A . Quantitative RT - PCR detection of CAV1 , CAV2 and CAVIN1 in primary human umbilical venous endothelial cells ( HUVECs ) upon Notch blockade ( dnMAML ) and induction ( NICD ) . n = 3 , data represent mean ± SEM , unpaired t - test .

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A. Quantitative RT-PCR detection of CAV1, CAV2 and CAVIN1 in primary human umbilical venous endothelial cells (HUVECs) upon Notch blockade (dnMAML) and induction (NICD). n=3, data represent mean ± SEM, unpaired t-test.


B . Representative western blot of CAV1 expression in HUVECs upon Notch manipulation . C . Densitometric analysis of western blot in B . Data represented as fold change over GFP control . n = 3 , data represent mean ± SEM , unpaired t - test .

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B. Representative western blot of CAV1 expression in HUVECs upon Notch manipulation. C. Densitometric analysis of western blot in B. Data represented as fold change over GFP control. n=3, data represent mean ± SEM, unpaired t-test.


D . Representative western blot of CAVIN1 expression in HUVECs upon Notch induction . E . Densitometric analysis of western blot in D . Data represented as fold change over LacZ control . n = 5 , data represent mean ± SEM , unpaired t - test .

D. Representative western blot of CAVIN1 expression in HUVECs upon Notch induction. E. Densitometric analysis of western blot in D. Data represented as fold change over LacZ control. n=5, data represent mean ± SEM, unpaired t-test.


F . Expression of CAV1 in HUVECs upon Notch manipulation and HEY1 induction . n = 3 , data represent mean ± SEM , unpaired t - test .

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F. Expression of CAV1 in HUVECs upon Notch manipulation and HEY1 induction. n=3, data represent mean ± SEM, unpaired t-test.


G . Expression of Cav1 , Cav2 , Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of RbpjiΔEC mice compared to control mice . n = 5 , data represent mean ± SEM , unpaired t - test .

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G. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of RbpjiΔEC mice compared to control mice. n=5, data represent mean ± SEM, unpaired t-test.


H . Expression of Cav1 , Cav2 , Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of NICDiOE - EC mice compared to control mice . n = 4 , data represent mean ± SEM , unpaired t - test .

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H. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of NICDiOE-EC mice compared to control mice. n=4, data represent mean ± SEM, unpaired t-test.


I . Expression of Cav1 , Cav2 , Cavin1 in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet ( CD , 10 % fat , 70 % carbohydrates ) or high - fat diet ( HFD , 60 % fat , 20 % carbohydrates ) or high - fat and sugar diet ( HFS , 60 % fat , 20 % carbohydrates and 42 g / l sucrose in drinking water ) . n = 4 , data represent mean ± SEM , unpaired t - test .

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I. Expression of Cav1, Cav2, Cavin1 in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test.


J , K . Quantification of number of caveolae attached to apical membrane in EC of cardiac capillaries from NICDiOE - EC mice ( 6 segments of 1000 nm length from 3 hearts per genotype ) or RbpjiΔEC ( 6 segments of 1000 nm length from 8 hearts per genotype ) based on electron microscopy . Box plot depicts median and percentiles ( 10th , 25th , 75th , 90th ) , unpaired t - test , * means p < 0 . 05 .

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J, K. Quantification of number of caveolae attached to apical membrane in EC of cardiac capillaries from NICDiOE-EC mice (6 segments of 1000 nm length from 3 hearts per genotype) or RbpjiΔEC (6 segments of 1000 nm length from 8 hearts per genotype) based on electron microscopy. Box plot depicts median and percentiles (10th, 25th, 75th, 90th), unpaired t-test, * means p<0.05.


Representative immune - histochemistry of Cav1 staining in heart sections from control and RbpjiΔEC mice . n = 3 , scale bar 50 µm .

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Representative immune-histochemistry of Cav1 staining in heart sections from control and RbpjiΔEC mice. n=3, scale bar 50 µm.


Representative confocal images of Cav1 staining in heart sections from control and RbpjiΔEC mice . n = 3 , scale bar 50 µm .

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Representative confocal images of Cav1 staining in heart sections from control and RbpjiΔEC mice. n=3, scale bar 50 µm.


B . Blood glucose levels of control and NICDiOE - EC mice during the 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test .

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B. Blood glucose levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


C . Plasma insulin levels of control and NICDiOE - EC mice during the 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test .

C. Plasma insulin levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


D . 2 - DG uptake levels in skeletal muscle of control and NICDiOE - EC mice . n = 5 , data represent mean ± SEM , unpaired t - test . E . 2 - DG uptake levels in visceral white adipose tissue ( vWAT ) of control ( n = 4 ) and NICDiOE - EC ( n = 5 ) mice . Data represent mean ± SEM , unpaired t - test .

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D. 2-DG uptake levels in skeletal muscle of control and NICDiOE-EC mice. n=5, data represent mean ± SEM, unpaired t-test. E. 2-DG uptake levels in visceral white adipose tissue (vWAT) of control (n=4) and NICDiOE-EC (n=5) mice. Data represent mean ± SEM, unpaired t-test.


G . Blood glucose levels of control and RbpjiΔEC mice during the 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test .

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G. Blood glucose levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


H . Plasma insulin levels of control and RbpjiΔEC mice during the 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test .

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H. Plasma insulin levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


I . 2 - DG uptake levels in skeletal muscle of control and RbpjiΔEC mice . n = 4 , data represent mean ± SEM , unpaired t - test . J . 2 - DG uptake levels in vWAT of control and RbpjiΔEC mice . n = 5 , data represent mean ± SEM , unpaired t - test .

