HT - 29 were treated as indicated . release of IL - 8 in culture media was analyzed by ELISA ( H ) . Data are means ± of four ( H ) independent experiments ( * * * P < 0 . 001 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were treated as indicated. The release of IL-8 in cell culture media was analyzed by ELISA (H). Data are means ± SEM of four (H) independent experiments (***P<0.001, ****P<0.0001, ANOVA).


HT - 29 were treated as indicated . Cxcl8 mRNA relative to untreated NS samples were measured by qPCR after 6 hours of treatment ( I ) . Data are means ± of three ( I ) independent experiments ( * * P < 0 . 01 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were treated as indicated. Cxcl8 mRNA levels relative to untreated NS samples were measured by qPCR after 6 hours of treatment (I). Data are means ± SEM of three (I) independent experiments (**P<0.01, ****P<0.0001, ANOVA).


HT - 29 were treated as indicated . lysates were analyzed by Western blotting ( J ) . Data information : MLKL is indicated with an arrowhead .

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

HT-29 cells were treated as indicated. Cell lysates were analyzed by Western blotting (J). Data information: Cleaved MLKL is indicated with an arrowhead.


HT - 29 were treated as indicated . IL - 8 release was analyzed in culture media by ELISA ( K ) . Data are means ± of four ( K ) independent experiments ( * * P < 0 . 01 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were treated as indicated. IL-8 release was analyzed in cell culture media by ELISA (K). Data are means ± SEM of four (K) independent experiments (**P<0.01, ****P<0.0001, ANOVA).


A . Experimental demonstration . A1 . Illustration of mapping principle between ChR2 - expressed PV neurons ( green ) regular pyramidal neuron ( Pyr , orange ) . A2 . Acute slices were obtained recorded at ZT0 or ZT12 . A3 . Coronal brain atlas showing visual cortex ( Top left panel , modified from MBL mouse brain atlas ) . Yellow box highlights region of experiment . Top right panel , reference image taken with a 10x objective from a slice prepared from a PV : Ai32 mouse . 0 . 45 mm2 shaded cubic was divided into 15 x 15 grids for independent stimulation . arrowhead indicates position of recording electrode . Bottom panel , representative raw traces of - evoked IPSCs ( LEIPSCs ) recorded from one pyramidal neuron . A4 . spatial distribution of LEIPSCs from example in A3 . A5 . of LEIPSCs in A4 .

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

A. 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 . average heatmaps at ZT0 ZT12 after soma alignment . Soma position was indicated by blue arrow head . C . Comparison of cumulative distribution ( Kolmogorov - Smirnov [ K - S ] ; P = 0 . 002 ) mean ( Inset . ZT0 : 813 . 3 ± 53 . 13 pA ; ZT12 : 1306 ± 93 . 73 pA ; P < 0 . 0001 ; Mann - Whitney U ) of max LEIPSC amplitude between ZT0 ZT12 . D . Comparison of cumulative distribution ( K - S , P = 0 . 0016 ) mean ( Inset . ZT0 : 10 . 44 ± 1 . 44 nA ; ZT12 : 18 . 87 ± 1 . 44 nA ; P < 0 . 0001 ; Mann - Whitney U ) of sum LEIPSC amplitude between ZT0 ZT12 . Data information : Sample sizes used in this figure : ZT0 : 48 from 9 mice ; ZT12 : 51 from 9 mice . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

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 . average heatmaps of LEIPSCs after pia aligned to reference image in A3 . blue overlay traces on side of map depicted marginal distribution of sum LEIPSCs for ZT0 ( blue ) ZT12 ( blue ) . F . Comparison of cumulative distribution ( K - S , P = 0 . 0021 ) mean ( Inset , ZT0 : 0 . 067 ± 0 . 004 mm2 ; ZT12 : 0 . 089 ± 0 . 0036 mm2 ; P = 0 . 0003 ; Mann - Whitney U ) inhibitory input between ZT0 ZT12 . Data information : Sample sizes used in this figure : ZT0 : 48 from 9 mice ; ZT12 : 51 from 9 mice . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

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.


A . 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 ) . Bottom right : cumulative probability histogram of inter - event intervals ( K - S , 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 ) . Bottom right : cumulative probability histogram of inter - event intervals of mIPSCs ( K - S , P < 0 . 0001 ) . C . mEPSC amplitude kinetics did not differ between ZT0 ZT12 . Top : average traces of well - isolated events . Bottom left : mEPSC amplitude ( ZT0 : 20 . 51 ± 0 . 78pA ; ZT12 : 21 . 26 ± 0 . 75pA ; P = 0 . 34 ; Mann - Whitney U ) . Bottom right : distribution of mEPSC amplitude of events ( K - S , P < 0 . 01 ) . D . mIPSC amplitude kinetics did not differ between ZT0 ZT12 . Top : average traces of well - isolated mIPSCs . Bottom left : mIPSC amplitude ( ZT0 : 41 . 31 ± 1 . 91pA ; ZT12 : 44 . 39 ± 1 . 31pA ; P = 0 . 35 ; Mann - Whitney U ) . Bottom right : cumulative distribution of mIPSC amplitudes ( K - S , P < 0 . 0001 Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 .

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

A. 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.


B . Charges of sEPSCs recorded at phase were higher compared to those at 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 ) . A copy of 24 - h sEPSCs was drawn with a dashed to demonstrate daily rhythm . C . Charges of sIPSCs oscillate during / 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 ) . A copy of 24 - h sEPSCs was drawn with a dashed to demonstrate daily rhythm . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

B. 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 . exposure prevented increase in sEPSCs that normally occurs at ZT12 ( ZT12 as reported in B ; - ZT12 : 5 . 46 ± 0 . 43 nC ; P = 0 . 0082 ; Mann - Whitney U ) . F . exposure did not significantly affect reduction of sIPSCs ( ZT12 as reported in C ; - ZT12 : 33 . 43 ± 1 . 74 nC ; P = 0 . 07 ; Mann - Whitney U ) . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

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 ) . I . Sleep deprivation abolished reduction of sIPSC ( ZT4 as reported in C ; SD - ZT4 : 47 . 13 ± 3 . 39 nC ; P < 0 . 0001 ; Mann - Whitney U ) . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

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.


B - E . Slices were obtained at ZT12 ( B - C ) or ZT0 ( D - E ) pre - incubated with 50 µM CARB for more than 1h before recording mIPSCs in presence of drug . Slices from same 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 ) , but not ZT0 ( CTL : 11 . 79 ± 1 . 00 Hz ; CARB : 11 . 47 ± 1 . 60 Hz ; P = 0 . 39 ; Mann - Whitney U ) . 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 ) . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

B-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 ) amplitude ( G , CTL : 45 . 88 ± 2 . 87 pA ; Atr : 43 . 64 ± 2 . 40 pA ; P = 0 . 70 ; Wilcoxon signed - rank ) recorded at ZT0 with acute atropine ( Atr ) wash on . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

F-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 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 ) 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 is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

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 ) 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 is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

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.


A1 . Diagram illustrating in vivo two - photon imaging timeline . A2 . GCaMP6s ( green ) was expressed in V1 of PV : Ai9 mice ( red : td - Tomato ) . Scale : 500 µm . A3 . Schematics of calcium imaging showing a mouse that was head - fixed free running on an air - suspended arena during 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

A1. 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 ZT12 . Bottom panels , Heatmaps showing spontaneous ΔF / F0 time - series traces of PV neurons imaged at ZT0 ZT12 . color code represents magnitude of ΔF / F0 ( % ) . C . Cumulative probability distribution of sum ΔF / F0 of PV neurons imaged at ZT0 ZT12 ( K - S ; P = 0 . 02 ) . Inset : mean sum ΔF / F0 ( ZT0 : 401 . 6 ± 19 . 55 ; ZT12 : 559 . 1 ± 35 . 29 ; P = 0 . 0002 ; Mann - Whitney U ) . 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 is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

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.


