Figure 4 ( A ) Endogenous p62 / SQSTM1 level in HCT - 116 ATG9A - HA KI - ATG13 WT , ATG13 KO , ATG101 KO , ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated antibodies . Cells were grown in full DMEM media , treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting ( left ) . The graph on right shows quantification of normalized p62 infrared signal . Mean ± SEM , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA test followed by Fisher ' s LSD tests ( right ) . ( B ) Endogenous p62 / SQSTM1 level in HEK - 293T ATG13 WT , ATG13 KO , ATG101 KO , ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated proteins . Cells were grown in full DMEM media , treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting . Mean ± SEM , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA test followed by Fisher ' s LSD tests ( right ) . ( C ) Confocal images of ATG9A colocalization with p62 / SQSTM1 . HCT - 116 ATG9 - HA KI - ATG13 WT , ATG13 KO , ATG101 KO , ATG9A KO and FIP200 KO cells were grown in full DMEM media , fixed , labeled with antibodies for HA and p62 / SQSTM1 and imaged ( Scale bar 10 µm ) . ATG101 KO was stained and quantified separately . Images are from the same single independent experiment ( D ) Quantification of average surface area of p62 / SQSTM1 puncta in C . Mean ± SEM , n = 1 independent experiment with 30 technical replicates . ATG101 KO was stained and quantified separately . Mean ± SEM , n = 1 independent experiment with 30 technical replicates . A break was inserted in the Y axis to accommodate the wide range of p62 / SQSTM1 puncta sizes . ( E ) Quantification of ATG9A colocalization with p62 / SQSTM1 . Mean ± SEM , n = 1 independent experiment with 30 technical replicates . ATG101 KO was stained and quantified separately . Mean ± SEM , n = 1 independent experiment with 30 technical replicates .

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

Figure 4(A) Endogenous p62/SQSTM1 level in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated antibodies. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting (left). The graph on right shows quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) Endogenous p62/SQSTM1 level in HEK-293T ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated proteins. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right).(C) Confocal images of ATG9A colocalization with p62/SQSTM1. HCT-116 ATG9-HA KI-ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO cells were grown in full DMEM media, fixed, labeled with antibodies for HA and p62/SQSTM1 and imaged (Scale bar 10 µm). ATG101 KO was stained and quantified separately. Images are from the same single independent experiment (D) Quantification of average surface area of p62/SQSTM1 puncta in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates. ATG101 KO was stained and quantified separately. Mean ± SEM, n=1 independent experiment with 30 technical replicates. A break was inserted in the Y axis to accommodate the wide range of p62/SQSTM1 puncta sizes. (E) Quantification of ATG9A colocalization with p62/SQSTM1. Mean ± SEM, n=1 independent experiment with 30 technical replicates. ATG101 KO was stained and quantified separately. Mean ± SEM, n=1 independent experiment with 30 technical replicates.


Figure 5 ( A ) Endogenous p62 / SQSTM1 level was measured in HCT - 116 ATG9A - HA KI - ATG13 WT , ATG13 KO OR ATG13 KO cells reconstituted with ATG13 WT , ATG13 ΔHORMA and ATG13 Δ2AA by immunoblotting with indicated proteins ( left ) . Cells were grown in full DMEM media and whole cell lysates were subjected to immunoblotting ( left ) . Quantification of normalized p62 infrared signal . Mean ± SEM , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA test followed by Fisher ' s LSD tests ( right ) . ( B ) HCT - 116 cells in A were incubated in EBSS for 4 hours then processed and analyzed as in A . ( C ) Confocal images of ATG9A colocalization with p62 / SQSTM1 in HCT - 116 ATG9A - HA KI - ATG13 WT , ATG13 KO or ATG13 KO cells reconstituted with ATG13 WT , ATG13 Δ2AA and ATG13 ΔHORMA . Cells were grown in full DMEM media , fixed , labelled with antibodies for HA and p62 / SQSTM1 and imaged ( Scale bar = 10 µm ) . Images are from the same single independent experiment ( D ) Quantification of average surface area of p62 / SQSTM1 puncta in C . Mean ± SEM , n = 1 independent experiment with 30 technical replicates . A break was inserted in the Y axis to accommodate the wide range of p62 / SQSTM1 puncta sizes . ( E ) Quantification of ATG9A colocalization with p62 / SQSTM1 in C . Mean ± SEM , n = 1 independent experiment with 30 technical replicates .

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

Figure 5(A) Endogenous p62/SQSTM1 level was measured in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13 KO OR ATG13 KO cells reconstituted with ATG13 WT, ATG13 ΔHORMA and ATG13 Δ2AA by immunoblotting with indicated proteins (left). Cells were grown in full DMEM media and whole cell lysates were subjected to immunoblotting (left). Quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) HCT-116 cells in A were incubated in EBSS for 4 hours then processed and analyzed as in A.(C) Confocal images of ATG9A colocalization with p62/SQSTM1 in HCT-116 ATG9A-HA KI- ATG13 WT, ATG13 KO or ATG13 KO cells reconstituted with ATG13 WT, ATG13 Δ2AA and ATG13 ΔHORMA. Cells were grown in full DMEM media, fixed, labelled with antibodies for HA and p62/SQSTM1 and imaged (Scale bar=10 µm). Images are from the same single independent experiment (D) Quantification of average surface area of p62/SQSTM1 puncta in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates. A break was inserted in the Y axis to accommodate the wide range of p62/SQSTM1 puncta sizes. (E) Quantification of ATG9A colocalization with p62/SQSTM1 in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates.


Figure 6 ( B ) Confocal images of Ubiquitin colocalization with p62 / SQSTM1 in HCT116 ATG13 WT and HCT116 ATG13 KO cells . Cells were grown in full DMEM media , fixed , labelled with antibodies for Ubiquitin and p62 / SQSTM1 and imaged ( Scale bar = 10 µm ) ( left ) . Quantification of Ubiquitin colocalization with p62 / SQSTM1 ( right ) . Mean ± SEM , n = 1 independent experiment with 30 technical replicates . ( C ) Modified pulse chase experiment . HEK293T ATG13 WT and HEK293T ATG13 KO cells stably expressing HA ATG9A - BirA * were pulse labeled with biotin for 24 hours , media was replaced with full DMEM and streptavidin pulldown was performed at indicated time points . Quantification of normalized p62 infrared signal . Mean ± SEM , n = 3 ( biological replicates ) . Significance measured using Student ' s t test ( bottom ) . ns = p > 0 . 05 , * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , * * * = p ≤ 0 . 001 , * * * * = p ≤ 0 . 0001 .

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

Figure 6(B) Confocal images of Ubiquitin colocalization with p62/SQSTM1 in HCT116 ATG13 WT and HCT116 ATG13 KO cells. Cells were grown in full DMEM media, fixed, labelled with antibodies for Ubiquitin and p62/SQSTM1 and imaged (Scale bar=10 µm) (left). Quantification of Ubiquitin colocalization with p62/SQSTM1 (right). Mean ± SEM, n=1 independent experiment with 30 technical replicates.(C) Modified pulse chase experiment. HEK293T ATG13 WT and HEK293T ATG13 KO cells stably expressing HA ATG9A-BirA* were pulse labeled with biotin for 24 hours, media was replaced with full DMEM and streptavidin pulldown was performed at indicated time points. Quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using Student's t test (bottom). ns=p>0.05, *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****= p≤0.0001.


Figure 7 ( C ) Confocal images of Venus colocalization with ATG9A and ULK1 in HCT116 ATG9A - HA KI ATG13 - ATG101 double KO cells stably expressing VenusC - ATG101 - 3X FLAG and VenusN - ATG13 - Myc or VenusN - ATG13 Δ2AA - Myc . Cells were grown in full DMEM media , fixed , labelled with antibodies for HA and ULK1 and imaged ( Scale bar = 10 µm ) . Quantification of overlap shown as percent of total Venus puncta ( bottom ) . Mean ± SEM , n = 1 independent experiment with 10 technical replicates . ( E ) Confocal images of EGFP - p62 / SQSTM1 colocalization with HA ATG9A and ATG13 in HCT116 ATG9A - HA KI cells stably expressing EGFP - p62 / SQSTM1 . Cells were grown in full DMEM media with or without 1 μM Wortmannin for time 4 hrs , fixed , labelled with antibodies for HA and ATG13 and imaged ( Scale bar = 10 µm ) .

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

Figure 7(C) Confocal images of Venus colocalization with ATG9A and ULK1 in HCT116 ATG9A-HA KI ATG13-ATG101 double KO cells stably expressing VenusC-ATG101-3X FLAG and VenusN-ATG13-Myc or VenusN-ATG13 Δ2AA-Myc. Cells were grown in full DMEM media, fixed, labelled with antibodies for HA and ULK1 and imaged (Scale bar=10 µm). Quantification of overlap shown as percent of total Venus puncta (bottom). Mean ± SEM, n=1 independent experiment with 10 technical replicates.(E) Confocal images of EGFP-p62/SQSTM1 colocalization with HA ATG9A and ATG13 in HCT116 ATG9A-HA KI cells stably expressing EGFP-p62/SQSTM1. Cells were grown in full DMEM media with or without 1 μM Wortmannin for time 4 hrs, fixed, labelled with antibodies for HA and ATG13 and imaged (Scale bar=10 µm).


