Figure 8 ( B ) Representative sequencing chromatograms of PIK3CA and TEK mutant VM - derived ECs . Arrows show the detected point mutations . ( C ) Representative confocal images of PIK3CA and TEK patient - derived ECs immunostained for VE - cadherin ( EC - specific junctional protein ) and ERG ( EC - specific transcription factor ) . Cell nuclei were visualised with DAPI . Scale bars : 30 μm . ( D ) Immunoblot showing the activation of PI3K / AKT / mTORC1 pathway ( by assessing the levels of p - AKT and p - S6 ) among different PIK3CA and TEK patient - derived ECs . Primary HUVEC were used as wild - type control . ( E - F ) Immunoblot showing the impact of miransertib at increasing doses on PI3K / AKT / mTORC1 pathway ( by assessing p - AKT and p - S6 levels ) in ( E ) PIK3CA and ( F ) TEK mutant patient - derived ECs . ( G ) Wild - type ( HUVECs ) , PIK3CA and TEK mutant EC viability upon the treatment with miransertib for 72h at different doses assessed by MTS assay . Fitting curves and 95 % CI IC50 values for wild - type ( HUVECs ) , PIK3CA and TEK ECs are shown . For PIK3CA and TEK - mutant ECs n = 3 biological replicates ( patients ) ; for wild - type ECs ( HUVECs ) n = 4 technical replicates PIK3CA - mutant vs . TEK - mutant p = 0 . 501 ; PIK3CA - mutant vs . HUVECs p = 0 . 0815 ; TEK - mutant vs . HUVECs : p = 0 . 4085 . Statistical analysis was performed by comparison of best - fit values using the extra sum - of - squares F test .

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

Figure 8(B) Representative sequencing chromatograms of PIK3CA and TEK mutant VM-derived ECs. Arrows show the detected point mutations.(C) Representative confocal images of PIK3CA and TEK patient-derived ECs immunostained for VE-cadherin (EC-specific junctional protein) and ERG (EC-specific transcription factor). Cell nuclei were visualised with DAPI. Scale bars: 30 μm.(D) Immunoblot showing the activation of PI3K/AKT/mTORC1 pathway (by assessing the levels of p-AKT and p-S6) among different PIK3CA and TEK patient-derived ECs. Primary HUVEC were used as wild-type control.(E-F) Immunoblot showing the impact of miransertib at increasing doses on PI3K/AKT/mTORC1 pathway (by assessing p-AKT and p-S6 levels) in (E) PIK3CA and (F) TEK mutant patient-derived ECs.(G) Wild-type (HUVECs), PIK3CA and TEK mutant EC viability upon the treatment with miransertib for 72h at different doses assessed by MTS assay. Fitting curves and 95% CI IC50 values for wild-type (HUVECs), PIK3CA and TEK ECs are shown. For PIK3CA and TEK-mutant ECs n = 3 biological replicates (patients); for wild-type ECs (HUVECs) n = 4 technical replicates PIK3CA-mutant vs. TEK-mutant p = 0.501; PIK3CA-mutant vs. HUVECs p = 0.0815; TEK-mutant vs. HUVECs: p = 0.4085. Statistical analysis was performed by comparison of best-fit values using the extra sum-of-squares F test.


Figure 1A . Experimental demonstration . A1 . Illustration of the mapping principle between ChR2 - expressed PV neurons ( green ) and regular pyramidal neuron ( Pyr , orange ) . A2 . Acute slices were obtained and recorded at ZT0 or ZT12 . A3 . Coronal brain atlas showing the primary visual cortex ( Top left panel , modified from MBL mouse brain atlas ) . Yellow box highlights the region of experiment . Top right panel , the reference image taken with a 10x objective from a slice prepared from a PV : Ai32 mouse . The 0 . 45 mm2 shaded cubic area was divided into 15 x 15 grids for independent light stimulation . The white arrowhead indicates the position of the recording electrode . Bottom panel , representative raw traces of light - evoked IPSCs ( LEIPSCs ) recorded from one pyramidal neuron . A4 . The spatial distribution of the LEIPSCs from the example in A3 . A5 . The heatmap of the LEIPSCs in A4 . B . The average heatmaps at ZT0 and ZT12 after soma alignment . Soma position was indicated by the blue arrow head . C . Comparison of the cumulative distribution ( Kolmogorov - Smirnov test [ K - S test ] ; P = 0 . 002 ) and the mean ( Inset . ZT0 : 813 . 3 ± 53 . 13 pA ; ZT12 : 1306 ± 93 . 73 pA ; P < 0 . 0001 ; Mann - Whitney U test ) of the max LEIPSC amplitude between ZT0 and ZT12 . D . Comparison of the cumulative distribution ( K - S test , P = 0 . 0016 ) and the mean ( Inset . ZT0 : 10 . 44 ± 1 . 44 nA ; ZT12 : 18 . 87 ± 1 . 44 nA ; P < 0 . 0001 ; Mann - Whitney U test ) of the sum LEIPSC amplitude between ZT0 and ZT12 . Data information : Sample sizes used in this figure : ZT0 : 48 cells from 9 mice ; ZT12 : 51 cells from 9 mice . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . E . The average heatmaps of LEIPSCs after pia aligned to the reference image in A3 . Dark and light blue overlay traces on the side of the map depicted the marginal distribution of the sum LEIPSCs for ZT0 ( Dark blue ) and ZT12 ( Light blue ) . F . Comparison of the cumulative distribution ( K - S test , P = 0 . 0021 ) and the mean ( Inset , ZT0 : 0 . 067 ± 0 . 004 mm2 ; ZT12 : 0 . 089 ± 0 . 0036 mm2 ; P = 0 . 0003 ; Mann - Whitney U test ) inhibitory input size between ZT0 and ZT12 . Data information : Sample sizes used in this figure : ZT0 : 48 cells from 9 mice ; ZT12 : 51 cells from 9 mice . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

Figure 1A. Experimental demonstration. A1. Illustration of the mapping principle between ChR2-expressed PV neurons (green) and regular pyramidal neuron (Pyr, orange). A2. Acute slices were obtained and recorded at ZT0 or ZT12. A3. Coronal brain atlas showing the primary visual cortex (Top left panel, modified from MBL mouse brain atlas). Yellow box highlights the region of experiment. Top right panel, the reference image taken with a 10x objective from a slice prepared from a PV:Ai32 mouse. The 0.45 mm2 shaded cubic area was divided into 15 x 15 grids for independent light stimulation. The white arrowhead indicates the position of the recording electrode. Bottom panel, representative raw traces of light-evoked IPSCs (LEIPSCs) recorded from one pyramidal neuron. A4. The spatial distribution of the LEIPSCs from the example in A3. A5. The heatmap of the LEIPSCs in A4.B. The average heatmaps at ZT0 and ZT12 after soma alignment. Soma position was indicated by the blue arrow head. C. Comparison of the cumulative distribution (Kolmogorov-Smirnov test [K-S test]; P = 0.002) and the mean (Inset. ZT0: 813.3 ± 53.13 pA; ZT12: 1306 ± 93.73 pA; P < 0.0001; Mann-Whitney U test) of the max LEIPSC amplitude between ZT0 and ZT12. D. Comparison of the cumulative distribution (K-S test, P = 0.0016) and the mean (Inset. ZT0: 10.44 ± 1.44 nA; ZT12: 18.87 ± 1.44 nA; P < 0.0001; Mann-Whitney U test) of the sum LEIPSC amplitude between ZT0 and ZT12. Data information: Sample sizes used in this figure: ZT0: 48 cells from 9 mice; ZT12: 51 cells from 9 mice. Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.E. The average heatmaps of LEIPSCs after pia aligned to the reference image in A3. Dark and light blue overlay traces on the side of the map depicted the marginal distribution of the sum LEIPSCs for ZT0 (Dark blue) and ZT12 (Light blue). F. Comparison of the cumulative distribution (K-S test, P = 0.0021) and the mean (Inset, ZT0: 0.067 ± 0.004 mm2; ZT12: 0.089 ± 0.0036 mm2; P = 0.0003; Mann-Whitney U test) inhibitory input size between ZT0 and ZT12. Data information: Sample sizes used in this figure: ZT0: 48 cells from 9 mice; ZT12: 51 cells from 9 mice. Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


