Figure 4B . Effect of oligo # 5 on pre - mRNA splicing of the reporter gene , stably transfected into HeLa cells . C . Effect of oligo # 5 on the truncated 5HT2C protein . Stable cell lines expressing the construct shown in panel ( A ) were treated with oligo # 3 and oligo # 5 . After 48 hrs cells were separated in membrane containing fractions and soluble cytosolic supernatant . The fractions were analyzed by Western Blot using the anti - RNA1 antiserum ( Figure EV1 ) . The protein samples were on the same membrane , but the supernatant fractions were exposed about ten times longer . D . Effect of oligo # 5 on the full - length 5HT2C protein . The protein was prepared as in panel ( C ) and analyzed using a polyclonal anti - GFP antiserum . E . Quantification of panels C and D from three independent experiments . The ratio of protein signal after oligo # 3 and oligo # 5 treatment is shown for RNA1 ( p = 0 . 002 ) and RNA2 ( p = 0 . 0003 ) , n = 3 , respectively . F , G . Effect of oligo # 5 on localization of the EGFP - tagged serotonin 2C receptor . Confocal microscopy images of three representative pictures are shown . H . Quantitification of GFP signal found on the membrane after oligo # 5 addition . The signal around the peripheral region was determined by visual inspection and calculated as a fraction of total signal using NIH image . 200 cells from three individual experiments were analyzed , p < 0 . 008 , n = 200 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4B. Effect of oligo#5 on pre-mRNA splicing of the reporter gene, stably transfected into HeLa cells.C. Effect of oligo#5 on the truncated 5HT2C protein. Stable cell lines expressing the construct shown in panel (A) were treated with oligo#3 and oligo#5. After 48 hrs cells were separated in membrane containing fractions and soluble cytosolic supernatant. The fractions were analyzed by Western Blot using the anti-RNA1 antiserum (Figure EV1). The protein samples were on the same membrane, but the supernatant fractions were exposed about ten times longer.D. Effect of oligo#5 on the full-length 5HT2C protein. The protein was prepared as in panel (C) and analyzed using a polyclonal anti-GFP antiserum.E. Quantification of panels C and D from three independent experiments. The ratio of protein signal after oligo#3 and oligo#5 treatment is shown for RNA1 (p=0.002) and RNA2 (p=0.0003), n=3, respectively.F, G. Effect of oligo#5 on localization of the EGFP-tagged serotonin 2C receptor. Confocal microscopy images of three representative pictures are shown.H. Quantitification of GFP signal found on the membrane after oligo#5 addition. The signal around the peripheral region was determined by visual inspection and calculated as a fraction of total signal using NIH image. 200 cells from three individual experiments were analyzed, p<0.008, n=200.


Figure 5A . Accumulation of Cy3 - labeled oligo # 5 in the area around the 3rd ventricle one hour post injection , shown is the ventral part of a coronal section - 1 . 34 mm from Bregma . The injection coordinates were AP : - 1 . 82 mm , DV : - 4 . 5 mm , ML : 0 . B . Uptake of oligo # 5 in the arcuate nucleus region ( arc ) , enlarged from panel A . C . Oligo # 5 enters neurons . Enlargement of hypothalamus region taking up oligo # 5 ( red ) , stained for the neuronal marker NeuN ( green ) . Most of the oligo # 5 signal is in neurons , but some is also in NeuN negative cells ( arrow ) , likely glia . D . Oligo # 5 enters some glia cells . Enlargement of a hypothalamic region adjacent to the 3rd ventricle one hour after oligo # 5 injection . The oligo ( red ) can be found in some GFAP - positive glia cells ( arrow ) , but numerous GFAP - positive cells ( green ) show no oligo # 5 uptake . E . Effect of oligo # 5 on RNA1 / RNA2 ratios in the injected area . The area marked by Cy3 staining in the dorsal hypothalamus , consisting mainly of the arcuate nucleus was microdissected , RNA isolated and analyzed by qPCR . NS : non - significant changes ( 3 hrs : p = 0 . 01 ; 6 hrs : p = 0 . 000001 ; 9 hrs : p = 0 . 02 ; 12 hrs : p = 0 . 06 ; n = 3 ) F . Oligo # 5 does not change the RNA2 - editing pattern . The hypothalamic area showing oligo # 5 staining was microdissected 6 hrs post injection and RNA2 was amplified and the PCR product subjected to deep sequencing . The percent of non - edited reads in total reads is shown ( at least 20 , 000 reads per lane ) . G . Effect of oligo # 5 on POMC expression . The RNA from panel D was analyzed using qPCR with primers against POMC , normalized to GAPDH ( 3 hrs : p = 0 . 0009 ; 6 hrs : p = 0 . 02 ; 9 hrs : p = 0 . 1 ; 12 hrs : p = 0 . 3 , n = 3 ) . H . Effect of oligo # 5 on food uptake in wild - type mice . C57BL / 6 wild - type mice received a guide canulae and did not have access to food for 16 hours . After injection of 2 µg oligonucleotides , they could freely access food . The food consumption was measured by weighing the food used . A total of 52 animals were used , p = 0 . 001 for the difference between oligo # 5 and control oligonucleotides . Each of the six individual experiments showed statistically significant differences between oligo # 5 and control ( p < 0 . 01 - 0 . 001 ) . Control oligo : an oligonucleotide against human SMN2 . I . Effect of oligo # 5 on food uptake in ob / ob mice . Ob / obmice received oligo # 5 through ICV injection , similar to panel F . However , the mice could freely access food at all times ( p = 0 . 04 , n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 5A. Accumulation of Cy3-labeled oligo#5 in the area around the 3rd ventricle one hour post injection, shown is the ventral part of a coronal section -1.34 mm from Bregma. The injection coordinates were AP: -1.82 mm, DV: -4.5 mm, ML: 0.B. Uptake of oligo#5 in the arcuate nucleus region (arc), enlarged from panel A.C. Oligo#5 enters neurons. Enlargement of hypothalamus region taking up oligo#5 (red), stained for the neuronal marker NeuN (green). Most of the oligo#5 signal is in neurons, but some is also in NeuN negative cells (arrow), likely glia.D. Oligo#5 enters some glia cells. Enlargement of a hypothalamic region adjacent to the 3rd ventricle one hour after oligo#5 injection. The oligo (red) can be found in some GFAP-positive glia cells (arrow), but numerous GFAP-positive cells (green) show no oligo#5 uptake.E. Effect of oligo#5 on RNA1/RNA2 ratios in the injected area. The area marked by Cy3 staining in the dorsal hypothalamus, consisting mainly of the arcuate nucleus was microdissected, RNA isolated and analyzed by qPCR. NS: non-significant changes (3 hrs: p=0.01; 6 hrs: p=0.000001; 9 hrs: p=0.02; 12 hrs: p=0.06; n=3)F. Oligo#5 does not change the RNA2-editing pattern. The hypothalamic area showing oligo#5 staining was microdissected 6 hrs post injection and RNA2 was amplified and the PCR product subjected to deep sequencing. The percent of non-edited reads in total reads is shown (at least 20,000 reads per lane).G. Effect of oligo#5 on POMC expression. The RNA from panel D was analyzed using qPCR with primers against POMC, normalized to GAPDH (3 hrs: p=0.0009; 6 hrs: p=0.02; 9 hrs: p=0.1; 12 hrs: p=0.3, n=3).H. Effect of oligo#5 on food uptake in wild-type mice. C57BL/6 wild-type mice received a guide canulae and did not have access to food for 16 hours. After injection of 2 µg oligonucleotides, they could freely access food. The food consumption was measured by weighing the food used. A total of 52 animals were used, p=0.001 for the difference between oligo#5 and control oligonucleotides. Each of the six individual experiments showed statistically significant differences between oligo#5 and control (p<0.01 - 0.001). Control oligo: an oligonucleotide against human SMN2.I. Effect of oligo#5 on food uptake in ob/ob mice. Ob/obmice received oligo#5 through ICV injection, similar to panel F. However, the mice could freely access food at all times (p=0.04, n=3).


Figure 6A . 50 µg of cy3 - labelled - oligo # 5 was delivered through a carotid artery catheter and one hour later the cy3 signal was detected . ChP : choroid plexus , RSGc : retrosplenial granular cx , c , 3V : 3rd ventricle ; D3V : dorsal 3rd ventricle . B . Change of RNA2 / RNA1 ratios after carotid artery injection . The area around the arcuate nucleus was microdissected at the times indicated post injection . The ration of RNA1 / RNA2 was determined using real - time PCR . Changes became apparent statistically significant after 4 and 8 hrs ( 2 hrs : p = 0 . 3 ; 4 hrs : p = 0 . 003 , 8 hrs : p = 0 . 0006 , n = 5 ) C . Effect of oligo # 5 delivered through a carotid artery catheter on food uptake in wild - type mice . C57BL / 6 wild - type mice with a pre - implanted cranial dosing carotid catheter received 50 or 100 µg of the oligonucleotides indicated . D . Long term effect of oligo # 5 action . Food intake was measured for 70 hrs after one injection of 100 µg oligo # 5 through a carotid catheter at time zero .

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

Figure 6A. 50 µg of cy3-labelled-oligo#5 was delivered through a carotid artery catheter and one hour later the cy3 signal was detected. ChP: choroid plexus, RSGc: retrosplenial granular cx,c, 3V: 3rd ventricle; D3V: dorsal 3rd ventricle.B. Change of RNA2/RNA1 ratios after carotid artery injection.The area around the arcuate nucleus was microdissected at the times indicated post injection. The ration of RNA1/RNA2 was determined using real-time PCR. Changes became apparent statistically significant after 4 and 8 hrs ( 2 hrs: p=0.3; 4 hrs: p=0.003, 8 hrs: p=0.0006, n=5)C. Effect of oligo#5 delivered through a carotid artery catheter on food uptake in wild-type mice. C57BL/6 wild-type mice with a pre-implanted cranial dosing carotid catheter received 50 or 100 µg of the oligonucleotides indicated.D. Long term effect of oligo#5 action. Food intake was measured for 70 hrs after one injection of 100 µg oligo#5 through a carotid catheter at time zero.


Figure 1 ( B ) SEC - MALLS traces of different TRIM25 and ( C ) TRIM32 constructs . The proteins are colour coded and the domain architecture is reported next to the respective MALLS curve . The constructs were analysed over different concentration ranges and the data are reported for : T25R , 4 mg / mL ; T25RB1 , 3 . 2 mg / mL ; T25RB1B2 , 3 . 6 mg / mL ; T25RBCC , 0 . 5 mg / mL ; T32R , 3 mg / mL ; T32RB , 4 mg / mL ; T32RBCC , 4 mg / mL .

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Figure 1(B) SEC-MALLS traces of different TRIM25 and(C) TRIM32 constructs. The proteins are colour coded and the domain architecture is reported next to the respective MALLS curve. The constructs were analysed over different concentration ranges and the data are reported for: T25R, 4 mg/mL; T25RB1, 3.2 mg/mL; T25RB1B2, 3.6 mg/mL; T25RBCC, 0.5 mg/mL; T32R, 3 mg/mL; T32RB, 4 mg/mL; T32RBCC, 4 mg/mL.


Figure 2 ( A , C ) UBE2D3 ~ Ub discharge assays with different TRIM25 and TRIM32 constructs , respectively . Assays were carried out with TRIM constructs as indicated and the reaction was monitored over 30 min . The asterisk indicates the band for the TRIM construct . ( B , D ) Quantification of the discharge assays using UBE2D1 ~ UbAtto and TRIM25 or TRIM32 , respectively . The loss of UBE2D1 ~ UbAtto is plotted as the average of experimental triplicates ( ± s . d . ) . ( E , G ) K63 poly - ubiquitination assays using UBE2N / UBE2V1 and TRIM25 and TRIM32 , respectively . Reactions were incubated for 30 minutes and samples taken at indicated times . The asterisk indicates the band for the TRIM construct . ( F , H ) Assays were quantified ( see Materials and Methods ) by supplementing the reaction with 1 uM UbAtto and integrating the loss of free UbAtto for TRIM25 and TRIM32 , respectively . Assays were carried out in triplicate .

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

Figure 2(A, C) UBE2D3~Ub discharge assays with different TRIM25 and TRIM32 constructs, respectively. Assays were carried out with TRIM constructs as indicated and the reaction was monitored over 30 min. The asterisk indicates the band for the TRIM construct.(B, D) Quantification of the discharge assays using UBE2D1~UbAtto and TRIM25 or TRIM32, respectively. The loss of UBE2D1~UbAtto is plotted as the average of experimental triplicates (± s.d.).(E, G) K63 poly-ubiquitination assays using UBE2N/UBE2V1 and TRIM25 and TRIM32, respectively. Reactions were incubated for 30 minutes and samples taken at indicated times. The asterisk indicates the band for the TRIM construct.(F, H) Assays were quantified (see Materials and Methods) by supplementing the reaction with 1 uM UbAtto and integrating the loss of free UbAtto for TRIM25 and TRIM32, respectively. Assays were carried out in triplicate.


Figure 3 ( A ) Structure of the TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan and blue and the Zn2 + ions as grey spheres . ( B ) Overlap of the RING dimers of TRIM32 ( cyan , 5FEY . pdb ) , TRIM25 ( yellow , 5FER . pdb ) and TRIM5 ( orange , 4TKP . pdb ) . The structures were overlapped on the circled RING domain . This overlap shows that the structures of the RINGs are very similar but that there are differences in the relative orientations of the two RINGs . ( C ) Structure of the TRIM25 RING / E2 ~ Ub complex with the RING domains in the same colour scheme as in ( A ) , UBE2D1 in grey and the ubiquitin molecules in salmon and red .

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 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

Figure 3(A) Structure of the TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan and blue and the Zn2+ ions as grey spheres.(B) Overlap of the RING dimers of TRIM32 (cyan, 5FEY.pdb), TRIM25 (yellow, 5FER.pdb) and TRIM5 (orange, 4TKP.pdb). The structures were overlapped on the circled RING domain. This overlap shows that the structures of the RINGs are very similar but that there are differences in the relative orientations of the two RINGs.(C) Structure of the TRIM25 RING/E2~Ub complex with the RING domains in the same colour scheme as in (A), UBE2D1 in grey and the ubiquitin molecules in salmon and red.


Figure 4 ( A ) Close - up of the TRIM25 RING dimer interface highlighting the hydrophobic interactions made between the four helices ( left ) . Close - up of the interface between each RING monomer and the proximal ubiquitin ( right ) . ( B ) UBE2D1 ~ UbAtto discharge assays with TRIM25 wild - type RING , the fused RING constructs and different mutants important for dimerization or the interaction with ubiquitin . Time point zero for the T25 - R Linker and T25 - R Linker V72R samples was taken before the addition of E3 as discharge is very fast . The loss of UBE2D1 ~ UbAtto is plotted as the average of experimental duplicates ( ± s . d . ) . ( C ) UBE2D3 ~ Ub discharge assays with the same mutants as in ( B ) , stained with InstantBlue . ( D ) K63 poly - ubiquitination assays using UBE2N / UBE2V1 . The asterisk indicates the band for the TRIM construct .

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

Figure 4(A) Close-up of the TRIM25 RING dimer interface highlighting the hydrophobic interactions made between the four helices (left). Close-up of the interface between each RING monomer and the proximal ubiquitin (right).(B) UBE2D1~UbAtto discharge assays with TRIM25 wild-type RING, the fused RING constructs and different mutants important for dimerization or the interaction with ubiquitin. Time point zero for the T25-R Linker and T25-R Linker V72R samples was taken before the addition of E3 as discharge is very fast. The loss of UBE2D1~UbAtto is plotted as the average of experimental duplicates (± s.d.).(C) UBE2D3~Ub discharge assays with the same mutants as in (B), stained with InstantBlue. (D) K63 poly-ubiquitination assays using UBE2N/UBE2V1. The asterisk indicates the band for the TRIM construct.


Figure 5 ( A ) Role of E10 in the RING of TRIM25 ( blue ) in stabilizing the closed E2 ~ Ub conformation by contacting K11 from the proximal ubiquitin ( salmon ) and N71 of the opposite RING ( cyan ) which in turn contacts K33 of the same ubiquitin . ( B ) E2 ~ Ub discharge and K63 poly - ubiquitination assays to test the role of E9 and E10 in TRIM25 activity . Substitution of Glu9 with Arg has no significant effect on activity , whereas the E10R mutation almost completely abolishes catalytic activity . ( C ) Discharge and K63 poly - ubiquitination assays to test the role of the equivalent residue E16 in TRIM32 and the role of I85R . Mutation of E16R abolishes catalytic activity indicating that the role of the glutamate is conserved . ( D and E ) Quantification of UBE2D1 ~ UbAtto discharge assays . The loss of E2 ~ Ub is plotted as the average of experimental duplicates ( ± s . 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 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 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

Figure 5(A) Role of E10 in the RING of TRIM25 (blue) in stabilizing the closed E2~Ub conformation by contacting K11 from the proximal ubiquitin (salmon) and N71 of the opposite RING (cyan) which in turn contacts K33 of the same ubiquitin.(B) E2~Ub discharge and K63 poly-ubiquitination assays to test the role of E9 and E10 in TRIM25 activity. Substitution of Glu9 with Arg has no significant effect on activity, whereas the E10R mutation almost completely abolishes catalytic activity.(C) Discharge and K63 poly-ubiquitination assays to test the role of the equivalent residue E16 in TRIM32 and the role of I85R. Mutation of E16R abolishes catalytic activity indicating that the role of the glutamate is conserved.(D and E) Quantification of UBE2D1~UbAtto discharge assays. The loss of E2~Ub is plotted as the average of experimental duplicates (± s.d.).


Figure 1 ( G ) Representative immunofluorescence staining of ∆ FosB ( green ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the lower row . Scale bar , 25μM . ( H ) Quantification of ∆ FosB + nuclei in cultured MSNs after the indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± SEM of % of MAP2 + cells displaying ∆ FosB in the nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates . ( I ) Representative immunofluorescence staining of ∆ FosB ( green ) in cultured MSNs exposed to consecutive stimulation , with different 1st and 2nd treatments with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the right column . Scale bar , 25μM . ( J ) Quantification of ∆ FosB + nuclei in cultured MSNs after different 1st and 2nd treatments with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Results are expressed as average ± SEM of % of MAP2 + cells displaying ∆ FosB in the nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates . ( K ) Representative immunofluorescence staining of ∆ FosB ( green ) in cultured MSNs extracted from mutant mice lacking ALK4 in GABAergic neurons ( Gad67Cre ; Alk4fl / fl ) and control mice ( Alk4fl / fl ) after exposure to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the second and fourth rows . Scale bar , 25μM . ( L ) Quantification of ∆ FosB + nuclei in cultured MSNs from mutant and control mice after the indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± SEM of % of MAP2 + cells displaying ∆ FosB in the nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological 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 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 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

Figure 1(G) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM. (H) Quantification of ∆FosB+ nuclei in cultured MSNs after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(I) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation, with different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the right column. Scale bar, 25μM. (J) Quantification of ∆FosB+ nuclei in cultured MSNs after different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(K) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs extracted from mutant mice lacking ALK4 in GABAergic neurons (Gad67Cre;Alk4fl/fl) and control mice (Alk4fl/fl) after exposure to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the second and fourth rows. Scale bar, 25μM. (L) Quantification of ∆FosB+ nuclei in cultured MSNs from mutant and control mice after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


Figure 2 ( A ) Western blot analysis of Protein Kinase A phospho - substrates ( PKA Ⓟ - subs ) in PCBP1 immunoprecipitates from HeLa cells exposed to sequential stimulation ( same treatment in both treatment days ) with forskolin ( Fsk ) in the absence or presence of the PKA inhibitor PKI14 - 22 ( PKI ) . Reprobing with anti - PCBP1 antibodies is shown in the middle blot . Lower blot shows the total amount of PCBP1 in the cell lysates . Quantification of three independent experiments is shown on the right . Results are expressed as average ± SEM of fold increase over vehicle normalized to PCBP1 levels . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates . ( B ) Nuclear localization of PCBP1 analyzed by Western blotting of extracts from HeLa cells exposed to sequential stimulation ( same treatment in both treatment days ) with activin A ( Act ) , forskolin ( Fsk ) , PKA inhibitor ( PKI ) and the ALK4 inhibitor SB431542 ( SB ) as indicated . Blots of nuclear and cytosolic fractions were reprobed with anti - PCNA and anti - αTubulin ( αTub ) antibodies , respectively . Quantification of three independent experiments is shown on the right . Results are expressed as average ± SEM of fold increase over vehicle ( first column ) normalized to PCBP1 levels . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates . ( C ) Western blot analysis of Smad3 in PCBP1 immunoprecipitates from HeLa cells exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , forskolin ( Fsk ) or their combination as indicated . The Act + Fsk extracts were also immuoprecipitated using control IgG antibodies as additional control . Reprobing with anti - PCBP1 antibodies is shown in the middle blot . The lower blot shows unaltered Smad3 levels across all conditions . Quantification of four independent experiments ( biological replicates ) is shown on the right . Results are expressed as average ± SEM of fold increase of Smad3 / PCBP1 co - immunoprecipitation ( coIP ) over vehicle ( first column ) normalized to PCBP1 levels . ( D ) Western blot analysis of PCBP1 in Smad3 immunoprecipitates from HeLa cells using conditions identical to panel ( C ) . The lower blot shows unaltered PCBP1 levels across all conditions . Quantification of four independent experiments ( biological replicates ) is shown on the right . Results are expressed as average ± SEM of fold increase of PCBP1 / Smad3 co - immunoprecipitation ( coIP ) over vehicle ( first column ) normalized to Smad3 levels . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates . ( E ) Representative immunofluorescence staining of Smad3 ( green ) and PCBP1 ( red ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the lower row . Scale bar , 25μM . ( F , G ) Quantification of PCBP1 + ( F ) or Smad3 + ( G ) nuclei in cultured MSNs after the indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± SEM of % of MAP2 + cells displaying PCBP1 ( F ) or Smad3 ( G ) in the nucleus . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates . ( H ) Quantification of PCBP1 / Smad3 co - localization in nuclei of cultured MSNs using conditions identical to panel ( F ) . Results are expressed as average ± SEM ( biological replicates ) of % of MAP2 + cells displaying co - localization of PCBP1 and Smad3 in the nucleus . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates . ( I ) RT - qPCR analysis of PCBP1 mRNA in cultured MSNs exposed to consecutive stimulation as indicated ( same treatment in both treatment days ) . Results are expressed as average ± SEM of fold change over vehicle . No significant differences were found by one - way ANOVA followed with Tukey ' s multiple comparisons test ( p ≥ 0 . 05 ; N = 4 ) . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates . ( J ) Representative images of PLA ( red ) between Smad3 and PCBP1 ( S3 / PCBP1 ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) , PKA inhibitor ( PKI ) or SB431542 ( SB ) as indicated . Counterstaining with MAP2 and DAPI is shown in the lower row . Scale bar , 25μM . ( K ) Quantification of PLA between Smad3 and PCBP1 in cultured MSNs after the indicated treatments . Results are expressed as average ± SEM of number of PLA dots per cell . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Western blot analysis of Protein Kinase A phospho-substrates (PKA Ⓟ-subs) in PCBP1 immunoprecipitates from HeLa cells exposed to sequential stimulation (same treatment in both treatment days) with forskolin (Fsk) in the absence or presence of the PKA inhibitor PKI14-22 (PKI). Reprobing with anti-PCBP1 antibodies is shown in the middle blot. Lower blot shows the total amount of PCBP1 in the cell lysates. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle normalized to PCBP1 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(B) Nuclear localization of PCBP1 analyzed by Western blotting of extracts from HeLa cells exposed to sequential stimulation (same treatment in both treatment days) with activin A (Act), forskolin (Fsk), PKA inhibitor (PKI) and the ALK4 inhibitor SB431542 (SB) as indicated. Blots of nuclear and cytosolic fractions were reprobed with anti-PCNA and anti-αTubulin (αTub) antibodies, respectively. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle (first column) normalized to PCBP1 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(C) Western blot analysis of Smad3 in PCBP1 immunoprecipitates from HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), forskolin (Fsk) or their combination as indicated. The Act+Fsk extracts were also immuoprecipitated using control IgG antibodies as additional control. Reprobing with anti-PCBP1 antibodies is shown in the middle blot. The lower blot shows unaltered Smad3 levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of Smad3/PCBP1 co-immunoprecipitation (coIP) over vehicle (first column) normalized to PCBP1 levels. (D) Western blot analysis of PCBP1 in Smad3 immunoprecipitates from HeLa cells using conditions identical to panel (C). The lower blot shows unaltered PCBP1 levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of PCBP1/Smad3 co-immunoprecipitation (coIP) over vehicle (first column) normalized to Smad3 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(E) Representative immunofluorescence staining of Smad3 (green) and PCBP1 (red) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM.(F, G) Quantification of PCBP1+ (F) or Smad3+ (G) nuclei in cultured MSNs after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying PCBP1 (F) or Smad3 (G) in the nucleus. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(H) Quantification of PCBP1/Smad3 co-localization in nuclei of cultured MSNs using conditions identical to panel (F). Results are expressed as average ± SEM (biological replicates) of % of MAP2+ cells displaying co-localization of PCBP1 and Smad3 in the nucleus. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(I) RT-qPCR analysis of PCBP1 mRNA in cultured MSNs exposed to consecutive stimulation as indicated (same treatment in both treatment days). Results are expressed as average ± SEM of fold change over vehicle. No significant differences were found by one-way ANOVA followed with Tukey's multiple comparisons test (p≥0.05; N=4). Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(J) Representative images of PLA (red) between Smad3 and PCBP1 (S3/PCBP1) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF), PKA inhibitor (PKI) or SB431542 (SB) as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM. (K) Quantification of PLA between Smad3 and PCBP1 in cultured MSNs after the indicated treatments. Results are expressed as average ± SEM of number of PLA dots per cell. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


Figure 3 ( D ) Representative images of RNA - PLA ( green ) between PCBP1 ( left ) or Smad3 ( right ) and sequences in exon 4 ( pE4 ) of FosB mRNA , respectively , in HeLa cells exposed to consecutive stimulation ( same treatment in both treatment days ) as indicated . Counterstaining with phalloidin and DAPI is shown in the second and fourth columns . Scale bars , 20μM . ( E , F ) Quantification of RNA - PLA between PCBP1 ( E ) or Smad3 ( F ) and pE4 in HeLa cells after the indicated treatments . Results are expressed as average ± SEM of number of RNA - PLA dots per cell . Only nuclear dots were quantified in this analysis . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates . ( G ) Representative images of RNA - PLA ( green ) between PCBP1 ( left ) or Smad3 ( right ) and sequences in exon 4 ( pE4 ) of FosB mRNA , respectively , in cultured MSNs exposed to consecutive stimulation as indicated . Counterstaining with phalloidin and DAPI is shown in the second and fourth columns . Scale bars , 20μM . ( H , I ) Quantification of RNA - PLA between PCBP1 ( E ) or Smad3 ( F ) and pE4 in cultured MSNs after the indicated treatments . Results are expressed as average ± SEM of number of RNA - PLA dots per cell . Only nuclear dots were quantified in this analysis . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological 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 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

Figure 3(D) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM. (E, F) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in HeLa cells after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(G) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in cultured MSNs exposed to consecutive stimulation as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM. (H, I) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in cultured MSNs after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


Figure 4 ( D ) Representative immunofluorescence staining of ∆ FosB ( red ) in cultured MSNs treated with scrambled and shPCBP1 lentiviruses and exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with green fluorescent protein ( GFP , expressed by the lentiviruses ) and DAPI is shown in the second and fourth rows . Scale bar , 25μM . ( E ) Quantification of ∆ FosB + nuclei in cultured MSNs treated with scrambled and shPCBP1 lentiviruses followed by the indicated treatments . Results are expressed as average ± SEM of % of GFP + cells displaying ∆ FosB in the nucleus . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; two - way ANOVA followed with Tukey ' s multiple comparisons test in E ) . N denotes biological 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

Figure 4(D) Representative immunofluorescence staining of ∆FosB (red) in cultured MSNs treated with scrambled and shPCBP1 lentiviruses and exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with green fluorescent protein (GFP, expressed by the lentiviruses) and DAPI is shown in the second and fourth rows. Scale bar, 25μM. (E) Quantification of ∆FosB+ nuclei in cultured MSNs treated with scrambled and shPCBP1 lentiviruses followed by the indicated treatments. Results are expressed as average ± SEM of % of GFP+ cells displaying ∆FosB in the nucleus. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; two-way ANOVA followed with Tukey's multiple comparisons test in E). N denotes biological replicates.


Figure 6 ( A - F ) RT - qPCR analysis of FosB ( A , D ) , c - Fos ( B , E ) and ∆ FosB ( C , F ) mRNAs in NAc of Gad67Cre ; Alk4fl / fl ( A - C ) mutant mice 1 hour ( A - C ) or 24 hour ( D - F ) after the second saline ( Sal ) or cocaine ( Coc ) injection of TIPS . Results are expressed as average ± SEM of fold increase over the saline group ( Sal / Sal ) of Gad67Cre mice . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates . ( G ) Representative immunofluorescence staining of ∆ FosB in Nac of control ( Gad67Cre ) and mutant ( Gad67Cre ; Alk4fl / fl ) mice , 24 hours after the second injection of the TIPS protocol . Scale bars 20 μm . ( H ) Quantification of ∆ FosB positive cells 24 hours after the second injection of TIPS protocol in the Nac of Gad67Cre and Gad67Cre ; Alk4fl / fl mice . Results are expressed as average ± SEM of the number of ∆ FosB + cells per mm2 . At least 10 images from both left and right hemispheres were analyzed per mouse and condition . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates . ( I ) Western blot analysis of FosB and ∆ FosB in in NAc of Gad67Cre and Gad67Cre ; Alk4fl / fl mice 24 hour after the second injection of TIPS . Blots were probed with an antibody that recognizes both FosB ( upper band , 48kDa ) and ∆ FosB ( lower band , 38 kDa ) . Lower blot shows reprobing with anti - β - actin . ( J , K ) Quantification of FosB ( J ) and ∆ FosB ( K ) levels in NAc of Gad67Cre and Gad67Cre ; Alk4fl / fl mice 24 hour after the second injection of TIPS . Results are expressed as average ± SEM of fold increase of FosB and ∆ FosB over the saline group ( Sal / Sal ) of Gad67Cre mice normalized to β - actin levels . N = 4s . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological 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 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 O

Figure 6(A-F) RT-qPCR analysis of FosB (A, D), c-Fos (B, E) and ∆FosB (C, F) mRNAs in NAc of Gad67Cre;Alk4fl/fl (A-C) mutant mice 1 hour (A-C) or 24 hour (D-F) after the second saline (Sal) or cocaine (Coc) injection of TIPS. Results are expressed as average ± SEM of fold increase over the saline group (Sal/Sal) of Gad67Cre mice. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(G) Representative immunofluorescence staining of ∆FosB in Nac of control (Gad67Cre) and mutant (Gad67Cre;Alk4fl/fl) mice, 24 hours after the second injection of the TIPS protocol. Scale bars 20 μm. (H) Quantification of ∆FosB positive cells 24 hours after the second injection of TIPS protocol in the Nac of Gad67Cre and Gad67Cre;Alk4fl/fl mice. Results are expressed as average ± SEM of the number of ∆FosB+ cells per mm2. At least 10 images from both left and right hemispheres were analyzed per mouse and condition. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(I) Western blot analysis of FosB and ∆FosB in in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Blots were probed with an antibody that recognizes both FosB (upper band, 48kDa) and ∆FosB (lower band, 38 kDa). Lower blot shows reprobing with anti-β-actin. (J, K) Quantification of FosB (J) and ∆FosB (K) levels in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Results are expressed as average ± SEM of fold increase of FosB and ∆FosB over the saline group (Sal/Sal) of Gad67Cre mice normalized to β-actin levels. N=4s. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


Figure 1A . KD efficiency of three different Syt11 shRNAs in DRG neurons . Cultured DRG neurons were infected with control or shRNA - expressing lentiviruses at DIV 1 , and Western blotting for Syt11 , Syt1 , Syt4 , synaptobrevin 2 ( Syb2 ) , SNAP25 , complexin 1 / 2 ( Cpx1 , Cpx2 ) , clathrin , adaptor protein 2 ( AP - 2 ) , and β - actin were performed at DIV 6 - 7 . N = 4 independent experiments . B , C . Representative Cm traces induced by 200 - ms pulse depolarization ( arrows ) ib DRG neurons . DRG neurons were transfected with plasmids expressing shSyt11 - 2 ( Syt11 KD , KD ) or scrambled shRNA ( Sc ) and Cm recording was performed 5 days after transfection . Endo - 5s represents the Cm decay 5 s after stimulation . Insets show Ca2 + currents recorded in the same neurons . D . Representative Cm overshoot recorded from an Syt11 KD neuron . About 45 % of the KD neurons showed excessive membrane retrieval after a 200 - ms depolarizing pulse . E . Averaged Cm traces recorded from Control ( Ctrl ) , Sc , KD ( from all three shRNAs , n = 39 for shSyt11 - 2 , n = 7 for shSyt11 - 1 , n = 14 for shSyt11 - 3 ) , and rescued ( with the shSyt11 - 2 - resistant form of Syt11 ) DRG neurons . F - H . Statistics of Cm jumps , Endo - 5s , and Ca2 + current recorded from DRGneurons as in ( B - E ) . Data were collected from 4 ( Sc , Rescue ) , 8 ( Ctrl ) , and 16 ( KD ) independent experiments . I . Normalized Cm changes induced by two pulses of 200 - ms depolarization ( arrows ) at a 1 - s interval . Data were normalized to the capacitance jump induced by the first depolarization . J . Paired - pulse ratios of ΔCm with different interstimulus intervals as in ( I ) . DRG neurons were stimulated with two 200 - ms pulses at different intervals as indicated , and the paired - pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one .

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

Figure 1A. KD efficiency of three different Syt11 shRNAs in DRG neurons. Cultured DRG neurons were infected with control or shRNA-expressing lentiviruses at DIV 1, and Western blotting for Syt11, Syt1, Syt4, synaptobrevin 2 (Syb2), SNAP25, complexin 1/2 (Cpx1, Cpx2), clathrin, adaptor protein 2 (AP-2), and β-actin were performed at DIV 6-7. N = 4 independent experiments.B, C. Representative Cm traces induced by 200-ms pulse depolarization (arrows) ib DRG neurons. DRG neurons were transfected with plasmids expressing shSyt11-2 (Syt11 KD, KD) or scrambled shRNA (Sc) and Cm recording was performed 5 days after transfection. Endo-5s represents the Cm decay 5 s after stimulation. Insets show Ca2+ currents recorded in the same neurons.D. Representative Cm overshoot recorded from an Syt11 KD neuron. About 45% of the KD neurons showed excessive membrane retrieval after a 200-ms depolarizing pulse.E. Averaged Cm traces recorded from Control (Ctrl), Sc, KD (from all three shRNAs, n = 39 for shSyt11-2, n = 7 for shSyt11-1, n = 14 for shSyt11-3), and rescued (with the shSyt11-2-resistant form of Syt11) DRG neurons.F-H. Statistics of Cm jumps, Endo-5s, and Ca2+ current recorded from DRGneurons as in (B-E). Data were collected from 4 (Sc, Rescue), 8 (Ctrl), and 16 (KD) independent experiments.I. Normalized Cm changes induced by two pulses of 200-ms depolarization (arrows) at a 1-s interval. Data were normalized to the capacitance jump induced by the first depolarization.J. Paired-pulse ratios of ΔCm with different interstimulus intervals as in (I). DRG neurons were stimulated with two 200-ms pulses at different intervals as indicated, and the paired-pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one.


Figure 2A , B . FM 1 - 43 uptake into DRG neurons stimulated by 100 mM K + for 30 s . The right - shifted cumulative frequency in ( B ) indicates the increased uptake level in KD neurons . Scale bars , 5 μm . C . Quantification of KCl - evoked FM 1 - 43 uptake with different stimulation times . D . Representative micrographs showing the increased constitutive FM 1 - 43 uptake ( for 5 min ) in KD neurons . Scale bars , 5 μm . E . Quantification of constitutive FM 1 - 43 uptake with different loading times . F . Basal [ Ca2 + ] i in DRG neurons measured with Fura - 2 . G . FM1 - 43 uptake by Syt11 KD ( RFP - positive ) and control ( RFP - negative ) hippocampal neurons stimulated by 100 mM K + for 2 min . Quantitative data are shown in the lower - right panel . Scale bars , 20 μm . H . Representative images showing the preloaded FM4 - 64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz . Scale bars , 5 μm . I . Time - course of FM4 - 64 unloading from control , Syt11 KD , and rescued nerve terminals within the same field of view ( 6 coverslips from at least 3 biological repeats each ) .

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

Figure 2A, B. FM 1-43 uptake into DRG neurons stimulated by 100 mM K+ for 30 s. The right-shifted cumulative frequency in (B) indicates the increased uptake level in KD neurons. Scale bars, 5 μm.C. Quantification of KCl-evoked FM 1-43 uptake with different stimulation times.D. Representative micrographs showing the increased constitutive FM 1-43 uptake (for 5 min) in KD neurons. Scale bars, 5 μm.E. Quantification of constitutive FM 1-43 uptake with different loading times.F. Basal [Ca2+]i in DRG neurons measured with Fura-2.G. FM1-43 uptake by Syt11 KD (RFP-positive) and control (RFP-negative) hippocampal neurons stimulated by 100 mM K+ for 2 min. Quantitative data are shown in the lower-right panel. Scale bars, 20 μm.H. Representative images showing the preloaded FM4-64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz. Scale bars, 5 μm.I. Time-course of FM4-64 unloading from control, Syt11 KD, and rescued nerve terminals within the same field of view (6 coverslips from at least 3 biological repeats each).


Figure 3A . Averaged ΔCm traces induced by 200 - ms depolarization in the presence and absence of dynasore . Endocytic inhibition by dynasore ( Dyna ) was estimated by comparison of Cm traces recorded during 1 - 3 min ( KD ) and 5 - 10 min ( KD + Dyna and Ctrl + Dyna ) after whole - cell dialysis . B , C . Endo - 5s and Cm jumps recorded as in ( A ) . D . Averaged ΔCm traces induced by 200 - ms depolarization in the presence and absence of dynole - 34 - 2TM ( Dynole ) . Cm traces and endocytic rate were measured as in ( A ) except that 10 μM dynole - 34 - 2TM with 0 . 1 % DMSO was dialyzed into patched cells . E , F . Endo - 5s and Cm jumps as in ( D ) . G , H . Averaged ∆ Cm traces and Endo - 5s recorded from KD neurons under control conditions ( 0 . 1 % DMSO ) . I . KCl - evoked FM 1 - 43 uptake in DRG neurons after 10 - min pre - incubation with 0 . 1 % DMSO ( control ) or with 100 μM dynasore .

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

Figure 3A. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynasore. Endocytic inhibition by dynasore (Dyna) was estimated by comparison of Cm traces recorded during 1-3 min (KD) and 5-10 min (KD + Dyna and Ctrl + Dyna) after whole-cell dialysis.B, C. Endo-5s and Cm jumps recorded as in (A).D. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynole-34-2TM (Dynole). Cm traces and endocytic rate were measured as in (A) except that 10 μM dynole-34-2TM with 0.1% DMSO was dialyzed into patched cells.E, F. Endo-5s and Cm jumps as in (D).G, H. Averaged ∆Cm traces and Endo-5s recorded from KD neurons under control conditions (0.1 % DMSO).I. KCl-evoked FM 1-43 uptake in DRG neurons after 10-min pre-incubation with 0.1% DMSO (control) or with 100 μM dynasore.


Figure 4A . DRG neurons expressing Myc - Syt11 were immunostained for Myc - Syt11 , dynamin ( Dyn ) , AP - 2 , clathrin ( CL ) , TGN46 , synaptobrevin 2 ( Syb2 ) , and LAMP1 . For dynamin , the box along the plasma membrane was straightened , enlarged , and is shown in the upper right panels with arrows indicating co - localized puncta . An enlarged inset of the inside of the cell ( box ) is shown in the lower panel . For localization of the transferrin receptor , EGFP - transferrin receptor ( TfR ) was expressed in DRG neurons . Scale bars , 10 μm . B . Analysis of co - localization of Myc - Syt11 with dynamin , AP - 2 , clathrin , TGN46 , synaptobrevin 2 , transferrin receptor , and LAMP1 . N = 3 independent experiments .

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

Figure 4A. DRG neurons expressing Myc-Syt11 were immunostained for Myc-Syt11, dynamin (Dyn), AP-2, clathrin (CL), TGN46, synaptobrevin 2 (Syb2), and LAMP1. For dynamin, the box along the plasma membrane was straightened, enlarged, and is shown in the upper right panels with arrows indicating co-localized puncta. An enlarged inset of the inside of the cell (box) is shown in the lower panel. For localization of the transferrin receptor, EGFP-transferrin receptor (TfR) was expressed in DRG neurons. Scale bars, 10 μm.B. Analysis of co-localization of Myc-Syt11 with dynamin, AP-2, clathrin, TGN46, synaptobrevin 2, transferrin receptor, and LAMP1. N = 3 independent experiments.


Figure 5A . Transferrin ( Tf , red ) uptake in Syt11 KD ( GFP - positive ) and control neurons . Quantitative data are on the right . Scale bars , 20 μm . B . Tf uptake in Syt11 over - expressing and control neurons . Quantitative data are on the right . Scale bars , 20 μm . C . Normalized ∆ Cm induced by 200 - ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double - exponential decay function ( solid black and red , fitted curves ) . D . Fast and slow time - constants of endocytosis in Syt11 KD neurons recorded as in ( C ) . E . KCl - evoked FM 1 - 43 uptake in DRG neurons after 1 - h pre - incubation with 0 . 1 % DMSO or 100 μM MDC in the bath solution .

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

Figure 5A. Transferrin (Tf, red) uptake in Syt11 KD (GFP-positive) and control neurons. Quantitative data are on the right. Scale bars, 20 μm.B. Tf uptake in Syt11 over-expressing and control neurons. Quantitative data are on the right. Scale bars, 20 μm.C. Normalized ∆Cm induced by 200-ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double-exponential decay function (solid black and red, fitted curves).D. Fast and slow time-constants of endocytosis in Syt11 KD neurons recorded as in (C).E. KCl-evoked FM 1-43 uptake in DRG neurons after 1-h pre-incubation with 0.1% DMSO or 100 μM MDC in the bath solution.