Figure 1 ( A ) BH3 ‐ only BIK displaces a subset of BCL ‐ 2 complexes at the ER . Purified LM from H1299 HA ‐ BCL ‐ 2b5 cells either mock infected or infected with Ad ‐ BIKb5 was subjected to cross ‐ linking with BMH and visualized by immunoblot . Asterisks ( * ) denote BCL ‐ 2 cross ‐ linked products , which are displaced by BIK . Bold arrow denotes region containing cross ‐ linked NAF ‐ 1 / BCL ‐ 2 . ( D ) Endogenous NAF ‐ 1 cross ‐ links with BCL ‐ 2 at the ER . LM purified from H1299 neo and HA ‐ BCL ‐ 2b5 cells were subjected to cross ‐ linking by BMH and immunoprecipitation with anti ‐ NAF ‐ 1 antibody . Precipitates were analysed by immunoblot with anti ‐ BCL ‐ 2 . ( E ) NAF ‐ 1 is displaced from BCL ‐ 2 by BH3 ‐ only BIK . H1299 HA ‐ BCL ‐ 2b5 cells were either mock infected or infected with Ad ‐ BIK . Purified LM was treated as in ( D ) .

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

Figure 1(A) BH3‐only BIK displaces a subset of BCL‐2 complexes at the ER. Purified LM from H1299 HA‐BCL‐2b5 cells either mock infected or infected with Ad‐BIKb5 was subjected to cross‐linking with BMH and visualized by immunoblot. Asterisks (*) denote BCL‐2 cross‐linked products, which are displaced by BIK. Bold arrow denotes region containing cross‐linked NAF‐1/BCL‐2.(D) Endogenous NAF‐1 cross‐links with BCL‐2 at the ER. LM purified from H1299 neo and HA‐BCL‐2b5 cells were subjected to cross‐linking by BMH and immunoprecipitation with anti‐NAF‐1 antibody. Precipitates were analysed by immunoblot with anti‐BCL‐2.(E) NAF‐1 is displaced from BCL‐2 by BH3‐only BIK. H1299 HA‐BCL‐2b5 cells were either mock infected or infected with Ad‐BIK. Purified LM was treated as in (D).


Figure 2 ( A ) Co ‐ immunoprecipitation of endogenous NAF ‐ 1 and BCL ‐ 2 . Lysates from SK ‐ Mel5 cells were collected and immunoprecipitation was performed with anti ‐ NAF ‐ 1 antibody . The precipitate was subjected to immunoblotting with anti ‐ BCL ‐ 2 and anti ‐ NAF ‐ 1 . ( B ) H1299 cells were fixed and double stained with anti ‐ NAF ‐ 1 and anti ‐ Calnexin or anti ‐ Cytochrome c antibodies . Scale bar represents 10 μm . ( C ) Co ‐ immunoprecipitation of NAF ‐ 1 ‐ Flag and HA ‐ BCL ‐ 2b5 . Lysates from H1299 HA ‐ BCL ‐ 2b5 cells infected with Ad ‐ NAF ‐ 1 ‐ Flag were collected and treated as in ( A ) . ( D ) Mutations in the CDGSH iron ‐ binding domain of NAF ‐ 1 interfere with NAF ‐ 1 binding to BCL ‐ 2 . H1299 HA ‐ BCL ‐ 2b5 cells were infected with either Ad ‐ rtTA , Ad ‐ NAF ‐ 1 ‐ Flag , or Ad ‐ NAF ‐ 1 ‐ mut ‐ Flag ( C99S C101S C110S H114Q ) . Lysates were treated as in ( A ) . Densitometric analysis was performed using Scion Image software to quantify expression and co ‐ precipitated levels of NAF ‐ 1 ‐ Flag and NAF ‐ 1 ‐ mut ‐ Flag . Graph depicts the ratio of co ‐ precipitated protein to expression level . ( E ) A functional CDGSH iron ‐ binding domain is necessary , but not sufficient for the interaction between the cytosolic domains of NAF ‐ 1 and BCL ‐ 2 . HA ‐ BCL ‐ 2 ΔTM was in vitro translated in rabbit reticulocyte lysate and equivalent aliquots were added to each GST pull ‐ down reaction . GST ‐ fusion proteins used were GST alone , GST ‐ NAF1 ‐ C , GST ‐ NAF1 ‐ C ‐ mut ( C99S C101S C110S H114Q ) , and GST ‐ MitoNEET ‐ C . The proteins were detected using anti ‐ HA and anti ‐

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

Figure 2(A) Co‐immunoprecipitation of endogenous NAF‐1 and BCL‐2. Lysates from SK‐Mel5 cells were collected and immunoprecipitation was performed with anti‐NAF‐1 antibody. The precipitate was subjected to immunoblotting with anti‐BCL‐2 and anti‐NAF‐1.(B) H1299 cells were fixed and double stained with anti‐NAF‐1 and anti‐Calnexin or anti‐Cytochrome c antibodies. Scale bar represents 10 μm.(C) Co‐immunoprecipitation of NAF‐1‐Flag and HA‐BCL‐2b5. Lysates from H1299 HA‐BCL‐2b5 cells infected with Ad‐NAF‐1‐Flag were collected and treated as in (A).(D) Mutations in the CDGSH iron‐binding domain of NAF‐1 interfere with NAF‐1 binding to BCL‐2. H1299 HA‐BCL‐2b5 cells were infected with either Ad‐rtTA, Ad‐NAF‐1‐Flag, or Ad‐NAF‐1‐mut‐Flag (C99S C101S C110S H114Q). Lysates were treated as in (A). Densitometric analysis was performed using Scion Image software to quantify expression and co‐precipitated levels of NAF‐1‐Flag and NAF‐1‐mut‐Flag. Graph depicts the ratio of co‐precipitated protein to expression level.(E) A functional CDGSH iron‐binding domain is necessary, but not sufficient for the interaction between the cytosolic domains of NAF‐1 and BCL‐2. HA‐BCL‐2 ΔTM was in vitro translated in rabbit reticulocyte lysate and equivalent aliquots were added to each GST pull‐down reaction. GST‐fusion proteins used were GST alone, GST‐NAF1‐C, GST‐NAF1‐C‐mut (C99S C101S C110S H114Q), and GST‐MitoNEET‐C. The proteins were detected using anti‐HA and anti‐GST


Figure 3 ( A ) H1299 neo and HA ‐ BCL ‐ 2b5 cells treated with control ( CTRL ) or NAF ‐ 1 shRNA were either mock infected or infected with Ad ‐ BIK in the absence or presence of 50 μM zVAD ‐ fmk . Cell lysates were analysed by immunoblot . ( B ) Caspase activity was measured using the fluorescent substrate DEVD ‐ AMC . The results represent the average ± s . d . of three independent experiments . ( C ) Prolonged BIK expression and caspase inhibition induces autophagy , which is enhanced by knockdown of NAF ‐ 1 . H1299 neo and HA ‐ BCL ‐ 2b5 cells treated with CTRL or NAF ‐ 1 shRNA were infected with Ad ‐ BIK in the presence of zVAD ‐ fmk for the indicated periods of time . Cell lysates were analysed by immunoblot . Levels of LC3 II were normalized to actin levels by densitometry analysis . Graph depicts normalized LC3 II levels of each lane .

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

Figure 3(A) H1299 neo and HA‐BCL‐2b5 cells treated with control (CTRL) or NAF‐1 shRNA were either mock infected or infected with Ad‐BIK in the absence or presence of 50 μM zVAD‐fmk. Cell lysates were analysed by immunoblot.(B) Caspase activity was measured using the fluorescent substrate DEVD‐AMC. The results represent the average±s.d. of three independent experiments.(C) Prolonged BIK expression and caspase inhibition induces autophagy, which is enhanced by knockdown of NAF‐1. H1299 neo and HA‐BCL‐2b5 cells treated with CTRL or NAF‐1 shRNA were infected with Ad‐BIK in the presence of zVAD‐fmk for the indicated periods of time. Cell lysates were analysed by immunoblot. Levels of LC3 II were normalized to actin levels by densitometry analysis. Graph depicts normalized LC3 II levels of each lane.


Figure 4 ( A ) Effect of NAF ‐ 1 knockdown on starvation ‐ induced autophagy . H1299 cells infected with CTRL or NAF ‐ 1 shRNA were starved for 4 h in EBSS with DMSO ( vehicle ) or Baf A1 ( 100 nM ) . Cell lysates were analysed by immunoblot . ( B ) Representative images of GFP ‐ LC3 staining in H1299 GFP ‐ LC3 cells transfected with LUC or NAF ‐ 1 siRNA , untreated and starved . Scale bar represents 10 μm . ( C ) Quantification of autophagy is expressed as the percentage of GFP ‐ LC3 ‐ expressing cells displaying punctate GFP ‐ LC3 . A minimum of 100 cells per sample were counted ; results represent the average ± s . d . of three independent experiments . Cell lysates were analysed by immunoblot .

