( D ) Loss of AP - 2µ significantly decreases the number of spines on granule neurons ( WT : 12 . 63 ± 0 . 88 , KO : 8 . 07 ± 0 . 45 , p = 0 . 004 , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(D) Loss of AP-2µ significantly decreases the number of spines on granule neurons (WT: 12.63±0.88, KO: 8.07±0.45, p=0.004, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( E , F ) Overlap coefficient between the presynaptic marker Bassoon and the postsynaptic marker PSD95 is significantly decreased in the entorhinal cortex of AP - 2µ KO mice compared to the WT ( WT : 0 . 45 ± 0 . 02 , KO : 0 . 33 ± 0 . 06 , p = 0 . 034 , N = 3 biological replicates ) . Scale bar , 750nm . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(E, F) Overlap coefficient between the presynaptic marker Bassoon and the postsynaptic marker PSD95 is significantly decreased in the entorhinal cortex of AP-2µ KO mice compared to the WT (WT: 0.45±0.02, KO: 0.33±0.06, p=0.034, N=3 biological replicates). Scale bar, 750nm. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Lost preference for the novel object ( nov ) comparing to the familiar object ( fam ) in AP - 2µ KO mice tested 24h after training in NOR Representative heatmaps are shown in ( G ) .

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

Lost preference for the novel object (nov) comparing to the familiar object (fam) in AP-2µ KO mice tested 24h after training in NOR Representative heatmaps are shown in (G).


Lost preference for the novel object ( nov ) comparing to the familiar object ( fam ) in AP - 2µ KO mice tested 24h after training in NOR ( WTfam : 27 . 51 ± 6 . 77 , WTnov : 72 . 49 ± 6 . 77 , pWTfam vs WTnov = 0 . 000 ; KOfam : 43 . 9 ± 13 . 12 , KOnov : 59 . 91 ± 13 . 12 , pKOfam vs KOnov = 0 . 469 , N = 7 WT and 8 KO mice ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

Lost preference for the novel object (nov) comparing to the familiar object (fam) in AP-2µ KO mice tested 24h after training in NOR (WTfam: 27.51±6.77, WTnov: 72.49±6.77, pWTfam vs WTnov=0.000; KOfam: 43.9±13.12, KOnov: 59.91±13.12, pKOfam vs KOnov=0.469, N=7 WT and 8 KO mice). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( I , J ) AP - 2µ KO mice are significantly more active in the open field compared to the WT ( WT : 1663 ± 139 . 08 % , KO : 3976 ± 802 . 30 % , p = 0 . 020 , N = 7 WT and 8 KO mice ) . Representative heatmaps are shown in ( I ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(I, J) AP-2µ KO mice are significantly more active in the open field compared to the WT (WT: 1663±139.08%, KO: 3976±802.30%, p=0.020, N=7 WT and 8 KO mice). Representative heatmaps are shown in (I). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( K , L ) Overlap coefficient between Bassoon and PSD95 is rescued upon BACE1 KD in AP - 2µ KO neurons ( WTscr : 0 . 39 ± 0 . 02 ; KOscr : 0 . 24 ± 0 . 01 ; WTshBACE1 : 0 . 37 ± 0 . 02 ; KOshBACE1 : 0 . 33 ± 0 . 02 ; pWTscr vs pKOscr < 0 . 000 ; pKOscr vs pKOshBACE1 = 0 . 010 ; pWTscr vs pWTshBACE1 = 0 . 813 ; pWTshBACE1 vs pKOshBACE1 = 0 . 623 , 22 - 24 neurons for each condition , N = 3 ) . Scale bars : 10µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(K, L) Overlap coefficient between Bassoon and PSD95 is rescued upon BACE1 KD in AP-2µ KO neurons (WTscr: 0.39±0.02; KOscr: 0.24±0.01; WTshBACE1: 0.37±0.02; KOshBACE1: 0.33±0.02; pWTscr vs pKOscr<0.000; pKOscr vs pKOshBACE1=0.010; pWTscr vs pWTshBACE1=0.813; pWTshBACE1 vs pKOshBACE1=0.623, 22-24 neurons for each condition, N=3). Scale bars: 10µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Neuronal loss is rescued in the entorhinal cortex of AP - 2µ mice haploinsufficient for BACE1 ( AP - 2WT / BACEWT : 2 . 25 ± 0 . 08 N = 9 sections from N = 3 for each genotype ) . Scale bar : 100 μm .

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

Neuronal loss is rescued in the entorhinal cortex of AP-2µ mice haploinsufficient for BACE1 (AP-2WT/BACEWT: 2.25±0.08 N=9 sections from N=3 for each genotype). Scale bar: 100 μm.


Neuronal loss is rescued in the entorhinal cortex of AP - 2µ KO mice haploinsufficient for BACE1 AP - 2KO / BACEWT : 1 . 51 ± 0 . 08 , AP - 2WT / BACEHET : 1 . 80 ± 0 . 07 , AP - 2KO / BACEHET : 2 . 00 ± 0 . 07 ; pAP - 2WT / BACE1WT vs pAP - 2KO / BACE1WT < 0 . 000 ; pAP - 2WT / BACE1WT vs pAP - 2WT / BACE1HET = 0 . 001 ; pAP - 2KO / BACE1WT vs pAP - 2KO / BACE1HET = 0 . 000 ; p AP - 2WT / BACE1HET vs AP - 2KO / BACE1HET = 0 . 268 , N = 9 sections from N = 3 for each genotype ) . Scale bar : 100 μm . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

Neuronal loss is rescued in the entorhinal cortex of AP-2µ KO mice haploinsufficient for BACE1 AP-2KO/BACEWT: 1.51±0.08, AP-2WT/BACEHET: 1.80±0.07, AP-2KO/BACEHET: 2.00±0.07; pAP-2WT/BACE1WT vs pAP-2KO/BACE1WT<0.000; pAP-2WT/BACE1WT vs pAP-2WT/BACE1HET=0.001; pAP-2KO/BACE1WT vs pAP-2KO/BACE1HET=0.000; p AP-2WT/BACE1HET vs AP-2KO/BACE1HET=0.268, N=9 sections from N=3 for each genotype). Scale bar: 100 μm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( P , Q ) APP - CTF levels are rescued in the entorhinal cortex of AP - 2µ KO mice haploinsufficient for BACE1 ( AP - 2WT / BACEWT : 427574 ± 24021 , AP - 2KO / BACEWT : 538895 ± 34662 , AP - 2WT / BACEHET : 389870 ± 19842 , AP - 2KO / BACEHET : 395389 ± 21853 ; pAP - 2WT / BACE1WT vs pAP - 2KO / BACE1WT = 0 . 017 ; pAP - 2KO / BACE1WT vs pAP - 2KO / BACE1HET = 0 . 001 ; p AP - 2WT / BACE1HET vs AP - 2KO / BACE1HET = 0 . 979 ; 13 - 15 sections , N = 3 mice for each genotype ) . Scale bars : 40µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(P, Q) APP-CTF levels are rescued in the entorhinal cortex of AP-2µ KO mice haploinsufficient for BACE1 (AP-2WT/BACEWT:427574±24021, AP-2KO/BACEWT: 538895±34662, AP-2WT/BACEHET: 389870±19842, AP-2KO/BACEHET: 395389±21853; pAP-2WT/BACE1WT vs pAP-2KO/BACE1WT=0.017; pAP-2KO/BACE1WT vs pAP-2KO/BACE1HET=0.001; p AP-2WT/BACE1HET vs AP-2KO/BACE1HET=0.979; 13-15 sections, N=3 mice for each genotype). Scale bars: 40µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Proteoliposomes containing human ADP / ATP carrier were loaded with either 0 . 10 mM ( red traces ) , 0 . 25 mM ( orange traces ) , 0 . 50 mM ( green traces ) , 1 . 00 mM ( light blue traces ) or 2 . 50 mM ( dark blue traces ) ATP ( ATPint ) , and transport was initiated by the externally added radiolabeled [ 33P ] - ATP ( ATPext ) at either 0 . 50 μM ( filled circles ) , 0 . 67 μM ( filled squares ) , 1 . 00 μM ( filled upward triangles ) , 1 . 50 μM ( filled downward triangles ) , 2 . 50 μM ( open circles ) , 5 . 00 μM ( open squares ) or 15 . 0 μM ( open upward triangles ) . A schematic representation of the transport assays is shown . Data information : The data are represented by the average and standard deviation of three biological repeats ( each the average of three technical repeats ) for the assays with 0 . 10 , 0 . 25 , 0 . 5 and 1 . 0 mM of internal ATP , and one biological repeat ( based on the average of three technical repeats ) for the 2 . 50 mM ATP internal assay .

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

Proteoliposomes containing human ADP/ATP carrier were loaded with either 0.10 mM (red traces), 0.25 mM (orange traces), 0.50 mM (green traces), 1.00 mM (light blue traces) or 2.50 mM (dark blue traces) ATP (ATPint), and transport was initiated by the externally added radiolabeled [33P]-ATP (ATPext) at either 0.50 μM (filled circles), 0.67 μM (filled squares), 1.00 μM (filled upward triangles), 1.50 μM (filled downward triangles), 2.50 μM (open circles), 5.00 μM (open squares) or 15.0 μM (open upward triangles). A schematic representation of the transport assays is shown. Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for the assays with 0.10, 0.25, 0.5 and 1.0 mM of internal ATP, and one biological repeat (based on the average of three technical repeats) for the 2.50 mM ATP internal assay.


