Figure 3 ( A , B ) Representative fluorescence images ( A ) and corresponding kymographs ( B ) from time - lapse videos of control neurons transfected with HA - BACE1 - eGFP and co - transfected with AP - 2μ - mCherry . Scale bars : ( A ) 2μm , 1 . 6µm inserts , ( B ) x = 2μm , y = 5s . Arrowheads in ( A ) correspond to BACE1 carriers indicated by the same arrowheads in ( B ) . ( C ) Representative fluorescence images and corresponding kymographs from time - lapse videos of WT and AP - 2μ KO neurons transfected with HA - BACE1 - eGFP . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels . ( D ) Loss of AP - 2μ significantly decreases the BACE1 motility ( WT : 37 . 42 ± 3 . 79 % , KO : 23 . 07 ± 3 . 07 % , p = 0 . 003 , WT = 31 , KO = 32 neurons , 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 ) Loss of AP - 2μ significantly decreases the anterograde and retrograde BACE1 velocity ( WTAntero : 0 . 37 ± 0 . 02 μm / s , KOAntero : 0 . 22 ± 0 . 03 μm / s , pWTAntero vs pKOAntero < 0 . 000 ; WTRetro : 0 . 35 ± 0 . 02 μm / s , KORetro : 0 . 25 ± . 03 μm / s , pWTRetro vs pKORetro = 0 . 006 , WT = 35 and KO = 32 neurons , 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 . ( F ) Representative fluorescence images and corresponding kymographs from time - lapse videos of WT and AP - 2μ KO neurons co - transfected with HA - BACE1 - GFP and AP - 2μ - mRFP . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels . ( G ) Bi - directional mobility of BACE1 carriers in WT and AP - 2μ ΚΟ neurons , co - expressing the AP - 2μ - mRFP ( WT : 37 . 60 ± 2 . 20 % , KO : 25 . 61 ± 2 . 67 % , WTAP - 2μ - mRFP : 45 . 04 ± 3 . 27 % , KOAP - 2μ - mRFP : 40 . 07 ± 2 . 37 % , pWT vs KO = 0 . 005 , pKO vs KO + AP - 2μ - mRFP < 0 . 000 , pWT vs WT + AP - 2μ - mRFP = 0 . 186 , pWT + AP - 2μ - mRFP vs KO + AP - 2μ - mRFP = 0 . 570 , 43 - 53 neurons per condition , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA in ( G ) . n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( H ) Representative kymographs of WT and AP - 2μ KO neurons co - expressing the HA - BACE1 - GFP and tagRFP - LC3B . Scale bars : x = 2μm , y = 5s . ( I ) Reduced bi - directional mobility of BACE1 - containing autophagosomes in AP - 2µ KO neurons . ( WT : 33 . 53 ± 4 . 19 % , KO : 19 . 45 ± 5 . 14 % , p = 0 . 000 , WT = 37 and KO = 35 neurons , 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 . ( J ) Representative kymographs of WT neurons co - expressing HA - BACE1 - GFP and AP2 - αΑ WT or AP2 - αΑ Μut . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels . ( K ) Mobility of BACE1 carriers is significantly decreased in neurons overexpressing the AP2 - αΑ Mut compared to neurons expressing the AP2 - αΑ WT ( WT : 52 . 23 ± 4 . 16 % , Mut : 27 . 95 ± 3 . 31 , p = < 0 . 000 , WT = 26 and Mut = 29 neurons , 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 . ( L ) Representative fluorescence images and corresponding kymographs from time - lapse videos of WT neurons transfected with either HA - BACE1 - GFP or HA - BACE1 - LL / AA - GFP . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels . ( M ) Reduced mobility of BACE1 - LL / AA - positive vesicles , when compared to WT BACE1 ( WT : 38 . 97 ± 1 . 40 % , LL / AA : 19 . 89 ± 1 . 77 % , p < 0 . 000 , WT = 31 and LL / AA = 33 neurons , 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 . ( N ) Reduced velocity of BACE1 - LL / AA - positive vesicles , when compared to WT BACE1 ( WTAntero : 0 . 