Figure 3A . HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts ( left ) as well as IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( right ) , post - doxycycline addition . Data information : data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . The half - lives of the respective transcripts are indicated in brackets . B . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT in HEK293T cells transfected with either empty plasmids , plasmids bearing REL - OPT or REL - WT . The immunoblot is representative of 3 independent experiments . ACTB is shown as the loading controls . C . ELISA of secreted IL6 concentrations of IL6 - OPT , IL6 - WT and IL6 - DE from HEK293T cells transfected with plasmids bearing IL6 - OPT , IL6 - WT and IL6 - DE . Data information : In ( C ) , the data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A one - way ANOVA with Tukey ' s multiple comparisons was performed between samples where , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . D . Fold changes of REL - OPT and REL - WT transcript levels ( top ) relative to their abundances from fraction 1 as detected by qPCR across polysome fractions ( below ) . Data represents the mean ± SD for 3 biological replicates . Data information data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . The half - lives of the respective transcripts are indicated in brackets .

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 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 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 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

Figure 3A. HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (left) as well as IL6-OPT, IL6-WT and IL6-DE transcripts (right), post-doxycycline addition. Data information: data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.B. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells transfected with either empty plasmids, plasmids bearing REL-OPT or REL-WT. The immunoblot is representative of 3 independent experiments. ACTB is shown as the loading controls.C. ELISA of secreted IL6 concentrations of IL6-OPT, IL6-WT and IL6-DE from HEK293T cells transfected with plasmids bearing IL6-OPT, IL6-WT and IL6-DE. Data information: In (C), the data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A one-way ANOVA with Tukey's multiple comparisons was performed between samples where, p<0.01 (**) and p<0.001 (***).D. Fold changes of REL-OPT and REL-WT transcript levels (top) relative to their abundances from fraction 1 as detected by qPCR across polysome fractions (below). Data represents the mean ± SD for 3 biological replicates. Data information data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.


Figure 4HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts under vehicle ( DMSO ) - and cycloheximide ( CHX ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( B ) , under vehicle ( DMSO ) - and cycloheximide ( CHX ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts ( C ) under vehicle ( PBS ) - and anisomycin ( ANI ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( D ) , under vehicle ( PBS ) - and anisomycin ( ANI ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . HEK293 Tet - off experiments showing the degradation of REL - OPT , REL - OPT ( + 1 Frameshift ) and REL - WT transcripts ( E ) post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT , IL6 - DE and IL6 - OPT ( + 1 Frameshift ) transcripts ( F ) post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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 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 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 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 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 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

Figure 4HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (B), under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (C) under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (D), under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).HEK293 Tet-off experiments showing the degradation of REL-OPT, REL-OPT (+1 Frameshift) and REL-WT transcripts (E) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT, IL6-DE and IL6-OPT (+1 Frameshift) transcripts (F) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


Figure 5Volcano plots showing the enrichment of RBPs which bind to REL - WT relative to REL - OPT transcripts ( A ) Data information : vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and p < 0 . 05 . Volcano plots showing the enrichment of RBPs which bind to , IL6 - DE relative to IL6 - WT transcripts ( B ) Data information : vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and p < 0 . 05 . Volcano plots showing the enrichment of RBPs which bind to IL6 - WT relative to IL6 - OPT transcripts ( C ) . Data information : , vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and 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 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 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

Figure 5Volcano plots showing the enrichment of RBPs which bind to REL-WT relative to REL-OPT transcripts (A) Data information: vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.Volcano plots showing the enrichment of RBPs which bind to , IL6-DE relative to IL6-WT transcripts (B) Data information: vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.Volcano plots showing the enrichment of RBPs which bind to IL6-WT relative to IL6-OPT transcripts (C). Data information: , vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.


Figure 6A . Cumulative distribution plots showing the difference in distribution of transcript optimality between upregulated and downregulated transcripts in K562 cells subject to ILF2 CRISPR interference targeting ILF2 . Transcript quantities are indicated in the figure legend . Data information : Wilcoxon signed rank tests were performed on the upregulated and downregulated groups against the control group . P - values are denoted ( right ) . B , C . HEK293 Tet - off experiments showing the degradation of REL - OPT ( B ) and REL - WT ( C ) transcripts with ILF2 and ILF3 siRNA and Control ( CTR ) siRNA treatment , post - doxycycline addition . Data information : data is representative of 3 independent experiments in which the data represents the mean ± SD for 3 biological replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) . D . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT expressed in HEK293T cells under ILF2 and ILF3 siRNA treatment . The immunoblot is representative of 3 independent experiments . ACTB is shown as loading controls . E . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT in HEK293T cells co - expressed with two different isoforms of ILF2 . The immunoblot is representative of 3 independent experiments . ACTB is shown as loading controls .

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 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 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 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

Figure 6A. Cumulative distribution plots showing the difference in distribution of transcript optimality between upregulated and downregulated transcripts in K562 cells subject to ILF2 CRISPR interference targeting ILF2. Transcript quantities are indicated in the figure legend. Data information: Wilcoxon signed rank tests were performed on the upregulated and downregulated groups against the control group. P-values are denoted (right).B, C. HEK293 Tet-off experiments showing the degradation of REL-OPT (B) and REL-WT (C) transcripts with ILF2 and ILF3 siRNA and Control (CTR) siRNA treatment, post-doxycycline addition. Data information: data is representative of 3 independent experiments in which the data represents the mean ± SD for 3 biological replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**).D. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT expressed in HEK293T cells under ILF2 and ILF3 siRNA treatment. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls.E. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells co-expressed with two different isoforms of ILF2. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls.


Figure 1a , b . The amount of FDP - lysine , which was major protein - bound acrolein , in the sera of healthy controls and PD patients was determined by an ELISA ( a . all patients , ( control n = 22 ; PD n = 94 ) b . staged by the H & Y scale , a five - steps index of PD severity . Larger number indicates more severe condition . n = 94 ; H & Y stage I ( n = 18 ) , stage II ( n = 42 ) stage III ( n = 23 ) , stage IV ( n = 7 ) , and stage V ( n = 4 ) .

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

Figure 1a, b. The amount of FDP-lysine, which was major protein-bound acrolein, in the sera of healthy controls and PD patients was determined by an ELISA (a. all patients, (control n= 22; PD n=94) b. staged by the H&Y scale, a five-steps index of PD severity. Larger number indicates more severe condition. n=94; H&Y stage I (n=18), stage II (n=42) stage III (n=23), stage IV (n=7), and stage V (n=4).


Figure 2SH - SY5Y cells were treated with the indicated chemicals at 50 μM for 4 h ( a ) Cells were fixed and stained with an anti - LAMP2 ( green ) antibody as a lysosomal marker . Nuclei were stained with DAPI ( blue ) . Cell membrane is indicated by the white line . Clustered relative to whole lysosomes were quantified as lysosomal clustering value ( a , right ) . e . SH - SY5Y cells were treated with acrolein for 2 h . Cell lysates were immunoblotted with the indicated antibodies . h . SH - SY5Y cells were treated with acrolein for 2 h . Cell lysates were immunoblotted with the indicated antibodies .

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 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 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

Figure 2SH-SY5Y cells were treated with the indicated chemicals at 50 μM for 4 h (a) Cells were fixed and stained with an anti-LAMP2 (green) antibody as a lysosomal marker. Nuclei were stained with DAPI (blue). Cell membrane is indicated by the white line. Clustered relative to whole lysosomes were quantified as lysosomal clustering value (a, right).e. SH-SY5Y cells were treated with acrolein for 2 h. Cell lysates were immunoblotted with the indicated antibodies.h. SH-SY5Y cells were treated with acrolein for 2 h. Cell lysates were immunoblotted with the indicated antibodies.


Figure 3d . SH - SY5Y cells were transfected with the indicated siRNAs for 48 h and then treated with 40 µM acrolein for 2 h . ( upper ) Cells were fixed and stained with an anti - LAMP2 antibody ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 µm . ( lower ) Clustered relative to whole lysosomes were quantified as lysosomal clustering value .

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

Figure 3d. SH-SY5Y cells were transfected with the indicated siRNAs for 48 h and then treated with 40 µM acrolein for 2 h. (upper) Cells were fixed and stained with an anti-LAMP2 antibody (green). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. (lower) Clustered relative to whole lysosomes were quantified as lysosomal clustering value.