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

Figure 4(A) Effect of NAF‐1 knockdown on starvation‐induced autophagy. H1299 cells infected with CTRL or NAF‐1 shRNA were starved for 4 h in EBSS with DMSO (vehicle) or Baf A1 (100 nM). Cell lysates were analysed by immunoblot.(B) Representative images of GFP‐LC3 staining in H1299 GFP‐LC3 cells transfected with LUC or NAF‐1 siRNA, untreated and starved. Scale bar represents 10 μm.(C) Quantification of autophagy is expressed as the percentage of GFP‐LC3‐expressing cells displaying punctate GFP‐LC3. A minimum of 100 cells per sample were counted; results represent the average±s.d. of three independent experiments. Cell lysates were analysed by immunoblot.


Figure 5 ( A ) NAF ‐ 1 contributes to the interaction between BCL ‐ 2 and Beclin 1 . H1299 HA ‐ BCL ‐ 2b5 cells were transfected with Flag ‐ Beclin 1 and either LUC or NAF ‐ 1 siRNA . Cells were lysed and subjected to immunoprecipitation with anti ‐ BCL ‐ 2 antibody . Precipitates were subjected to analysis by immunoblot using anti ‐ Beclin 1 and anti ‐ BCL ‐ 2 . All lanes are derived from the same gel and of the same exposure . Thin white lines indicate where lanes have been removed . ( B ) Loss of NAF ‐ 1 prevents BCL ‐ 2b5 from antagonizing starvation ‐ induced autophagy . H1299 GFP ‐ LC3 and BCL ‐ 2b5 / GFP ‐ LC3 cells were transfected with either LUC or NAF ‐ 1 siRNA and starved for 4 h . Cells were analysed as in Figure 4C . ( C ) Representative electron micrographs of H1299 neo and HA ‐ BCL ‐ 2b5 cells treated with either LUC or NAF ‐ 1 siRNA , with or without subsequent starvation . Scale bar represents 10 μm . Cell lysates were analysed by immunoblot . All lanes are derived from the same gel and of the same exposure . Thin white line indicates where lanes have been removed . Autophagy levels observed by electron microscopy ( C ) were quantified and expressed as either the percentage of cells containing autophagic vacuoles ( D ) or the number of autophagosomes per cell ( E ) . A minimum of 100 cells per sample were counted ; results represent the average ± s . e . m .

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

Figure 5(A) NAF‐1 contributes to the interaction between BCL‐2 and Beclin 1. H1299 HA‐BCL‐2b5 cells were transfected with Flag‐Beclin 1 and either LUC or NAF‐1 siRNA. Cells were lysed and subjected to immunoprecipitation with anti‐BCL‐2 antibody. Precipitates were subjected to analysis by immunoblot using anti‐Beclin 1 and anti‐BCL‐2. All lanes are derived from the same gel and of the same exposure. Thin white lines indicate where lanes have been removed.(B) Loss of NAF‐1 prevents BCL‐2b5 from antagonizing starvation‐induced autophagy. H1299 GFP‐LC3 and BCL‐2b5/GFP‐LC3 cells were transfected with either LUC or NAF‐1 siRNA and starved for 4 h. Cells were analysed as in Figure 4C.(C) Representative electron micrographs of H1299 neo and HA‐BCL‐2b5 cells treated with either LUC or NAF‐1 siRNA, with or without subsequent starvation. Scale bar represents 10 μm. Cell lysates were analysed by immunoblot. All lanes are derived from the same gel and of the same exposure. Thin white line indicates where lanes have been removed. Autophagy levels observed by electron microscopy (C) were quantified and expressed as either the percentage of cells containing autophagic vacuoles (D) or the number of autophagosomes per cell (E). A minimum of 100 cells per sample were counted; results represent the average±s.e.m.


Figure 6 ( A ) H1299 neo and HA ‐ BCL ‐ 2b5 cells treated with either CTRL or NAF ‐ 1 shRNA were loaded with Fura ‐ 2AM , and ER Ca2 + stores were measured as the difference in cytoplasmic Ca2 + concentration before and after addition of TG ( 2 μM ) . Shown are representative traces of Fura ‐ 2AM fluorescence measured at 340 / 380 nm excitation wavelength ratio at 510 nm wavelength emission . Arrow indicates time at which TG was added , delta values indicate TG ‐ sensitive ER Ca2 + stores . ( B ) Differences in ER Ca2 + stores are shown as the average ± s . e . m . of three independent experiments as described in ( A ) . ( C ) Co ‐ immunoprecipitation of endogenous NAF ‐ 1 and endogenous IP3 receptor type I . Lysate from H1299 HA ‐ BCL ‐ 2b5 cells was collected and immunoprecipitation was performed with anti ‐ IP3R1 antibody . The resulting precipitate was analysed by immunoblot .

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Figure 6(A) H1299 neo and HA‐BCL‐2b5 cells treated with either CTRL or NAF‐1 shRNA were loaded with Fura‐2AM, and ER Ca2+ stores were measured as the difference in cytoplasmic Ca2+ concentration before and after addition of TG (2 μM). Shown are representative traces of Fura‐2AM fluorescence measured at 340/380 nm excitation wavelength ratio at 510 nm wavelength emission. Arrow indicates time at which TG was added, delta values indicate TG‐sensitive ER Ca2+ stores.(B) Differences in ER Ca2+ stores are shown as the average±s.e.m. of three independent experiments as described in (A).(C) Co‐immunoprecipitation of endogenous NAF‐1 and endogenous IP3 receptor type I. Lysate from H1299 HA‐BCL‐2b5 cells was collected and immunoprecipitation was performed with anti‐IP3R1 antibody. The resulting precipitate was analysed by immunoblot.


Figure 2A , B cRNA or water as a control was injected into oocytes and uptake was assessed two days after injection for 14h in N free MBS . Data information : biological replicates : n = 5 with 2 oocytes in each sample . Asterisks denote significant difference : * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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Figure 2A, B cRNA or water as a control was injected into oocytes and uptake was assessed two days after injection for 14h in N free MBS. Data information: biological replicates: n = 5 with 2 oocytes in each sample. Asterisks denote significant difference: **P<0.01, *P<0.05 (Student's t-test).


Figure 3A , B The oocytes were exposed to different concentrations of NO3 - in N free MBS for 1 . 5 h at pH5 . 5 . Net NO3 - accumulation was fitted with both Michaelis - Menten and Linear Regression in A for high affinity range and Linear Regression in B for low affinity . Values are the mean ± SE ( biological replicates : n = 3 ) with 3 - 4 oocytes in each sample .

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Figure 3A, B The oocytes were exposed to different concentrations of NO3- in N free MBS for 1.5 h at pH5.5. Net NO3- accumulation was fitted with both Michaelis-Menten and Linear Regression in A for high affinity range and Linear Regression in B for low affinity. Values are the mean ± SE (biological replicates: n=3) with 3-4 oocytes in each sample.


Figure 4A Histochemical stained roots from composite plants transformed with proMtNPF6 . 5 : GUS ( left column ) , proMtNPF6 . 7 : GUS ( middle column ) , proMtNPF6 . 6 : GUS ( right column ) . B Expression of MtNPFs in roots after transfer to media containing different levels of NO3 - for 30 minutes . C = KCl control , N = KNO3 ( Biological replicates : n = 3 ( 3 pooled root organs in each replicate ) , asterisk denotes significance p < 0 . 05 , Student ' s t - test ) . The means of all samples were compared to that of their concentration - matched KCl controls . Expression is shown relative to Ubiquitin .

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Figure 4A Histochemical stained roots from composite plants transformed with proMtNPF6.5:GUS (left column), proMtNPF6.7:GUS (middle column), proMtNPF6.6:GUS (right column).B Expression of MtNPFs in roots after transfer to media containing different levels of NO3- for 30 minutes. C=KCl control, N=KNO3 (Biological replicates: n = 3 (3 pooled root organs in each replicate), asterisk denotes significance p<0.05, Student's t-test). The means of all samples were compared to that of their concentration-matched KCl controls. Expression is shown relative to Ubiquitin.