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I. 2-DG uptake levels in skeletal muscle of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test. J. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=5, data represent mean ± SEM, unpaired t-test.


B . Blood glucose levels for glucose tolerance test ( GTT ) of control ( n = 4 ) or RbpjiΔEC ( n = 5 ) mice kept on high - fat diet ( HFD ) . Data represent unpaired t - test , mean ± SEM . C . Quantification of area under curve ( AUC ) for GTT in B . Data represent mean ± SEM , unpaired t - test .

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B. Blood glucose levels for glucose tolerance test (GTT) of control (n=4) or RbpjiΔEC (n=5) mice kept on high-fat diet (HFD). Data represent unpaired t-test, mean ± SEM. C. Quantification of area under curve (AUC) for GTT in B. Data represent mean ± SEM, unpaired t-test.


D . Blood glucose levels for insulin tolerance test ( ITT ) of control ( n = 4 ) or RbpjiΔEC ( n = 3 ) mice kept on HFD . Data represent unpaired t - test , mean ± SEM . E . Quantification of AUC for ITT in D . Data represent mean ± SEM , unpaired t - test .

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D. Blood glucose levels for insulin tolerance test (ITT) of control (n=4) or RbpjiΔEC (n=3) mice kept on HFD. Data represent unpaired t-test, mean ± SEM. E. Quantification of AUC for ITT in D. Data represent mean ± SEM, unpaired t-test.


F . HbA1c levels of control ( n = 4 ) or RbpjiΔEC ( n = 6 ) mice kept on HFD seven weeks after recombination . Data represent mean ± SEM , unpaired t - test .

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F. HbA1c levels of control (n=4) or RbpjiΔEC (n=6) mice kept on HFD seven weeks after recombination. Data represent mean ± SEM, unpaired t-test.


H . Blood glucose levels of control and RbpjiΔEC mice on HFD during 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test .

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H. Blood glucose levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


I . Plasma insulin levels of control and RbpjiΔEC mice on HFD during 2 - DG uptake assay . n = 5 , data represent mean ± SEM , unpaired t - test .

I. Plasma insulin levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


J . 2 - DG uptake levels in skeletal muscle of control ( n = 5 ) and RbpjiΔEC ( n = 4 ) mice on HFD . Data represent mean ± SEM , unpaired t - test . K . 2 - DG uptake levels in vWAT of control and RbpjiΔEC mice . n = 4 , data represent mean ± SEM , unpaired t - test .

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J. 2-DG uptake levels in skeletal muscle of control (n=5) and RbpjiΔEC (n=4) mice on HFD. Data represent mean ± SEM, unpaired t-test. K. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test.


( C - D ) RNA - Seq data from GTEx of the normalized expression of human CSAG1 ( C ) and CSAG2 ( D ) in the indicated tissues . Number of biological replicates are as follows : testis 361 , brain 255 , small intestine 187 , white adipose 663 , muscle 803 , skin 604 , esophagus 555 , stomach 359 , liver 226 , heart 429 . blood vessel 663 , lung 578 , colon 373 , nerve 619 , pituitary 283 , blood 755 , adrenal gland 258 , kidney 85 , prostate 245 , salivary gland 162 , ovary 180 , breast 459 , pancreas 328 , vagina 156 , uterus 142 , spleen 241 , fallopian tube 9 , bladder 21 , cervix 10 . Central band indicates median , boxes define 25 and 75 percentiles , and whiskers define 5 and 95 percentiles .

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(C-D) RNA-Seq data from GTEx of the normalized expression of human CSAG1 (C) and CSAG2 (D) in the indicated tissues. Number of biological replicates are as follows: testis 361, brain 255, small intestine 187, white adipose 663, muscle 803, skin 604, esophagus 555, stomach 359, liver 226, heart 429. blood vessel 663, lung 578, colon 373, nerve 619, pituitary 283, blood 755, adrenal gland 258, kidney 85, prostate 245, salivary gland 162, ovary 180, breast 459, pancreas 328, vagina 156, uterus 142, spleen 241, fallopian tube 9, bladder 21, cervix 10. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.


( E - F ) Expression of CSAG1 ( E ) and CSAG2 ( F ) in normal adjacent ( blue ) or tumor tissue ( red ) from TCGA RNA - Seq data . N . D . represents normal adjacent is not available . Data visualized by firebrowse . Number of biological replicates are as follows : ACC 41 , BLCA 307 , BRCA 524 , CESC 175 , CHOL 11 , COAD 153 , COADREAD 232 , DLBC 36 , ESCA 121 , HNSC 416 , KICH 46 , KIPAN 515 , KIRC 411 , KIRP 58 , LAML 7 , LGG 528 , LUAD 301 , LUSC 400 , MESO 31 , OV 183 , PCPG 70 , PRAD 204 , READ 79 , SARC 156 , SKCM 422 , STAD 288 , STES 409 , TGCT 126 , THCA 408 , THYM 75 , UCEC 306 , and UCS 37 . Central band indicates median , boxes define 25 and 75 percentiles , and whiskers define 5 and 95 percentiles .

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(E-F) Expression of CSAG1 (E) and CSAG2 (F) in normal adjacent (blue) or tumor tissue (red) from TCGA RNA-Seq data. N.D. represents normal adjacent is not available. Data visualized by firebrowse. Number of biological replicates are as follows: ACC 41, BLCA 307, BRCA 524, CESC 175, CHOL 11, COAD 153, COADREAD 232, DLBC 36, ESCA 121, HNSC 416, KICH 46, KIPAN 515, KIRC 411, KIRP 58, LAML 7, LGG 528, LUAD 301, LUSC 400, MESO 31, OV 183, PCPG 70, PRAD 204, READ 79, SARC 156, SKCM 422, STAD 288, STES 409, TGCT 126, THCA 408, THYM 75, UCEC 306, and UCS 37. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.