B . Heatmaps showing spontaneous ΔF / F0 time - series traces of PV neurons imaged at ZT4 SD _ ZT4 . color code represents magnitude of ΔF / F0 ( % ) . C . Cumulative probability distribution of sum ΔF / F0 of PV neurons imaged at ZT4 SD _ ZT4 ( K - S ; P = 0 . 0001 ) . Inset : mean sum ΔF / F0 ( ZT4 : 322 . 4 ± 18 . 64 ; SD _ ZT4 : 424 . 0 ± 21 . 54 ; P = 0 . 0004 ; Mann - Whitney U ) . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

B. 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 spontaneous ΔF / F0 time - series traces of PV neurons imaged at SD _ ZT4 _ SD . color code represents magnitude of ΔF / F0 ( % ) . E . Cumulative probability distribution of sum ΔF / F0 of PV neurons imaged at SD _ ZT4 _ SD ( K - S ; P = 0 . 015 ) . Inset : mean sum ΔF / F0 ( SD _ ZT4 : 375 . 1 ± 20 . 19 ; _ SD : 293 . 8 ± 16 . 55 ; P = 0 . 0022 ; Mann - Whitney U ) . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

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.


A . Top panel , Example spontaneous ΔF / F0 traces from three putative pyramidal neurons repetitively imaged at ZT0 ZT12 . Bottom panels , Heatmaps showing spontaneous ΔF / F0 time - series traces of putative pyramidal neurons imaged at ZT0 ZT12 . B . cumulative distribution of sum ΔF / F0 of putative pyramidal neurons judged by skewness ( K - S ; P < 0 . 0001 ) . Inset : means of sum ΔF / F0 ( ZT0 : 679 . 5 ± 38 . 22 ; ZT12 : 485 . 8 ± 32 . 22 ; P < 0 . 0001 ; Mann - Whitney U ) . C . Mean activity of putative pyramidal neurons indicated by Z score imaged at four timepoints . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

A. 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 showing GCaMP6s ( green ) ChR2 - mCherry ( red ) expression in V1 dLGN respectively . optical fiber track was also labeled . ( Scale : 500 µm ) . D3 . Response profile of an example neurons for dLGN stimulation during / cycle . Horizontal blue brief lines represent stimulus frequency .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 responsive neurons ( % ΔF / F0 ) . Bottom : mean Ca2 + response waveform for mouse ( dotted color lines ) mice ( thick black gray lines ) . Trials were aligned to LED onset time ( gray solid lines ) . Data information : Sample is indicated as ( mice , ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 . fraction of neurons responsive to dLGN stimulus in ZT0 ZT12 . Each gray represents one imaging mouse . Black circles error bars represent mean ( ZT0 : 8 . 51 ± 1 . 86 ; ZT12 : 5 . 96 ± 1 . 39 ; P = 0 . 015 ; Wilcoxon signed - rank ; n = 9 mice ) . H . Response fidelity of each in ZT0 ZT12 . Black circles error bars represent mean ( ZT0 : 0 . 42 ± 0 . 022 ; ZT12 : 0 . 31 ± 0 . 023 ; P < 0 . 0001 ; Wilcoxon signed - rank ; n = 195 ) . I . Comparison of peak evoked response of each in ZT0 ZT12 . Black circles error bars represent mean ( ZT0 : 81 . 01 ± 4 . 79 ; ZT12 : 48 . 42 ± 3 . 82 ; P < 0 . 0001 ; Wilcoxon signed - rank ; n = 195 ) . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at * P < 0 .

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

G. 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


Effects of DC Beva treatment in 786 - O orthoxenograft . Evaluation of progression ( average ± SD ) Box plots indicate median , Q1 / Q3 max / min value whiskers in Control ( black ) , DC101 ( red ) Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney , Mantel - Cox for survival where * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 DC * p < 0 . 05 Beva # p < 0 . 05 vs Control ) .

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

Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of tumor progression (average±SD) Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).


Effects of DC Beva treatment in 786 - O orthoxenograft . Evaluation of ( C ) at sacrifice , ( D ) vessel number , Box plots indicate median , Q1 / Q3 max / min value whiskers in Control ( black ) , DC101 ( red ) Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney , Mantel - Cox for survival where * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 DC * p < 0 . 05 Beva # p < 0 . 05 vs Control ) .

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

Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of (C) tumor weight at sacrifice, (D) vessel number, Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).


Effects of DC Beva treatment in 786 - O orthoxenograft . Evaluation of of necrotic by histology , Box plots indicate median , Q1 / Q3 max / min value whiskers in Control ( black ) , DC101 ( red ) Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney , Mantel - Cox for survival where * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 DC * p < 0 . 05 Beva # p < 0 . 05 vs Control ) .

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

Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of percentage of necrotic area by histology, Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).


Effects of DC Beva treatment in 786 - O orthoxenograft . Evaluation of survival by Kaplan Meier plot in control treated . Box plots indicate median , Q1 / Q3 max / min value whiskers in Control ( black ) , DC101 ( red ) Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney , Mantel - Cox for survival where * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 DC * p < 0 . 05 Beva # p < 0 . 05 vs Control ) .

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

Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of survival by Kaplan Meier plot in control and treated animals. Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).


G ) Representative invasive front profile of 786 - O control short - term DC Beva treated tumors in vimentin stained sections ( 4X ) . Front ( red ) rear ( ) edges of protrusion into kidney determine depth of invasion ( green arrow ) perpendicularly connecting red lines . Box plot shows of invasion indicating median , Q1 / Q3 max / min value whiskers of fold - invasion ( ∆ invasion ) in treated vs control tumors ( 7 samples / group ; * p < 0 . 05 by Mann - Whitney ) .

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

G) Representative invasive front profile of 786-O control and short-term DC and Beva treated tumors in vimentin stained sections (4X). Front (red line) and rear (white line) edges of tumor protrusion into kidney determine the depth of invasion (green arrow) perpendicularly connecting red and white lines. Box plot shows the quantification of invasion indicating median, Q1/Q3 and max/min value whiskers of fold-invasion (∆ invasion) in treated vs control tumors (7 samples/group; *p < 0.05 by Mann-Whitney test).


H ) Evaluation of lung micrometastases in survival experiments . Incidence ( pie chart , * p < 0 . 05 by Chi - square ) proportion of mice with 0 , 1 , 2 or > 3 metastases ( mets ) per lungs ( * p < 0 . 05 by Mann - Whitney ) are shown .

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

H) Evaluation of lung micrometastases in survival experiments. Incidence (pie chart, *p < 0.05 by Chi-square) and proportion of mice with 0, 1, 2 or > 3 metastases (mets) per lungs (*p < 0.05 by Mann-Whitney test) are shown.


Overall mass architecture of paired human specimens Ren - PDOX tumors observed by HE ( 20X ) . TNM staging Fuhrman grade ( F ) of implanted tumors are reported . For Ren - PDOXs , lung micrometastases in HE vimentin stained sections are shown ( 40X ) .

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

Overall mass architecture of paired human specimens and Ren-PDOX tumors observed by HE staining (20X). TNM staging and Fuhrman grade (F) of implanted tumors are reported. For Ren-PDOXs, lung micrometastases in HE and vimentin stained sections are shown (40X).