Initial rates were estimated by fitting the data with the model in Equation 1 . A . Michaelis - Menten plots of 0 . 10 mM ( red traces ) , 0 . 25 mM ( orange traces ) , 0 . 50 mM ( green traces ) , 1 . 00 mM ( light blue traces ) and 2 . 50 mM ( dark blue traces ) internally - loaded ATP . Data information : The data are represented by the average and standard deviation of three biological repeats ( each the average of three technical repeats ) for all internal concentrations but 2 . 50 mM ATP , which is the average of three technical repeats .

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

Initial rates were estimated by fitting the data with the model in Equation 1. A. Michaelis-Menten plots of 0.10 mM (red traces), 0.25 mM (orange traces), 0.50 mM (green traces), 1.00 mM (light blue traces) and 2.50 mM (dark blue traces) internally-loaded ATP. Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for all internal concentrations but 2.50 mM ATP, which is the average of three technical repeats.


Initial rates were estimated by fitting the data with the model in Equation 1 . B . Lineweaver - Burk analysis of [ 33P ] - ATP / ATP homo - exchange ( same color scheme as A ) . Data information : The data are represented by the average and standard deviation of three biological repeats ( each the average of three technical repeats ) for all internal concentrations but 2 . 50 mM ATP , which is the average of three technical repeats .

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

Initial rates were estimated by fitting the data with the model in Equation 1. B. Lineweaver-Burk analysis of [33P]-ATP/ATP homo-exchange (same color scheme as A). Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for all internal concentrations but 2.50 mM ATP, which is the average of three technical repeats.


Initial rates were estimated by fitting the data with the model in Equation 1 . C . Km plotted against Vmax for the various substrate gradients . The kinetic parameters were determined by fitting of the Michaelis - Menten curves through iteration . Data information : The data are represented by the average and standard deviation of three biological repeats ( each the average of three technical repeats ) for all internal concentrations but 2 . 50 mM ATP , which is the average of three technical repeats .

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

Initial rates were estimated by fitting the data with the model in Equation 1. C. Km plotted against Vmax for the various substrate gradients. The kinetic parameters were determined by fitting of the Michaelis-Menten curves through iteration. Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for all internal concentrations but 2.50 mM ATP, which is the average of three technical repeats.


( F ) TIRF images of Ramos cells transduced with endophilin A2 - GFP and mCherry - ClathrinLC on antigen - loaded PMS . Scale bar = 5 μm . White arrowheads highlight endophilin A2 colocalization with antigen clusters .

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

(F) TIRF images of Ramos cells transduced with endophilin A2-GFP and mCherry-ClathrinLC on antigen-loaded PMS. Scale bar = 5 μm. White arrowheads highlight endophilin A2 colocalization with antigen clusters.


( J ) IgM - BCR immunoprecipitation to investigate recruitment of endophilin A2 to activated BCRs . Anti - endophilin A2 blots detect overexpressed endophilin A2 - GFP in streptavidin immunoprecipitates and total supernatants from anti - IgM - biotin - stimulated Ramos and DG75 cells . Representative experiment and quantification from 3 independent pulldowns in WT , BLNK - or GRB2 - knockout ( KO ) DG75 cells . Pulldown intensity is calculated as a percentage of total endophilin A2 protein and normalized to WT in each experiment . Data show mean and SEM ; p values calculated using one - way ANOVA .

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

(J) IgM-BCR immunoprecipitation to investigate recruitment of endophilin A2 to activated BCRs. Anti-endophilin A2 blots detect overexpressed endophilin A2-GFP in streptavidin immunoprecipitates and total supernatants from anti-IgM-biotin-stimulated Ramos and DG75 cells. Representative experiment and quantification from 3 independent pulldowns in WT, BLNK- or GRB2-knockout (KO) DG75 cells. Pulldown intensity is calculated as a percentage of total endophilin A2 protein and normalized to WT in each experiment. Data show mean and SEM; p values calculated using one-way ANOVA.


( E ) Quantification of surface CD86 and CD69 mean fluorescence intensity ( MFI ) . Data show mean and SEM from 6 mice across 2 experiments .

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

(E) Quantification of surface CD86 and CD69 mean fluorescence intensity (MFI). Data show mean and SEM from 6 mice across 2 experiments.


( F ) Fractions of mCherry + cells in GC , PC , IgG1 + class - switched and follicular B cells at 4 - , 8 - and 14 - days post SRBC - HEL immunization . N = 8 - 10 mice ; data shows mean and SEM . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 using 2 - way ANOVA with multiple comparisons .

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

(F) Fractions of mCherry+ cells in GC, PC, IgG1+ class-switched and follicular B cells at 4-, 8- and 14-days post SRBC-HEL immunization. N = 8-10 mice; data shows mean and SEM. *p<0.05; ***p<0.001; ****p<0.0001 using 2-way ANOVA with multiple comparisons.


( C ) Surface levels of MHCII , BAFFR , CD40 and LPAM1 in Sh3gl1 - / - and WT littermates . N = 4 - 5 mice . P , statistical significance from unpaired t tests . Data show mean ± SEM .

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

(C) Surface levels of MHCII, BAFFR, CD40 and LPAM1 in Sh3gl1-/- and WT littermates. N = 4-5 mice. P, statistical significance from unpaired t tests. Data show mean ± SEM.


( D ) Quantification of B220 + Fas + CD38 - GC population as percentage of total splenic B cells and NP - specific cells as a percentage of the GC population 14 days after NP - CGG immunization in alum . Data show mean ± SEM from N = 6 mice , p , significance in unpaired t tests .

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

(D) Quantification of B220+Fas+CD38- GC population as percentage of total splenic B cells and NP-specific cells as a percentage of the GC population 14 days after NP-CGG immunization in alum. Data show mean ± SEM from N = 6 mice, p, significance in unpaired t tests.


( E - F ) Serum antibodies collected pre - immunization and 7 - or 14 - days post NP - CGG immunization , detected by NP7 - BSA ELISA using isotype - specific secondary antibodies . Data show mean ± SEM from N = 3 experiments with statistical significance from 2 - way ANOVA with multiple comparisons .

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

(E-F) Serum antibodies collected pre-immunization and 7- or 14-days post NP-CGG immunization, detected by NP7-BSA ELISA using isotype-specific secondary antibodies. Data show mean ± SEM from N = 3 experiments with statistical significance from 2-way ANOVA with multiple comparisons.


( H ) Numbers of Tfh cells 14 days post - immunization , identified as a CD4 + CD44 + PD - 1 + CXCR5 + population . Mean and SEM of N = 4 mice .

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

(H) Numbers of Tfh cells 14 days post-immunization, identified as a CD4+CD44+PD-1+CXCR5+ population. Mean and SEM of N = 4 mice.


( A ) Soluble anti - IgM internalization in CRISPR - targeted follicular B cells . N = 11 - 12 mice . Data show mean ± SEM . P , statistical significance from two - way ANOVA . ( B ) Soluble anti - IgD internalization . N = 3 mice . Data show mean ± SEM . P , statistical significance from two - way ANOVA . ( C ) Soluble anti - IgG internalization in GC B cells from SRBC - immunized mice . N = 4 mice . Data show mean ± SEM . P , statistical significance from two - way ANOVA .

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

(A) Soluble anti-IgM internalization in CRISPR-targeted follicular B cells. N = 11- 12 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA. (B) Soluble anti-IgD internalization. N = 3 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA. (C) Soluble anti-IgG internalization in GC B cells from SRBC-immunized mice. N = 4 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA.


( F ) IgM degradation following total cell surface biotinylation , measured as loss of biotin - labelled membrane IgM ( mIgM ) in cell lysates over time . ( G ) Western densitometry quantifying IgM degradation assay . Proportion of biotin detected is normalized to the initial WT condition ( left ) or to the initial amount for each genotype ( right ) . Data show mean ± SEM from three independent tests . P , statistical significance from two - way ANOVA .

(F) IgM degradation following total cell surface biotinylation, measured as loss of biotin-labelled membrane IgM (mIgM) in cell lysates over time. (G) Western densitometry quantifying IgM degradation assay. Proportion of biotin detected is normalized to the initial WT condition (left) or to the initial amount for each genotype (right). Data show mean ± SEM from three independent tests. P, statistical significance from two-way ANOVA.


( K ) Proliferation ( CFSE dilution ) of OTII CD4 cells in co - cultures with anti - Igκ - OVA pulsed B cells ; and quantification from 6 - 8 mice . Data show mean ± SEM .

(K) Proliferation (CFSE dilution) of OTII CD4 cells in co-cultures with anti-Igκ-OVA pulsed B cells; and quantification from 6-8 mice. Data show mean ± SEM.


( C - D ) Isolated naïve B cells from CRISPR - targeted chimeras after 3 - day culture in stated cytokines . ( C ) mCherry percentage in targeted primary cell cultures . N = 15 mice . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 using two - way ANOVA . ( D ) AnnexinV surface stain in targeted cells . N = 3 - 4 mice . Data information : All data show means ± SEM .

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

(C-D) Isolated naïve B cells from CRISPR-targeted chimeras after 3-day culture in stated cytokines. (C) mCherry percentage in targeted primary cell cultures. N = 15 mice. *p<0.05, **p<0.01, ****p<0.0001 using two-way ANOVA. (D) AnnexinV surface stain in targeted cells. N = 3-4 mice. Data information: All data show means ± SEM.