Figure 5A , B I - V responses in oocytes ( subtracted of the water injected controls ) exposed to 40 mM Cl at pH5 . 8 ( A ) and pH7 . 4 ( B ) . The currents were measured while the oocyte plasma membrane was clamped at - 60 mv to between 20 and - 140 mv for 120 ms with - 20 mv increments for each Cl - concentration . The Cl - - elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6 . 5 injected oocytes at each Cl - concentration Data information : Value = mean ± SEM , biological replicates : n = 4C , D I - V responses in the ( C ) water and ( D ) MtNPF6 . 5 ( subtracted of the water injected controls ) injected oocytes exposed to different concentrations of Cl - at pH5 . 8 . The currents were measured while the oocyte plasma membrane was clamped at - 60 mv to between 20 and - 140 mv for 120 ms with - 20 mv increments for each Cl - concentration . The Cl - - elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6 . 5 injected oocytes at each Cl - concentration . Data information : Value = mean ± SEM , biological replicates : n =

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

Figure 5A, B I-V responses in oocytes (subtracted of the water injected controls) exposed to 40 mM Cl at pH5.8 (A) and pH7.4 (B). The currents were measured while the oocyte plasma membrane was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl- concentration. The Cl- -elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6.5 injected oocytes at each Cl- concentration Data information: Value = mean ± SEM, biological replicates: n = 4C, D I-V responses in the (C) water and (D) MtNPF6.5 (subtracted of the water injected controls) injected oocytes exposed to different concentrations of Cl- at pH5.8. The currents were measured while the oocyte plasma membrane was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl- concentration. The Cl- -elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6.5 injected oocytes at each Cl- concentration. Data information: Value = mean ± SEM, biological replicates: n = 6


Figure 6C Oocytes were exposed to 1 . 17 mM 36Cl - for 2h with 1 and 10 mM NO3 - . Biological replicates : n = 6 ( 4 oocytes in each sample ) . D The same data as in C is shown without AtNPF6 . 3 to allow for better comparison between MtNPFs . Biological replicates : n = 6 ( 4 oocytes in each sample ) . Data information : Asterisks denote significant difference : * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . In the boxplots , horizontal black bars represent the median , and black cross represent the mean . Boxes represent the middle 50 % of the distribution , and whiskers represent the entire spread of the data . Means were compared using Student ' s t - test , α = 0 . 05 .

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Figure 6C Oocytes were exposed to 1.17 mM 36Cl- for 2h with 1 and 10 mM NO3-. Biological replicates: n=6 (4 oocytes in each sample). D The same data as in C is shown without AtNPF6.3 to allow for better comparison between MtNPFs. Biological replicates: n=6 (4 oocytes in each sample). Data information: Asterisks denote significant difference: **P<0.01, *P<0.05 (Student's t-test). In the boxplots, horizontal black bars represent the median, and black cross represent the mean. Boxes represent the middle 50% of the distribution, and whiskers represent the entire spread of the data. Means were compared using Student's t-test, α= 0.05.


Figure 7A , B Relative expression of NPFs in M . truncatula roots grown in FP and exposed to different NaCl treatments . Data information : * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) ± SEMD Relative expression of NPFs in M . truncatula roots grown in FP and exposed to 100mM KCl treatments for different times . Data information : * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) ± SEM

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Figure 7A, B Relative expression of NPFs in M. truncatula roots grown in FP and exposed to different NaCl treatments. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEMD Relative expression of NPFs in M. truncatula roots grown in FP and exposed to 100mM KCl treatments for different times. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEM


Figure 8A Seedling Cl - content determined by ion chromatography 2 h after treatment with 50mM NaCl . * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 4 . B Cl - content determined by ion chromatography of seedlings supplemented with 5mM KNO3 or NH4NO3 . * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 4 . C On the left , relative Cl - content determined by XRF of NO3 - starved seedlings after KNO3 supplementation . * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 - 5 . On the right , a representative XRF image of Cl - signal across WT and mutant roots at the 0h timepoint . Five roots per genotype are shown . The outlines indicate the measurement areas . The color legend indicates relative Cl - concentration .

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Figure 8A Seedling Cl- content determined by ion chromatography 2 h after treatment with 50mM NaCl. *p<0.05, Student's t-test; biological replicates: n=4. B Cl- content determined by ion chromatography of seedlings supplemented with 5mM KNO3 or NH4NO3. *p<0.05, Student's t-test; biological replicates: n=4. C On the left, relative Cl- content determined by XRF of NO3- starved seedlings after KNO3 supplementation. *p<0.05, Student's t-test; biological replicates: n=3-5. On the right, a representative XRF image of Cl- signal across WT and mutant roots at the 0h timepoint. Five roots per genotype are shown. The outlines indicate the measurement areas. The color legend indicates relative Cl- concentration.


Figure 9A Biomass ( fresh weight ) of seedlings grown on FP or FP supplemented with 50 or 100 mM NaCl . * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 ( 2 - 5 pooled organs in each replicate ) . B Image of seedlings grown on FP or 50 mM NaCl . Data information : roots were re - arranged to better display their length , they did not exhibit curling / agravitropic growth .

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Figure 9A Biomass (fresh weight) of seedlings grown on FP or FP supplemented with 50 or 100 mM NaCl. *p<0.05, Student's t-test; biological replicates: n=3 (2-5 pooled organs in each replicate).B Image of seedlings grown on FP or 50 mM NaCl. Data information: roots were re-arranged to better display their length, they did not exhibit curling/agravitropic growth.


Figure 10B Changes in NO3 - and Cl - content ( mol / g dry weight ) of NO3 - starved plants after NO3 - provision Data information : , * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 4 ± SEM . E Relative MtNPF expression following treatment with 5mM KNO3 + 100mM NaCl Data information : expression shown is relative to Ubiquitin ; * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) ± SEM .

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Figure 10B Changes in NO3- and Cl- content (mol/g dry weight) of NO3- starved plants after NO3- provision Data information: , *p< 0.05, Student's t-test; biological replicates: n=4 ±SEM.E Relative MtNPF expression following treatment with 5mM KNO3 +100mM NaCl Data information: expression shown is relative to Ubiquitin; *p< 0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate) ±SEM.


Figure 11A The induction of MtNPF6 . 7 and repression of MtNPF6 . 5 by NO3 - requires MtNLP1 . Plants were grown on FP plates and then treated with 5 mM KNO3 . expression shown is relative to UBQ ; * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) . B The switch from Cl - to NO3 - as the major anion upon NO3 - addition is lost in mtnlp1 . Plants were grown on FP plates and then treated with 5 mM KNO3 treatment . * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 4 ( 3 pooled root organs in each replicate ) .

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Figure 11A The induction of MtNPF6.7 and repression of MtNPF6.5 by NO3- requires MtNLP1. Plants were grown on FP plates and then treated with 5 mM KNO3. expression shown is relative to UBQ; *p<0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate).B The switch from Cl- to NO3- as the major anion upon NO3- addition is lost in mtnlp1. Plants were grown on FP plates and then treated with 5 mM KNO3 treatment. *p< 0.05, Student's t-test; biological replicates: n=4 (3 pooled root organs in each replicate).C


Figure 1B , CPP score during the pre - test and CPP test . Two - way ANOVA with Sidak ' s multiple comparisons test . Test of ASE - sdC mice , t = 1 . 306 , p = 0 . 3586 versus Pre - test of ASE - sdC mice ; Test of ACE - sdC mice , t = 10 . 91 , p < 0 . 0001 versus Pre - test of ACE - sdC mice . ASE - sdC group , n = 18 mice ; ACE - sdC group , n = 24 mice . Data information : ASE - sdC , adolescent saline - exposed mice with subthreshold dose of cocaine treatment during adulthood ; ACE - sdC , adolescent cocaine - exposed mice with subthreshold dose of cocaine treatment during adulthood . The data were presented as the mean ± SEM . Statistical analyses were performed using Two - way ANOVA with Sidak ' s multiple comparisons test or unpaired t - tests . N . S . , p > 0 . 05 , # # , p < 0 . 01 versus baseline ( pre - test CPP score ) ; N . S . , p > 0 . 05 , * * , p < 0 . 01 versus ASE - sdC mice .

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Figure 1B, CPP score during the pre-test and CPP test. Two-way ANOVA with Sidak's multiple comparisons test. Test of ASE-sdC mice, t = 1.306, p = 0.3586 versus Pre-test of ASE-sdC mice; Test of ACE-sdC mice, t = 10.91, p < 0.0001 versus Pre-test of ACE-sdC mice. ASE-sdC group, n = 18 mice; ACE-sdC group, n = 24 mice. Data information: ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA with Sidak's multiple comparisons test or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, **, p < 0.01 versus ASE-sdC mice.


Figure 2A , The number of c - Fos - positive neurons after CPP test in mPFC and its subregions of Cg1 , PrL , IL . The c - Fos ( red ) and NeuN ( green ) are used as markers for evaluating cell activity and labeling neuron , respectively . Two - tailed unpaired t test . mPFC , t = 2 . 89 , p = 0 . 0161 ; Cg1 , t = 2 . 912 , p = 0 . 0155 ; PrL , t = 2 . 311 , p = 0 . 0435 ; IL , t = 0 . 8092 , p = 0 . 4372 . n = 6 mice per group . Scale bar , 400 μm / 100 μm . Data information : Cg1 , cingulate cortex ; PrL , prelimbic cortex ; IL , infralimbic cortex ; ASE - sdC , adolescent saline - exposed mice with subthreshold dose of cocaine treatment during adulthood ; ACE - sdC , adolescent cocaine - exposed mice with subthreshold dose of cocaine treatment during adulthood . B , The correlation of number of mPFC c - Fos - positive neurons with the corresponding ΔCPP score in ACE - sdC mice . mPFC , r = 0 . 9048 , p = 0 . 0132 ; Cg1 , r = 0 . 9206 , p = 0 . 0092 ; PrL , r = 0 . 8646 , p = 0 . 0262 ; IL , r = 0 . 4181 , p = 0 . 4094 . n = 6 mice . Data information : Cg1 , cingulate cortex ; PrL , prelimbic cortex ; IL , infralimbic cortex ; ACE - sdC , adolescent cocaine - exposed mice with subthreshold dose of cocaine treatment during adulthood . The data were presented as the mean ± SEM . Statistical analyses were performed using unpaired t - tests . N . S . , p > 0 . 05 , * , p < 0 . 05 versus ASE - sdC mice . E , Quantification of ΔF / F . ASE - sdC , adolescent saline - exposed mice with subthreshold dose of cocaine treatment during adulthood ; ACE - sdC , adolescent cocaine - exposed mice with subthreshold dose of cocaine treatment during adulthood . The data were presented as the mean ± SEM . Statistical analyses were performed using unpaired t - tests . N . S . , p > 0 . 05 , * , p < 0 . 05 versus ASE - sdC mice .