Human to mouse stroma substitution . CD34 / CD31 for respectively human ( h ) mouse ( m ) blood vessels in original human specimens paired Ren13 28 - PDOX are shown ( 20X ) . Ren - PDOX OT referred to generated from first implantation of human specimen in mice . Insets show human mouse kidney as positive / negative controls of ( 20X ) .

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

Human to mouse stroma substitution. CD34/CD31 staining for respectively human (h) and mouse (m) blood vessels in original human specimens and paired Ren13 and 28-PDOX are shown (20X). Ren-PDOX OT referred to tumor generated from first implantation of human specimen in mice. Insets show human and mouse kidney as positive/negative controls of staining (20X).


Frequency histogram of SNV / or indels found in Ren - PDOX by high - throughput sequencing in Ren13 , Ren28 , Ren36 , Ren86 ( targeted - NGS by Haloplex ) Ren50 - PDOX ( WES ) . Only non - synonymous / stop gain SNVs frameshift indels novel or with MAF < 0 . 01 are included .

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Frequency histogram of SNV and/or indels found in Ren-PDOX by high-throughput sequencing in Ren13, Ren28, Ren36, Ren86 (targeted-NGS analysis by Haloplex) and Ren50-PDOX (WES). Only non-synonymous/stop gain SNVs and frameshift indels novel or with MAF<0.01 are included.


Effects of DC Beva treatments on Ren13 , Ren86 , Ren28 Ren50 - PDOX . Evaluation of progression average SD in control ( black ) compared to DC ( red , * p < 0 . 05 ) Beva ( blue , # p < 0 . 05 ) treatments from 5 - 13 per treatment group by Mann Whitney .

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Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Evaluation of tumor progression average and SD in control (black) compared to DC (red, *p < 0.05) and Beva (blue, # p < 0.05) treatments from 5-13 animals per tumor and treatment group by Mann Whitney test.


Effects of DC Beva treatments on Ren13 , Ren86 , Ren28 Ren50 - PDOX . of in control treated tumors from 4 - 5 samples / / treatment group by Mann Whitney . Box plots indicate median , Q1 / Q3 max / min value whiskers .

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Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of tumor weight in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers.


Effects of DC Beva treatments on Ren13 , Ren86 , Ren28 Ren50 - PDOX . of vessel number in control treated tumors from 4 - 5 samples / / treatment group by Mann Whitney . Box plots indicate median , Q1 / Q3 max / min value whiskers .

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Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of vessel number in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers.


Effects of DC Beva treatments on Ren13 , Ren86 , Ren28 Ren50 - PDOX . of necrosis in control treated tumors from 4 - 5 samples / / treatment group by Mann Whitney . Box plots indicate median , Q1 / Q3 max / min value whiskers .

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Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of tumor necrosis in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers.


Effects of DC Beva treatments on Ren13 , Ren86 , Ren28 Ren50 - PDOX . Evaluation of overall survival comparing Control ( black ) , DC101 ( red ) Beva ( blue ) treated mice from 4 - 13 mice / / treatment group by Mantel - Cox where * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Evaluation of overall survival comparing Control (black), DC101 (red) and Beva (blue) treated mice from 4-13 mice/tumor/treatment group by Mantel-Cox test where *p < 0.05, **p < 0.01 and ***p < 0.001.


Effects of DC Beva treatment on Ren13 , Ren86 , Ren28 Ren50 - PDOX on capsular invasion Incidence of Capsular invasion ( stripes ) in each model treatment . 5 - 8 per model treatment group were analyzed by Chi - square where * p < 0 . 05 , * * p < 0 . 01 .

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Effects of DC and Beva treatment on Ren13, Ren86, Ren28 and Ren50-PDOX on capsular invasion Incidence of Capsular invasion (stripes) in each model and treatment. 5-8 animals per model and treatment group were analyzed by Chi-square test where *p < 0.05, **p < 0.01.


C ) Effects of DC Beva treatment on Ren13 , Ren86 , Ren28 Ren50 - PDOX on front of invasion Representative of Vimentin ( 4X ) , as described in Figure 1 , box plot of fold - invasion ( ∆ invasion ) of treated vs control tumors indicating median , Q1 / Q3 max / min value whiskers . 6 - 12 per model treatment group were analyzed by Mann - Whitney where * p < 0 . 05 , * * p < 0 . 01 .

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C) Effects of DC and Beva treatment on Ren13, Ren86, Ren28 and Ren50-PDOX on tumor front of invasion Representative images of Vimentin staining (4X), as described in Figure 1, and box plot quantification of fold-invasion (∆ invasion) of treated vs control tumors indicating median, Q1/Q3 and max/min value whiskers. 6-12 animals per model and treatment group were analyzed by Mann-Whitney test where *p < 0.05, **p < 0.01.


D ) Effects of sunitinib treatment on Ren13BM Ren28 on capsular invasion front invasion . Bar graphs show incidence of capsular invasion ( stripes ) in control sunitinib treated tumors . 3 - 9 per model treatment group were analyzed by Chi - square where * p < 0 . 05 , * * p < 0 . 01 . Box plots represent median , Q1 / Q3 max / min value whiskers of fold - invasion ( ∆ invasion ) of sunitinib - treated vs control tumors . 5 - 8 per model treatment group were analyzed by Mann - Whitney . * p < 0 . 05 , * * p < 0 . 01 .

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D) Effects of sunitinib treatment on Ren13BM and Ren28 on capsular invasion and tumor front invasion. Bar graphs show incidence of capsular invasion (stripes) in control and sunitinib treated tumors. 3-9 animals per model and treatment group were analyzed by Chi-square test where *p < 0.05, **p < 0.01. Box plots represent median, Q1/Q3 and max/min value whiskers of fold-invasion (∆ invasion) of sunitinib-treated vs control tumors. 5-8 animals per model and treatment group were analyzed by Mann-Whitney test. *p < 0.05, **p < 0.01.


Evaluation of lung micrometastases using HE vimentin - stained lung sections upon antiangiogenic therapies in Ren13BM Representative of lung metastasis in Ren13BM upon antiangiogenic therapies .

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Evaluation of lung micrometastases using HE and vimentin-stained lung sections upon antiangiogenic therapies in Ren13BM Representative images of lung metastasis in Ren13BM upon antiangiogenic therapies.


Evaluation of lung micrometastases using HE vimentin - stained lung sections upon antiangiogenic therapies in Ren13BM Ren28 . proportion of mice with 0 , 1 , 2 or > 3 metastases ( mets ) per lungs ( * p < 0 . 05 by Mann - Whitney ) are shown .

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Evaluation of lung micrometastases using HE and vimentin-stained lung sections upon antiangiogenic therapies in Ren13BM and Ren28. proportion of mice with 0, 1, 2 or > 3 metastases (mets) per lungs (*p < 0.05 by Mann-Whitney test) are shown.


specific ( human ) enriched gene sets in Ren13 Ren50 Ren - PDOX tumors , after GSEA from RNAseq data . Gene sets most significantly enriched in Ren13 or Ren50 are indicated in table ( left ) , including their normalized enrichment score ( NES ) , p - value false discovery rate ( FDR q - value ) . Heatmaps illustrate expression of genes responsible for enrichment of KRAS signaling up down in Ren13 Ren50 , respectively ( right ) .

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Tumor specific (human) enriched gene sets in Ren13 and Ren50 Ren-PDOX tumors, after GSEA analysis from RNAseq data. Gene sets most significantly enriched in Ren13 or Ren50 are indicated in the table (left), including their normalized enrichment score (NES), p-value and false discovery rate (FDR q-value). Heatmaps illustrate expression levels of the genes responsible for the enrichment of KRAS signaling up and down in Ren13 and Ren50, respectively (right).