Figure 2A . mEPSC frequency was higher at ZT12 than ZT0 . Top : example traces of mEPSC recordings . Bottom left : mEPSC frequency ( ZT0 : 12 . 17 ± 0 . 94Hz ; ZT12 : 17 . 03 ± 1 . 20Hz ; P = 0 . 0036 ; Mann - Whitney U test ) . Bottom right : cumulative probability histogram of the inter - event intervals ( K - S test , P < 0 . 0001 ) . B . mIPSC frequency was lower at ZT12 than ZT0 . Top : example traces . Bottom left : mIPSC frequency ( ZT0 : 19 . 15 ± 0 . 99Hz ; ZT12 : 15 . 19 ± 0 . 63Hz ; P = 0 . 0042 ; Mann - Whitney U test ) . Bottom right : cumulative probability histogram of the inter - event intervals of mIPSCs ( K - S test , P < 0 . 0001 ) . C . mEPSC amplitude and kinetics did not differ between ZT0 and ZT12 . Top : average traces of the well - isolated events . Bottom left : mEPSC amplitude ( ZT0 : 20 . 51 ± 0 . 78pA ; ZT12 : 21 . 26 ± 0 . 75pA ; P = 0 . 34 ; Mann - Whitney U test ) . Bottom right : The distribution of mEPSC amplitude of all events ( K - S test , P < 0 . 01 ) . D . mIPSC amplitude and kinetics did not differ between ZT0 and ZT12 . Top : average traces of the well - isolated mIPSCs . Bottom left : mIPSC amplitude ( ZT0 : 41 . 31 ± 1 . 91pA ; ZT12 : 44 . 39 ± 1 . 31pA ; P = 0 . 35 ; Mann - Whitney U test ) . Bottom right : The cumulative distribution of mIPSC amplitudes ( K - S test , P < 0 . 0001 Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. mEPSC frequency was higher at ZT12 than ZT0. Top: example traces of mEPSC recordings. Bottom left: mEPSC frequency (ZT0: 12.17 ± 0.94Hz; ZT12: 17.03 ± 1.20Hz; P = 0.0036; Mann-Whitney U test). Bottom right: cumulative probability histogram of the inter-event intervals (K-S test, P < 0.0001). B. mIPSC frequency was lower at ZT12 than ZT0. Top: example traces. Bottom left: mIPSC frequency (ZT0: 19.15 ± 0.99Hz; ZT12: 15.19 ± 0.63Hz; P = 0.0042; Mann-Whitney U test). Bottom right: cumulative probability histogram of the inter-event intervals of mIPSCs (K-S test, P < 0.0001). C. mEPSC amplitude and kinetics did not differ between ZT0 and ZT12. Top: average traces of the well-isolated events. Bottom left: mEPSC amplitude (ZT0: 20.51 ± 0.78pA; ZT12: 21.26 ± 0.75pA; P = 0.34; Mann-Whitney U test). Bottom right: The distribution of mEPSC amplitude of all events (K-S test, P < 0.01). D. mIPSC amplitude and kinetics did not differ between ZT0 and ZT12. Top: average traces of the well-isolated mIPSCs. Bottom left: mIPSC amplitude (ZT0: 41.31 ± 1.91pA; ZT12: 44.39 ± 1.31pA; P = 0.35; Mann-Whitney U test). Bottom right: The cumulative distribution of mIPSC amplitudes (K-S test, P < 0.0001 Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01.


Figure 3B . Charges of sEPSCs recorded at light phase were higher compared to those at dark phase ( ZT0 : 4 . 21 ± 0 . 28 nC ; ZT4 : 7 . 05 ± 0 . 61 nC ; ZT8 : 7 . 74 ± 0 . 63 nC ; ZT12 : 7 . 81 ± 0 . 59 nC ; ZT18 : 5 . 25 ± 0 . 53 nC ; One - way ANOVA , F ( 4 , 88 ) = 8 . 78 , P < 0 . 0001 , Holm - Sidak post - hoc analysis ) . A copy of the 24 - h sEPSCs was drawn with a dashed line to demonstrate the daily rhythm . C . Charges of sIPSCs oscillate during the light / dark cycle ( ZT0 : 45 . 73 ± 2 . 99 nC ; ZT4 : 28 . 85 ± 1 . 76 nC ; ZT8 : 28 . 25 ± 2 . 77 nC ; ZT12 : 28 . 04 ± 2 . 15 nC ; ZT18 : 43 . 37 ± 2 . 42 nC ; One - way ANOVA , F ( 4 , 106 ) = 13 . 65 , P < 0 . 0001 , Holm - Sidak post - hoc analysis ) . A copy of the 24 - h sEPSCs was drawn with a dashed line to demonstrate the daily rhythm . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . E . Dark exposure prevented the increase in sEPSCs that normally occurs at ZT12 ( ZT12 as reported in B ; Dark - ZT12 : 5 . 46 ± 0 . 43 nC ; P = 0 . 0082 ; Mann - Whitney U test ) . F . Dark exposure did not significantly affect the reduction of sIPSCs ( ZT12 as reported in C ; Dark - ZT12 : 33 . 43 ± 1 . 74 nC ; P = 0 . 07 ; Mann - Whitney U test ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . H . Sleep deprivation further increased sEPSCs compared to ad lib sleep mice ( ZT4 as reported in B ; SD - ZT4 : 11 . 22 ± 0 . 92 nC ; P = 0 . 0013 ; Mann - Whitney U test ) . I . Sleep deprivation abolished the reduction of sIPSC ( ZT4 as reported in C ; SD - ZT4 : 47 . 13 ± 3 . 39 nC ; P < 0 . 0001 ; Mann - Whitney U test ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O 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 3B. Charges of sEPSCs recorded at light phase were higher compared to those at dark phase (ZT0: 4.21 ± 0.28 nC; ZT4: 7.05 ± 0.61 nC; ZT8: 7.74 ± 0.63 nC; ZT12: 7.81 ± 0.59 nC; ZT18: 5.25 ± 0.53 nC; One-way ANOVA, F(4,88) = 8.78, P <0.0001, Holm-Sidak post-hoc analysis). A copy of the 24-h sEPSCs was drawn with a dashed line to demonstrate the daily rhythm. C. Charges of sIPSCs oscillate during the light/dark cycle (ZT0: 45.73 ± 2.99 nC; ZT4: 28.85 ± 1.76 nC; ZT8: 28.25 ± 2.77 nC; ZT12: 28.04 ± 2.15 nC; ZT18: 43.37 ± 2.42 nC; One-way ANOVA, F(4,106) = 13.65, P <0.0001, Holm-Sidak post-hoc analysis). A copy of the 24-h sEPSCs was drawn with a dashed line to demonstrate the daily rhythm. Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.E. Dark exposure prevented the increase in sEPSCs that normally occurs at ZT12 (ZT12 as reported in B; Dark-ZT12: 5.46 ± 0.43 nC; P = 0.0082; Mann-Whitney U test). F. Dark exposure did not significantly affect the reduction of sIPSCs (ZT12 as reported in C; Dark-ZT12: 33.43 ± 1.74 nC; P = 0.07; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.H. Sleep deprivation further increased sEPSCs compared to ad lib sleep mice (ZT4 as reported in B; SD-ZT4: 11.22 ± 0.92 nC; P = 0.0013; Mann-Whitney U test). I. Sleep deprivation abolished the reduction of sIPSC (ZT4 as reported in C; SD-ZT4: 47.13 ± 3.39 nC; P < 0.0001; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