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

Figure 2A, The number of c-Fos-positive neurons after CPP test in mPFC and its subregions of Cg1, PrL, IL. The c-Fos (red) and NeuN (green) are used as markers for evaluating cell activity and labeling neuron, respectively. Two-tailed unpaired t test. mPFC, t = 2.89, p = 0.0161; Cg1, t = 2.912, p = 0.0155; PrL, t = 2.311, p = 0.0435; IL, t = 0.8092, p = 0.4372. n = 6 mice per group. Scale bar, 400 μm/100 μm. Data information: Cg1, cingulate cortex; PrL, prelimbic cortex; IL, infralimbic cortex; ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood.B, The correlation of number of mPFC c-Fos-positive neurons with the corresponding ΔCPP score in ACE-sdC mice. mPFC, r = 0.9048, p = 0.0132; Cg1, r = 0.9206, p = 0.0092; PrL, r = 0.8646, p = 0.0262; IL, r = 0.4181, p = 0.4094. n = 6 mice. Data information: Cg1, cingulate cortex; PrL, prelimbic cortex; IL, infralimbic cortex ; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05 versus ASE-sdC mice.E, Quantification of ΔF/F. ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05 versus ASE-sdC mice.


Figure 3A , Volcano plot illustration showed the upregulated , downregulated and unchanged genes in response to cocaine administration in mPFC . Each dot represents one gene . Red , blue and gray dots represent the upregulated , downregulated and not changed genes , respectively . C , Dusp1 mRNA levels on P74 . Two - tailed unpaired t test . t = 0 . 3691 , p = 0 . 7307 . n = 3 biological replicates per group . Data information : ASE , adolescent saline - exposed mice during adulthood ; ACE , adolescent cocaine - exposed mice during adulthood The data were presented as the mean ± SEM . Statistical analyses were performed using unpaired t - tests . N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus ASE miceD , DUSP1 protein levels on P74 . Two - tailed unpaired t test . t = 1 . 251 , p = 0 . 2792 . n = 3 biological replicates per group . Data information : ASE , adolescent saline - exposed mice during adulthood ; ACE , adolescent cocaine - exposed mice during adulthood ; The data were presented as the mean ± SEM . Statistical analyses were performed using unpaired t - tests . N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus ASE miceI , Mean gray value of DUSP1 in CaMKII - positive neurons . Two - tailed unpaired t test . t = 5 . 751 , p = 0 . 0045 . n = 3 mice per group . Scale bar , 400 μm / 100 μm . Data information ASE - sdC , adolescent saline - exposed mice with subthreshold dose of cocaine treatment during adulthood ; ACE - sdC , adolescent cocaine - exposed mice with subthreshold dose of cocaine treatment during adulthood . The data were presented as the mean ± SEM . Statistical analyses were performed using unpaired t - tests . N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus ASE - sdC mice . J , Levels of p - ERK / ERK , p - ERK and ERK proteins on P78 . Two - tailed unpaired t test . p - ERK to ERK : t = 3 . 338 , p = 0 . 0289 ; p - ERK : t = 3 . 143 , p = 0 . 0348 ; ERK1 / 2 : t = 1 . 030 , p = 0 . 3611 . n = 3 biological replicates per group . Data information : ASE - sdC , adolescent saline - exposed mice with subthreshold dose of cocaine treatment during adulthood ; ACE - sdC , adolescent cocaine - exposed mice with subthreshold dose of cocaine treatment during adulthood . The data were presented as the mean ± SEM . Statistical analyses were performed using unpaired t - tests . N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus ASE - sdC mice .

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

Figure 3A, Volcano plot illustration showed the upregulated, downregulated and unchanged genes in response to cocaine administration in mPFC. Each dot represents one gene. Red, blue and gray dots represent the upregulated, downregulated and not changed genes, respectively.C, Dusp1 mRNA levels on P74. Two-tailed unpaired t test. t = 0.3691, p = 0.7307. n = 3 biological replicates per group. Data information: ASE, adolescent saline-exposed mice during adulthood; ACE, adolescent cocaine-exposed mice during adulthood The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE miceD, DUSP1 protein levels on P74. Two-tailed unpaired t test. t = 1.251, p = 0.2792. n = 3 biological replicates per group. Data information: ASE, adolescent saline-exposed mice during adulthood; ACE, adolescent cocaine-exposed mice during adulthood; The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE miceI, Mean gray value of DUSP1 in CaMKII-positive neurons. Two-tailed unpaired t test. t = 5.751, p = 0.0045. n = 3 mice per group. Scale bar, 400 μm/100 μm. Data information ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE-sdC mice.J, Levels of p-ERK/ERK, p-ERK and ERK proteins on P78. Two-tailed unpaired t test. p-ERK to ERK: t = 3.338, p = 0.0289; p-ERK: t = 3.143, p = 0.0348; ERK1/2: t = 1.030, p = 0.3611. n = 3 biological replicates per group. Data information: ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE-sdC mice.


Figure 4B , Dusp1 mRNA levels on P78 . Two - tailed unpaired t test . t = 4 . 413 , p = 0 . 0116 . n = 3 biological replicates per group . Data information : Ctrl , control virus - treated mice ; OE , overexpression virus - treated mice . The data were presented as the mean ± SEM . Statistical analyses were performed using Two - way ANOVA or unpaired t - tests . N . S . , p > 0 . 05 , # , p < 0 . 05 , versus baseline ( pre - test CPP score ) ; N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus Ctrl virus - treated mice . C , DUSP1 protein levels on P78 . Two - tailed unpaired t test . t = 4 . 071 , p = 0 . 0152 . n = 3 biological replicates per group . Data information : Ctrl , control virus - treated mice ; OE , overexpression virus - treated mice . The data were presented as the mean ± SEM . Statistical analyses were performed using Two - way ANOVA or unpaired t - tests . N . S . , p > 0 . 05 , # , p < 0 . 05 , versus baseline ( pre - test CPP score ) ; N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus Ctrl virus - treated mice . G , The total distance traveled by mice during CPP test . Two - way ANOVA with Sidak ' s multiple comparisons test . Ctrl virus - treated ACE - sdC mice , t = 0 . 4813 , p = 0 . 9975 versus Ctrl virus - treated ASE - sdC mice ; OE virus - treated ASE - sdC mice , t = 1 . 068 , p = 0 . 8734 versus Ctrl virus - treated ASE - sdC mice ; OE virus - treated ACE - sdC mice , t = 1 . 425 , p = 0 . 6527 versus Ctrl virus - treated ACE - sdC mice . ASE - sdC group , n = 10 mice per group ; ACE - sdC group , n = 13 mice per group . Data information : Ctrl , control virus - treated mice ; OE , overexpression virus - treated mice . The data were presented as the mean ± SEM . Statistical analyses were performed using Two - way ANOVA or unpaired t - tests . N . S . , p > 0 . 05 , # , p < 0 . 05 , versus baseline ( pre - test CPP score ) ; N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus Ctrl virus - treated mice . I , The percentage of c - Fos - positive neurons in virus - transfected neurons . Two - tailed unpaired t test . t = 3 . 624 , p = 0 . 0047 . n = 6 mice per group . Scale bar , 200 μm / 100 μm . Data information : Ctrl , control virus - treated mice ; OE , overexpression virus - treated mice . The data were presented as the mean ± SEM . Statistical analyses were performed using Two - way ANOVA or unpaired t - tests . N . S . , p > 0 . 05 , # , p < 0 . 05 , versus baseline ( pre - test CPP score ) ; N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus Ctrl virus - treated mice . J , Levels of p - ERK / ERK , p - ERK and ERK proteins on P78 . Two - tailed unpaired t test . p - ERK to ERK , t = 3 . 503 , p = 0 . 0248 ; p - ERK , t = 9 . 063 , p = 0 . 0008 ; ERK , t = 0 . 1728 , p = 0 . 8712 . n = 3 biological replicates . Data information : Ctrl , control virus - treated mice ; OE , overexpression virus - treated mice . The data were presented as the mean ± SEM . Statistical analyses were performed using Two - way ANOVA or unpaired t - tests . N . S . , p > 0 . 05 , # , p < 0 . 05 , versus baseline ( pre - test CPP score ) ; N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus Ctrl virus - treated mice .