Validation of candidate gene expression in unique series of 39 RCC ( GSE29609 ) , where microvascular invasion ( MVI ) renal vein involvement ( RV ) are specifically annotated . Progressive invasion relates to three progressive degrees of invasion toward vena cava in this series : 1st , no invasion ; 2nd , microvascular invasion ; 3rd , renal vein involvement . gene expression is shown for each variable condition in box plots representing median , Q1 / Q3 max / min value whiskers , * p < 0 . 05 * * p < 0 . 01 by Mann - Whitney .

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Validation of candidate gene expression in unique series of 39 RCC patients (GSE29609), where microvascular invasion (MVI) and renal vein involvement (RV) are specifically annotated. Progressive invasion relates to the three progressive degrees of invasion toward the vena cava in this series: 1st, no invasion; 2nd, microvascular invasion; and 3rd, renal vein involvement. The gene expression is shown for each variable and condition in box plots representing median, Q1/Q3 and max/min value whiskers, *p < 0.05 **p < 0.01 by Mann-Whitney test.


Representative of ALDH1A3 expression on pro - invasive ( Ren13 Ren86 ) non - pro - invasive ( Ren28 Ren50 ) tumors .

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Representative images of ALDH1A3 expression on pro-invasive (Ren13 and Ren86) and non-pro-invasive (Ren28 and Ren50) tumors.


Confirmation of ALDH1A3 mRNA of gene expression in pro - invasive ( Ren13 Ren86 ) non - pro - invasive ( Ren28 Ren50 ) tumors . Average ± SD of 4 - 6 independent replicates per group were analyzed by Mann - Whitney * p < 0 . 05 * * p < 0 . 01 .

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Confirmation of ALDH1A3 mRNA levels of gene expression in pro-invasive (Ren13 and Ren86) and non-pro-invasive (Ren28 and Ren50) tumors. Average ± SD of 4-6 independent replicates per group were analyzed by Mann-Whitney test *p < 0.05 **p < 0.01.


Association of pre - treatment ALDH1A3 by on a series of Ren - PDOX models treated with sunitinib . ( C ) Representation of ALDH1A3 expression on pro - invasive non - pro - invasive Ren - PDOX after suntinib treatment ( * * p < 0 . 01 by Chi - Square ) .

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Association of pre-treatment ALDH1A3 protein levels by IHC on a series of Ren-PDOX models treated with sunitinib. (C) Representation of ALDH1A3 expression on pro-invasive and non-pro-invasive Ren-PDOX after suntinib treatment (**p < 0.01 by Chi-Square test).


D ) Association of pre - treatment ALDH1A3 by on a series of Ren - PDOX models treated with sunitinib . ( D ) Correlation of pre - treatment ALDH1A3 by with increased invasion found after sunitinib treatment . n = 7 , Spearman ' s non - parametric correlation , p = 0 . 0095 .

D) Association of pre-treatment ALDH1A3 protein levels by IHC on a series of Ren-PDOX models treated with sunitinib. (D) Correlation of pre-treatment ALDH1A3 protein levels by IHC with increased tumor invasion found after sunitinib treatment. n=7, Spearman's non-parametric correlation, p=0.0095.


F ) Representation of ALDH1A3 score ( 0 = absent , 1 = low , 2 = high expression ) on pre - treatment samples of pro - aggressive non - pro - aggressive ccRCC described above . n = 15 , Chi - square for independence p = 0 . 015 , Chi - square for trend p = 0 . 030 ( * ) .

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F) Representation of ALDH1A3 score (0=absent, 1=low, 2=high expression) on pre-treatment samples of pro-aggressive and non-pro-aggressive ccRCC patients described above. n=15, Chi-square test for independence p=0.015, and Chi-square test for trend p=0.030 (*).


( A ) of amount of LC3 ‐ II in mouse Rab ‐ knockdown . Representative blots are shown in supplementary Fig S1C online . Error bars represent means s . e . m . of three independent experiments .

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(A) Quantification of the amount of LC3‐II protein in mouse Rab‐knockdown cells. Representative blots are shown in supplementary Fig S1C online. Error bars represent the means and s.e.m. of three independent experiments.


( B ) of amount of p62 in candidates Rab ‐ knockdown . Representative blots are shown in supplementary Fig S1D online . Error bars represent means s . e . m . of three independent experiments .

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(B) Quantification of the amount of p62 protein in candidates Rab‐knockdown cells. Representative blots are shown in supplementary Fig S1D online. Error bars represent the means and s.e.m. of three independent experiments.


( C , D ) Control Rab12 ‐ knockdown MEFs were cultured under N or S conditions , fixed then immunostained with antibodies indicated . Representative are shown in supplementary Fig S2 online . mean numbers of LC3 ‐ positive ( C ) or Atg16L1 ‐ positive ( D ) dots per are shown . Error bars represent means s . e . m . of representative data ( n ≥ 80 ) from three independent experiments .

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(C,D) Control and Rab12‐knockdown MEFs were cultured under N or S conditions, fixed and then immunostained with the antibodies indicated. Representative images are shown in supplementary Fig S2 online. The mean numbers of LC3‐positive (C) or Atg16L1‐positive (D) dots per cell are shown. Error bars represent the means and s.e.m. of representative data (n≥80) from three independent experiments.


( E ) MEFs that had been transfected with control siRNA or Rab12 siRNA were cultured as in ( C , D ) , their lysates were analysed by immunoblotting with antibodies indicated .

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(E) MEFs that had been transfected with the control siRNA or Rab12 siRNA were cultured as in (C,D), and their lysates were analysed by immunoblotting with the antibodies indicated.


( G ) Control Rab12 ‐ knockdown MEFs were cultured under N or S conditions in absence or presence of 100 μM bafilomycin A1 for 1 h . lysates were analysed by immunoblotting with antibodies indicated , of LC3 ‐ II band was quantified . normalized amount ( arbitrary units ) of LC3 ‐ II in lanes 1 , 2 , 3 , 4 , 5 6 is 1 . 0 , 2 . 3 , 4 . 3 , 0 . 9 , 1 . 6 3 . 8 , respectively .

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(G) Control and Rab12‐knockdown MEFs were cultured under N or S conditions in the absence or presence of 100 μM bafilomycin A1 for 1 h. Cell lysates were analysed by immunoblotting with the antibodies indicated, and the intensity of the LC3‐II band was quantified. The normalized amount (arbitrary units) of LC3‐II in lanes 1, 2, 3, 4, 5 and 6 is 1.0, 2.3, 4.3, 0.9, 1.6 and 3.8, respectively.


( H ) MEFs transiently expressing mStr ‐ Rab12 were cultured under starved conditions immunostained with antibodies indicated . Scale bar , 20 μm . MEFs , mouse embryonic fibroblasts ; N , nutrient ‐ rich ; S , starved ; siRNA , short interfering . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 .

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(H) MEFs transiently expressing mStr‐Rab12 were cultured under starved conditions and immunostained with the antibodies indicated. Scale bar, 20 μm. MEFs, mouse embryonic fibroblasts; N, nutrient‐rich; S, starved; siRNA, short interfering RNA. *P0.05; **P0.01; ***P0.005.


( A ) Control Rab12 ‐ knockdown MEFs were cultured under nutrient ‐ rich or starved conditions , fixed then immunostained with antibodies indicated . Scale bar , 20 μm .

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(A) Control and Rab12‐knockdown MEFs were cultured under nutrient‐rich or starved conditions, fixed and then immunostained with the antibodies indicated. Scale bar, 20 μm.