( G ) Oxygen consumption rate ( OCR ) in CRISPR - targeted Ramos cells . N = 2 independent infections . P , two - way ANOVA of basal and maximum OCR . ( H ) OCR in B cells isolated from Sh3gl1 - / - and WT littermates . N = 9 mice . P , two - way ANOVA of basal and maximum OCR .

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

(G) Oxygen consumption rate (OCR) in CRISPR-targeted Ramos cells. N = 2 independent infections. P, two-way ANOVA of basal and maximum OCR. (H) OCR in B cells isolated from Sh3gl1-/- and WT littermates. N = 9 mice. P, two-way ANOVA of basal and maximum OCR.


Cell cultures with full RPMI media supplemented with 50 μg / ml FeSO4 or 5 μM hemin chloride . ( J ) SH3GL1 - targeted Ramos cells . N = 4 independent infections . P , statistical significance from two - way ANOVA from both treatments versus RPMI . ( K ) Control sgRNA - targeted Ramos cells .

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

Cell cultures with full RPMI media supplemented with 50 μg/ml FeSO4 or 5 μM hemin chloride. (J) SH3GL1-targeted Ramos cells. N = 4 independent infections. P, statistical significance from two-way ANOVA from both treatments versus RPMI. (K) Control sgRNA-targeted Ramos cells.


( O ) TfR internalization assay in Ramos cells using biotinylated anti - CD71 . 4 independent CRISPR infections in 2 experiments . P , significance calculated using two - way ANOVA and indicated in corresponding color .

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

(O) TfR internalization assay in Ramos cells using biotinylated anti-CD71. 4 independent CRISPR infections in 2 experiments. P, significance calculated using two-way ANOVA and indicated in corresponding color.


( P , Q ) Mouse transferrin internalization in B cells from SRBC - immunized WT and Sh3gl1 - / - littermates . N = 7 mice per genotype in 2 experiments . P , statistical significance analyzed using two - way ANOVA . ( P ) Naïve B cells . ( Q ) GC B cells .

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

(P,Q) Mouse transferrin internalization in B cells from SRBC-immunized WT and Sh3gl1-/- littermates. N = 7 mice per genotype in 2 experiments. P, statistical significance analyzed using two-way ANOVA. (P) Naïve B cells. (Q) GC B cells.


B : Scatter Plot of mass spectrometry assay . Dots depict protein intensity ( IBAQ ) and relative significance obtained from mass spectrometry assay . X - axis : Difference of Che - 1 intensity versus IP control ; on the y - axis are the corresponding − log10 P - values . Significant Che - 1 interacting proteins relevant in this study are represented in blue .

B: Scatter Plot of mass spectrometry assay. Dots depict protein intensity (IBAQ) and relative significance obtained from mass spectrometry assay. X-axis: Difference of Che-1 intensity versus IP control; on the y-axis are the corresponding −log10 P-values. Significant Che-1 interacting proteins relevant in this study are represented in blue.


C : Nuclear extracts from Kms27 MM cells treated or not with RNase A were subjected to immunoprecipitation with the indicated antibodies ( abs ) and analyzed by western blot ( WB ) with the indicated abs . Input corresponds to 5 % of the total extract used for immunoprecipitation .

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

C: Nuclear extracts from Kms27 MM cells treated or not with RNase A were subjected to immunoprecipitation with the indicated antibodies (abs) and analyzed by western blot (WB) with the indicated abs. Input corresponds to 5% of the total extract used for immunoprecipitation.


D : ( Top ) qRT - PCR approach described in the schematic representation . ( Bottom ) RIP - qPCR analysis with anti - Che - 1 specific antibody or control rabbit IgG of Kms27 MM cells . Data are expressed as percent of Input . Error bars represent the standard error of three different biological experiments performed in technical duplicate ( Two Tailed T test * * * P < 0 . 005 , * * * * P < 0 . 001 , n . s . = not significant ) .

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

D: (Top) qRT-PCR approach described in the schematic representation. (Bottom) RIP-qPCR analysis with anti-Che-1 specific antibody or control rabbit IgG of Kms27 MM cells. Data are expressed as percent of Input. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test ***P<0.005, ****P<0.001, n.s. = not significant).


E : Colocalization assays in Kms27 and RPMI8226 MM cell lines by combining In Situ Hybridization ( FISH ) staining of NEAT1 ( Stellaris ) with immunofluorescence of Che - 1 . Scale bar 10 μm .

E: Colocalization assays in Kms27 and RPMI8226 MM cell lines by combining In Situ Hybridization (FISH) staining of NEAT1 (Stellaris) with immunofluorescence of Che-1. Scale bar 10 μm.


F : ( Left ) RT - qPCR analysis of NEAT1 expression levels in Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides . Values were normalized to Actin expression . Error bars represent the standard error of three different biological experiments performed in technical duplicate ( Two Tailed T test * * * * P < 0 . 001 ) . ( Right ) Nuclear extracts from KMS27 MM cells were immunoprecipitated with the indicated abs and analyzed by WB with the indicated abs .

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

F: (Left) RT-qPCR analysis of NEAT1 expression levels in Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in technical duplicate (Two Tailed T test ****P<0.001). (Right) Nuclear extracts from KMS27 MM cells were immunoprecipitated with the indicated abs and analyzed by WB with the indicated abs.


A : Quantitative RT - PCR ( RT - qPCR ) of Kms27 MM cells transiently transfected with two different LNA GapmeR NEAT1 or GapmeR Control oligonucleotides . Values were normalized to Actin expression . Error bars represent the standard error of three different experiments performed in duplicate ( Two Tailed T test * * * * P < 0 . 001 ) .

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

A: Quantitative RT-PCR (RT-qPCR) of Kms27 MM cells transiently transfected with two different LNA GapmeR NEAT1 or GapmeR Control oligonucleotides. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments performed in duplicate (Two Tailed T test ****P<0.001).


B : WB analysis with indicated abs of total extracts from Kms27 MM cells transiently transfected as in A .

B: WB analysis with indicated abs of total extracts from Kms27 MM cells transiently transfected as in A.


C : Representative confocal images of Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A , GapmeR NEAT1B or GapmeR Control oligonucleotides and immunostained with anti - Che - 1 antibody . Scale bar 10 μm .

C: Representative confocal images of Kms27 MM cells transiently transfected with LNA GapmeR NEAT1A, GapmeR NEAT1B or GapmeR Control oligonucleotides and immunostained with anti-Che-1 antibody. Scale bar 10 μm.


D : Isolated cell compartments from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides , were subjected to WB with the indicated abs .

D: Isolated cell compartments from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides, were subjected to WB with the indicated abs.


E : Che - 1 ChIP - seq signal intensity of LNA GapmeR NEAT1A ( 2 replicates on right ) and GapmeR Control ( 2 replicates on left ) and at co - localizing Che - 1 / NEAT1 sites ( N = 14654 sites ) . Signal intensity from 1 ( weak ) to 12 ( strongest ) .

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

E: Che-1 ChIP-seq signal intensity of LNA GapmeR NEAT1A (2 replicates on right) and GapmeR Control (2 replicates on left) and at co-localizing Che-1/NEAT1 sites (N=14654 sites). Signal intensity from 1 (weak) to 12 (strongest).


F : FISH images of Kms27 MM cells depleted or not for Che - 1 expression ( siChe - 1A ) labeled with Hoechst ( cell nuclei ) and Quasar 670 ( NEAT1 RNA ) . Scale bar 10 μm .

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

F: FISH images of Kms27 MM cells depleted or not for Che-1 expression (siChe-1A) labeled with Hoechst (cell nuclei) and Quasar 670 (NEAT1 RNA). Scale bar 10 μm.


G : NEAT1 ChIRP - seq signal intensity of Kms27 MM cells transiently transfected with siChe - 1A or siControl at co - localizing Che - 1 / NEAT1 sites ( N = 14654 sites ) . Signal intensity from 1 ( weak ) to 14 ( strongest ) .

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

G: NEAT1 ChIRP-seq signal intensity of Kms27 MM cells transiently transfected with siChe-1A or siControl at co-localizing Che-1/NEAT1 sites (N=14654 sites). Signal intensity from 1 (weak) to 14 (strongest).


B : Chromatin extracts from Kms27 and RPMI8226 MM cells treated or not with RNase H , were subjected to immunoprecipitation with S9 . 6 antibody and analyzed by WB with the indicated abs . Input corresponds to 5 % of the total extract used for immunoprecipitation .

O O O O O O O O O O O O O O O O O O O O O O O O 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: Chromatin extracts from Kms27 and RPMI8226 MM cells treated or not with RNase H, were subjected to immunoprecipitation with S9.6 antibody and analyzed by WB with the indicated abs. Input corresponds to 5% of the total extract used for immunoprecipitation.


C : Representative confocal images showing the interaction between Che - 1 and S9 . 6 revealed by PLA in Kms27 MM cells . Negative controls were performed by omitting Che - 1 or S9 . 6 primary antibody . Scale bar 10 μm .

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

C: Representative confocal images showing the interaction between Che-1 and S9.6 revealed by PLA in Kms27 MM cells. Negative controls were performed by omitting Che-1 or S9.6 primary antibody. Scale bar 10 μm.


D : Chromatin extracts from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides were immunoprecipitated with S9 . 6 antibody and analyzed by WB with the indicated antibodies . Input corresponds to 5 % of the total extract used for immunoprecipitation .

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

D: Chromatin extracts from Kms27 MM cells transfected with LNA GapmeR NEAT1A or GapmeR Control oligonucleotides were immunoprecipitated with S9.6 antibody and analyzed by WB with the indicated antibodies. Input corresponds to 5% of the total extract used for immunoprecipitation.