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

Figure 4B, Dusp1 mRNA levels on P78. Two-tailed unpaired t test. t = 4.413, p = 0.0116. n = 3 biological replicates per group. Data information: Ctrl, control virus-treated mice; OE, overexpression virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, #, p < 0.05, versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.C, DUSP1 protein levels on P78. Two-tailed unpaired t test. t = 4.071, p = 0.0152. n = 3 biological replicates per group. Data information: Ctrl, control virus-treated mice; OE, overexpression virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, #, p < 0.05, versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.G, The total distance traveled by mice during CPP test. Two-way ANOVA with Sidak's multiple comparisons test. Ctrl virus-treated ACE-sdC mice, t = 0.4813, p = 0.9975 versus Ctrl virus-treated ASE-sdC mice; OE virus-treated ASE-sdC mice, t = 1.068, p = 0.8734 versus Ctrl virus-treated ASE-sdC mice; OE virus-treated ACE-sdC mice, t = 1.425, p = 0.6527 versus Ctrl virus-treated ACE-sdC mice. ASE-sdC group, n = 10 mice per group; ACE-sdC group, n = 13 mice per group. Data information: Ctrl, control virus-treated mice; OE, overexpression virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, #, p < 0.05, versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.I, The percentage of c-Fos-positive neurons in virus-transfected neurons. Two-tailed unpaired t test. t = 3.624, p = 0.0047. n = 6 mice per group. Scale bar, 200 μm/100 μm. Data information: Ctrl, control virus-treated mice; OE, overexpression virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, #, p < 0.05, versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.J, Levels of p-ERK/ERK, p-ERK and ERK proteins on P78. Two-tailed unpaired t test. p-ERK to ERK, t = 3.503, p = 0.0248; p-ERK, t = 9.063, p = 0.0008; ERK, t = 0.1728, p = 0.8712. n = 3 biological replicates. Data information: Ctrl, control virus-treated mice; OE, overexpression virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, #, p < 0.05, versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.


Figure 5B , Dusp1 mRNA levels on P78 . Two - tailed unpaired t test . t = 9 . 250 , p < 0 . 0001 . n = 4 biological replicates per group . Data information : Ctrl , control virus - treated mice ; KD , knocking - down virus - treated mice . The data were presented as the mean ± SEM . Statistical analyses were performed using Two - way ANOVA or unpaired t - tests . N . S . , p > 0 . 05 , # # , p < 0 . 01 versus baseline ( pre - test CPP score ) ; N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus Ctrl virus - treated mice . C , DUSP1 protein levels on P78 . Two - tailed unpaired t test . t = 11 . 00 , p = 0 . 0004 . n = 3 biological replicates per group . Data information : Ctrl , control virus - treated mice ; KD , knocking - down virus - treated mice . The data were presented as the mean ± SEM . Statistical analyses were performed using Two - way ANOVA or unpaired t - tests . N . S . , p > 0 . 05 , # # , p < 0 . 01 versus baseline ( pre - test CPP score ) ; N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus Ctrl virus - treated mice . G , Average distance traveled during CPP test . Two - way ANOVA with Sidak ' s multiple comparisons test . Ctrl virus - treated ACE - sdC mice , t = 0 . 6755 , p = 0 . 9851 versus Ctrl virus - treated ASE - sdC mice ; KD virus - treated ASE - sdC mice , t = 0 . 02773 , p > 0 . 9999 versus Ctrl virus - treated ASE - sdC mice ; KD virus - treated ACE - sdC mice , t = 1 . 670 , p = 0 . 4832 versus Ctrl virus - treated ACE - sdC mice . ASE - sdC group , n = 10 mice per group ; ACE - sdC group , Ctrl , n = 7 mice ; KD , n = 11 mice . Data information : Ctrl , control virus - treated mice ; KD , knocking - down virus - treated mice . The data were presented as the mean ± SEM . Statistical analyses were performed using Two - way ANOVA or unpaired t - tests . N . S . , p > 0 . 05 , # # , p < 0 . 01 versus baseline ( pre - test CPP score ) ; N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus Ctrl virus - treated mice . I , The percentage of c - Fos - positive neurons in virus - transfected neurons . Two - tailed unpaired t test . t = 3 . 976 , p = 0 . 0073 . n = 4 mice per group . Scale bar , 200 μm / 100 μm . Data information : Ctrl , control virus - treated mice ; KD , knocking - down virus - treated mice . The data were presented as the mean ± SEM . Statistical analyses were performed using Two - way ANOVA or unpaired t - tests . N . S . , p > 0 . 05 , # # , p < 0 . 01 versus baseline ( pre - test CPP score ) ; N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus Ctrl virus - treated mice . J , Levels of p - ERK / ERK , p - ERK and ERK proteins on P78 . Two - tailed unpaired t test . p - ERK to ERK , t = 3 . 293 , p = 0 . 0301 ; p - ERK , t = 3 . 903 , p = 0 . 0175 ; ERK , t = 0 . 1856 , p = 0 . 8618 . n = 3 biological replicates per group . Data information : Ctrl , control virus - treated mice ; KD , knocking - down virus - treated mice . The data were presented as the mean ± SEM . Statistical analyses were performed using Two - way ANOVA or unpaired t - tests . N . S . , p > 0 . 05 , # # , p < 0 . 01 versus baseline ( pre - test CPP score ) ; N . S . , p > 0 . 05 , * , p < 0 . 05 , * * , p < 0 . 01 versus Ctrl virus - treated mice .

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

Figure 5B, Dusp1 mRNA levels on P78. Two-tailed unpaired t test. t = 9.250, p < 0.0001. n = 4 biological replicates per group. Data information: Ctrl, control virus-treated mice; KD, knocking-down virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.C, DUSP1 protein levels on P78. Two-tailed unpaired t test. t = 11.00, p = 0.0004. n = 3 biological replicates per group. Data information: Ctrl, control virus-treated mice; KD, knocking-down virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.G, Average distance traveled during CPP test. Two-way ANOVA with Sidak's multiple comparisons test. Ctrl virus-treated ACE-sdC mice, t =0.6755, p = 0.9851 versus Ctrl virus-treated ASE-sdC mice; KD virus-treated ASE-sdC mice, t = 0.02773, p > 0.9999 versus Ctrl virus-treated ASE-sdC mice; KD virus-treated ACE-sdC mice, t = 1.670, p = 0.4832 versus Ctrl virus-treated ACE-sdC mice. ASE-sdC group, n = 10 mice per group; ACE-sdC group, Ctrl, n = 7 mice; KD, n = 11 mice. Data information: Ctrl, control virus-treated mice; KD, knocking-down virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.I, The percentage of c-Fos-positive neurons in virus-transfected neurons. Two-tailed unpaired t test. t = 3.976, p = 0.0073. n = 4 mice per group. Scale bar, 200 μm/100 μm. Data information: Ctrl, control virus-treated mice; KD, knocking-down virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.J, Levels of p-ERK/ERK, p-ERK and ERK proteins on P78. Two-tailed unpaired t test. p-ERK to ERK, t = 3.293, p = 0.0301; p-ERK, t = 3.903, p = 0.0175; ERK, t = 0.1856, p = 0.8618. n = 3 biological replicates per group. Data information: Ctrl, control virus-treated mice; KD, knocking-down virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.


Figure 1Expression pattern of Rgcc in the embryonic day 14 . 5 ( E14 . 5 ) mouse cortex as detected by in situ hybridization . The right panels showed the expression patterns of NSC markers , including Pax6 , Sox2 , and Ki67 , along the ventricular zone ( VZ ) of the E14 . 5 mouse cortex . Scale bar : 20 μm .

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

Figure 1Expression pattern of Rgcc in the embryonic day 14.5 (E14.5) mouse cortex as detected by in situ hybridization. The right panels showed the expression patterns of NSC markers, including Pax6, Sox2, and Ki67, along the ventricular zone (VZ) of the E14.5 mouse cortex. Scale bar: 20 μm.


Figure 2Immunofluorescent staining of coronal sections of the E14 . 5 mouse cortex 1 day post IUE . ShRNA targeting Rgcc and control scramble - shRNA were nucleofected separately . Ki67 were co - stained with GFP . To quantify the ratio between NSC markers and GFP positive cells , only the cells in VZ and SVZ are taken into account . And ROIs of 800 * 1900 pixel were randomly picked for counting . Scale bars : 20 μm . Quantification of the relative ratio of Ki67 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E14 . 5 mouse cortex 1 day post IUE . Sox2 were co - stained with GFP . Scale bars : 20 μm . Quantification of the relative ratio of Sox2 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E14 . 5 mouse cortex 1 day post IUE . Pax6 were co - stained with GFP . Scale bars : 20 μm . Quantification of the relative ratio of Pax6 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E17 . 5 mouse cortex 5 days post IUE . ShRNA targeting Rgcc and control scramble - shRNA were transfected separately at E12 . 5 . All the brains were harvest at E17 . 5 . Tbr1 were co - stained with GFP . And ROIs of 300 * 300 pixel were randomly picked along Tbr1 positive region for counting . Scale bars : 100 μm . Quantification of the relative ratio of Tbr1 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E17 . 5 mouse cortex 4 days post IUE . ShRNA targeting Rgcc and control scramble - shRNA were transfected separately at E13 . 5 . All the brains were harvest at E17 . 5 . Ctip2 were co - stained with GFP . And ROIs of 300 * 300 pixel were randomly picked along Ctip2 positive region for counting . Scale bars : 100 μm . Quantification of the relative ratio of Ctip2 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E17 . 5 mouse cortex 4 days post IUE . ShRNA targeting Rgcc and control scramble - shRNA were transfected separately at E13 . 5 . All the brains were harvest at E17 . 5 . Satb2 were co - stained with GFP . And ROIs of 300 * 300 pixel were randomly picked along ventricle for counting . Scale bars : 100 μm . Quantification of the relative ratio of Satb2 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E17 . 5 mouse cortex 4 days post IUE . Scale bars : 100 μm . The ratio of GFP positive cells in VZ / SVZ , IZ , and CP were counted for migration analysis .