Figure 4a . Cells were transfected with the TMEM55B - myc - DDK plasmid for 24 h and then treated with 40 μM acrolein for 2 h . Cell lysates were immunoprecipitated with flag magnetic beads and immunoblotted with the indicated antibodies . b . SH - SY5Y cells were treated with 40 μM acrolein for 2 h . Cells were fixed and stained with anti - JIP4 ( green ) and anti - LAMP2 ( red ) antibodies . Nuclei were stained with DAPI ( blue ) . c . SH - SY5Y cells treated with the indicated concentration of acrolein for 2 h were lysed , subjected to Phos - tag PAGE , and immunoblotted with an anti - JIP4 antibody . d . SH - SY5Y cells were pre - treated with 10 µM Jak3 inhibitor VI ( Jak3 i VI ) for 1 h and then treated with 40 µM acrolein for an additional 2 h . Cells lysates were subjected to Phos - tag PAGE and immunoblotted with an anti - JIP4 antibody . e . SH - SY5Y cells were pre - treated with 10 µM Jak3 inhibitor VI for 1 h and then treated with 40 µM acrolein for an additional 2 h . Cells were fixed and stained with anti - JIP4 ( green ) and anti - LAMP2 ( red ) antibodies . Nuclei were stained with DAPI ( blue ) . SH - SY5Y cells transfected with indicated siRNAs for 48 h were treated with 40 μM acrolein for 2 h . Cell lysates were subjected to Phos - tag PAGE and immunoblotted with anti - JIP4 antibody ( f , left ) . CaMK2G knockdown efficiency was confirmed by qRT - PCR . ; * * * p < 0 . 001 , n = 3 technical replicates . Values are mean ± SD . At least three experiments were replicated ( f , right ) . j . His6 - tagged recombinant truncated JIP4 ( 100 - 300 aa ) was purified from E . coli and incubated with recombinant CaMK2G with / without Ca2 + and calmodulin at 30 ° C for 1 h . The reactant was subjected to Phos - tag PAGE and immunoblotted with anti - His antibody . Jak3 inhibitor VI was pre - reacted with CaMK2G for 30 min .

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 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 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 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 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 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

Figure 4a. Cells were transfected with the TMEM55B-myc-DDK plasmid for 24 h and then treated with 40 μM acrolein for 2 h. Cell lysates were immunoprecipitated with flag magnetic beads and immunoblotted with the indicated antibodies.b. SH-SY5Y cells were treated with 40 μM acrolein for 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue).c. SH-SY5Y cells treated with the indicated concentration of acrolein for 2 h were lysed, subjected to Phos-tag PAGE, and immunoblotted with an anti-JIP4 antibody. d. SH-SY5Y cells were pre-treated with 10 µM Jak3 inhibitor VI (Jak3 i VI) for 1 h and then treated with 40 µM acrolein for an additional 2 h. Cells lysates were subjected to Phos-tag PAGE and immunoblotted with an anti-JIP4 antibody.e. SH-SY5Y cells were pre-treated with 10 µM Jak3 inhibitor VI for 1 h and then treated with 40 µM acrolein for an additional 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue).SH-SY5Y cells transfected with indicated siRNAs for 48 h were treated with 40 μM acrolein for 2 h. Cell lysates were subjected to Phos-tag PAGE and immunoblotted with anti-JIP4 antibody (f, left). CaMK2G knockdown efficiency was confirmed by qRT-PCR.; ***p <0.001, n=3 technical replicates. Values are mean ± SD. At least three experiments were replicated (f, right).j. His6-tagged recombinant truncated JIP4 (100-300 aa) was purified from E. coli and incubated with recombinant CaMK2G with/without Ca2+ and calmodulin at 30°C　for 1 h. The reactant was subjected to Phos-tag PAGE and immunoblotted with anti-His antibody. Jak3 inhibitor VI was pre-reacted with CaMK2G for 30 min.


Figure 5c . GFP or GFP - ALG2 transfected TRPML1 - mCherry stably expressing cells were treated with acrolein for 2 h . Cell lysates were immunoprecipitated with anti - RFP magnetic beads and immunoblotted with the indicated antibodies . f . SH - SY5Y cells transfected with GFP or GFP - ALG2 for 24 h were treated with acrolein for 2 h and then spatial proximity of JIP4 and GFP was detected by a PLA using JIP4 and GFP antibodies .

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

Figure 5c. GFP or GFP-ALG2 transfected TRPML1-mCherry stably expressing cells were treated with acrolein for 2 h. Cell lysates were immunoprecipitated with anti-RFP magnetic beads and immunoblotted with the indicated antibodies.f. SH-SY5Y cells transfected with GFP or GFP-ALG2 for 24 h were treated with acrolein for 2 h and then spatial proximity of JIP4 and GFP was detected by a PLA using JIP4 and GFP antibodies.


Figure 6Parental and JIP4 KO SH - SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 for 2 h ( a ) . Cell lysates were immunoblotted with the indicated antibodies . Autophagic flux was determined as the LC3 - II production rate calculated as the level of LC3 - II with bafilomycin A1 treatment . SH - SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 in the presence of 10 µM Jak3 inhibitor VI ( Jak3 i VI ) for 2 h ( b ) . Cell lysates were immunoblotted with the indicated antibodies . Autophagic flux was determined as the LC3 - II production rate calculated as the level of LC3 - II with bafilomycin A1 treatment . d . ( upper ) SH - SY5Y cells were pretreated with 10 µM BAPTA - AM or 10 µM Jak3 inhibitor VI for 1 h and then treated with 500 µM H2O2 for an additional 2 h or cultured in starvation medium for an additional 2 h . Cells were fixed and stained with anti - JIP4 ( green ) and anti - LAMP2 ( red ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar , 20 μm . ( lower ) Clustered relative to whole lysosomes were quantified as lysosomal clustering value . e . SH - SY5Y cells were treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h . Cells were lysed , subjected to Phos - tag PAGE , and immunoblotted with anti - JIP4 antibody . f . ( left ) SH - SY5Y cells were transfected with TMEM55B siRNAs for 48 h and then treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h . Cells were fixed and stained with anti - γ - tubulin ( green ) and anti - LAMP2 ( red ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar , 20 μm . ( right ) Clustered relative to whole lysosomes were quantified as lysosomal clustering value . * * *

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 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 O O O O 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 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

Figure 6Parental and JIP4 KO SH-SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 for 2 h (a). Cell lysates were immunoblotted with the indicated antibodies. Autophagic flux was determined as the LC3-II production rate calculated as the level of LC3-II with bafilomycin A1 treatment.SH-SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 in the presence of 10 µM Jak3 inhibitor VI (Jak3 i VI) for 2 h (b). Cell lysates were immunoblotted with the indicated antibodies. Autophagic flux was determined as the LC3-II production rate calculated as the level of LC3-II with bafilomycin A1 treatment.d. (upper) SH-SY5Y cells were pretreated with 10 µM BAPTA-AM or 10 µM Jak3 inhibitor VI for 1 h and then treated with 500 µM H2O2 for an additional 2 h or cultured in starvation medium for an additional 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (lower) Clustered relative to whole lysosomes were quantified as lysosomal clustering value.e. SH-SY5Y cells were treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h. Cells were lysed, subjected to Phos-tag PAGE, and immunoblotted with anti-JIP4 antibody.f. (left) SH-SY5Y cells were transfected with TMEM55B siRNAs for 48 h and then treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h. Cells were fixed and stained with anti-γ-tubulin (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (right) Clustered relative to whole lysosomes were quantified as lysosomal clustering value. ***


Figure 7b . Parental and JIP4 KO SH - SY5Y cells were treated with various concentrations of acrolein for 24 h . Cytotoxicity was measured by an LDH assay . c . SH - SY5Y cells were transfected with TRPML1 siRNAs for 48 h and then treated with various concentrations of acrolein for 24 h . Cytotoxicity was measured by an LDH assay .

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

Figure 7b. Parental and JIP4 KO SH-SY5Y cells were treated with various concentrations of acrolein for 24 h. Cytotoxicity was measured by an LDH assay. c. SH-SY5Y cells were transfected with TRPML1 siRNAs for 48 h and then treated with various concentrations of acrolein for 24 h. Cytotoxicity was measured by an LDH assay.


Figure 1C . TIRF microscopy revealed time courses of the CAR - GFP microcluster formation as CAR T cells spread on the CD19 and ICAM - 1 - coated SLBs . D . Enrichment of phosphor - tyrosine ( pY ) at CAR microclusters . CAR T cells were fixed 20 min after being plated on the SLB , followed by staining with an anti - pan phosphor - tyrosine antibody . No CD19 was included in the control experiment ( only ICAM - 1 ) . Scale bar : 2 μm .