38 ± 0 . 03 μm / s , LL / AAAntero : 0 . 15 ± 0 . 03 μm / s , pWTAntero vs pLL / AAAntero < 0 . 000 ; WTRetro : 0 . 39 ± 0 . 03 μm / s , LL / AARetro : 0 . 25 ± . 03 μm / s , pWTRetro vs pLL / AARetro = 0 . 000 , WT = 31 and LL / AA = 33 neurons , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A, B) Representative fluorescence images (A) and corresponding kymographs (B) from time-lapse videos of control neurons transfected with HA-BACE1-eGFP and co-transfected with AP-2μ-mCherry. Scale bars: (A) 2μm, 1.6µm inserts, (B) x=2μm, y=5s. Arrowheads in (A) correspond to BACE1 carriers indicated by the same arrowheads in (B).(C) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT and AP-2μ KO neurons transfected with HA-BACE1-eGFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.(D) Loss of AP-2μ significantly decreases the BACE1 motility (WT: 37.42±3.79%, KO: 23.07±3.07%, p=0.003, WT=31, KO=32 neurons, 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) Loss of AP-2μ significantly decreases the anterograde and retrograde BACE1 velocity (WTAntero: 0.37±0.02 μm/s, KOAntero: 0.22±0.03 μm/s, pWTAntero vs pKOAntero<0.000; WTRetro: 0.35±0.02 μm/s, KORetro: 0.25±.03 μm/s, pWTRetro vs pKORetro =0.006, WT=35 and KO=32 neurons, 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.(F) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT and AP-2μ KO neurons co-transfected with HA-BACE1-GFP and AP-2μ-mRFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.(G) Bi-directional mobility of BACE1 carriers in WT and AP-2μ ΚΟ neurons, co-expressing the AP-2μ-mRFP (WT: 37.60±2.20%, KO: 25.61±2.67%, WTAP-2μ-mRFP: 45.04±3.27%, KOAP-2μ-mRFP: 40.07±2.37%, pWT vs KO=0.005, pKO vs KO+AP-2μ-mRFP<0.000, pWT vs WT+AP-2μ-mRFP=0.186, pWT+AP-2μ-mRFP vs KO+AP-2μ-mRFP=0.570, 43-53 neurons per condition, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in (G). n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(H) Representative kymographs of WT and AP-2μ KO neurons co-expressing the HA-BACE1-GFP and tagRFP-LC3B. Scale bars: x=2μm, y=5s.(I) Reduced bi-directional mobility of BACE1-containing autophagosomes in AP-2µ KO neurons. (WT: 33.53±4.19%, KO: 19.45±5.14%, p=0.000, WT=37 and KO=35 neurons, 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.(J) Representative kymographs of WT neurons co-expressing HA-BACE1-GFP and AP2-αΑ WT or AP2-αΑ Μut. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.(K) Mobility of BACE1 carriers is significantly decreased in neurons overexpressing the AP2-αΑ Mut compared to neurons expressing the AP2-αΑ WT (WT: 52.23±4.16%, Mut: 27.95±3.31, p=<0.000, WT=26 and Mut=29 neurons, 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.(L) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT neurons transfected with either HA-BACE1-GFP or HA-BACE1-LL/AA-GFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.(M) Reduced mobility of BACE1-LL/AA-positive vesicles, when compared to WT BACE1 (WT: 38.97±1.40%, LL/AA: 19.89±1.77%, p<0.000, WT=31 and LL/AA=33 neurons, 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.(N) Reduced velocity of BACE1-LL/AA-positive vesicles, when compared to WT BACE1 (WTAntero: 0.38±0.03 μm/s, LL/AAAntero: 0.15±0.03 μm/s, pWTAntero vs pLL/AAAntero<0.000; WTRetro: 0.39±0.03 μm/s, LL/AARetro: 0.25±.03 μm/s, pWTRetro vs pLL/AARetro =0.000, WT=31 and LL/AA=33 neurons, 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.