Figure 4B - E . Slices were obtained at ZT12 ( B - C ) or ZT0 ( D - E ) and pre - incubated with 50 µM CARB for more than 1h before recording mIPSCs in the presence of drug . Slices from the same animals were used as controls . CARB increased mIPSC frequency at ZT12 ( CTL : 7 . 74 ± 0 . 55 Hz ; CARB : 12 . 41 ± 1 . 03 Hz ; P = 0 . 0001 ; Mann - Whitney U test ) , but not ZT0 ( CTL : 11 . 79 ± 1 . 00 Hz ; CARB : 11 . 47 ± 1 . 60 Hz ; P = 0 . 39 ; Mann - Whitney U test ) . mIPSC amplitude was unchanged at either time ( C , ZT12 : CTL : 40 . 94 ± 2 . 08 pA ; CARB : 40 . 64 ± 1 . 66 pA ; P = 0 . 99 ; E , ZT0 : CTL : 34 . 73 ± 2 . 14 pA ; CARB : 33 . 61 ± 2 . 39 pA ; P = 0 . 85 ; Mann - Whitney U test ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . F - G . mIPSC frequency ( F , CTL : 9 . 82 ± 0 . 74 Hz ; Atr : 7 . 70 ± 0 . 57 Hz ; P = 0 . 0009 ; Wilcoxon signed - rank test ) and amplitude ( G , CTL : 45 . 88 ± 2 . 87 pA ; Atr : 43 . 64 ± 2 . 40 pA ; P = 0 . 70 ; Wilcoxon signed - rank test ) recorded at ZT0 with acute atropine ( Atr ) wash on . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . H - I . Effect of acute wash - on and wash - off of pirenzepine ( Pir ) on mIPSC frequency ( H , CTL : 9 . 00 ± 1 . 24 Hz ; Pir : 7 . 94 ± 1 . 07 Hz ; Washout : 8 . 99 ± 1 . 14 Hz ; One - way repeated measures ANOVA , F ( 2 , 20 ) = 7 . 483 , P = 0 . 0037 ; Holm - Sidak ' s multiple comparison test ) and amplitude ( I , CTL : 61 . 23 ± 3 . 72 pA ; Pir : 58 . 85 ± 4 . 06 pA ; Washout : 58 . 25 ± 4 . 20 pA ; One - way repeated measures ANOVA , F ( 2 , 20 ) = 3 . 197 , P = 0 . 0624 ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . J - K . Effect of pirenzepine or mecamylamine ( MEC ) on carbachol - induced elevation in mIPSC frequency at ZT12 ( J , CARB : 10 . 02 ± 0 . 64 Hz ; CARB + MEC : 10 . 95 ± 0 . 78 Hz ; CARB + Pir : 7 . 75 ± 0 . 79 Hz ; One - way ANOVA , F ( 2 , 25 ) = 4 . 848 , P = 0 . 0166 ; Holm - Sidak ' s multiple comparison test ) and amplitude ( K , CARB : 39 . 85 ± 2 . 60 pA ; CARB + MEC : 42 . 17 ± 1 . 65 pA ; CARB + Pir : 40 . 67 ± 4 . 34 pA ; One - way ANOVA , F ( 2 , 25 ) = 0 . 1501 , P = 0 . 8614 ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 4B-E. Slices were obtained at ZT12 (B-C) or ZT0 (D-E) and pre-incubated with 50 µM CARB for more than 1h before recording mIPSCs in the presence of drug. Slices from the same animals were used as controls. CARB increased mIPSC frequency at ZT12 (CTL: 7.74 ± 0.55 Hz; CARB: 12.41 ± 1.03 Hz; P = 0.0001; Mann-Whitney U test), but not ZT0 (CTL:11.79 ± 1.00 Hz; CARB: 11.47 ± 1.60 Hz; P = 0.39; Mann-Whitney U test). mIPSC amplitude was unchanged at either time (C, ZT12: CTL: 40.94 ± 2.08 pA; CARB: 40.64 ± 1.66 pA; P = 0.99; E, ZT0: CTL: 34.73 ± 2.14 pA; CARB: 33.61 ± 2.39 pA; P = 0.85; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.F-G. mIPSC frequency (F, CTL: 9.82± 0.74 Hz; Atr: 7.70 ± 0.57 Hz; P = 0.0009; Wilcoxon signed-rank test) and amplitude (G, CTL: 45.88 ± 2.87 pA; Atr: 43.64 ± 2.40 pA; P = 0.70; Wilcoxon signed-rank test) recorded at ZT0 with acute atropine (Atr) wash on. Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.H-I. Effect of acute wash-on and wash-off of pirenzepine (Pir) on mIPSC frequency (H, CTL: 9.00 ± 1.24 Hz; Pir: 7.94 ± 1.07 Hz; Washout: 8.99 ± 1.14 Hz; One-way repeated measures ANOVA, F(2, 20) = 7.483, P = 0.0037; Holm-Sidak's multiple comparison test) and amplitude (I, CTL: 61.23 ± 3.72 pA; Pir: 58.85 ± 4.06 pA; Washout: 58.25 ± 4.20 pA; One-way repeated measures ANOVA, F(2, 20) = 3.197, P = 0.0624). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.J-K. Effect of pirenzepine or mecamylamine (MEC) on carbachol-induced elevation in mIPSC frequency at ZT12 (J, CARB: 10.02 ± 0.64 Hz; CARB+MEC: 10.95 ± 0.78 Hz; CARB+Pir: 7.75 ± 0.79 Hz; One-way ANOVA, F(2, 25) = 4.848, P = 0.0166; Holm-Sidak's multiple comparison test) and amplitude (K, CARB: 39.85 ± 2.60 pA; CARB+MEC: 42.17 ± 1.65 pA; CARB+Pir: 40.67 ± 4.34 pA; One-way ANOVA, F(2, 25) = 0.1501, P = 0.8614). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.


Figure 5A1 . Diagram illustrating the in vivo two - photon imaging timeline . A2 . GCaMP6s ( green ) was expressed in V1 of the PV : Ai9 mice ( red : td - Tomato ) . Scale : 500 µm . A3 . Schematics of the calcium imaging showing a mouse that was head - fixed and free running on an air - suspended arena during the imaging section . Locomotor activity was simultaneously monitored ( bottom left ) along with Ca2 + imaging ( Top right . Scale : 100 µm ) . Example spontaneous ΔF / F0 traces ( bottom right ) . B . Top panel , Example spontaneous ΔF / F0 traces from three PV neurons repetitively imaged at ZT0 and ZT12 . Bottom panels , Heatmaps showing the spontaneous ΔF / F0 time - series traces of all PV neurons imaged at ZT0 and ZT12 . The color code represents the magnitude of ΔF / F0 ( % ) . C . Cumulative probability distribution of the sum ΔF / F0 of all PV neurons imaged at ZT0 and ZT12 ( K - S test ; P = 0 . 02 ) . Inset : the mean sum ΔF / F0 ( ZT0 : 401 . 6 ± 19 . 55 ; ZT12 : 559 . 1 ± 35 . 29 ; P = 0 . 0002 ; Mann - Whitney U test ) . D . Left panel , diagram depicting repetitive imaging protocol . Right panel , mean activity of PV neurons indicated by Z score imaged at four timepoints . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A1. Diagram illustrating the in vivo two-photon imaging timeline. A2. GCaMP6s (green) was expressed in V1 of the PV:Ai9 mice (red: td-Tomato). Scale: 500 µm. A3. Schematics of the calcium imaging showing a mouse that was head-fixed and free running on an air-suspended arena during the imaging section. Locomotor activity was simultaneously monitored (bottom left) along with Ca2+ imaging (Top right. Scale: 100 µm). Example spontaneous ΔF/F0 traces (bottom right).B. Top panel, Example spontaneous ΔF/F0 traces from three PV neurons repetitively imaged at ZT0 and ZT12. Bottom panels, Heatmaps showing the spontaneous ΔF/F0 time-series traces of all PV neurons imaged at ZT0 and ZT12. The color code represents the magnitude of ΔF/F0(%). C. Cumulative probability distribution of the sum ΔF/F0 of all PV neurons imaged at ZT0 and ZT12 (K-S test; P = 0.02). Inset: the mean sum ΔF/F0 (ZT0: 401.6 ± 19.55; ZT12: 559.1 ± 35.29; P = 0.0002; Mann-Whitney U test). D. Left panel, diagram depicting repetitive imaging protocol. Right panel, mean activity of PV neurons indicated by Z score imaged at four timepoints. Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.


Figure 6B . Heatmaps showing the spontaneous ΔF / F0 time - series traces of all PV neurons imaged at ZT4 and SD _ ZT4 . The color code represents the magnitude of ΔF / F0 ( % ) . C . Cumulative probability distribution of the sum ΔF / F0 of all PV neurons imaged at ZT4 and SD _ ZT4 ( K - S test ; P = 0 . 0001 ) . Inset : the mean sum ΔF / F0 ( ZT4 : 322 . 4 ± 18 . 64 ; SD _ ZT4 : 424 . 0 ± 21 . 54 ; P = 0 . 0004 ; Mann - Whitney U test ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . D . Heatmaps showing the spontaneous ΔF / F0 time - series traces of all PV neurons imaged at SD _ ZT4 and Dark _ SD . The color code represents the magnitude of ΔF / F0 ( % ) . E . Cumulative probability distribution of the sum ΔF / F0 of all PV neurons imaged at SD _ ZT4 and Dark _ SD ( K - S test ; P = 0 . 015 ) . Inset : the mean sum ΔF / F0 ( SD _ ZT4 : 375 . 1 ± 20 . 19 ; Dark _ SD : 293 . 8 ± 16 . 55 ; P = 0 . 0022 ; Mann - Whitney U test ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * 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 O O O O O O O O O O O O O O O O O O O O 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

Figure 6B. Heatmaps showing the spontaneous ΔF/F0 time-series traces of all PV neurons imaged at ZT4 and SD_ZT4. The color code represents the magnitude of ΔF/F0 (%). C. Cumulative probability distribution of the sum ΔF/F0 of all PV neurons imaged at ZT4 and SD_ZT4 (K-S test; P = 0.0001). Inset: the mean sum ΔF/F0 (ZT4: 322.4 ± 18.64; SD_ZT4: 424.0 ± 21.54; P = 0.0004; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.D. Heatmaps showing the spontaneous ΔF/F0 time-series traces of all PV neurons imaged at SD_ZT4 and Dark_SD. The color code represents the magnitude of ΔF/F0(%). E. Cumulative probability distribution of the sum ΔF/F0 of all PV neurons imaged at SD_ZT4 and Dark_SD (K-S test; P = 0.015). Inset: the mean sum ΔF/F0 (SD_ZT4: 375.1 ± 20.19; Dark_SD: 293.8 ± 16.55; P = 0.0022; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.