E : ( Left ) Representative confocal images of Kms27 MM cells treated or not with RNase H , transiently transfected with siChe - 1A or siControl and immunostained with S9 . 6 antibody and . Scale bar 10 μm . ( Right ) Box plot showing S9 . 6 signal nuclear intensity per cell ( Arbitrary Unit - AU ) . siChe - 1A cells are described in green color , while siControl in grey color . 100 cells were counted in each replicate . Each dot represents a cell . Median scores per sample are written at the top of the median red line . P value : < 1 . 02 - 18 . ANOVA test was performed to evaluate statistical significance . The red center line denotes the median value ( 50th percentile ) while the black box contains the 25th to 75th percentiles of data . The whiskers mark the 5th and 95th percentiles .

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

E: (Left) Representative confocal images of Kms27 MM cells treated or not with RNase H, transiently transfected with siChe-1A or siControl and immunostained with S9.6 antibody and. Scale bar 10 μm. (Right) Box plot showing S9.6 signal nuclear intensity per cell (Arbitrary Unit-AU). siChe-1A cells are described in green color, while siControl in grey color. 100 cells were counted in each replicate. Each dot represents a cell. Median scores per sample are written at the top of the median red line. P value: <1.02-18. ANOVA test was performed to evaluate statistical significance. The red center line denotes the median value (50th percentile) while the black box contains the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.


F : ( Top ) S9 . 6 antibody was used for dot blot analysis to evaluate RNA : DNA hybrids formation in Kms27 MM cells by serial dilutions of genomic DNA starting at 1 . 5 micrograms . ( Bottom ) Quantification of S9 . 6 levels by densitometric analysis . Values were normalized to a Methylene blue loading control . Error bars represent the standard error of three different biological experiments ( Two Tailed T test * * * P < 0 . 005 , * * * * P < 0 . 001 ) .

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

F: (Top) S9.6 antibody was used for dot blot analysis to evaluate RNA:DNA hybrids formation in Kms27 MM cells by serial dilutions of genomic DNA starting at 1.5 micrograms. (Bottom) Quantification of S9.6 levels by densitometric analysis. Values were normalized to a Methylene blue loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ***P<0.005, ****P<0.001).


G : Enrichment density of DRIP - seq signals at NEAT1 / Che1 co - localizing sites in Kms27 MM cells identified in siRNA control ( siControl ) ( grey ) and siRNA Che - 1 ( siChe - 1A ) ( dark green ) samples . Enrichment density was calculated on the relative size of each peak ( start to end of each fragment ) extended 2000 bp upstream and downstream . Right frame represents the signal obtained from 14654 randomly selected sites from the same experiments .

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

G: Enrichment density of DRIP-seq signals at NEAT1/Che1 co-localizing sites in Kms27 MM cells identified in siRNA control (siControl) (grey) and siRNA Che-1 (siChe-1A) (dark green) samples. Enrichment density was calculated on the relative size of each peak (start to end of each fragment) extended 2000 bp upstream and downstream. Right frame represents the signal obtained from 14654 randomly selected sites from the same experiments.


D : ( Top ) Representative confocal images of Kms27 MM cells transfected and treated as in C and immunostained with S9 . 6 antibody . Scale bar 10 μm . ( Bottom ) Box plot showing S9 . 6 signal nuclear intensity per cell ( Arbitrary Unit - AU ) . siChe - 1A cells are described in dark grey , siChe1A plus CX5461 in green color , while siControl in grey color . Median scores per sample are written at the top of the median red line . P value : < 1 . 30 - 54 . ANOVA test was performed to evaluate statistical significance . The red center line denotes the median value ( 50th percentile ) while the black box contains the 25th to 75th percentiles of data . The whiskers mark the 5th and 95th percentiles .

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

D: (Top) Representative confocal images of Kms27 MM cells transfected and treated as in C and immunostained with S9.6 antibody. Scale bar 10 μm. (Bottom) Box plot showing S9.6 signal nuclear intensity per cell (Arbitrary Unit-AU). siChe-1A cells are described in dark grey, siChe1A plus CX5461 in green color, while siControl in grey color. Median scores per sample are written at the top of the median red line. P value: <1.30-54. ANOVA test was performed to evaluate statistical significance. The red center line denotes the median value (50th percentile) while the black box contains the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.


E : ( Top ) Dot blot analysis of decreasing amounts of genomic DNA extracts from Kms27 MM cells depleted or not for Che - 1 expression ( siChe - 1A ) and treated with CX5461 , were probed using abs directed against RNA : DNA hybrids . ( Bottom ) Quantification of S9 . 6 levels by densitometric analysis . Values were normalized to a Methylene blue loading control . Error bars represent the standard error of three different biological experiments ( Two Tailed T test * * * * P < 0 . 001 ) .

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

E: (Top) Dot blot analysis of decreasing amounts of genomic DNA extracts from Kms27 MM cells depleted or not for Che-1 expression (siChe-1A) and treated with CX5461, were probed using abs directed against RNA:DNA hybrids. (Bottom) Quantification of S9.6 levels by densitometric analysis. Values were normalized to a Methylene blue loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ****P<0.001).


A : Differential analysis of siControl vs . siChe1A transcriptome in Kms27 MM cells . Volcano plot shows 135 significantly upregulated ( dark green ) and 392 downregulated genes ( dark grey ) . x - axis reports the base - 2 logarithm of fold change which is approximated to the B value coming from the statistical Wald test . y - axis reports the base - 10 logarithm of Q value of significant genes .

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

A: Differential analysis of siControl vs. siChe1A transcriptome in Kms27 MM cells. Volcano plot shows 135 significantly upregulated (dark green) and 392 downregulated genes (dark grey). x-axis reports the base-2 logarithm of fold change which is approximated to the B value coming from the statistical Wald test. y-axis reports the base-10 logarithm of Q value of significant genes.


D : WB analysis of total extracts of Kms27 MM cells depleted or not for Che - 1 ( siChe - 1A ) and probed for the indicated abs .

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

D: WB analysis of total extracts of Kms27 MM cells depleted or not for Che-1 (siChe-1A) and probed for the indicated abs.


E : ( Left ) Luciferase activity of human NF - kB promoter was measured in Kms27 MM cells transiently transfected with siChe - 1A or siControl oligonucleotides or with empty vector or p65 / RelA protein expressing vector . Error bars represent the standard error of three different biological experiments performed in duplicate ( Two Tailed T test * * * P < 0 . 005 , * * * * P < 0 . 001 ) . ( Right ) WB analysis of total extracts of Kms27 MM cells transiently transfected as described in E and incubated with the indicated antibodies .

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

E: (Left) Luciferase activity of human NF-kB promoter was measured in Kms27 MM cells transiently transfected with siChe-1A or siControl oligonucleotides or with empty vector or p65/RelA protein expressing vector. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005, ****P<0.001). (Right) WB analysis of total extracts of Kms27 MM cells transiently transfected as described in E and incubated with the indicated antibodies.


RT - qPCR analysis of the levels of the indicated genes in Kms27 MM cells transiently transfected with siChe - 1A or siControl . Values were normalized to Actin expression . Error bars represent the standard error of three different experiments in duplicate ( Two Tailed T test * * * P < 0 . 001 , * * * * P < 0 . 001 ) .

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

RT-qPCR analysis of the levels of the indicated genes in Kms27 MM cells transiently transfected with siChe-1A or siControl. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments in duplicate (Two Tailed T test ***P<0.001, ****P<0.001).


G : RT - qPCR analysis of the levels of the indicated genes in Kms27 MM cells transiently transfected with siChe - 1A or siControl . Values were normalized to Actin expression . Error bars represent the standard error of three different experiments in duplicate ( Two Tailed T test * * * P < 0 . 001 , * * * * P < 0 . 001 ) .

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

G: RT-qPCR analysis of the levels of the indicated genes in Kms27 MM cells transiently transfected with siChe-1A or siControl. Values were normalized to Actin expression. Error bars represent the standard error of three different experiments in duplicate (Two Tailed T test ***P<0.001, ****P<0.001).


H : WB analysis for the indicated antibodies of total extracts from Kms27 MM cells with siChe - 1A or siControl and where indicated with GFP - tagged RNaseH expression vectors .

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

H: WB analysis for the indicated antibodies of total extracts from Kms27 MM cells with siChe-1A or siControl and where indicated with GFP-tagged RNaseH expression vectors.


I : RT - qPCR analysis of IFNB and IFNG levels in Kms27 MM cells transiently transfected as in H . Values were normalized to Actin expression . Error bars represent the standard error of three different biological experiments performed in duplicate ( Two Tailed T test * * P < 0 . 01 , * * * P < 0 . 005 . )

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

I: RT-qPCR analysis of IFNB and IFNG levels in Kms27 MM cells transiently transfected as in H. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test **P<0.01, ***P<0.005.)


A : Representative S9 . 6 immunofluorescence levels in healthy and tumor CD138 + plasma cells purified from patient bone marrow . Nuclei were visualized by staining with Hoechst dye . Scale bar 10 μm .

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

A: Representative S9.6 immunofluorescence levels in healthy and tumor CD138+ plasma cells purified from patient bone marrow. Nuclei were visualized by staining with Hoechst dye. Scale bar 10 μm.