( B ) Control Rab12 ‐ knockdown MEFs were cultured as in ( A ) , their lysates were analysed by immunoblotting with antibodies indicated .

(B) Control and Rab12‐knockdown MEFs were cultured as in (A), and their lysates were analysed by immunoblotting with the antibodies indicated.


( C ) Lysates of MEFs that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with antibodies indicated .

(C) Lysates of MEFs that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated.


( D , E ) of phospho ‐ S6K phospho ‐ Akt shown in ( C ) . MEFs , mouse embryonic fibroblasts ; N , nutrient ‐ rich ; S , starved ; siRNA , short interfering . * P0 . 05 ; * * P0 . 01 .

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(D,E) Quantification of the phospho‐S6K levels and phospho‐Akt levels shown in (C). MEFs, mouse embryonic fibroblasts; N, nutrient‐rich; S, starved; siRNA, short interfering RNA. *P0.05; **P0.01.


( A ) MEFs transiently co ‐ expressing Myc ‐ PAT4 mStr ‐ Rab12 were immunostained with anti ‐ Myc antibody .

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(A) MEFs transiently co‐expressing Myc‐PAT4 and mStr‐Rab12 were immunostained with anti‐Myc antibody.


( B ) MEFs transiently expressing Myc ‐ PAT4 were immunostained with antibodies indicated . Scale bars , 20 μm .

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(B) MEFs transiently expressing Myc‐PAT4 were immunostained with the antibodies indicated. Scale bars, 20 μm.


( C ) Total lysates surface biotinylated from MEFs stably expressing HA ‐ PAT4 were analysed by immunoblotting with antibodies indicated .

(C) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA‐PAT4 were analysed by immunoblotting with the antibodies indicated.


( D ) Lysates of MEFs stably expressing HA ‐ PAT4 that had been transfected with control or Rab12 siRNAs were analysed by immunoblotting with antibodies indicated .

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(D) Lysates of MEFs stably expressing HA‐PAT4 that had been transfected with control or Rab12 siRNAs were analysed by immunoblotting with the antibodies indicated.


(E) Quantification of (D). ***P0.005.


( F ) PAT4 mRNA from MEFs transfected with control or Rab12 siRNA as revealed by reverse ‐ transcription PCR analyses . Gapdh was used as an internal control .

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(F) PAT4 mRNA levels from MEFs transfected with control or Rab12 siRNA as revealed by reverse‐transcription PCR analyses. Gapdh was used as an internal control.


( G ) Total lysates surface biotinylated from MEFs stably expressing HA ‐ PAT4 that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with antibodies indicated . Note that amounts of plasma ‐ ‐ localized PAT4 TfR were higher in Rab12 ‐ knockdown MEFs , whereas total amounts of EGFR plasma ‐ localized EGFR in these were slightly lower , but mechanisms responsible for these changes are unknown . GAPDH , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ; MEFs , mouse embryonic fibroblasts ; PAT4 , proton ‐ coupled ‐ acid transporter 4 ; siRNA , short interfering .

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(G) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA‐PAT4 that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated. Note that the amounts of plasma‐membrane‐localized PAT4 protein and TfR protein were higher in Rab12‐knockdown MEFs, whereas the total amounts of EGFR protein and plasma membrane‐localized EGFR protein in these cells were slightly lower, but the mechanisms responsible for these changes are unknown. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; MEFs, mouse embryonic fibroblasts; PAT4, proton‐coupled amino‐acid transporter 4; siRNA, short interfering RNA.


( A ) Lysates of MEFs transfected with siRNAs indicated were analysed by immunoblotting with antibodies indicated .

(A) Lysates of MEFs transfected with the siRNAs indicated were analysed by immunoblotting with the antibodies indicated.


(B) Quantification of the phospho‐S6K levels shown in (A).


( C ) MEFs transfected with siRNAs indicated were cultured under nutrient ‐ rich ( N ) or starved ( S ) conditions were immunostained with anti ‐ LC3 antibody . mean numbers of LC3 ‐ positive dots per are shown .

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(C) MEFs transfected with the siRNAs indicated were cultured under nutrient‐rich (N) or starved (S) conditions and were immunostained with anti‐LC3 antibody. The mean numbers of LC3‐positive dots per cell are shown.


A ) BW5147 reporter stably expressing human FcγR - ζ chain chimeras or BW5147 parental ( grey / dashed ) were stained with FcγR specific conjugated mAbs as indicated measured for surface expression of FcγRs via flow cytometry . Bar graphs show means of three independent experiments performed in technical duplicates . Error bars = SD .

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A) BW5147 reporter cells stably expressing human FcγR-ζ chain chimeras or BW5147 parental cells (grey/dashed) were stained with FcγR specific conjugated mAbs as indicated and measured for surface expression of FcγRs via flow cytometry. Bar graphs show means of three independent experiments performed in technical duplicates. Error bars = SD.


C ) Immobilized IC , immobilized IgG IgG opsonized represent qualitatively similar ligands for FcγRs . Response curves of human FcγRs activated by opsonized ( 293T stably expressing CD20 + Rituximab [ Rtx ] ) , immobilized IC ( rec . soluble CD20 + Rtx ) immobilized IgG ( Rtx ) . sICs formed using monovalent antigen ( rec . soluble CD20 + Rtx ) do not activate human FcγRs . X - Axis shows sample determined by antibody molarity . Y - Axis shows FcγR activation determined by reporter mouse IL - 2 production ( OD 450nm ) . Two independent experiments performed in technical duplicates . Error bars = SD .

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C) Immobilized IC, immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs. Response curves of human FcγRs activated by opsonized cells (293T cells stably expressing CD20 + Rituximab [Rtx]), immobilized IC (rec. soluble CD20 + Rtx) and immobilized IgG (Rtx). sICs formed using monovalent antigen (rec. soluble CD20 + Rtx) do not activate human FcγRs. X-Axis shows sample concentration determined by antibody molarity. Y-Axis shows FcγR activation determined by reporter cell mouse IL-2 production (OD 450nm). Two independent experiments performed in technical duplicates. Error bars = SD.


Ultra - pure antigen ( Ag , TNF - α ) mixed with therapy - grade mAb ( infliximab , Ifx ) was used to generate sICs . X - Axis : concentrations of stimulant expressed as molarity of either mAb or Ag monomer IC ( expressed as mAb molarity ) at a mAb : Ag of 1 : 2 . Soluble antigen or soluble antibody alone served as negative controls were not sufficient to activate human FcγRs . Immobilized IgG ( Rtx ) or immobilized FcγR - specific mAbs served as positive controls . Two independent experiments performed in technical duplicates . Error bars = SD . Error bars smaller than symbols are not shown . Left panel : IL - 2 16 h after reporter activation . Background ( blank ) was subtracted ( dashed ) . IL - 2 was measured via anti - IL - 2 ELISA ( A450nm ) IL - 2 concentrations were calculated from an IL - 2 standard . Right panel : Reporter CD69 expression 4 h post trigger was measured using flow cytometry . MFI were normalized to untreated ( ctrl . ) are presented as fold - change increase .

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Ultra-pure antigen (Ag, TNF-α) mixed with therapy-grade mAb (infliximab, Ifx) was used to generate sICs. X-Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. Immobilized IgG (Rtx) or immobilized FcγR-specific mAbs served as positive controls. Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown. Left panel: IL-2 quantification 16 h after reporter cell activation. Background (blank) was subtracted (dashed line). IL-2 was measured via anti-IL-2 ELISA (A450nm) and IL-2 concentrations were calculated from an IL-2 standard. Right panel: Reporter cell CD69 expression 4 h post trigger was measured using flow cytometry. MFI were normalized to untreated cells (ctrl.) and are presented as fold-change increase.