Figure 4 ( A , B ) The eGFP / mCherry signal intensity ratio is significantly increased in mCherry - APP - P1 - eGFP - expressing KO neurons comparing to the WT ( WT : 0 . 94 ± 0 . 06 % , KO : 1 . 65 ± 0 . 18 , p = 0 . 000 , WT = 39 and KO = 41 neurons , N = 5 biological replicates ) . Scale bars : 2μm . 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 . Confocal images of WT and AP - 2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP - CTFs . Scale bars : 100 µm ( C ) Confocal images of WT and AP - 2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP - CTFs . Scale bars : 20µm ( D ) . ( E - G ) Levels of APP - CTFs are significantly increased in AP - 2µ KO cortex compared to the WT set to 100 % ( KOC99 : 156 . 65 ± 8 . 37 % , p = 0 . 004 , N = 4 biological replicates ; KOC83 : 154 . 79 ± 15 . 6 % , p = 0 . 012 ; N = 5 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( H ) Levels of full - length ( FL ) APP are unaltered in AP - 2µ KO cortex ( KOAPP FL : 106 . 64 ± 12 . 96 % , p = 0 . 315 , N = 6 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( I ) Levels of Nicastrin ( NCT ) are unaltered in the AP - 2µ KO cortex ( J , K ) Intracellular levels of Aβ1 - 40 are significantly increased in AP - 2µ KO axons compared to the WT ( WTAβ1 - 40 : 1 . 82 ± 0 . 12 , KOAβ1 - 40 : 2 . 45 ± 0 . 12 , p = 0 . 000 , WT = 24 and KO = 24 neurons , N = 3 biological replicates ) . Scale bars , 5µm . 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 . Intracellular levels of Aβ1 - 42 are significantly increased in AP - 2µ KO axons ( WTAβ1 - 42 : 0 . 65 ± 0 . 08 , KOAβ1 - 42 : 0 . 91 ± 0 . 10 , p = 0 . 046 , WT = 38 and KO = 38 neurons , N = 3 biological replicates ) . Scale bar : 5 µm . Intracellular levels of Aβ1 - 42 are significantly increased in AP - 2µ KO axons ( WTAβ1 - 42 : 0 . 65 ± 0 . 08 , KOAβ1 - 42 : 0 . 91 ± 0 . 10 , p = 0 . 046 , WT = 38 and KO = 38 neurons , N = 3 biological replicates ) . Scale bar : 5 µm . 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 . ( N ) Significantly increased ratio of Αβ1 - 42 / Αβ1 - 40 measured by ELISA in the media of cultured AP - 2μ KO neurons compared to the WT set to 100 % ( KO : 124 . 57 ± 10 . 97 % , p = 0 . 037 , N = 6 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 . Accumulation of intracellular Aβ1 − 42 in neurons expressing the AP2 - α − WT ( WT : 1 . 14 ± 0 . 18 , WT = 28 and Mut = 33 neurons , N = 4 biological replicates ) . Scale bars : 20µm , 2µm ( inserts ) . Accumulation of intracellular Aβ1 − 42 in neurons overexpressing the AP2 - α - Mut comparing to neurons expressing the AP2 - α − WT ( WT : 1 . 14 ± 0 . 18 , Mut : 2 . 06 ± 0 . 29 , p = 0 . 011 , WT = 28 and Mut = 33 neurons , N = 4 biological replicates ) . Scale bars : 20µm , 2µm ( inserts ) . 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 . Accumulation of intracellular Aβ1 - 42 is rescued upon BACE1 knock - down ( WTScr : 2 . 76 ± 0 . 20 WTshBACE1 : 2 . 52 ± 0 . 12 27 to 39 neurons per condition , N = 3 biological replicates ) . Scale bar : 10µm . Accumulation of intracellular Aβ1 - 42 is rescued upon BACE1 knock - down in AP - 2µ KO neurons ( WTScr : 2 . 76 ± 0 . 20 , KOScr : 3 . 94 ± 0 . 21 ; WTshBACE1 : 2 . 52 ± 0 . 12 ; KOshBACE1 : 2 . 74 ± 0 . 21 ; pWTScr vs pKOScr = 0 . 000 , pKOscr vs pKOshBACE1 = 000 ; pWTscr vs pWTshBACE1 = 0 . 826 ; pWTshBACE1 vs pKOshBACE1 = 0 . 863 ; 27 to 39 neurons per condition , N = 3 biological replicates ) . Scale bar : 10µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA in ( T ) n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( U , V ) Levels of intracellular Aβ1 − 42 are significantly upregulated in neurons overexpressing the HA - BACE1 - LL / AA - GFP comparing to neurons expressing the HA - BACE1 - GFP ( BACE1 : 0 . 78 ± 0 . 90 , BACE1 - LL - AA : 1 . 99 ± 0 . 33 , p = 000 , 28 - 29 neurons per condition , N = 3 biological replicates ) . Scale bar , 50µm . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A, B) The eGFP/mCherry signal intensity ratio is significantly increased in mCherry-APP-P1-eGFP-expressing KO neurons comparing to the WT (WT: 0.94±0.06%, KO: 1.65±0.18, p=0.000, WT=39 and KO=41 neurons, N=5 biological replicates). Scale bars: 2μm. 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.Confocal images of WT and AP-2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP-CTFs. Scale bars: 100 µm (C)Confocal images of WT and AP-2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP-CTFs. Scale bars: 20µm (D).(E-G) Levels of APP-CTFs are significantly increased in AP-2µ KO cortex compared to the WT set to 100% (KOC99: 156.65±8.37%, p=0.004, N=4 biological replicates; KOC83: 154.79±15.6%, p=0.012; N=5 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(H) Levels of full-length (FL) APP are unaltered in AP-2µ KO cortex (KOAPP FL: 106.64±12.96%, p=0.315, N=6 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(I) Levels of Nicastrin (NCT) are unaltered in the AP-2µ KO cortex(J, K) Intracellular levels of Aβ1-40 are significantly increased in AP-2µ KO axons compared to the WT (WTAβ1-40: 1.82±0.12, KOAβ1-40: 2.45±0.12, p=0.000, WT=24 and KO=24 neurons, N=3 biological replicates). Scale bars, 5µm. 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.Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 µm.Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 µm. 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.(N) Significantly increased ratio of Αβ1-42/ Αβ1-40 measured by ELISA in the media of cultured AP-2μ KO neurons compared to the WT set to 100% (KO:124.57±10.97%, p=0.037, N=6 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.Accumulation of intracellular Aβ1−42 in neurons expressing the AP2-α−WT (WT: 1.14±0.18 , WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts).Accumulation of intracellular Aβ1−42 in neurons overexpressing the AP2-α-Mut comparing to neurons expressing the AP2-α−WT (WT: 1.14±0.18, Mut: 2.06±0.29, p=0.011, WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts). 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.Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down (WTScr: 2.76±0.20 WTshBACE1: 2.52±0.12 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm.Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down in AP-2µ KO neurons (WTScr: 2.76±0.20, KOScr: 3.94±0.21; WTshBACE1: 2.52±0.12; KOshBACE1: 2.74±0.21; pWTScr vs pKOScr=0.000, pKOscr vs pKOshBACE1=000; pWTscr vs pWTshBACE1=0.826; pWTshBACE1 vs pKOshBACE1=0.863; 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in (T) n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(U, V) Levels of intracellular Aβ1−42 are significantly upregulated in neurons overexpressing the HA-BACE1-LL/AA-GFP comparing to neurons expressing the HA-BACE1-GFP (BACE1: 0.78±0.90, BACE1-LL-AA: 1.99±0.33, p=000, 28-29 neurons per condition, N=3 biological replicates). Scale bar, 50µm. 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.