( G ) Xq28 MAGE and CSAG genes are frequently co - expressed in lung squamous carcinoma tumors ( n = 184 ) . RNA - Seq gene expression data analyzed from TCGA . Statistics shown were calculated by linear regression analysis .

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(G) Xq28 MAGE and CSAG genes are frequently co-expressed in lung squamous carcinoma tumors (n=184). RNA-Seq gene expression data analyzed from TCGA. Statistics shown were calculated by linear regression analysis.


( H - K ) Expression of CSAG1 ( H - I ) or CSAG2 ( J - K ) in patients from indicated cancer types correlates with poor overall survival . Data is from TCGA and visualized by kmplotter .

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(H-K) Expression of CSAG1 (H-I) or CSAG2 (J-K) in patients from indicated cancer types correlates with poor overall survival. Data is from TCGA and visualized by kmplotter.


( A - B ) The effect of stable knockdown CSAG2 in HCT116 ( A ) or A375 ( B ) were assayed for clonogenic growth on plastic for 7 days . The number of colonies was quantified and is shown . Data are represented as the mean ± SD , n = 3 biological replicates .

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(A-B) The effect of stable knockdown CSAG2 in HCT116 (A) or A375 (B) were assayed for clonogenic growth on plastic for 7 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.


( C - D ) The effect of stable knockdown CSAG2 in HCT116 ( C ) or A375 ( D ) were determined for anchorage - independent growth in soft agar colony formation assays for 14 days . The number of colonies was quantified and is shown . Data are represented as the mean ± SD , n = 3 biological replicates .

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(C-D) The effect of stable knockdown CSAG2 in HCT116 (C) or A375 (D) were determined for anchorage-independent growth in soft agar colony formation assays for 14 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.


( E - F ) Knockdown of CSAG2 in A375 ( F ) or HCT116 ( E ) cells decreases xenograft tumor growth in mice . Cells with doxycycline - inducible shRNAs were implanted into NOD SCID gamma mice . Doxycycline ( 2 mg / mL ) was administered via drinking water . Tumor growth was monitored over time . Data are represented as the mean ± SD , n = 6 mice for each group .

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(E-F) Knockdown of CSAG2 in A375 (F) or HCT116 (E) cells decreases xenograft tumor growth in mice. Cells with doxycycline-inducible shRNAs were implanted into NOD SCID gamma mice. Doxycycline (2 mg/mL) was administered via drinking water. Tumor growth was monitored over time. Data are represented as the mean ± SD, n=6 mice for each group.


( G - H ) Stable expression of CSAG2 in HCT116 ( G ) and CSAG2 - negative H460 ( H ) cells increases anchorage - independent growth in soft agar colony formation assays , n = 3 biological replicates . Data are represented as the mean ± SD .

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(G-H) Stable expression of CSAG2 in HCT116 (G) and CSAG2-negative H460 (H) cells increases anchorage-independent growth in soft agar colony formation assays, n=3 biological replicates. Data are represented as the mean ± SD.


( A ) HEK293 cells stably expressing TAP - vector , TAP - CSAG1 , or TAP - CSAG2 were subjected to pull - down followed by SDS - PAGE and LC - MS / MS . Spectral counts ( SC ) of the identified proteins in TAP - CSAG1 and TAP - CSAG2 pulldowns , but absence in TAP - vector control , are shown .

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(A) HEK293 cells stably expressing TAP-vector, TAP-CSAG1, or TAP-CSAG2 were subjected to pull-down followed by SDS-PAGE and LC-MS/MS. Spectral counts (SC) of the identified proteins in TAP-CSAG1 and TAP-CSAG2 pulldowns, but absence in TAP-vector control, are shown.


( B ) HEK293FT cells stably expressing HA - SIRT1 were transfected with Myc - CSAG1 or Myc - CSAG2 for 48 h before IP with anti - HA followed by SDS - PAGE and immunoblotting for indicated proteins .

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(B) HEK293FT cells stably expressing HA-SIRT1 were transfected with Myc-CSAG1 or Myc-CSAG2 for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for indicated proteins.


( C ) Recombinant GST - SIRT1 , but not GST , binds in vitro translated Myc - CSAG2 . Myc - tagged CSAG2 was in vitro translated and GST - pulldown experiments were performed with recombinant GST or GST - SIRT1 followed by SDS - PAGE and immunoblotting for anti - Myc .

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(C) Recombinant GST-SIRT1, but not GST, binds in vitro translated Myc-CSAG2. Myc-tagged CSAG2 was in vitro translated and GST-pulldown experiments were performed with recombinant GST or GST-SIRT1 followed by SDS-PAGE and immunoblotting for anti-Myc.


Expression of CSAG2 decreased p53 ac - K382 levels and downregulated the expression of downstream p53 target genes in H460 cells . Cells were treated by either 1 μM doxorubicin ( D - E ) with 0 . 4 μM TSA for the indicated times . Cells were then harvested and blotted for the indicated proteins . Quantitation of expression levels is shown ( E Ac - p53 K382 levels normalized to total p53 , whereas all other proteins were normalized to GAPDH .

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Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by either 1 μM doxorubicin (D-E) with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown (E Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.


