of dissociation events ( D ) .

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A scatter plot showing the probability of Cas9 transitioning between targets before dissociation. The values are averages from five measurements and error bars represent standard error of the mean. The transition probability (p = T / (T+D)) was calculated by summing up all the number of FRET transitions (T) and dividing T by the sum of T and the total number of dissociation events (D).


A histogram showing all dwelltimes from single - target controls and tandem - target experiments . τ1 is similar for single - target control and tandem target experiments . τ2 only appears in tandem target experiments and is over 20 - fold higher than τ1 or τst . The values represent the mean dwelltimes that were obtained by fitting dwelltime distributions by either single or double exponential decay function from five measurements . Error bars represent standard error of the mean .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 histogram showing all dwelltimes from single-target controls and tandem-target experiments. τ1 is similar for single-target control and tandem target experiments. τ2 only appears in tandem target experiments and is over 20-fold higher than τ1 or τst. The values represent the mean dwelltimes that were obtained by fitting dwelltime distributions by either single or double exponential decay function from five measurements. Error bars represent standard error of the mean.


Histogram showing the percentage of binding events starting at either target . The values are averages of five measurements over three different days . Error bars represent standard error of the mean .

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

Histogram showing the percentage of binding events starting at either target. The values are averages of five measurements over three different days. Error bars represent standard error of the mean.


An example trace showing the addition of Cy3 - labeled DNA target and the first binding event . Δτon is indicated .

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

An example trace showing the addition of Cy3-labeled DNA target and the first binding event. Δτon is indicated.


Scatter plot showing the on - target binding rate ( kon - target ) for each construct . Binding rate decreases moderately with increasing number of adjacent PAMs . The values are averages of four measurements over three days . Error bars represent standard error of the mean . kon - target was calculated using the equation kon - target = 1 / ( CxΔτon ) where C is the molarity of DNA target in the channel .

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

Scatter plot showing the on-target binding rate (kon-target) for each construct. Binding rate decreases moderately with increasing number of adjacent PAMs. The values are averages of four measurements over three days. Error bars represent standard error of the mean. kon-target was calculated using the equation kon-target=1/ (CxΔτon) where C is the molarity of DNA target in the channel.


B . Two - hybrid interaction between GBD - AC40 and WT or mutant GAD - IN1578 - 635 . Alanine substitutions in IN1578 - 635 are indicated . Cells were plated in two - fold serial dilutions on DO - Leu - Trp - His plates . No growth or protein expression defects were detected ( Figs EV1A - B ) . + , interaction ; - , no interaction .

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B. Two-hybrid interaction between GBD-AC40 and WT or mutant GAD-IN1578-635. Alanine substitutions in IN1578-635 are indicated. Cells were plated in two-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected (Figs EV1A-B). +, interaction; -, no interaction.


C . Two - hybrid interaction between GBD - AC40 and different IN1 regions fused to GAD , as indicated . Cells were plated in ten - fold serial dilutions on DO - Leu - Trp - His plates . No growth or protein expression defects were detected ( Figs EV1C - D ) . + , interaction ; - , no interaction .

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C. Two-hybrid interaction between GBD-AC40 and different IN1 regions fused to GAD, as indicated. Cells were plated in ten-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected (Figs EV1C-D). +, interaction; -, no interaction.


D . In vitro interaction between AC40 and IN1 proteins co - expressed in E . coli . Co - precipitation of WT or mutated ( K617A , S621A , or L622A ) IN1 - C - tag using AC40 - Twin - Strep - tag as bait from bacterial protein extracts co - expressing ( + ) or not ( - ) these proteins . Expected sizes are 41 kDa for AC40 - Twin - Strep - tag and 100 kDa for IN1 - C - tag ( WT and mutants ) .

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

D. In vitro interaction between AC40 and IN1 proteins co-expressed in E. coli. Co-precipitation of WT or mutated (K617A, S621A, or L622A) IN1-C-tag using AC40-Twin-Strep-tag as bait from bacterial protein extracts co-expressing (+) or not (-) these proteins. Expected sizes are 41 kDa for AC40-Twin-Strep-tag and 100 kDa for IN1-C-tag (WT and mutants).


A . Localization of GFP2 - IN1 bNLS variants in yeast cells by direct fluorescence microscopy . Nup49 - mCherry signal was used to visualize the location of the nuclear envelope . Corresponding DIC images are shown . Scale bar is 5 μm .

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A. Localization of GFP2-IN1 bNLS variants in yeast cells by direct fluorescence microscopy. Nup49-mCherry signal was used to visualize the location of the nuclear envelope. Corresponding DIC images are shown. Scale bar is 5 μm.


B . Retrotransposition frequency ( log scale ) of pGAL1 - Ty1his3AI bearing the indicated substitutions of conserved residues in a spt3 - 101 rad52 ∆ strain . IN1 catalytic core domain mutant D154A is defective for integration and NLS mutant ( KKR628 - 630GGT ) for nuclear import . Values are mean ± SD , n = 4 experiments , each performed with four independent colonies . * * * p ≤ 0 . 001 , otherwise not significant ( Student t - test ) .

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B. Retrotransposition frequency (log scale) of pGAL1-Ty1his3AI bearing the indicated substitutions of conserved residues in a spt3-101 rad52∆ strain. IN1 catalytic core domain mutant D154A is defective for integration and NLS mutant (KKR628-630GGT) for nuclear import. Values are mean±SD, n=4 experiments, each performed with four independent colonies. ***p ≤ 0.001, otherwise not significant (Student t-test).


A . Co - immunoprecipitation of ectopic IN1 using C160 - HA as bait , from yeast protein extracts expressing WT or mutated IN1 - Strep ( K617A , S621A , or L622A ) from the GAL1 promoter in the presence of galactose . Expected sizes are 160 kDa for C160 - HA and 100 kDa for IN1 - Strep ( WT and mutants ) .