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

Figure 2Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. ShRNA targeting Rgcc and control scramble-shRNA were nucleofected separately. Ki67 were co-stained with GFP. To quantify the ratio between NSC markers and GFP positive cells, only the cells in VZ and SVZ are taken into account. And ROIs of 800*1900 pixel were randomly picked for counting. Scale bars: 20 μm. Quantification of the relative ratio of Ki67 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. Sox2 were co-stained with GFP. Scale bars: 20 μm. Quantification of the relative ratio of Sox2 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. Pax6 were co-stained with GFP. Scale bars: 20 μm. Quantification of the relative ratio of Pax6 and GFP double positive cells versus GFP positive cells.Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 5 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E12.5. All the brains were harvest at E17.5. Tbr1 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along Tbr1 positive region for counting. Scale bars: 100 μm. Quantification of the relative ratio of Tbr1 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E13.5. All the brains were harvest at E17.5. Ctip2 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along Ctip2 positive region for counting. Scale bars: 100 μm. Quantification of the relative ratio of Ctip2 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E13.5. All the brains were harvest at E17.5. Satb2 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along ventricle for counting. Scale bars: 100 μm. Quantification of the relative ratio of Satb2 and GFP double positive cells versus GFP positive cells.Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. Scale bars: 100 μm. The ratio of GFP positive cells in VZ/SVZ, IZ, and CP were counted for migration analysis.


Figure 3Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with SOX2 . Scale bar : 200 μm Quantification of the VZ - like SOX2 - positive rosette area . Each plot represented an individual rosette . H9 , n = 19 ; # 6 , n = 24 ; # 23 , n = 19 from 2 independent experiments . Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with PAX6 and Phospho - Histone 3 ( PH3 ) . Scale bar : 200 μm Quantification of the ratio of PH3 and PAX6 double positive cells versus the total number of PAX6 positive cells of each rosette . Each plot represented an individual organoid ( H9 , n = 6 ; # 6 , n = 8 ; # 23 , n = 8 from 2 independent experiments ) . Three to five rosettes were analyzed for each organoid . Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with Ki67 . Scale bar : 200 μm . Quantification of the VZ - like Ki67 positive rosette area . Each plot represented an individual rosette . H9 , n = 60 ; # 6 , n = 54 ; # 23 , n = 96 from 2 independent experiments . Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with TUJ1 and PAX6 . Scale bar : 200 μm . Quantification of the ratio of TUJ1 area versus PAX6 area . ( H9 , n = 15 ; # 6 , n = 6 ; # 23 , n = 9 from 2 independent experiment ) . Each plot represented an individual organoid . Three to five rosettes were analyzed for each organoid . Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with TBR1 and PAX6 . Scale bar : 200 μm . Quantification of the ratio of TBR1 - positive area around PAX6 - positive area versus PAX6 - positive area in each rosette . ( H9 , n = 10 ; # 6 , n = 10 ; # 23 , n = 7 from 2 independent experiment ) . Each plot represented an individual organoid . Three to five rosettes were analyzed for each organoid . Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with CTIP2 and PAX6 . Scale bar : 200 μm . Quantification of the ratio of CTIP2 area versus PAX6 area . ( H9 , n = 8 ; # 6 , n = 8 ; # 23 , n = 8 from 2 independent experiment ) . Each plot represented an individual organoid . Three to five rosettes were analyzed for each organoid .

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

Figure 3Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with SOX2. Scale bar: 200 μm Quantification of the VZ-like SOX2-positive rosette area. Each plot represented an individual rosette. H9, n=19; #6, n=24; #23, n=19 from 2 independent experiments. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with PAX6 and Phospho-Histone 3 (PH3). Scale bar: 200 μm Quantification of the ratio of PH3 and PAX6 double positive cells versus the total number of PAX6 positive cells of each rosette. Each plot represented an individual organoid (H9, n=6; #6, n=8; #23, n=8 from 2 independent experiments). Three to five rosettes were analyzed for each organoid. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with Ki67. Scale bar: 200 μm. Quantification of the VZ-like Ki67 positive rosette area. Each plot represented an individual rosette. H9, n=60; #6, n=54; #23, n=96 from 2 independent experiments.Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with TUJ1 and PAX6. Scale bar: 200 μm. Quantification of the ratio of TUJ1 area versus PAX6 area. (H9, n=15; #6, n=6; #23, n=9 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with TBR1 and PAX6. Scale bar: 200 μm. Quantification of the ratio of TBR1-positive area around PAX6-positive area versus PAX6-positive area in each rosette. (H9, n=10; #6, n=10; #23, n=7 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with CTIP2 and PAX6. Scale bar: 200 μm. Quantification of the ratio of CTIP2 area versus PAX6 area. (H9, n=8; #6, n=8; #23, n=8 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid.


Figure 5Immunofluorescent staining of coronal sections of the E14 . 5 mouse cortex one - day post IUE . ShRNA targeting Rgcc and control scramble - shRNA were nucleofected separately . PH3 were co - stained with GFP . The punctuated PH3 cells were at G2 - phase , while PH3 cells with continuous pattern were at M - phase . To quantify the ratio between PH3 and GFP double positive cells and GFP cells , only the cells in VZ and SVZ are taken into account . Scale bar : 20 μm . n ≥ 3 mouse brains for each group . Data are presented as mean ± SEM ; n ≥ 3 mouse brains for each group ; Statistical significance was tested by Student ' s t - test . * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Representative pictures of mitotic NSC marked by P - Vimentin and PH3 revealed by DAPI at the apical surface of the E14 . 5 mouse cortex after IUE . A representative vertically dividing cell was shown in upper panel , and a representative horizontally dividing cell was shown in lower panel . White dash line indicates the apical surface and splitting plane . Scale bar : 5 μm .

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

Figure 5Immunofluorescent staining of coronal sections of the E14.5 mouse cortex one-day post IUE. ShRNA targeting Rgcc and control scramble-shRNA were nucleofected separately. PH3 were co-stained with GFP. The punctuated PH3 cells were at G2-phase, while PH3 cells with continuous pattern were at M-phase. To quantify the ratio between PH3 and GFP double positive cells and GFP cells, only the cells in VZ and SVZ are taken into account. Scale bar: 20 μm. n≥3 mouse brains for each group. Data are presented as mean ± SEM; n≥3 mouse brains for each group; Statistical significance was tested by Student's t-test. **, p<0.01; ***, p<0.001;Representative pictures of mitotic NSC marked by P-Vimentin and PH3 revealed by DAPI at the apical surface of the E14.5 mouse cortex after IUE. A representative vertically dividing cell was shown in upper panel, and a representative horizontally dividing cell was shown in lower panel. White dash line indicates the apical surface and splitting plane. Scale bar: 5 μm.


Figure 6RGCC was co - stained with PCNT on 3D hCO slices . RGCC was with stronger expression in NSCs during M - phase . prometaphase ( B ' ) , metaphase ( B ' ' ) anaphase ( B ' ' ' ) and telophase ( B ' ' ' ' ) . RGCC was expressed around nucleus in interphase ( B ' ' ' ' ' ) . NSCs undergoing prometaphase , metaphase , anaphase and telophase were pointed out by yellow arrow . Interphase were pointed out by white arrow ( B ' ' ' ' ' ) . Immunostaining of 2D - differentiated NSCs in metaphase showed partial colocalization of RGCC with α - TUBULIN in the spindle ( B ' ' ' ' ' ' , yellow arrow ) . Scale bar : 5 μm . The Western blots results . Proteins were collected from WT hCOs and RGCC KO hCOs # 6 at day 45 . B - ACTIN were used as references . PCNT was co - stained with β - TUBULIN on WT NSCs and RGCC KO NSCs differentiated by 2D cultured dual - SMAD inhibition method . Scale bar : 5μm .