Figure 7A . Top panel , Example spontaneous ΔF / F0 traces from three putative pyramidal neurons repetitively imaged at ZT0 and ZT12 . Bottom panels , Heatmaps showing the spontaneous ΔF / F0 time - series traces of all putative pyramidal neurons imaged at ZT0 and ZT12 . B . The cumulative distribution of the sum ΔF / F0 of putative pyramidal neurons judged by skewness ( K - S test ; P < 0 . 0001 ) . Inset : the means of sum ΔF / F0 ( ZT0 : 679 . 5 ± 38 . 22 ; ZT12 : 485 . 8 ± 32 . 22 ; P < 0 . 0001 ; Mann - Whitney U test ) . C . Mean activity of putative pyramidal neurons indicated by Z score imaged at four timepoints . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . D1 . Schematic showing in vivo dLGN - evoked Ca2 + reponses in V1 . D2 . Immunofluorescent staining showing the GCaMP6s ( green ) and ChR2 - mCherry ( red ) expression in V1 and dLGN respectively . The optical fiber track was also labeled . ( Scale : 500 µm ) . D3 . Response profile of an example neurons for dLGN stimulation during the light / dark cycle . Horizontal blue brief lines represent stimulus frequency . E - F . Top : color - coded calcium signals of all responsive neurons ( % ΔF / F0 ) . Bottom : The mean Ca2 + response waveform for individual mouse ( dotted color lines ) and all mice ( thick black and gray lines ) . Trials were aligned to LED onset time ( gray solid lines ) . Data information : Sample size is indicated as ( mice , cells ) . G . The fraction of neurons responsive to dLGN stimulus in ZT0 and ZT12 . Each gray line represents one imaging mouse . Black and white circles and error bars represent the mean and SEM ( ZT0 : 8 . 51 ± 1 . 86 ; ZT12 : 5 . 96 ± 1 . 39 ; P = 0 . 015 ; Wilcoxon signed - rank test ; n = 9 mice ) . H . The Response fidelity of each cell in ZT0 and ZT12 . Black and white circles and error bars represent the mean and SEM ( ZT0 : 0 . 42 ± 0 . 022 ; ZT12 : 0 . 31 ± 0 . 023 ; P < 0 . 0001 ; Wilcoxon signed - rank test ; n = 195 cell ) . I . Comparison of peak evoked response of each cell in ZT0 and ZT12 . Black and white circles and error bars represent the mean and SEM ( ZT0 : 81 . 01 ± 4 . 79 ; ZT12 : 48 . 42 ± 3 . 82 ; P < 0 . 0001 ; Wilcoxon signed - rank test ; n = 195 cell ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at * P < 0 .

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

Figure 7A. Top panel, Example spontaneous ΔF/F0 traces from three putative pyramidal neurons repetitively imaged at ZT0 and ZT12. Bottom panels, Heatmaps showing the spontaneous ΔF/F0 time-series traces of all putative pyramidal neurons imaged at ZT0 and ZT12. B. The cumulative distribution of the sum ΔF/F0 of putative pyramidal neurons judged by skewness (K-S test; P < 0.0001). Inset: the means of sum ΔF/F0 (ZT0: 679.5 ± 38.22; ZT12: 485.8 ± 32.22; P < 0.0001; Mann-Whitney U test). C. Mean activity of putative pyramidal neurons indicated by Z score imaged at four timepoints. Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.D1. Schematic showing in vivo dLGN-evoked Ca2+ reponses in V1. D2. Immunofluorescent staining showing the GCaMP6s (green) and ChR2-mCherry (red) expression in V1 and dLGN respectively. The optical fiber track was also labeled. (Scale: 500 µm). D3. Response profile of an example neurons for dLGN stimulation during the light/dark cycle. Horizontal blue brief lines represent stimulus frequency.E-F. Top: color-coded calcium signals of all responsive neurons (% ΔF/F0). Bottom: The mean Ca2+ response waveform for individual mouse (dotted color lines) and all mice (thick black and gray lines). Trials were aligned to LED onset time (gray solid lines). Data information: Sample size is indicated as (mice, cells).G. The fraction of neurons responsive to dLGN stimulus in ZT0 and ZT12. Each gray line represents one imaging mouse. Black and white circles and error bars represent the mean and SEM (ZT0: 8.51 ± 1.86; ZT12: 5.96 ± 1.39; P = 0.015; Wilcoxon signed-rank test; n = 9 mice). H. The Response fidelity of each cell in ZT0 and ZT12. Black and white circles and error bars represent the mean and SEM (ZT0: 0.42 ± 0.022; ZT12: 0.31 ± 0.023; P < 0.0001; Wilcoxon signed-rank test; n = 195 cell). I. Comparison of peak evoked response of each cell in ZT0 and ZT12. Black and white circles and error bars represent the mean and SEM (ZT0: 81.01 ± 4.79; ZT12: 48.42 ± 3.82; P < 0.0001; Wilcoxon signed-rank test; n = 195 cell). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at *P < 0.05


Figure 1A . Protein lysates from the melanocytic - like melanoma cells 501mel , MM011 , MM074 and MM117 or the mesenchymal - like melanoma cells MM029 , MM047 and MM099 were immuno - blotted for proteins as indicated . Molecular mass of the proteins is indicated ( kDa ) . B . Melanoma cells were treated with increasing concentrations of THZ1 as indicated for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . IC50 for each cell line is indicated . Melanocytic - type ( MITF - High , proliferative ) melanoma cells are shown in red , while mesenchymal - like ( MITF - low , invasive ) melanoma cells are shown in blue . Data information : data are presented as mean values + Standard Deviation ( SD ) for three replicates ( n = 3 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Protein lysates from the melanocytic-like melanoma cells 501mel, MM011, MM074 and MM117 or the mesenchymal-like melanoma cells MM029, MM047 and MM099 were immuno-blotted for proteins as indicated. Molecular mass of the proteins is indicated (kDa).B. Melanoma cells were treated with increasing concentrations of THZ1 as indicated for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. IC50 for each cell line is indicated. Melanocytic-type (MITF-High, proliferative) melanoma cells are shown in red, while mesenchymal-like (MITF-low, invasive) melanoma cells are shown in blue. Data information: data are presented as mean values + Standard Deviation (SD) for three replicates (n=3).


Figure 2D . Protein lysates from MM074 , MM074BRAFi - R or MM074CDK7i - R were immuno - blotted for indicated proteins . Molecular sizes of the proteins are indicated ( kDa ) . The numbers below the gel lanes represent relative protein level , which was determined from the band intensity using ImageJ software normalized to each relative Vinculin control .

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

Figure 2D. Protein lysates from MM074, MM074BRAFi-R or MM074CDK7i-R were immuno-blotted for indicated proteins. Molecular sizes of the proteins are indicated (kDa). The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software normalized to each relative Vinculin control.


Figure 3B . Protein lysates from the indicated cells were immuno - blotted for the indicated proteins . Molecular sizes of the proteins are indicated in kDa . The numbers below the gel lanes represent relative protein level , which was determined from the band intensity using ImageJ software normalized relative to each relative ACTIN control . E - H . qRT - PCR analysis showing average TBP - normalized expression of GATA6 ( E ) , AMIGO2 ( F ) , SERPINE1 ( G ) and ABCG2 ( H ) in the indicated cells treated with either siCTL or siGATA6 for 72h . Data information : data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * * < 0 . 01 , * * * < 0 . 005 and ns = non - significantJ . MM029 ( left ) and MM099 ( right ) were pre - treated with either siCTL or siGATA6 for 48h and treated with increasing concentrations of THZ1 for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . Data information : data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * * < 0 . 01 , * * * < 0 . 005 and ns = non -

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

Figure 3B. Protein lysates from the indicated cells were immuno-blotted for the indicated proteins. Molecular sizes of the proteins are indicated in kDa. The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software normalized relative to each relative ACTIN control.E-H. qRT-PCR analysis showing average TBP-normalized expression of GATA6 (E), AMIGO2 (F), SERPINE1 (G) and ABCG2 (H) in the indicated cells treated with either siCTL or siGATA6 for 72h. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, **<0.01, ***<0.005 and ns=non-significantJ. MM029 (left) and MM099 (right) were pre-treated with either siCTL or siGATA6 for 48h and treated with increasing concentrations of THZ1 for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, **<0.01, ***<0.005 and ns=non-significant


Figure 4C - D . MM099 ( c ) and MM029 ( d ) were pre - treated with either siCTL or siABCG2 as indicated and treated with increasing concentrations of THZ1 for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . E - F . MM099 ( e ) and MM029 ( f ) were pre - treated with either siCTL or siABCG2 as indicated and treated with increasing doses of Vemurafenib for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . D Data information : data are presented as mean values + SD for three replicates ( n = 3 ) . IC50 for each cell line is indicated .

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Figure 4C-D. MM099 (c) and MM029 (d) were pre-treated with either siCTL or siABCG2 as indicated and treated with increasing concentrations of THZ1 for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. E-F. MM099 (e) and MM029 (f) were pre-treated with either siCTL or siABCG2 as indicated and treated with increasing doses of Vemurafenib for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. D Data information: data are presented as mean values + SD for three replicates (n=3). IC50 for each cell line is indicated.


Figure 5C - E . qRT - PCR analysis showing average TBP - normalized fold expression of MITF ( C ) , SOX10 ( D ) and GATA6 ( E ) in 501mel treated either with DMSO / THZ1 ( 50nM ) ( upper ) or DMSO / JQ1 ( 10μM ) ( lower ) for 24h . Data information : data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * < 0 . 05 .