Figure 5 ( A , B ) AP - 2α levels normalized to GAPDH are significantly decreased in lysates of iPSC - derived neurons from late - onset AD patients carrying the TREM2 p . R47H ( AD - TREM2 - 2 and AD - TREM2 - 4 ) variant compared to healthy controls ( CON8 and CON9 ) set to 100 % ( ADAP - 2α : 52 . 42 ± 8 . 04 % , p = 0 . 020 , N = 3 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( C ) Confocal images of either AP - 2 WT ( AAV - GFPCamkIIα - labelled ) or AP - 2 KO ( AAV - Cre - GFPCamkIIα - labelled ) granule neurons in the dentate gyrus of 13 - week - old mice . Scale bars : 20µm , 2µm ( inserts ) . ( 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 . 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 . ( 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 . 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A, B) AP-2α levels normalized to GAPDH are significantly decreased in lysates of iPSC-derived neurons from late-onset AD patients carrying the TREM2 p.R47H (AD-TREM2-2 and AD-TREM2-4) variant compared to healthy controls (CON8 and CON9) set to 100% (ADAP-2α: 52.42±8.04%, p=0.020, N=3 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(C) Confocal images of either AP-2 WT (AAV-GFPCamkIIα-labelled) or AP-2 KO (AAV-Cre-GFPCamkIIα-labelled) granule neurons in the dentate gyrus of 13-week-old mice. Scale bars: 20µm, 2µm (inserts).(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.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.(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.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.(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.


Figure 1Proteoliposomes 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Proteoliposomes 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.


Figure 2Initial 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Initial 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.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.


Figure 2 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.


Figure 3 ( 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 .

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Figure 3(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.


Figure 4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.


Figure 5 ( 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 . ( 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 .

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Figure 5(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. (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.


Figure 6 ( 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 . 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 . ( 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 .

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Figure 6(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. 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.(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.


Figure 1B : 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B: 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.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.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.


Figure 3A : 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A: 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.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.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.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).


Figure 4B : 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B: 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.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.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.


Figure 5D : ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5D: (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).


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

Figure 6A: 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.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).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.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.)


Figure 7A : 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A: 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.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.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.


Figure 8A : 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A: 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.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.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).


Figure 1A . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 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.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.


Figure 2A . 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. 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.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.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.G. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC2-KO cells treated with SA. Data information: Scale bars, 10 µm.