Expression of CSAG2 decreased p53 ac - K382 levels and downregulated the expression of downstream p53 target genes in H460 cells . Cells were treated by 20 μM etoposide with 0 . 4 μM TSA for the indicated times . Ac - p53 K382 levels normalized to total p53 , whereas all other proteins were normalized to GAPDH .

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Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by 20 μM etoposide with 0.4 μM TSA for the indicated times. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.


Expression of CSAG2 decreased p53 ac - K382 levels and downregulated the expression of downstream p53 target genes in H460 cells . Cells were treated by 20 μM etoposide with 0 . 4 μM TSA for the indicated times . Cells were then harvested and blotted for the indicated proteins . Quantitation of expression levels is shown G ) as the mean ± SD , n = 3 biological replicates . Ac - p53 K382 levels normalized to total p53 , whereas all other proteins were normalized to GAPDH .

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Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by 20 μM etoposide with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown G) as the mean ± SD, n=3 biological replicates. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.


Knockdown of endogenous CSAG2 in HCT116 ( H - I ) cells increased p53 ac - K382 levels . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cells were then harvested and blotted for the indicated proteins . Quantitation of ac - p53 K382 levels relative to total p53 is shown ( I as the mean ± SD , n = 3 biological replicates .

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Knockdown of endogenous CSAG2 in HCT116 (H-I) cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown (I as the mean ± SD, n=3 biological replicates.


Knockdown of endogenous CSAG2 in A375 cells increased p53 ac - K382 levels . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cells were then harvested and blotted for the indicated proteins .

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Knockdown of endogenous CSAG2 in A375 cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins.


Knockdown of endogenous CSAG2 in A375 cells increased p53 ac - K382 levels . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cells were then harvested and blotted for the indicated proteins . Quantitation of ac - p53 K382 levels relative to total p53 is shown K ) as the mean ± SD , n = 3 biological replicates .

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Knockdown of endogenous CSAG2 in A375 cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown K) as the mean ± SD, n=3 biological replicates.


( L - M ) Re - expression of CSAG2 rescued p53 ac - K382 levels in CSAG2 knockdown stable cell lines . Myc - CSAG2 was transfected in HCT116 ( L ) or A375 ( M ) CSAG2 knockdown stable cell lines . Cells were treated with 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr and then blotted for the indicated proteins . Quantitation of ac - p53 K382 levels relative to total p53 is shown as the mean ± SD , n = 3 biological replicates .

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(L-M) Re-expression of CSAG2 rescued p53 ac-K382 levels in CSAG2 knockdown stable cell lines. Myc-CSAG2 was transfected in HCT116 (L) or A375 (M) CSAG2 knockdown stable cell lines. Cells were treated with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr and then blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown as the mean ± SD, n=3 biological replicates.


( N ) CSAG2 does not enhance anchorage - independent growth of HCT116 p53 - null cells . CSAG2 was stably expressed in isogenic HCT116 p53 - wild - type or p53 - null cells . Anchorage - independent growth in soft agar colony formation assays was determined after 14 days . The number of colonies was quantified , normalized to each vector control cell line , and is shown as the mean ± SD , n = 3 biological replicates .

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(N) CSAG2 does not enhance anchorage-independent growth of HCT116 p53-null cells. CSAG2 was stably expressed in isogenic HCT116 p53-wild-type or p53-null cells. Anchorage-independent growth in soft agar colony formation assays was determined after 14 days. The number of colonies was quantified, normalized to each vector control cell line, and is shown as the mean ± SD, n=3 biological replicates.


( A ) Knockdown of SIRT1 abolished CSAG2 - mediated decrease in ac - p53 K382 . CSAG2 over - expressing HCT116 were transfected with SIRT1 siRNA for 72 hr before doxorubicin / TSA treatment for 6 hr . Cell lysates were blotted for the indicated proteins .

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(A) Knockdown of SIRT1 abolished CSAG2-mediated decrease in ac-p53 K382. CSAG2 over-expressing HCT116 were transfected with SIRT1 siRNA for 72 hr before doxorubicin/TSA treatment for 6 hr. Cell lysates were blotted for the indicated proteins.


( B ) Inhibition of SIRT1 enzymatic activity blocked CSAG2 - induced decrease in p53 ac - K382 . CSAG2 over - expressing HCT116 cells were treated with 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . Cell lysates were blotted for the indicated proteins .

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(B) Inhibition of SIRT1 enzymatic activity blocked CSAG2-induced decrease in p53 ac-K382. CSAG2 over-expressing HCT116 cells were treated with 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cell lysates were blotted for the indicated proteins.


Inhibition of SIRT1 enzymatic activity prevents CSAG2 - knockdown increase in p53 ac - K382 . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . Cells were then harvested and blotted for the indicated proteins ( C )

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Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins (C)


Inhibition of SIRT1 enzymatic activity prevents CSAG2 - knockdown increase in p53 ac - K382 . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . Cells were then harvested and blotted for the indicated proteins and quantitated ( D ; n = 3 biological replicates ) . Data shown as mean ± SD .

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Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins and quantitated (D; n=3 biological replicates). Data shown as mean ± SD.


( E ) CSAG2 binds catalytically inactive SIRT1 H363Y . Cells were transfected with the indicated constructs before anti - Myc IP and blotting for the indicated proteins .

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(E) CSAG2 binds catalytically inactive SIRT1 H363Y. Cells were transfected with the indicated constructs before anti-Myc IP and blotting for the indicated proteins.


( F ) SIRT1 H363Y blocks CSAG2 regulation of p53 ac - K382 levels . HCT116 cells were transfected with the indicated constructs and blotted for the indicated proteins .