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

Figure 5C-E. qRT-PCR analysis showing average TBP-normalized fold expression of MITF (C), SOX10 (D) and GATA6 (E) in 501mel treated either with DMSO/THZ1 (50nM) (upper) or DMSO/JQ1 (10μM) (lower) for 24h. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, *<0.05.


Figure 6A . qRT - PCR analysis showing average TBP - normalized fold expression of SOX10 , MITF and GATA6 in 501mel treated with either siCTL or siSOX10 for 48h . Data information : , data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * < 0 . 05 .

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

Figure 6A. qRT-PCR analysis showing average TBP-normalized fold expression of SOX10, MITF and GATA6 in 501mel treated with either siCTL or siSOX10 for 48h. Data information: , data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, *<0.05.


Figure 7D . MM099MITF - SOX10 - PAX3 expressing inducible MITF - SOX10 - PAX3 genes was treated or not with Doxycycline ( 1μg / ml ) for 24h and protein lysates were immuno - blotted for the indicated protein . The numbers below the gel lanes represent relative protein level , which was determined from the band intensity using ImageJ software , and normalized relative to VINCULIN . E . qRT - PCR analysis showing average TBP - normalized fold expression of GATA6 , ABCG2 , AMIGO2 or SERPINE1 in MM099MITF - SOX10 - PAX3 treated or not with Doxycycline ( 1μg / ml ) for 24h . Data information : data are presented as mean values + SD for three biological triplicates . The p - value ( Student ' s t - test ) is indicated , * < 0 . 05 , * * < 0 . 01 ; * * * < 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 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

Figure 7D. MM099MITF-SOX10-PAX3 expressing inducible MITF-SOX10-PAX3 genes was treated or not with Doxycycline (1μg/ml) for 24h and protein lysates were immuno-blotted for the indicated protein. The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software, and normalized relative to VINCULIN.E. qRT-PCR analysis showing average TBP-normalized fold expression of GATA6, ABCG2, AMIGO2 or SERPINE1 in MM099MITF-SOX10-PAX3 treated or not with Doxycycline (1μg/ml) for 24h. Data information: data are presented as mean values + SD for three biological triplicates. The p-value (Student's t-test) is indicated, *<0.05, **<0.01; ***<0.001.


Figure 8A . Tumor sections were immuno - labelled ( IHC ) with anti - GATA6 ( red ) and anti - SOX10 ( green ) antibodies and images were captured by confocal microscopy at the indicated magnification . We analysed six tumor sections of metastases and observed significant GATA6 expression in only one of them . Scale bar 250 μM for 40x , 100μM for 100x . D - E . Graphs showing the average expression of GATA6 ( d ) and MITF ( e ) ( RPKM ) for each phenotype cluster in T0 ( drug naïve ) ( blue ) , phases 1 - 2 ( MDR ) ( green and yellow ) and phase 3 ( drug - resistance ) ( red ) . Data information : data are presented as mean values + SEM ( n = 6 , 574 cells from 5 PDX ) .

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

Figure 8A. Tumor sections were immuno-labelled (IHC) with anti-GATA6 (red) and anti-SOX10 (green) antibodies and images were captured by confocal microscopy at the indicated magnification. We analysed six tumor sections of metastases and observed significant GATA6 expression in only one of them. Scale bar 250 μM for 40x, 100μM for 100x.D-E. Graphs showing the average expression of GATA6 (d) and MITF (e) (RPKM) for each phenotype cluster in T0 (drug naïve) (blue), phases 1-2 (MDR) (green and yellow) and phase 3 (drug-resistance) (red). Data information: data are presented as mean values + SEM (n=6,574 cells from 5 PDX).


Figure 1A , Live images of neurons co - transfected with SyGCaMP5 and MtDsRed before and during 10 Hz field stimulation . The full arrow indicates a mitochondrially occupied terminal , and the empty arrow a terminal without a mitochondrion . Scale bar , 10 m . B , Average ΔF / F0 SyGCaMP5 traces from n = 11 neurons ( 98 terminals ) plotting the average of terminals without a mitochondrion ( black trace ) and with a mitochondrion ( red trace ) . Stimulation occurred for 10s ( t = 20 - 30 ) at 10 Hz . C , Average Ca2 + response following stimulation in terminals with or without a mitochondrion ( average of ΔF measurement taken for a t = 20 - 30s of B ) , ΔF / F0 = 1 . 9 0 . 3 with and 3 . 5 0 . 4 without mitochondria , paired t - test , * * * p < 0 . 0001 . D E , However , there is no significant difference in the presynaptic Ca2 + response in terminals with and without a mitochondrion in response to single action potentials . D , Example trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5 . 1 ms stimulation pulses were applied every 5 s . The red trace represents an average of the terminals occupied with a mitochondrion in this neuron , whereas the black trace represents the averaged response of terminals without a mitochondrion . E , Maximal fluorescence intensity in terminals without a mitochondrion ( left ) or with a mitochondrion ( right ) within the same axon ( dots connected by a line ) ( n = 8 neurons , 44 terminals , paired t - test , p = 0 . 55 ) . F , Average ΔF / F0 SyGCaMP5 traces for 80 stimuli ( n = 7 neurons , 21 terminals , t = 20 - 28s , ΔF / F0 = 1 . 3 0 . 2 with and 2 . 2 0 . 5 without mitochondria , paired t - test , * p < 0 . 05 ) , 40 stimuli ( n = 5 neurons , 25 terminals , t = 20 - 24s , ΔF / F0 = 0 . 9 0 . 3 with and 2 . 0 0 . 4 without mitochondria , paired t - test , * p < 0 . 05 ) , 20 stimuli ( n = 10 neurons , 55 terminals , t = 20 - 22s , ΔF / F0 = 0 . 8 0 . 4 with and 1 . 8 0 . 4 without mitochondria , paired t - test , * p < 0 . 05 ) and 10 stimuli ( n = 4 neurons , 21 terminals , t = 20 - 21s , ΔF / F0 = 0 . 4 0 . 3 with and 0 . 4 0 . 1 without mitochondria , paired t - test , p = 0 . 90 ) , all delivered at 10 Hz . Experiments performed in E18 or P0 rat hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent SEM .

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

Figure 1A, Live images of neurons co-transfected with SyGCaMP5 and MtDsRed before and during 10 Hz field stimulation. The full arrow indicates a mitochondrially occupied terminal, and the empty arrow a terminal without a mitochondrion. Scale bar, 10 m.B, Average ΔF/F0 SyGCaMP5 traces from n=11 neurons (98 terminals) plotting the average of terminals without a mitochondrion (black trace) and with a mitochondrion (red trace). Stimulation occurred for 10s (t=20-30) at 10 Hz.C, Average Ca2+ response following stimulation in terminals with or without a mitochondrion (average of ΔF measurement taken for a t=20-30s of B), ΔF/F0 = 1.9 0.3 with and 3.5 0.4 without mitochondria, paired t-test, ***p<0.0001.D E, However, there is no significant difference in the presynaptic Ca2+ response in terminals with and without a mitochondrion in response to single action potentials. D, Example trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5. 1 ms stimulation pulses were applied every 5 s. The red trace represents an average of the terminals occupied with a mitochondrion in this neuron, whereas the black trace represents the averaged response of terminals without a mitochondrion. E, Maximal fluorescence intensity in terminals without a mitochondrion (left) or with a mitochondrion (right) within the same axon (dots connected by a line) (n=8 neurons, 44 terminals, paired t-test, p=0.55).F, Average ΔF/F0 SyGCaMP5 traces for 80 stimuli (n=7 neurons, 21 terminals, t= 20-28s, ΔF/F0 = 1.3 0.2 with and 2.2 0.5 without mitochondria, paired t-test, *p<0.05), 40 stimuli (n=5 neurons, 25 terminals, t= 20-24s, ΔF/F0 = 0.9 0.3 with and 2.0 0.4 without mitochondria, paired t-test, *p<0.05), 20 stimuli (n=10 neurons, 55 terminals, t= 20-22s, ΔF/F0 = 0.8 0.4 with and 1.8 0.4 without mitochondria, paired t-test, *p<0.05) and 10 stimuli (n=4 neurons, 21 terminals, t= 20-21s, ΔF/F0 = 0.4 0.3 with and 0.4 0.1 without mitochondria, paired t-test, p=0.90), all delivered at 10 Hz. Experiments performed in E18 or P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


Figure 2A , Average trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5 imaged after incubation in ACSF for 30 min . Neurons were stimulated using field stimulation for 10s at 10 Hz . Red trace = average of the terminals occupied with a mitochondrion , black trace = averaged response of terminals without a mitochondrion . B , as in A , but after 30 min treatment with 10 µM Ru360 . C , Summary bar graph of the control neurons ( corresponds to the traces shown in panel A ) . Average Ca2 + response ( t = 20 - 30s ) following stimulation for terminals occupied by a mitochondrion ( ΔF / F0 = 3 . 5 0 . 4 ) and unoccupied terminals ( ΔF / F0 = 4 . 9 0 . 5 , n = 15 neurons , 96 terminals , * * * p < 0 . 001 , paired t - test ) . D , Summary bar graph of the Ru360 - treated neurons ( corresponds to the traces shown in panel B ) . Average Ca2 + response ( t = 20 - 30s ) following stimulation for terminals occupied by a mitochondrion ( ΔF / F0 = 4 . 1 0 . 5 ) and unoccupied terminals ( ΔF / F0 = 3 . 8 0 . 9 , n = 13 neurons , 79 terminals , p = 0 . 53 , paired t - test ) . Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent SEM .

