( H , I ) Experiments conducted as in ( C ) , but in cells arrested in G1 with alfa - factor ( H ) or in metaphase with nocodazole ( I ) for 2 h at 26oC and for one additional hour at 26oC or 35oC .

(H, I) Experiments conducted as in (C), but in cells arrested in G1 with alfa-factor (H) or in metaphase with nocodazole (I) for 2 h at 26oC and for one additional hour at 26oC or 35oC.


( A - E ) DNA topology of the indicated minichromosomes of increasing DNA length ( kb ) before ( 26oC ) and after inactivation of condensin ( 35oC ) in smc2 - 8 cells . In each case , the first 2D gel resolves the Lk topoisomers ( Lk ) , the second 2D gel uncovers the knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare the Pkn before and after the inactivation of condensin ( mean ± SD from three independent experiments ) . P - values ( Student ' s t test ) : ns , p > 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of condensin (35oC) in smc2-8 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of condensin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05; **p < 0.01; ***p < 0.001.


( F ) Plot of Pkn values of minichromosomes of increasing DNA length ( including YEp13 ) before and after inactivation of condensin .

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

(F) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of condensin.


( A - D ) DNA topology of the indicated minichromosomes of increasing DNA length ( kb ) before ( 26oC ) and after inactivation of cohesin ( 35oC ) in scc1 - 73 cells . In each case , the first 2D gel resolves the Lk topoisomers ( Lk ) , the second 2D gel uncovers the knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare the Pkn before and after the inactivation of cohesin ( mean ± SD from three independent experiments ) . P - values ( Student ' s t test ) : ns , p > 0 . 05 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of cohesin (35oC) in scc1-73 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of cohesin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05.


( E ) Plot of Pkn values of minichromosomes of increasing DNA length ( including YEp13 ) before and after inactivation of cohesin . P - values ( Student ' s t test ) : * * p < 0 . 01 .

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

(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of cohesin. P-values (Student's t test): **p < 0.01.


( A - D ) DNA topology of the indicated minichromosomes of increasing DNA length ( kb ) before ( 26oC ) and after inactivation of Smc5 / 6 complex ( 35oC ) in smc6 - 9 cells . In each case , the first 2D gel resolves the Lk topoisomers ( Lk ) , the second 2D gel uncovers the knotted forms ( Kn ) . Gel signals are denoted as in Fig 2 . Bottom graphs compare the Pkn before and after the inactivation of Smc5 / 6 complex ( mean ± SD from two independent experiments ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of Smc5/6 complex (35oC) in smc6-9 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of Smc5/6 complex (mean ± SD from two independent experiments).


( E ) Plot of Pkn values of minichromosomes of increasing DNA length ( including YEp13 ) before and after inactivation of Smc5 / 6 complex .

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

(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of Smc5/6 complex.


A Measurement of urinary albumin / creatinine by ELISA ( albumin - to - creatinine ratio : day 14 : Phb2fl / fl 0 . 09 ± 0 . 03 mg / mg , n = 4 , versus Phb2pko 0 . 37 ± 0 . 21 mg / mg , n = 4 , P = 0 . 2350 ; day 21 : Phb2fl / fl 0 . 65 ± 0 . 16 mg / mg , n = 4 , versus Phb2pko 193 . 10 ± 26 . 81 mg / mg , n = 4 , * * * P = 0 . 003 ; day 28 : Phb2fl / fl 0 . 57 ± 0 . 07 mg / mg , n = 4 , versus Phb2pko 470 . 40 ± 131 . 30 mg / mg , n = 4 , * P = 0 . 0117 ) .

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A Measurement of urinary albumin/creatinine by ELISA (albumin-to-creatinine ratio: day 14: Phb2fl/fl 0.09 ± 0.03 mg/mg, n = 4, versus Phb2pko 0.37 ± 0.21 mg/mg, n = 4, P = 0.2350; day 21: Phb2fl/fl 0.65 ± 0.16 mg/mg, n = 4, versus Phb2pko 193.10 ± 26.81 mg/mg, n = 4, ***P = 0.003; day 28: Phb2fl/fl 0.57 ± 0.07 mg/mg, n = 4, versus Phb2pko 470.40 ± 131.30 mg/mg, n = 4, *P = 0.0117).


B Appearance of Phb2fl / fl and Phb2pko mice at day 28 .

B Appearance of Phb2fl/fl and Phb2pko mice at day 28.


C Analysis of body weight from postnatal day 14 until day 32 ( P - values for Phb2fl / fl or Phb2het versus Phb2pko : * P = 0 . 0372 at day 27 , * P = 0 . 0311 at day 28 , * P = 0 . 0157 at day 29 , * * P = 0 . 008 at day 30 , * * P = 0 . 0013 at day 31 , * * P = 0 . 0035 at day 32 , n = 3 for Phb2fl / fl and n = 4 for Phb2het and Phb2pko ) .

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C Analysis of body weight from postnatal day 14 until day 32 (P-values for Phb2fl/fl or Phb2het versus Phb2pko: *P = 0.0372 at day 27, *P = 0.0311 at day 28, *P = 0.0157 at day 29, **P = 0.008 at day 30, **P = 0.0013 at day 31, **P = 0.0035 at day 32, n = 3 for Phb2fl/fl and n = 4 for Phb2het and Phb2pko).


D Measurement of serum creatinine levels of mice in their fifth week of life ( Phb2fl / fl / het 14 . 74 ± 3 . 75 μmol / l versus Phb2pko 74 . 73 ± 2 . 20 μmol / l , n = 3 for both groups ; * * * P = 0 . 0002 ) .

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D Measurement of serum creatinine levels of mice in their fifth week of life (Phb2fl/fl/het 14.74 ± 3.75 μmol/l versus Phb2pko 74.73 ± 2.20 μmol/l, n = 3 for both groups; ***P = 0.0002).


E Measurement of serum urea levels of mice in their fifth week of life ( Phb2fl / fl / het 44 . 00 ± 7 . 02 mg / dl versus Phb2pko 493 . 70 ± 45 . 34 mg / dl , n = 3 for both groups ; * * * P = 0 . 006 ) .

E Measurement of serum urea levels of mice in their fifth week of life (Phb2fl/fl/het 44.00 ± 7.02 mg/dl versus Phb2pko 493.70 ± 45.34 mg/dl, n = 3 for both groups; ***P = 0.006).


F Kaplan - Meier survival curve ( n = 5 for Phb2fl / fl / het and n = 6 for Phb2pko ) .

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F Kaplan-Meier survival curve (n = 5 for Phb2fl/fl/het and n = 6 for Phb2pko).


Phb2pko mice develop glomerulosclerosisA PAS staining of kidney sections ( scale bar : 20 μm ) . B Immunohistochemistry for podocin on kidney sections ( scale bar : 20 μm ) . C Analysis of podocyte foot processes in electron micrographs ( scale bar : 0 . 7 μm ) . FP , podocyte foot processes ; GBM , glomerular basement membrane ; EC , endothelial cells . D Analysis of mitochondrial morphology in electron micrographs ( arrows ; scale bar : 0 . 3 μm ) . E High - power view of mitochondria in electron micrographs ( scale bar : 0 . 1 μm ) .

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Phb2pko mice develop glomerulosclerosisA PAS staining of kidney sections (scale bar: 20 μm).B Immunohistochemistry for podocin on kidney sections (scale bar: 20 μm).C Analysis of podocyte foot processes in electron micrographs (scale bar: 0.7 μm). FP, podocyte foot processes; GBM, glomerular basement membrane; EC, endothelial cells.D Analysis of mitochondrial morphology in electron micrographs (arrows; scale bar: 0.3 μm).E High-power view of mitochondria in electron micrographs (scale bar: 0.1 μm).


A PAS staining of tPod - Phb2pko and control mice 2 . 5 weeks after the end of tamoxifen treatment ( scale bar : 20 μm ) . B Immunohistochemistry for podocin on kidney sections of tPod - Phb2pko and control mice 2 . 5 weeks after the end of tamoxifen treatment ( scale bar : 20 μm ) .

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A PAS staining of tPod-Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm).B Immunohistochemistry for podocin on kidney sections of tPod-Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm).


C Coomassie stain of urinary samples of tPod - Phb2pko and control mice .

C Coomassie stain of urinary samples of tPod-Phb2pko and control mice.


A Kaplan - Meier survival curve showed that an additional knockout of the insulin receptor ( Insr ) prolonged survival of Phb2pko mice ( n = 7 for Phb2pko / Insrpko , n = 4 for Phb2pko / Insrhet , and n = 6 for Phb2pko ) .

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A Kaplan-Meier survival curve showed that an additional knockout of the insulin receptor (Insr) prolonged survival of Phb2pko mice (n = 7 for Phb2pko/Insrpko, n = 4 for Phb2pko/Insrhet, and n = 6 for Phb2pko).