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A. Co-immunoprecipitation of ectopic IN1 using C160-HA as bait, from yeast protein extracts expressing WT or mutated IN1-Strep (K617A, S621A, or L622A) from the GAL1 promoter in the presence of galactose. Expected sizes are 160 kDa for C160-HA and 100 kDa for IN1-Strep (WT and mutants).


B . Quantitative ChIP analysis of HA - IN1 enrichment at Pol III - transcribed genes . Immunoprecipitated DNA from yeast cells producing ectopic IN1 is expressed as a value relative to that of the input . Pol III transcribed - genes : tDNA - Ile and tDNA - Leu families ( 16 and 22 genes , respectively ) and the unique SCR1 gene . GAL1 ORF serves as a control . Data represent means ± SD ( n ≥ 3 ) .

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

B. Quantitative ChIP analysis of HA-IN1 enrichment at Pol III-transcribed genes. Immunoprecipitated DNA from yeast cells producing ectopic IN1 is expressed as a value relative to that of the input. Pol III transcribed-genes: tDNA-Ile and tDNA-Leu families (16 and 22 genes, respectively) and the unique SCR1 gene. GAL1 ORF serves as a control. Data represent means ± SD (n ≥ 3).


C . Fold enrichment of WT HA - IN1 over input at all the genes where HA - IN1 has been detected by ChIP - seq analysis . Each dot represents a gene . Grey , tDNAs ; Red , other Pol III - transcribed genes ; Blue , Pol II - transcribed genes . The dashed line indicates the separation between Pol II and Pol III - transcribed genes .

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C. Fold enrichment of WT HA-IN1 over input at all the genes where HA-IN1 has been detected by ChIP-seq analysis. Each dot represents a gene. Grey, tDNAs ; Red, other Pol III-transcribed genes ; Blue, Pol II-transcribed genes. The dashed line indicates the separation between Pol II and Pol III-transcribed genes.


D . WT and mutant IN1 association with all Pol III - transcribed genes . Values obtained from ChIP seq analysis have been normalized in log2 RPKM ( reads per kilobase per million mapped reads ) . Control is anti - HA immunoprecipitation of chromatin extracts in cells expressing IN1 - Strep . Immunopurified DNA from 3 - 4 independent experiments was combined in one replicate . Data are presented as boxplots showing the median , the first quartile , and the third quartile . Error bars show minimum and maximum values .

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

D. WT and mutant IN1 association with all Pol III-transcribed genes. Values obtained from ChIP seq analysis have been normalized in log2 RPKM (reads per kilobase per million mapped reads). Control is anti-HA immunoprecipitation of chromatin extracts in cells expressing IN1-Strep. Immunopurified DNA from 3-4 independent experiments was combined in one replicate. Data are presented as boxplots showing the median, the first quartile, and the third quartile. Error bars show minimum and maximum values.


E . Genome - wide occupancy profiles ( top ) and heatmaps ( bottom ) of WT and HA - IN1 mutants over a 1kb window upstream and downstream of all tDNAs . Pearson correlation to WT values corresponding to Fig EV2B are indicated .

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E. Genome-wide occupancy profiles (top) and heatmaps (bottom) of WT and HA-IN1 mutants over a 1kb window upstream and downstream of all tDNAs. Pearson correlation to WT values corresponding to Fig EV2B are indicated.


F . WT and mutant IN1 association to all Pol - II transcribed genes respectively . Control , replicates and data presentation , as described for panel D . Regions used for calculation are gene bodies .

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F. WT and mutant IN1 association to all Pol-II transcribed genes respectively. Control, replicates and data presentation, as described for panel D. Regions used for calculation are gene bodies.


G . Recruitment of IN1 along the SCR1 gene locus . Top . ChIP ‐ qPCR analysis as described for panel Fig 3B . Data represent means ± SD ( n = 3 ) . Bottom . Schematic representation of the SCR1 locus with DNA amplicon positions . TATA , A and B boxes and terminator of transcription ( T ) are indicated .

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G. Recruitment of IN1 along the SCR1 gene locus. Top. ChIP‐qPCR analysis as described for panel Fig 3B. Data represent means ± SD (n = 3). Bottom. Schematic representation of the SCR1 locus with DNA amplicon positions. TATA, A and B boxes and terminator of transcription (T) are indicated.


A . Co - immunoprecipitation of ectopic IN1 using TAP - tagged - A190 as bait from yeast protein extracts expressing WT or the indicated IN1 - HBH mutants from a pTet - Off promoter in the absence of doxycycline . Expected sizes are 204 kDa for A190 - HA and 82 kDa for IN1 - Strep ( WT and mutants ) .

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A. Co-immunoprecipitation of ectopic IN1 using TAP-tagged-A190 as bait from yeast protein extracts expressing WT or the indicated IN1-HBH mutants from a pTet-Off promoter in the absence of doxycycline. Expected sizes are 204 kDa for A190-HA and 82 kDa for IN1-Strep (WT and mutants).


B . Genome browser visualization of HA - IN1 occupancy at the RDN1 locus . Occupancy of WT HA - IN1 and K617A , S621A and L622A HA - IN1 mutants is represented in each panel . Control , as described for panel 3D .

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B. Genome browser visualization of HA-IN1 occupancy at the RDN1 locus. Occupancy of WT HA-IN1 and K617A, S621A and L622A HA-IN1 mutants is represented in each panel. Control, as described for panel 3D.


C . Top . IN1 is recruited at Pol I - transcribed genes . ChIP ‐ qPCR analysis as described for panel 3B . Data represent means ± SD ( n = 3 ) . Bottom . Schematic representation of the RDN1 locus with DNA amplicon positions .