A ) Negatively selected NK purified from PBMCs of three healthy donors were tested for activation markers using flow cytometry . NK were incubated with immobilized IgG ( mepolizumab , Mpz ) , soluble IC ( Mpz : IL - 5 = 1 : 1 ) , soluble Mpz or soluble IL - 5 ( at 200 nM , 106 ) . Incubation with PMA Ionomycin ( Iono ) served as a positive control . Incubation with medium alone served as a negative control . Each dot represents one donor measured in technical duplicates . Error bars = SD . One - way ANOVA ( Tukey ) ; * p < 0 . 05 , * * * * p < 0 . 0001 .

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A) Negatively selected primary NK cells purified from PBMCs of three healthy donors were tested for activation markers using flow cytometry. NK cells were incubated with immobilized IgG (mepolizumab, Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 106 cells). Incubation with PMA and Ionomycin (Iono) served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one donor measured in technical duplicates. Error bars = SD. One-way ANOVA (Tukey); *p<0.05, ****p<0.0001.


B ) Negatively selected neutrophils purified from whole blood of four healthy donors were tested for adhesion activation markers using flow cytometry . Neutrophils were incubated with immobilized IgG ( Mpz ) , soluble IC ( Mpz : IL - 5 = 1 : 1 ) , soluble Mpz or soluble IL - 5 ( at 200 nM , 2 * 105 ) . Incubation with PMA or immobilized rituximab served as positive controls . Incubation with medium served as a negative control . Immobilized FcγRII FcγRIII specific mAbs served as functional controls . Mean florescence ( MFI ) values at t = 30 minutes of incubation are presented as increase over t = 0 min . Each dot represents one donor measured in technical duplicates . Error bars = SD . Two - way ANOVA compared to medium ( Dunnett ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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B) Negatively selected primary neutrophils purified from whole blood of four healthy donors were tested for adhesion and activation markers using flow cytometry. Neutrophils were incubated with immobilized IgG (Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 2*105 cells). Incubation with PMA or immobilized rituximab served as positive controls. Incubation with medium served as a negative control. Immobilized FcγRII and FcγRIII specific mAbs served as functional controls. Mean florescence intensity (MFI) values at t=30 minutes of incubation are presented as increase over t=0 min. Each dot represents one donor measured in technical duplicates. Error bars = SD. Two-way ANOVA compared to medium (Dunnett); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


B ) sICs of different were generated by cross - titration according to AF4 measurement . Reporter were incubated with fixed amounts of either mAb ( infliximab , left ) or Ag ( rhTNF - α , right ) titrated amounts of antigen or antibody , respectively . X - Axis shows titration of either antigen or antibody , respectively ( TNF - α calculated as monomer ) . Two independent experiments performed in technical duplicates . Error bars = SD .

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B) sICs of different size were generated by cross-titration according to the AF4 measurement. Reporter cells were incubated with fixed amounts of either mAb (infliximab, left) or Ag (rhTNF-α, right) and titrated amounts of antigen or antibody, respectively. X-Axis shows titration of either antigen or antibody, respectively (TNF-α calculated as monomer). Two independent experiments performed in technical duplicates. Error bars = SD.


C ) Purified NK from three different donors were incubated with cross - titrated sICs as in B . NK were measured for MIP - 1β expression ( % positivity ) . Incubation with PMA Ionomycin served as a positive control . Incubation with medium alone served as a negative control . Each dot represents one experimental replicate , dashed represents mean values .

C) Purified primary NK cells from three different donors were incubated with cross-titrated sICs as in B. NK cells were measured for MIP-1β expression (% positivity). Incubation with PMA and Ionomycin served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one experimental replicate, the dashed line represents the mean values.


A ) Experiments shown for an exemplary group of 8 SLE two healthy individuals . Dose - dependent reactivity of FcγRs IIIA IIB / C was observed only for SLE sera not for sera from healthy individuals . Each titration represents one donor measured in technical duplicates .

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A) Experiments shown for an exemplary group of 8 SLE patients and two healthy individuals. Dose-dependent reactivity of FcγRs IIIA and IIB/C was observed only for SLE patient sera and not for sera from healthy individuals. Each titration represents one donor measured in technical duplicates.


B ) Activation of FcγRs IIB / C IIIA by serum is mediated by serum derived sICs . serum samples were depleted of sICs by PEG precipitation supernatant ( SN ) was compared to untreated serum regarding FcγR activation ( left ) . Shown is one exemplary experiment performed in technical duplicates . Error bars indicate reproducibility as range . IgG in precipitate ( PC ) , supernatant ( SN ) or unfractionated serum respectively is shown in bar graph ( right ) .

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B) Activation of FcγRs IIB/C and IIIA by patient serum is mediated by serum derived sICs. Patient serum samples were depleted of sICs by PEG precipitation and the supernatant (SN) was compared to untreated serum regarding FcγR activation (left). Shown is one exemplary experiment performed in technical duplicates. Error bars indicate reproducibility as range. IgG concentration in the precipitate (PC), supernatant (SN) or unfractionated serum respectively is shown in the bar graph (right).


C - D ) Correlation of FcγR activation data from A with ( C ) SLEDAI or ( D ) conventional SLE disease markers ( α - dsDNA indicated as IU / ml or C3d concentrations indicated as mg / L ) . Left panel : FcγRIIIA ; right panel : FcγRIIB / C . FcγR activation from a dose - response curve as in A was calculated as under curve ( AUC ) for each SLE represented by dots . AUC from SLE are expressed as fold change compared to healthy control mean ( n = 4 ) . SLE with α - dsDNA below 50 IU / ml C3d values below 6 mg / L were excluded . One - Spearman ' s . Dotted lines show 95 % confidence bands .

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C-D) Correlation of FcγR activation data from A with (C) patient SLEDAI or (D) conventional SLE disease markers (α-dsDNA levels indicated as IU/ml or C3d concentrations indicated as mg/L). Left panel: FcγRIIIA; right panel: FcγRIIB/C. FcγR activation from a dose-response curve as in A was calculated as area under curve (AUC) for each SLE patient represented by individual dots. AUC from SLE patients are expressed as fold change compared to the healthy control mean (n=4). SLE patients with α-dsDNA levels below 50 IU/ml and C3d values below 6 mg/L were excluded. One-tailed Spearman's. Dotted lines show 95% confidence bands.


A ) Reporter expressing mFcγRIII , mFcγRIV or parental BW5147 were incubated with titrated amounts of synthetic sICs generated from rhTNF - α mouse - anti - hTNF - α at a 1 : 1 by mass . One experiment performed in technical duplicates . Error bars indicate reproducibility as range .

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A) Reporter cells expressing mFcγRIII, mFcγRIV or parental BW5147 cells were incubated with titrated amounts of synthetic sICs generated from rhTNF-α and mouse-anti-hTNF-α at a 1:1 ratio by mass. One experiment performed in technical duplicates. Error bars indicate reproducibility as range.


B ) Titrations of three mouse sera per group ( C57BL / 6 , K / BxN or NZB / WF1 ) were incubated with mFcγR reporter FcγR activation was assessed as described above . Sera from BL / 6 mice served as negative control . Each symbol represents mean values of one mouse measured in technical duplicates . Error bars = Mean with SD .

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B) Titrations of three mouse sera per group (C57BL/6, K/BxN or NZB/WF1) were incubated with mFcγR reporter cells and FcγR activation was assessed as described above. Sera from BL/6 mice served as negative control. Each symbol represents mean values of one mouse measured in technical duplicates. Error bars = Mean with SD.