B Kaplan - Meier survival curve revealed that the survival time of Phb2pko mice is not changed by an additional Igf1r deficiency ( n = 6 for Phb2pko / Igf1rpko , n = 4 for Phb2pko / Igf1rhet , and n = 6 for Phb2pko ) .

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B Kaplan-Meier survival curve revealed that the survival time of Phb2pko mice is not changed by an additional Igf1r deficiency (n = 6 for Phb2pko/Igf1rpko, n = 4 for Phb2pko/Igf1rhet, and n = 6 for Phb2pko).


C Statistical analysis comparing all genotypes with Phb2pko mice .

C Statistical analysis comparing all genotypes with Phb2pko mice.


D Kaplan - Meier survival curve revealed prolonged survival of Phb2pko / Insrpko / Igf1rpko and Phb2pko / Insrpko / Igf1rhet mice ( n = 19 for Phb2pko / Insrpko / Igf1rpko , n = 9 for Phb2pko / Insrhet / Igf1rpko , n = 11 for Phb2pko / Insrpko / Igf1rhet , n = 9 for Phb2pko / Insrhet / Igf1rhet , and n = 6 for Phb2pko ) . E Statistical analysis comparing all genotypes with Phb2pko mice .

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D Kaplan-Meier survival curve revealed prolonged survival of Phb2pko/Insrpko/Igf1rpko and Phb2pko/Insrpko/Igf1rhet mice (n = 19 for Phb2pko/Insrpko/Igf1rpko, n = 9 for Phb2pko/Insrhet/Igf1rpko, n = 11 for Phb2pko/Insrpko/Igf1rhet, n = 9 for Phb2pko/Insrhet/Igf1rhet, and n = 6 for Phb2pko).E Statistical analysis comparing all genotypes with Phb2pko mice.


A Serum creatinine and urea levels in the fifth week of life ( left graph : Phb2pko 74 . 73 ± 2 . 20 μmol / l versus Phb2pko / Insrpko / Igf1rpko 37 . 36 ± 8 . 83 μmol / l , n = 3 for Phb2pko and n = 4 for Phb2pko / Insrpko / Igf1rpko , * P = 0 . 0169 ; right graph : Phb2pko 493 . 70 ± 45 . 34 mg / dl versus Phb2pko / Insrpko / Igf1rpko 272 . 80 ± 49 . 29 mg / dl , n = 3 for Phb2pko and n = 4 for Phb2pko / Insrpko / Igf1rpko , * P = 0 . 0247 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Serum creatinine and urea levels in the fifth week of life (left graph: Phb2pko 74.73 ± 2.20 μmol/l versus Phb2pko/Insrpko/Igf1rpko 37.36 ± 8.83 μmol/l, n = 3 for Phb2pko and n = 4 for Phb2pko/Insrpko/Igf1rpko, *P = 0.0169; right graph: Phb2pko 493.70 ± 45.34 mg/dl versus Phb2pko/Insrpko/Igf1rpko 272.80 ± 49.29 mg/dl, n = 3 for Phb2pko and n = 4 for Phb2pko/Insrpko/Igf1rpko, *P = 0.0247).


B Abundance of deleted Phb2 gene in isolated glomeruli of Phb2fl / fl , Phb2het , Phb2pko and Phb2pko / Insrpko / Igf1rpko mice compared to the floxed Phb2 gene ( left graph ; quantified by qPCR : n = 3 for all groups , P = 0 . 83 for Phb2pko / Insrpko / Igf1rpko versus Phb2pko ) and compared to a reference gene ( right graph ; quantified by qPCR : n = 3 for all groups , P = 0 . 58 for Phb2pko / Insrpko / Igf1rpko versus Phb2pko ) .

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B Abundance of deleted Phb2 gene in isolated glomeruli of Phb2fl/fl, Phb2het, Phb2pko and Phb2pko/Insrpko/Igf1rpko mice compared to the floxed Phb2 gene (left graph; quantified by qPCR: n = 3 for all groups, P = 0.83 for Phb2pko/Insrpko/Igf1rpko versus Phb2pko) and compared to a reference gene (right graph; quantified by qPCR: n = 3 for all groups, P = 0.58 for Phb2pko/Insrpko/Igf1rpko versus Phb2pko).


C Analysis of mitochondrial morphology in electron micrographs ( left image : scale bar , 1 μm ; right image : scale bar , 0 . 1 μm ) .

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C Analysis of mitochondrial morphology in electron micrographs (left image: scale bar, 1 μm; right image: scale bar, 0.1 μm).


A Immunofluorescence staining of mPhb2 shRNA and control podocytes with a Tom20 antibody revealed a disturbed mitochondrial network in Phb2 - deficient podocytes ( scale bar : 10 μm ) . B Quantification of tubular and fragmented mitochondrial morphology mPhb2 shRNA versus control podocytes seen in ( A ) ( mitochondrial network of n = 100 individual cells of mPhb2 shRNA podocytes and n = 108 individual cells of scrambled shRNA podocytes was assessed ) . C Morphometric image analysis revealed an increased ratio of mitochondrial count versus mitochondrial area in Phb2 - deficient podocytes compared to control podocytes ( n = 75 cell patches for scrambled shRNA , n = 58 cell patches for mPhb2 shRNA ) . D Morphometric image analysis showed a decrease in branch numbers in Phb2 - deficient podocytes compared to control podocytes ( n = 75 cell patches for scrambled shRNA , n = 58 cell patches for mPhb2 shRNA ) . E Morphometric image analysis detected a decrease in mitochondrial size in Phb2 - deficient podocytes compared to control podocytes ( n = 75 cell patches for scrambled shRNA , n = 58 cell patches for mPhb2 shRNA ) .

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A Immunofluorescence staining of mPhb2 shRNA and control podocytes with a Tom20 antibody revealed a disturbed mitochondrial network in Phb2-deficient podocytes (scale bar: 10 μm).B Quantification of tubular and fragmented mitochondrial morphology mPhb2 shRNA versus control podocytes seen in (A) (mitochondrial network of n = 100 individual cells of mPhb2 shRNA podocytes and n = 108 individual cells of scrambled shRNA podocytes was assessed).C Morphometric image analysis revealed an increased ratio of mitochondrial count versus mitochondrial area in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).D Morphometric image analysis showed a decrease in branch numbers in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).E Morphometric image analysis detected a decrease in mitochondrial size in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).


F Measurements of mitochondrial respiration with complex II substrates ( succinate and glycerol - 3 - phosphate ) did not show significant differences between Phb2 - deficient and control podocytes ( n = 6 ) .

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F Measurements of mitochondrial respiration with complex II substrates (succinate and glycerol-3-phosphate) did not show significant differences between Phb2-deficient and control podocytes (n = 6).


G Quantification of mtDNA as a measure for mitochondrial mass showed similar levels of mtDNA in Phb2 - deficient podocytes compared to control podocytes ( n = 3 ) .

G Quantification of mtDNA as a measure for mitochondrial mass showed similar levels of mtDNA in Phb2-deficient podocytes compared to control podocytes (n = 3).


H FACS analysis of MitoTracker - and MitoSOX - stained Phb2 - deficient podocytes and control podocytes . I Quantification of mean fluorescence intensity of MitoTracker and MitoSOX by FACS analysis revealed the same mitochondrial mass and the same ROS levels in Phb2 - deficient podocytes compared to control podocytes ( n = 3 , P = 0 . 7782 for MitoTracker , P = 0 . 1088 for MitoSOX ) .

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H FACS analysis of MitoTracker- and MitoSOX-stained Phb2-deficient podocytes and control podocytes.I Quantification of mean fluorescence intensity of MitoTracker and MitoSOX by FACS analysis revealed the same mitochondrial mass and the same ROS levels in Phb2-deficient podocytes compared to control podocytes (n = 3, P = 0.7782 for MitoTracker, P = 0.1088 for MitoSOX).


A Phb2 - deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes , which could be blocked by treatment with rapamycin ( n = 5 , bars represent mean ± SEM , Student ' s t - test , * * P = 0 . 0036 ) . pS6RP , phosphorylated S6 ribosomal protein ; S6RP , S6 ribosomal protein .

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A Phb2-deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes, which could be blocked by treatment with rapamycin (n = 5, bars represent mean ± SEM, Student's t-test, **P = 0.0036). pS6RP, phosphorylated S6 ribosomal protein; S6RP, S6 ribosomal protein.


B Immunohistochemistry for phosphorylated S6 ribosomal protein ( pS6RP ) on kidney sections revealed increased levels of pS6RP in glomeruli of Phb2pko compared to Phb2fl / fl mice ( arrows point to cells showing a signal for pS6RP ; scale bar : 20 μm ) .

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B Immunohistochemistry for phosphorylated S6 ribosomal protein (pS6RP) on kidney sections revealed increased levels of pS6RP in glomeruli of Phb2pko compared to Phb2fl/fl mice (arrows point to cells showing a signal for pS6RP; scale bar: 20 μm).