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

Figure 2A, Average trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5 imaged after incubation in ACSF for 30 min. Neurons were stimulated using field stimulation for 10s at 10 Hz. Red trace = average of the terminals occupied with a mitochondrion, black trace = averaged response of terminals without a mitochondrion. B, as in A, but after 30 min treatment with 10 µM Ru360.C, Summary bar graph of the control neurons (corresponds to the traces shown in panel A). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 3.5 0.4) and unoccupied terminals (ΔF/F0 = 4.9 0.5, n=15 neurons, 96 terminals, ***p<0.001, paired t-test). D, Summary bar graph of the Ru360-treated neurons (corresponds to the traces shown in panel B). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 4.1 0.5) and unoccupied terminals (ΔF/F0 = 3.8 0.9, n=13 neurons, 79 terminals, p=0.53, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


Figure 3A , Example images of hippocampal neurons co - transfected with Mito - mKate2 and VGlut1pHluorin before and during field stimulation . The white arrow indicates a terminal occupied with a mitochondrion , while the empty arrow indicates a terminal without a mitochondrion . B , Average traces of presynaptic terminals of hippocampal neurons transfected with VGlut1pHluorin and Mito - mKate2 and stimulated using field stimulation at 10 Hz for 20s ( 40 - 60s ) . Terminals with a mitochondrion are represented by the red trace and terminals without are represented by the black trace . C , Summary graph corresponds to the traces shown in panel B ) . Mean stimulation fluorescence ( time points 60 - 70s ) comparing terminals of the same axon which contain mitochondria ( right , ΔF / F0 = 2 . 3 0 . 9 ) compared to terminals without mitochondria ( left , ΔF / F0 = 3 . 2 0 . 7 ) ( n = 9 neurons , 66 terminals , * p < 0 . 05 , paired t - test ) . Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent SEM .

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

Figure 3A, Example images of hippocampal neurons co-transfected with Mito-mKate2 and VGlut1pHluorin before and during field stimulation. The white arrow indicates a terminal occupied with a mitochondrion, while the empty arrow indicates a terminal without a mitochondrion. B, Average traces of presynaptic terminals of hippocampal neurons transfected with VGlut1pHluorin and Mito-mKate2 and stimulated using field stimulation at 10 Hz for 20s (40-60s). Terminals with a mitochondrion are represented by the red trace and terminals without are represented by the black trace. C, Summary graph corresponds to the traces shown in panel B). Mean stimulation fluorescence (time points 60-70s) comparing terminals of the same axon which contain mitochondria (right, ΔF/F0 = 2.3 0.9) compared to terminals without mitochondria (left, ΔF/F0 = 3.2 0.7) (n=9 neurons, 66 terminals, *p<0.05, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


Figure 4A , C E , Confocal images of neuronal processes of fixed neurons transfected with MtDsRed and SYN - GFP and C , myc - Miro1 ( Miro OE ) or E , myc - Miro1ΔEF ( Miro ΔEF ) . Neurons are either non - treated ( NT ) , TTX treated ( 1 M , 48h ) , PTX treated ( 100 M , 48h ) , or DMSO treated ( 1 : 2000 , as PTX ) . Scale bar , 10 m . B , D F , Fraction of SYN - GFP clusters co - localising with mitochondria in cultures in B , control conditions , DMSO 28 . 7 % 3 . 9 , TTX 16 . 6 % 3 . 1 , PTX 50 . 0 % 7 . 2 , ANOVA * * p0 . 001 , n = 8 - 9 neurons , D , with the expression of myc - Miro1 , DMSO 33 . 9 % 4 . 7 , TTX 35 . 9 % 5 . 4 , PTX 46 . 7 % 4 . 0 , ANOVA , * p0 . 05 , n = 8 - 9 , F , with the expression of myc - Miro1ΔEF , ANOVA p0 . 05 , n = 9 - 12 . Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent SEM .

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

Figure 4A, C E, Confocal images of neuronal processes of fixed neurons transfected with MtDsRed and SYN-GFP and C, myc-Miro1 (Miro OE) or E, myc-Miro1ΔEF (Miro ΔEF). Neurons are either non-treated (NT), TTX treated (1 M, 48h), PTX treated (100 M, 48h), or DMSO treated (1:2000, as PTX). Scale bar, 10 m. B, D F, Fraction of SYN-GFP clusters co-localising with mitochondria in cultures in B, control conditions, DMSO 28.7% 3.9, TTX 16.6% 3.1, PTX 50.0% 7.2, ANOVA **p0.001, n=8-9 neurons, D, with the expression of myc-Miro1, DMSO 33.9% 4.7, TTX 35.9% 5.4, PTX 46.7% 4.0, ANOVA, *p0.05, n=8-9, F, with the expression of myc-Miro1ΔEF, ANOVA p0.05, n=9-12. Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


Figure 5A , Live images of neurons co - transfected with SyGCaMP5 and MtDsRed or myc - ΔEF - Miro1 - IRES - MtDsRed ( ΔEF Miro ) before and during 10 Hz field stimulation with and without TTX treatments . Scale bar , 10 m . B , Average ΔF / F0 SyGCaMP5 traces from terminals treated with TTX ( red trace , ΔF / F0 = 2 . 5 0 . 5 ) , n = 10 neurons ( 62 terminals ) and non - treated terminals ( black trace , ΔF / F0 = 1 . 3 0 . 6 ) , n = 11 neurons ( 69 terminals ) co - transfected with MtDsRed , * p < 0 . 05 , t - test . C , Average ΔF / F0 SyGCaMP5 traces from terminals treated with TTX ( red trace , ΔF / F0 = 1 . 8 0 . 3 ) in n = 7 neurons ( 33 terminals ) and non - treated terminals ( black trace , ΔF / F0 = 2 . 3 0 . 6 ) in n = 7 neurons ( 32 terminals ) co - transfected with myc - ΔEF - Miro1 - IRES - MtDsRed ; p = 0 . 5 , t - test . Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent SEM .

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

Figure 5A, Live images of neurons co-transfected with SyGCaMP5 and MtDsRed or myc-ΔEF-Miro1-IRES-MtDsRed (ΔEF Miro) before and during 10 Hz field stimulation with and without TTX treatments. Scale bar, 10 m. B, Average ΔF/F0 SyGCaMP5 traces from terminals treated with TTX (red trace, ΔF/F0 = 2.5 0.5), n=10 neurons (62 terminals) and non-treated terminals (black trace, ΔF/F0 = 1.3 0.6), n=11 neurons (69 terminals) co-transfected with MtDsRed, *p<0.05, t-test. C, Average ΔF/F0 SyGCaMP5 traces from terminals treated with TTX (red trace, ΔF/F0 = 1.8 0.3) in n=7 neurons (33 terminals) and non-treated terminals (black trace, ΔF/F0 = 2.3 0.6) in n=7 neurons (32 terminals) co-transfected with myc-ΔEF-Miro1-IRES-MtDsRed; p=0.5, t-test. Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


Figure 1A . MS / MS analysis of peptides corresponding to amino acids 380 - 399 of STAT2 . 293T cells expressing flag - tagged STAT2 were used . B . Abundance of T387 phosphorylated STAT2 in 293T cells expressing flag - tagged STAT2 .

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Figure 1A. MS/MS analysis of peptides corresponding to amino acids 380-399 of STAT2. 293T cells expressing flag-tagged STAT2 were used.B. Abundance of T387 phosphorylated STAT2 in 293T cells expressing flag-tagged STAT2.


Figure 2A . U6A cells expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) . Cells were harvested after 4 h and total RNAs were analyzed by real - time PCR . Values are the means ± SD from three independent experiments . B . U6A cells expressing wild - type , T387A , or T387D STAT2 were treated with IFN - β ( 100 IU / ml ) . Cells were harvested after 4 h and total RNAs were analyzed by real - time PCR . Values are the means ± SD from three independent experiments . C , D . U6A cells expressing wild - type or T387A STAT2 were treated with IFN - β for 0 , 4 , 8 , or 24 h . Total RNAs were analyzed by using an Illumina HumanHT - 12 v4 Expression BeadChip array . The average signal for each probe was used to determine expression levels . Genes with average signals below 25 and detection P values greater than 0 . 01 in the untreated or treated cells were excluded from the analysis . Inductions of less than 2 - fold were not scored . The Venn diagram shows the numbers of genes induced by 1 . 2 fold or more in U6A cells expressing T387A STAT2 , relative to untreated control cells . The numbers in the table are fold changes calculated from the ratios T387A STAT2 treated / untreated and wild - type STAT2 treated / untreated .