D , E Immunostaining of MCT2 SMCT1 in tumors of KIC mice treated with βOHB ( 100mg / kg / day , i . p . ) or 0 . 9 % NaCl ( i . p . ) ( n = 5 mice / group ) . Areas of MCT2 ( upper panel ) SMCT1 ( lower panel ) stainings ( D ) are expressed as mean of of total tissue ± . Significance was defined by Mann - Whitney . * : p < 0 . 05 , * * p < 0 . 01 . Representative of MCT2 SMCT1 stainings ( E ) in tumors from KIC mice treated with βOHB or NaCl . Scale bar : 100 µm .

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D, E Immunostaining of MCT2 and SMCT1 in tumors of KIC mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=5 mice/group). Areas of MCT2 (upper panel) and SMCT1 (lower panel) stainings (D) are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01. Representative images of MCT2 and SMCT1 stainings (E) in tumors from KIC mice treated with βOHB or NaCl. Scale bar: 100 µm.


B - D [ U - 13C ] Glucose ( n = 3 mice ) ( B ) , [ U - 13C ] Glutamine ( n = 5 mice ) ( C ) [ U - 13C ] Acetate ( n = 6 mice ) ( D ) tracing into βOHB in poorly differentiated PDA explants from KIC mice . Data are expressed as mean ± .

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B-D [U-13C]Glucose (n=3 mice) (B), [U-13C]Glutamine (n=5 mice) (C) and [U-13C]Acetate (n=6 mice) (D) tracing into βOHB in poorly differentiated PDA explants from KIC mice. Data are expressed as mean ± SEM.


G Volcano plot illustrating significantly under - or over - represented in sg - CTRL sg - HMGCL PANC - 1 spheroids ( n = 3 independent experiments ) . Significance was defined by one - Student ' s t - . with a q - value < 0 . 05 ( horizontal axis ) a fold change < - 1 . 5 or > + 1 . 5 ( vertical axis ) are considered as significantly down or up - regulated in sg - HMGCL PANC - 1 spheroids .

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G Volcano plot illustrating proteins significantly under- or over-represented in sg-CTRL and sg-HMGCL PANC-1 spheroids (n=3 independent experiments). Significance was defined by one-tailed Student's t-test. Protein levels with a q-value<0.05 (horizontal axis) and a fold change <-1.5 or >+1.5 (vertical axis) are considered as significantly down or up-regulated in sg-HMGCL PANC-1 spheroids.


A , B of volume with representative ( A ) ( B ) of sg - CTRL ( n = 15 for A n = 9 for B ) , sg HMGCL # 2 ( n = 19 for A n = 8 for B ) , # 3 ( n = 7 for A B ) pancreatic tumors . Data are expressed as mean of volume or ± . Significance was defined by Mann - Whitney . * : p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

A, B Quantification of volume with representative images (A) and weight (B) of sg-CTRL (n=15 for A and n=9 for B), sg HMGCL #2 (n=19 for A and n=8 for B), #3 (n=7 for A and B) pancreatic tumors. Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01, ***p<0.001.


C Extracellular matrix following trichrome in sg - CTRL sg - HMGCL # 2 pancreatic tumors sections ( n = 8 9 mice / group respectively , left panel ) . Data are expressed as mean of of total tissue ± . Significance was defined by Mann - Whitney . * * : p < 0 . 01 . Representative of trichrome in sg - CTRL sg - HMGCL pancreatic tumors . Scale bar : 100 µm ( right panel ) .

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C Extracellular matrix quantification following trichrome staining in sg-CTRL and sg-HMGCL #2 pancreatic tumors sections (n=8 and 9 mice/group respectively, left panel). Data are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. **: p<0.01. Representative images of trichrome staining in sg-CTRL and sg-HMGCL pancreatic tumors. Scale bar: 100 µm (right panel).


D , E of volume ( D ) ( E ) of sg - CTRL pancreatic tumors treated with 0 . 9 % NaCl ( i . p . ) ( n = 4 ) , pancreatic tumors from two different clones of sg - HMGCL treated with 0 . 9 % NaCl ( i . p . ) or βOHB ( 100mg / kg / bi - weekly , i . p . ) ( n = 10 / group ) . Data are expressed as mean of volume or ± . Significance was defined by Mann - Whitney . ns : not significant , * * p < 0 . 01 .

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D, E Quantification of volume (D) and weight (E) of sg-CTRL pancreatic tumors treated with 0.9% NaCl (i.p.) (n=4), and pancreatic tumors from two different clones of sg-HMGCL treated with 0.9% NaCl (i.p.) or βOHB (100mg/kg/bi-weekly, i.p.) (n=10/group). Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. ns: not significant, **p<0.01.


H [ U - 13C ] βOHB tracing into TCA intermediate : citrate in sg - CTRL sg - HMGCL # 2 # 3 PANC - 1 cultured in indicated glucose concentrations . Data are expressed as mean ± . ( n = 2 independent experiments ) . Significance was defined by one - way ANOVA followed by a Bonferroni ' s multiple comparisons , only significances between sg - HMGCL # 2 , # 3 PANC - 1 sg - CTRL PANC - 1 under same culture condition are mentioned . * * * p < 0 . 001 .

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H [U-13C]βOHB tracing into TCA intermediate: citrate in sg-CTRL and sg-HMGCL #2 and #3 PANC-1 cells cultured in indicated glucose concentrations. Data are expressed as mean ± SEM. (n=2 independent experiments). Significance was defined by one-way ANOVA followed by a Bonferroni's multiple comparisons test, only significances between sg-HMGCL #2, #3 PANC-1 cells and sg-CTRL PANC-1 cells under the same culture condition are mentioned. ***p<0.001.


( B ) Box - - whisker plots of ketone bodies ( KB ) metagene score defined as first component of PCA of genes in tumors ( n = 728 ) , in metastases ( n = 76 ) , in liver metastases specifically ( n = 35 ) . Box - - whisker plot were defined with default parameters by median value ( central band at 50th percentile ) , interquartile ranges ( IQR , box limited by 25th 75th percentile ) whisker boundaries defined at 1 . 5x IQR . Significance was defined by Student ' s t - .

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(B) Box-and-whisker plots of ketone bodies (KB) metagene score defined as first component of PCA of genes in primary tumors (n=728), in all metastases (n=76), and in liver metastases specifically (n=35). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined at 1.5x IQR. Significance was defined by Student's t-test.


D - F Effect of βOHB treatment on liver metastatic incidence , status . Representative HPS of liver lobes from metastatic mice treated with βOHB ( 100mg / kg / day , i . p . ) or 0 . 9 % NaCl ( i . p . ) ( n = 8 mice / group ) . Metastatic areas are separated from liver by yellow dotted lines . Scale bar : 1mm 500 µm for insets ( D ) . Number of metastasis per lobe classified in small , medium large ( E ) . Pathological status of metastasis from mice treated with βOHB or NaCl ( n = 13 or 5 lobes / group , respectively ) . Data are expressed as of total metastasis in liver lobes presenting metastasis ( F ) .

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D-F Effect of βOHB treatment on liver metastatic incidence, size and status. Representative HPS staining of liver lobes from metastatic mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=8 mice/group). Metastatic areas are separated from liver by yellow dotted lines. Scale bar: 1mm and 500 µm for insets (D). Number and size of metastasis per lobe and classified in small, medium and large size (E). Pathological status of metastasis from mice treated with βOHB or NaCl (n=13 or 5 lobes/group, respectively). Data are expressed as percentage of total metastasis in all liver lobes presenting metastasis (F).