C Kaplan - Meier survival curve revealed that treatment of Phb2pko mice with rapamycin prolonged survival for several days compared to vehicle - treated Phb2pko mice ( n = 11 for Phb2fl / fl + rapamycin , n = 15 for Phb2pko + vehicle , n = 16 for Phb2pko + rapamycin ) . D Statistical analysis comparing rapamycin - treated Phb2pko mice with vehicle - treated Phb2pko mice .

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C Kaplan-Meier survival curve revealed that treatment of Phb2pko mice with rapamycin prolonged survival for several days compared to vehicle-treated Phb2pko mice (n = 11 for Phb2fl/fl + rapamycin, n = 15 for Phb2pko + vehicle, n = 16 for Phb2pko + rapamycin).D Statistical analysis comparing rapamycin-treated Phb2pko mice with vehicle-treated Phb2pko mice.


D - G Culture dynamics of phage ‐ infected wild ‐ type ( black ) versus BREX ‐ containing ( red ) strains of B . subtilis BEST7003 . Bacterial strains were exposed to phage at time = 0 h , and optical density measurements were read in a 96 ‐ well plate format . Each experiment was performed three times with three technical triplicates for each biological replicate . Error bars represent SEM . Shown are representative results for three of the ten phages tested ; data for the remaining seven phages are found in Supplementary Fig S2 .

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D-G Culture dynamics of phage‐infected wild‐type (black) versus BREX‐containing (red) strains of B. subtilis BEST7003. Bacterial strains were exposed to phage at time = 0 h, and optical density measurements were read in a 96‐well plate format. Each experiment was performed three times with three technical triplicates for each biological replicate. Error bars represent SEM. Shown are representative results for three of the ten phages tested; data for the remaining seven phages are found in Supplementary Fig S2.


A Culture dynamics over an extended period ( > 30 h ) for SPO1 ‐ infected wild ‐ type ( black ) versus BREX ‐ containing ( red ) strains of B . subtilis BEST7003 . Each curve represents a single technical replicate grown in a single well on a 96 ‐ well plate . Culture decline is temporally reproducible for the BREX ‐ lacking strain but occurs later , at apparently stochastic time points , for the BREX ‐ containing strain . Re ‐ growth following culture crash represents phage ‐ resistant mutants .

A Culture dynamics over an extended period (> 30 h) for SPO1‐infected wild‐type (black) versus BREX‐containing (red) strains of B. subtilis BEST7003. Each curve represents a single technical replicate grown in a single well on a 96‐well plate. Culture decline is temporally reproducible for the BREX‐lacking strain but occurs later, at apparently stochastic time points, for the BREX‐containing strain. Re‐growth following culture crash represents phage‐resistant mutants.


B Phage production during a one ‐ step phage growth curve experiment with wild ‐ type ( black ) and BREX ‐ containing ( red ) strains of B . subtilis BEST7003 infected with SPO1 . Error bars represent SD . Y ‐ axis represents absolute phage concentrations . Black and red arrows point to the time point of maximal burst for BREX ‐ lacking and BREX ‐ containing strains , respectively .

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B Phage production during a one‐step phage growth curve experiment with wild‐type (black) and BREX‐containing (red) strains of B. subtilis BEST7003 infected with SPO1. Error bars represent SD. Y‐axis represents absolute phage concentrations. Black and red arrows point to the time point of maximal burst for BREX‐lacking and BREX‐containing strains, respectively.


C Phage production during a one ‐ step phage growth curve experiment with wild ‐ type ( black ) and BREX ‐ containing ( red ) strains of B . subtilis BEST7003 infected with Φ3T . Error bars represent SD . Y ‐ axis represents relative phage concentrations normalized to the value at the beginning of the infection .

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C Phage production during a one‐step phage growth curve experiment with wild‐type (black) and BREX‐containing (red) strains of B. subtilis BEST7003 infected with Φ3T. Error bars represent SD. Y‐axis represents relative phage concentrations normalized to the value at the beginning of the infection.


D Multiplex PCR assay showing lysogeny during a phage infection time course in the strain lacking BREX ( black ) , but not in BREX ‐ containing strain ( red ) , or uninfected ( U ) strains . Amplicons for the bacterial DNA , phage DNA , and lysogen ‐ specific DNA are 293 , 485 , and 1 , 218 bp , respectively .

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

D Multiplex PCR assay showing lysogeny during a phage infection time course in the strain lacking BREX (black), but not in BREX‐containing strain (red), or uninfected (U) strains. Amplicons for the bacterial DNA, phage DNA, and lysogen‐specific DNA are 293, 485, and 1,218 bp, respectively.


A BREX does not work via an abortive infection mechanism . Increasing the MOI of the Φ3T infection from 0 . 05 to 5 shortens the time to culture crash for the BREX ‐ lacking strain , but does not result in culture decline for BREX ‐ containing strain . Error bars represent SD of technical triplicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 BREX does not work via an abortive infection mechanism. Increasing the MOI of the Φ3T infection from 0.05 to 5 shortens the time to culture crash for the BREX‐lacking strain, but does not result in culture decline for BREX‐containing strain. Error bars represent SD of technical triplicates.


B The system does not interfere with phage adsorption to bacterial cells . Strains either containing ( red ) or lacking ( black ) the BREX system were infected with phage Φ3T and then chloroform ‐ treated 15 min following infection . The culture was plated on Φ3T ‐ sensitive B . subtilis cells and plaques , representing extracellular , unadsorbed phages , were counted .

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

B The system does not interfere with phage adsorption to bacterial cells. Strains either containing (red) or lacking (black) the BREX system were infected with phage Φ3T and then chloroform‐treated 15 min following infection. The culture was plated on Φ3T‐sensitive B. subtilis cells and plaques, representing extracellular, unadsorbed phages, were counted.


C Phage DNA replication does not occur in BREX ‐ containing cells ( red ) , but is observed in the BREX ‐ lacking strain ( black ) . Y ‐ axis represents relative phage concentrations normalized to the value at the beginning of the infection , as measured by Illumina sequencing .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Phage DNA replication does not occur in BREX‐containing cells (red), but is observed in the BREX‐lacking strain (black). Y‐axis represents relative phage concentrations normalized to the value at the beginning of the infection, as measured by Illumina sequencing.


C , D Culture dynamics of non ‐ infected ( C ) or phage ‐ infected ( D ) cultures . Curves depict culture dynamics of strains lacking BREX ( black ) and BREX ‐ containing ( red ) strains of B . subtilis BEST7003 , as well as a BREX ‐ containing strain where the pglX methylase was deleted ( green ) . Axes and error bars are as in Fig .

C, D Culture dynamics of non‐infected (C) or phage‐infected (D) cultures. Curves depict culture dynamics of strains lacking BREX (black) and BREX‐containing (red) strains of B. subtilis BEST7003, as well as a BREX‐containing strain where the pglX methylase was deleted (green). Axes and error bars are as in Fig .


E Southern blot analysis of phage Φ3T genome during infection . Numbers indicate time ( in min ) following infection ; U , uninfected . Probe was designed to match positions 94 , 645 - 95 , 416 in the phage genome . Each lane contains 200 ng total DNA .

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

E Southern blot analysis of phage Φ3T genome during infection. Numbers indicate time (in min) following infection; U, uninfected. Probe was designed to match positions 94,645-95,416 in the phage genome. Each lane contains 200 ng total DNA.


( A ) MCF - 7 cells were co - transfected with either MIR - CNT ( control plasmid ) or MIR376A and GFP - LC3 plasmid , and GFP - LC3 dot formation was analyzed . White arrows indicate clusters of the GFP - LC3 dots in cells . ( B ) Quantification of the experiments in A . MIR376A overexpression , but not MIR - CNT expression , blocked starvation ( STV ) - induced autophagy ( mean ± S . D . of independent experiments , n = 4 , * * * p < 0 , 01 ) . NON STV , non -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MCF-7 cells were co-transfected with either MIR-CNT (control plasmid) or MIR376A and GFP-LC3 plasmid, and GFP-LC3 dot formation was analyzed. White arrows indicate clusters of the GFP-LC3 dots in cells. (B) Quantification of the experiments in A. MIR376A overexpression, but not MIR-CNT expression, blocked starvation (STV)-induced autophagy (mean ± S.D. of independent experiments, n = 4, ***p<0,01). NON STV, non-starved


( C ) Overexpression of MIR376A resulted in a decrease in the autophagic activity of MCF - 7 cells . Starvation - induced conversion of LC3 - I to LC3 - II in MCF - 7 cells was analyzed . Tests were performed in the presence or absence of E64d ( 10 µg / ml ) and Pepstatin A ( 10 µg / ml ) ( E + P ) . LC3 - II / LC3 - I densitometric ratios were marked . ACTB was used as a loading control .

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

(C) Overexpression of MIR376A resulted in a decrease in the autophagic activity of MCF-7 cells. Starvation-induced conversion of LC3-I to LC3-II in MCF-7 cells was analyzed. Tests were performed in the presence or absence of E64d (10 µg/ml) and Pepstatin A (10 µg/ml) (E+P). LC3-II/LC3-I densitometric ratios were marked. ACTB was used as a loading control.