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Figure 2A. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.B. U6A cells expressing wild-type, T387A, or T387D STAT2 were treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.C, D. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β for 0, 4, 8, or 24 h. Total RNAs were analyzed by using an Illumina HumanHT-12 v4 Expression BeadChip array. The average signal for each probe was used to determine expression levels. Genes with average signals below 25 and detection P values greater than 0.01 in the untreated or treated cells were excluded from the analysis. Inductions of less than 2-fold were not scored. The Venn diagram shows the numbers of genes induced by 1.2 fold or more in U6A cells expressing T387A STAT2, relative to untreated control cells. The numbers in the table are fold changes calculated from the ratios T387A STAT2 treated / untreated and wild-type STAT2 treated / untreated.


Figure 3A . HME cells expressing wild - type , T387A , or T387D STAT2 were seeded in 96 - well plates at 8000 cells / well . The cells were exposed to vesicular stomatitis virus ( VSV ) for 2 h , with or without pre - treatment with IFN - β ( 100 IU / ml ) . After 20 h , the amounts of the VSV - M and STAT2 proteins were analyzed by the Western method . B , C . HME cells expressing wild - type , T387A , or T387D STAT2 were seeded in 96 - well plates at 8000 cells / well . On the second day , the cells were treated with IFN - β ( 10 IU / ml ) for 2 h . The cells were then exposed to different multiplicities of infection of VSV for 2 h , followed by fresh media containing 10 IU / ml of IFN - β . The cells were analyzed by staining with crystal violet ( B ) after 48 h . B , C . HME cells expressing wild - type , T387A , or T387D STAT2 were seeded in 96 - well plates at 8000 cells / well . On the second day , the cells were treated with IFN - β ( 10 IU / ml ) for 2 h . The cells were then exposed to different multiplicities of infection of VSV for 2 h , followed by fresh media containing 10 IU / ml of IFN - β . The cells were analyzed by the MTT assay ( C ) after 48 h . D . U6A cells expressing wild - type , T387A , or T387D STAT2 were placed into 96 well plates ( 2000 cells / well ) and the cells were treated with IFN - β ( 1000 IU / ml ) for 96 h . Cell survival was analyzed by the MTT assay . Each experiment was carried out two independent times , with results similar to the representative examples that are shown .

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

Figure 3A. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. The cells were exposed to vesicular stomatitis virus (VSV) for 2 h, with or without pre-treatment with IFN-β (100 IU/ml). After 20 h, the amounts of the VSV-M and STAT2 proteins were analyzed by the Western method.B, C. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. On the second day, the cells were treated with IFN-β (10 IU/ml) for 2 h. The cells were then exposed to different multiplicities of infection of VSV for 2 h, followed by fresh media containing 10 IU/ml of IFN-β. The cells were analyzed by staining with crystal violet (B) after 48 h.B, C. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. On the second day, the cells were treated with IFN-β (10 IU/ml) for 2 h. The cells were then exposed to different multiplicities of infection of VSV for 2 h, followed by fresh media containing 10 IU/ml of IFN-β. The cells were analyzed by the MTT assay (C) after 48 h.D. U6A cells expressing wild-type, T387A, or T387D STAT2 were placed into 96 well plates (2000 cells/well) and the cells were treated with IFN-β (1000 IU/ml) for 96 h. Cell survival was analyzed by the MTT assay. Each experiment was carried out two independent times, with results similar to the representative examples that are shown.


Figure 4A . An immuno - precipitation was performed using whole cell lysates from cells expressing Flag - tagged wild - type STAT2 , and samples were analyzed by the Western method . The numbers in the chart are the proportions of T387 phosphorylated STAT2 in each cell type , calculated in comparison with 293T cells . B . HME cells expressing wild - type STAT2 were treated with IFN - β ( 100 IU / ml ) . Whole cell lysates harvested at the times indicated were analyzed by the Western method ; C . HME cells expressing wild - type STAT2 were seeded in complete medium . On the second day , the cells were re - fed with medium lacking supplements . On the third day , the cells were treated with each supplement for 4 h , and total lysates were analyzed by the Western method . 1 . EGF , 2 . Insulin , 3 . Hydrocortisone , 4 . GA - 1000 , 5 . BPE . D . HME cells were pretreated with hydrocortisone ( 0 . 04mg / ml and 0 . 4mg / ml ) for 3 h and then treated with IFN - β ( 100 IU / ml ) . Cells were harvested after 4 h and total RNAs were analyzed by real - time PCR . Values are the means ± SD from three independent experiments . F . HCC827 and LnCAP cells expressing wild - type STAT2 were treated with CDK inhibitors ( 1 µM ) for 4 h and whole - cell lysates were analyzed by the Western method . 1 . Flavopiridol , 2 . AT7519 , 3 . Dinaciclib .

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

Figure 4A. An immuno-precipitation was performed using whole cell lysates from cells expressing Flag-tagged wild-type STAT2, and samples were analyzed by the Western method. The numbers in the chart are the proportions of T387 phosphorylated STAT2 in each cell type, calculated in comparison with 293T cells.B. HME cells expressing wild-type STAT2 were treated with IFN-β (100 IU/ml). Whole cell lysates harvested at the times indicated were analyzed by the Western method;C. HME cells expressing wild-type STAT2 were seeded in complete medium. On the second day, the cells were re-fed with medium lacking supplements. On the third day, the cells were treated with each supplement for 4 h, and total lysates were analyzed by the Western method. 1. EGF, 2. Insulin, 3. Hydrocortisone, 4. GA-1000, 5. BPE.D. HME cells were pretreated with hydrocortisone (0.04mg/ml and 0.4mg/ml) for 3 h and then treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.F. HCC827 and LnCAP cells expressing wild-type STAT2 were treated with CDK inhibitors (1 µM) for 4 h and whole-cell lysates were analyzed by the Western method. 1. Flavopiridol, 2. AT7519, 3. Dinaciclib.


Figure 5A . U6A cells expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 30 min or were untreated . The cells were washed with PBS and the media was replaced with fresh media containing staurosporine ( 500 nM ) . Whole - cell lysates were harvested and analyzed by the Western method ; B . U6A cells expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 4 h or were untreated . Whole - cell lysates were used for immunoprecipitations of IRF9 and Flag - STAT2 . C . U2A cells expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 4 h or were untreated . Whole - cell lysates were used for immunoprecipitation of Flag - STAT2 . D . U3A cells expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 4 h or were untreated . Whole - cell lysates were used for immunoprecipitation of Flag - tagged STAT2 . E . EMSAs using an ISRE probe . Extracts from U6A cells expressing wild - type or T387A STAT2 treated with IFN - β for 1 h were analyzed . The position of ISGF3 is labeled .

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 O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5A. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 30 min or were untreated. The cells were washed with PBS and the media was replaced with fresh media containing staurosporine (500 nM). Whole-cell lysates were harvested and analyzed by the Western method;B. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitations of IRF9 and Flag-STAT2.C. U2A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitation of Flag-STAT2.D. U3A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitation of Flag-tagged STAT2.E. EMSAs using an ISRE probe. Extracts from U6A cells expressing wild-type or T387A STAT2 treated with IFN-β for 1 h were analyzed. The position of ISGF3 is labeled.


Figure 1DICER - KO B cells were co - cultured for 24 hours with OTII - derived T cells in the presence or absence of OVA . Representative confocal microscopy images of CMAC - stained conjugates formed between B lymphocytes ( isolated from DICER - KO mouse splenocytes and pre - activated with a mixture of LPS plus IL - 4 ) and CD4 + T cells ( from OVA - specific OT - II transgenic mice ) ; conjugates were formed either in the presence or in the absence of OVA . A representative experiment of at least 3 independent experiments is shown . DICER - KO B cells were co - cultured for 24 hours with OTII - derived T cells in the presence or absence of OVA . showing the percentage of B cells forming conjugatesDICER - KO B cells were co - cultured for 24 hours with OTII - derived T cells in the presence or absence of OVA . the accumulation at the IS of the TCR Conjugates were assessed using the Image J plug - in for IS analysisDICER - KO B cells were co - cultured for 24 hours with OTII - derived T cells in the presence or absence of OVA . the accumulation of the actin at the IS Conjugates were assessed using the Image J plug - in for IS analysisDICER - KO B cells were co - cultured for 24 hours with OTII - derived T cells in the presence or absence of OVA . the distance from the CD4 + T cell MTOC to the contact site with the B cell MTOC - B - cell distance was measured with Imaris analysis software . DICER - KO B cells were co - cultured for 24 hours with OTII - derived T cells in the presence or absence of OVA . Flow cytometry analysis of B lymphocytes co - cultured with OT - II isolated T cells in the presence or absence of OVA , showing IgG1 expression after 24h of co - culture , DICER - KO B cells were co - cultured for 24 hours with OTII - derived T cells in the presence or absence of OVA . Flow cytometry analysis of B lymphocytes co - cultured with OT - II isolated T cells in the presence or absence of OVA B cell proliferation after 24h , 48h and 72h co - culture ( Ki67 expression in live B cells and cell violet tracer dilution , respectively ) DICER - KO B cells were co - cultured for 24 hours with OTII - derived T cells in the presence or absence of OVA . B cell proliferation after 24h , 48h and 72h co - culture ( Ki67 expression in live B cells and cell violet tracer dilution , respectively ) , DICER - KO B cells were co - cultured for 24 hours with OTII - derived T cells in the presence or absence of OVA . Flow cytometry analysis of B lymphocytes co - cultured with OT - II isolated T cells in the presence or absence of OVA survival ( staining with the live marker DAPI ) DICER - KO B cells were co - cultured for 24 hours with OTII - derived T cells in the presence or absence of OVA . Flow cytometry analysis of B lymphocytes co - cultured with OT - II isolated T cells in the presence or absence of OVA apoptosis ( cleaved Caspase - 3 expression ) 24h , 48h and 72h after IS formation .

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 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 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 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 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

Figure 1DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Representative confocal microscopy images of CMAC-stained conjugates formed between B lymphocytes (isolated from DICER-KO mouse splenocytes and pre-activated with a mixture of LPS plus IL-4) and CD4+ T cells (from OVA-specific OT-II transgenic mice); conjugates were formed either in the presence or in the absence of OVA. A representative experiment of at least 3 independent experiments is shown.DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. showing the percentage of B cells forming conjugatesDICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. the accumulation at the IS of the TCR Conjugates were assessed using the Image J plug-in for IS analysisDICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. the accumulation of the actin at the IS Conjugates were assessed using the Image J plug-in for IS analysisDICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. the distance from the CD4+ T cell MTOC to the contact site with the B cell MTOC-B-cell distance was measured with Imaris analysis software.DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA, showing IgG1 expression after 24h of co-culture,DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA B cell proliferation after 24h, 48h and 72h co-culture (Ki67 expression in live B cells and cell violet tracer dilution, respectively)DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. B cell proliferation after 24h, 48h and 72h co-culture (Ki67 expression in live B cells and cell violet tracer dilution, respectively),DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA survival (staining with the live marker DAPI)DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA apoptosis (cleaved Caspase-3 expression) 24h, 48h and 72h after IS formation.


Figure 2CD4 + T and DICER - KO B cells were co - cultured for 24 hours with or without OVA , and flow - cytometry sorted B cells were analyzed by small RNA next generation sequencing ( NGS ) . Heat map showing microRNAs significantly upregulated in B cells after co - culture with T cells in the presence of OVA . Data are from 3 independent experiments ( samples 1 - 3 ) , and only microRNAs with an adjusted P - value < 0 . 02 are shown . Fold increase in the B cell content of upregulated microRNAs in the presence of OVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Heat map showing NGS analysis of microRNAs differentially expressed in activated WT C57BL / 6 CD4 + T cells and their secreted exosomes . Data are from 3 independent experiments ( samples a - c ) , and only microRNAs with an adjusted P - value < 0 . 02 are shown . Mean tags per million ( TPMs ) for the differentially expressed microRNAs under study in exosomes from activated CD4 + T cells . Bars represent the mean ± SEM . Quantitative real - time PCR of upregulated microRNAs after co - culture of OT - II derived CD4 + T cells with DICER - KO B cells activated with LPS + IL - 4 Bars represent the mean ± SEM of at least three independent experiments and data were obtained by the 2 - ∆ ∆ Ct method using Biogazelle software . Results are expressed as a proportion of the microRNA expression in non - OVA containing co - cultures , with normalization to the small nucleolar RNAs RNU1A1 and RNU5G . Quantitative real - time PCR of upregulated microRNAs after co - culture of OT - II derived CD4 + T cells with DICER - KO B cells activated with CD40 + IgM Bars represent the mean ± SEM of at least three independent experiments and data were obtained by the 2 - ∆ ∆ Ct method using Biogazelle software . Results are expressed as a proportion of the microRNA expression in non - OVA containing co - cultures , with normalization to the small nucleolar RNAs RNU1A1 and RNU5G . Quantitative real - time PCR showing the expression of down - modulated mRNA targets upon IS formation between OT - II CD4 + T cells and DICER - KO B cells pre - activated with LPS + IL - 4 Results are the means ± SEM of at least four independent experiments and are expressed as a proportion of mRNA expression in the non - OVA condition , with normalization to GAPDH . Quantitative real - time PCR showing the expression of down - modulated mRNA targets upon IS formation between OT - II CD4 + T cells and DICER - KO B cells pre - activated with CD40 + IgM Results are the means ± SEM of at least four independent experiments and are expressed as a proportion of mRNA expression in the non - OVA condition , with normalization to GAPDH . Fluorescently tagged mimics of the microRNAs mmu - miR - 20a - 5p , mmu - miR - 25 - 3p and mmu - miR - 155 - 3p were nucleofected into activated B lymphocytes from C57 / BL6 mice . Quantitative real - time PCR showing expression of nucleofected microRNA mimics in FACS - sorted FAM + B cells with normalization to RNU1A1 and RNU5GFluorescently tagged mimics of the microRNAs mmu - miR - 20a - 5p , mmu - miR - 25 - 3p and mmu - miR - 155 - 3p were nucleofected into activated B lymphocytes from C57 / BL6 mice . Quantitative real - time PCR showing expression of Bcl2l11 and Pten mRNA targets in mimic - nucleofected B cells with normalization to GAPDHFlow cytometry analysis of mimic - nucleofected B lymphocytes . Dot - plots show B220 + IgG1 + B cells 48h after mimic nucleofection from at least 4 independent experiments and bar charts show mean values ± SEM of 4 independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 2CD4+ T and DICER-KO B cells were co-cultured for 24 hours with or without OVA, and flow-cytometry sorted B cells were analyzed by small RNA next generation sequencing (NGS). Heat map showing microRNAs significantly upregulated in B cells after co-culture with T cells in the presence of OVA. Data are from 3 independent experiments (samples 1-3), and only microRNAs with an adjusted P-value < 0.02 are shown. Fold increase in the B cell content of upregulated microRNAs in the presence of OVA; *P<0.05, **P<0.01, ***P<0.001.Heat map showing NGS analysis of microRNAs differentially expressed in activated WT C57BL/6 CD4+ T cells and their secreted exosomes. Data are from 3 independent experiments (samples a-c), and only microRNAs with an adjusted P-value < 0.02 are shown.Mean tags per million (TPMs) for the differentially expressed microRNAs under study in exosomes from activated CD4+ T cells. Bars represent the mean ± SEM.Quantitative real-time PCR of upregulated microRNAs after co-culture of OT-II derived CD4+ T cells with DICER-KO B cells activated with LPS+IL-4 Bars represent the mean ± SEM of at least three independent experiments and data were obtained by the 2-∆∆Ct method using Biogazelle software. Results are expressed as a proportion of the microRNA expression in non-OVA containing co-cultures, with normalization to the small nucleolar RNAs RNU1A1 and RNU5G.Quantitative real-time PCR of upregulated microRNAs after co-culture of OT-II derived CD4+ T cells with DICER-KO B cells activated with CD40+IgM Bars represent the mean ± SEM of at least three independent experiments and data were obtained by the 2-∆∆Ct method using Biogazelle software. Results are expressed as a proportion of the microRNA expression in non-OVA containing co-cultures, with normalization to the small nucleolar RNAs RNU1A1 and RNU5G.Quantitative real-time PCR showing the expression of down-modulated mRNA targets upon IS formation between OT-II CD4+ T cells and DICER-KO B cells pre-activated with LPS+IL-4 Results are the means ± SEM of at least four independent experiments and are expressed as a proportion of mRNA expression in the non-OVA condition, with normalization to GAPDH.Quantitative real-time PCR showing the expression of down-modulated mRNA targets upon IS formation between OT-II CD4+ T cells and DICER-KO B cells pre-activated with CD40+IgM Results are the means ± SEM of at least four independent experiments and are expressed as a proportion of mRNA expression in the non-OVA condition, with normalization to GAPDH.Fluorescently tagged mimics of the microRNAs mmu-miR-20a-5p, mmu-miR-25-3p and mmu-miR-155-3p were nucleofected into activated B lymphocytes from C57/BL6 mice. Quantitative real-time PCR showing expression of nucleofected microRNA mimics in FACS-sorted FAM+ B cells with normalization to RNU1A1 and RNU5GFluorescently tagged mimics of the microRNAs mmu-miR-20a-5p, mmu-miR-25-3p and mmu-miR-155-3p were nucleofected into activated B lymphocytes from C57/BL6 mice. Quantitative real-time PCR showing expression of Bcl2l11 and Pten mRNA targets in mimic-nucleofected B cells with normalization to GAPDHFlow cytometry analysis of mimic-nucleofected B lymphocytes. Dot-plots show B220+ IgG1+ B cells 48h after mimic nucleofection from at least 4 independent experiments and bar charts show mean values ± SEM of 4 independent experiments.