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 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

Figure 1C. TIRF microscopy revealed time courses of the CAR-GFP microcluster formation as CAR T cells spread on the CD19 and ICAM-1-coated SLBs.D. Enrichment of phosphor-tyrosine (pY) at CAR microclusters. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-pan phosphor-tyrosine antibody. No CD19 was included in the control experiment (only ICAM-1). Scale bar: 2 μm.


Figure 2T cells expressing GFP - CAR were plated on CD19 and ICAM - 1 coated SLBs . TIRF microscopy was implemented to visualize CAR localization . A . CAR microclusters underwent centripetal movement to form cSMAC ( upper panel ) or remained separated ( lower panel ) . B . Quantification of the categories regarding the formation of CAR cSMAC . Unstable cSMACs are defined by a cSMAC duration below 12 min . Data are shown as mean ± SD . n = 3 independent experiments . 37 , 38 , and 47 cells were quantified in individual experiments . C . Quantification of time from cell landing to the start of retrograde flow . Data are shown as mean ± SD . n = 33 ( stable ) , 31 ( unstable ) , 10 ( kinapse ) , and 41 ( no cSMAC ) cells . Results are pooled from three independent experiments . D . Localization of CAR and CD45 in the immunological synapse . CAR T cells were fixed 20 min after being plated on the SLB , followed by staining with an anti - CD45 antibody . E . Line scan of CAR and CD45 across the long axis of the immunological synapse . Shown are the normalized fluorescence intensity .

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 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

Figure 2T cells expressing GFP-CAR were plated on CD19 and ICAM-1 coated SLBs. TIRF microscopy was implemented to visualize CAR localization. A. CAR microclusters underwent centripetal movement to form cSMAC (upper panel) or remained separated (lower panel). B. Quantification of the categories regarding the formation of CAR cSMAC. Unstable cSMACs are defined by a cSMAC duration below 12 min. Data are shown as mean ± SD. n = 3 independent experiments. 37, 38, and 47 cells were quantified in individual experiments. C. Quantification of time from cell landing to the start of retrograde flow. Data are shown as mean ± SD. n = 33 (stable), 31 (unstable), 10 (kinapse), and 41 (no cSMAC) cells. Results are pooled from three independent experiments. D. Localization of CAR and CD45 in the immunological synapse. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-CD45 antibody. E. Line scan of CAR and CD45 across the long axis of the immunological synapse. Shown are the normalized fluorescence intensity.


Figure 3A . WT or LAT - deficient Jurkat cells ( Jcam2 . 5 ) expressing CAR - GFP were stimulated on SLB - coated with OKT3 ( anti - TCR antibody ) or CD19 ( CAR antigen ) ; in both conditions , the lipid bilayers were also coated with ICAM - 1 to facilitate cell attachment . TIRF microscopy revealed clustering of Ax647 - labeled streptavidin - Biotin - OKT3 or CD19 , which serves as a probe for TCR or CAR respectively . Scale bar , 2 µm . B . Quantification of clustering as normalized variance . n = 100 cells . Shown are the mean ± SD . Statistical test : unpaired two - tailed t - test . * * * * p < 0 . 0001 . * * 0 . 005 < 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 O O O O O O O O O O O O O O O O O O

Figure 3A. WT or LAT-deficient Jurkat cells (Jcam2.5) expressing CAR-GFP were stimulated on SLB-coated with OKT3 (anti-TCR antibody) or CD19 (CAR antigen); in both conditions, the lipid bilayers were also coated with ICAM-1 to facilitate cell attachment. TIRF microscopy revealed clustering of Ax647-labeled streptavidin-Biotin-OKT3 or CD19, which serves as a probe for TCR or CAR respectively. Scale bar, 2 µm. B. Quantification of clustering as normalized variance. n = 100 cells. Shown are the mean ± SD. Statistical test: unpaired two-tailed t-test. **** p < 0.0001. ** 0.005 < p < 0.05.


Figure 4B . WT or LAT - deficient ( Jcam2 . 5 ) cells expressing the 3rd or 1st generation CAR were stimulated on supported lipid bilayers coated with CD19 and ICAM - 1 . TIRF microscopy revealed clustering of CAR and Ax647 - labeled streptavidin - Biotin - CD19 . Scale bar , 2 µm . C . Quantification of clustering level of Ax647 - labeled streptavidin - Biotin - CD19 . Shown are means ± SD . Clustering is quantified as normalized variance . N = 50 cells for CAR 3rd WT and 1st WT , 63 cells for CAR 3rd lat - , and 51 cells for CAR 1st lat - . * * : 0 . 0005 < p < 0 . 005 . Statistical test : unpaired two - tailed t - test .

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 4B. WT or LAT-deficient (Jcam2.5) cells expressing the 3rd or 1st generation CAR were stimulated on supported lipid bilayers coated with CD19 and ICAM-1. TIRF microscopy revealed clustering of CAR and Ax647-labeled streptavidin-Biotin-CD19. Scale bar, 2 µm. C. Quantification of clustering level of Ax647-labeled streptavidin-Biotin-CD19. Shown are means ± SD. Clustering is quantified as normalized variance. N = 50 cells for CAR 3rd WT and 1st WT, 63 cells for CAR 3rd lat-, and 51 cells for CAR 1st lat-. **: 0.0005 < p < 0.005. Statistical test: unpaired two-tailed t-test.


Figure 5CAR ( 3rd generation ) T cells were plated on supported lipid bilayers coated with CD19 and ICAM - 1 and imaged by TIRF microscopy . A . Gads was recruited to CAR microclusters in both the wild - type and LAT - deficient ( Jcam2 . 5 ) cells . n = 33 or 34 cells . Shown are the means ± SE . n . s . , p = 0 . 58 by unpaired two - tailed t - test . As a control , Gads is not recruited to LAT - deficient cells activated by OKT3 . CAR ( 3rd generation ) T cells were plated on supported lipid bilayers coated with CD19 and ICAM - 1 and imaged by TIRF microscopy . B . Phosphorylated SLP76 , recognized by an anti - SLP76 pY145 antibody , was enriched in CAR microclusters in both the wild - type and LAT - deficient cells . In contrast , phosphorylated SLP76 was not enriched in TCR microclusters in LAT - deficient cells . n = 50 cells . Shown are the means ± SE . n . s . , p = 0 . 95 by unpaired two - tailed t - test . CAR ( 3rd generation ) T cells were plated on supported lipid bilayers coated with CD19 and ICAM - 1 and imaged by TIRF microscopy . C . Actin remodeling was induced in LAT - deficient cells by CD19 - , but not OKT3 - coated SLB . No ICAM - 1 was supplemented to SLB in this experiment to rule out the costimulation of actin via the LFA - 1 / ICAM - 1 pathway . Actin network was revealed by F - tractin - mScarlet . n = 6 cells . Shown are the means ± SD . n . s . , p = 0 . 68 , and 0 . 98 by unpaired two - tailed t - test .

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 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 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

Figure 5CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. A. Gads was recruited to CAR microclusters in both the wild-type and LAT-deficient (Jcam2.5) cells. n = 33 or 34 cells. Shown are the means ± SE. n.s., p = 0.58 by unpaired two-tailed t-test. As a control, Gads is not recruited to LAT-deficient cells activated by OKT3. CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. B. Phosphorylated SLP76, recognized by an anti-SLP76 pY145 antibody, was enriched in CAR microclusters in both the wild-type and LAT-deficient cells. In contrast, phosphorylated SLP76 was not enriched in TCR microclusters in LAT-deficient cells. n = 50 cells. Shown are the means ± SE. n.s., p = 0.95 by unpaired two-tailed t-test.CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. C. Actin remodeling was induced in LAT-deficient cells by CD19-, but not OKT3-coated SLB. No ICAM-1 was supplemented to SLB in this experiment to rule out the costimulation of actin via the LFA-1/ICAM-1 pathway. Actin network was revealed by F-tractin-mScarlet. n = 6 cells. Shown are the means ± SD. n.s., p = 0.68, and 0.98 by unpaired two-tailed t-test.