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C. Top. IN1 is recruited at Pol I-transcribed genes. ChIP‐qPCR analysis as described for panel 3B. Data represent means ± SD (n = 3). Bottom. Schematic representation of the RDN1 locus with DNA amplicon positions.


D . Recruitment of WT and mutant HA - IN1 ( K617A , S621A , or L622A ) at the 18S rDNA locus transcribed by Pol I . ChIP ‐ qPCR analysis as described for panel 3B . Data represent means ± SD ( n = 3 ) .

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D. Recruitment of WT and mutant HA-IN1 (K617A, S621A, or L622A) at the 18S rDNA locus transcribed by Pol I. ChIP‐qPCR analysis as described for panel 3B. Data represent means ± SD (n = 3).


A . Detection of de novo Ty1 insertions upstream of the SUF16 and SEO1 genes by PCR using a primer in HIS3 ( red triangle ) and a primer in the locus of interest ( blue triangle ) . Ty1 retrotransposition was induced in cells transformed with plasmids expressing WT or mutant ( IN1 K617A , S621A and L622A ) Ty1his3AI from the GAL1 promoter . Total genomic DNA was extracted from His + cells obtained from independent cultures .

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A. Detection of de novo Ty1 insertions upstream of the SUF16 and SEO1 genes by PCR using a primer in HIS3 (red triangle) and a primer in the locus of interest (blue triangle). Ty1 retrotransposition was induced in cells transformed with plasmids expressing WT or mutant (IN1 K617A, S621A and L622A) Ty1his3AI from the GAL1 promoter. Total genomic DNA was extracted from His+ cells obtained from independent cultures.


B . Genome - Wide Ty1 insertion frequencies at each genomic feature are clustered in a heatmap . Score is computed in column Z - score . ORFs , all Pol II - transcribed genes except gene at subtelomeres ; Pol I , one RDN37 copy ; up 1kb Pol III , 1kb upstream of all Pol III transcribed genes ; Subtelomeres , genomic coordinates corresponding to chromatin covered by Sir2 and Sir3 , when they are co - overexpressed ( Hocher et al , 2018 ) ; Random , 100 000 random Ty1 computed insertions in the genome .

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B. Genome-Wide Ty1 insertion frequencies at each genomic feature are clustered in a heatmap. Score is computed in column Z-score. ORFs, all Pol II-transcribed genes except gene at subtelomeres; Pol I, one RDN37 copy; up 1kb Pol III, 1kb upstream of all Pol III transcribed genes; Subtelomeres, genomic coordinates corresponding to chromatin covered by Sir2 and Sir3, when they are co-overexpressed (Hocher et al, 2018); Random, 100 000 random Ty1 computed insertions in the genome.


C . Detection of de novo Ty1 insertions upstream of RDN5 and in RDN37 genes by PCR as described for panel 5A . Four total genomic DNA preparation from panel A were randomly tested by PCR .

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C. Detection of de novo Ty1 insertions upstream of RDN5 and in RDN37 genes by PCR as described for panel 5A. Four total genomic DNA preparation from panel A were randomly tested by PCR.


D . Ty1 insertion profile upstream of tDNAs . Total genomic DNA extracted in ( B ) was prepared for Ty1 de novo integration event sequencing . Ty1 insertions are computed in a 1kb window upstream of all the 275 nuclear tDNAs ( position 0 in the graph ) . Each position is divided by the number of insertions at this position ( weight ) . The Smoothing curves indicate the general trend . Nucleosome center positions for the three first nucleosomes upstream of all tDNAs are from ( Brogaard et al , 2012 ) .

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D. Ty1 insertion profile upstream of tDNAs. Total genomic DNA extracted in (B) was prepared for Ty1 de novo integration event sequencing. Ty1 insertions are computed in a 1kb window upstream of all the 275 nuclear tDNAs (position 0 in the graph). Each position is divided by the number of insertions at this position (weight). The Smoothing curves indicate the general trend. Nucleosome center positions for the three first nucleosomes upstream of all tDNAs are from (Brogaard et al, 2012).


E . Ty1 insertion frequencies for each left and right subtelomere of chromosomes are clustered in a heatmap . Score is computed in row Z - score . Random , as described for panel B . High level of WT Ty1 - HIS3 integration in II - L is due to the presence of the tRNA gene tF ( GAA ) B .

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E. Ty1 insertion frequencies for each left and right subtelomere of chromosomes are clustered in a heatmap. Score is computed in row Z-score. Random, as described for panel B. High level of WT Ty1-HIS3 integration in II-L is due to the presence of the tRNA gene tF(GAA)B.


A . Two - hybrid interaction between GBD - AC40 ( left ) or GBD - Sir4 ( right ) and different GAD - IN5 or GAD - IN1 constructions . Cells were plated in five - fold serial dilutions on DO - Leu - Trp - His plates . No growth or protein expression defects were

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A. Two-hybrid interaction between GBD-AC40 (left) or GBD-Sir4 (right) and different GAD-IN5 or GAD-IN1 constructions. Cells were plated in five-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected


B . Genome - wide occupancy profiles ( top ) and heatmaps ( bottom ) of IN5 , WT , and indicated mutants , + / - 1kb upstream and downstream of all tDNAs .

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B. Genome-wide occupancy profiles (top) and heatmaps (bottom) of IN5, WT, and indicated mutants, +/- 1kb upstream and downstream of all tDNAs.


C . Genome browser visualization of different HA - IN occupancy for chromosome V ( chrV : 431129 . . 443275 ) . Occupancy of the indicated integrase is represented in each panel . Control , as panel 3D . The region contains tH ( GUG ) E2 , tK ( CUU ) E2 , tV ( ACC ) E1 , tI ( AAU ) E1 , SNR52 and SCR1 , all transcribed by Pol III .