B : Dot blot performed to evaluate the enrichment of RNA : DNA hybrids formation using the S9 . 6 antibody by serial dilutions of genomic DNA starting at 1 . 0 micrograms . Methylene blue normalization of S9 . 6 signal image acquired by using Alliance Mini HD6 system by UVITEC Ltd .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Dot blot performed to evaluate the enrichment of RNA:DNA hybrids formation using the S9.6 antibody by serial dilutions of genomic DNA starting at 1.0 micrograms. Methylene blue normalization of S9.6 signal image acquired by using Alliance Mini HD6 system by UVITEC Ltd.


C : Boxplot showing relative S9 . 6 nuclear mean intensity in CD138 + plasma cells from 15 MM patients ( red ) and 5 healthy ( light blue ) . Dot plot representing each cell considered overlay the boxplot . Y - axis : S9 . 6 Nuclear Mean Intensity ( AU ) . The center line denotes the median value ( 50th percentile ) while the boxes contain the 25th to 75th percentiles of data . The whiskers mark the 5th and 95th percentiles .

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

C: Boxplot showing relative S9.6 nuclear mean intensity in CD138+ plasma cells from 15 MM patients (red) and 5 healthy (light blue). Dot plot representing each cell considered overlay the boxplot. Y-axis: S9.6 Nuclear Mean Intensity (AU). The center line denotes the median value (50th percentile) while the boxes contain the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.


D : Representative WB analysis with the indicated antibodies of healthy and neoplastic CD138 + purified plasma cells from patients .

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

D: Representative WB analysis with the indicated antibodies of healthy and neoplastic CD138+ purified plasma cells from patients.


F : Unsupervised clustering heatmap showing enrichment profiles of 9 IFN signature genes . From the top : ISS stages 1 , 2 , 3 of each patient ( N = 687 ) . IFN signature High ( red , N = 71 ) and Low ( white , N = 614 ) assigned to each patient . Color scheme representing up - ( red ) and down - ( blue ) regulation of the selected genes across the patient dataset . Colors are proportional to normalized ( cpm ) read enrichment - the relative read enrichment median of each gene within the full patient cohort .

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

F: Unsupervised clustering heatmap showing enrichment profiles of 9 IFN signature genes. From the top: ISS stages 1,2,3 of each patient (N=687). IFN signature High (red, N=71) and Low (white, N=614) assigned to each patient. Color scheme representing up- (red) and down- (blue) regulation of the selected genes across the patient dataset. Colors are proportional to normalized (cpm) read enrichment - the relative read enrichment median of each gene within the full patient cohort.


A : WB analysis of total extracts from Kms27 and U266 human MM cells with the indicated abs .

A: WB analysis of total extracts from Kms27 and U266 human MM cells with the indicated abs.


B : ( Left ) Kms27 and U266 MM cells were subjected to immunostaining with S9 . 6 antibody Scale bar 10 μm . ( Right ) Dot plot showing relative S9 . 6 nuclear mean intensity cell in the nucleus . 100 cells were counted in each replicate . Median scores per sample are written at the top of the median red line . P value : 3 . 3 - 13 . T - test was performed to evaluate statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: (Left) Kms27 and U266 MM cells were subjected to immunostaining with S9.6 antibody Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity cell in the nucleus. 100 cells were counted in each replicate. Median scores per sample are written at the top of the median red line. P value: 3.3-13. T-test was performed to evaluate statistical significance.


C : Different expression of IFNG and IFNB in Kms27 and U266 MM cells was assessed by RT - qPCR . Values were normalized to Actin expression . Error bars represent the standard error of three different biological experiments performed in duplicate ( Two Tailed T test * * * P < 0 . 005 ) .

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

C: Different expression of IFNG and IFNB in Kms27 and U266 MM cells was assessed by RT-qPCR. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005).


D : WB analysis with the indicated abs of total extracts from U266 MM cells treated or not with Thapsigargin ( THAP ) 0 . 5 μg / ml for 16 hours . G : WB with indicated abs of total extracts from Kms27 MM cells treated or not with ISRIB 1μM for 6 hours .

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

D: WB analysis with the indicated abs of total extracts from U266 MM cells treated or not with Thapsigargin (THAP) 0.5 μg/ml for 16 hours. G: WB with indicated abs of total extracts from Kms27 MM cells treated or not with ISRIB 1μM for 6 hours.


E : ( Left ) U266 MM cells treated or not with THAP as in D and immunostained with S9 . 6 antibody Scale bar 10 μm . ( Right ) Dot plot showing relative S9 . 6 nuclear mean intensity per cell . 100 cells were counted in each replicate . Median scores per sample are written at the top of the median red line . P value : 1 . 1 - 20 . T - test was performed to evaluate statistical significance . H : ( Left ) Immunostaining with S9 . 6 antibody of Kms27 MM cells treated or not with ISRIB as in G Scale bar 10 μm . ( Right ) Dot plot showing relative S9 . 6 nuclear mean intensity per cell . 100 cells were counted in each replicate Median scores per sample are written at the top of the median red line . P value : 4 . 7 - 23 . T - test was performed to evaluate statistical significance .

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

E: (Left) U266 MM cells treated or not with THAP as in D and immunostained with S9.6 antibody Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity per cell. 100 cells were counted in each replicate. Median scores per sample are written at the top of the median red line. P value: 1.1-20. T-test was performed to evaluate statistical significance. H: (Left) Immunostaining with S9.6 antibody of Kms27 MM cells treated or not with ISRIB as in G Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity per cell. 100 cells were counted in each replicate Median scores per sample are written at the top of the median red line. P value: 4.7-23. T-test was performed to evaluate statistical significance.


F : RT - qPCR analysis of IFNG and IFNB in U266 MM cells treated or not with THAP . Values were normalized to Actin expression . Error bars represent the standard error of three different biological experiments performed in duplicate ( Two Tailed T test * * * P < 0 . 005 , * * * * P < 0 . 001 ) I : RT - qPCR analysis of IFNG and IFNB levels in Kms27 MM cells treated as in G . Values were normalized to Actin expression . Error bars represent the standard error of three different biological experiments performed in duplicate ( Two Tailed T test * * * * P < 0 . 001 ) .

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

F: RT-qPCR analysis of IFNG and IFNB in U266 MM cells treated or not with THAP. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005, ****P<0.001) I: RT-qPCR analysis of IFNG and IFNB levels in Kms27 MM cells treated as in G. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ****P<0.001).


A . Localization of TRAPP complex components after sodium arsenite ( SA ) treatment in HeLa cells treated with 300 µM SA for 30 min . Fluorescence microscopy of fixed cells using antibodies against TRAPPC2 and eIF3 ( to label SGs ) at steady state and after SA treatment ( top two rows ) . Other panels show localization of GFP - TRAPPC3 and endogenous TRAPPC1 to eIF3 - labeled SGs and of endogenous TRAPPC9 and TRAPPC8 to G3BP - labeled SGs . DAPI ( blue ) . Scale bar , 10 µm .

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

A. Localization of TRAPP complex components after sodium arsenite (SA) treatment in HeLa cells treated with 300 µM SA for 30 min. Fluorescence microscopy of fixed cells using antibodies against TRAPPC2 and eIF3 (to label SGs) at steady state and after SA treatment (top two rows). Other panels show localization of GFP-TRAPPC3 and endogenous TRAPPC1 to eIF3-labeled SGs and of endogenous TRAPPC9 and TRAPPC8 to G3BP-labeled SGs. DAPI (blue). Scale bar, 10 µm.


B . Representative images of a time course analysis of TRAPPC2 redistribution to SGs . Cells were treated The graph ( B ' ) shows quantification of TRAPPC2 localization at SGs over time as the ratio between TRAPPC2 ( mean fluorescence intensity ) in SG puncta and cytosolic TRAPPC2 . Mean ± s . e . m . n = 50 cells per experiment , N = 3 . Scale bar , 10 µm .

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

B. Representative images of a time course analysis of TRAPPC2 redistribution to SGs. Cells were treated The graph (B') shows quantification of TRAPPC2 localization at SGs over time as the ratio between TRAPPC2 (mean fluorescence intensity) in SG puncta and cytosolic TRAPPC2. Mean ± s.e.m. n = 50 cells per experiment, N = 3. Scale bar, 10 µm.


C . Analysis of TRAPPC2 ( green ) , TRAPPC8 ( blue ) and TRAPPC9 ( orange ) localization at SGs upon TRAPPC2 , TRAPPC4 TRAPPC8 and TRAPPC9 depletion . Mean ± s . e . m . n > 100 . * p < 0 . 05 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison test .

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

C. Analysis of TRAPPC2 (green), TRAPPC8 (blue) and TRAPPC9 (orange) localization at SGs upon TRAPPC2, TRAPPC4 TRAPPC8 and TRAPPC9 depletion. Mean ± s.e.m. n>100. *p<0.05; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.


E . Analysis of TRAPPC2 localization at SGs upon CLIC1 , CRYAB and ENO1 depletion . Mean ± s . e . m . n > 100 . * * * * p < 0 . 0001 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison test .

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

E. Analysis of TRAPPC2 localization at SGs upon CLIC1, CRYAB and ENO1 depletion. Mean ± s.e.m. n>100. ****p<0.0001; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.


A . HeLa cells , untreated or treated with SA , were fixed and visualized by fluorescence microscopy using anti - Sec24C Ab , anti - eIF3 Ab , and DAPI ( blue ) . Data information : Scale bars , 10 µm .