( D ) MIR376A blocked starvation induced SQSTM1 degradation in MCF - 7 cells . ACTB was used as a loading control . SQSTM1 / ACTIN densitometric ratios were marked .

O O O O O O O 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) MIR376A blocked starvation induced SQSTM1 degradation in MCF-7 cells. ACTB was used as a loading control. SQSTM1/ACTIN densitometric ratios were marked.


( A ) MIR376A blocked GFP - LC3 dot formation under starvation condition .

(A) MIR376A blocked GFP-LC3 dot formation under starvation condition.


( A ) MIR376A blocked GFP - LC3 dot formation under starvation condition . ( B ) Quantitative analysis of experiments in A ( mean ± S . D . of independent experiments , n = 3 , * * * p < 0 , 01 ) .

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(A) MIR376A blocked GFP-LC3 dot formation under starvation condition. (B) Quantitative analysis of experiments in A (mean ± S.D. of independent experiments, n = 3, ***p<0,01).


( C ) Overexpression of MIR376A resulted in decreased autophagic flux in Huh - 7 cells . Starvation - induced conversion of LC3 - I to LC3 - II was analyzed . Tests were performed in the presence or absence of E64d ( 10 µg / ml ) and Pepstatin A ( 10 µg / ml ) ( E + P ) . LC3 - II / LC3 - I densitometric ratios were marked . ACTB was used as a loading control .

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

(C) Overexpression of MIR376A resulted in decreased autophagic flux in Huh-7 cells. Starvation-induced conversion of LC3-I to LC3-II was analyzed. Tests were performed in the presence or absence of E64d (10 µg/ml) and Pepstatin A (10 µg/ml) (E+P). LC3-II/LC3-I densitometric ratios were marked. ACTB was used as a loading control.


( C and D ) qPCR analysis of ATG4C and BECN1 mRNA expression in control miRNA ( MIR - CONT ) or MIR376A overexpressing cells under non - starved ( NON STV ) or starved ( STV ) condition ( mean ± S . D . of independent experiments , n = 3 , * p < 0 , 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) qPCR analysis of ATG4C and BECN1 mRNA expression in control miRNA (MIR-CONT) or MIR376A overexpressing cells under non-starved (NON STV) or starved (STV) condition (mean ± S.D. of independent experiments, n = 3, *p<0,05).


( E and F ) Immunoblots showing ATG4C and BECN1 protein levels following control miRNA ( MIR - CONT ) or MIR376A overexpression . ACTB was used as a loading control . ATG4C / ACTB or BECN1 / ACTB densitometric ratios were marked .

O O O O O O O O O O O O O O O O O O O O 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 and F) Immunoblots showing ATG4C and BECN1 protein levels following control miRNA (MIR-CONT) or MIR376A overexpression. ACTB was used as a loading control. ATG4C/ACTB or BECN1/ACTB densitometric ratios were marked.


( A and B ) qPCR analysis of ATG4C and BECN1 mRNA levels in MCF - 7 cells transfected with control antagomirs ( CNT - Ant ) or antagomir - 376a ( Ant - 376a ) ( mean ± S . D . of independent experiments , n = 3 , * * p < 0 , 03 for ATG4C , and n = 3 , * * p < 0 , 03 for BECN1 ) . Results were expressed as fold changes against GAPDH mRNA levels .

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

(A and B) qPCR analysis of ATG4C and BECN1 mRNA levels in MCF-7 cells transfected with control antagomirs (CNT-Ant) or antagomir-376a (Ant-376a) (mean ± S.D. of independent experiments, n = 3, **p<0,03 for ATG4C, and n = 3, **p<0,03 for BECN1). Results were expressed as fold changes against GAPDH mRNA levels.


( C and D ) ATG4C and BECN1 protein levels were increased following antagomir - 376a ( Ant - 376a ) transfection . ATG4C / ACTB or BECN1 / ACTB densitometric ratios were marked .

O O O O O O O O O O O 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 and D) ATG4C and BECN1 protein levels were increased following antagomir-376a (Ant-376a) transfection. ATG4C/ACTB or BECN1/ACTB densitometric ratios were marked.


( C and D ) Normalized luciferase activity measuremets of lysates from 293T cells that were co - transfected with either MIR - CNT or MIR376A plasmids together with the wild - type or mutant luciferase constructs ( mean ± S . D . of independent experiments , n = 3 for ATG4C tests , * p < 0 , 05 and n = 5 for BECN1 tests , * * * p < 0 , 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) Normalized luciferase activity measuremets of lysates from 293T cells that were co-transfected with either MIR-CNT or MIR376A plasmids together with the wild-type or mutant luciferase constructs (mean ± S.D. of independent experiments, n = 3 for ATG4C tests, *p<0,05 and n = 5 for BECN1 tests, ***p<0,01).


( A ) Blockage of endogenous MIR376A by Ant - 376a , but not CNT - Ant further stimulated starvation ( STV ) - activated LC3 - I to LC3 - II conversion in MCF - 7 cells . ACTB was used as a loading control . LC3 - II / LC3 - I densitometric ratios were marked .

(A) Blockage of endogenous MIR376A by Ant-376a, but not CNT-Ant further stimulated starvation (STV)-activated LC3-I to LC3-II conversion in MCF-7 cells. ACTB was used as a loading control. LC3-II/LC3-I densitometric ratios were marked.


( B ) Ant - 376a , but not CNT - Ant resulted in further activation of SQSTM1 protein degradation following starvation in MCF - 7 cells . SQSTM1 / ACTB ratios were marked .

O O O O O O O O O O O O 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) Ant-376a, but not CNT-Ant resulted in further activation of SQSTM1 protein degradation following starvation in MCF-7 cells. SQSTM1/ACTB ratios were marked.


MIR376A and MIR376B level changes in response to starvation stress . ( A and B ) TaqMan quantitative PCR ( qPCR ) analysis of endogenous MIR376A ( A ) and MIR376B ( B ) levels under control ( CNT , no starvation ) or starvation ( STV , 1 to 8 hours ) conditions . TaqMan qPCR data was normalized using U6 small nuclear 1 ( RNU6 - 1 ) ( U6 ) mRNA levels ( mean ± S . D . of independent experiments , n = 5 for both A and B , * * * p < 0 , 01 ) .

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

MIR376A and MIR376B level changes in response to starvation stress. (A and B) TaqMan quantitative PCR (qPCR) analysis of endogenous MIR376A (A) and MIR376B (B) levels under control (CNT, no starvation) or starvation (STV, 1 to 8 hours) conditions. TaqMan qPCR data was normalized using U6 small nuclear 1 (RNU6-1) (U6) mRNA levels (mean ± S.D. of independent experiments, n = 5 for both A and B, ***p<0,01).


C . Dissociation constants ( Kd ) of Ago2 - miRNA complexes ( containing an AU - rich supplementary region ) binding to target RNAs with bridging regions of various lengths . D . Dissociation constants ( Kd ) of Ago2 - miRNA complexes ( containing an GC - rich supplementary region ) binding to target RNAs with bridging regions of various lengths . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Dissociation constants (Kd) of Ago2-miRNA complexes (containing an AU-rich supplementary region) binding to target RNAs with bridging regions of various lengths. D. Dissociation constants (Kd) of Ago2-miRNA complexes (containing an GC-rich supplementary region) binding to target RNAs with bridging regions of various lengths. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


E . Fold repression of a luciferase reporter ( relative to no target site control ) plotted versus affinity ( 1 / Kd ) values measured for the same sites in vitro . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM .

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

E. Fold repression of a luciferase reporter (relative to no target site control) plotted versus affinity (1/Kd) values measured for the same sites in vitro. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


A . Top : schematic of miR - 122 isomiRs paired with a target complementary to the seed region only . Bottom : release of the seed - paired target from Ago2 - miR complexes was assessed by monitoring the fraction of the total target RNA bound as a function of time . B . Top : schematic of miR - 122 isomiRs paired with a target complementary to the seed and supplementary ( g13 - g17 ) regions . Bottom : fraction target bound to the Ago2 - miR complexes as a function of time . C . Top : schematic of miR - 122 isomiRs paired with a target complementary to the seed and shifted supplementary ( g15 - g19 ) regions . . Bottom : fraction target bound to the Ago2 - miR complexes as a function of time . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM .

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A. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed region only. Bottom: release of the seed-paired target from Ago2-miR complexes was assessed by monitoring the fraction of the total target RNA bound as a function of time. B. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed and supplementary (g13-g17) regions. Bottom: fraction target bound to the Ago2-miR complexes as a function of time. C. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed and shifted supplementary (g15-g19) regions. . Bottom: fraction target bound to the Ago2-miR complexes as a function of time. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


E . Release half times ( t1 / 2 ) of target RNAs in panels ( A - C ) plotted against isomiR length . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM .

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E. Release half times (t1/2) of target RNAs in panels (A-C) plotted against isomiR length. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


Overall structure of the RBD - ACE2 - B0AT1 complex . ( A ) Cryo - EM map of the RBD - ACE2 - B0AT1 complex . Left : Overall reconstruction of the ternary complex at 2 . 9 Å . Inset : focused refined map of RBD .

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Overall structure of the RBD-ACE2-B0AT1 complex.(A) Cryo-EM map of the RBD-ACE2-B0AT1 complex. Left: Overall reconstruction of the ternary complex at 2.9 Å. Inset: focused refined map of RBD.


Overall structure of the RBD - ACE2 - B0AT1 complex . The complex is colored by subunits , with the protease domain ( PD ) and the Collectrin - like domain ( CLD ) colored cyan and blue in one of the ACE2 protomers , respectively . The glycosylation moieties are shown as sticks .

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

Overall structure of the RBD-ACE2-B0AT1 complex. The complex is colored by subunits, with the protease domain (PD) and the Collectrin-like domain (CLD) colored cyan and blue in one of the ACE2 protomers, respectively. The glycosylation moieties are shown as sticks.


Cryo - EM density of the interface between RBD and ACE2 . The density , shown as pink meshes , is contoured at 12 σ .

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Cryo-EM density of the interface between RBD and ACE2.The density, shown as pink meshes, is contoured at 12 σ.


Impact of MB on cytotoxicity of OT - 1 CTLs against EG7 or EG7 - L1 . Splenocytes from OT - 1 mice in culture were stimulated with 10 nM of OVA257 - 264 ( SIINFEKL ) for 3 days to generate mature CTLs . CTLs were incubated with CFSE labeled EG7 - L1 cells in the presence of MB at indicated concentrations . Cytotoxicity was determined by flow cytometry analysis . Data are representative of three independent experiments . ( effector to target ratio = 5 : 1 , unpaired t - test ) . EG7 - L1 : EG7 overexpressing PD - L1 . Statistical results of ( B ) . ELISA measured of LDH release to assess the cytotoxicity of OT - I CTLs on EG7 - L1 in the presence of MB . Anti - PD1 antibody ( aPD - 1 ) served as positive control . Data information : Data are representative of three independent experiments . Unpaired t - test ; Error bars denote s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

Impact of MB on cytotoxicity of OT-1 CTLs against EG7 or EG7-L1. Splenocytes from OT-1 mice in culture were stimulated with 10 nM of OVA257-264 (SIINFEKL) for 3 days to generate mature CTLs. CTLs were incubated with CFSE labeled EG7-L1 cells in the presence of MB at indicated concentrations. Cytotoxicity was determined by flow cytometry analysis. Data are representative of three independent experiments. (effector to target ratio = 5:1, unpaired t-test). EG7-L1: EG7 overexpressing PD-L1.   Statistical results of (B).   ELISA measured of LDH release to assess the cytotoxicity of OT-I CTLs on EG7-L1 in the presence of MB. Anti-PD1 antibody (aPD-1) served as positive control.   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001.


Impact of MB on cytotoxicity of PD - 1 - / - ( PD - 1KO ) OT - 1 CTLs against EG7 or PD - L1 expressing EG7 stable cell line . Data are representative of three independent experiments . ( unpaired t - test ) . Statistical results of ( E ) . Data information : Data are representative of three independent experiments . Unpaired t - test ; Error bars denote s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

Impact of MB on cytotoxicity of PD-1-/- (PD-1KO) OT-1 CTLs against EG7 or PD-L1 expressing EG7 stable cell line. Data are representative of three independent experiments. (unpaired t-test). Statistical results of (E). Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001.


Cytotoxicity of OT - I CTLs against B16 - F10 - OVA cells ( designated B16 - OVA ) in the presence of 1 μΜ of MB or DMSO . Cytotoxicity was determined by the relative area unoccupied by cells examined under microscope ( scale bar = 50 μm ) . Data are representative of three independent experiments , and were analyzed by unpaired t - test . Statistical results of ( G ) . Data information : Data are representative of three independent experiments . Unpaired t - test ; Error bars denote s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

Cytotoxicity of OT-I CTLs against B16-F10-OVA cells (designated B16-OVA) in the presence of 1 μΜ of MB or DMSO. Cytotoxicity was determined by the relative area unoccupied by cells examined under microscope (scale bar = 50 μm). Data are representative of three independent experiments, and were analyzed by unpaired t-test.   Statistical results of (G).   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001.


Effect of MB on the proliferation of WT CTLs , PD - 1KO CTLs . Splenic cells were stained with 5 μM of CFSE and seeded into aCD3 / aCD28 coated 96 - well plates . 10 μg / ml of PD - L1 were administered in media . Cell proliferation were checked by monitoring dilution of CFSE 48 hours after CD3 / CD28 stimulation by gating on CD8 positive population through FACS analysis . WT : splenic cell from wildtype C57BL / J mice . PD - 1KO : splenic cell from PD - 1 knockout mice . Statistical results of ( A ) Data information : Data are representative of three independent experiments . Unpaired t - test ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

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

Effect of MB on the proliferation of WT CTLs, PD-1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28 coated 96-well plates. 10 μg/ml of PD-L1 were administered in media. Cell proliferation were checked by monitoring dilution of CFSE 48 hours after CD3/CD28 stimulation by gating on CD8 positive population through FACS analysis. WT: splenic cell from wildtype C57BL/J mice. PD-1KO: splenic cell from PD-1 knockout mice.   Statistical results of (A)   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


FACS analysis of the effect of MB on the activation of OT - I CTLs . OT - I CTLs were stimulated with precoated aCD3 / aCD28 and 10 μg / ml of mouse PD - L1 protein in the presence of 100 nM MB or 10 μg / ml aPD1 ( served as positive control ) . After 24 hours , surface expression of CD25 and CD69 on OT - 1 CTLs were determined through FACS analysis . Statistical results of ( C ) Data information : Data are representative of three independent experiments . Unpaired t - test ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FACS analysis of the effect of MB on the activation of OT-I CTLs. OT-I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD-L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 hours, surface expression of CD25 and CD69 on OT-1 CTLs were determined through FACS analysis.   Statistical results of (C)   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


Effect of MB on the activation status of Jurkat T cells . Luciferase activity is suppressed in JP - luc by co - culture with Raji - L1 preloaded with 1 μg / ml superantigen ( SEE ) . Treatment with MB enhanced luciferase expression ( SEE - Loaded Raji ( PD - L1 negative ) served as positive control ) . IC50 is calculated to be 117 nM . JP - luc : Jurkat cell harboring NFAT - luciferase transgene and overexpressing PD - 1 ; Raji - L1 : Raji overexpressing PD - L1 Data information : Data are representative of three independent experiments . Unpaired t - test ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

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

Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP-luc by co-culture with Raji-L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE-Loaded Raji (PD-L1 negative) served as positive control). IC50 is calculated to be 117 nM. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1 Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


Impact of MB on luciferase activity of various engineered Jurkat T cells . J - luc : Jurkat cell harboring NFAT - luciferase transgene ; J - luc - sgPD - 1 : J - luc cells treated with lentivirus expressing sgPD - 1 / CAS9 simultaneously Data information : Data are representative of three independent experiments . Unpaired t - test ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

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

Impact of MB on luciferase activity of various engineered Jurkat T cells. J-luc: Jurkat cell harboring NFAT-luciferase transgene; J-luc-sgPD-1: J-luc cells treated with lentivirus expressing sgPD-1/CAS9 simultaneously Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


qRT - PCR analysis of IL - 2 mRNA level in JP - luc cells stimulated with precoated aCD3 / aCD28 ( 10 μg / ml ) in the presence of 10 μg / ml of PD - L1 and MB at indicated concentrations Data information : Data are representative of three independent experiments . Unpaired t - test ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

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

qRT-PCR analysis of IL-2 mRNA level in JP-luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD-L1 and MB at indicated concentrations Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


MB enhancing IL - 2 expression by JP - luc stimulated with Raji - L1 for 24 - hour quantified through ELISA analysis ( aPD1 served as positive control ) . MB enhancing production of cytokine and cytolytic granule by OT - I CTLs . CTLs were incubated with EG7 - L1 cells in the presence of protein transport inhibitor ( PTI ) and MB at indicated concentrations . Expression of cytokine and cytolytic granule were determined by flow cytometry . aPD1 served as positive control . EG7 - L1 : EG7 overexpressing PD - L1 . Statistical results of ( I ) Data information : Data are representative of three independent experiments . Unpaired t - test ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

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

MB enhancing IL-2 expression by JP-luc stimulated with Raji-L1 for 24-hour quantified through ELISA analysis (aPD1 served as positive control).   MB enhancing production of cytokine and cytolytic granule by OT-I CTLs. CTLs were incubated with EG7-L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule were determined by flow cytometry. aPD1 served as positive control. EG7-L1: EG7 overexpressing PD-L1.   Statistical results of (I)   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


Confocal cell images of Jurkat cells co - expressing PD - 1 - EGFP and SHP2 - mCherry . Jurkat cells co - expressing PD - 1 - EGFP and SHP2 - mCherry were incubated with 10 μg / ml of human PD - L1 protein in the presence of 100 nM and 1 μM of MB for 2 minutes , fixed with 4 % paraformaldehyde and stained with DAPI . Colocalization signal were examined with confocal microscope ( scale bar = 2 µm )

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

Confocal cell images of Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry. Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry were incubated with 10 μg/ml of human PD-L1 protein in the presence of 100 nM and 1 μM of MB for 2 minutes, fixed with 4% paraformaldehyde and stained with DAPI. Colocalization signal were examined with confocal microscope (scale bar = 2 µm)


Fluorescence complementation analysis of the effect of MB on the interaction between PD - 1 with SHP2 . Jurkat cells co - expressing PD1 - C - Luc or PD1Y248F - C - Luc and SHP2 - N - luc were seeded in a 96 - well precoated with 10 μg / ml of aCD3 / aCD28 with media supplemented with 10 μg / ml human PD - L1 protein in the presenc of MB at indicated concentrations for 6 hours followed by analysis of luciferase activity Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001

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

Fluorescence complementation analysis of the effect of MB on the interaction between PD-1 with SHP2. Jurkat cells co-expressing PD1-C-Luc or PD1Y248F-C-Luc and SHP2-N-luc were seeded in a 96-well precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD-L1 protein in the presenc of MB at indicated concentrations for 6 hours followed by analysis of luciferase activity Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001


Co - IP analysis of impact of MB on interaction between PD - 1 and SHP2 . 293T cells co - expressing PD - 1 and SHP2 - FLAG were treated with MB at indicated concentration for 6 hours

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 B-CONTROLLED_VAR O O O O O O

Co-IP analysis of impact of MB on interaction between PD-1 and SHP2. 293T cells co-expressing PD-1 and SHP2-FLAG were treated with MB at indicated concentration for 6 hours


Ability of MB to block interaction between PD - 1 with SHP2 revealed through ELISA . Y248 phosphorylated human PD - 1 ITSM peptide was coated at 4 μg / ml in 96 plate overnight . Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 hours . Bound SHP2 were measured by monitoring the activity of horse - peroxidase conjugated on secondary antibody targeting SHP2 Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001

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

Ability of MB to block interaction between PD-1 with SHP2 revealed through ELISA. Y248 phosphorylated human PD-1 ITSM peptide was coated at 4 μg/ml in 96 plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 hours. Bound SHP2 were measured by monitoring the activity of horse-peroxidase conjugated on secondary antibody targeting SHP2 Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001


Representative western blot showing the levels of total and phosphorylated ZAP70 , PKCθ and PLCγ1 in the lysates of stimulated CTL , inhibited by PD - L1 in the presence of 1μΜ of MB

O O O O O O O O O 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

Representative western blot showing the levels of total and phosphorylated ZAP70, PKCθ and PLCγ1 in the lysates of stimulated CTL, inhibited by PD-L1 in the presence of 1μΜ of MB


Representative western blot showing the levels CD28 phosphorylation . CTL were stimulated with EG7 cells ( parental ) , EG7 - L1 and EG7 - L1 in the presence 100 nM of MB for 2 hours . Protein immunoprecipitated ( IP ) with Anti - pY from the lysates of the indicated CTL - EG7 co - culture were subjected to SDS - PAGE separation and blotted with aCD28 . β - actin served as loading control . EG7 - L1 : EG7 overexpressing PD - L1 Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001

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

Representative western blot showing the levels CD28 phosphorylation. CTL were stimulated with EG7 cells (parental), EG7-L1 and EG7-L1 in the presence 100 nM of MB for 2 hours. Protein immunoprecipitated (IP) with Anti-pY from the lysates of the indicated CTL-EG7 co-culture were subjected to SDS-PAGE separation and blotted with aCD28. β-actin served as loading control. EG7-L1: EG7 overexpressing PD-L1 Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001


Phos - tag analysis of CD28 phosphorylation in CTL . CTLs were stimulated with EG7 or EG7 - L1 in the presence of 1μΜ MB . Cell lysate were then separated by SDS - PAGE containing 50 μM of Phos - tag and blotted with aCD2 antibody . Phosphorylated CD28 ( slow - moving ) species were visualized through

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

Phos-tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7-L1 in the presence of 1μΜ MB. Cell lysate were then separated by SDS-PAGE containing 50 μM of Phos-tag and blotted with aCD2 antibody. Phosphorylated CD28 (slow-moving) species were visualized through exposure


Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85 . Jurkat or Jurkat - PD - 1 cells were co - transfected with CD28 - C - Luc or CD28Y191F - C - Luc and P85 - N - Luc . Cells were seeded in a 96 - well plate coated with 10 μg / ml of aCD3 / aCD28 in the presence of human 10 μg / ml of PD - L1 protein and 100 nM of MB for 6 hours . Luciferase activity was measured as readout for CD28 - P85 interaction Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001

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

Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat-PD-1 cells were co-transfected with CD28-C-Luc or CD28Y191F-C-Luc and P85-N-Luc. Cells were seeded in a 96-well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD-L1 protein and 100 nM of MB for 6 hours. Luciferase activity was measured as readout for CD28-P85 interaction Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001


Schematic of the xenograft mouse model for MB treatment . C57BL / 6J mice were inoculated with EG7 - L1 cells ( 2 × 106 cells , s . c . ) on the right flank on day 1 , followed by injection ( 2 × 106 cells , i . v . ) of CD45 . 1 + CTL on day 3 and day 6 respectively . The mice were randomized into 3 groups ( n = 5 ) and treated with vehicle , MB ( 20 mg / kg / day , i . g . ) , aPD1 ( 10 mg / kg , every other day , i . p . ) served as positive control . Tumor size was measured through calipering every other day . EG7 - L1 : EG7 overexpressing PD - L1 . Tumor growth curve for ( A ) was shown as mean ± s . e . m . Representative image of the tumor on day 10 mentioned in ( A ) . Tumor weight on day 10 ( n = 5 ) mentioned in ( A ) . Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

Schematic of the xenograft mouse model for MB treatment. C57BL/6J mice were inoculated with EG7-L1 cells (2 × 106 cells, s.c.) on the right flank on day 1, followed by injection (2 × 106 cells, i.v.) of CD45.1+ CTL on day 3 and day 6 respectively. The mice were randomized into 3 groups (n = 5) and treated with vehicle, MB (20 mg/kg/day, i.g.), aPD1 (10 mg/kg, every other day, i.p.) served as positive control. Tumor size was measured through calipering every other day. EG7-L1: EG7 overexpressing PD-L1.   Tumor growth curve for (A) was shown as mean ±s.e.m.   Representative image of the tumor on day 10 mentioned in (A).   Tumor weight on day 10 (n=5) mentioned in (A).   Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.


Immunohistochemical analysis of cell proliferation marker ki - 67 and CD8 in tumor nodules on day 10 ( n = 5 ) Statistical results of ( E ) . Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

Immunohistochemical analysis of cell proliferation marker ki-67 and CD8 in tumor nodules on day 10 (n=5) Statistical results of (E). Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.


Expression of Perforin , GZMB and IL - 2 by CD8 + CD45 . 1 + TILs were analyzed by flow cytometry . Statistical results of ( G ) . Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

Expression of Perforin, GZMB and IL-2 by CD8+ CD45.1+ TILs were analyzed by flow cytometry.   Statistical results of (G).   Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.


Schematic of EGFR L858R mice treated with MB . Tumor burdens recorded through Computed tomography ( CT ) scanning for EGFR L858R mice ( n = 5 , lung section = 3 ) . Mice were treated with MB ( 20 mg / kg / day , i . g . ) for 1 week , followed by imaged with CT again to record tumor burden . Statistical results of ( J ) . Haematoxylin and Eosin staining of lung section of EGFR L858R mice ( n = 5 , lung section = 3 ) in ( J ) . Statistical results of ( L ) . Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

Schematic of EGFR L858R mice treated with MB.   Tumor burdens recorded through Computed tomography (CT) scanning for EGFR L858R mice (n=5, lung section = 3). Mice were treated with MB (20 mg/kg/day, i.g.) for 1 week, followed by imaged with CT again to record tumor burden.   Statistical results of (J).   Haematoxylin and Eosin staining of lung section of EGFR L858R mice (n = 5, lung section = 3) in (J).   Statistical results of (L).   Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.


Effect of MB on proliferation of human CD8 + T cells . CFSE labeled human peripheral blood mononuclear cells ( PBMC ) were pre - incubated with DMSO , MB , 25 μg / ml of pembrolizumab or 20 μg / ml of Nivolumab for 1 h and then seeded in the 96 - well plate precoated with 10 μg / ml of aCD3 / aCD28 in the presence of 10 μg / ml human PD - L1 Protein . Cell proliferation was analyzed through FACS analysis of dilution of CFSE . Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * * P < 0 . 01 ; * * * * P < 0 . 0001 .

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-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of MB on proliferation of human CD8+ T cells. CFSE labeled human peripheral blood mononuclear cells (PBMC) were pre-incubated with DMSO, MB, 25 μg/ml of pembrolizumab or 20 μg/ml of Nivolumab for 1 h and then seeded in the 96-well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD-L1 Protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.


Cytokine and cytolytic granule production in CD8 + T cells . Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg / ml of phytohaemagglutinin for 2 days and then rested for 1 day . Cells were pretreated with DMSO , MB , 25 μg / ml of pembrolizumab ( Pem ) or 20 μg / ml of nivolumab ( Niv ) for 1 h and then seeded in a 96 - well precoated with 10 μg / ml aCD3 / aCD28 , with media supplemented with 10 μg / ml of human PD - L1 Protein . Cytokine and cytolytic granule production were determined by intracellular flow cytometry by gating on CD8 + populations . Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * * P < 0 . 01 ; * * * * P < 0 . 0001 .

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

Cytokine and cytolytic granule production in CD8+ T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96-well precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD-L1 Protein. Cytokine and cytolytic granule production were determined by intracellular flow cytometry by gating on CD8+ populations. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.


Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell . JP - luc cells were stimulated with Raji - L1 or parental Raji cells preloaded with 1 μg / ml of superantigen ( SEE ) in the presence of MB at indicated concentration or 25μg / ml of pembrolizumab ( Pem ) or 20μg / ml of nivolumab ( Niv ) for 6 hours . Luciferase activity was measured by luminometer . Data are representative of three independent experiments , and were analyzed by unpaired t - test . JP - luc : Jurkat cell harboring NFAT - luciferase transgene and overexpressing PD - 1 ; Raji - L1 : Raji overexpressing PD - L1 . Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * * P < 0 . 01 ; * * * * P < 0 . 0001 .

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

Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP-luc cells were stimulated with Raji-L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25μg/ml of pembrolizumab (Pem) or 20μg/ml of nivolumab (Niv) for 6 hours. Luciferase activity was measured by luminometer. Data are representative of three independent experiments, and were analyzed by unpaired t-test. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.


C Expression of F9 mRNA and protein in hepatic tissue of indicated mouse strains . Data represent means ± SE . M : Marker ; arrowhead : FIX protein ; Note : the bands of FIX in F9383STOP lanes are faint with small molecular weight smears .

O O O O O O O O O O O O O O O O O O O O O O O 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 Expression of F9 mRNA and protein in hepatic tissue of indicated mouse strains. Data represent means ± SE. M: Marker; arrowhead: FIX protein; Note: the bands of FIX in F9383STOP lanes are faint with small molecular weight smears.


D Measurement of coagulation activity by aPTT in mice at 8 weeks of age . n = 5 for each group , Data represent means ± SE .

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D Measurement of coagulation activity by aPTT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE.


E Measurement of coagulation activity by PT in mice at 8 weeks of age . n = 5 for each group , Data represent means ± SE .

E Measurement of coagulation activity by PT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE.


F Measurement of blood loss over a 5 - minute period after tail transection in mice at 8 weeks of age . Wild - type , n = 5 ; F9Y381S , n = 6 ; F9Y381D , n = 13 ; F9383STOP , n = 5 .

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F Measurement of blood loss over a 5-minute period after tail transection in mice at 8 weeks of age. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5.


G Survival rate of mice after the tail - clip challenge . The mice were monitored for two days after tail clipping . Wild - type , n = 5 ; F9Y381S , n = 6 ; F9Y381D , n = 13 ; F9383STOP , n = 5 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 Survival rate of mice after the tail-clip challenge. The mice were monitored for two days after tail clipping. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5.


B Test of clotting activity by aPTT and PT at 8 weeks following hydrodynamic tail vein injection of 120 µg pX458 and 120 µg donor plasmids ( or 120 µg ssODN ) per mouse .

O O O O O O O O O O O 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 Test of clotting activity by aPTT and PT at 8 weeks following hydrodynamic tail vein injection of 120 µg pX458 and 120 µg donor plasmids (or 120 µg ssODN) per mouse.


C Survival rate of mice after the tail - clip challenge . F9Y381D mice were treated with or without Cas9 / sgRNA / donor DNA for 3 months . After tail - clip challenge , the survival rate of each group over 2 days was determined . Wild - type , n = 9 ; untreated F9Y381D , n = 13 ; DNA treated F9Y381D , n = 14 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Survival rate of mice after the tail-clip challenge. F9Y381D mice were treated with or without Cas9/sgRNA/donor DNA for 3 months. After tail-clip challenge, the survival rate of each group over 2 days was determined. Wild-type, n=9; untreated F9Y381D, n=13; DNA treated F9Y381D, n=14.


D Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing ( Cas9 / donor vector treated group ) or by TA - clone sequencing ( Cas9 / ssODN group ) .

D Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing (Cas9/donor vector treated group) or by TA-clone sequencing (Cas9/ssODN group).


F The genome editing efficiency of individual mice is presented .

F The genome editing efficiency of individual mice is presented.


G Plasma aspartate aminotransferase ( AST ) and alanine aminotransferase ( ALT ) level was determined in F9Y381D mice 8 weeks after HTV injection ( n = 5 ) .

O O O O O O 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 Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) level was determined in F9Y381D mice 8 weeks after HTV injection (n=5).


H mRNA levels of inflammatory cytokines from liver tissue of naked DNA treated mice were determined by real - time PCR . Data represent means ± SE .

H mRNA levels of inflammatory cytokines from liver tissue of naked DNA treated mice were determined by real-time PCR. Data represent means ± SE.


B In vivo infection efficiency 4 days after Adv injection . Inserts : negative control from untreated mouse liver tissue . Scale bar : 100 μm .

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B In vivo infection efficiency 4 days after Adv injection. Inserts: negative control from untreated mouse liver tissue. Scale bar: 100 μm.


C T7E1 assay was performed on liver DNA obtained from samples in ( B ) , and the frequency of Cas9 - induced indels is indicated as ' % ' below the lane .

O O O O O O O O O O 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 T7E1 assay was performed on liver DNA obtained from samples in (B), and the frequency of Cas9-induced indels is indicated as '%' below the lane.


D Measurement of clotting activity by aPTTs and PTs 8 weeks after tail vein injection with indicated vectors . Data represent means ± SE .

O 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 Measurement of clotting activity by aPTTs and PTs 8 weeks after tail vein injection with indicated vectors. Data represent means ± SE.


E In vivo genome editing efficiency of whole liver tissues from mice treated with Adv ( Cas9 + G / T ) . ( Left ) HDR donor used for correction of Y381D mutation , and Illumina sequencing reads ( rn ) are presented . Red text indicates the correction of the F9 mutation , whereas blue text indicates the intended synonymous mutations . ( Right ) The percentage of indel or HDR corrections obtained through sequencing of 235 individual TA - clones ( low dose ) or Illumina sequencing ( high dose ) of treated mouseliver tissues .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vivo genome editing efficiency of whole liver tissues from mice treated with Adv(Cas9+G/T). (Left) HDR donor used for correction of Y381D mutation, and Illumina sequencing reads (rn) are presented. Red text indicates the correction of the F9 mutation, whereas blue text indicates the intended synonymous mutations. (Right) The percentage of indel or HDR corrections obtained through sequencing of 235 individual TA-clones (low dose) or Illumina sequencing (high dose) of treated mouseliver tissues.


A . Volcano plot depicting differential expression of nearly 18000 unique genes ( minimum expression of 1 read per kilobase per million , RPKM ) in response to AMV infection . In red , upregulated genes ( log2FC ≥ 1 ; adjusted p - value ≤ 0 . 05 ) ; in blue , downregulated genes ( log2FC ≤ - 1 ; adjusted p - value ≤ 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Volcano plot depicting differential expression of nearly 18000 unique genes (minimum expression of 1 read per kilobase per million, RPKM) in response to AMV infection. In red, upregulated genes (log2FC ≥ 1; adjusted p-value ≤ 0.05); in blue, downregulated genes (log2FC ≤ -1; adjusted p-value ≤ 0.05


D . RT - qPCR analysis of expression of four upregulated m6A pathway - related genes in response to AMV infection ( three biological replicates ) . Error bars represent the standard error of the mean ( SEM ) . Asterisks indicate a p < 0 . 05 applying Student ' s t - test for ΔCt mean values ( n = 3 ) .

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

D. RT-qPCR analysis of expression of four upregulated m6A pathway-related genes in response to AMV infection (three biological replicates). Error bars represent the standard error of the mean (SEM). Asterisks indicate a p < 0.05 applying Student's t-test for ΔCt mean values (n = 3).


A - D . RNA blots were hybridized to AMV - specific probes to reveal accumulation of viral RNA in double ( de ) and triple ( te ) ect2 / 3 / 4 ( A , B ) or ect2 / 3 / 5 ( C , D ) mutants compared to WT plants . A and C show local infection at 3 days post - inoculation ( dpi ) ; B and D show systemic infection in aerial tissue at 7 dpi . Each panel shows a representative RNA blot displaying AMV RNAs 1 - 4 ( left ) and its quantification histogram ( right ) . Dashed lines indicate non - contiguous samples analyzed on the same membrane . Ethidium bromide staining of ribosomal RNAs ( EB ) was used as RNA loading control . Error bars indicate standard deviations . Asterisks indicate significant differences from the WT ( * : p < 0 . 05 ; * * ; p < 0 . 01 ) using the Student ' s t - test ( n = 4 ) . AU , arbitrary units .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. RNA blots were hybridized to AMV-specific probes to reveal accumulation of viral RNA in double (de) and triple (te) ect2/3/4 (A, B) or ect2/3/5 (C, D) mutants compared to WT plants. A and C show local infection at 3 days post-inoculation (dpi); B and D show systemic infection in aerial tissue at 7 dpi. Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Dashed lines indicate non-contiguous samples analyzed on the same membrane. Ethidium bromide staining of ribosomal RNAs (EB) was used as RNA loading control. Error bars indicate standard deviations. Asterisks indicate significant differences from the WT (*: p < 0.05; **; p < 0.01) using the Student's t-test (n = 4). AU, arbitrary units.


A , RNA - blot analysis of AMV systemic infection in plants expressing ECT2WT - mCherry ( WT ) or ECT2W464A - mCherry ( W464A ) in the de23 background ( A ) at 7 days post - inoculation . Each panel shows a representative RNA blot displaying AMV RNAs 1 - 4 ( left ) and its quantification histogram ( right ) . Ethidium bromide staining of rRNAs ( EB ) was used as RNA loading control . Genotypes are indicated on the top of each northern blot . In A , upper and lower panels show independent complementation transgenic lines , and different letters indicate statistical differences according to Fisher ' s Least Significant Difference ( LSD ) ( p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, RNA-blot analysis of AMV systemic infection in plants expressing ECT2WT-mCherry (WT) or ECT2W464A-mCherry (W464A) in the de23 background (A) at 7 days post-inoculation. Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. In A, upper and lower panels show independent complementation transgenic lines, and different letters indicate statistical differences according to Fisher's Least Significant Difference (LSD) (p < 0.05).


B . RNA - blot analysis of AMV systemic infection in plants expressing in vir - 1 plants ( B ) at 7 days post - inoculation . Each panel shows a representative RNA blot displaying AMV RNAs 1 - 4 ( left ) and its quantification histogram ( right ) . Ethidium bromide staining of rRNAs ( EB ) was used as RNA loading control . Genotypes are indicated on the top of each northern blot . In B , dashed line indicates non - contiguous samples that are analyzed on the same membrane . Error bars indicate standard deviations . Asterisks indicate significant differences from the WT ( * * : p < 0 . 01 ) applying Student ' s t - test ( n = 4 ) . AU , arbitrary units .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RNA-blot analysis of AMV systemic infection in plants expressing in vir-1 plants (B) at 7 days post-inoculation. Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. In B, dashed line indicates non-contiguous samples that are analyzed on the same membrane. Error bars indicate standard deviations. Asterisks indicate significant differences from the WT (**: p < 0.01) applying Student's t-test (n = 4). AU, arbitrary units.


A . ADAR expression ( TPM - Transcripts Per Kilobase Million - mapping ) in five samples of ECT2 - FLAG - ADAR ( S1 - S5 ) and FLAG - ADAR ( S6 - S10 ) transgenic lines .

O O O O O O O O O O O O O O O O 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. ADAR expression (TPM - Transcripts Per Kilobase Million - mapping) in five samples of ECT2-FLAG-ADAR (S1-S5) and FLAG-ADAR (S6-S10) transgenic lines.


B . Percentage of total reads mapping to the AMV RNAs in the different samples .

B. Percentage of total reads mapping to the AMV RNAs in the different samples.


A , Systemic infection of rosettes at 7 days post - inoculation ( dpi ) ( A ) Each panel shows a representative RNA blot displaying AMV RNAs 1 - 4 ( left ) and its quantification histogram ( right ) . Ethidium bromide staining of rRNAs ( EB ) was used as RNA loading control . Genotypes are indicated on the top of each northern blot . # 1 and # 2 correspond to the progeny of two different quadruple homozygous F2 siblings derived from the alkbh9b x te235 cross . Error bars show standard deviations . AU , arbitrary units . Different letters indicate statistical differences according to Fisher ' s Least Significant Difference ( LSD ) ( p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Systemic infection of rosettes at 7 days post-inoculation (dpi) (A) Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. #1 and #2 correspond to the progeny of two different quadruple homozygous F2 siblings derived from the alkbh9b x te235 cross. Error bars show standard deviations. AU, arbitrary units. Different letters indicate statistical differences according to Fisher's Least Significant Difference (LSD) (p < 0.05).


B . Systemic infection of rosettes and floral stems at 20 dpi ( B ) . Each panel shows a representative RNA blot displaying AMV RNAs 1 - 4 ( left ) and its quantification histogram ( right ) . Ethidium bromide staining of rRNAs ( EB ) was used as RNA loading control . Genotypes are indicated on the top of each northern blot . # 1 and # 2 correspond to the progeny of two different quadruple homozygous F2 siblings derived from the alkbh9b x te235 cross . Error bars show standard deviations . AU , arbitrary units . Different letters indicate statistical differences according to Fisher ' s Least Significant Difference ( LSD ) ( p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Systemic infection of rosettes and floral stems at 20 dpi (B). Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. #1 and #2 correspond to the progeny of two different quadruple homozygous F2 siblings derived from the alkbh9b x te235 cross. Error bars show standard deviations. AU, arbitrary units. Different letters indicate statistical differences according to Fisher's Least Significant Difference (LSD) (p < 0.05).


A . Schematic representations of the annotated ECT2 gene ( AT3G13460 . 1 , top ) or protein ( bottom ) showing the deletion of the Tyr - rich region ( ΔN5 ) in ECT2ΔN5 - mCherry lines .

O O O O O O O O O O O O O O O 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. Schematic representations of the annotated ECT2 gene (AT3G13460.1, top) or protein (bottom) showing the deletion of the Tyr-rich region (ΔN5) in ECT2ΔN5-mCherry lines.


C . Protein blot developed with mCherry antisera to show accumulation of ECT2WT - mCherry or ECT2ΔN5 - mCherry fusion proteins in pools of the same samples used in panel B . Proteins on the membrane were stained with Coomassie - blue as loading control ( lower panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Protein blot developed with mCherry antisera to show accumulation of ECT2WT-mCherry or ECT2ΔN5-mCherry fusion proteins in pools of the same samples used in panel B. Proteins on the membrane were stained with Coomassie-blue as loading control (lower panel).


E . Phenotypes of seedlings and young rosettes of the indicated genotypes at 9 and 16 days after germination ( DAG ) . Scale bars : black , 1 mm ; white , 1 cm .

O O O O O O O O O O O O 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. Phenotypes of seedlings and young rosettes of the indicated genotypes at 9 and 16 days after germination (DAG). Scale bars: black, 1 mm; white, 1 cm.


( A ‑ B ) BMDCs were plated at different densities and stimulated with poly ( I : C ) for 24h or left unstimulated . DC activation was evaluated based on surface expression of CD86 and CD40 molecules as measured by flow cytometry . ( A ) Representative dot plots showing CD86 and CD40 expression . ( B ) Quantification of CD86 and CD40 upregulation as a fold change in geometric mean fluorescence intensity ( gMFI ) over unstimulated controls . Data are pooled from 3 independent experiments .

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 O O O O O O O O O O O O O O O O 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‑B) BMDCs were plated at different densities and stimulated with poly(I:C) for 24h or left unstimulated. DC activation was evaluated based on surface expression of CD86 and CD40 molecules as measured by flow cytometry. (A) Representative dot plots showing CD86 and CD40 expression. (B) Quantification of CD86 and CD40 upregulation as a fold change in geometric mean fluorescence intensity (gMFI) over unstimulated controls. Data are pooled from 3 independent experiments.


( A ) IFN ‑ α ( left ) and IFN ‑ β ( right ) production by WT and Irf3 - / - Irf7 - / - BMDCs plated at high density ( 2x105 cells / cm2 ) and stimulated with poly ( I : C ) ( blue dots ) or left unstimulated ( grey dots ) . Dotted lines represent the ELISA threshold of detection . Data are representative of 2 independent 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 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

(A) IFN‑α (left) and IFN‑β (right) production by WT and Irf3-/-Irf7-/- BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) (blue dots) or left unstimulated (grey dots). Dotted lines represent the ELISA threshold of detection. Data are representative of 2 independent experiments.