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Figure 6RGCC was co-stained with PCNT on 3D hCO slices. RGCC was with stronger expression in NSCs during M-phase. prometaphase (B'), metaphase (B'') anaphase (B''') and telophase (B''''). RGCC was expressed around nucleus in interphase (B''''') . NSCs undergoing prometaphase, metaphase, anaphase and telophase were pointed out by yellow arrow. Interphase were pointed out by white arrow (B'''''). Immunostaining of 2D-differentiated NSCs in metaphase showed partial colocalization of RGCC with α-TUBULIN in the spindle (B'''''', yellow arrow). Scale bar: 5 μm.The Western blots results. Proteins were collected from WT hCOs and RGCC KO hCOs #6 at day 45. B-ACTIN were used as references.PCNT was co-stained with β -TUBULIN on WT NSCs and RGCC KO NSCs differentiated by 2D cultured dual-SMAD inhibition method. Scale bar: 5μm.


Figure 1A . Infection by a L . pneumophila strain overexpressing MvcA but not the inactive mutant MvcAC83A interferes with UBE2N modification . Raw264 . 7 cells were infected with the indicated bacterial strains at an MOI of 5 for 2 h . Saponin soluble proteins resolved by SDS - PAGE were probed with a UBE2N - specific antibody ( top panel ) . The delivery of MavC and MvcA into infected cells was probed with antibodies specific to each of these two proteins . Note that MavC but not MvcA is detectable in cells infected with wild type bacteria ( middle panels labeled as " translocation " ) . Tubulin was probed as a loading control . Protein levels of MavC and MvcA associated with the bacteria were similarly probed in the saponin insoluble fraction ( lower panels labeled as " expression " ) . The bacterial metabolism enzyme isocitrate dehydrogenase ( ICDH ) was probed as a loading control . Note that endogenous MvcA was not detectable in bacteria grown in bacteriological medium ( the middle panel of the lower portion ) . B . Coexpression of MvcA with MavC causes accumulation of unmodified UBE2N in mammalian cells . HEK293T cells were transfected with combinations of empty vector and plasmid directing the expression of MavC or MvcA . 16 h after transfection , modification of endogenous UBE2N was detected by immunoblotting ( upper level ) . The canonical ubiquitin deamidase CifYpt from Yersinia pseudotuberculosis ( Ypt ) , which cannot modify UBE2N , was included as a control . Note that coexpression of MvcA led to accumulation of unmodified UBE2N ( 4th lane ) in samples that expressed MavC . The expression of MavC MvcA and Cif was detected using a Flag - specific antibody ( lower panel ) . Tubulin was probed as a loading control ( lower panel ) . C . MvcA interferes with MavC - induced UBE2N ubiquitination . Reactions containing the indicated combinations of proteins were allowed to proceed for 30 min and samples resolved by SDS - PAGE were detected by Coomassie Brilliant blue ( CBB ) staining . Note that MvcA ( inclusion of MvcA with MavC , 7th lane . Compare to MavC alone in 3rd lane ) but not the MvcAC83A mutant ( 8th lane ) abolished MavC - induced UBE2N modification . D . Dose - dependent modification of UBE2N by MavC and MvcA . A series of reactions containing MavC and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min . Samples resolved by SDS - PAGE were detected by CBB staining .

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Figure 1A. Infection by a L. pneumophila strain overexpressing MvcA but not the inactive mutant MvcAC83A interferes with UBE2N modification. Raw264.7 cells were infected with the indicated bacterial strains at an MOI of 5 for 2 h. Saponin soluble proteins resolved by SDS-PAGE were probed with a UBE2N-specific antibody (top panel). The delivery of MavC and MvcA into infected cells was probed with antibodies specific to each of these two proteins. Note that MavC but not MvcA is detectable in cells infected with wild type bacteria (middle panels labeled as "translocation"). Tubulin was probed as a loading control. Protein levels of MavC and MvcA associated with the bacteria were similarly probed in the saponin insoluble fraction (lower panels labeled as "expression"). The bacterial metabolism enzyme isocitrate dehydrogenase (ICDH) was probed as a loading control. Note that endogenous MvcA was not detectable in bacteria grown in bacteriological medium (the middle panel of the lower portion).B. Coexpression of MvcA with MavC causes accumulation of unmodified UBE2N in mammalian cells. HEK293T cells were transfected with combinations of empty vector and plasmid directing the expression of MavC or MvcA. 16 h after transfection, modification of endogenous UBE2N was detected by immunoblotting (upper level). The canonical ubiquitin deamidase CifYpt from Yersinia pseudotuberculosis (Ypt), which cannot modify UBE2N, was included as a control. Note that coexpression of MvcA led to accumulation of unmodified UBE2N (4th lane) in samples that expressed MavC. The expression of MavC MvcA and Cif was detected using a Flag-specific antibody (lower panel). Tubulin was probed as a loading control (lower panel).C. MvcA interferes with MavC-induced UBE2N ubiquitination. Reactions containing the indicated combinations of proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by Coomassie Brilliant blue (CBB) staining. Note that MvcA (inclusion of MvcA with MavC, 7th lane. Compare to MavC alone in 3rd lane) but not the MvcAC83A mutant (8th lane) abolished MavC-induced UBE2N modification.D. Dose-dependent modification of UBE2N by MavC and MvcA. A series of reactions containing MavC and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min. Samples resolved by SDS-PAGE were detected by CBB staining.


Figure 2A . The hydrolytic cleavage of UBE2N - Ub by MvcA into UBE2N and ubiquitin ( Q40E variant ) . Reactions containing the indicated proteins were allowed to proceed for 30 min and samples resolved by SDS - PAGE were detected by CBB staining ( left ) . Mass spectrometric analysis of the protein band corresponding to ubiquitin produced by MvcA from UBE2N - Ub ( right ) . The ubiquitin product is the Glu40 variant of ubiquitin . C . Dose - dependent cleavage of UBE2N - Ub by MvcA . A series of reactions containing UBE2N - Ub and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min before SDS - PAGE and CBB staining . Note that the activity was detectable in a reaction starting at the molar ratio of between UBE2N - Ub and MvcA is 2048 : 1 . D . Time - dependent cleavage of UBE2N - Ub by MvcA . UBE2N - Ub was incubated with MvcA at the molar ratio of 128 . Samples taken at the indicated time points were resolved by SDS - PAGE and detected by CBB staining . E . UBE2N produced from UBE2N - Ub via MvcA catalyzed deubiquitination is active in catalyzing the formation of polyubiquitin chains . UBE2N - Ub was preincubated with MvcA or its inactive mutant for 30 min . Then a cocktail containing E1 , UBE2V2 , 3xHA - ubiquitin , the E3 enzyme TRAF6 and ATP was added into reactions . Ubiquitination was allowed to proceed for 30 min . Proteins in samples resolved by SDS - PAGE were transferred onto nitrocellulose membranes and blotted with the HA specific antibody . Note the robust formation of polyubiquitin chains in the reaction receiving MvcA ( middle lane ) .

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Figure 2A. The hydrolytic cleavage of UBE2N-Ub by MvcA into UBE2N and ubiquitin (Q40E variant). Reactions containing the indicated proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by CBB staining (left). Mass spectrometric analysis of the protein band corresponding to ubiquitin produced by MvcA from UBE2N-Ub (right). The ubiquitin product is the Glu40 variant of ubiquitin.C. Dose-dependent cleavage of UBE2N-Ub by MvcA. A series of reactions containing UBE2N-Ub and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min before SDS-PAGE and CBB staining. Note that the activity was detectable in a reaction starting at the molar ratio of between UBE2N-Ub and MvcA is 2048:1.D. Time-dependent cleavage of UBE2N-Ub by MvcA. UBE2N-Ub was incubated with MvcA at the molar ratio of 128. Samples taken at the indicated time points were resolved by SDS-PAGE and detected by CBB staining.E. UBE2N produced from UBE2N-Ub via MvcA catalyzed deubiquitination is active in catalyzing the formation of polyubiquitin chains. UBE2N-Ub was preincubated with MvcA or its inactive mutant for 30 min. Then a cocktail containing E1, UBE2V2, 3xHA-ubiquitin, the E3 enzyme TRAF6 and ATP was added into reactions. Ubiquitination was allowed to proceed for 30 min. Proteins in samples resolved by SDS-PAGE were transferred onto nitrocellulose membranes and blotted with the HA specific antibody. Note the robust formation of polyubiquitin chains in the reaction receiving MvcA (middle lane).


Figure 3H . Mutational analysis of residues important for substrate recognition by MvcA predicted by structural analysis . For deubiquitination reactions , the indicated amounts of MvcA or its mutants were incubated with 2 . 0 μg UBE2N for 5 min in 25 μl reactions . For ubiquitin deamidase activity , the indicated amounts of MvcA or its mutants were incubated with 10 μg of ubiquitin for 60 min in 25 μl reactions . The ratio of cleavage was analyzed by SDS - PAGE and then densitometry . Each bar represents the ratio of cleavage or deamidation obtained from three independent experiments . Bars and error bars : means and standard error of the mean ( SEM ) .

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Figure 3H. Mutational analysis of residues important for substrate recognition by MvcA predicted by structural analysis. For deubiquitination reactions, the indicated amounts of MvcA or its mutants were incubated with 2.0 μg UBE2N for 5 min in 25 μl reactions. For ubiquitin deamidase activity, the indicated amounts of MvcA or its mutants were incubated with 10 μg of ubiquitin for 60 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage or deamidation obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM).