H ) Quantification of the interaction of DRP1 variants with BAX based on the % cells with detectable RA - BAX / GB - DRP1 fluorescence signal ( interacting cells ) normalized to the number of mito - BFP positive cells . Box plots represent the interquartile ( box ) , median ( line ) and SD ( whiskers ) of n = 3 independent experiments ( with n = 100 cells each ) . Significance was tested using paired two - tailed Student ´ s t - test ( n . s . p > 0 . 05 ) compared to DRP1 WT .

H) Quantification of the interaction of DRP1 variants with BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Significance was tested using paired two-tailed Student´s t-test (n.s. p > 0.05) compared to DRP1 WT.


C ) Forced dimerization of BAX and DRP1 in healthy cells induces their translocation to mitochondria and foci formation . Confocal microscopy images of U2OS BAX / BAK DKO cells transfected with FKBP - mCherry - DRP1 ( red ) and FRB - EGFP - BAX ( green ) and stained with MitoTracker Deep Red FM ( cyan ) were acquired before ( 0 min ) and after induction of BAX / DRP1 dimerization ( 5 - 60 min ) . From top to down , the individual rows correspond to fluorescence signal of BAX and DRP1 and the merge of both together with the MitoTracker signal , respectively . Scale bar 20 µm .

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

C) Forced dimerization of BAX and DRP1 in healthy cells induces their translocation to mitochondria and foci formation. Confocal microscopy images of U2OS BAX/BAK DKO cells transfected with FKBP-mCherry-DRP1 (red) and FRB-EGFP-BAX (green) and stained with MitoTracker Deep Red FM (cyan) were acquired before (0 min) and after induction of BAX/DRP1 dimerization (5-60 min). From top to down, the individual rows correspond to fluorescence signal of BAX and DRP1 and the merge of both together with the MitoTracker signal, respectively. Scale bar 20 µm.


E ) Induced dimerization of BAX and DRP1 causes mitochondrial depolarization . U2OS BAX / BAK DKO cells were transfected as described in C ) and stained with the mitochondrial membrane potential - sensitive dye MitoSpy NIR ( cyan ) . Confocal microscopy images were acquired before ( 0 min ) and after induced dimerization of BAX and DRP1 ( 10 min ) . Scale Bar 20 µm . F ) Quantification of mitochondrial depolarization normalized to the number of transfected cells before ( Ctrl ) and after induced dimerization of BAX and DRP1 ( Dim ) . Significance was tested using paired two - tailed Student ´ s t - test ( * p < 0 . 05 ) compared to non - induced control .

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

E) Induced dimerization of BAX and DRP1 causes mitochondrial depolarization. U2OS BAX/BAK DKO cells were transfected as described in C) and stained with the mitochondrial membrane potential-sensitive dye MitoSpy NIR (cyan). Confocal microscopy images were acquired before (0 min) and after induced dimerization of BAX and DRP1 (10 min). Scale Bar 20 µm. F) Quantification of mitochondrial depolarization normalized to the number of transfected cells before (Ctrl) and after induced dimerization of BAX and DRP1 (Dim). Significance was tested using paired two-tailed Student´s t-test (* p < 0.05) compared to non-induced control. G


G - K ) Induced dimerization of DRP1 with itself ( G ) , BAX with itself ( H ) , BAX with TOM20 ( I ) in U2OS BAX / BAK DKO cells , or BAX with DRP1 in HCT OctaKO cells ( J , K ) , transected with FKBP - and FRB - mCherry DRP1 ( G , red ) , FKBP - and FRB - EGFP - BAX ( H , green ) , TOM20 - mCherry - FKBP ( red ) and FRB - EGFP - BAX ( green , I ) , respectively . Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR ( cyan ) as indicated . Scale bar 20 µm ( G - I ) and 10 µm ( J , K ) . All images are representative of n = 3 independent experiments .

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

G-K) Induced dimerization of DRP1 with itself (G), BAX with itself (H), BAX with TOM20 (I) in U2OS BAX/BAK DKO cells, or BAX with DRP1 in HCT OctaKO cells (J,K), transected with FKBP- and FRB-mCherry DRP1 (G, red), FKBP- and FRB-EGFP-BAX (H, green), TOM20-mCherry-FKBP (red) and FRB-EGFP-BAX (green, I), respectively. Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR (cyan) as indicated. Scale bar 20 µm (G-I) and 10 µm (J,K ). All images are representative of n=3 independent experiments.


Confocal micrographs show CK7 ( green ) and vimentin ( red ) in patient - derived HGSC cells cultured in 3D collagen for 7 d . Scale bars : 50 μm .

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

Confocal micrographs show CK7 (green) and vimentin (red) in patient-derived HGSC cells cultured in 3D collagen for 7 d. Scale bars: 50 μm.


Confocal micrographs of EphA2 ( red ) in HGSC cells cultured in 3D collagen for 7 d and treated for 72 h with 10 μM cisplatin ( 5 μM for OCKI _ p01 ) . The intensity of EphA2 is comparable only between mock and treatment conditions for each patient . Scale bars : 20 μm .

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

Confocal micrographs of EphA2 (red) in HGSC cells cultured in 3D collagen for 7 d and treated for 72 h with 10 μM cisplatin (5 μM for OCKI_p01). The intensity of EphA2 is comparable only between mock and treatment conditions for each patient. Scale bars: 20 μm.


Chart illustrates EphA2 fold change after treatment . Mock is set to one . N = 3 .

Chart illustrates EphA2 fold change after treatment. Mock is set to one. N = 3.


EphA2 ( total and phosphorylated at S897 or Y588 ) in OVCAR3 , OVCAR4 and OVCAR8 after treatment with 0 - 20 μM cisplatin for 72 h was assessed by immunoblotting ( A ) and quantified for pS897 / pY588 ratio ( B ) . N = 4 .

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

EphA2 (total and phosphorylated at S897 or Y588) in OVCAR3, OVCAR4 and OVCAR8 after treatment with 0-20 μM cisplatin for 72 h was assessed by immunoblotting (A) and quantified for pS897/pY588 ratio (B). N = 4.


EphA2 ( total and phosphorylated ) in corresponding untreated cells .

EphA2 (total and phosphorylated) in corresponding untreated cells.


Cytotoxicity assay results after cell treatment with 0 - 20 μM cisplatin for 72 h . N = 6 .

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

Cytotoxicity assay results after cell treatment with 0-20 μM cisplatin for 72 h. N = 6.


Quantitative assessment of EphA2 ( total and phosphorylated ) and pS897 / pY588 ratio in early passage patient - derived HGSC cultures treated with 0 - 20 μM cisplatin for 72 h ( see immunoblots in Appendix Fig S2A ) . N = 6 patients , pooled .

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

Quantitative assessment of EphA2 (total and phosphorylated) and pS897/pY588 ratio in early passage patient-derived HGSC cultures treated with 0-20 μM cisplatin for 72 h (see immunoblots in Appendix Fig S2A). N = 6 patients, pooled.


Corresponding EphA2 pS897 / pY588 ratios for individual patient cells .

Corresponding EphA2 pS897/pY588 ratios for individual patient cells.


EphA2 ( total and phosphorylated ) in OVCAR4 and OVCAR8 after treatment with 0 - 80 μM carboplatin for 72 h ( G ) along with pS897 / pY588 quantification ( H ) . N = 3 .

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

EphA2 (total and phosphorylated) in OVCAR4 and OVCAR8 after treatment with 0-80 μM carboplatin for 72 h (G) along with pS897/pY588 quantification (H). N = 3.


Bioluminescence images visualize mock and carboplatin treated OVCAR4 xenograft tumors ( day 53 ; after 13 d i . p . treatment ) . Color scale unit : p / sec / cm2 / sr . Charts illustrate the tumor growth ( B ) and ascites volumes at the end of the follow - up ( C ) . Carboplatin treatment started on day 35 . Bioluminescent unit : p / sec / cm2 / sr . N = 5 mice / group .

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

Bioluminescence images visualize mock and carboplatin treated OVCAR4 xenograft tumors (day 53; after 13 d i.p. treatment). Color scale unit: p/sec/cm2/sr. Charts illustrate the tumor growth (B) and ascites volumes at the end of the follow-up (C). Carboplatin treatment started on day 35. Bioluminescent unit: p/sec/cm2/sr. N = 5 mice/group.


Representative micrographs of hematoxylin - eosin staining in the xenografts tumors . Scale bars : 100

Representative micrographs of hematoxylin-eosin staining in the xenografts tumors. Scale bars: 100 μm


EphA2 - pS897 immunofluorescence in the xenografts tumors . Scale bars : 10

EphA2-pS897 immunofluorescence in the xenografts tumors. Scale bars: 10 μm


Quantitative assessment of tumor EphA2 ( F ) and EphA2 - pS897 ( G ) . N = 5 mice / group .

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

Quantitative assessment of tumor EphA2 (F) and EphA2-pS897 (G). N = 5 mice/group.


Confocal micrographs of tumor EphA2 - pS897 ( red ) and cleaved caspase - 3 ( H ; clCasp3 , green ) and corresponding clCasp3 quantification ( I ) . Scale bars : 20 μm . N = 5 mice / group .

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

Confocal micrographs of tumor EphA2-pS897 (red) and cleaved caspase-3 (H; clCasp3, green) and corresponding clCasp3 quantification (I). Scale bars: 20 μm. N = 5 mice/group.


Immunoblot images show EphA2 ( A ; total , pS897 and pY588 ) and corresponding pS897 / pY588 ratios ( B ) of TYK - nu and TYK - nu . R after treatment with 0 - 10 μM cisplatin for 72 h . N = 5 .

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

Immunoblot images show EphA2 (A; total, pS897 and pY588) and corresponding pS897/pY588 ratios (B) of TYK-nu and TYK-nu.R after treatment with 0-10 μM cisplatin for 72 h . N = 5.


EphA2 ( total and phosphorylated ) in the cells treated with 0 - 80 μM carboplatin for 72 h .

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

EphA2 (total and phosphorylated) in the cells treated with 0-80 μM carboplatin for 72 h.


EphA2 ( total and phosphorylated ) in the

EphA2 (total and phosphorylated) in the cells


EphA2 ( total and pS897 ) in TYK - nu after treatment with inhibitors against MEK ( UO126 , 10 μM ) , PKA ( H89 . 2HCl , 10 μM ) , PI3K ( LY294002 , 10 μM ) or its alpha subunit ( BYL - 719 , 5 μM ) and RSK ( BI - D1870 , 10 μM ) in combination with 0 - 10 μM cisplatin for 72 h ( E ) . Corresponding EphA2 - pS897 quantification for BI - D1870 treatment ( F ) N = 5 .

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

EphA2 (total and pS897) in TYK-nu after treatment with inhibitors against MEK (UO126, 10 μM), PKA (H89.2HCl, 10 μM), PI3K (LY294002, 10 μM) or its alpha subunit (BYL-719, 5 μM) and RSK (BI-D1870, 10 μM) in combination with 0-10 μM cisplatin for 72 h (E). Corresponding EphA2-pS897 quantification for BI-D1870 treatment (F) N = 5.


EphA2 ( total and pS897 ) in TYK - nu . R treated with 0 - 20 μM cisplatin without or with 10 μM BI - D1870 for 72 h .

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

EphA2 (total and pS897) in TYK-nu.R treated with 0-20 μM cisplatin without or with 10 μM BI-D1870 for 72 h.


EphA2 , RSK and ERK1 / 2 ( total and phosphorylated ) in TYK - nu ( H ) and OVCAR4 ( I ) treated with 0 - 30 μM carboplatin without or with 50 μM LJH685 for 72 h .

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

EphA2, RSK and ERK1/2 (total and phosphorylated) in TYK-nu (H) and OVCAR4 (I) treated with 0-30 μM carboplatin without or with 50 μM LJH685 for 72 h.


Charts show OC cell viability after 72 h treatment with 0 - 20 μM cisplatin and 10 μM BI -

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

Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 10 μM BI-D1870


Charts show OC cell viability after 72 h treatment with 0 - 20 μM cisplatin and 50 μM

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

Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 50 μM LJH685


RSK and EphA2 ( total and phosphorylated ) in OC cells treated with 10 µM cisplatin and 25 µM LJH685 as indicated for 72 h .

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

RSK and EphA2 (total and phosphorylated) in OC cells treated with 10 µM cisplatin and 25 µM LJH685 as indicated for 72 h.


Charts illustrate the corresponding quantified inhibition of EphA2 - pS897 and increase of EphA2 - pY588 shown in ( C ) .

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

Charts illustrate the corresponding quantified inhibition of EphA2-pS897 and increase of EphA2-pY588 shown in (C).


Immunoblot images ( A ) and quantification ( B ) show GPRC5A 46 / 41 kDa ratios in OC cells treated with 0 - 10 µM cisplatin and with 25 µM LJH685 alone or as a combination for 72 h . The ratio in mock cells was set to 1 . N = 4 .

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

Immunoblot images (A) and quantification (B) show GPRC5A 46/41 kDa ratios in OC cells treated with 0-10 µM cisplatin and with 25 µM LJH685 alone or as a combination for 72 h. The ratio in mock cells was set to 1. N = 4.


Confocal micrographs of EphA2 ( red ) and GPRC5A ( green ) in TYK - nu and TYK - nu . R treated with 5 µM cisplatin and 10 μM LJH685 as indicated for 72 h . Arrows point co - localization of the receptors . Scale bars : 20 μm .

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

Confocal micrographs of EphA2 (red) and GPRC5A (green) in TYK-nu and TYK-nu.R treated with 5 µM cisplatin and 10 μM LJH685 as indicated for 72 h. Arrows point co-localization of the receptors. Scale bars: 20 μm.


EphA2 ( total and phosphorylated ) , RSK1 / 2 and GPRC5A in TYK - nu were assessed by immunoblotting after RSK1 / 2 knockdown for 2 d and following treatment with 0 - 5 μM cisplatin for 72 h . Asterisks indicate unspecific bands .

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

EphA2 (total and phosphorylated), RSK1/2 and GPRC5A in TYK-nu were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands.


EphA2 ( total and phosphorylated ) , RSK1 / 2 and GPRC5A and these proteins coupled with PARP and PCNA in TYK - nu . R were assessed by immunoblotting after RSK1 / 2 knockdown for 2 d and following treatment with 0 - 5 μM cisplatin for 72 h . Asterisks indicate unspecific bands .

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

EphA2 (total and phosphorylated), RSK1/2 and GPRC5A and these proteins coupled with PARP and PCNA in TYK-nu.R were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands.


Chart illustrates the viability of the RSK1 / 2 depleted TYK - nu . R after cisplatin treatment . N = 3 .

Chart illustrates the viability of the RSK1/2 depleted TYK-nu.R after cisplatin treatment. N = 3.


Confocal micrographs show cleaved caspase - 3 ( clCasp3 , green ) , mRFP ( orange ) and phalloidin ( F - actin , red ; only shown for CAF ) in 3D OVCAR8 - RFP and OCKI _ p22 CAF mono - and co - cultures treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h . Scale bars : 100 μm .

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

Confocal micrographs show cleaved caspase-3 (clCasp3, green), mRFP (orange) and phalloidin (F-actin, red; only shown for CAF) in 3D OVCAR8-RFP and OCKI_p22 CAF mono- and co-cultures treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 100 μm.


Quantitative assessment of clCasp3 + cells in the 3D cultures . N = 4 .

Quantitative assessment of clCasp3+ cells in the 3D cultures. N = 4.


Confocal micrographs show cytokeratin 7 ( CK7 , green ) and phalloidin ( F - actin , red ) in 3D patient OCKI _ p13 mono - and co - culture with OCKI _ p22 CAF treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h . Scale bars : 50 μm . Quantitative assessment of clCasp3 + OCKI _ p13 cells in 3D co - cultures of patient - derived cells . N = 4 .

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

Confocal micrographs show cytokeratin 7 (CK7, green) and phalloidin (F-actin, red) in 3D patient OCKI_p13 mono- and co-culture with OCKI_p22 CAF treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 50 μm. Quantitative assessment of clCasp3+ OCKI_p13 cells in 3D co-cultures of patient-derived cells. N = 4.


Immunohistochemistry images and quantifications of TUNEL in OVCAR4 xenografts in mock ( N = 4 ) , carboplatin ( N = 4 ) and carboplatin + BI - D1870 ( N = 5 ) treated mice . Scale bars : 100 μm .

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

Immunohistochemistry images and quantifications of TUNEL in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.


Immunohistochemistry images and quantifications of Ki67 in OVCAR4 xenografts in mock ( N = 4 ) , carboplatin ( N = 4 ) and carboplatin + BI - D1870 ( N = 5 ) treated mice . Scale bars : 100 μm .

Immunohistochemistry images and quantifications of Ki67 in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.


Representative confocal micrographs show EphA2 ( red ) , RSK ( green : top ) and GPRC5A ( green : bottom ) in frozen sections of HGSC patient tumors . S indicates the stroma . Scale bars : 50 μm .

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

Representative confocal micrographs show EphA2 (red), RSK (green: top) and GPRC5A (green: bottom) in frozen sections of HGSC patient tumors. S indicates the stroma. Scale bars: 50 μm.


Indicated proteins and EphA2 phosphorylation were assessed by immunoblotting in patient - derived HGSC cells and CAFs .

Indicated proteins and EphA2 phosphorylation were assessed by immunoblotting in patient-derived HGSC cells and CAFs.


GPRC5A in earlier ( top ) and later passage ( bottom ) HGSC cells . See normalized GPRC5A relative to OCKI _ p01 below the immunoblot images .

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

GPRC5A in earlier (top) and later passage (bottom) HGSC cells. See normalized GPRC5A relative to OCKI_p01 below the immunoblot images.


Charts illustrate EphA2 - pS897 inhibition ( circles ) and EphA2 - pY588 increase ( squares ) by 10 µM BI - D1870 alone ( unbroken line ) or in combination with 0 - 10 µM cisplatin ( dotted line ) in the GPRC5Ahigh HGSC cells . See Appendix S7B for immunoblots .

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

Charts illustrate EphA2-pS897 inhibition (circles) and EphA2-pY588 increase (squares) by 10 µM BI-D1870 alone (unbroken line) or in combination with 0-10 µM cisplatin (dotted line) in the GPRC5Ahigh HGSC cells. See Appendix S7B for immunoblots.


Cell viability of the GPRC5Ahigh OCKI _ p02 and OCKI _ p06 after treatment with a combination of 0 - 20 µM cisplatin and 10 µM BI - D1870 for 72 h . N = 3 .

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

Cell viability of the GPRC5Ahigh OCKI_p02 and OCKI_p06 after treatment with a combination of 0-20 µM cisplatin and 10 µM BI-D1870 for 72 h. N = 3.


Cell viability upon 72 h combinatorial treatment with 0 - 20 µM cisplatin and 25 µM LJH685 in patient - derived

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

Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 25 µM LJH685 in patient-derived CAFs


Cell viability upon 72 h combinatorial treatment with 0 - 20 µM cisplatin and 10 µM BI - D1870 in mesothelial cells ( G ; N = 3 ) .

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

Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 10 µM BI-D1870 in mesothelial cells (G; N = 3).


Chart illustrates the changes of EphA2 - pS897 and EphA2 - pY588 by BI - D1870 alone and with cisplatin in mesothelial cells . Upon combination with 10 µM cisplatin for 72 h , 10 µM BI - D1870 decreased EphA2 - pS897 and EphA2 - pY588 . See Appendix Fig S7J for immunoblot .

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

Chart illustrates the changes of EphA2-pS897 and EphA2-pY588 by BI-D1870 alone and with cisplatin in mesothelial cells. Upon combination with 10 µM cisplatin for 72 h, 10 µM BI-D1870 decreased EphA2-pS897 and EphA2-pY588. See Appendix Fig S7J for immunoblot.


Confocal micrographs of EphA2 ( red ) and GPRC5A ( green ) in primary OCKI _ p06 patient cells cultured in 3D collagen for 4 d before 72 h treatment with or without 10 µM cisplatin . Scale bars : 20 μm .

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

Confocal micrographs of EphA2 (red) and GPRC5A (green) in primary OCKI_p06 patient cells cultured in 3D collagen for 4 d before 72 h treatment with or without 10 µM cisplatin. Scale bars: 20 μm.


Representative immunostainings for GPRC5A scoring in HGSC primary and metastatic tumors . For maximum intensity scores an optical 4 - point scale ( 0 : negative , 1 : mild , 2 : moderate , 3 : high ) was used . For survival analyses a 2 - point scale ( low : scores 0 - 2 , high : score 3 ) was used . Scale bars : 200 μm .

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

Representative immunostainings for GPRC5A scoring in HGSC primary and metastatic tumors. For maximum intensity scores an optical 4-point scale (0: negative, 1: mild, 2: moderate, 3: high) was used. For survival analyses a 2-point scale (low: scores 0-2, high: score 3) was used. Scale bars: 200 μm.


Kaplan Meier survival curves illustrate the overall ( OS ) and progression - free ( PFS ) survival of patients with high or low GPRC5A . In primary tumors , mean OS for GPRC5Ahigh was 39 months vs 56 months for GPRC5Alow ( C ) . In patients with metastatic tumors , mean OS for GPRC5Ahigh was 37 months vs 60 months for GPRC5Alow . Mean PFS in metastatic tumors was 20 months for GPRC5Ahigh vs 35 months for GPRC5Alow ( D ) . No significant difference in mean PFS in primary tumor .

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

Kaplan Meier survival curves illustrate the overall (OS) and progression-free (PFS) survival of patients with high or low GPRC5A. In primary tumors, mean OS for GPRC5Ahigh was 39 months vs 56 months for GPRC5Alow (C). In patients with metastatic tumors, mean OS for GPRC5Ahigh was 37 months vs 60 months for GPRC5Alow. Mean PFS in metastatic tumors was 20 months for GPRC5Ahigh vs 35 months for GPRC5Alow (D). No significant difference in mean PFS in primary tumor.


Kaplan Meier survival estimates for the PFS of patients with high or low ( top 25 % vs bottom 25 % ) GPRC5A ( F ) , EPHA2 ( G ) , GPRC5A + EPHA2 ( H ) and GPRC5A + EPHA2 + RSK1 ( RPS6KA1 ) + RSK2 ( RPS6KA3 ) combined ( I ) mRNA expression . Mean PFS for GPRC5A + EPHA2high was 15 months vs 26 months for GPRC5A + EPHA2low ( H ) . Mean PFS for GPRC5A + EPHA2 + RPS6KA1 / 3 high was 14 months vs 31 months for GPRC5A + EPHA2 + RPS6KA1 / 3low ( I ) .

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

Kaplan Meier survival estimates for the PFS of patients with high or low (top 25% vs bottom 25%) GPRC5A (F), EPHA2 (G), GPRC5A+EPHA2 (H) and GPRC5A+EPHA2+RSK1(RPS6KA1)+RSK2(RPS6KA3) combined (I) mRNA expression. Mean PFS for GPRC5A+EPHA2high was 15 months vs 26 months for GPRC5A+EPHA2low (H). Mean PFS for GPRC5A+EPHA2+RPS6KA1/3 high was 14 months vs 31 months for GPRC5A+EPHA2+RPS6KA1/3low (I).


D H & E ( hematoxylin - eosin ) staining ( left ) and oil red O staining ( right ) of livers . Scale bar , 50μm .

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

D H&E (hematoxylin-eosin) staining (left) and oil red O staining (right) of livers. Scale bar, 50μm.


E Serum cholesterol concentrations of each group . Liver cholesterol concentrations ( F ) of each group were examined .

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

E Serum cholesterol concentrations of each group. Liver cholesterol concentrations (F) of each group were examined.


H and I Cholesterol levels in p53 + / + and p53 - / - HepG2 cells ( H ) or HCT116 cells ( I ) were examined . Protein expression was shown by western blotting ( bottom panel ) .

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

H and I Cholesterol levels in p53+/+ and p53-/- HepG2 cells (H) or HCT116 cells (I) were examined. Protein expression was shown by western blotting (bottom panel).


C mRNA and protein levels of SQLE in different tissues of p53 + / + or p53 - / - mice were determined by qRT - PCR and western blot respectively .

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

C mRNA and protein levels of SQLE in different tissues of p53+/+ or p53-/- mice were determined by qRT-PCR and western blot respectively.


F and G p53 + / + and p53 - / - HepG2 cells were cultured for 48 hours in medium containing fetal bovine serum ( Serum ) or lipoprotein - depleted FBS ( LPDS ) . Protein expression was shown by western blotting ( F ) . mRNA levels of SQLE were examined by qRT - PCR ( G ) . P : premature SREBP2 ; M : mature SREBP2 .

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

F and G p53+/+ and p53-/- HepG2 cells were cultured for 48 hours in medium containing fetal bovine serum (Serum) or lipoprotein-depleted FBS (LPDS). Protein expression was shown by western blotting (F). mRNA levels of SQLE were examined by qRT-PCR (G). P: premature SREBP2; M: mature SREBP2.


Protein expression and mRNA levels of p53 + / + and p53 - / - HepG2 cells treated with control siRNA or three separated sets of SREBP2 siRNAs for 72 hours as indicated .

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

Protein expression and mRNA levels of p53+/+ and p53-/- HepG2 cells treated with control siRNA or three separated sets of SREBP2 siRNAs for 72 hours as indicated.


F Luciferase reporter constructs containing RE1 , RE2 , RE1mut or RE2mut were transfected into p53 - / - HCT116 cells together with control ( PRK5 - flag vector ) or p53 expression vector ( PRK5 - flag p53 ) for 48 hours . Renilla vector pRL - CMV was used as a transfection internal control . Relative levels of luciferase are shown . Data represent three independent experiments . Protein expression is shown G Luciferase reporter constructs containing RE1 , RE2 , RE1mut or RE2mut were transfected into HEK293T cells together with control ( PRK5 - flag vector ) or p53 expression vector ( PRK5 - flag p53 ) for 48 hours . Renilla vector pRL - CMV was used as a transfection internal control . Relative levels of luciferase are shown . Protein expression is shown .

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

F Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into p53-/- HCT116 cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data represent three independent experiments. Protein expression is shown G Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into HEK293T cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Protein expression is shown.


A - C Squalene ( A ) , lanosterol ( B ) and cholesterol ( C ) abundance in p53 + / + and p53 - / - HepG2 cells transfected with control or SQLE siRNA for 48 hours were determined via ultra - high pressure liquid chromatography coupled to mass spectrometry ( UHPLC - MS ) . Protein levels were analyzed by western blotting with specific antibodies ( A bottom panel ) .

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

A-C Squalene (A), lanosterol (B) and cholesterol (C) abundance in p53+/+ and p53-/- HepG2 cells transfected with control or SQLE siRNA for 48 hours were determined via ultra-high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS). Protein levels were analyzed by western blotting with specific antibodies (A bottom panel).


A Protein expression of SQLE in human liver tumors ( C ) and non - cancerous adjacent tissue ( N ) were analyzed by western blotting .

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

A Protein expression of SQLE in human liver tumors (C) and non-cancerous adjacent tissue (N) were analyzed by western blotting.


B SQLE gene expression in HCC and adjacent normal tissue was analyzed in TCGA Liver Hepatocellular Carcinoma ( normal [ n = 50 ] versus tumors [ n = 371 ] ) . C SQLE gene expression in COAD and adjacent normal tissue was analyzed in TCGA Colon Cancer ( normal [ n = 41 ] versus tumors [ n = 286 ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SQLE gene expression in HCC and adjacent normal tissue was analyzed in TCGA Liver Hepatocellular Carcinoma (normal [n=50] versus tumors [n=371]). C SQLE gene expression in COAD and adjacent normal tissue was analyzed in TCGA Colon Cancer (normal [n=41] versus tumors [n=286]).


J - M p53 + / + HCT116 cells ( J and K ) and p53 + / + HepG2 cells ( L and M ) were transfected with SQLE siRNA or control siRNA in the presence or absence of exogenous SQLE cDNA for 48 hours . Cholesterol concentration ( upper panel ) and protein expression ( bottom panel ) were determined ( J and L ) . Cell proliferation is shown ( K and M ) .

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

J - M p53+/+ HCT116 cells (J and K) and p53+/+ HepG2 cells (L and M) were transfected with SQLE siRNA or control siRNA in the presence or absence of exogenous SQLE cDNA for 48 hours. Cholesterol concentration (upper panel) and protein expression (bottom panel) were determined (J and L). Cell proliferation is shown (K and M).


C Growth of p53 + / + and p53 - / - HepG2 cells transfected with and without exogenous SQLE ( HA - SQLE ) in LPDS medium . Protein expression is assayed after transfection 24 hours .

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

C Growth of p53+/+ and p53-/- HepG2 cells transfected with and without exogenous SQLE (HA-SQLE) in LPDS medium. Protein expression is assayed after transfection 24 hours.


G and H Cholesterol levels , protein expression ( G ) and cell growth ( H ) of SK - HEP - 1 cells stably expressing control shRNA or p53 shRNA in LPDS medium in presence or absence of 5μg cholesterol .

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

G and H Cholesterol levels, protein expression (G) and cell growth (H) of SK-HEP-1 cells stably expressing control shRNA or p53 shRNA in LPDS medium in presence or absence of 5μg cholesterol.


L Percentage of SA - β - gal - positive cells of p53 + / + and p53 - / - HepG2 cells expressing SQLE siRNA or control siRNA ( upper panel ) . Protein expression ( middle panel ) and representative images ( lower panel ) are shown . Sacle bar , 100 μm .

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

L Percentage of SA-β-gal-positive cells of p53+/+ and p53-/- HepG2 cells expressing SQLE siRNA or control siRNA (upper panel). Protein expression (middle panel) and representative images (lower panel) are shown . Sacle bar, 100 μm.


p53 - / - C57BL / 6N male mice ( n = 7 ) were injected with a single dose of diethylnitrosamine ( DEN ) ( 25mg / kg ) at two weeks and then fed with HFD diet at 4 weeks . Oral administration of terbinafine were given at 13 weeks old as indicated . Shown are liver tumor incidence ( L ) and tumor numbers ( M ) .

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

p53-/- C57BL/6N male mice (n=7) were injected with a single dose of diethylnitrosamine (DEN) (25mg/kg) at two weeks and then fed with HFD diet at 4 weeks. Oral administration of terbinafine were given at 13 weeks old as indicated. Shown are liver tumor incidence (L) and tumor numbers (M).


( N ) H & E ( left ) , Ki - 67 ( middle ) and Oil Red O ( right ) staining of livers of mice .

(N) H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice.


( O ) Total liver cholesterol concentrations of mice were examined .

(O) Total liver cholesterol concentrations of mice were examined.


representative liver photos ( C , left ) , liver tumor numbers ( C , right ) , representative images ( D , left ) and quantification ( D , right ) of Ki - 67 staining are shown . Red arrows indicate the tumors in the liver ( C ) .

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

representative liver photos (C, left), liver tumor numbers (C, right), representative images (D, left) and quantification (D, right) of Ki-67 staining are shown. Red arrows indicate the tumors in the liver (C).


E H & E ( left ) , Ki - 67 ( middle ) and Oil Red O ( right ) staining of livers of

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

E H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice


A . Serum levels of 15 - keto - dihydro - PGF2α in type 2 diabetic patients without diabetic retinopathy ( no DR ) , with non - proliferative diabetic retinopathy ( NPDR ) , and with proliferative diabetic retinopathy ( PDR ) ( n = 20 - 24 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Kruskal - Wallis test with Dunn ' s multiple comparisons test ( A ) , Data are represented as mean ± SEM .

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

A. Serum levels of 15-keto-dihydro-PGF2α in type 2 diabetic patients without diabetic retinopathy (no DR), with non-proliferative diabetic retinopathy (NPDR), and with proliferative diabetic retinopathy (PDR) (n=20-24). Data information: n.s. stands for "not significant." Data were analyzed by Kruskal-Wallis test with Dunn's multiple comparisons test (A), Data are represented as mean ± SEM.


C . Retinal PGF2α production in OIR mice during postnatal days 12 - 17 ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by , two - way ANOVA with Tukey ' s multiple comparisons test ( C , Data are represented as mean ± SEM .

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

C. Retinal PGF2α production in OIR mice during postnatal days 12-17 (n=4). Data information: n.s. stands for "not significant." Data were analyzed by , two-way ANOVA with Tukey's multiple comparisons test (C, Data are represented as mean ± SEM.


D . mRNA expression of PGF2α synthases in the retinas of OIR mice during postnatal days 12 - 17 ( n = E . mRNA expression of Ptgfr in the retinas of OIR mice during postnatal days 12 - 17 ( n = 6 - 8 ) . Data information : n . s . stands for " not significant . " Data were analyzed by , two - way ANOVA with Tukey ' s multiple comparisons test D , E , Data are represented as mean ± SEM .

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

D. mRNA expression of PGF2α synthases in the retinas of OIR mice during postnatal days 12-17 (n= E. mRNA expression of Ptgfr in the retinas of OIR mice during postnatal days 12-17 (n=6-8). Data information: n.s. stands for "not significant." Data were analyzed by , two-way ANOVA with Tukey's multiple comparisons test D, E, Data are represented as mean ± SEM.


F . mRNA expression of Ptgfr in each retinal layer from normoxic and OIR mice on postnatal day 16 ( n = 6 ) . The left - hand image displays representative cross - sections from OIR and normoxic retinas , with isolectin B4 - stained vessels in green , DAPI - stained nuclei in blue , and LCM isolation locations circled by white dashed circles . INL , inner nuclear layer ; ONL , outer nuclear layer ; RGC , retinal ganglion cells . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test F ) Scale bar : 20 μm . Data are represented as mean ± SEM .

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

F. mRNA expression of Ptgfr in each retinal layer from normoxic and OIR mice on postnatal day 16 (n=6). The left-hand image displays representative cross-sections from OIR and normoxic retinas, with isolectin B4-stained vessels in green, DAPI-stained nuclei in blue, and LCM isolation locations circled by white dashed circles. INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cells. Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test F) Scale bar: 20 μm. Data are represented as mean ± SEM.


G . Ptgfr mRNA expression in retinal endothelial cells ( CD31 + CD45 - ) sorted by MACS from normoxic and OIR mice on postnatal day 16 ( n = 5 ) . Data information : n . s . stands for " not significant . " Data were analyzed Mann - Whitney test ( G ) . Scale bar : 20 μm . Data are represented as mean ± SEM .

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

G. Ptgfr mRNA expression in retinal endothelial cells (CD31+CD45-) sorted by MACS from normoxic and OIR mice on postnatal day 16 (n=5). Data information: n.s. stands for "not significant." Data were analyzed Mann-Whitney test (G). Scale bar: 20 μm. Data are represented as mean ± SEM.


B . Ptgfr mRNA levels in retinal microvessels of CKO - T and control mice ( n = 6 ) . INL , inner nuclear layer ; ONL , outer nuclear layer ; RGC , retinal ganglion cells . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test ( B , Data are represented as mean ± SEM .

B. Ptgfr mRNA levels in retinal microvessels of CKO-T and control mice (n=6). INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cells. Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, Data are represented as mean ± SEM.


C . Representative images of OIR retinas from CKO - T and control mice on postnatal day 17 . The green color shows the isolectin B4 - stained vessels . The second - row panels are the enlarged images of white boxes from the first - row panels . The third - row images show neovascular tufts ( NV , white ) and the fourth - row images show the vaso - obliteration ( VO , white ) area . D . Quantitation of oxygen - induced retinal neovascularization in CKO - T and control mice ( n = 12 ) . E . Quantitation of retinal vaso - obliteration in CKO - T and control mice ( n = 12 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test D , E ) . Scale bars : 500 μm ( unmagnified image ) , 150 μm ( magnified image ) . Data are represented as mean ± SEM .

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

C. Representative images of OIR retinas from CKO-T and control mice on postnatal day 17. The green color shows the isolectin B4-stained vessels. The second-row panels are the enlarged images of white boxes from the first-row panels. The third-row images show neovascular tufts (NV, white) and the fourth-row images show the vaso-obliteration (VO, white) area. D. Quantitation of oxygen-induced retinal neovascularization in CKO-T and control mice (n=12). E. Quantitation of retinal vaso-obliteration in CKO-T and control mice (n=12). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test D, E). Scale bars: 500 μm (unmagnified image), 150 μm (magnified image). Data are represented as mean ± SEM.


A . Representative images of scratch - induced migration of PGF2α - treated HRMECs . Yellow solid lines represent scratch locations , dashed lines represent post - migration locations , and arrows indicate migration direction . B . Quantitation of the cell migration distance in A ( n = 4 ) . C . Effect of PGF2α treatment on HRMEC proliferation in a dose - dependent manner . ( n = 6 ) . Data information : One data point represented the mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney test ( B , Kruskal - Wallis test with Dunn ' s multiple comparisons test ( C ) . Scale bars : 100 μm ( A ) Data are represented as mean ± SEM .

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

A. Representative images of scratch-induced migration of PGF2α-treated HRMECs. Yellow solid lines represent scratch locations, dashed lines represent post-migration locations, and arrows indicate migration direction. B. Quantitation of the cell migration distance in A (n=4). C. Effect of PGF2α treatment on HRMEC proliferation in a dose-dependent manner. (n=6). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test (B, Kruskal-Wallis test with Dunn's multiple comparisons test (C). Scale bars: 100 μm (A) Data are represented as mean ± SEM.


D . Representative images of staining for BrdU incorporation in PGF2α - treated HRMECs . E . Quantification of BrdU + cells in D ( n = 4 ) . Data information : One data point represented the mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney test Data are represented as mean ± SEM .

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

D. Representative images of staining for BrdU incorporation in PGF2α-treated HRMECs. E. Quantification of BrdU+ cells in D (n=4). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test Data are represented as mean ± SEM.


F . Representative tube formation images of PGF2α - or vehicle ( DMSO ) - treated HRMECs . G - J . Quantitation of total tubule length , number of junctions , number of meshes , and percentage of mesh area ( n = 4 ) . Data information : One data point represented the mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney test G , H , I , J , Scale bars : 200 μm ( F , Data are represented as mean ± SEM .

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

F. Representative tube formation images of PGF2α-or vehicle (DMSO)-treated HRMECs. G-J. Quantitation of total tubule length, number of junctions, number of meshes, and percentage of mesh area (n=4). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test G, H, I, J, Scale bars: 200 μm (F, Data are represented as mean ± SEM.


K . Representative images of endothelial cell sprouting of PGF2α - treated mouse aortic rings . Yellow lines indicate the EC sprouting area . L . Quantification of the EC sprouting area ( n = 6 ) . Data information : One data point represented the mean value of three technical replicates from one independent biological replicate . Data were analyzed by Mann - Whitney test L ) Scale bars : 200 μm K ) . Data are represented as mean ± SEM .

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

K. Representative images of endothelial cell sprouting of PGF2α-treated mouse aortic rings. Yellow lines indicate the EC sprouting area. L. Quantification of the EC sprouting area (n=6). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test L) Scale bars: 200 μm K). Data are represented as mean ± SEM.


A . Heat map of upregulated genes in PGF2α - treated HRMECs ( n = 3 samples ) .

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

A. Heat map of upregulated genes in PGF2α-treated HRMECs (n=3 samples).


C . Effect of PGF2α treatment on ELR + CXC chemokine expression in HRMECs ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired Student ' s t - test ( C ) Data in C .

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

C. Effect of PGF2α treatment on ELR+ CXC chemokine expression in HRMECs (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired Student's t-test (C) Data in C.


D - G . Relative expression levels of enriched ELR + CXC chemokines in retinal microvascular ECs from patients with PDR ( n = 4 ) compared with those from normal subjects ( n = 7 ) ( Gene Expression Omnibus Database dataset GSE94019 ) . TPM stands for " Transcripts per kilobase million . " Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test ( D , E , F , G ) , Data are presented as the mean + SD .

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

D-G. Relative expression levels of enriched ELR+ CXC chemokines in retinal microvascular ECs from patients with PDR (n=4) compared with those from normal subjects (n=7) (Gene Expression Omnibus Database dataset GSE94019). TPM stands for "Transcripts per kilobase million." Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (D, E, F, G), Data are presented as the mean + SD.


H . Effect of CXCR2 inhibitor SB265610 ( 1 μM ) on PGF2α - stimulated HRMEC migration . The yellow solid line indicates the original position of the scratch , the dashed line indicates the position after cell migration , and the arrows indicate the migratory direction . I . Quantitation of scratch - induced HRMEC migration in H ( n = 4 ) . J . Effect of SB265610 ( 1 μM ) on PGF2α - stimulated HRMEC proliferation ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test ( I , J , Data in I . J . are represented as mean ± SEM . Scale bar : 100 μm ( H )

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

H. Effect of CXCR2 inhibitor SB265610 (1 μM) on PGF2α-stimulated HRMEC migration. The yellow solid line indicates the original position of the scratch, the dashed line indicates the position after cell migration, and the arrows indicate the migratory direction. I. Quantitation of scratch-induced HRMEC migration in H (n=4). J. Effect of SB265610 (1 μM) on PGF2α-stimulated HRMEC proliferation (n=4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (I, J, Data in I. J. are represented as mean ± SEM. Scale bar: 100 μm (H)


K . Effect of CXCR2 inhibitor SB265610 ( 1μM ) on tube formation in PGF2α - treated HRMECs . L . Statistical graph of tube formation experiments in K , comparing the total tubule length , number of junctions , number of meshes , and percentage of mesh area ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test L ) . Data in L are represented as mean ± SEM . Scale bar : 200 μm ( K ) .

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

K. Effect of CXCR2 inhibitor SB265610 (1μM) on tube formation in PGF2α-treated HRMECs. L. Statistical graph of tube formation experiments in K, comparing the total tubule length, number of junctions, number of meshes, and percentage of mesh area (n=4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test L). Data in L are represented as mean ± SEM. Scale bar: 200 μm (K).


A . Effect of CXCR2 inhibitor SB265610 ( 1 μM ) on PGF2α - promoted sprouting of aortic rings . Yellow lines show the sprouting area . B . Quantitation of the sprouting areas in A ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test ( B , Scale bar : 200 μm ( A ) , image ) . Data are represented as mean ± SEM .

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

A. Effect of CXCR2 inhibitor SB265610 (1 μM) on PGF2α-promoted sprouting of aortic rings. Yellow lines show the sprouting area. B. Quantitation of the sprouting areas in A (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (B, Scale bar: 200 μm (A), image).Data are represented as mean ± SEM.


C . mRNA expression of ELR + CXC chemokines in retinas from OIR mice during postnatal days 12 - 17 ( n = 4 - 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test image ) . Data are represented as mean ± SEM .

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

C. mRNA expression of ELR+ CXC chemokines in retinas from OIR mice during postnatal days 12-17 (n=4-6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test image).Data are represented as mean ± SEM.


D . Effect of PTGFR deficiency in ECs on ELR + CXC chemokine expression in retinas from OIR mice on postnatal day 16 ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test image ) . Data are represented as mean ± SEM .

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

D. Effect of PTGFR deficiency in ECs on ELR+ CXC chemokine expression in retinas from OIR mice on postnatal day 16 (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test image).Data are represented as mean ± SEM.


F . Retinal CXCL1 protein - expression levels in Cxcl1 - expressing lentivirus - injected CKO mice ( n = 3 ) . G . Quantitation of retinal CXCL1 protein in F ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test G , image ) . Data are represented as mean ± SEM .

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

F. Retinal CXCL1 protein-expression levels in Cxcl1-expressing lentivirus-injected CKO mice (n=3). G. Quantitation of retinal CXCL1 protein in F (n=3). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test G, image).Data are represented as mean ± SEM.


H . Representative images of oxygen - induced retinal angiogenesis in Cxcl1 - expressing lentivirus - injected CKO mice on postnatal day 17 . Green represents the isolectin B4 - stained vessels , the second - row panels display the enlarged images of white boxes in the first - row panels , the third - row images show neovascular tufts ( NV , white ) , and the fourth - row images show the vaso - obliteration ( VO , white ) area . I . Quantitation of oxygen - induced retinal neovascularization in H ( n = 8 ) . J . Quantitation of retinal vaso - obliteration in H ( n = 8 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test , I , J ) . Scale bar : 500 μm ( H , unmagnified image ) , 150 μm ( H , magnified image ) . Data are represented as mean ± SEM .

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

H. Representative images of oxygen-induced retinal angiogenesis in Cxcl1-expressing lentivirus-injected CKO mice on postnatal day 17. Green represents the isolectin B4-stained vessels, the second-row panels display the enlarged images of white boxes in the first-row panels, the third-row images show neovascular tufts (NV, white), and the fourth-row images show the vaso-obliteration (VO, white) area. I. Quantitation of oxygen-induced retinal neovascularization in H (n=8). J. Quantitation of retinal vaso-obliteration in H (n=8). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test , I, J). Scale bar: 500 μm (H, unmagnified image), 150 μm (H, magnified image).Data are represented as mean ± SEM.


A . Heat map of differentially expressed transcription factors in HRMECs in response to 500 nM PGF2α ( n = 3 ) .

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

A. Heat map of differentially expressed transcription factors in HRMECs in response to 500 nM PGF2α (n=3).


B . Relative expression levels of FOS in retinal microvascular ECs from patients with PDR ( n = 4 ) compared with the corresponding expression levels from normal subjects ( n = 7 ) ( Gene Expression Omnibus Database dataset GSE94019 ) . TPM stands for " Transcripts per kilobase million . " Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test ( B , Data in B are expressed as the mean + SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Relative expression levels of FOS in retinal microvascular ECs from patients with PDR (n=4) compared with the corresponding expression levels from normal subjects (n=7) (Gene Expression Omnibus Database dataset GSE94019). TPM stands for "Transcripts per kilobase million." Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, Data in B are expressed as the mean + SD.


C . Effect of FOS knockdown on 500 nM PGF2α - induced CXCL8 expression in HRMECs ( n = 4 ) . D . Effect of FOS knockdown on 500 nM PGF2α - induced HRMEC CXCL8 secretion in the culture medium ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test C , D , Data in C , D , are represented as mean ± SEM .

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

C. Effect of FOS knockdown on 500 nM PGF2α-induced CXCL8 expression in HRMECs (n=4). D. Effect of FOS knockdown on 500 nM PGF2α-induced HRMEC CXCL8 secretion in the culture medium (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test C, D, Data in C, D, are represented as mean ± SEM.


E . and F . Effect of RA ( 1 μM , AP - 1 dimer inhibitor ) , SN - 50 ( 100 μg / mL , NFκB inhibitor ) , and Inca - 6 ( 2 . 5 μM , NFAT inhibitor ) on 500 nM PGF2α - induced CXCL8 expression in HRMECs and HRMEC CXCL8 secretion in the culture medium ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test E , F , Data in E , F , are represented as mean ± SEM .

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

E. and F. Effect of RA (1 μM, AP-1 dimer inhibitor), SN-50 (100 μg/mL, NFκB inhibitor), and Inca-6 (2.5 μM, NFAT inhibitor) on 500 nM PGF2α-induced CXCL8 expression in HRMECs and HRMEC CXCL8 secretion in the culture medium (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test E, F, Data in E, F, are represented as mean ± SEM.


G . Representative images of scratch - induced migration of 500 nM PGF2α - treated HRMECs with or without FOS knockdown . The yellow solid line indicates the original position of the scratch , the dashed line indicates the position of the cells after migration , and the arrows represent the migratory direction . H . Quantitation of the cell - migration distance in H ( n = 4 ) . I . Effect of FOS knockdown on 500 nM PGF2α - induced HRMEC proliferation ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test ( H , I Scale bar : 100 μm ( G ) Data in H , I , are represented as mean ± SEM .

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

G. Representative images of scratch-induced migration of 500 nM PGF2α-treated HRMECs with or without FOS knockdown. The yellow solid line indicates the original position of the scratch, the dashed line indicates the position of the cells after migration, and the arrows represent the migratory direction. H. Quantitation of the cell-migration distance in H (n=4). I. Effect of FOS knockdown on 500 nM PGF2α-induced HRMEC proliferation (n=4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (H, I Scale bar: 100 μm (G) Data in H, I, are represented as mean ± SEM.


J . Representative images of tube formation by 500 nM PGF2α - treated HRMECs with or without FOS knockdown . K . Quantitation of total tube length , number of junctions , number of meshes , and percentage of mesh area in J ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test K , Scale bar : 200 μm ( J , Data in K , are represented as mean ± SEM .

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

J. Representative images of tube formation by 500 nM PGF2α-treated HRMECs with or without FOS knockdown. K. Quantitation of total tube length, number of junctions, number of meshes, and percentage of mesh area in J (n=4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test K, Scale bar: 200 μm (J, Data in K, are represented as mean ± SEM.


L . Fos mRNA levels in retinas from OIR mice during postnatal days 12 - 17 ( n = 3 - 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test L , Data in , L , are represented as mean ± SEM .

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

L. Fos mRNA levels in retinas from OIR mice during postnatal days 12-17 (n=3-4). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test L, Data in , L, are represented as mean ± SEM.


M . Effect of PTGFR deficiency in ECs on Fos expression in the retina from OIR mice on postnatal day 16 ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test M , Data in M , are represented as mean ± SEM .

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

M. Effect of PTGFR deficiency in ECs on Fos expression in the retina from OIR mice on postnatal day 16 (n=6). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test M, Data in M, are represented as mean ± SEM.


N . Effect of FOS inhibitor SR11302 ( 1 μM ) on 500 nM PGF2α - promoted sprouting of mouse aortic rings . Data information : Scale bar : 200 μm N ) .

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

N. Effect of FOS inhibitor SR11302 (1 μM) on 500 nM PGF2α-promoted sprouting of mouse aortic rings. Data information: Scale bar: 200 μm N).


O . Quantitation of sprouting areas in N ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test O ) . Data in O , are represented as mean ± SEM .

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

O. Quantitation of sprouting areas in N (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test O). Data in O, are represented as mean ± SEM.


P . Effect of FOS inhibitor SR11302 ( 1 μM ) on 500 nM PGF2α - promoted CXCL1 secretion in mouse aortic ring culture medium ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test P ) Data in P are represented as mean ± SEM .

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

P. Effect of FOS inhibitor SR11302 (1 μM) on 500 nM PGF2α-promoted CXCL1 secretion in mouse aortic ring culture medium (n=6). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test P) Data in P are represented as mean ± SEM.


B . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , Y27632 ( 5 μM ) , and Ly294002 ( 15 μM ) treatment on 500 nM PGF2α - induced FOS gene expression in HRMECs ( n = 4 ) . C . Effect of KN93 ( 10 μM ) and RO - 318220 ( 250 nM ) treatment on 500 nM PGF2α - induced FOS expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test ( B , C Data are represented as mean ± SEM .

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

B. Effect of U73122 (10 μM), BAPTA (50 μM), Y27632 (5 μM), and Ly294002 (15 μM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs (n=4). C. Effect of KN93 (10 μM) and RO-318220 (250 nM) treatment on 500 nM PGF2α-induced FOS expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test (B, C Data are represented as mean ± SEM.


D . Effect of SB203580 ( 2 μM ) , PD98059 ( 20 μM ) , and SP600125 ( 60 nM ) treatment on 500 nM PGF2α - induced FOS gene expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test Data are represented as mean ± SEM .

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

D. Effect of SB203580 (2 μM), PD98059 (20 μM), and SP600125 (60 nM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test Data are represented as mean ± SEM.


E . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , and KN93 ( 10 μM ) treatment on 500 nM PGF2α - induced p38 phosphorylation in HRMECs . F . Quantitation of the ratio of p38 phosphorylation to total p38 protein in E ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test ( F , Data are represented as mean ± SEM .

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

E. Effect of U73122 (10 μM), BAPTA (50 μM), and KN93 (10 μM) treatment on 500 nM PGF2α-induced p38 phosphorylation in HRMECs. F. Quantitation of the ratio of p38 phosphorylation to total p38 protein in E (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test (F, Data are represented as mean ± SEM.


G . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , and SB203580 ( 2 μM ) on the phosphorylation of CAMK2 isoforms in 500 nM PGF2α - treated HRMECs . H . Quantitation of the ratio of CAMK2G phosphorylation to total CAMK2G in G ( n = 3 ) . I . Quantitation of the ratio of phosphorylated CAMK2D to total CAMK2D in G ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test H , I , Data are represented as mean ± SEM .

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

G. Effect of U73122 (10 μM), BAPTA (50 μM), and SB203580 (2 μM) on the phosphorylation of CAMK2 isoforms in 500 nM PGF2α-treated HRMECs. H. Quantitation of the ratio of CAMK2G phosphorylation to total CAMK2G in G (n=3). I. Quantitation of the ratio of phosphorylated CAMK2D to total CAMK2D in G (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test H, I, Data are represented as mean ± SEM.


J . Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α - triggered p38 phosphorylation in HRMECs . K . Quantitation of the ratio of p38 phosphorylation to total p38 protein in J ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test Data are represented as mean ± SEM .

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

J. Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α-triggered p38 phosphorylation in HRMECs. K. Quantitation of the ratio of p38 phosphorylation to total p38 protein in J (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test Data are represented as mean ± SEM.


L . Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α - induced FOS expression in HRMECs ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Mann - Whitney test L ) Data are represented as mean ± SEM .

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

L. Effect of CAMK2G or CAMK2D knockdown on 500 nM PGF2α-induced FOS expression in HRMECs (n=4). Data information: n.s. stands for "not significant." Data were analyzed by Mann-Whitney test L) Data are represented as mean ± SEM.


M . Effect of U73122 ( 10 μM ) , BAPTA ( 50 μM ) , KN93 ( 10 μM ) , and SB203580 ( 2 μM ) on ELK - 1 phosphorylation in 500 nM PGF2α - treated HRMECs . N . Quantitation of the ratio of phosphorylated ELK - 1 to total ELK - 1 in M ( n = 3 ) . Data information : n . s . stands for " not significant . " Data were analyzed by unpaired student ' s t - test N ) . Data are represented as mean ± SEM .

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

M. Effect of U73122 (10 μM), BAPTA (50 μM), KN93 (10 μM), and SB203580 (2 μM) on ELK-1 phosphorylation in 500 nM PGF2α-treated HRMECs. N. Quantitation of the ratio of phosphorylated ELK-1 to total ELK-1 in M (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired student's t-test N). Data are represented as mean ± SEM.


B . Representative images of OIR retinas in WT and Cxcr2 - / - mice with or without AL8810 treatment . The green color shows the isolectin B4 - stained vessels , the second - row panels display the enlarged images of white boxes in the first - row panels , the third - row images show neovascular tufts ( NV , white ) , and the fourth - row images show the vaso - obliteration ( VO , white ) area . C . Quantitation of oxygen - induced retinal neovascularization in WT and Cxcr2 - / - mice with or without AL8810 treatment ( n = 12 ) . D . Quantitation of retinal vaso - obliteration in WT and Cxcr2 - / - mice with or without AL8810 treatment ( n = 12 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test ( C , D ) Scale bar : 500 μm ( unmagnified image ) , 150 μm ( magnified image ) . Data are represented as mean ± SEM .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of OIR retinas in WT and Cxcr2-/- mice with or without AL8810 treatment. The green color shows the isolectin B4-stained vessels, the second-row panels display the enlarged images of white boxes in the first-row panels, the third-row images show neovascular tufts (NV, white), and the fourth-row images show the vaso-obliteration (VO, white) area. C. Quantitation of oxygen-induced retinal neovascularization in WT and Cxcr2-/- mice with or without AL8810 treatment (n=12). D. Quantitation of retinal vaso-obliteration in WT and Cxcr2-/- mice with or without AL8810 treatment (n=12). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (C, D) Scale bar: 500 μm (unmagnified image), 150 μm (magnified image). Data are represented as mean ± SEM.


E . Effect of AL8810 on retinal Fos expression in WT and Cxcr2 - / - OIR mice on postnatal day 16 ( n = 6 ) . F . Effect of AL8810 on retinal Cxcl1 expression in WT and Cxcr2 - / - OIR mice on postnatal day 16 ( n = 6 ) . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Mann - Whitney test ( E , F ) . Data are represented as mean ± SEM .

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

E. Effect of AL8810 on retinal Fos expression in WT and Cxcr2-/- OIR mice on postnatal day 16 (n=6). F. Effect of AL8810 on retinal Cxcl1 expression in WT and Cxcr2-/- OIR mice on postnatal day 16 (n=6). Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Mann-Whitney test (E, F). Data are represented as mean ± SEM.


b , Representative confocal images of NeuN ( green ) and Npas4 ( red ) immunostaining performed 1h after the last injection of cocaine or saline ( scale bar : 50 µm ) .

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

b, Representative confocal images of NeuN (green) and Npas4 (red) immunostaining performed 1h after the last injection of cocaine or saline (scale bar: 50 µm).


c , Quantifications of Npas4 - positive cells ( fold change normalized to the 30min saline control group ) in the NAc and dStr . Mean + SEM ; n = 4 mice per group . 10 images ( 5 per hemisphere ) per mouse for the NAc and 10 images ( 5 per hemisphere ) for the dStr . p * * < 0 . 01 , unpaired Student ' s t - test .

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

c, Quantifications of Npas4-positive cells (fold change normalized to the 30min saline control group) in the NAc and dStr. Mean + SEM; n = 4 mice per group. 10 images (5 per hemisphere) per mouse for the NAc and 10 images (5 per hemisphere) for the dStr. p** < 0.01, unpaired Student's t-test.


c , MSN cultures at DIV11 were treated for 2h with either 10 µM of the D1 - agonist SKF - 38393 ( SKF ) alone or with SKF and 10 µM of the selective D1 - antagonist SCH - 23390 ( SCH ) . qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos and Arc . Mean + SEM ; one - way ANOVA with Holm - Sidak post - hoc correction ; p * * < 0 . 01 , p * * * * < 0 . 0001 ; n = 3 independent cultures as biological replicates .

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

c, MSN cultures at DIV11 were treated for 2h with either 10 µM of the D1-agonist SKF-38393 (SKF) alone or with SKF and 10 µM of the selective D1-antagonist SCH-23390 (SCH). qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos and Arc. Mean + SEM; one-way ANOVA with Holm-Sidak post-hoc correction; p** < 0.01, p**** < 0.0001; n = 3 independent cultures as biological replicates.


d , MSNs were depolarized with potassium chloride ( HiK , 55 mM ) or treated with the D1 - agonist SKF - 38393 ( 10 µM ) for 1h , 2h or 4h . Subsequently , qPCR was performed to assess immediate early gene mRNA levels . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test ; indicated p - values for induction over control for each treatment ; p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 ; n = 4 independent cultures as biological replicates .

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

d, MSNs were depolarized with potassium chloride (HiK, 55 mM) or treated with the D1-agonist SKF-38393 (10 µM) for 1h, 2h or 4h. Subsequently, qPCR was performed to assess immediate early gene mRNA levels. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test; indicated p-values for induction over control for each treatment; p* < 0.05, p** < 0.01, p***<0.001; n = 4 independent cultures as biological replicates.


g , Differential induction of Npas4 through HiK and not forskolin in hiPSC - derived forebrain organoids . Organoids were treated with HiK ( 55 mM ) or the PKA activator forskolin plus TTX ( Fsk , 10 µM ; TTX 1 µM ) for 1h before qPCR analysis was performed to assess mRNA levels of Npas4 and Arc . Mean + SEM ; n = 7 organoids in control condition , n = 5 organoids for HiK , n = 5 organoids for Fsk / TTX . One - way ANOVA with Tukey ' s post - hoc test , ns = not significant , p * < 0 . 05 , p * * * < 0 . 001 .

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

g, Differential induction of Npas4 through HiK and not forskolin in hiPSC-derived forebrain organoids. Organoids were treated with HiK (55 mM) or the PKA activator forskolin plus TTX (Fsk, 10 µM; TTX 1 µM) for 1h before qPCR analysis was performed to assess mRNA levels of Npas4 and Arc. Mean + SEM; n = 7 organoids in control condition, n = 5 organoids for HiK, n = 5 organoids for Fsk/TTX. One-way ANOVA with Tukey's post-hoc test, ns = not significant, p* < 0.05, p***<0.001.


MSN cultures were stimulated with HiK ( 55 mM ) for 1h in the presence of various pharmacologicals to dissect the synapse - to - nucleus cascades mediating transcription of Npas4 , Fosb and Arc . Values were normalized to the induced condition to compare for differences in induction between treatments . Mean + SEM and one - way ANOVA with Bonferroni post - hoc correction were used in all experiments . p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 , p * * * * < 0 . 0001 . n - number indicated in each panel . a , co - incubation with the NMDA receptor blocker APV ( 100 mM , applied 5min before ) reveals that all IEG inductions through HiK in MSNs depend on NDMA receptor activity . Mean + SEM ; n = 4 independent cultures as biological replicates . b , blockade of voltage - dependent calcium channels through verapamil ( 30 µM , 5min before ) and TTX ( 1 µM , 5min , to prevent verapamil - induced bursting ) leads to significant reduction in gene - inductions for Npas4 and Fosb . Mean + SEM ; n = 4 independent cultures as biological replicates . c , inhibition of ERK1 / 2 through UO126 ( 10 µM , 20min before ) leads to significant blockade of mRNA induction for Arc and Fosb , but not Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates . d , inhibition of p38alpha and p38beta through SB203580 or SB202190 respectively ( both 10 µM and 20min before ) leads to significant reductions in mRNA induction for Arc and Fosb , but not Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates . e , blockade of PKA activity through H - 89 ( 10 µM , 20min before ) leads to reduced induction of Arc , unaffected induction of Fosb and a superinduction of Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates . f , inhibition of calcineurin through co - application of Cyclosporin and FK506 ( 1 µM each , 20min before ) leads to unaffected induction of mRNAs for Arc and Fosb , but significantly reduced levels of Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates .

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

MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of Npas4, Fosb and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. a, co-incubation with the NMDA receptor blocker APV (100 mM, applied 5min before) reveals that all IEG inductions through HiK in MSNs depend on NDMA receptor activity. Mean + SEM; n = 4 independent cultures as biological replicates. b, blockade of voltage-dependent calcium channels through verapamil (30 µM, 5min before) and TTX (1 µM, 5min, to prevent verapamil-induced bursting) leads to significant reduction in gene-inductions for Npas4 and Fosb. Mean + SEM; n =4 independent cultures as biological replicates. c, inhibition of ERK1/2 through UO126 (10 µM, 20min before) leads to significant blockade of mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. d, inhibition of p38alpha and p38beta through SB203580 or SB202190 respectively (both 10 µM and 20min before) leads to significant reductions in mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. e, blockade of PKA activity through H-89 (10 µM, 20min before) leads to reduced induction of Arc, unaffected induction of Fosb and a superinduction of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. f, inhibition of calcineurin through co-application of Cyclosporin and FK506 (1 µM each, 20min before) leads to unaffected induction of mRNAs for Arc and Fosb, but significantly reduced levels of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates.


MSN cultures were stimulated with HiK ( 55 mM ) for 1h in the presence of various pharmacologicals to dissect the synapse - to - nucleus cascades mediating transcription of Npas4 , Fosb and Arc . Values were normalized to the induced condition to compare for differences in induction between treatments . Mean + SEM and one - way ANOVA with Bonferroni post - hoc correction were used in all experiments . p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 , p * * * * < 0 . 0001 . n - number indicated in each panel . g , blockade of CaMKII / IV through application of KN - 93 ( 1 µM , 20min before ) leads to significantly reduced mRNA inductions of Arc , Fosb and Npas4 . Mean + SEM ; n = 4 independent cultures as biological replicates . h , co - application of HiK and SKF reveals synergistic effects on mRNA induction of Fosb but not Arc or Npas4 , Mean + SEM ; n = 6 independent cultures as biological replicates .

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

MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of Npas4, Fosb and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. g, blockade of CaMKII/IV through application of KN-93 (1 µM, 20min before) leads to significantly reduced mRNA inductions of Arc, Fosb and Npas4. Mean + SEM; n = 4 independent cultures as biological replicates. h, co-application of HiK and SKF reveals synergistic effects on mRNA induction of Fosb but not Arc or Npas4, Mean + SEM; n = 6 independent cultures as biological replicates.


investigating the role of nuclear calcium signaling in mRNA induction of Arc , Fosb and Npas4 . k , immunohistochemical analysis of MSNs infected with mCherry - NLS as control or the competitive nuclear Ca2 + / CaM antagonist CaMBP4 - mCherry . Infection on DIV4 , fixation on DIV11 . D32 = anti - Darpp - 32 . scale bar = 10 µm . l , MSNs expressing rAAV - mCherry - control or CaMBP4 ( BP4 ) were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test , ns = not significant , p * * * * < 0 . 0001 . n = 5 independent cultures as biological replicates . m , immunohistochemical analysis of MSNs infected with mCherry - NLS as control or the nuclear Ca2 + - buffer Parvalbumin - NLS - mCherry . Infection on DIV4 , fixation on DIV11 . D32 = anti - Darpp - 32 . n , MSNs expressing rAAV - mCherry - control or PV - NLS were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test , p * < 0 . 05 , p * * < 0 . 01 , p * * * * < 0 . 0001 . n = 3 independent cultures as biological replicates .

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

investigating the role of nuclear calcium signaling in mRNA induction of Arc, Fosb and Npas4. k, immunohistochemical analysis of MSNs infected with mCherry-NLS as control or the competitive nuclear Ca2+/CaM antagonist CaMBP4-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. scale bar = 10 µm. l, MSNs expressing rAAV-mCherry-control or CaMBP4 (BP4) were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, ns = not significant, p****<0.0001. n = 5 independent cultures as biological replicates. m, immunohistochemical analysis of MSNs infected with mCherry-NLS as control or the nuclear Ca2+-buffer Parvalbumin-NLS-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. n, MSNs expressing rAAV-mCherry-control or PV-NLS were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p** < 0.01, p****<0.0001. n = 3 independent cultures as biological replicates.


e , Npas4 controls activity - dependent upregulation of genes for glutamatergic synapse formation and Ca2 + channels . MSNs were infected with rAAVs expressing shRNA - scrambled ( control ) or shNpas4 ( Npas4 knockdown ) on DIV5 . On DIV 12 , cultures were stimulated with HiK ( 55 mM ) for 1h or 6h and mRNA levels were analysed by qPCR . mRNA level = fold change in mRNA compared to shscrambled unstimulated control . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test , p * < 0 . 05 , p * * * < 0 . 001 . n = 5 independent cultures as biological replicates .

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

e, Npas4 controls activity-dependent upregulation of genes for glutamatergic synapse formation and Ca2+ channels. MSNs were infected with rAAVs expressing shRNA-scrambled (control) or shNpas4 (Npas4 knockdown) on DIV5. On DIV 12, cultures were stimulated with HiK (55 mM) for 1h or 6h and mRNA levels were analysed by qPCR. mRNA level = fold change in mRNA compared to shscrambled unstimulated control. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p***<0.001. n = 5 independent cultures as biological replicates.


e , Confocal images of tdTomato - labelled D1 - MSNs expressing shscr - GFP or shNpas4 - GFP ( left panels ; scale bar : 20 µm ) . 3D volume - renderings of MSN dendrites from the NAc shell ( right panels ; scale bar : 2 µm ) .

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

e, Confocal images of tdTomato-labelled D1-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm).


g , Confocal images of tdTomato - labelled D2 - MSNs expressing shscr - GFP or shNpas4 - GFP ( left panels ; scale bar : 20 µm ) . 3D volume - renderings of MSN dendrites from the NAc shell ( right panels ; scale bar : 2 µm ) .

g, Confocal images of tdTomato-labelled D2-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm).


a - d , Scatter plot of passive and active membrane properties of MSNs in ex vivo slice preparations from shRNA - AAV - infected mice , plotted for MSNs as D1 - positive ( D1 + ) , D1 - negative ( D1 - ) and those two populations pooled ( all ) . a , rheobase ; mean ± SEM . b , firing rate estimated at twice the value of the rheobase ; mean ± SEM . c , input / output gain values recorded in shscr - or shNpas4 - injected mice , mean ± SEM . The increased rheobase and lower input - output gain indicate a lower excitability for MSNs in the Npas4 - knockdown group . d , facilitation at corticostriatal synapses examined in MSNs with paired - pulse stimulation at 20 and 4 Hz . Representative current traces and scatter plots of paired - pulse experiments illustrate a potent facilitation in MSNs in mice injected with scr - shRNA and a significant decrease of the facilitation in mice injected with shNpas4 , with a stronger effect in D1 - negative MSNs . Mean ± SEM . Data information : For n - numbers , please see Table EV1 . Mann - Whitney - Test , p * < 0 . 05 , p * * < 0 . 01 .

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

a-d, Scatter plot of passive and active membrane properties of MSNs in ex vivo slice preparations from shRNA-AAV-infected mice, plotted for MSNs as D1-positive (D1+), D1-negative (D1-) and those two populations pooled (all). a, rheobase; mean ± SEM. b, firing rate estimated at twice the value of the rheobase; mean ± SEM. c, input/output gain values recorded in shscr- or shNpas4-injected mice, mean ± SEM. The increased rheobase and lower input-output gain indicate a lower excitability for MSNs in the Npas4-knockdown group. d, facilitation at corticostriatal synapses examined in MSNs with paired-pulse stimulation at 20 and 4 Hz. Representative current traces and scatter plots of paired-pulse experiments illustrate a potent facilitation in MSNs in mice injected with scr-shRNA and a significant decrease of the facilitation in mice injected with shNpas4, with a stronger effect in D1-negative MSNs. Mean ± SEM. Data information: For n-numbers, please see Table EV1. Mann-Whitney-Test, p*<0.05, p**<0.01.


b , cocaine - induced hyperlocomotion is attenuated by Npas4 - knockdown while locomotor behaviour after saline injection is unaffected on all days . Mean + SEM ; two - way ANOVA followed by Bonferroni post hoc test . p * < 0 . 05 , p * * < 0 . 01 , ns = not significant . n = 12 - 16 animals per condition .

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

b, cocaine-induced hyperlocomotion is attenuated by Npas4-knockdown while locomotor behaviour after saline injection is unaffected on all days. Mean + SEM; two-way ANOVA followed by Bonferroni post hoc test. p* < 0.05, p** < 0.01, ns = not significant. n = 12 - 16 animals per condition.