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(F) SIRT1 H363Y blocks CSAG2 regulation of p53 ac-K382 levels. HCT116 cells were transfected with the indicated constructs and blotted for the indicated proteins.


( G ) Inhibition of SIRT1 activity abolished CSAG2 - induced anchorage - independent growth . Soft agar colony formation assays were performed in control or CSAG2 over - expressing HCT116 cells with or without EX - 527 treatment . Data are represented as the mean ± SD , n = 3 biological replicates .

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(G) Inhibition of SIRT1 activity abolished CSAG2-induced anchorage-independent growth. Soft agar colony formation assays were performed in control or CSAG2 over-expressing HCT116 cells with or without EX-527 treatment. Data are represented as the mean ± SD, n=3 biological replicates.


( H ) Summary of region on CSAG2 required for SIRT1 interaction . HEK293FT cells stably expressing HA - SIRT1 were transfected with indicated CSAG2 constructs for 48 h before IP with anti - HA followed by SDS - PAGE and immunoblotting for anti - Myc .

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(H) Summary of region on CSAG2 required for SIRT1 interaction. HEK293FT cells stably expressing HA-SIRT1 were transfected with indicated CSAG2 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.


( I ) CSAG2 Δ37 mutant fails to interact with SIRT1 in vitro . Myc - tagged CSAG2 38 - 127 fragment was in vitro translated and followed by in vitro binding assay with recombinant GST - SIRT1 , SDS - PAGE , and immunoblotting for anti - Myc .

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(I) CSAG2 Δ37 mutant fails to interact with SIRT1 in vitro. Myc-tagged CSAG2 38-127 fragment was in vitro translated and followed by in vitro binding assay with recombinant GST-SIRT1, SDS-PAGE, and immunoblotting for anti-Myc.


( J ) CSAG2 Δ37 mutant does not regulate p53 ac - K382 levels . Cells were transfected with the indicated Myc - CSAG2 constructs for 48 hr before being treated with 1 μM doxorubicin / 0 . 4 μM TSA . Cell lysates were blotted for the indicated proteins .

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(J) CSAG2 Δ37 mutant does not regulate p53 ac-K382 levels. Cells were transfected with the indicated Myc-CSAG2 constructs for 48 hr before being treated with 1 μM doxorubicin/ 0.4 μM TSA. Cell lysates were blotted for the indicated proteins.


( K ) CSAG2 Δ37 fails to promote anchorage - independent growth of H460 cells . Soft agar colony formation assays were performed in CSAG2 - negative H460 cells stably expressing CSAG2 wild - type or CSAG2 Δ37 mutant ( n = 3 biological replicates ) . Data are represented as the mean ± SD .

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(K) CSAG2 Δ37 fails to promote anchorage-independent growth of H460 cells. Soft agar colony formation assays were performed in CSAG2-negative H460 cells stably expressing CSAG2 wild-type or CSAG2 Δ37 mutant (n=3 biological replicates). Data are represented as the mean ± SD.


Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin ( A ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin (A) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of H2O2 ( B ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of H2O2 (B) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin ( C ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin (C) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 cells were treated with the indicated concentrations of H2O2 ( D ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of H2O2 (D) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


( E - F ) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cell lysates were collected and blotted for the indicated proteins ( E ) . Quantitation ( n = 3 biological replicates ) of normalized cleaved PARP levels is shown ( F ) .

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(E-F) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cell lysates were collected and blotted for the indicated proteins (E). Quantitation (n=3 biological replicates) of normalized cleaved PARP levels is shown (F).


( G ) H460 cells were transfected with wild type , Δ37 , or Δ63 CSAG2 for 48 hr . Cells were then treated with the indicated concentrations of H2O2 for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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(G) H460 cells were transfected with wild type, Δ37, or Δ63 CSAG2 for 48 hr. Cells were then treated with the indicated concentrations of H2O2 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


( H ) Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress . CSAG2 over - expressing H460 cells were treated with the indicated concentrations of H2O2 with or without 1 μM EX - 527 for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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(H) Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress. CSAG2 over-expressing H460 cells were treated with the indicated concentrations of H2O2 with or without 1 μM EX-527 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing HCT116 p53 - / - cells were treated with the indicated concentrations of H2O2 ( I ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (I) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing HCT116 p53 - / - cells were treated with the indicated concentrations of doxorubicin ( J ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (J) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 p53 - / - cells were treated with the indicated concentrations of H2O2 ( K ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (K) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 p53 - / - cells were treated with the indicated concentrations of doxorubicin ( L ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (L) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing H1299 ( p53 mutant ) cells were treated with the indicated concentrations of H2O2 ( M ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of H2O2 (M) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing H1299 ( p53 mutant ) cells were treated with the indicated concentrations of doxorubicin ( N ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of doxorubicin (N) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


( A ) CSAG2 does not alter SIRT1 protein levels . HEK293FT cells were transfected with different amounts of Myc - CSAG2 for 48 hr before cells lysates were harvested and immunoblotted for indicated proteins .

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(A) CSAG2 does not alter SIRT1 protein levels. HEK293FT cells were transfected with different amounts of Myc-CSAG2 for 48 hr before cells lysates were harvested and immunoblotted for indicated proteins.


( B ) CSAG2 does not alter SIRT1 subcellular location . U2OS cells were transfected with or without mcherry - CSAG2 for 48 hr before fixation , SIRT1 immunostaining , and imaging . Scale bar : 10 µm .

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(B) CSAG2 does not alter SIRT1 subcellular location. U2OS cells were transfected with or without mcherry-CSAG2 for 48 hr before fixation, SIRT1 immunostaining, and imaging. Scale bar: 10 µm.


( C ) Summary of results mapping interaction region of SIRT1 recognized by CSAG2 . HEK293FT cells stably expressing HA - CSAG2 were transfected with indicated Myc - SIRT1 constructs for 48 h before IP with anti - HA followed by SDS - PAGE and immunoblotting for anti - Myc .

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(C) Summary of results mapping interaction region of SIRT1 recognized by CSAG2. HEK293FT cells stably expressing HA-CSAG2 were transfected with indicated Myc-SIRT1 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.


( D ) CSAG2 directly binds SIRT1 catalytic domain in vitro . HA - tagged SIRT1 fragments were in vitro translated . In vitro binding assays with recombinant GST or GST - CSAG2 were performed followed by SDS - PAGE and immunoblotting for anti - HA .

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(D) CSAG2 directly binds SIRT1 catalytic domain in vitro. HA-tagged SIRT1 fragments were in vitro translated. In vitro binding assays with recombinant GST or GST-CSAG2 were performed followed by SDS-PAGE and immunoblotting for anti-HA.


( E ) CSAG2 enhances SIRT1 activity in vitro . In vitro SIRT1 activity towards p53 ac - K382 peptide was determined in the presence of GST or GST - CSAG2 , n = 3 biological replicates . Nicotinamide ( 1 mM ) was added to the reaction were indicated to block SIRT1 activity .

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(E) CSAG2 enhances SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST or GST-CSAG2, n=3 biological replicates. Nicotinamide (1 mM) was added to the reaction were indicated to block SIRT1 activity.


( F ) CSAG2 Δ37 mutant that does not bind SIRT1 does not enhance SIRT1 activity in vitro . In vitro SIRT1 activity towards p53 ac - K382 peptide was determined in the presence of GST , GST - CSAG2 or GST - CSAG2 38 - 127 , n = 3 biological replicates .

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(F) CSAG2 Δ37 mutant that does not bind SIRT1 does not enhance SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST, GST-CSAG2 or GST-CSAG2 38-127, n=3 biological replicates.


( G ) CSAG2 enhances SIRT1 deacetylation of p53 ac - K382 peptide through increasing kcat , but not Km . not affecting the binding ability with its substrate p53 . SIRT1 activity assays were performed as described above , except varying amounts of p53 ac - K382 fluorometric peptide were used in the presence of saturating NAD + ( 3 mM ) , n = 3 biological replicates .

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(G) CSAG2 enhances SIRT1 deacetylation of p53 ac-K382 peptide through increasing kcat, but not Km. not affecting the binding ability with its substrate p53. SIRT1 activity assays were performed as described above, except varying amounts of p53 ac-K382 fluorometric peptide were used in the presence of saturating NAD+ (3 mM), n=3 biological replicates.


( H - I ) U2OS cells were transfected with Myc - Vector or Myc - CSAG2 for 48 hr before being treated with or without 0 . 4 μM TSA for 6 hr . Cell lysates were blotted for the indicated proteins ( H ) . Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD , n = 3 biological replicates . White circles indicate Myc - Vector and black circles indicate Myc - CSAG2 .

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(H-I) U2OS cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr before being treated with or without 0.4 μM TSA for 6 hr. Cell lysates were blotted for the indicated proteins (H). Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.


( J ) HeLa , U2OS , or HCT116 cells were transfected with Myc - Vector or Myc - CSAG2 for 48 hr . Cell lysates were blotted for the indicated proteins . Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD , n = 3 biological replicates . White circles indicate Myc - Vector and black circles indicate Myc - CSAG2 .

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(J) HeLa, U2OS, or HCT116 cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.


( K ) Knockdown of endogenous CSAG2 in A375 cells increased ac - H4K16 levels . Cell lysates were blotted for the indicated proteins . Quantitation of expression levels of ac - H4K16 relative to total H4 is shown as the mean ± SD , n = 3 biological replicates .

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(K) Knockdown of endogenous CSAG2 in A375 cells increased ac-H4K16 levels. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of ac-H4K16 relative to total H4 is shown as the mean ± SD, n=3 biological replicates.


A . Four representative images of RHOB immunostaining in human non - small - cell lung cancers . Figures correspond to the percentage of tumor analyzed .

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A. Four representative images of RHOB immunostaining in human non-small-cell lung cancers. Figures correspond to the percentage of tumor analyzed.


B . Characteristics of 96 patients with an EGFR - activating mutation who were treated with EGFR - TKI . Each column corresponds to one patient . Data include general characteristics ( stage at diagnosis , age , sex , tobacco usage , position of EGFR mutation , TKI type ) , progression - free survival ( PFS ; lower graph ) and intensity of RHOB staining as determined by immunohistochemistry ( null : 0 , weak : + , moderate : + + and high : + + + ) .

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B. Characteristics of 96 patients with an EGFR-activating mutation who were treated with EGFR-TKI. Each column corresponds to one patient. Data include general characteristics (stage at diagnosis, age, sex, tobacco usage, position of EGFR mutation, TKI type), progression-free survival (PFS; lower graph) and intensity of RHOB staining as determined by immunohistochemistry (null: 0, weak: +, moderate: ++ and high: +++).


C . Representative scans of patients with low or high RHOB - expressing lung tumors , before and after erlotinib treatment . Red arrows indicate lung tumors .

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C. Representative scans of patients with low or high RHOB-expressing lung tumors, before and after erlotinib treatment. Red arrows indicate lung tumors.