Figure 6A , B . Cm traces with bulk endocytosis ( arrowheads ) in control ( A ) and KD ( B ) neurons induced by 200 - ms depolarization . Expanded bulk events are shown on the right . C , D . Frequency distribution and bulk endocytic events / cell within 16 s after stimulation . E , F . Size and duration of Cm decay during bulk endocytosis . G , H . Representative z - projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons . Neurons were loaded with 50 μM tetramethylrhodamine - dextran ( 40 kD ) by 2 - min exposure to 100 mM KCl . Scale bars , 10 μm .

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

Figure 6A, B. Cm traces with bulk endocytosis (arrowheads) in control (A) and KD (B) neurons induced by 200-ms depolarization. Expanded bulk events are shown on the right.C, D. Frequency distribution and bulk endocytic events/cell within 16 s after stimulation.E, F. Size and duration of Cm decay during bulk endocytosis.G, H. Representative z-projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons. Neurons were loaded with 50 μM tetramethylrhodamine-dextran (40 kD) by 2-min exposure to 100 mM KCl. Scale bars, 10 μm.


Figure 7A . Representative electron micrographs showing a small / clear vesicle ( SV , arrow ) , dense - core vesicles ( DCVs , arrowheads ) and a clathrin - coated pit ( hatched arrow ) in DRG neurons . B , C . Diameter distributions of SVs and DCVs ( statistics from 6 control cells and 9 KD cells ) in DRG neurons . The red and green lines are curves fitted to a Gaussian function . D , E . Diameters of SVs and DCVs in Syt11 KD and control neurons with ( 100K , statistics from 6 control cells and 9 KD cells ) or without 100 mM K + stimulation ( SE , statistics from 6 cells for each group ) .

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

Figure 7A. Representative electron micrographs showing a small/clear vesicle (SV, arrow), dense-core vesicles (DCVs, arrowheads) and a clathrin-coated pit (hatched arrow) in DRG neurons.B, C. Diameter distributions of SVs and DCVs (statistics from 6 control cells and 9 KD cells) in DRG neurons. The red and green lines are curves fitted to a Gaussian function.D, E. Diameters of SVs and DCVs in Syt11 KD and control neurons with (100K, statistics from 6 control cells and 9 KD cells) or without 100 mM K+ stimulation (SE, statistics from 6 cells for each group).


Figure 8A . Representative electron micrographs of HRP uptake from standard ( SE ) or 100 mM K + ( 100K ) bath solution . Scale bar , 2 μm . B - D . HRP - labeled small vesicles ( filled arrows ) and endosomes ( arrowheads ) in DRG neurons . Quantitative data are shown in ( C ) and ( D ) . Scale bar , 200 nm . B - D . HRP - labeled small vesicles ( filled arrows ) and endosomes ( arrowheads ) in DRG neurons . Quantitative data are shown in ( C ) and ( D ) . Scale bar , 200 nm . E . Representative docked vesicles ( arrows ) in a DRG neuron . Quantitative data are on the right . Scale bar , 200 nm . F - H . Clathrin - coated pits ( CCPs , hatched arrows ) and bulk - endocytosis - related structures of invaginating membrane ( open arrowhead ) in DRGneurons . Quantitative data are shown in ( G ) and ( H ) . Scale bars , 200 nm . F - H . Clathrin - coated pits ( CCPs , hatched arrows ) and bulk - endocytosis - related structures of invaginating membrane ( open arrowhead ) in DRGneurons . Quantitative data are shown in ( G ) and ( H ) . Scale bars , 200 nm . I , J . Cumulative frequencies of diameter distributions of small vesicles and endosomes ( statistics from 6 control cells and 9 KD cells ) . Kolmogorov - Smirnov test , P = 0 . 304 for ( I ) and P = 0 . 553 for ( J ) .

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

Figure 8A. Representative electron micrographs of HRP uptake from standard (SE) or 100 mM K+ (100K) bath solution. Scale bar, 2 μm.B-D. HRP-labeled small vesicles (filled arrows) and endosomes (arrowheads) in DRG neurons. Quantitative data are shown in (C) and (D). Scale bar, 200 nm.B-D. HRP-labeled small vesicles (filled arrows) and endosomes (arrowheads) in DRG neurons. Quantitative data are shown in (C) and (D). Scale bar, 200 nm.E. Representative docked vesicles (arrows) in a DRG neuron. Quantitative data are on the right. Scale bar, 200 nm.F-H. Clathrin-coated pits (CCPs, hatched arrows) and bulk-endocytosis-related structures of invaginating membrane (open arrowhead) in DRGneurons. Quantitative data are shown in (G) and (H). Scale bars, 200 nm.F-H. Clathrin-coated pits (CCPs, hatched arrows) and bulk-endocytosis-related structures of invaginating membrane (open arrowhead) in DRGneurons. Quantitative data are shown in (G) and (H). Scale bars, 200 nm.I, J. Cumulative frequencies of diameter distributions of small vesicles and endosomes (statistics from 6 control cells and 9 KD cells). Kolmogorov-Smirnov test, P = 0.304 for (I) and P = 0.553 for (J).


Figure 9Figure 9 . Functional domains of Syt11 in endocytosisA . Diagram of the mutant forms of Syt11 used for rescue experiments . B - D . Statistics of FM4 - 64 ( 5 min ) , Tf , and large dextran ( 40 - KD ) uptake into KD neurons with or without rescue by indicated forms of Syt11 . All data are presented as mean ± s . e . m . of 3 independent experiments . One - way ANOVA , values labeled with different letters ( a , b , and c ) are significantly different from each other , * * * P < 0 . 001 . Student ' s t - test for the comparison between KD and control cells in ( B - D ) , * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 9Figure 9. Functional domains of Syt11 in endocytosisA. Diagram of the mutant forms of Syt11 used for rescue experiments.B-D. Statistics of FM4-64 (5 min), Tf, and large dextran (40-KD) uptake into KD neurons with or without rescue by indicated forms of Syt11.All data are presented as mean ± s.e.m. of 3 independent experiments. One-way ANOVA, values labeled with different letters (a, b, and c) are significantly different from each other, ***P < 0.001. Student's t-test for the comparison between KD and control cells in (B-D), **P < 0.01, ***P < 0.001.


Figure 1Colorectal tumor outcomes in WT ( n = 7 ) , IL - 33KO ( n = 4 ) and ST2KO ( n = 3 ) mice at the completion of the AOM / DSS carcinogenesis protocol . ( A ) Tumor volumes at the endpoint ( each dot represents a tumor , mice with no tumor are marked as zero ) , Skin tumor outcomes in WT ( n = 7 ) , IL - 33KO ( n = 6 ) and ST2KO ( n = 7 ) mice at the completion of the DMBA / DNFB carcinogenesis protocol . ( D ) Tumor volumes at the endpoint ( each dot represents a tumor , mice with no tumor are marked as zero ) , H ) Quantification of nuclear IL - 33 + epidermal and dermal cells in the DNFB - versus acetone - treated WT skin . Dots represent cell counts from three randomly selected high power field ( HPF ) images per sample ( n = 5 per group ) . J ) Quantification of nuclear IL - 33 + epithelial and stromal cells of colon treated with AOM / DSS versus no treatment . Dots represent cell counts from three randomly selected HPF images per sample ( n = 4 per group ) . Data information : Graphs show mean + SD , unpaired t - test .

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Figure 1Colorectal tumor outcomes in WT (n=7), IL-33KO (n=4) and ST2KO (n=3) mice at the completion of the AOM/DSS carcinogenesis protocol. (A) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero), Skin tumor outcomes in WT (n=7), IL-33KO (n=6) and ST2KO (n=7) mice at the completion of the DMBA/DNFB carcinogenesis protocol. (D) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero),H) Quantification of nuclear IL-33+ epidermal and dermal cells in the DNFB- versus acetone-treated WT skin. Dots represent cell counts from three randomly selected high power field (HPF) images per sample (n=5 per group). J) Quantification of nuclear IL-33+ epithelial and stromal cells of colon treated with AOM/DSS versus no treatment. Dots represent cell counts from three randomly selected HPF images per sample (n=4 per group). Data information: Graphs show mean + SD, unpaired t-test.


Figure 2 ( H ) ChIP - qPCR assay for Smad6 in the presence of IL - 33 full length or cytokine domain using an anti - RUNX2 antibody . After Pam212 cells were transfected with IL - 33 full length or cytokine domain for 24 hours , cell lysates were subjected to chromatin - immunoprecipitation with anti - RUNX2 antibody and eluted RUNX2 - bound chromatin was used for qPCR with primers against Smad6 promoter region . Anti - IgG ChIP - qPCR results are shown as negative controls ( n = 4 per group ) .

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Figure 2(H) ChIP-qPCR assay for Smad6 in the presence of IL-33 full length or cytokine domain using an anti-RUNX2 antibody. After Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours, cell lysates were subjected to chromatin-immunoprecipitation with anti-RUNX2 antibody and eluted RUNX2-bound chromatin was used for qPCR with primers against Smad6 promoter region. Anti-IgG ChIP-qPCR results are shown as negative controls (n=4 per group).