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

A. HeLa cells, untreated or treated with SA, were fixed and visualized by fluorescence microscopy using anti-Sec24C Ab, anti-eIF3 Ab, and DAPI (blue). Data information: Scale bars, 10 µm.


B . Quantification of Sec24C redistribution to SGs over time after SA treatment ( the ratio between Sec24C ( mean fluorescence intensity ) in SG puncta and cytosolic Sec24C ) . Mean ± s . e . m . n = 50 cells per experiment , N = 3 .

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

B. Quantification of Sec24C redistribution to SGs over time after SA treatment (the ratio between Sec24C (mean fluorescence intensity) in SG puncta and cytosolic Sec24C). Mean ± s.e.m. n = 50 cells per experiment, N = 3.


C . Quantification of residual Sec24C in ERES after SA treatment . The data are expressed as percentage of steady state values ( CTRL ) . Mean ± s . e . m . n = 100 cells per experiment , N = 3 .

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

C. Quantification of residual Sec24C in ERES after SA treatment. The data are expressed as percentage of steady state values (CTRL). Mean ± s.e.m. n = 100 cells per experiment, N = 3.


D . Representative images of TRAPPC2 localization in Sec23AB - KD and Sec24ABCD - KD cells treated with SA . Cells were fixed and visualized by fluorescence microscopy using anti - TRAPPC2 Ab , anti - G3BP Ab ( to label SGs ) and DAPI ( blue ) . Data information : Scale bars , 10 µm .

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

D. Representative images of TRAPPC2 localization in Sec23AB-KD and Sec24ABCD-KD cells treated with SA. Cells were fixed and visualized by fluorescence microscopy using anti-TRAPPC2 Ab, anti-G3BP Ab (to label SGs) and DAPI (blue). Data information: Scale bars, 10 µm.


E . Quantification of TRAPPC2 redistribution to SGs after KD of the indicated Sec23 and Sec24 combinations , calculated as the ratio between TRAPPC2 ( mean intensity ) in SG puncta and cytosolic TRAPPC2 and expressed as % of CTRL . Mean ± s . e . m . , n = 40 - 60 cells per experiment , N = 3 . ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison test .

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

E. Quantification of TRAPPC2 redistribution to SGs after KD of the indicated Sec23 and Sec24 combinations, calculated as the ratio between TRAPPC2 (mean intensity) in SG puncta and cytosolic TRAPPC2 and expressed as % of CTRL. Mean ± s.e.m., n = 40-60 cells per experiment, N = 3. ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.


F . Representative images of Sec24C localization at SGs ( stained for G3BP ) in TRAPPC3 - KD and TRAPPC2 - KD cells treated with SA . Depletion of the entire TRAPP complex ( via TRAPPC3 depletion ) or of only TRAPPC2 reduces Sec24C recruitment . Graph , quantification of Sec24C at SGs , calculated as in ( E ) . Mean ± s . e . m . , n = 40 - 60 cells per experiment , N = 3 . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test . Data information : Scale bars , 10 µm .

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

F. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC3-KD and TRAPPC2-KD cells treated with SA. Depletion of the entire TRAPP complex (via TRAPPC3 depletion) or of only TRAPPC2 reduces Sec24C recruitment. Graph, quantification of Sec24C at SGs, calculated as in (E). Mean ± s.e.m., n = 40-60 cells per experiment, N = 3. ****p< 0.0001, One-way ANOVA with Dunnett's multiple comparison test. Data information: Scale bars, 10 µm.


G . Representative images of Sec24C localization at SGs ( stained for G3BP ) in TRAPPC2 - KO cells treated with SA . Data information : Scale bars , 10 µm .

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

G. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC2-KO cells treated with SA. Data information: Scale bars, 10 µm.


A . TRAPP and COPII associate more stably with SGs than with ERES . The membrane association of Sec24C was evaluated in non - permeabilized or permeabilized cells with or without SA treatment , as indicated . G3BP was used as an SG marker . Left panel insets , G3BP staining in non - permeabilized cells . Dashed white lines show the outline of permeabilized SA - untreated cells . Blue , DAPI . Data information ; Scale bars , 10 µm .

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

A. TRAPP and COPII associate more stably with SGs than with ERES. The membrane association of Sec24C was evaluated in non-permeabilized or permeabilized cells with or without SA treatment, as indicated. G3BP was used as an SG marker. Left panel insets, G3BP staining in non-permeabilized cells. Dashed white lines show the outline of permeabilized SA-untreated cells. Blue, DAPI. Data information; Scale bars, 10 µm.


B . Stabilizing TRAPP and COPII at the ERES prevents their relocalization to SGs . HeLa cells overexpressing GFP - Sar1H79G were treated with SA and immunostained for Sec24C , TRAPPC2 , and eIF3 , as indicated . Blue , DAPI . Graphs show quantification of Sec24C or TRAPPC2 at SGs in GFP - Sar1H79G - expressing cells . Data are the ratio between Sec24C or TRAPPC2 mean intensity in SG puncta and Sec24C or TRAPPC2 mean intensity at ERES , expressed as a percentage of the non - transfected ( NT ) cells . Mean ± s . e . m . n = 60 - 70 , three independent experiments . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information ; Scale bars , 10 µm .

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

B. Stabilizing TRAPP and COPII at the ERES prevents their relocalization to SGs. HeLa cells overexpressing GFP-Sar1H79G were treated with SA and immunostained for Sec24C, TRAPPC2, and eIF3, as indicated. Blue, DAPI. Graphs show quantification of Sec24C or TRAPPC2 at SGs in GFP-Sar1H79G-expressing cells. Data are the ratio between Sec24C or TRAPPC2 mean intensity in SG puncta and Sec24C or TRAPPC2 mean intensity at ERES, expressed as a percentage of the non-transfected (NT) cells. Mean ± s.e.m. n = 60-70, three independent experiments. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.


C . Effect of Sec31A depletion on TRAPPC2 recruitment to SGs . Cells were mock - treated or KD for Sec31 , treated with SA , and then immunostained for TRAPPC2 and eIF3 as indicated . Graphs , quantification of TRAPPC2 at ERES and SGs after SA treatment . Mean ± s . e . m . , one representative experiment ; n = 60 - 80 . * * p < 0 . 001 , * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information ; Scale bars , 10 µm .

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

C. Effect of Sec31A depletion on TRAPPC2 recruitment to SGs. Cells were mock-treated or KD for Sec31, treated with SA, and then immunostained for TRAPPC2 and eIF3 as indicated. Graphs, quantification of TRAPPC2 at ERES and SGs after SA treatment. Mean ± s.e.m., one representative experiment; n=60-80. ** p<0.001, ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.


D . Effect of Sec31A depletion on Sec24C recruitment to SGs . Cells were mock - treated or KD for Sec31 and then immunostained for Sec24C and cTAGE5 ( to stain ERES , top panels ) and for eIF3 ( bottom panels ) . Insets show eIF3 . Graphs , quantification of Sec24C at ERES and in SGs after SA treatment . n = 80 . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information ; Scale bars , 10 µm .

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

D. Effect of Sec31A depletion on Sec24C recruitment to SGs. Cells were mock-treated or KD for Sec31 and then immunostained for Sec24C and cTAGE5 (to stain ERES, top panels) and for eIF3 (bottom panels). Insets show eIF3. Graphs, quantification of Sec24C at ERES and in SGs after SA treatment. n=80. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.


A . Cells were treated with 273 kinase inhibitors ( 10 µM ) from the Sellekchem library and with SA , and Sec24C localization in G3BP puncta ( % of total G3BP area ) was analyzed by automated microscopy under High content screening and is reported as a percentage of the control ( cells treated with SA alone ) . Gray dashed line , mean of control ; gray box , control standard deviation ( ± 18 . 2 % ) ; red line , threshold of positive hits ; red box : positive hits . B . Enrichment Analysis of the positive hits ; for each class of inhibitor , the enrichment ( in percentage ) of compounds falling into positive hits and the enrichment ( in percentage ) in the total number of compounds was calculated and expressed as a ratio .

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

A. Cells were treated with 273 kinase inhibitors (10 µM) from the Sellekchem library and with SA, and Sec24C localization in G3BP puncta (% of total G3BP area) was analyzed by automated microscopy under High content screening and is reported as a percentage of the control (cells treated with SA alone). Gray dashed line, mean of control; gray box, control standard deviation (± 18.2%); red line, threshold of positive hits; red box: positive hits. B. Enrichment Analysis of the positive hits; for each class of inhibitor, the enrichment (in percentage) of compounds falling into positive hits and the enrichment (in percentage) in the total number of compounds was calculated and expressed as a ratio.


C . Evaluation of CDK kinase activity upon SA treatment . Western blot analysis of phosphoSer - CDK substrates in control , SA ( 300µM , 30 min ) , Dinaciclib ( 10 µM , 180 min ) and SA + Dinaciclib ( 150 min Dinaciclib and 30 min SA ) . CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition . Left , Western blot , right : Ponceau was used as loading control . Image , one representative experiment out of 3 independent replicates .

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

C. Evaluation of CDK kinase activity upon SA treatment. Western blot analysis of phosphoSer-CDK substrates in control, SA (300µM, 30 min), Dinaciclib (10 µM, 180 min) and SA+Dinaciclib (150 min Dinaciclib and 30 min SA). CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition. Left, Western blot, right: Ponceau was used as loading control. Image, one representative experiment out of 3 independent replicates.


D . Drug specificity from high ( + + + + ) to low ( + ) of the different CDKs as described by Selleckchem , and Sec24C recruitment to SGs as a percentage of control .

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

D. Drug specificity from high (++++) to low (+) of the different CDKs as described by Selleckchem, and Sec24C recruitment to SGs as a percentage of control.


E . Analysis of Sec24C recruitment to SGs in control and CDK1 + 2 - KD HeLa cells . Different levels of CDK1 + 2 KD were achieved by transfecting cells with different amounts of siRNA for different times ( 2 nM 24 h ; 5 nM 24 h ; 5 nM 48 h ; 20 nM 48 h ) to give 50 , 30 , 20 and 10 % , respectively , of the CDK1 + 2 levels in the control . CDK1 + 2 levels measured by qRT - PCR and Sec24C recruitment to SGs were evaluated from parallel cultures . Sec24C recruitment to SGs was performed by automated microscopy as described in ( A ) and is expressed as a percentage of mock ( CTRL , set at 100 % ) ± s . d . N = 8 .

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

E. Analysis of Sec24C recruitment to SGs in control and CDK1+2-KD HeLa cells. Different levels of CDK1+2 KD were achieved by transfecting cells with different amounts of siRNA for different times (2 nM 24 h; 5 nM 24 h; 5 nM 48 h; 20 nM 48 h) to give 50, 30, 20 and 10%, respectively, of the CDK1+2 levels in the control. CDK1+2 levels measured by qRT-PCR and Sec24C recruitment to SGs were evaluated from parallel cultures. Sec24C recruitment to SGs was performed by automated microscopy as described in (A) and is expressed as a percentage of mock (CTRL, set at 100%) ± s.d. N=8.


F . Representative images of HeLa cells treated with the three best hits ( Flavopiridol , SNS - 032 , Dinaciclib ; 1µM 150 min ) and then exposed to SA ( 300 µM , 30 min ) , followed by immunostaining for Sec24C and G3BP . Scale bar , 10 µm .

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

F. Representative images of HeLa cells treated with the three best hits (Flavopiridol, SNS-032, Dinaciclib; 1µM 150 min) and then exposed to SA (300 µM, 30 min), followed by immunostaining for Sec24C and G3BP. Scale bar, 10 µm.


G . HeLa cells were treated with Flavopiridol , SNS - 032 , or Dinaciclib for 150 min at the indicated concentrations , subsequently treated with SA ( 300 µM , 30 min ) , and then immunostained for Sec24C and G3BP and imaged by OPERA . BS - 181 , a specific CDK7 inhibitor , was used as negative control . Sec24C localization to SGs is expressed as a percentage of the control ( cells treated with SA alone ) . Dashed red line , mean of control ; yellow box , standard deviation of control ( ± 9 . 8 % ) , N = 12 .

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

G. HeLa cells were treated with Flavopiridol, SNS-032, or Dinaciclib for 150 min at the indicated concentrations, subsequently treated with SA (300 µM, 30 min), and then immunostained for Sec24C and G3BP and imaged by OPERA. BS-181, a specific CDK7 inhibitor, was used as negative control. Sec24C localization to SGs is expressed as a percentage of the control (cells treated with SA alone). Dashed red line, mean of control; yellow box, standard deviation of control (± 9.8%), N=12.


H . Cells were treated as in ( G ) and immunostained for TRAPPC2 and eIF3 . Scale bar , 10 µm . The graph shows the quantification of TRAPPC2 localization in SG puncta ( mean intensity ) . Data are mean ± s . e . m . expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol , SNS - 032 or Dinaciclib treatment compared to the control ( cells treated with SA alone ) . N = 3 , three independent experiments , n = 60 - 80 cells per experiment . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

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

H. Cells were treated as in (G) and immunostained for TRAPPC2 and eIF3. Scale bar, 10 µm. The graph shows the quantification of TRAPPC2 localization in SG puncta (mean intensity). Data are mean ± s.e.m. expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol, SNS-032 or Dinaciclib treatment compared to the control (cells treated with SA alone). N=3, three independent experiments, n=60-80 cells per experiment. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


A . Immunofluorescence images of Sec24C and TRAPPC2 in vehicle - treated and SNS - 032 - treated ( 1 µM , 3 h ) cells . Graphs show the quantification of Sec24C and TRAPPC2 at ERES ( mean intensity ) normalized in SNS - 032 - treated cells relative to vehicle - treated cells ( set as 100 % ) . Mean ± s . e . m . of three independent experiments . * * p < 0 . 05 , * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information : Scale bars , 10 µm .

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

A. Immunofluorescence images of Sec24C and TRAPPC2 in vehicle-treated and SNS-032-treated (1 µM, 3 h) cells. Graphs show the quantification of Sec24C and TRAPPC2 at ERES (mean intensity) normalized in SNS-032-treated cells relative to vehicle-treated cells (set as 100%). Mean ± s.e.m. of three independent experiments. **p<0.05, ****p<0.0001, Student's unpaired two-tailed t-test. Data information: Scale bars, 10 µm.


B . HeLa cells were treated with Dinaciclib ( 1 µM , 3 h ) and permeabilized or not with digitonin A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency . Upper panels , non - permeabilized ( NP ) control cells ; middle panels , permeabilized control cells ; lower panels , permeabilized Dinaciclib - treated cells . Data information : Scale bars , 10 µm .

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

B. HeLa cells were treated with Dinaciclib (1 µM, 3 h) and permeabilized or not with digitonin A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency. Upper panels, non-permeabilized (NP) control cells; middle panels, permeabilized control cells; lower panels, permeabilized Dinaciclib-treated cells. Data information: Scale bars, 10 µm.


C . Quantification of Sec24C membrane association after CDK inhibitor treatment . Digitonin - permeabilized cells treated with vehicle ( CTRL ) or the indicated CDK inhibitor ( 1 µM , 3 h ) were immunostained for Sec24C . The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug - treated cells is expressed as fold change compared to the control ; n = 60 - 80 ; mean ± s . e . m . of three independent experiments * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

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

C. Quantification of Sec24C membrane association after CDK inhibitor treatment. Digitonin-permeabilized cells treated with vehicle (CTRL) or the indicated CDK inhibitor (1 µM, 3 h) were immunostained for Sec24C. The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug-treated cells is expressed as fold change compared to the control; n= 60-80; mean ± s.e.m. of three independent experiments ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


D . HeLa cells transfected with HA - hnRNPK were left untreated ( Steady state ) or treated with SA ( 300µM , 30 min ) or SA + Dinaciclib ( 10 µM , 150 min ) and then immunostained with anti - HA and anti - G3BP antibodies . Data information Scale bars , 10 µm .

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

D. HeLa cells transfected with HA-hnRNPK were left untreated (Steady state) or treated with SA (300µM, 30 min) or SA+Dinaciclib (10 µM, 150 min) and then immunostained with anti-HA and anti-G3BP antibodies. Data information Scale bars, 10 µm.


E . TRAPPC2 interacts with hnRNPK . ( Top ) SDS - PAGE and Western blot analysis of endogenous hnRNPK after immunoprecipitation with an anti - TRAPPC2 Ab or IgG , and ( bottom ) after immunoprecipitation with an anti - HA Ab from cells transfected ( + ) or not ( - ) with hnRNPK - HA detected with the indicated antibodies .

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

E. TRAPPC2 interacts with hnRNPK. (Top) SDS-PAGE and Western blot analysis of endogenous hnRNPK after immunoprecipitation with an anti-TRAPPC2 Ab or IgG, and (bottom) after immunoprecipitation with an anti-HA Ab from cells transfected (+) or not (-) with hnRNPK-HA detected with the indicated antibodies.


F . Mock or hnRNPK KD cells treated with SA were immunostained for TRAPPC2 and eIF3 . Data information : Scale bars , 10 µm .

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

F. Mock or hnRNPK KD cells treated with SA were immunostained for TRAPPC2 and eIF3. Data information: Scale bars, 10 µm.


G . Mock or hnRNPK KD cells treated with SA were immunostained for Sec24C and G3BP . Data information : Scale bars , 10 µm .

G. Mock or hnRNPK KD cells treated with SA were immunostained for Sec24C and G3BP. Data information: Scale bars, 10 µm.


H . Graphs show quantification of TRAPPC2 and Sec24C redistribution to SGs in mock treated ( CTRL ) and hnRNPK KD cells . n > 100 , N = 3 mean ± s . d . * * * p < 0 . 001 . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test .

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

H. Graphs show quantification of TRAPPC2 and Sec24C redistribution to SGs in mock treated (CTRL) and hnRNPK KD cells. n>100, N=3 mean ± s.d. ***p<0.001.****p<0.0001, Student's unpaired two-tailed t-test.


A . HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA ( 30 min , 300 µM ) , and immunostained for Sec24C or TRAPPC2 and eIF3 . Scale bar 10 µm .

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

A. HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA (30 min, 300 µM), and immunostained for Sec24C or TRAPPC2 and eIF3. Scale bar 10 µm.


B . Analysis of the proliferation status of the cells after 8 h starvation in HBSS . Top , representative images of starved and non - starved ( CTRL ) cells using EdU incorporation ( see Materials and Methods ) . Scale bar 100 µm . Bottom , quantification of EdU incorporation in starved cells as a percentage of incorporation in control fed cells , N = 3 , mean ± s . d .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of the proliferation status of the cells after 8 h starvation in HBSS. Top, representative images of starved and non-starved (CTRL) cells using EdU incorporation (see Materials and Methods). Scale bar 100 µm. Bottom, quantification of EdU incorporation in starved cells as a percentage of incorporation in control fed cells, N=3, mean ± s.d.


C . HeLa cells were seeded at different confluency , treated with SA and stained for Sec24C and G3BP as an SG marker . Scale bar , 10 µm . Flow cytometry ( FACS ) analysis ( right panels ) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency . The graph shows quantification of Sec24C mean intensity in SG puncta ( normalized for the cytosolic Sec24C ) at the indicated cell confluency . Mean ± s . e . m . of a representative experiment out of 5 biological replicates . n = 50 - 80 . ns : not significant ; * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

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

C. HeLa cells were seeded at different confluency, treated with SA and stained for Sec24C and G3BP as an SG marker. Scale bar, 10 µm. Flow cytometry (FACS) analysis (right panels) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency. The graph shows quantification of Sec24C mean intensity in SG puncta (normalized for the cytosolic Sec24C) at the indicated cell confluency. Mean ± s.e.m. of a representative experiment out of 5 biological replicates. n= 50-80. ns: not significant; ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


D . Differentiation of podocytes . Western blot of growing and differentiated podocytes . Synaptopodin ( SYNPO ) was used as a differentiation marker , GAPDH as a loading control .

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

D. Differentiation of podocytes. Western blot of growing and differentiated podocytes. Synaptopodin (SYNPO) was used as a differentiation marker, GAPDH as a loading control.


E . Growing and differentiated podocytes were treated with SA ( 300 µM , 30 min ) and stained for TRAPPC2 or Sec24C and eIF3 . Scale bar , 10 µm . The graphs show quantification of TRAPPC2 and Sec24C ( mean intensity ) in SGs . Mean ± s . e . m . of three independents experiments . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test .

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

E. Growing and differentiated podocytes were treated with SA (300 µM, 30 min) and stained for TRAPPC2 or Sec24C and eIF3. Scale bar, 10 µm. The graphs show quantification of TRAPPC2 and Sec24C (mean intensity) in SGs. Mean ± s.e.m. of three independents experiments. ****p<0.0001, Student's unpaired two-tailed t-test.


F . Western blot analysis of total ( RB ) and phosphorylated ( p - RB ) retinoblastoma in growing ( non - differentiated ) versus differentiated podocytes . ß - actin was used as loading control .

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

F. Western blot analysis of total (RB) and phosphorylated (p-RB) retinoblastoma in growing (non-differentiated) versus differentiated podocytes. ß-actin was used as loading control.


G . CDK kinase activity in growing and differentiated podocytes . Western blot using a specific antibody recognizing phosphoSer - CDK substrates was used on total cell lysates .

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

G. CDK kinase activity in growing and differentiated podocytes. Western blot using a specific antibody recognizing phosphoSer-CDK substrates was used on total cell lysates.


A . SG area in mock , TRAPPC2 TRAPPC3 , TRAPPC8 , and TRAPPC9 - depleted cells treated with SA . Data are shown in box - and - whisker plots : Box - plot central line , median ; box limits , upper and lower quartiles ; whiskers show the minimum to maximum . N = 3 . * * * p < 0 . 001 , * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

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

A. SG area in mock, TRAPPC2 TRAPPC3, TRAPPC8, and TRAPPC9-depleted cells treated with SA. Data are shown in box-and-whisker plots: Box-plot central line, median; box limits, upper and lower quartiles; whiskers show the minimum to maximum. N=3. ***p<0.001, ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


B . HeLa cells microinjected with control IgG or a TRAPPC3 - specific antibody ( right panels in green ) were treated with SA . The TRAPPC3 Ab disrupts the Golgi ( Yu et al , 2006 ) , monitored using an anti - TGN46 Ab . Anti - G3BP was used to stain SGs . The graph shows quantification of the SG area . Mean ± s . e . m . three independent experiments ; n > 80 . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Scale bar , 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HeLa cells microinjected with control IgG or a TRAPPC3-specific antibody (right panels in green) were treated with SA. The TRAPPC3 Ab disrupts the Golgi (Yu et al, 2006), monitored using an anti-TGN46 Ab. Anti-G3BP was used to stain SGs. The graph shows quantification of the SG area. Mean ± s.e.m. three independent experiments; n>80. ****p<0.0001, Student's unpaired two-tailed t-test. Scale bar, 10 µm.


C . Structured Illumination Microscopy ( SIM ) - Super resolution ( SR ) images of endogenous TRAPPC2 and GFP - Sec23 localizing at SGs , stained for G3BP . Scale bar 5 µm . Right , magnification of boxed area . Scale bar , 0 . 5 µm .

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

C. Structured Illumination Microscopy (SIM)-Super resolution (SR) images of endogenous TRAPPC2 and GFP-Sec23 localizing at SGs, stained for G3BP. Scale bar 5 µm. Right, magnification of boxed area. Scale bar, 0.5 µm.


Localization of Raptor ( D ) in mock , TRAPPC3 - KD and TRAPPC2 - KD HeLa cells treated with SA . G3BP was used to stain SGs . Scale bar , 10 µm . Each graph shows the quantification ( mean intensity ) of the respective protein in SG spots expressed as a percentage of the mock ( TRL ) . Mean ± S . D . three independent replicates . * p < 0 . 02 ; * * p < 0 . 009 in ( D ) , One - way ANOVA with Dunnett ' s multiple comparison test .

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

Localization of Raptor (D) in mock, TRAPPC3-KD and TRAPPC2-KD HeLa cells treated with SA. G3BP was used to stain SGs. Scale bar, 10 µm. Each graph shows the quantification (mean intensity) of the respective protein in SG spots expressed as a percentage of the mock (TRL). Mean ± S.D. three independent replicates. *p<0.02; **p<0.009 in (D) , One-way ANOVA with Dunnett's multiple comparison test.


Localization of RACK1 ( E ) in mock , TRAPPC3 - KD and TRAPPC2 - KD HeLa cells treated with SA . G3BP was used to stain SGs . Scale bar , 10 µm . Each graph shows the quantification ( mean intensity ) of the respective protein in SG spots expressed as a percentage of the mock ( TRL ) . Mean ± S . D . independent replicates . , * p < 0 . 05 ; * * * * p < 0 . 0001 in ( E ) , One - way ANOVA with Dunnett ' s multiple comparison test .

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

Localization of RACK1 (E) in mock, TRAPPC3-KD and TRAPPC2-KD HeLa cells treated with SA. G3BP was used to stain SGs. Scale bar, 10 µm. Each graph shows the quantification (mean intensity) of the respective protein in SG spots expressed as a percentage of the mock (TRL). Mean ± S.D. independent replicates. , *p<0.05; ****p<0.0001 in (E), One-way ANOVA with Dunnett's multiple comparison test.


Immunoprecipitation ( IP ) with an anti - TRAPPC2 Ab or IgG from cells with or without SA treatment , as indicated , followed by SDS - PAGE and Western blot with ant - Raptor ( F ) Abs . Input : cell lysate ( 40 µg for TRAPPC2 , 10 µg for Raptor ) .

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

Immunoprecipitation (IP) with an anti-TRAPPC2 Ab or IgG from cells with or without SA treatment, as indicated, followed by SDS-PAGE and Western blot with ant-Raptor (F) Abs. Input: cell lysate (40 µg for TRAPPC2, 10 µg for Raptor).


Immunoprecipitation ( IP ) with an anti - TRAPPC2 Ab or IgG from cells with or without SA treatment , as indicated , followed by SDS - PAGE and Western blot with anti - RACK1 ( G ) Abs . Input : cell lysate ( 40 µg for TRAPPC2 , 10 µg for

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

Immunoprecipitation (IP) with an anti-TRAPPC2 Ab or IgG from cells with or without SA treatment, as indicated, followed by SDS-PAGE and Western blot with anti-RACK1 (G) Abs. Input: cell lysate (40 µg for TRAPPC2, 10 µg for RACK1


Localization of Raptor ( H ) untreated cells or cells pre - treated with the indicated CDK inhibitor ( 1 µM , 150 min ) and then with SA ( 300 µM , 30 min ) . G3BP was used to stain SGs . Scale bar , 10 µm . Graphs show quantification of the localization of the respective protein with SGs , expressed as a percentage of the control . Mean ± s . e . m . of one representative experiment out of three independent replicates , n = 60 - 80 . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

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

Localization of Raptor (H) untreated cells or cells pre-treated with the indicated CDK inhibitor (1 µM, 150 min) and then with SA (300 µM, 30 min). G3BP was used to stain SGs. Scale bar, 10 µm. Graphs show quantification of the localization of the respective protein with SGs, expressed as a percentage of the control. Mean ± s.e.m. of one representative experiment out of three independent replicates, n=60-80. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


Localization of RACK1 ( I ) in untreated cells or cells pre - treated with the indicated CDK inhibitor ( 1 µM , 150 min ) and then with SA ( 300 µM , 30 min ) . G3BP was used to stain SGs . Scale bar , 10 µm . Graphs show quantification of the localization of the respective protein with SGs , expressed as a percentage of the control . Mean ± s . e . m . of one representative experiment out of three independent replicates , n = 60 - 80 . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

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

Localization of RACK1 (I) in untreated cells or cells pre-treated with the indicated CDK inhibitor (1 µM, 150 min) and then with SA (300 µM, 30 min). G3BP was used to stain SGs. Scale bar, 10 µm. Graphs show quantification of the localization of the respective protein with SGs, expressed as a percentage of the control. Mean ± s.e.m. of one representative experiment out of three independent replicates, n=60-80. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.