( A ) Mouse were immunostained with anti - neurofilament antibody ( blue ) , showing mesencephalic projection . Dispersed axon projections ( arrows point to of superior colliculus ) decreased axon ( arrowheads point to of pretectal commissure ) were observed in Plcg1f / f ; Nes - Cre mice at E11 . 5 ( n = 20 ) . areas of superior colliculus is shown magnified in each inset ( scale bar = 200 μm )

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(A) Mouse embryos were immunostained with anti-neurofilament antibody (blue), showing mesencephalic projection. Dispersed axon projections (arrows point to the area of the superior colliculus) and decreased axon fibers (arrowheads point to the area of the pretectal commissure) were observed in Plcg1f/f;Nes-Cre mice at E11.5 (n = 20). The areas of the superior colliculus is shown magnified in each inset (scale bar = 200 μm)


( B ) Representative between posterior anterior borderlines of superior colliculus ( a b ) . red boxes were drawn as rostrocaudal region of interest ( ROI ) on upper part of pretectal commissure in embryonic mesencephalon ( scale bar = 100 μm )

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(B) Representative images between the posterior and anterior borderlines of the superior colliculus (a and b). The red boxes were drawn as rostrocaudal region of interest (ROI) based on the upper part of the pretectal commissure in the embryonic mesencephalon (scale bar = 100 μm)


( C D ) axons passing through ROI was quantified by measuring in ROI , distribution of axons was statistically analyzed ( n = 5 ; t - ; * * P < 0 . 005 )

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(C and D) The axons passing through the ROI was quantified by measuring the fluorescence intensity in the ROI, and the distribution of axons was statistically analyzed (n = 5; t-test; **P < 0.005)


( E ) Whole - mount immunostaining show neurofilament ( red ) , netrin - 1 ( purple ) , DCC ( green ) , respectively . An E11 . 5 Plcg1f / f ; Nes - Cre showed shortened dorsal ramus opthalmic nerves ( yellow boxes ) . Scale bar : 200

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(E) Whole-mount immunostaining images show neurofilament (red), netrin-1 (purple), and DCC (green), respectively. An E11.5 Plcg1f/f;Nes-Cre embryo showed shortened dorsal ramus and opthalmic nerves (white and yellow boxes). Scale bar: 200 μm


( F - G ) Enlarged views of opthalmic nerve ( yellow arrow heads ) dorsal ramus ( arrow heads ) ( scale bar : 50 μm )

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(F - G) Enlarged views of the opthalmic nerve (yellow arrow heads) and dorsal ramus (white arrow heads) (scale bar: 50 μm)


( H ) Nerve measurements ; Op : ophthalmic nerve ; DR : dorsal ramus ( n = 3 ; t - ; * * P < 0 . 005 ) . Data are presented as mean ± s . e .

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(H) Nerve length measurements; Op: ophthalmic nerve; DR: dorsal ramus (n = 3; t-test; **P < 0.005). Data are presented as the mean ± s.e.m


( I ) Double immunostaining . pattern of expression colocalization of DCC ( green ) - TH ( red ) - positive axon bundles extending toward alar thalamus . Scale bar : 100

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(I) Double immunostaining images. The pattern of expression and colocalization of DCC (green)- and TH (red)-positive axon bundles extending toward the alar thalamus. Scale bar: 100 μm


( A ) In mesencephalic neurons , phosphorylation of PLCγ1 on Y783 was increased proportionally to netrin - 1 . lower graph presents of band intensities in ( A ) ( n = 3 )

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(A) In primary mesencephalic neurons, the phosphorylation of PLCγ1 on Y783 was increased proportionally to the netrin-1 concentration. The lower graph presents quantification of the band intensities in (A) (n = 3)


( B ) Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of DCC receptor by siRNA ( siDcc ) ( N = 3 )

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(B) Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of the DCC receptor by siRNA (siDcc) (N = 3)


( C ) Western blot for NTN1 secretion . Lane # 1 : transfection - free lysate , # 2 : transfection - free supernatant , # 3 : NTN1 - transfected lyasate , # 3 # 4 : NTN - 1 - transfected

(C) Western blot for the NTN1 secretion test. Lane #1: transfection-free lysate, #2: transfection-free supernatant, #3: NTN1-transfected cell lyasate, #3 and #4: NTN-1- transfected supernatant


( D ) Ventral mesencephalic ( A9 ) cultures ( daggers ) derived from control or Plcg1f / f ; Nes - Cre mice , were co - cultured with netrin - 1 - releasing 293T ( asterisks ; scale bar = 100 μm ) . areas delineated by boxes between mesencephalon tissue 293T are magnified in respective insets ( n = 4 )

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(D) Ventral mesencephalic (A9) cultures (daggers) derived from control or Plcg1f/f;Nes-Cre mice, were co-cultured with netrin-1-releasing 293T cell aggregates (asterisks; scale bar = 100 μm). The areas delineated by the white boxes between the mesencephalon tissue and 293T cell aggregates are magnified in the respective insets (n = 4)


(F) Mean angle of the axon extension


( G ) of ventral mesencephalic axons . bar graphs indicate percentages of proper targeting of ventral mesencephalic axons correctly targeting netrin - 1 - releasing ( n = 200 ) . Each experimental set was performed in

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(G) Quantification of the ventral mesencephalic axons. The bar graphs indicate the percentages of proper targeting of ventral mesencephalic axons correctly targeting the netrin-1-releasing cell aggregates (n = 200). Each experimental set was performed in quadruplicate


( H ) Anti - tyrosine hydroxylase ( TH ) immunostaining in ventral mesencephalic ( VM ) tissue . ( scale bar = 200 μm )

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(H) Anti-tyrosine hydroxylase (TH) immunostaining in ventral mesencephalic (VM) tissue. (scale bar = 200 μm)


( I ) mesencephalic neuron culture in absence or presence of NTN1 - conditioned media ( n = 100 ; scale bar : 500μm )

(I) Primary mesencephalic neuron culture in the absence or presence of NTN1-conditioned media (n = 100; scale bar: 500μm)


( K ) Mean of neurites ( F - value : 1 . 82 , t - , P * * < 0 . 005 )

(K) Mean length of neurites (F-value:1.82, t-test, P**<0.005)


( A ) Co - immunoprecipitation data showed that PLCγ1 did not interact with DCC receptor ( n = 2 , N = 3 ) .

(A) Co-immunoprecipitation data showed that PLCγ1 did not interact with the DCC receptor (n = 2, N = 3).


( B ) Immunoprecipitation showed that Src kinase interacted with PLCγ1 . Rac1 is a positive interacting control for PLCγ1 ( N = 3 ) .

(B) Immunoprecipitation assay showed that Src kinase interacted with PLCγ1. Rac1 is a positive interacting control for PLCγ1 (N = 3).


( C ) In vitro kinase confirmed that Src kinase directly phosphorylated Y783 of PLCγ1 . ( D ) of band intensities in ( C ) ( N = 3 ) .

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(C) In vitro kinase assay confirmed that Src kinase directly phosphorylated Y783 of PLCγ1. (D) Quantification of the band intensities in (C) (N = 3).


( E ) Src kinase inhibitor reduced specific phosphorylation of PLCγ1 on Y783 ( arrow ; Src I IC50 conc = 44 nM ; N = 3 ) .

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(E) Src kinase inhibitor reduced the specific phosphorylation of PLCγ1 on Y783 (arrow; Src I IC50 conc = 44 nM; N = 3).


( F ) Rac1 activity ( N = 3 ) . ( G ) of Rac1 activity in ( F ) .

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(F) Rac1 activity assay (N = 3). (G) Quantification of Rac1 activity levels in (F).