Figure 3 ( D ) Immunoblot of p - SMAD2 / 3 and SMAD2 / 3 proteins in response to TGF - β and poly ( I : C ) treatment . Pam212 cells were treated with poly ( I : C ) or PBS followed by incubation with and without TGF - β ( 5nM ) . Data represent three independent experiments with similar results . ( E ) Immunoblot of p - SMAD2 / 3 and SMAD2 / 3 proteins in response to TGF - β + poly ( I : C ) after knocking down of IL - 33 . Pam212 cells were transfected with siIl33 knockdown construct or siRNA control ( siCon ) for 30 hours followed by incubation with poly ( I : C ) and TGF - β . Data represent three independent experiments with similar results . ( F ) Immunoblot of p - SMAD2 / 3 and SMAD2 / 3 proteins upon the expression of IL - 33 full length or cytokine domain . Pam212 cells were transfected with IL - 33 full length or cytokine domain for 24 hours followed by incubation with and without TGF - β . Data represent three independent experiments with similar results . Data information : GAPDH is used as the control housekeeping protein in D - F ) . Graphs show mean + SD , NS : not significant , unpaired t - test . ( G ) Impact of IL - 33 knockdown on cell proliferation in response to TGF - β . Pam212 cells were treated with siIl33 or siCon followed by TGF - β treatment ( n = 7 in each group ) . ( H ) Impact of IL - 33 knockdown and SB431542 treatment on cell proliferation . Pam212 cells were treated with siIl33 or siCon in combination with SB431542 or DMSO ( carrier control , n = 7 in each group ) .

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Figure 3(D) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β and poly (I:C) treatment. Pam212 cells were treated with poly (I:C) or PBS followed by incubation with and without TGF-β (5nM). Data represent three independent experiments with similar results. (E) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β + poly (I:C) after knocking down of IL-33. Pam212 cells were transfected with siIl33 knockdown construct or siRNA control (siCon) for 30 hours followed by incubation with poly (I:C) and TGF-β. Data represent three independent experiments with similar results. (F) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins upon the expression of IL-33 full length or cytokine domain. Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours followed by incubation with and without TGF-β. Data represent three independent experiments with similar results. Data information: GAPDH is used as the control housekeeping protein in D-F). Graphs show mean + SD, NS: not significant, unpaired t-test.(G) Impact of IL-33 knockdown on cell proliferation in response to TGF-β. Pam212 cells were treated with siIl33 or siCon followed by TGF-β treatment (n=7 in each group). (H) Impact of IL-33 knockdown and SB431542 treatment on cell proliferation. Pam212 cells were treated with siIl33 or siCon in combination with SB431542 or DMSO (carrier control, n=7 in each group).


Figure 4 ( I ) SMAD signaling proteins levels in WT and K14 - IL33tg , ST2KO skin treated with acetone ( control ) at the completion of the DMBA / TPA carcinogenesis protocol . Tissue lysates prepared from the whole back skin of the animals were subjected to immunoblot with p - SMAD2 / 3 , p - SMAD1 / 5 , SMAD6 , SMAD2 / 3 and SMAD1 antibodies . GAPDH is used as the control housekeeping protein ( n = 5 in each group ) . ( J ) Representative images of IL - 33 and p - SMAD2 / 3 immunostaining on the adjacent sections of human squamous cell carcinoma ( SCC ) and normal skin ( scale bar : 100 μm ) . Data information : Graphs show mean + SD , NS : not significant , unpaired t - test .

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Figure 4(I) SMAD signaling proteins levels in WT and K14-IL33tg,ST2KO skin treated with acetone (control) at the completion of the DMBA/TPA carcinogenesis protocol. Tissue lysates prepared from the whole back skin of the animals were subjected to immunoblot with p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 antibodies. GAPDH is used as the control housekeeping protein (n=5 in each group).(J) Representative images of IL-33 and p-SMAD2/3 immunostaining on the adjacent sections of human squamous cell carcinoma (SCC) and normal skin (scale bar: 100 μm). Data information: Graphs show mean + SD, NS: not significant, unpaired t-test.


Figure 5 ( B ) Il33 and Smad6 mRNA levels in caerulein - treated WT pancreas compared with PBS - treated controls at the completion of chronic pancreatitis protocol ( n = 8 in caerulein and n = 9 in PBS group for Il33 , n = 9 in caerulein and n = 10 in PBS group for Smad6 , unpaired t - test ) . ( D ) Pancreatic tumor - free survival of WT ( n = 4 ) , IL - 33KO ( n = 5 ) and ST2KO ( n = 6 ) mice at the completion of the DMBA / caerulein carcinogenesis protocol ( log - rank test ) . ( G , H ) Quantification of nuclear ( G ) IL - 33 + and ( H ) p - SMAD2 / 3 + epithelial cells per HPF in the matched human pancreatic tissues . Each dot represents the average cell counts across three randomly selected HPF images per sample ( n = 18 patients , paired t - test ) .

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Figure 5(B) Il33 and Smad6 mRNA levels in caerulein-treated WT pancreas compared with PBS-treated controls at the completion of chronic pancreatitis protocol (n=8 in caerulein and n=9 in PBS group for Il33, n=9 in caerulein and n=10 in PBS group for Smad6, unpaired t-test).(D) Pancreatic tumor-free survival of WT (n=4), IL-33KO (n=5) and ST2KO (n=6) mice at the completion of the DMBA/caerulein carcinogenesis protocol (log-rank test).(G, H) Quantification of nuclear (G) IL-33+ and (H) p-SMAD2/3+ epithelial cells per HPF in the matched human pancreatic tissues. Each dot represents the average cell counts across three randomly selected HPF images per sample (n=18 patients, paired t-test).


Figure 1C . Western blot for POT1 and DDR markers upon POT1 deletion induced with 0 . 5 μM 4 - OHT . Cl75 - parental cell line . gRNA1 - POT1 knock - out in the population by transient transfection with pSpCas9 ( BB ) - 2A - puro containing gRNA1 sequence . EV - corresponding empty vector . D . Time course of telomere length changes upon POT1 removal in clone 35 . d ( days ) TP0 - time point 0 , no 4OHT . E . Growth curve for POT1 WT and POT1 knockout cells . Population doublings ( PDL ) are represented as mean ± SD ( three biological replicates ) . F . Quantification for TIFs in EdU positive and EdU negative cells upon POT1 removal in clone 35 . The bars show percentage of cells containing more than 5 TIFs ± SD . Experiments were performed in triplicate . At least 120 cells were analyzed per condition per replicate . Significance was determined using two - way ANOVA . p - values are indicated on the graph .

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Figure 1C. Western blot for POT1 and DDR markers upon POT1 deletion induced with 0.5 μM 4-OHT. Cl75 - parental cell line. gRNA1 - POT1 knock-out in the population by transient transfection with pSpCas9(BB)-2A-puro containing gRNA1 sequence. EV - corresponding empty vector.D. Time course of telomere length changes upon POT1 removal in clone 35. d (days) TP0 - time point 0, no 4OHT.E. Growth curve for POT1 WT and POT1 knockout cells. Population doublings (PDL) are represented as mean ± SD (three biological replicates).F. Quantification for TIFs in EdU positive and EdU negative cells upon POT1 removal in clone 35. The bars show percentage of cells containing more than 5 TIFs ± SD. Experiments were performed in triplicate. At least 120 cells were analyzed per condition per replicate. Significance was determined using two-way ANOVA. p-values are indicated on the graph.


Figure 2A . G - overhang assay for clone 35 upon POT1 removal . The bars show relative amounts of single stranded telomeric DNA ± SD . Experiments were performed in triplicate . B . 2D gels for telomeric DNA from clone 35 cells with POT1 ( NT ) and upon POT1 removal for 7 days ( 7d 4OHT ) . C . Examples of metaphase spreads for non - treated ( NT ) clone 35 cells and clone 35 treated with 4 - OHT for 4 and 7 days . White arrowheads indicate fragile telomeres , the red arrowhead indicates a sister - chromatid fusion . Scale bar equals 6 µm . D . Quantification of fragile telomeres ( left graph ) , sister chromatid fusions ( middle graph ) and chromosome fusions ( right graph ) as a percentage of events per metaphase . Experiments were performed in triplicate . At least 25 metaphases were examined per condition per replicate . The mean is displayed and error bars represent ± SD . Significance was determined using one - way ANOVA . p - values are indicated on the graph . E . Quantification and representative images of anaphases with bridges and micronuclei for non - treated ( NT ) clone 35 cells and clone 35 treated with 4 - OHT for 4 and 7 days . Scale bar equals 6 µm . Experiments were performed in triplicate . The bars show percentage of cells containing anaphases , anaphases with bridges or micronuclei + SD . At least 650 cells were examined per condition per replicate . Significance was determined using one - way ANOVA . p - values are indicated on the graph . In all experiments , POT1 deletion was induced with 0 . 5 μM 4 - OHT .

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Figure 2A. G-overhang assay for clone 35 upon POT1 removal. The bars show relative amounts of single stranded telomeric DNA ± SD. Experiments were performed in triplicate.B. 2D gels for telomeric DNA from clone 35 cells with POT1 (NT) and upon POT1 removal for 7 days (7d 4OHT).C. Examples of metaphase spreads for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. White arrowheads indicate fragile telomeres, the red arrowhead indicates a sister-chromatid fusion. Scale bar equals 6 µm. D. Quantification of fragile telomeres (left graph), sister chromatid fusions (middle graph) and chromosome fusions (right graph) as a percentage of events per metaphase. Experiments were performed in triplicate. At least 25 metaphases were examined per condition per replicate. The mean is displayed and error bars represent ± SD. Significance was determined using one-way ANOVA. p-values are indicated on the graph. E. Quantification and representative images of anaphases with bridges and micronuclei for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Scale bar equals 6 µm. Experiments were performed in triplicate. The bars show percentage of cells containing anaphases, anaphases with bridges or micronuclei + SD. At least 650 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT.


Figure 3A . Western blot for POT1 and DDR markers upon POT1 removal in clone 35 and overexpression of ectopic WT POT1 . B . Telomere length and G - overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic WT POT1 ( constant - field gel electrophoresis ) . D , E Telomere length and G - overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic POT1 variants carrying disease - associated mutations ( constant - field gel electrophoresis ) .

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Figure 3A. Western blot for POT1 and DDR markers upon POT1 removal in clone 35 and overexpression of ectopic WT POT1.B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic WT POT1 (constant-field gel electrophoresis).D, E Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic POT1 variants carrying disease-associated mutations (constant-field gel electrophoresis).


Figure 4B . Quantification of precipitated telomeric DNA in 2 - step QTIP ( % of input ) and fold enrichment of precipitated telomeric DNA compared with precipitated Alu repeat DNA ( based on dot blot analyses ) . Bars represent data from three independent experiments ± SD . C . Scatter plots representing immunoprecipitation specificity ( 2 - step QTIP / IgG ratio ) versus difference between non treated cells ( NT ) and cells at day 4 upon 4 - OHT induction ( 4d 4OHT / NT ratio ) or difference between non treated cells ( NT ) and cells at day 7 upon 4 - OHT induction ( 7d 4OHT / NT ratio ) respectively ( data from Table EV1 ) . MRN complex in red , 9 - 1 - 1 complex in blue , shelterin complex in orange except for POT1 in black . Values are the average of the 3 biological replicates . D . Heat map representing changes in levels of selected proteins involved in DNA damage ( MRN complex in red , 9 - 1 - 1 complex in blue ) and HDR proteins ( purple ) after POT1 knockout induction . Proteins marked with * pass t - test , but not t - test with Benjamini - Hochberg correction ( p < 0 . 05 , q < 0 . 09 ) . Data information : 4 - OHT - 4 - hydroxytamoxifen , NT - non - treated cells .

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

Figure 4B. Quantification of precipitated telomeric DNA in 2-step QTIP (% of input) and fold enrichment of precipitated telomeric DNA compared with precipitated Alu repeat DNA (based on dot blot analyses). Bars represent data from three independent experiments ± SD.C. Scatter plots representing immunoprecipitation specificity (2-step QTIP/IgG ratio) versus difference between non treated cells (NT) and cells at day 4 upon 4-OHT induction (4d 4OHT/NT ratio) or difference between non treated cells (NT) and cells at day 7 upon 4-OHT induction (7d 4OHT/NT ratio) respectively (data from Table EV1). MRN complex in red, 9-1-1 complex in blue, shelterin complex in orange except for POT1 in black. Values are the average of the 3 biological replicates.D. Heat map representing changes in levels of selected proteins involved in DNA damage (MRN complex in red, 9-1-1 complex in blue) and HDR proteins (purple) after POT1 knockout induction. Proteins marked with * pass t-test, but not t-test with Benjamini-Hochberg correction (p < 0.05, q < 0.09). Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells.


Figure 6A . Telomere length for clone 35 upon POT1 removal and depletion of selected recombination proteins with siRNA ( pulsed - field gel electrophoresis ) . B . Telomere length and G - overhang length analysis for clone 35 upon POT1 removal and inhibition of telomerase with 1 µM GRN163L ( pulsed - field gel electrophoresis ) . C . DRIP assay for DNA - RNA hybrid detection at telomeres in clone 35 upon POT1 removal with S9 . 6 antibody . Dot blots represent two replicates . Bars represent data from three independent experiments ± SD . The IgG signal was subtracted . Significance was determined using one - way ANOVA , p - values are indicated on the graph . D . C - circle ( CC ) assay for clone 35 upon POT1 removal . Bars represent data from three independent experiments ± SD . DNA from ALT positive cell line U2OS was used as positive control . Significance was determined using one - way ANOVA , p - values are indicated on the graph . In all experiments , POT1 deletions was induced with 0 . 5 μM 4 - OHT for 4 and 7 days . E . C - overhang length analysis for clone 35 upon POT1 removal ( pulsed - field gel electrophoresis , hybridization with Telo - G probe ) . F . Quantification and representative pictures for visualization of the PML protein ( IF , green ) and telomeres ( FISH , red ) in non - treated ( NT ) clone 35 cells and clone 35 treated with 4 - OHT for 4 and 7 days . Arrows indicate co - localizations . Scale bar equals 10 µm . Bars represent data from three independent experiments ± SD . At least 150 cells were examined per condition per replicate . Significance was determined using one - way ANOVA . p - values are indicated on the graph . In all experiments , POT1 deletion was induced with 0 . 5 μM 4 - OHT . Data information : 4 - OHT - 4 - hydroxytamoxifen , NT - non - treated cells , GRN163L - telomerase inhibitor Imitelstat , siNT - non - tagreting siRNA , negative control .

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

Figure 6A. Telomere length for clone 35 upon POT1 removal and depletion of selected recombination proteins with siRNA (pulsed-field gel electrophoresis).B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and inhibition of telomerase with 1 µM GRN163L (pulsed-field gel electrophoresis).C. DRIP assay for DNA-RNA hybrid detection at telomeres in clone 35 upon POT1 removal with S9.6 antibody. Dot blots represent two replicates. Bars represent data from three independent experiments ± SD. The IgG signal was subtracted. Significance was determined using one-way ANOVA, p-values are indicated on the graph.D. C-circle (CC) assay for clone 35 upon POT1 removal. Bars represent data from three independent experiments ± SD. DNA from ALT positive cell line U2OS was used as positive control. Significance was determined using one-way ANOVA, p-values are indicated on the graph. In all experiments, POT1 deletions was induced with 0.5 μM 4-OHT for 4 and 7 days.E. C-overhang length analysis for clone 35 upon POT1 removal (pulsed-field gel electrophoresis, hybridization with Telo-G probe).F. Quantification and representative pictures for visualization of the PML protein (IF, green) and telomeres (FISH, red) in non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Arrows indicate co-localizations. Scale bar equals 10 µm. Bars represent data from three independent experiments ± SD. At least 150 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT. Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells, GRN163L - telomerase inhibitor Imitelstat, siNT -non-tagreting siRNA, negative control.


Figure 1Differences in clinical variables including ( B ) liver fat , plasma levels of ( C ) ALT , ( D ) AST , ( E ) uric acid and ( F ) creatinine are presented in the CMA and placebo groups on Days 0 , 14 and 70 after weight loss adjustment . Adj . P indicates p value after weight loss adjustment . Statistical significance is defined based on paired Student ' s t test . p < 0 . 05 . The boxes show the distribution of the clinical parameters in different groups . The bottom and top of the boxes represent the 25th and 75th percentiles . The central band represents the median value . The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values . The sample sizes on Days 0 , 14 or 70 were marked in each boxplot .

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

Figure 1Differences in clinical variables including (B) liver fat, plasma levels of (C) ALT, (D) AST, (E) uric acid and (F) creatinine are presented in the CMA and placebo groups on Days 0, 14 and 70 after weight loss adjustment. Adj.P indicates p value after weight loss adjustment. Statistical significance is defined based on paired Student's t test. p< 0.05. The boxes show the distribution of the clinical parameters in different groups. The bottom and top of the boxes represent the 25th and 75th percentiles. The central band represents the median value. The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values. The sample sizes on Days 0, 14 or 70 were marked in each boxplot.