Figure 1Impact 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Impact 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.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.


Figure 2 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 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 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

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

Figure 2 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  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  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


Figure 3 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 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  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  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  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


Figure 4Schematic 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Schematic 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.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.


Figure 5Effect 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 . 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 .

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Figure 5Effect 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.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.


Figure 2C 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 . 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 . 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 .

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Figure 2C 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.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.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.


Figure 3B 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 . 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 . 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 .

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Figure 3B 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.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.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.


Figure 4B 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 . 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 .

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Figure 4B 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.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.


Figure 1A . 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 . 05D . 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 ) .

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Figure 1A. 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 ≤ 0.05D. 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).


Figure 2A - 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 .

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Figure 2A-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.


Figure 3A , 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 .

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Figure 3A, 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.


Figure 4A . 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 .

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Figure 4A. 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.


Figure 5A , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, 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).


Figure 6A . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. 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).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.


Figure 2 ( 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 .

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Figure 2(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.


Figure 3 ( 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 . ( B ) CD86 and CD40 expression was quantified on WT ( blue dots ) and Irf3 - / - Irf7 - / - ( orange dots ) BMDCs plated at high density ( 2x105 cells / cm2 ) and stimulated with poly ( I : C ) or left unstimulated . Data are pooled from 2 independent experiments . ( C ) CD86 and CD40 expression on Irf3 - / - Irf7 - / - BMDCs plated at 2x105 cells / cm2 and stimulated with poly ( I : C ) ( dark blue dots ) , IFN - α ( light blue dots ) or a combination of both ( green dots ) . Data are representative of 2 independent experiments .

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Figure 3(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.(B) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3-/-Irf7-/- (orange dots) BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) or left unstimulated. Data are pooled from 2 independent experiments. (C) CD86 and CD40 expression on Irf3-/-Irf7-/- BMDCs plated at 2x105 cells/cm2 and stimulated with poly(I:C) (dark blue dots), IFN-α (light blue dots) or a combination of both (green dots). Data are representative of 2 independent experiments.


Figure 4 ( A ) CD86 and CD40 expression was quantified on WT ( blue dots ) and Irf3 ‑ / ‑ Irf7 ‑ / ‑ ( orange dots ) DCs in non ‑ draining ( ndLN ) and draining ( dLN ) popliteal lymph nodes of WT and Irf3 ‑ / ‑ Irf7 ‑ / ‑ mice injected with poly ( I : C ) or PBS . Data are pooled data from 2 independent experiments . ( D ) Representative dot plots of CD86 and CD40 expression on WT ( blue dots ) and Irf3 ‑ / ‑ Irf7 ‑ / ‑ ( orange dots ) DCs of chimeras injected with PBS or poly ( I : C ) .

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Figure 4(A) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs in non‑draining (ndLN) and draining (dLN) popliteal lymph nodes of WT and Irf3‑/‑Irf7‑/‑ mice injected with poly(I:C) or PBS. Data are pooled data from 2 independent experiments.(D) Representative dot plots of CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs of chimeras injected with PBS or poly(I:C).


Figure 5 ( B ) Representative dot plots showing CD86 and CD40 expression by low ‑ density BMDCs stimulated with poly ( I : C ) for different periods of IFN ‑ α exposure .

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Figure 5(B) Representative dot plots showing CD86 and CD40 expression by low‑density BMDCs stimulated with poly(I:C) for different periods of IFN‑α exposure.


Figure 6Monocyte recruitment in the draining lymph node requires collective DC activation ( A ) Representative dot plots showing the percentage of monocytes ( CD11b + Ly6Chigh cells , black box ) present in popliteal dLN of WT : Irf3 ‑ / ‑ Irf7 ‑ / ‑ chimeric mice injected with poly ( I : C ) ( middle and right ) or with PBS ( left ) . CD11b + Ly6Clow / int cells corresponded to neutrophils and were not included in the analysis . Collective DC activation is essential for the generation of T cell responses . ( G ) Representative dot plots showing the percentage of CD44high tetramer H ‑ 2Kb ‑ OVA + CD8 + T cells in the draining lymph node of chimeric mice injected with PBS or a mixture of OVA + poly ( I : C ) . Collective DC activation is essential for the generation of T cell responses . ( H ) Flow cytometric quantification of CD44high H ‑ 2Kb ‑ OVA tetramer + CD8 + T cells in the draining lymph node of 90 : 10 and 5 : 95 ( WT : Irf3 ‑ / ‑ Irf7 ‑ / ‑ ) chimeric mice injected with PBS or OVA + poly ( I : C ) . Data are pooled from 2 independent experiments .

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Figure 6Monocyte recruitment in the draining lymph node requires collective DC activation (A) Representative dot plots showing the percentage of monocytes (CD11b+ Ly6Chigh cells, black box) present in popliteal dLN of WT:Irf3‑/‑Irf7‑/‑ chimeric mice injected with poly(I:C) (middle and right) or with PBS (left). CD11b+ Ly6Clow/int cells corresponded to neutrophils and were not included in the analysis.Collective DC activation is essential for the generation of T cell responses. (G) Representative dot plots showing the percentage of CD44high tetramer H‑2Kb‑OVA+ CD8+ T cells in the draining lymph node of chimeric mice injected with PBS or a mixture of OVA+poly(I:C).Collective DC activation is essential for the generation of T cell responses. (H) Flow cytometric quantification of CD44high H‑2Kb‑OVA tetramer+ CD8+ T cells in the draining lymph node of 90:10 and 5:95 (WT:Irf3‑/‑Irf7‑/‑) chimeric mice injected with PBS or OVA+poly(I:C). Data are pooled from 2 independent experiments.


Figure 1B . Immunoblot of parental wild - type ( WT ) and UPF3B mutant cell lines showing levels of proteins on the right . In 3BΔ2BD , a smaller UPF3B protein with deletion of amino acids 20 - 155 ( UPF3BΔ20 - 155 ) is expressed . Relative Expression ( Rel Exp ) of this deletion protein as compared to the full - length UPF3B along with standard error of mean ( SEM ) are indicated below lane 2 . UPF3B antibody recognizes antigen outside the deleted region in 3BΔ2BD . HNRNPA1 is used as a loading control . ND = UPF3B levels not determined . C . Cumulative Distribution Function ( CDF ) plots of PTC + isoforms and PTC - isoforms from the same set of genes . X - axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown ( siNC ) . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown . D . Bar plots from isoform specific RT - qPCR analysis showing average fold change ( y - axis ) of PTC + and PTC - isoforms from genes indicated on the bottom in WT and the two UPF3B mutant cells identified in the legend on the top right . For each isoform , levels in mutant cells are compared to the levels in WT cells ( set to 1 ) . E . Cumulative Distribution Function ( CDF ) plots of NMD + isoforms and NMD - isoforms . X - axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown ( siNC ) . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown . F . Cumulative Distribution Function ( CDF ) plots of PTC + isoforms and PTC - isoforms from same set of genes . X - axis represents fold change in UPF1 knockdown ( siUPF1 ) versus control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown . G . Cumulative Distribution Function ( CDF ) plots of NMD + isoforms and NMD - isoforms . X - axis represents fold change in UPF1 knockdown ( siUPF1 ) versus control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Immunoblot of parental wild-type (WT) and UPF3B mutant cell lines showing levels of proteins on the right. In 3BΔ2BD, a smaller UPF3B protein with deletion of amino acids 20-155 (UPF3BΔ20-155) is expressed. Relative Expression (Rel Exp) of this deletion protein as compared to the full-length UPF3B along with standard error of mean (SEM) are indicated below lane 2. UPF3B antibody recognizes antigen outside the deleted region in 3BΔ2BD. HNRNPA1 is used as a loading control. ND = UPF3B levels not determined.C. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from the same set of genes. X-axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.D. Bar plots from isoform specific RT-qPCR analysis showing average fold change (y-axis) of PTC+ and PTC- isoforms from genes indicated on the bottom in WT and the two UPF3B mutant cells identified in the legend on the top right. For each isoform, levels in mutant cells are compared to the levels in WT cells (set to 1).E. Cumulative Distribution Function (CDF) plots of NMD+ isoforms and NMD- isoforms. X-axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown. F. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from same set of genes. X-axis represents fold change in UPF1 knockdown (siUPF1) versus control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown. G. Cumulative Distribution Function (CDF) plots of NMD+ isoforms and NMD- isoforms. X-axis represents fold change in UPF1 knockdown (siUPF1) versus control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.


Figure 2A . Immunoblots showing levels of proteins on the right in input or FLAG immunoprecipitates ( IP ) from WT and UPF3B mutant cells expressing endogenously FLAG - tagged UPF1 protein as indicated above each lane . The presence of RNase A during FLAG - IP is indicated above each lane . B . Immunoblots showing levels of proteins ( right ) in input and IP with normal rabbit IgG ( IgG - IP ) or antibody targeting EIF4A3 ( EIF4A3 - IP ) from WT and UPF3B mutant cells . C , D . CDF plots of ( C ) PTC + and PTC - isoforms , ( D ) NMD + and NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in WT cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . E , F . CDF plots of ( E ) PTC + and PTC - isoforms , ( F ) NMD + and NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . G , H . CDF plots of UPF3B - dependent ( G ) and - independent ( H ) PTC + isoforms and their respective PTC - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . I . CDF plots of UPF3B - dependent and UPF3B - independent NMD + isoforms as compared to NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . J . Bar plot showing average fold change as measured by isoform specific RT - qPCR of PTC + and PTC - isoform from genes indicated on the bottom in WT and two independent clones of 3AKO , 3BΔ2BD , 3BKO , and 3DKO cells . Relative levels from each replicate are shown by white circles . Error bars indicate standard errors of means .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Immunoblots showing levels of proteins on the right in input or FLAG immunoprecipitates (IP) from WT and UPF3B mutant cells expressing endogenously FLAG-tagged UPF1 protein as indicated above each lane. The presence of RNase A during FLAG-IP is indicated above each lane.B. Immunoblots showing levels of proteins (right) in input and IP with normal rabbit IgG (IgG-IP) or antibody targeting EIF4A3 (EIF4A3-IP) from WT and UPF3B mutant cells.C, D. CDF plots of (C) PTC+ and PTC-isoforms, (D) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in WT cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. E, F. CDF plots of (E) PTC+ and PTC-isoforms, (F) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. G, H. CDF plots of UPF3B-dependent (G) and -independent (H) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.I. CDF plots of UPF3B-dependent and UPF3B-independent NMD+ isoforms as compared to NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.J. Bar plot showing average fold change as measured by isoform specific RT-qPCR of PTC+ and PTC- isoform from genes indicated on the bottom in WT and two independent clones of 3AKO, 3BΔ2BD, 3BKO, and 3DKO cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means.


Figure 3B . Immunoblot showing levels of EJC and UPF proteins ( on the right ) in input or FLAG - IP samples from 3DKO # 2 cells stably expressing different FLAG - tagged proteins indicated above each lane . D . Immunoblot showing EJC / UPF proteins ( on the right ) in input or FLAG - IP samples from 3DKO # 2 cells stably expressing FLAG - tagged proteins given above each lane ( hUPF3B = human UPF3B ; hUPF3B - h3AC = human UPF3B protein with C - terminal domain from human UPF3A ; hUPF3B - m3AC = human UPF3B protein with C - terminal domain from mouse UPF3A ) .

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Figure 3B. Immunoblot showing levels of EJC and UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing different FLAG-tagged proteins indicated above each lane. D. Immunoblot showing EJC/UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing FLAG-tagged proteins given above each lane (hUPF3B = human UPF3B; hUPF3B-h3AC = human UPF3B protein with C-terminal domain from human UPF3A; hUPF3B-m3AC = human UPF3B protein with C-terminal domain from mouse UPF3A).


Figure 4A . Northern blots showing levels of β - globin reporter mRNAs in wild - type HeLa Tet - off cells and UPF3B knockout HeLa Tet - off cells . β39 is a tetracycline ( Tet ) - inducible reporter with a PTC at codon 39 whose levels are shown at different timepoints after transcriptional shut - off ( chase ) as indicated above each lane . β - GAP is a stable , constitutively - expressed , longer β - globin mRNA used as transfection control . Proteins overexpressed ( OE ) in each condition are indicated on top and reporter mRNA half - lives ( t1 / 2 ) along with standard errors of means are on the bottom . C . Northern blot showing steady - state levels of β39 NMD reporter and β - GAP control in HeLa Tet - off UPF3B knockout cells upon transient overexpression of wild - type UPF3 proteins or different UPF3A chimeric proteins indicated above each lane . Below each lane , relative fold - change ( Rel FC ) indicates β39 reporter levels ( normalized to β - GAP control ) as compared to the normalized β39 reporter levels in UPF3B expressing cells . D . Immunoblot showing levels of EJC proteins or HNRNPA1 in input or EIF4A3 - IP from 3DKO # 2 cells stably expressing different UPF3 proteins or EGFP as a control as indicated above each lane . Relative IP of FLAG - tagged proteins are quantified against EIF4A3 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Northern blots showing levels of β-globin reporter mRNAs in wild-type HeLa Tet-off cells and UPF3B knockout HeLa Tet-off cells. β39 is a tetracycline (Tet)-inducible reporter with a PTC at codon 39 whose levels are shown at different timepoints after transcriptional shut-off (chase) as indicated above each lane. β-GAP is a stable, constitutively-expressed, longer β-globin mRNA used as transfection control. Proteins overexpressed (OE) in each condition are indicated on top and reporter mRNA half-lives (t1/2) along with standard errors of means are on the bottom.C. Northern blot showing steady-state levels of β39 NMD reporter and β-GAP control in HeLa Tet-off UPF3B knockout cells upon transient overexpression of wild-type UPF3 proteins or different UPF3A chimeric proteins indicated above each lane. Below each lane, relative fold-change (Rel FC) indicates β39 reporter levels (normalized to β-GAP control) as compared to the normalized β39 reporter levels in UPF3B expressing cells.D. Immunoblot showing levels of EJC proteins or HNRNPA1 in input or EIF4A3-IP from 3DKO#2 cells stably expressing different UPF3 proteins or EGFP as a control as indicated above each lane. Relative IP of FLAG-tagged proteins are quantified against EIF4A3.


Figure 5A , B . CDF plots of ( A ) PTC + and PTC - isoforms , ( B ) NMD + and NMD - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . D , E . CDF plots of UPF3A / 3B - independent ( UPF3A / 3B - indep . , D ) and - dependent ( UPF3A / 3B - dep . , E ) PTC + isoforms and their respective PTC - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . F . CDF plots of UPF3A / 3B - dependent ( UPF3A / 3B - dep . ) , UPF3A / 3B - independent ( UPF3A / 3B - indep . ) NMD + isoforms and NMD - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, B. CDF plots of (A) PTC+ and PTC-isoforms, (B) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.D, E. CDF plots of UPF3A/3B-independent (UPF3A/3B-indep., D) and -dependent (UPF3A/3B-dep., E) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.F. CDF plots of UPF3A/3B-dependent (UPF3A/3B-dep.), UPF3A/3B-independent (UPF3A/3B-indep.) NMD+ isoforms and NMD- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


Figure 6A , B . Western blots showing levels of EJC proteins or HNRNPA1 ( control ) in input , IgG IP or EIF4A3 IP following overexpression ( OE ) of CASC3 wild - type ( WT ) and EJC binding deficient ( HDAA ) mutant proteins in ( A ) HeLa Tet - off cells , and ( B ) 3BKO # 3 HeLa Tet - off cells . Ramps above lanes indicate expression levels of the CASC3 proteins . C . Western blots showing levels of EJC / UPF proteins and HNRNPA1 in input and FLAG followed by EIF4A3 tandem IP from HCT116 cells expressing the FLAG - tagged endogenous protein indicated above each lane . Quantifications of UPF3B and CASC3 protein enrichment from two replicates are shown at the bottom . D . Meta - exon plot showing read distributions within the 100 nucleotide ( nt ) window from the exon 3 ′ end in RIPiT - Seq replicates of MAGOH : EIF4A3 , UPF3B : EIF4A3 , and CASC3 : EIF4A3 . The black vertical line indicates the - 24 nt position . F . Scatter plot comparing log2 - transformed fold change in occupancy of CASC3 : EIF4A3 EJC as compared to MAGOH : EIF4A3 EJC ( x - axis ) and UPF3B : EIF4A3 EJC compared to MAGOH : EIF4A3 EJC . Each dot represents a gene where gene - level occupancy of each EJC composition was quantified at the canonical position for EJC footprints . Pearson correlation coefficient is shown on the top left . G . Box plots comparing occupancy of a specific EJC composition on mRNA ( indicated on y - axis ; normalized by gene expression from RNA - Seq ) between genes of different NMD efficiency . Up , no , and down stands for upregulation , no obvious change or downregulation of a representative PTC + isoform for each gene ( see material and methods ) that is used to reflect overall NMD efficiency for each gene . In the box plots , central band represents the median , boxes represent the interquartile range ( IQR ) , whiskers indicate maximum ( top ) and minimum ( bottom ) values ( 1 . 5 - times the highest or lowest IQR values , respectively ) , and circles represent the outliers . p - values are from Wilcoxon signed - rank test comparing the two indicated distributions . H . Box plots as in G comparing specific EJC composition occupancy relative to EJC core occupancy ( normalized by MAGOH : EIF4A3 RIPiT - Seq ) between genes of different NMD efficiency . Up , no , down , central band , boxes , and whiskers are as in G . I . Bar plots showing fold changes measured by isoform specific RT - qPCR of PTC + and PTC - isoform from genes indicated on the bottom in WT and CASC3 - KO HCT116 cells . Relative levels from each replicate are shown by white circles . Error bars indicate standard error of means .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A, B. Western blots showing levels of EJC proteins or HNRNPA1 (control) in input, IgG IP or EIF4A3 IP following overexpression (OE) of CASC3 wild-type (WT) and EJC binding deficient (HDAA) mutant proteins in (A) HeLa Tet-off cells, and (B) 3BKO#3 HeLa Tet-off cells. Ramps above lanes indicate expression levels of the CASC3 proteins.C. Western blots showing levels of EJC/UPF proteins and HNRNPA1 in input and FLAG followed by EIF4A3 tandem IP from HCT116 cells expressing the FLAG-tagged endogenous protein indicated above each lane. Quantifications of UPF3B and CASC3 protein enrichment from two replicates are shown at the bottom.D. Meta-exon plot showing read distributions within the 100 nucleotide (nt) window from the exon 3′ end in RIPiT-Seq replicates of MAGOH:EIF4A3, UPF3B:EIF4A3, and CASC3:EIF4A3. The black vertical line indicates the -24 nt position.F. Scatter plot comparing log2-transformed fold change in occupancy of CASC3:EIF4A3 EJC as compared to MAGOH:EIF4A3 EJC (x-axis) and UPF3B:EIF4A3 EJC compared to MAGOH:EIF4A3 EJC. Each dot represents a gene where gene-level occupancy of each EJC composition was quantified at the canonical position for EJC footprints. Pearson correlation coefficient is shown on the top left.G. Box plots comparing occupancy of a specific EJC composition on mRNA (indicated on y-axis; normalized by gene expression from RNA-Seq) between genes of different NMD efficiency. Up, no, and down stands for upregulation, no obvious change or downregulation of a representative PTC+ isoform for each gene (see material and methods) that is used to reflect overall NMD efficiency for each gene. In the box plots, central band represents the median, boxes represent the interquartile range (IQR), whiskers indicate maximum (top) and minimum (bottom) values (1.5-times the highest or lowest IQR values, respectively), and circles represent the outliers. p-values are from Wilcoxon signed-rank test comparing the two indicated distributions.H. Box plots as in G comparing specific EJC composition occupancy relative to EJC core occupancy (normalized by MAGOH:EIF4A3 RIPiT-Seq) between genes of different NMD efficiency. Up, no, down, central band, boxes, and whiskers are as in G.I. Bar plots showing fold changes measured by isoform specific RT-qPCR of PTC+ and PTC- isoform from genes indicated on the bottom in WT and CASC3-KO HCT116 cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard error of means.


Figure 1C ) Representative immunoblots of brain extracts of wild - type , Ttll1 - / - and Ttll7 - / - mice . Polyglutamylation of α - tubulin , detected with polyE and GT335 , is specifically lost in Ttll1 - / - brain . By contrast , β - tubulin glutamylation , which is specifically detected with β ‑ monoE , is absent from brains of Ttll7 - / - mice . D ) Representative immunoblots of extracts of wild - type , Ttll1 ‑ / - and Ttll7 - / - primary hippocampal neurons at DIV4 . As in brain ( C ) , α - tubulin polyglutamylation is specifically lost in Ttll1 - / - neurons , while β - tubulin glutamylation lacks in Ttll7 - / - neurons . E F , G ) Immunocytochemistry of DIV4 primary hippocampal neurons from wild type , Ttll1 - / - and Ttll7 ‑ / - mice . Microtubules in Ttll1 - / - cells have reduced levels of polyE signal , indicative of lower α - tubulin polyglutamylation ( F ) , Ttll7 ‑ / - neurons are specifically reduced in β ‑ monoE reactivity , indicating the lack of β ‑ tubulin glutamylation ( G ) .

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Figure 1C) Representative immunoblots of brain extracts of wild-type, Ttll1-/- and Ttll7-/- mice. Polyglutamylation of α-tubulin, detected with polyE and GT335, is specifically lost in Ttll1-/- brain. By contrast, β-tubulin glutamylation, which is specifically detected with β‑monoE, is absent from brains of Ttll7-/- mice. D) Representative immunoblots of extracts of wild-type, Ttll1‑/- and Ttll7-/- primary hippocampal neurons at DIV4. As in brain (C), α-tubulin polyglutamylation is specifically lost in Ttll1-/- neurons, while β-tubulin glutamylation lacks in Ttll7-/- neurons. E F,G) Immunocytochemistry of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7‑/- mice. Microtubules in Ttll1-/- cells have reduced levels of polyE signal, indicative of lower α-tubulin polyglutamylation (F), Ttll7‑/- neurons are specifically reduced in β‑monoE reactivity, indicating the lack of β‑tubulin glutamylation (G).


Figure 2A ) Representative histology images of cerebella from 3 - months - old mice stained with anti - calbindin antibody , which specifically labels Purkinje cells ( Sequier et al , 1988 ) . Note the presence of an intact Purkinje - cell layer in cerebella of wild - type , Ttll1 - / - , Ttll7 - / - and Ccp1 - / - Ttll1 - / - mice , in contrast to the complete absence of these cells in Ccp1 - / - and Ccp1 - / - Ttll7 - / - mice . B ) Histology of 25 - day old mice with partial degeneration of Purkinje Cells . Note that both , Ccp1 - / - and Ccp1 - / - Ttll7 - / - brains , show highly similar pattern of partial Purkinje - cell loss ( red arrow heads : gaps in the Purkinje - cell layer , indicating degenerated Purkinje cells ; orange arrow heads : swellings of Purkinje - cell axons , indicative of ongoing degeneration ) . C ) Representative immunoblots of cerebellum extracts of wild - type , Ccp1 - / - , Ttll7 - / - , Ccp1 - / - Ttll7 - / - , Ttll1 - / - and Ccp1 - / - Ttll1 - / - mice . Loss of CCP1 leads to hyperglutamylation of α - and β - tubulin as revealed with polyE and GT335 antibodies . Concomitant deletion of TTLL7 lead to a loss of glutamylation from β - tubulin ( β ‑ monoE , GT335 ) , while hyperglutamylation of α - tubulin persists ( polyE , GT335 ) . Concomitant loss of CCP1 and TTLL1 eliminates hyperglutamylation from both , α - and β - tubulin ( polyE , GT335 ) , while β - tubulin glutamylation is unaltered ( β - monoE ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) Representative histology images of cerebella from 3-months-old mice stained with anti-calbindin antibody, which specifically labels Purkinje cells (Sequier et al, 1988). Note the presence of an intact Purkinje-cell layer in cerebella of wild-type, Ttll1-/-, Ttll7-/- and Ccp1-/-Ttll1-/- mice, in contrast to the complete absence of these cells in Ccp1-/- and Ccp1-/-Ttll7-/- mice.B) Histology of 25-day old mice with partial degeneration of Purkinje Cells. Note that both, Ccp1-/- and Ccp1-/-Ttll7-/- brains, show highly similar pattern of partial Purkinje-cell loss (red arrow heads: gaps in the Purkinje-cell layer, indicating degenerated Purkinje cells; orange arrow heads: swellings of Purkinje-cell axons, indicative of ongoing degeneration).C) Representative immunoblots of cerebellum extracts of wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Loss of CCP1 leads to hyperglutamylation of α- and β-tubulin as revealed with polyE and GT335 antibodies. Concomitant deletion of TTLL7 lead to a loss of glutamylation from β-tubulin (β‑monoE, GT335), while hyperglutamylation of α-tubulin persists (polyE, GT335). Concomitant loss of CCP1 and TTLL1 eliminates hyperglutamylation from both, α- and β-tubulin (polyE, GT335), while β-tubulin glutamylation is unaltered (β-monoE).


Figure 3Semi - thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue . Note the smaller size of Ccp1 - / - and Ccp1 - / - Ttll7 - / - nerves ( A ) . Semi - thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue . These sections were quantified for ( B ) number of myelinated axons , ( C ) nerve area , ( D ) nerve perimeter ( Fig . EV2 ) of femoral quadriceps nerves from wild - type , Ccp1 - / - , Ttll7 - / - , Ccp1 - / - Ttll7 - / - , Ttll1 - / - and Ccp1 - / - Ttll1 - / - mice . Scatter dot plots in which each point represents one nerve of an individual mouse are shown . The black lines indicate means ± SEM . Significance was tested with one - way ANOVA and Holm - Šídák ' s multiple comparisons test . Note that all three parameters are decreased for Ccp1 - / - and Ccp1 - / - Ttll7 ‑ / - mice , while Ccp1 - / - Ttll1 - / - mice showed no difference to the Ttll1 - / - control . E , F ) Representative electron microscopy images of cross sections of femoral quadriceps nerves . ( E ) Nerves from Ttll1 - / - and Ccp1 - / - Ttll1 - / - mice show no abnormalities of myelinated axons ( green asterisks ) , indicating that loss of TTLL1 prevents axonal degeneration caused by the absence of CCP1 . ( F ) Ccp1 - / - and Ccp1 - / - Ttll7 - / - mice both show the presence of foamy macrophages ( magenta arrowheads ) , which are signs for myelin phagocytosis during axon degeneration . This indicates that loss of TTLL7 cannot prevent the CCP1 - induced degeneration of femoral quadriceps nerves .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Semi-thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue. Note the smaller size of Ccp1-/- and Ccp1-/-Ttll7-/- nerves (A).Semi-thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue. These sections were quantified for (B) number of myelinated axons, (C) nerve area, (D) nerve perimeter (Fig. EV2) of femoral quadriceps nerves from wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Scatter dot plots in which each point represents one nerve of an individual mouse are shown. The black lines indicate means ±SEM. Significance was tested with one-way ANOVA and Holm-Šídák's multiple comparisons test. Note that all three parameters are decreased for Ccp1-/- and Ccp1-/-Ttll7‑/- mice, while Ccp1-/-Ttll1-/- mice showed no difference to the Ttll1-/- control.E,F) Representative electron microscopy images of cross sections of femoral quadriceps nerves. (E) Nerves from Ttll1-/- and Ccp1-/-Ttll1-/- mice show no abnormalities of myelinated axons (green asterisks), indicating that loss of TTLL1 prevents axonal degeneration caused by the absence of CCP1. (F) Ccp1-/- and Ccp1-/-Ttll7-/- mice both show the presence of foamy macrophages (magenta arrowheads), which are signs for myelin phagocytosis during axon degeneration. This indicates that loss of TTLL7 cannot prevent the CCP1-induced degeneration of femoral quadriceps nerves.


Figure 4A ) Representative images of DIV4 primary hippocampal neurons from wild type , Ttll1 - / - and Ttll7 - / - mice Cells were incubated with MitoTracker ® ( black dots ) and mitochondria movements were recorded in the longest neurite ( orange lines ) . B ) Kymographs of one representative experiment representing mitochondrial movements C - F ) Analysis of one single experiment shown in ( B ) . Scatter dot plots of ( C ) run length and ( D ) speed distributions , where each point represents a single run extracted from the kymographs shown in ( B ) . The black lines indicate the median of the distribution , with whiskers at interquartile range . Numerical values of medians are indicated below each plot . ( E ) A bar graph showing the overall motility of mitochondria ( the fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s ) . ( F ) A bar graph showing mitochondrial density in analysed axons . Values of the overall motilities and mitochondrial densities are given below the bar plots .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) Representative images of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7-/- mice Cells were incubated with MitoTracker® (black dots) and mitochondria movements were recorded in the longest neurite (orange lines).B) Kymographs of one representative experiment representing mitochondrial movements C - F) Analysis of one single experiment shown in (B). Scatter dot plots of (C) run length and (D) speed distributions, where each point represents a single run extracted from the kymographs shown in (B). The black lines indicate the median of the distribution, with whiskers at interquartile range. Numerical values of medians are indicated below each plot. (E) A bar graph showing the overall motility of mitochondria (the fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s). (F) A bar graph showing mitochondrial density in analysed axons. Values of the overall motilities and mitochondrial densities are given below the bar plots.


Figure 2A . Typical fluorescence intensity trace collected over 10 seconds . B . Typical normalized FCS curves of Smc3 - GFP at different cell cycle phases . Dotted lines indicate fitting of the one - component anomalous diffusion model to the respective FCS curve . C - F . Diffusion times of Smc3 - GFP , Scc3 - GFP , and Scc1 - GFP at the G1 phase ( C ) , S phase ( D ) , G2 phase ( E ) , and mitosis ( F ) . Data shown as mean ± s . e . m . from at least three independent experiments . * : p < 0 . 01 , # : p < 10 - 13 with the Mann - Whitney test .

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Figure 2A. Typical fluorescence intensity trace collected over 10 seconds.B. Typical normalized FCS curves of Smc3-GFP at different cell cycle phases. Dotted lines indicate fitting of the one-component anomalous diffusion model to the respective FCS curve.C-F. Diffusion times of Smc3-GFP, Scc3-GFP, and Scc1-GFP at the G1 phase (C), S phase (D), G2 phase (E), and mitosis (F). Data shown as mean ± s.e.m. from at least three independent experiments. *: p<0.01, #: p<10-13 with the Mann-Whitney test.


Figure 3Representative normalized FCS curve of Scc1 - GFP at different cell cycle phases . Dotted lines indicate fitting of one - component anomalous diffusion models to the respective FCS curve . The diffusion times of Scc1 - GFP at S phase ( B ) were measured in strains yIO664 ( wild - type ) and yEB005 ( WPL1 ∆ ) , and in auxin - depleted yEB002 ( PDS5 - AID ) , yAM085 ( SCC3 - AID ) and yAM946 ( ECO1 - AID ) cells . Data are shown as mean ± s . e . m . from at least three independent experiments . * : p < 10 - 5 , * * : p < 10 - 13 . The diffusion times of Scc1 - GFP at G2 phase ( C ) and M phase ( D ) were measured in strains yIO664 ( wild - type ) and yEB005 ( WPL1 ∆ ) , and in auxin - depleted yEB002 ( PDS5 - AID ) , yAM085 ( SCC3 - AID ) and yAM946 ( ECO1 - AID ) cells . Data are shown as mean ± s . e . m . from at least three independent experiments . * : p < 10 - 5 , * * : p < 10 - 13 .

O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Representative normalized FCS curve of Scc1-GFP at different cell cycle phases. Dotted lines indicate fitting of one-component anomalous diffusion models to the respective FCS curve.The diffusion times of Scc1-GFP at S phase (B) were measured in strains yIO664 (wild-type) and yEB005 (WPL1∆), and in auxin-depleted yEB002 (PDS5-AID), yAM085 (SCC3-AID) and yAM946 (ECO1-AID) cells. Data are shown as mean ± s.e.m. from at least three independent experiments. *: p< 10-5, **: p< 10-13.The diffusion times of Scc1-GFP at G2 phase (C) and M phase (D) were measured in strains yIO664 (wild-type) and yEB005 (WPL1∆), and in auxin-depleted yEB002 (PDS5-AID), yAM085 (SCC3-AID) and yAM946 (ECO1-AID) cells. Data are shown as mean ± s.e.m. from at least three independent experiments. *: p< 10-5, **: p< 10-13.