Figure 6A . Cell - cell conjugation assay . Wild - type or Lat - null ( J . LAT ) Jurkat T cells expressing CAR - GFP were mixed with Raji cells expressing mCherry - CAAX at a 1 : 1 ratio for 1 hr before being fixed . Three independent experiments were performed and over 90 cells were scored in each experiment . Scale bar : 5 µm . B . CAR T cells were incubated with Raji B cells at a 1 : 1 ratio for 24 hrs and IL - 2 from the supernatant were measured by ELISA . C . CAR T cells were incubated with Raji B cells at a 1 : 1 ratio and fixed at 24hr , stained with an anti - CD69 antibody , and analyzed by FACS . CD69 positive cells were scored within the CAR - GFP + population .

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 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

Figure 6A. Cell-cell conjugation assay. Wild-type or Lat-null (J.LAT) Jurkat T cells expressing CAR-GFP were mixed with Raji cells expressing mCherry-CAAX at a 1:1 ratio for 1 hr before being fixed. Three independent experiments were performed and over 90 cells were scored in each experiment. Scale bar: 5 µm.B. CAR T cells were incubated with Raji B cells at a 1:1 ratio for 24 hrs and IL-2 from the supernatant were measured by ELISA. C. CAR T cells were incubated with Raji B cells at a 1:1 ratio and fixed at 24hr, stained with an anti-CD69 antibody, and analyzed by FACS. CD69 positive cells were scored within the CAR-GFP+ population.


Figure 7A . Western blot showed that LAT is efficiently depleted in human primary T cells using CRISPR . Shown are the means ± SD . n = 4 independent experiments . B . CAR T cells were stimulated on SLBs coated with biotin - OKT3 ( left ) or biotin - CD19 ( right ) supplemented with ICAM - 1 . TIRF microscopy revealed clustering of Alexa647 - labeled streptavidin - Biotin - OKT3 or CD19 . Scale bar , 5 µm . C . Quantification of clustering of OKT3 or CD19 as normalized variance . n > 200 cells from two independent donors were scored for each condition . Central band : mean ; Box : quartiles ; Whisker : rest of the distribution . * * * * , p < 0 . 0001 ; n . s . p = 0 . 48 . D . Immunofluorescence analysis revealed membrane - localized phosphorylated SLP76 . Cells were plated on supported lipid bilayer coated with ICAM - 1 only ( control ) or ICAM - 1 supplemented with CD19 or OKT3 as indicated . The white dashed circle indicated cell boundary . Scale bar , 5 µm . E . Quantification of pSLP76 level on the membrane . n > 200 cells were scored , and the cells were isolated from two independent donors . Central band : mean ; Box : quartiles ; Whisker : rest of the distribution . * * * * , p < 0 . 0001 ; n . s . p = 0 . 11 . F . T cells were incubated with Raji B cells at a 4 : 1 ratio and IL - 2 production was measured by ELISA at 24 hr . Data were pooled from three donors . * , 0 . 005 < p < 0 . 05 ; * * , 0 . 0005 < p < 0 . 005 . Unpaired two - tailed t - test was applied .

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 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 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

Figure 7A. Western blot showed that LAT is efficiently depleted in human primary T cells using CRISPR. Shown are the means ± SD. n = 4 independent experiments.B. CAR T cells were stimulated on SLBs coated with biotin-OKT3 (left) or biotin-CD19 (right) supplemented with ICAM-1. TIRF microscopy revealed clustering of Alexa647-labeled streptavidin-Biotin-OKT3 or CD19. Scale bar, 5 µm. C. Quantification of clustering of OKT3 or CD19 as normalized variance. n > 200 cells from two independent donors were scored for each condition. Central band: mean; Box: quartiles; Whisker: rest of the distribution. ****, p < 0.0001; n.s. p = 0.48. D. Immunofluorescence analysis revealed membrane-localized phosphorylated SLP76. Cells were plated on supported lipid bilayer coated with ICAM-1 only (control) or ICAM-1 supplemented with CD19 or OKT3 as indicated. The white dashed circle indicated cell boundary. Scale bar, 5 µm. E. Quantification of pSLP76 level on the membrane. n > 200 cells were scored, and the cells were isolated from two independent donors. Central band: mean; Box: quartiles; Whisker: rest of the distribution. ****, p < 0.0001; n.s. p = 0.11. F. T cells were incubated with Raji B cells at a 4:1 ratio and IL-2 production was measured by ELISA at 24 hr. Data were pooled from three donors. *, 0.005 < p < 0.05; **, 0.0005 < p < 0.005. Unpaired two-tailed t-test was applied.


Figure 1 ( B - C ) Representative time - lapse images of ( B ) Tet1 - 3 triple KD ( Tet - TKD ) , ( C ) Tet3 - KD and control embryos at 3 . 5 days post fertilization . Scale bar = 100 µm . ( D - E ) Developmental rates of ( D ) control ( control - MO injected ) , Tet1 - , Tet2 - and Tet3 - single KD and Tet - TKD embryos and ( E ) control ( control - MO + Tet2 mRNA co - injected ) , Tet2 - KD2 ( Tet2 MO2 injected ) , Tet2 - KD2 + GFP mRNA and Tet2 - KD2 + Tet2 mRNA embryos until blastocyst stage . Indicated p - values were calculated using log rank ( Mantel - Cox ) test against ( D ) control embryos or ( E ) Tet2 - KD2 embryos or as indicated ( ns = non - significant , * = p < 0 . 05 , * * * * = p < 0 . 0001 ; numbers of analyzed embryos for each condition are indicated in parenthesis ) .

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 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 1(B-C) Representative time-lapse images of (B) Tet1-3 triple KD (Tet-TKD), (C) Tet3-KD and control embryos at 3.5 days post fertilization. Scale bar = 100 µm.(D-E) Developmental rates of (D) control (control-MO injected), Tet1-, Tet2- and Tet3-single KD and Tet-TKD embryos and (E) control (control-MO + Tet2 mRNA co-injected), Tet2-KD2 (Tet2 MO2 injected), Tet2-KD2 + GFP mRNA and Tet2-KD2 + Tet2 mRNA embryos until blastocyst stage. Indicated p-values were calculated using log rank (Mantel-Cox) test against (D) control embryos or (E) Tet2-KD2 embryos or as indicated (ns = non-significant, * = p < 0.05, **** = p < 0.0001; numbers of analyzed embryos for each condition are indicated in parenthesis).


Figure 2 ( A - B ) Representative images of ( A ) 5hmC - and ( B ) 5mC and 5caC - IF of control ( control - MO injected ) , Tet1 + 2 - DKD and Tet - TKD zygotes at 12 hpf . Paternal and maternal pronuclei are indicated , Pb = polar body ; Sp = sperm ; Scale bar = 20 μm . ( C - E ) Quantification of paternal / maternal ( C ) 5mC - , ( D ) 5hmC - , and ( E ) 5caC - signal ratios of 12 hpf derived zygotes normalized against DNA signal . A total of 5 - 16 zygotes from two to three experiments per condition were analyzed . Significance to control zygotes was calculated using ordinary one - way ANOVA with Tukey ' s multiple comparisons test ( C , E ) or Welch ANOVA with Dunnett ' s T3 multiple comparisons test ( D ) and significant differences are indicated ( * = p < 0 . 05 , * * = < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 , data are represented as mean ± SD , with dots representing single zygotes ) .

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 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

Figure 2(A-B) Representative images of (A) 5hmC- and (B) 5mC and 5caC-IF of control (control-MO injected), Tet1+2-DKD and Tet-TKD zygotes at 12 hpf. Paternal and maternal pronuclei are indicated, Pb = polar body; Sp = sperm; Scale bar = 20 μm.(C-E) Quantification of paternal/maternal (C) 5mC-, (D) 5hmC-, and (E) 5caC-signal ratios of 12 hpf derived zygotes normalized against DNA signal. A total of 5-16 zygotes from two to three experiments per condition were analyzed. Significance to control zygotes was calculated using ordinary one-way ANOVA with Tukey's multiple comparisons test (C, E) or Welch ANOVA with Dunnett's T3 multiple comparisons test (D) and significant differences are indicated (* = p < 0.05, ** = < 0.01, *** = p < 0.001, **** = p < 0.0001, data are represented as mean ± SD, with dots representing single zygotes).


Figure 3 ( B ) MA plots of RNA - seq data from Tet3 - KD / control ( left ) and Tet - TKD / control ( right ) 2 - cell embryos . Significant differentially expressed genes are colored . The amount of up - and downregulated genes is indicated . Differentially expressed genes were determined using DEseq2 ( padj < 0 . 05 , | log2FC | > 0 . 7 ) .

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

Figure 3(B) MA plots of RNA-seq data from Tet3-KD/control (left) and Tet-TKD/control (right) 2-cell embryos. Significant differentially expressed genes are colored. The amount of up- and downregulated genes is indicated. Differentially expressed genes were determined using DEseq2 (padj<0.05, |log2FC|>0.7).


Figure 4 ( A ) Expression changes of different classes of TEs in Tet3 - KD and Tet - TKD . Shown are log2 - fold changes of KD embryos compared to control 2 - cell embryos of different repetitive elements subdivided in classes of TEs . Indicated p - values were calculated using a paired two - tailed t - test ( * * * = p < 0 . 001 , * * * * = p < 0 . 0001 , data are represented as medium smoothed violin plots with indicated median and quartiles as dotted lines ) . ( B - C ) Expression levels of ( B ) maternally expressed ERV3 - LTRs and ( C ) 2 - cell embryo specific ERV3 - LTRs in control , Tet3 - KD and Tet - TKD 2 - cell embryos ( p - values were calculated using DEseq2 ; * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 , data are represented as mean ± SD , dots represent single embryos ) . ( E ) Expression levels of important candidate factors in control , Tet3 - KD and Tet - TKD 2 - cell embryos , implicated in the generation of 2C - like cells and as important regulators of EGA ( p - values were calculated using DEseq2 ; * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 , data are represented as mean ± SD , dots represent single embryos ) .

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 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 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

Figure 4(A) Expression changes of different classes of TEs in Tet3-KD and Tet-TKD. Shown are log2-fold changes of KD embryos compared to control 2-cell embryos of different repetitive elements subdivided in classes of TEs. Indicated p-values were calculated using a paired two-tailed t-test (*** = p < 0.001, **** = p <0.0001, data are represented as medium smoothed violin plots with indicated median and quartiles as dotted lines).(B-C) Expression levels of (B) maternally expressed ERV3-LTRs and (C) 2-cell embryo specific ERV3-LTRs in control, Tet3-KD and Tet-TKD 2-cell embryos (p-values were calculated using DEseq2; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, data are represented as mean ± SD, dots represent single embryos).(E) Expression levels of important candidate factors in control, Tet3-KD and Tet-TKD 2-cell embryos, implicated in the generation of 2C-like cells and as important regulators of EGA (p-values were calculated using DEseq2; * = p < 0.05, ** = p < 0.01, **** = p < 0.0001, data are represented as mean ± SD, dots represent single embryos).


Figure 5 ( A ) % CpG methylation of 20 kb tiles in 5 % bins in control or Tet - TKD 2 - cell embryos . All samples from one condition were merged for this visualization . ( F ) Significant methylation changes of specific TEs subdivided in different classes of TEs ( calculated using logistic regression function of Seqmonk , padj < 0 . 05 ; data are represented as medium smoothed violin plot with indicated median and quartiles as dotted line ) . ( G ) Correlation of significant DNA methylation changes in TEs ( calculated with logistic regression function of Seqmonk , padj < 0 . 05 , methylation difference > 5 % ) , which are also significant differentially expressed ( DEseq2 , padj < 0 . 05 , | log2FC | > 0 . 7 ) . ( H ) Developmental rate of embryos derived from control , Tet2 - MO2 , Tet2 - MO2 + Tet2 full - length mRNA ( as shown in Fig . 1E ) and Tet2 - MO2 + Tet2 truncated mRNA ( no catalytical domain ) injected oocytes . Indicated p - values were calculated using the log rank ( Mantel - Cox ) test against Tet2 - MO2 KD or as indicated ( * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 , numbers of analyzed embryos for each condition is indicated in parenthesis ) .

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 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 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 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

Figure 5(A) % CpG methylation of 20 kb tiles in 5% bins in control or Tet-TKD 2-cell embryos. All samples from one condition were merged for this visualization.(F) Significant methylation changes of specific TEs subdivided in different classes of TEs (calculated using logistic regression function of Seqmonk, padj < 0.05; data are represented as medium smoothed violin plot with indicated median and quartiles as dotted line).(G) Correlation of significant DNA methylation changes in TEs (calculated with logistic regression function of Seqmonk, padj < 0.05, methylation difference >5%), which are also significant differentially expressed (DEseq2, padj < 0.05, |log2FC|>0.7).(H) Developmental rate of embryos derived from control, Tet2-MO2, Tet2-MO2 + Tet2 full-length mRNA (as shown in Fig. 1E) and Tet2-MO2 + Tet2 truncated mRNA (no catalytical domain) injected oocytes. Indicated p-values were calculated using the log rank (Mantel-Cox) test against Tet2-MO2 KD or as indicated (* = p < 0.05, *** = p < 0.001, **** = p < 0.0001, numbers of analyzed embryos for each condition is indicated in parenthesis).


Figure 1Nucleosomal array oligomers are globular . A Nucleosomal array oligomers were stained with DAPI and examined using FM ( fluorescence microscopy ) as described in the Materials and Methods section . Shown are representative images obtained in 4 . 5 mM and 10mM MgCl2 . B Control FM images obtained in 0 , 1 and 2 . 5 mM MgCl2 . C Nucleosomal array oligomers were negatively stained and visualized by TEM as described under Materials and Methods . Shown in the left panels are representative images obtained in 4 . 5 mM , and 10 mM MgCl2 . Shown in the right panels are images of the interior of the oligomers ( white arrows , left panels ) after cropping and re - scaling .

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 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 1Nucleosomal array oligomers are globular.A Nucleosomal array oligomers were stained with DAPI and examined using FM (fluorescence microscopy) as described in the Materials and Methods section. Shown are representative images obtained in 4.5 mM and 10mM MgCl2.B Control FM images obtained in 0, 1 and 2.5 mM MgCl2.C Nucleosomal array oligomers were negatively stained and visualized by TEM as described under Materials and Methods. Shown in the left panels are representative images obtained in 4.5 mM, and 10 mM MgCl2. Shown in the right panels are images of the interior of the oligomers (white arrows, left panels) after cropping and re-scaling.


Figure 2Figure 2Sedimentation velocity analysis of the salt - dependent assembly of nucleosomal array oligomers . A Representative experiment showing the second moment sedimentation coefficients of the oligomeric nucleosomal arrays as a function of MgCl2 . The second moment sedimentation coefficient is equivalent to the mass average sedimentation coefficient for the entire sample ( see Materials and Methods ) . The inset shows the mean second moment sedimentation coefficient the standard error for three replicated experiments . B Analysis of the same raw data as in panel ( A ) by the time derivative method to yield the sedimentation coefficient distribution , g ( s * ) .

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

Figure 2Figure 2Sedimentation velocity analysis of the salt-dependent assembly of nucleosomal array oligomers.A Representative experiment showing the second moment sedimentation coefficients of the oligomeric nucleosomal arrays as a function of MgCl2. The second moment sedimentation coefficient is equivalent to the mass average sedimentation coefficient for the entire sample (see Materials and Methods). The inset shows the mean second moment sedimentation coefficient the standard error for three replicated experiments.B Analysis of the same raw data as in panel (A) by the time derivative method to yield the sedimentation coefficient distribution, g(s*).


Figure 3A , B SAXS profiles of the nuclesomal arrays in 0 ( TE ) , 1 , 2 . 5 mM MgCl2 ( A ) and 5 , 10 mM MgCl2 ( B ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , intensity ; S , scattering vector ( 1 / nm ) ) .

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

Figure 3A, B SAXS profiles of the nuclesomal arrays in 0 (TE), 1, 2.5 mM MgCl2 (A) and 5, 10 mM MgCl2 (B) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).


Figure 4A 601 - nucleosomal arrays were incubated in 0 . 5 , 5 mM MgCl2 , 5 mM MgCl2 + MNase , and analyzed by the differential centrifugation assay to determine the fraction oligomeric . The amounts of DNA in the supernatant fraction were measured . Note that for the MNase - digested oligomers the supernatant fraction also includes the digested free linker DNA . Each value is the mean of three measurements and the error bars represent the standard deviation . B Verification of complete MNase digestion . DNA was purified from the nucleosomal arrays incubated in 5 mM MgCl2 or 5 mM MgCl2 + MNase , and then electrophoresed on agarose gel . The position of mono nucleosome is marked with a star symbol . C Nucleosomal array oligomers ( left ) or with MNase treatment ( right ) were stained with DAPI and examined using FM . Shown are representative images obtained . Note that the sizes of MNase - treated oligomers are much smaller than those of the control oligomers ( left and Fig . 1A ) .

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 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 4A 601-nucleosomal arrays were incubated in 0.5, 5 mM MgCl2, 5 mM MgCl2 + MNase, and analyzed by the differential centrifugation assay to determine the fraction oligomeric. The amounts of DNA in the supernatant fraction were measured. Note that for the MNase-digested oligomers the supernatant fraction also includes the digested free linker DNA. Each value is the mean of three measurements and the error bars represent the standard deviation.B Verification of complete MNase digestion. DNA was purified from the nucleosomal arrays incubated in 5 mM MgCl2 or 5 mM MgCl2 + MNase, and then electrophoresed on agarose gel. The position of mono nucleosome is marked with a star symbol.C Nucleosomal array oligomers (left) or with MNase treatment (right) were stained with DAPI and examined using FM. Shown are representative images obtained. Note that the sizes of MNase-treated oligomers are much smaller than those of the control oligomers (left and Fig. 1A).


Figure 5A Oligomers assembled from H1 - nucleosomal arrays were stained with DAPI and examined using FM . Shown are representative images obtained in 4 mM and 5 mM MgCl2 . B Control FM images obtained in 0 , 1 and 3 mM MgCl2 . C H1 - nucleosomal array oligomers were negatively stained and visualized by TEM . Shown in the left panel is a representative image obtained in 4 mM MgCl2 . Shown in the right panel is an image of the interior of the oligomer ( white arrow , left panel ) after cropping and re - scaling .

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 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

Figure 5A Oligomers assembled from H1-nucleosomal arrays were stained with DAPI and examined using FM. Shown are representative images obtained in 4 mM and 5 mM MgCl2.B Control FM images obtained in 0, 1 and 3 mM MgCl2.C H1-nucleosomal array oligomers were negatively stained and visualized by TEM. Shown in the left panel is a representative image obtained in 4 mM MgCl2. Shown in the right panel is an image of the interior of the oligomer (white arrow, left panel) after cropping and re-scaling.


Figure 6Figure 6Sedimentation analysis of salt - dependent H1 - oligomer assembly . A Representative experiment showing the second moment sedimentation coefficients of the H1 - oligomers as a function of MgCl2 . The dashed line indicates the upper limit of measurable sedimentation coefficients ( ~ 106 S ) . The white symbol is intended to show that the sedimentation coefficient of the H1 - oligomers in 5 mM MgCl2 is beyond the detectable limit . B Analysis of the same raw data as in panel ( A ) by the time derivative method to yield the sedimentation coefficient distribution , g ( s * ) . C The g ( s * ) profiles in 2 . 5 mM , 3 mM , and 3 . 5 mM from panel ( B ) are re - plotted on a smaller scale .

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

Figure 6Figure 6Sedimentation analysis of salt-dependent H1-oligomer assembly.A Representative experiment showing the second moment sedimentation coefficients of the H1-oligomers as a function of MgCl2. The dashed line indicates the upper limit of measurable sedimentation coefficients (~106 S). The white symbol is intended to show that the sedimentation coefficient of the H1-oligomers in 5 mM MgCl2 is beyond the detectable limit.B Analysis of the same raw data as in panel (A) by the time derivative method to yield the sedimentation coefficient distribution, g(s*).C The g(s*) profiles in 2.5 mM, 3 mM, and 3.5 mM from panel (B) are re-plotted on a smaller scale.


Figure 7A , B SAXS profiles of the H1 - nuclesomal arrays in 0 ( TE ) , 1 , 2 . 5 mM MgCl2 ( A ) and 5 , 10 mM MgCl2 ( B ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , intensity ; S , scattering vector ( 1 / nm ) ) . C , D SAXS profiles of the native chicken chromatin in 0 ( TE ) , 0 . 5 , 1 mM MgCl2 ( C ) and 2 . 5 , 5 mM MgCl2 ( D ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , intensity ; S , scattering vector ( 1 / nm ) ) .

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 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

Figure 7A, B SAXS profiles of the H1-nuclesomal arrays in 0 (TE), 1, 2.5 mM MgCl2 (A) and 5, 10 mM MgCl2 (B) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).C, D SAXS profiles of the native chicken chromatin in 0 (TE), 0.5, 1 mM MgCl2 (C) and 2.5, 5 mM MgCl2 (D) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).


Figure 8A FMimages of chromatin structure in the nuclei with 0 mM ( left ) , 1 mM ( center ) and 5 mM MgCl2 ( right ) . Insets show the intensity line profiles between the two marked arrow heads in the images . B SAXS profiles of the isolated HeLa nuclei . SAXS profiles of the nuclei in 0 mM ( left ) , 1 mM ( center ) and 5 mM MgCl2 ( right ) are shown in blue . For comparison , the scattering curves of H1 - arrays from Figs . 7A and B were overlaid as red lines ( left , 0 mM ; center and right , 10 mM MgCl2 ) .

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 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

Figure 8A FMimages of chromatin structure in the nuclei with 0 mM (left), 1 mM (center) and 5 mM MgCl2 (right). Insets show the intensity line profiles between the two marked arrow heads in the images.B SAXS profiles of the isolated HeLa nuclei. SAXS profiles of the nuclei in 0 mM (left), 1 mM (center) and 5 mM MgCl2 (right) are shown in blue. For comparison, the scattering curves of H1-arrays from Figs. 7A and B were overlaid as red lines (left, 0 mM; center and right, 10 mM MgCl2).


Figure 1 ( A , B ) Poly ( I : C ) treatment inhibits axonal growth at 3 DIV ( A ) and dendritic growth at 6 DIV ( B ) in WT neurons . Neurons were transfected with GFP at 1 or 4 DIV . One day later , neurons were treated with poly ( I : C ) for 24 h before harvest . ( C ) Axon and dendrite morphology of Tlr3 - / - neurons after poly ( I : C ) treatment . Data in A , B , C were analyzed by unpaired t - test . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . * * P < 0 . 001 , * * * P < 0 . 0001 . Scale bar , 20μm in A and B . ( D , E ) Neuronal morphology in saline - and poly ( I : C ) - treated Tlr3 + / + ; Thy1 - Yfp ( D ) and Tlr3 - / - ; Thy1 - Yfp ( E ) mouse brains . Total dendrite length , dendritic tip number and Sholl analysis of dendritic processes were used to examine the dendrite phenotype . Data of dendrite length and tip number were analyzed by unpaired t - test . The Sholl data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . The numbers of analyzed neurons and mice are presented in each column . Mean values SEM are shown . * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 0001 . Scale bar , 50 μm in D and E .

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 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

Figure 1(A, B) Poly(I:C) treatment inhibits axonal growth at 3 DIV (A) and dendritic growth at 6 DIV (B) in WT neurons. Neurons were transfected with GFP at 1 or 4 DIV. One day later, neurons were treated with poly(I:C) for 24 h before harvest. (C) Axon and dendrite morphology of Tlr3-/- neurons after poly(I:C) treatment. Data in A,B,C were analyzed by unpaired t-test. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. ** P < 0.001, *** P < 0.0001. Scale bar, 20μm in A and B.(D, E) Neuronal morphology in saline- and poly(I:C)-treated Tlr3+/+;Thy1-Yfp (D) and Tlr3-/-;Thy1-Yfp (E) mouse brains. Total dendrite length, dendritic tip number and Sholl analysis of dendritic processes were used to examine the dendrite phenotype. Data of dendrite length and tip number were analyzed by unpaired t-test. The Sholl data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. The numbers of analyzed neurons and mice are presented in each column. Mean values SEM are shown. * P < 0.05, ** P < 0.001, *** P < 0.0001. Scale bar, 50 μm in D and E.


Figure 2 ( A , B ) Quantitation of dendrite morphology of Trif mutant neurons ( A ) and Myd88 - / - neurons ( B ) after poly ( I : C ) stimulation . Trif mutant or Myd88 - / - neurons were transfected with a GFP construct at 4 DIV and treated with poly ( I : C ) one day later . Neuron morphology was analyzed at 6 DIV according to the GFP signal . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by unpaired t - test . ( C ) MYD88 but not TRIF expression reduces dendrite outgrowth . HA - tagged MYD88 and TRIF were co - transfected with a GFP construct into cultured wild - type ( WT ) neurons at 4 DIV , and cell morphology was analyzed at 6 DIV . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by one - way ANOVA with Bonferroni ' s multiple comparison test . ( D ) MYD88 knockdown , but not TRIF knockdown , robustly increases dendritic arbors and loses the response to poly ( I : C ) . Three DIV neurons were transfected with control shRNA ( shCtrl ) , MYD88 shRNA ( shMYD88 ) or TRIF shRNA ( shTRIF ) and poly ( I : C ) was applied into the culture at 5 DIV for 24 h before harvest . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . Scale bar , 20 μm . * * * P < 0 . 0001 .

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 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 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

Figure 2(A, B) Quantitation of dendrite morphology of Trif mutant neurons (A) and Myd88-/- neurons (B) after poly(I:C) stimulation. Trif mutant or Myd88-/- neurons were transfected with a GFP construct at 4 DIV and treated with poly(I:C) one day later. Neuron morphology was analyzed at 6 DIV according to the GFP signal. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test.(C) MYD88 but not TRIF expression reduces dendrite outgrowth. HA-tagged MYD88 and TRIF were co-transfected with a GFP construct into cultured wild-type (WT) neurons at 4 DIV, and cell morphology was analyzed at 6 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by one-way ANOVA with Bonferroni's multiple comparison test.(D) MYD88 knockdown, but not TRIF knockdown, robustly increases dendritic arbors and loses the response to poly(I:C). Three DIV neurons were transfected with control shRNA (shCtrl), MYD88 shRNA (shMYD88) or TRIF shRNA (shTRIF) and poly(I:C) was applied into the culture at 5 DIV for 24 h before harvest. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. *** P < 0.0001.


Figure 3 ( A ) Coimmunoprecipitation of TLR3 and MYD88 . HEK293T cells were transfected with the indicated constructs . Twenty - four h later , immunoprecipitation ( IP ) was performed using HA antibody . The precipitates were immunoblotted ( IB ) with Myc and HA antibodies as indicated . EC , TLR3 ectodomain . ( B ) Poly ( I : C ) treatment increases the interaction between TLR3 and MYD88 . One day after transfection , cells were treated with saline or poly ( I : C ) for 30 min before harvesting . ( C ) Upper , schematic of MYD88 constructs . DD , death domain ; ID , intermediate domain , TIR , TIR domain . Lower , coimmunoprecipitation of TLR3 and the N - terminal region of MYD88 . ( D ) Full - length and N - terminal MYD88 inhibit dendrite outgrowth . Myd88 - / - neurons were transfected with control vector ( Ctrl ) , HA - tagged MYD88 ( MYD88 ) , N - terminal MYD88 ( MYD88 - N ) , or C - terminal MYD88 ( MYD88 - C ) with a GFP construct . Poly ( I : C ) was applied into the culture at 5 DIV and neuronal morphology was analyzed at 6 DIV . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . Scale bar , 20 μm . ( E ) Expression patterns of full - length and truncated fragments of MYD88 in Myd88 - / - neurons . Representative images are shown .

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 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 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 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

Figure 3(A) Coimmunoprecipitation of TLR3 and MYD88. HEK293T cells were transfected with the indicated constructs. Twenty-four h later, immunoprecipitation (IP) was performed using HA antibody. The precipitates were immunoblotted (IB) with Myc and HA antibodies as indicated. EC, TLR3 ectodomain.(B) Poly(I:C) treatment increases the interaction between TLR3 and MYD88. One day after transfection, cells were treated with saline or poly(I:C) for 30 min before harvesting.(C) Upper, schematic of MYD88 constructs. DD, death domain; ID, intermediate domain, TIR, TIR domain. Lower, coimmunoprecipitation of TLR3 and the N-terminal region of MYD88.(D) Full-length and N-terminal MYD88 inhibit dendrite outgrowth. Myd88-/- neurons were transfected with control vector (Ctrl), HA-tagged MYD88 (MYD88), N-terminal MYD88 (MYD88-N), or C-terminal MYD88 (MYD88-C) with a GFP construct. Poly(I:C) was applied into the culture at 5 DIV and neuronal morphology was analyzed at 6 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm.(E) Expression patterns of full-length and truncated fragments of MYD88 in Myd88-/- neurons. Representative images are shown.


Figure 4 ( A ) qPCR analysis of proinflammatory cytokines Il - 6 , Tnfα , Il - 1β and antiviral cytokine Ifnβ in WT cultured cortical and hippocampal neurons . Data were analyzed by unpaired t - test . Il - 6 and Tnfα N = 8 ; Ifnβ and Il - 1β , N = 6 . ( B , C ) Poly ( I : C ) treatment inhibits dendrite outgrowth of Il - 6 - / - neurons ( B ) and Tnfα - / - neurons ( C ) . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by unpaired t - test . ( D ) Left , schematic of experiment using conditioned medium . Upper right , conditioned medium applied to WT neurons . Lower right , conditioned medium applied to Tlr3 - / - neurons . Dendrite morphology was analyzed one day after adding conditioned medium . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by unpaired t - test . * P < 0 . 05 , * * * P < 0 . 0001 .

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 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

Figure 4(A) qPCR analysis of proinflammatory cytokines Il-6, Tnfα, Il-1β and antiviral cytokine Ifnβ in WT cultured cortical and hippocampal neurons. Data were analyzed by unpaired t-test. Il-6 and Tnfα N=8; Ifnβ and Il-1β, N=6.(B, C) Poly(I:C) treatment inhibits dendrite outgrowth of Il-6-/- neurons (B) and Tnfα-/- neurons (C). Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test.(D) Left, schematic of experiment using conditioned medium. Upper right, conditioned medium applied to WT neurons. Lower right, conditioned medium applied to Tlr3-/- neurons. Dendrite morphology was analyzed one day after adding conditioned medium. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test. * P < 0.05, *** P < 0.0001.


Figure 5At 4 DIV , cultured cortical neurons were transfected with indicated plasmids . Three days later , neurons were treated with 10 μg / ml poly ( I : C ) for 24 h . Neuronal morphology was monitored by GFP signals at 8 DIV . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . Scale bar , 20 μm . * * * P < 0 . 0001 , ns : non - significant .

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

Figure 5At 4 DIV, cultured cortical neurons were transfected with indicated plasmids. Three days later, neurons were treated with 10 μg/ml poly(I:C) for 24 h. Neuronal morphology was monitored by GFP signals at 8 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. *** P < 0.0001, ns: non-significant.


Figure 6 ( A ) Expression of 12 neuropsychiatric disorder - related genes in saline - and poly ( I : C ) - treated WT neurons . ( B ) Expression of 9 neuropsychiatric disorder - related genes in poly ( I : C ) - treated Tlr3 - / - neurons . Relative expression levels compared with vehicle control are shown . In ( B - D ) , dashed lines indicate the level of saline control . ( C , D ) Expression of Auts2 , Disc1 , Fmr1 , Pten and Ube3a in poly ( I : C ) - treated Myd88 - / - neurons ( C ) and poly ( I : C ) - treated P5 WT mouse brains ( D ) . In ( B - D ) , dashed lines indicate the level of saline control . ( E ) DIV 5 WT neurons were treated with poly ( I : C ) for 24 h . The total neuronal lysates were subjected to immunoblotting with the indicated antibodies . VCP and β - actin were used as loading controls . The protein levels were normalized with β - actin . Data were analyzed by unpaired t - test . Numbers of experimental repeats ( N ) are shown in each column . * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 0001 .

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 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 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

Figure 6(A) Expression of 12 neuropsychiatric disorder-related genes in saline- and poly(I:C)-treated WT neurons.(B) Expression of 9 neuropsychiatric disorder-related genes in poly(I:C)-treated Tlr3-/- neurons. Relative expression levels compared with vehicle control are shown. In (B-D), dashed lines indicate the level of saline control.(C, D) Expression of Auts2, Disc1, Fmr1, Pten and Ube3a in poly(I:C)-treated Myd88-/- neurons (C) and poly(I:C)-treated P5 WT mouse brains (D). In (B-D), dashed lines indicate the level of saline control.(E) DIV 5 WT neurons were treated with poly(I:C) for 24 h. The total neuronal lysates were subjected to immunoblotting with the indicated antibodies. VCP and β-actin were used as loading controls. The protein levels were normalized with β-actin. Data were analyzed by unpaired t-test. Numbers of experimental repeats (N) are shown in each column. * P < 0.05, ** P < 0.001, *** P < 0.0001.


Figure 7 ( A ) Overexpression of WT DISC1 , but not the DISC1 L604F mutant , in cultured neurons suppresses dendrite withdrawal induced by TLR3 activation . Mean values SEM of representatives of three independent experiments are shown . The numbers of analyzed neurons in the representative experiments are indicated in each column . Data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . Scale bar , 20 μm . * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 0001 . ( B - C ) Overexpression of DISC1 in cortical neurons of mouse brain is resistant to poly ( I : C ) - triggered reduction of dendritic arborization . Vector control ( Ctrl ) ( B ) or Myc - DISC1 ( C ) was co - expressed with GFP in cortical layer 2 / 3 neurons in mouse brain . After poly ( I : C ) injection at P4 and P5 , the neuronal morphology was analyzed at P7 by tracing the GFP signals . The data of dendrite length and tip number were analyzed by unpaired t - test . The Sholl data were analyzed by two - way ANOVA with Bonferroni ' s multiple comparison test . The numbers of analyzed neurons collected from 3 - 4 mice of each group are indicated . Mean values SEM are shown . Scale bar , 30 μm . * P < 0 . 05 , * * * P < 0 . 0001 .

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 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

Figure 7(A) Overexpression of WT DISC1, but not the DISC1 L604F mutant, in cultured neurons suppresses dendrite withdrawal induced by TLR3 activation. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. * P < 0.05, ** P < 0.001, *** P < 0.0001.(B-C) Overexpression of DISC1 in cortical neurons of mouse brain is resistant to poly(I:C)-triggered reduction of dendritic arborization. Vector control (Ctrl) (B) or Myc-DISC1 (C) was co-expressed with GFP in cortical layer 2/3 neurons in mouse brain. After poly(I:C) injection at P4 and P5, the neuronal morphology was analyzed at P7 by tracing the GFP signals. The data of dendrite length and tip number were analyzed by unpaired t-test. The Sholl data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. The numbers of analyzed neurons collected from 3-4 mice of each group are indicated. Mean values SEM are shown. Scale bar, 30 μm. * P < 0.05, *** P < 0.0001.


Figure 8 ( A ) Cortical and hippocampal mixed neuronal cultures were transfected with a GFP construct at 12 DIV and treated with poly ( I : C ) ( pIC ) at 17 DIV for 24 h . Spine morphology was examined at 18 DIV . Three secondary dendrites of each neuron were selected to analyze the spine density , the width of the spine head and the spine length . Mean values SEM of representatives of three independent experiments are shown . For the saline group , 15 neurons / 45 dendrites were examined ; for the poly ( I : C ) group , 16 neurons / 48 dendrites were examined . Data were analyzed by unpaired t - test . Scale bar , 20 μm in right upper panel and 5 µm in lower left panel . * * * P < 0 . 0001 . ( C , D ) Spine morphology of P21 Thy1 - Yfp ( C ) and Tlr3 - / - ; Thy1 - Yfp mice ( D ) after poly ( I : C ) stimulation at P4 and P5 . One secondary dendrite of an apical dendrite of each somatosensory layer 5 cortical neuron was selected to examine the spine morphology . Three mice were analyzed for each group . Saline - treated Thy1 - Yfp , 44 neurons ; poly ( I : C ) - treated Thy1 - Yfp , 43 neurons ; saline - treated Tlr3 - / - ; Thy1 - Yfp , 46 neurons ; poly ( I : C ) - treated Tlr3 - / - ; Thy1 - Yfp , 50 neurons . Mean values SEM are shown . Data were analyzed by unpaired t - test . Scale bar , 5 μm . * * * P < 0 . 0001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8(A) Cortical and hippocampal mixed neuronal cultures were transfected with a GFP construct at 12 DIV and treated with poly(I:C) (pIC) at 17 DIV for 24 h. Spine morphology was examined at 18 DIV. Three secondary dendrites of each neuron were selected to analyze the spine density, the width of the spine head and the spine length. Mean values SEM of representatives of three independent experiments are shown. For the saline group, 15 neurons/45 dendrites were examined; for the poly(I:C) group, 16 neurons/48 dendrites were examined. Data were analyzed by unpaired t-test. Scale bar, 20 μm in right upper panel and 5 µm in lower left panel. *** P < 0.0001.(C, D) Spine morphology of P21 Thy1-Yfp (C) and Tlr3-/-; Thy1-Yfp mice (D) after poly(I:C) stimulation at P4 and P5. One secondary dendrite of an apical dendrite of each somatosensory layer 5 cortical neuron was selected to examine the spine morphology. Three mice were analyzed for each group. Saline-treated Thy1-Yfp, 44 neurons; poly(I:C)-treated Thy1-Yfp, 43 neurons; saline-treated Tlr3-/-;Thy1-Yfp, 46 neurons; poly(I:C)-treated Tlr3-/-;Thy1-Yfp, 50 neurons. Mean values SEM are shown. Data were analyzed by unpaired t-test. Scale bar, 5 μm. *** P < 0.0001.


Figure 1 ( A ) HeLa cells were infected with Listeria for 0 . 5 - 4 h , and lysates were analysed by blotting using indicated antibodies . ( B ) HeLa cells left unstimulated ( CTR ) or infected with Listeria for 1 or 3 h , analysed by IF using antibodies against mTOR and LAMP2 . ( C ) Percentage of Listeria ‐ infected cells displaying mTOR localization to LAMP2 + vesicles . Values are means ± s . e . m . n = 3 . * * * P0 . 001 and * * P0 . 01 over uninfected . ( D , E ) HeLa cells were infected with Listeria for 0 . 5 - 4 h , and lysates were analysed by blotting using indicated antibodies . ( F ) qPCR analysis of ATF3 induction following infection of HeLa cells with Listeria for 1 - 4 h . Values are means ± s . e . m . n = 3 . Scale bars : 10 μm . Source data for this figure is available on the online supplementary information page .

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 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 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 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

Figure 1(A) HeLa cells were infected with Listeria for 0.5-4 h, and lysates were analysed by blotting using indicated antibodies.(B) HeLa cells left unstimulated (CTR) or infected with Listeria for 1 or 3 h, analysed by IF using antibodies against mTOR and LAMP2.(C) Percentage of Listeria‐infected cells displaying mTOR localization to LAMP2+ vesicles. Values are means±s.e.m. n=3. ***P0.001 and **P0.01 over uninfected.(D, E) HeLa cells were infected with Listeria for 0.5-4 h, and lysates were analysed by blotting using indicated antibodies.(F) qPCR analysis of ATF3 induction following infection of HeLa cells with Listeria for 1-4 h. Values are means±s.e.m. n=3. Scale bars: 10 μm.Source data for this figure is available on the online supplementary information page.


Figure 2 ( A ) HeLa cells were infected with Listeria wild ‐ type ( WT ) or the LLO / PlcA / PlcB triple knockout ( TKO ) mutant strain for 0 . 5 - 4 h , and lysates were analysed by blotting using indicated antibodies . ( B ) qPCR analysis of ATF3 and IL ‐ 8 induction following infection of HeLa cells with Listeria WT or TKO for 1 - 4 h . Values are means ± s . e . m . n = 3 . ( C ) HeLa cells left unstimulated ( CTR ) or infected with Listeria TKO for 1 h , analysed by IF using antibodies against mTOR and LAMP2 . ( D , E ) Percentage of cells infected with either WT or TKO Listeria strains displaying one or several LAMP2 + ( D ) or NDP52 + ( E ) Listeria vesicles . Values are means ± s . e . m . n = 3 . * P0 . 05 ; * * P0 . 01 . Scale bars : 10 μm . Source data for this figure is available on the online supplementary information page .

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 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 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 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

Figure 2(A) HeLa cells were infected with Listeria wild‐type (WT) or the LLO/PlcA/PlcB triple knockout (TKO) mutant strain for 0.5-4 h, and lysates were analysed by blotting using indicated antibodies.(B) qPCR analysis of ATF3 and IL‐8 induction following infection of HeLa cells with Listeria WT or TKO for 1-4 h. Values are means±s.e.m. n=3.(C) HeLa cells left unstimulated (CTR) or infected with Listeria TKO for 1 h, analysed by IF using antibodies against mTOR and LAMP2.(D, E) Percentage of cells infected with either WT or TKO Listeria strains displaying one or several LAMP2+ (D) or NDP52+ (E) Listeria vesicles. Values are means±s.e.m. n=3. *P0.05; **P0.01. Scale bars: 10 μm.Source data for this figure is available on the online supplementary information page.