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C. Genome browser visualization of different HA-IN occupancy for chromosome V (chrV:431129..443275). Occupancy of the indicated integrase is represented in each panel. Control, as panel 3D. The region contains tH(GUG)E2, tK(CUU)E2, tV(ACC)E1, tI(AAU)E1, SNR52 and SCR1, all transcribed by Pol III.


D . Detection of Ty5 , Ty5ΔTD5 + bNLS or Ty1 de novo integration events at HMR and HML loci , SCR1 and upstream of all glycine tDNAs , by PCR using a primer in HIS3 and a primer in the locus of interest . Retrotransposition was induced in cells transformed with WT or mutated pGAL1 - Ty5his3AI ( Ty5 or Ty5ΔTD5 + bNLS ) and pGAL1 - Ty1his3AI ( Ty1 ) . Total genomic DNA was extracted from His + cells obtained from independent cultures . Nucleosome position ( green circles ) is indicated on the right of the panels ( Brogaard et al , 2012 ) .

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

D. Detection of Ty5, Ty5ΔTD5+bNLS or Ty1 de novo integration events at HMR and HML loci, SCR1 and upstream of all glycine tDNAs, by PCR using a primer in HIS3 and a primer in the locus of interest. Retrotransposition was induced in cells transformed with WT or mutated pGAL1-Ty5his3AI (Ty5 or Ty5ΔTD5+bNLS) and pGAL1-Ty1his3AI (Ty1). Total genomic DNA was extracted from His+ cells obtained from independent cultures. Nucleosome position (green circles) is indicated on the right of the panels (Brogaard et al, 2012).


( A ) Ribbon representation of the Atg12 - Atg5 conjugate bound to Atg16N . Atg12 , Atg5 and Atg6 are coloured salmon pink , green and orange , respectively . Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 are shown with a stick model . The disordered C ‐ terminal region of Atg12 ( residues 182 - 184 ) is indicated with a broken line . The left figure was obtained by a 90 ° rotation of the right figure along the vertical axis . The amino ‐ and carboxy ‐ termini are denoted as N and C , respectively . All of the figures representing the molecular structures were generated with PyMOL [ 26 ] .

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

(A) Ribbon representation of the Atg12-Atg5 conjugate bound to Atg16N. Atg12, Atg5 and Atg6 are coloured salmon pink, green and orange, respectively. Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 are shown with a stick model. The disordered C‐terminal region of Atg12 (residues 182-184) is indicated with a broken line. The left figure was obtained by a 90° rotation of the right figure along the vertical axis. The amino‐ and carboxy‐termini are denoted as N and C, respectively. All of the figures representing the molecular structures were generated with PyMOL [ 26].


( B ) Stereo view of the detailed interactions between Atg12 and Atg5 . The side chains of the residues involved in the Atg12 - Atg5 interaction are shown with a stick model , in which nitrogen and oxygen atoms are coloured red and blue , respectively .

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

(B) Stereo view of the detailed interactions between Atg12 and Atg5. The side chains of the residues involved in the Atg12-Atg5 interaction are shown with a stick model, in which nitrogen and oxygen atoms are coloured red and blue, respectively.


( C ) Electron density map of the isopeptide linkage between Atg12 Gly 186 and Atg5 Lys 149 . The simulated annealing Fo - Fc difference Fourier map was calculated by omitting Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 , and is shown with black meshes at 5 . 0σ .

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

(C) Electron density map of the isopeptide linkage between Atg12 Gly 186 and Atg5 Lys 149. The simulated annealing Fo-Fc difference Fourier map was calculated by omitting Atg12 Phe 185 and Gly 186 and Atg5 Lys 149, and is shown with black meshes at 5.0σ.


( D ) Superimposition of the Atg5 ‐ Atg16N complex structure ( PDB ID 2DYM ) on that of the Atg12 - Atg5 ‐ Atg16N complex . The Atg12 - Atg5 ‐ Atg16N complex is coloured red , while the Atg5 ‐ Atg16N complex is coloured grey . Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model .

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

(D) Superimposition of the Atg5‐Atg16N complex structure (PDB ID 2DYM) on that of the Atg12-Atg5‐Atg16N complex. The Atg12-Atg5‐Atg16N complex is coloured red, while the Atg5‐Atg16N complex is coloured grey. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model.


( E ) In vivo analyses of the Atg12 and Atg5 mutants for studying the functional significance of the non ‐ covalent Atg12 ‐ Atg5 interactions . Yeast cells with or without starvation were lysed and subjected to urea SDS - polyacrylamide gel electrophoresis ( PAGE ) followed by western blotting . Ape1 , aminopeptidase I ; mApe1 , mature form of Ape1 ; PE , phosphatidylethanolamine ; prApe1 , preform of Ape1 ; WT , wild ‐ type .

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

(E) In vivo analyses of the Atg12 and Atg5 mutants for studying the functional significance of the non‐covalent Atg12‐Atg5 interactions. Yeast cells with or without starvation were lysed and subjected to urea SDS-polyacrylamide gel electrophoresis (PAGE) followed by western blotting. Ape1, aminopeptidase I; mApe1, mature form of Ape1; PE, phosphatidylethanolamine; prApe1, preform of Ape1; WT, wild‐type.


( A ) In vitro pull ‐ down assay using recombinant plant Atg homologues . CBB , Coomassie Brilliant Blue staining ; GST , S ; HA , haemagglutinin ; PE , phosphatidylethanolamine ; WT , wild ‐ 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 O O O O O O O O O O O O O O O

(A) In vitro pull‐down assay using recombinant plant Atg homologues.CBB, Coomassie Brilliant Blue staining; GST, S; HA, haemagglutinin; PE, phosphatidylethanolamine; WT, wild‐type.


( B ) Mapping of the mutated residues on the Atg12 - Atg5 ‐ Atg16N complex structure . Atg5 and Atg16N are shown with a ribbon model , whereas Atg12 is shown with both surface and ribbon models . The side chains of Tyr 147 , Tyr 149 and Phe 154 of Atg12 are shown with a stick model and coloured grey ( Tyr 147 ) or blue ( Tyr 149 and Phe 154 ) . Corresponding AtAtg12b residues are indicated in parentheses . Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model .

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

(B) Mapping of the mutated residues on the Atg12-Atg5‐Atg16N complex structure. Atg5 and Atg16N are shown with a ribbon model, whereas Atg12 is shown with both surface and ribbon models. The side chains of Tyr 147, Tyr 149 and Phe 154 of Atg12 are shown with a stick model and coloured grey (Tyr 147) or blue (Tyr 149 and Phe 154). Corresponding AtAtg12b residues are indicated in parentheses. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model.


A : Relative expression of ELDR in OSCC patient samples compared to adjacent non - tumor tissues ( N = 20 ) analyzed by qRT - PCR . Technical triplicates were used from each sample . 18S rRNA was used as an internal control . Data were analyzed by Student ' s t test . Small bars indicate standard error ( * , p < 0 . 05 , * * , p < 0 . 01 ; * * * p < 0 . 001 ) .

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

A: Relative expression of ELDR in OSCC patient samples compared to adjacent non-tumor tissues (N=20) analyzed by qRT-PCR. Technical triplicates were used from each sample. 18S rRNA was used as an internal control. Data were analyzed by Student's t test. Small bars indicate standard error (*, p<0.05, **, p<0.01; *** p<0.001).


B : OSCC grade wise upregulation of ELDR expression was noted in the patient samples ( N = 20 ) . Correlation analysis showed signification positive correlation of ELDR expression with tumor grade ( R2 = 0 . 926 , significant P value = 0 . 01 ) . Pearson correlation coefficient and p value was calculated using socscistatistics . com . Well : well differentiated , Mod : moderately differentiated , Poor : poorly differentiated carcinoma . Data are represented as the mean ± SD ,

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

B: OSCC grade wise upregulation of ELDR expression was noted in the patient samples (N=20). Correlation analysis showed signification positive correlation of ELDR expression with tumor grade (R2= 0.926, significant P value = 0.01). Pearson correlation coefficient and p value was calculated using socscistatistics.com. Well: well differentiated, Mod: moderately differentiated, Poor: poorly differentiated carcinoma. Data are represented as the mean ± SD,


C : Representative images of FISH analysis with an RNA probe of ELDR ( red ) and DAPI staining ( blue ) in formalin fixed paraffin embedded OSCC tissue . An unrelated probe was used as negative control . Magnification : 40X ; inset magnification 100X and scale bar 10 µm .

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

C: Representative images of FISH analysis with an RNA probe of ELDR (red) and DAPI staining (blue) in formalin fixed paraffin embedded OSCC tissue. An unrelated probe was used as negative control. Magnification: 40X; inset magnification 100X and scale bar 10 µm.


D : Relative expression of ELDR in OSCC cell lines compared to normal oral keratinocytes ( NOK ) analyzed by qRT - PCR . 18S was used as internal control . Data were analyzed by Student ' s t test . Small bars indicate standard error ( * * , p < 0 . 01 ; * * * p < 0 . 001 ) . n = 4 biological replicates . Data are represented as the mean ± SD ,

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

D: Relative expression of ELDR in OSCC cell lines compared to normal oral keratinocytes (NOK) analyzed by qRT-PCR. 18S was used as internal control. Data were analyzed by Student's t test. Small bars indicate standard error (**, p<0.01; *** p<0.001). n = 4 biological replicates. Data are represented as the mean ± SD,


A : Control or ELDR plasmid DNA stably transfected NOKs were analyzed for cell proliferation using Trypan blue exclusion at the indicated time points . Live cell numbers are presented . n = 4 biological replicates . Data are represented as the mean ± SD ,

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

A: Control or ELDR plasmid DNA stably transfected NOKs were analyzed for cell proliferation using Trypan blue exclusion at the indicated time points. Live cell numbers are presented. n = 4 biological replicates. Data are represented as the mean ± SD,


B : Control or ELDR plasmid DNA overexpressed NOK ( 3 × 102 ) were seeded and allowed to form colonies . After two weeks , colonies were stained with crystal violet and counted . Representative images of colonies in control and ELDR overexpressed cells are presented . The right panel shows quantitation . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

B: Control or ELDR plasmid DNA overexpressed NOK (3×102) were seeded and allowed to form colonies. After two weeks, colonies were stained with crystal violet and counted. Representative images of colonies in control and ELDR overexpressed cells are presented. The right panel shows quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,


C : Cal27 , JHU029 and JHU022 cells were transfected with control or ELDR siRNA ( 50nM ) , and cell proliferation was analyzed at the indicated time points . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

C: Cal27, JHU029 and JHU022 cells were transfected with control or ELDR siRNA (50nM), and cell proliferation was analyzed at the indicated time points. n = 3 biological replicates. Data are represented as the mean ± SD,


D : Cell lysates from control or ELDR overexpressing NOKs , control or Cal27 and JHU029 cells treated with two different ELDR siRNAs were subjected to Western blot analysis for PCNA expression using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control for normalization . The bottom panel shows a quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

D: Cell lysates from control or ELDR overexpressing NOKs, control or Cal27 and JHU029 cells treated with two different ELDR siRNAs were subjected to Western blot analysis for PCNA expression using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control for normalization. The bottom panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


E : Cal27 and JHU029 cells were transfected with siELDR , ELDR plasmid DNA or both siELDR and ELDR plasmid DNA . Cell lysates were analyzed by Western blot for PCNA expression using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . The right panel shows quantitative representation of Western blot band intensities . n = 2 biological replicates . Data are represented as the mean ± SD ,

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

E: Cal27 and JHU029 cells were transfected with siELDR, ELDR plasmid DNA or both siELDR and ELDR plasmid DNA. Cell lysates were analyzed by Western blot for PCNA expression using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. The right panel shows quantitative representation of Western blot band intensities. n = 2 biological replicates. Data are represented as the mean ± SD,


A : Cal27 or JHU029 cells were transfected with control or ELDR siRNA , and EGFR mRNA expression was examined by qRT - PCR . 18S gene was used as internal control . n = 3 biological replicates and 3 technical repeates .

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

A: Cal27 or JHU029 cells were transfected with control or ELDR siRNA, and EGFR mRNA expression was examined by qRT-PCR. 18S gene was used as internal control. n = 3 biological replicates and 3 technical repeates.


B : Cell lysates from control and ELDR depleted Cal27 or JHU029 cells using two different siRNAs to ELDR were subjected to Western blot analysis for the EGFR using specific antibodies . The membrane was reprobed with an antibody against Actin as an internal control . The same actin blot was used in Fig . 2D ( for PCNA ) . The bottom panel shows a quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

B: Cell lysates from control and ELDR depleted Cal27 or JHU029 cells using two different siRNAs to ELDR were subjected to Western blot analysis for the EGFR using specific antibodies. The membrane was reprobed with an antibody against Actin as an internal control. The same actin blot was used in Fig. 2D (for PCNA). The bottom panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


C : Cal27 and JHU029 cells were transfected with siELDR , ELDR plasmid or both siELDR and ELDR plasmid . Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody . The membrane was reprobed with an antibody against Actin as internal control . The same actin blot was used in Fig . 2E ( for PCNA ) . n = 2 biological replicates .

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

C: Cal27 and JHU029 cells were transfected with siELDR, ELDR plasmid or both siELDR and ELDR plasmid. Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody. The membrane was reprobed with an antibody against Actin as internal control. The same actin blot was used in Fig. 2E (for PCNA). n = 2 biological replicates.


D : Cell lysates from control and ELDR overexpressed NOK were subjected to Western blot analysis for the EGFR using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . The right panel shows a quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

D: Cell lysates from control and ELDR overexpressed NOK were subjected to Western blot analysis for the EGFR using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. The right panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


E : Cell lysates from control and ELDR - depleted Cal27 and JHU029 cells were subjected to Western blot analysis for phsospho - STAT3 ( pSTAT3 Y - 705 ) and total STAT3 using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . Right panel shows a quantitative representation of Western blot band intensities . n = 2 biological replicates . Data are represented as the mean ± SD ,

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

E: Cell lysates from control and ELDR-depleted Cal27 and JHU029 cells were subjected to Western blot analysis for phsospho-STAT3 (pSTAT3 Y-705) and total STAT3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Right panel shows a quantitative representation of Western blot band intensities. n = 2 biological replicates. Data are represented as the mean ± SD,


C : Cell lysates from Cal27 and JHU022 cells were incubated with biotinylated ELDR sense and antisense RNA , pulled down and subjected to Western blot analysis for the ILF3 using specific antibodies . Bottom panel shows Western blot analysis of PUM - 1 ( an unrelated RNA binding protein ) .

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

C: Cell lysates from Cal27 and JHU022 cells were incubated with biotinylated ELDR sense and antisense RNA, pulled down and subjected to Western blot analysis for the ILF3 using specific antibodies. Bottom panel shows Western blot analysis of PUM-1 (an unrelated RNA binding protein).


D : Cal27 and JHU022 cell lysates were immunoprecipitated against control or ILF3 antibody and RNA was isolated from the precipitates . Relative expression of ELDR ( left penal ) and PCAT1 ( right panel ) was analyzed by qRT - PCR . n = 3 biological replicates and 3 technical replicates . Data are represented as the mean ± SD ,

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

D: Cal27 and JHU022 cell lysates were immunoprecipitated against control or ILF3 antibody and RNA was isolated from the precipitates. Relative expression of ELDR (left penal) and PCAT1 (right panel) was analyzed by qRT-PCR. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,


E : Cell lysates from control and ELDR depleted Cal27 and JHU029 cells were subjected to Western blot analysis for ILF3 using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . Right panel shows the quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

E: Cell lysates from control and ELDR depleted Cal27 and JHU029 cells were subjected to Western blot analysis for ILF3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Right panel shows the quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


F : Control , ELDR overexpressed and ELDR - depleted Cal27 cells were probed with an antibody against ILF3 ( red ) and DAPI ( blue ) . Representative confocal microscopic images show nuclear and cytoplasmic expression of ILF3 . Arrows indicate cytoplasmic expression . Magnifications 60X , scale bar 20 μ .

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

F: Control, ELDR overexpressed and ELDR-depleted Cal27 cells were probed with an antibody against ILF3 (red) and DAPI (blue). Representative confocal microscopic images show nuclear and cytoplasmic expression of ILF3. Arrows indicate cytoplasmic expression. Magnifications 60X, scale bar 20 μ.


A : Cal27 and JHU022 cell lysates were immunoprecipitated with control or ILF3 antibodies and RNA was isolated from the precipitates . Relative mRNA expression of EGFR was analyzed by qRT - PCR . n = 3 biological replicates and 3 technical replicates . Data are represented as the mean ± SD ,

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

A: Cal27 and JHU022 cell lysates were immunoprecipitated with control or ILF3 antibodies and RNA was isolated from the precipitates. Relative mRNA expression of EGFR was analyzed by qRT-PCR. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,


B : Cal27 and JHU029 cells were transfected with control or ILF3 siRNA and after 48hr cell lysates were subjected to Western blot analysis for EGFR and ILF3 using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . n = 3 biological replicates .

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

B: Cal27 and JHU029 cells were transfected with control or ILF3 siRNA and after 48hr cell lysates were subjected to Western blot analysis for EGFR and ILF3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. n = 3 biological replicates.


C : Cal27 and JHU029 cell lysates were transfected with either siELDR or siILF3 and relative mRNA expression of Cyclin E1 was analyzed by qRT - PCR . 18S was used as internal control . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

C: Cal27 and JHU029 cell lysates were transfected with either siELDR or siILF3 and relative mRNA expression of Cyclin E1 was analyzed by qRT-PCR. 18S was used as internal control. n = 3 biological replicates. Data are represented as the mean ± SD,


D : Control or ELDR depleted Cal27 and JHU029 cell lysates using two different siRNAs to ELDR were subjected to Western blot analysis for Cyclin E1 using specific antibody . The membrane was reprobed with an antibody against Actin as an internal control . Bottom panels show quantitation . n = 4 biological replicates . Data are represented as the mean ± SD ,

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

D: Control or ELDR depleted Cal27 and JHU029 cell lysates using two different siRNAs to ELDR were subjected to Western blot analysis for Cyclin E1 using specific antibody. The membrane was reprobed with an antibody against Actin as an internal control. Bottom panels show quantitation. n = 4 biological replicates. Data are represented as the mean ± SD,


E : Control or ELDR overexpressed NOK lysates were subjected to Western blot analysis for Cyclin E1 using specific antibody . The membrane was reprobed with an antibody against Actin as internal control . Bottom panel shows quantitation . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

E: Control or ELDR overexpressed NOK lysates were subjected to Western blot analysis for Cyclin E1 using specific antibody. The membrane was reprobed with an antibody against Actin as internal control. Bottom panel shows quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,


F : Control or ILF3depleted Cal27 and JHU029 cell lysates were subjected to Western blot analysis for Cyclin E1 using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . Bottom panels show quantitation . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

F: Control or ILF3depleted Cal27 and JHU029 cell lysates were subjected to Western blot analysis for Cyclin E1 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Bottom panels show quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,


G : Cal27 and JHU029 cells were transfected with either siELDR or siILF3 and after 48hr cell lysates were subjected to Western blot analysis for phospho - RB and total RB using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . Bottom panels show quantitation . n = 3 biological replicates . Data are represented as the mean ±

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

G: Cal27 and JHU029 cells were transfected with either siELDR or siILF3 and after 48hr cell lysates were subjected to Western blot analysis for phospho-RB and total RB using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Bottom panels show quantitation. n = 3 biological replicates. Data are represented as the mean ± SD


H : Control or ELDR knockdown synchronized Cal27 and JHU029 cells were harvested , fixed and stained with propidium iodide . DNA content was analyzed by flow cytometry . Results are represented cell population in G1 , S , and G2 / M phases of the cell cycle . Right panels show percentage of cell population in different phases of cell cycle . n = 4 biological replicates . Data are represented as the mean ± SD ,

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

H: Control or ELDR knockdown synchronized Cal27 and JHU029 cells were harvested, fixed and stained with propidium iodide. DNA content was analyzed by flow cytometry. Results are represented cell population in G1, S, and G2/M phases of the cell cycle. Right panels show percentage of cell population in different phases of cell cycle. n = 4 biological replicates. Data are represented as the mean ± SD,


A : Cal27 and JHU029 cells were transfected with control or ELDR siRNA and relative expression of miR - 7 was analyzed by qRT - PCR . U47 was used as an internal control . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

A: Cal27 and JHU029 cells were transfected with control or ELDR siRNA and relative expression of miR-7 was analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates. Data are represented as the mean ± SD,


B : Cal27 and JHU029 cells were transfected with control or ELDR plasmid and relative expression of miR - 7 was analyzed by qRT - PCR . U47 was used as an internal control . n = 3 biological replicates . Data are represented as the mean ± SD ,

B: Cal27 and JHU029 cells were transfected with control or ELDR plasmid and relative expression of miR-7 was analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates. Data are represented as the mean ± SD,


C : Cal27 and JHU029 cells were transfected with control or mimic miR - 7 and relative expression of ELDR was analyzed by qRT - PCR . 18S rRNA was used as an internal control . n = 2 biological replicates ; n = 3 technical replicates . Data are represented as the mean ± SD ,

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

C: Cal27 and JHU029 cells were transfected with control or mimic miR-7 and relative expression of ELDR was analyzed by qRT-PCR. 18S rRNA was used as an internal control. n = 2 biological replicates; n= 3 technical replicates. Data are represented as the mean ± SD,


D : Cal27 and JHU029 cells were transfected with miR - 7 mimic , ELDR plasmid or both miR - 7 and ELDR plasmid . Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody . The membrane was reprobed with an antibody against Actin as an internal control . Representative bands were cropped from same gel . Right panels show quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

D: Cal27 and JHU029 cells were transfected with miR-7 mimic, ELDR plasmid or both miR-7 and ELDR plasmid. Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody. The membrane was reprobed with an antibody against Actin as an internal control. Representative bands were cropped from same gel. Right panels show quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


E : Relative expression of miR - 7 in Cal27 and JHU029 cells compared to NOK cell line analyzed by qRT - PCR . U47 was used as an internal control . n = 3 biological replicates and 3 technical replicates . Data are represented as the mean ± SD ,

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E: Relative expression of miR-7 in Cal27 and JHU029 cells compared to NOK cell line analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,


A : Body weight was measured in control and siELDR treated mice . n = 5 animal per

A: Body weight was measured in control and siELDR treated mice. n=5 animal per group


B : Representative images of tumors in control and treatment groups .

B: Representative images of tumors in control and treatment groups.


C : Tumors were measured using a slide caliper and tumor volumes were calculated . Arrow indicates starting point siELDR treatment . n = 5 animal per group and tumor volume presented as the mean ±

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C: Tumors were measured using a slide caliper and tumor volumes were calculated. Arrow indicates starting point siELDR treatment. n=5 animal per group and tumor volume presented as the mean ± SD


D : ELDR knockdown was examined by analyzing expression of ELDR in treated tumors compared to the control tumors . 18S rRNA gene was used as internal control . n = 5 animal per group and 3 technical replicates from each tumor .

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D: ELDR knockdown was examined by analyzing expression of ELDR in treated tumors compared to the control tumors. 18S rRNA gene was used as internal control. n=5 animal per group and 3 technical replicates from each tumor.


E : Control or siELDR treated tumor lysates were subjected to Western blot analysis for EGFR and Cyclin E1expression using specific antibodies and representative bands are shown . The blot was reprobed with an antibody against Actin for normalization . Right panels show quantitation . n = 3 biological replicates from each

O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O 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: Control or siELDR treated tumor lysates were subjected to Western blot analysis for EGFR and Cyclin E1expression using specific antibodies and representative bands are shown. The blot was reprobed with an antibody against Actin for normalization. Right panels show quantitation. n= 3 biological replicates from each tumor


F : OSCC - PDX tissue was implanted subcutaneously into the flank of NSG mice . When the tumor volume reached > 70 mm3 , mice were divided into two groups . 10µg of siELDR or control oligoes were injected intratumorally as described above . Body weight was measured in control and siELDR treated mice . n = 5 animal per

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F: OSCC-PDX tissue was implanted subcutaneously into the flank of NSG mice. When the tumor volume reached >70 mm3, mice were divided into two groups. 10µg of siELDR or control oligoes were injected intratumorally as described above. Body weight was measured in control and siELDR treated mice. n=5 animal per group


G : Representative images of OSCC - PDX tumors in control and treatment groups .

G: Representative images of OSCC-PDX tumors in control and treatment groups.


H : PDX tumors were measured using a slide caliper and tumor volumes were calculated . n = 5 animal per group and tumor volume presented as the mean ±

O O O O O O 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: PDX tumors were measured using a slide caliper and tumor volumes were calculated. n=5 animal per group and tumor volume presented as the mean ± SD


I : ELDR knockdown was examined by analyzing expression of ELDR in treated OSCC - PDX tumors compared to the control tumors . 18S gene was used as internal control . n = 5 animal per group and 3 technical replicates presented as the mean ±

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I: ELDR knockdown was examined by analyzing expression of ELDR in treated OSCC-PDX tumors compared to the control tumors. 18S gene was used as internal control. n=5 animal per group and 3 technical replicates presented as the mean ± SD


Immunohistochemistry performed on 12 month - old THY - Tau22 mice showing AT100 - positive neurons ( green ) , CBP - positive nuclei ( red ) and DAPI - stained nuclei ( blue ) in the CA1 region of the dorsal hippocampus . A representative image is shown with a focus . Scale bars : 40µm and 20µm as noted . ( n = 5 mice ) . Arrows depict neurons bearing neurofibrillary tangles and do not display CBP immunostaining . The star depicts a ghost tangle ( AT100 - positive neuron and no nucleus )

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

Immunohistochemistry performed on 12 month-old THY-Tau22 mice showing AT100-positive neurons (green), CBP-positive nuclei (red) and DAPI-stained nuclei (blue) in the CA1 region of the dorsal hippocampus. A representative image is shown with a focus. Scale bars: 40µm and 20µm as noted. (n=5 mice). Arrows depict neurons bearing neurofibrillary tangles and do not display CBP immunostaining. The star depicts a ghost tangle (AT100-positive neuron and no nucleus)


Western blot analyses perfomed in the dorsal hippocampus of 12 month - old THY - Tau22 mice compared to age - matched controls . NeuN , actin , tubulin and total H2B levels are not changed . Phosphorylated tau on serine 404 ( Tau - Ph404 ) attest for samples from tauopathic mice . Quantification represents the ration of the protein level detected on the total amount of proteins on the membranes , with WT arbitrarily set at 1 ( fold induction ) . Bar graphs are mean ± SEM . n = 5 - 7 / group as noted , multiple t tests , CBP * p = 0 . 003 and GFAP * * p = 0 . 0001 for THY - Tau vs . WT

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

Western blot analyses perfomed in the dorsal hippocampus of 12 month-old THY-Tau22 mice compared to age-matched controls. NeuN, actin, tubulin and total H2B levels are not changed. Phosphorylated tau on serine 404 (Tau-Ph404) attest for samples from tauopathic mice. Quantification represents the ration of the protein level detected on the total amount of proteins on the membranes, with WT arbitrarily set at 1 (fold induction). Bar graphs are mean ± SEM. n=5-7/group as noted, multiple t tests, CBP * p=0.003 and GFAP **p=0.0001 for THY-Tau vs. WT mice


RNA - sequencing data performed in the hippocampus of 8 month - old mice . Functional analyses ( DAVID GOTERM ) performed on significantly deregulated genes in THY - Tau22 mice compared to WT mice . Representative genes are presented below . | log2 Fold Change | > 0 . 2 ; * indicates FDR < 0 . 05 . IEG , Immediate Early Genes . Predicted promoter motif ( right ) analyses performed with GREAT . Groups : WT mice ( n = 3 ) , THY - Tau22 mice ( n = 2 )

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

RNA-sequencing data performed in the hippocampus of 8 month-old mice. Functional analyses (DAVID GOTERM) performed on significantly deregulated genes in THY-Tau22 mice compared to WT mice. Representative genes are presented below. |log2 Fold Change| > 0.2; * indicates FDR<0.05. IEG, Immediate Early Genes. Predicted promoter motif (right) analyses performed with GREAT. Groups: WT mice (n=3), THY-Tau22 mice (n=2)