D . Progression - free survival of erlotinib - treated patients with EGFR - mutated lung tumors , according to RHOB expression , assessed by immunohistochemistry ( low RHOB group = negative + weak staining ; high RHOB group = moderate + high staining ) .

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D. Progression-free survival of erlotinib-treated patients with EGFR-mutated lung tumors, according to RHOB expression, assessed by immunohistochemistry (low RHOB group = negative + weak staining; high RHOB group = moderate + high staining).


E . Progression - free survival of patients who received EGFR - TKi as first - line therapy ( n = 63 ) . F . Progression - free survival of patients who received EGFR - TKi as 2nd - line ( n = 28 ) , 3rd - line ( n = 3 ) or 4th - line ( n = 2 ) therapy .

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E. Progression-free survival of patients who received EGFR-TKi as first-line therapy (n=63). F. Progression-free survival of patients who received EGFR-TKi as 2nd-line (n=28), 3rd-line (n=3) or 4th-line (n=2) therapy.


G . RHOB immunostaining score evolution in EGFR - mutated lung tumors before treatment and after EGFR - TKI relapse .

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G. RHOB immunostaining score evolution in EGFR-mutated lung tumors before treatment and after EGFR-TKI relapse.


A . Representative H & amp ; amp ; E staining of whole lungs from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days . Scale bar : 5 mm . B . Quantification of the tumor / lung ratio . n = 7 for each group except for EGFRL858R / Rhob + / - placebo ( n = 6 ) .

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A. Representative H&amp;E staining of whole lungs from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. Scale bar: 5 mm. B. Quantification of the tumor/lung ratio. n=7 for each group except for EGFRL858R/Rhob+/- placebo (n=6).


C . Representative Ki67 immunostaining of EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days ( scale bar : 50 µm ) , and the corresponding quantification ( D ) . Three independent zones / mice lung were used for quantification . n = 21 ( 7 mice ) for each group except for EGFRL858R / Rhob + / - placebo ( n = 18 ; 6 mice ) .

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C. Representative Ki67 immunostaining of EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D). Three independent zones/mice lung were used for quantification. n=21 (7 mice) for each group except for EGFRL858R/Rhob+/- placebo (n=18; 6 mice).


E . Immunostaining of cleaved caspase - 3 in lung tumors from EGFRL858R / Rhob + / + or EGFRL858R / Rhob - / - mice treated for 24 h with erlotinib at 12 . 5 mg / kg . Scale bar : 50 µm . ( * * : p < 0 . 001 vs . placebo ; * * * : p < 0 . 0001 vs . placebo ) .

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E. Immunostaining of cleaved caspase-3 in lung tumors from EGFRL858R/Rhob+/+ or EGFRL858R/Rhob-/- mice treated for 24 h with erlotinib at 12.5 mg/kg. Scale bar: 50 µm. (**: p<0.001 vs. placebo; ***: p<0.0001 vs. placebo).


HCC4006 cells were ( A ) transfected with two siRNA against RHOB ( siB1 , siB2 ) then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells .

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HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


HCC4006 cells were ( A ) transfected with two siRNA against RHOB ( siB1 , siB2 ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


HCC4006 cells were ( B ) transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells .

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HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


HCC4006 cells were ( B ) transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


HCC4006 cells were ( C ) both transfected with siRNA and transduced by adenoviruses ; then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells .

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HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


HCC4006 cells were ( C ) both transfected with siRNA and transduced by adenoviruses ; then treated with increasing doses of erlotinib . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


Erlotinib IC50 values were quantified in RHOB - overexpressing or RHOB - depleted ( D ) HCC827 cells , ( E ) HCC2935 cells and ( F ) H3255 cells , as determined by an MTS assay after 72 h treatment . RHOB overexpression or inhibition was monitored by western blotting for each condition .

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Erlotinib IC50 values were quantified in RHOB-overexpressing or RHOB-depleted (D) HCC827 cells, (E) HCC2935 cells and (F) H3255 cells, as determined by an MTS assay after 72 h treatment. RHOB overexpression or inhibition was monitored by western blotting for each condition.


G . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) at an increasing multiplicity of infection ( MOI ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


G . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) at an increasing multiplicity of infection ( MOI ) , then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed .

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G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI), then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed.


A . HCC4006 , HCC827 , HCC2935 and H3255 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line . The phosphorylation status of AKT , ERK1 / 2 and EGFR was assessed by western blotting and normalized according to total protein levels . RHOB overexpression was also monitored by western blotting .

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A. HCC4006, HCC827, HCC2935 and H3255 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line. The phosphorylation status of AKT, ERK1/2 and EGFR was assessed by western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by western blotting.


B . Representative immunostaining of phospho - AKT ( Ser473 ) , phospho ERK1 / 2 and their total protein amounts in lung tumors from EGFRL858R / Rhob - / - or EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days . The remaining hyperplastic areas were selected in erlotinib - treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes . Scale bar : 100 µm .

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B. Representative immunostaining of phospho-AKT (Ser473), phospho ERK1/2 and their total protein amounts in lung tumors from EGFRL858R/Rhob-/- or EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. The remaining hyperplastic areas were selected in erlotinib-treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bar: 100 µm.


C . HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant ( AKTmyr , myristoylated ) or an empty vector ( ø ) , and treated for 72 h with increasing concentrations of erlotinib . The surviving cell fraction was determined by an MTS assay . Data are representative of at least three independent experiments .

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C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay. Data are representative of at least three independent experiments.


C . HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant ( AKTmyr , myristoylated ) or an empty vector ( ø ) , and treated for 72 h with increasing concentrations of erlotinib . AKT overexpression and phosphorylation at Ser473 were assessed by western blotting . Data are representative of at least three independent experiments .

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C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. AKT overexpression and phosphorylation at Ser473 were assessed by western blotting. Data are representative of at least three independent experiments.


A . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for four hours with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . The phosphorylation status of GSK3β ( Ser9 ) , ERK1 / 2 and EGFR ( Tyr1173 ) was assessed by western blotting and normalized according to the total protein levels . RHOB overexpression was also monitored by western blotting .

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A. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2 and EGFR (Tyr1173) was assessed by western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by western blotting.


B . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 72 h with erlotinib alone ( black and red curves ) or in combination with the AKT inhibitor G594 at 100 nM ( green and blue curves ) . The surviving cell fraction was determined by an MTS assay .

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B. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay.


C . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594 . The surviving cell fraction was determined by an MTS assay , and erlotinib IC50 values were determined for each condition . ( * * : p < 0 . 001 vs . AdCont cells ; * * * : p < 0 . 0001 vs . AdCont cells ) . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs .

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C. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594. The surviving cell fraction was determined by an MTS assay, and erlotinib IC50 values were determined for each condition. (**: p<0.001 vs. AdCont cells; ***: p<0.0001 vs. AdCont cells). HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs.


HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . Apoptosis was then determined by quantification of the subG1 cell population ( D ) .

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HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by quantification of the subG1 cell population (D).


HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . Apoptosis was then determined by detection of cleaved PARP and caspase - 3 ( E ) .

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HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by detection of cleaved PARP and caspase-3 (E).


A . Representative H & amp ; amp ; E staining of lung tumors from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not during four days with erlotinib ( 12 . 5 mg / kg / day ) , with the AKT inhibitor G594 ( 25 mg / kg / day ) , or in combination with both drugs . Scale bar : 500 µm . B . Quantification of the tumor / lung ratio of mice treated or not with the individual drugs or with a combination of both .

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A. Representative H&amp;E staining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not during four days with erlotinib (12.5 mg/kg/day), with the AKT inhibitor G594 (25 mg/kg/day), or in combination with both drugs. Scale bar: 500 µm. B. Quantification of the tumor/lung ratio of mice treated or not with the individual drugs or with a combination of both.


C . Representative Ki67 immunostaining of lung tumors from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib alone ( 12 . 5 mg / kg / day ) or in combination with the AKT inhibitor G594 ( 25 mg / kg / day ) for four days ( scale bar : 50 µm ) , and the corresponding quantification ( D ) .

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C. Representative Ki67 immunostaining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib alone (12.5 mg/kg/day) or in combination with the AKT inhibitor G594 (25 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D).


A . - E . Staining for β - galactosidase in 3 - months old VEGF - Ahyper ( lacZ / wt ) mouseeyes . A . Low magnification image shows β - gal + cells ( in blue ) in the RPE ( white arrow ) , retina ( blue arrow ) , ciliary body ( black arrow ) , and lens ( orange arrow ) . Images of Figures 1B to 1E are higher magnification images of Figure 1A . Scale bar 200 μm . B . Strong expression of VEGF with β - gal + cells are observed in the RPE ( short white arrow ) and the inner nuclear layer ( INL ) ( long white arrow ) , while less staining is observed in the ganglion cell layer ( black arrow ) of the retina . Scale bar 100 μm . C . Ciliary body epithelial cells are β - gal + , similarly as RPE cells ( white arrow ) . Scale bar 100 μm . D . VEGF - A is strongly expressed in secondary nuclearfiber cells in the adult lens ( white arrow ) . Scale bar 50 μm . E . No β - gal + cells are observed in the corneas of adult VEGF - Ahyper ( lacZ / wt ) mouseeyes . White arrow shows the stroma of the cornea and the black arrow shows the corneal epithelium . Scale bar 100 μm .

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A.-E. Staining for β-galactosidase in 3-months old VEGF-Ahyper(lacZ/wt)mouseeyes.A. Low magnification image shows β-gal+ cells (in blue) in the RPE (white arrow), retina (blue arrow), ciliary body (black arrow), and lens (orange arrow). Images of Figures 1B to 1E are higher magnification images of Figure 1A. Scale bar 200 μm.B. Strong expression of VEGF with β-gal+ cells are observed in the RPE (short white arrow) and the inner nuclear layer (INL) (long white arrow), while less staining is observed in the ganglion cell layer (black arrow) of the retina. Scale bar 100 μm.C. Ciliary body epithelial cells are β-gal+, similarly as RPE cells (white arrow). Scale bar 100 μm.D. VEGF-A is strongly expressed in secondary nuclearfiber cells in the adult lens (white arrow). Scale bar 50 μm.E. No β-gal+ cells are observed in the corneas of adult VEGF-Ahyper(lacZ/wt)mouseeyes. White arrow shows the stroma of the cornea and the black arrow shows the corneal epithelium. Scale bar 100 μm.


G . RPE cells maintain strong expression of VEGF - A ( β - gal + cells [ white arrow ] ) in the retina with progressive age . Immunofluorescence for β - gal in the retina of a 24 - months old VEGF - Ahypermouse is shown . Ocular macrophages ( F4 / 80 + cells [ yellow arrow ] ) show no strong β - galactosidase expression , while some immunolabeling for β - galactosidase is detected in retinal cells of the inner nuclear layer ( orange arrow ) of the retina . RPE : retinal pigment epithelium ; ONL : outer nuclear layer ; INL : inner nuclear layer . Scale bar 50 μm .

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G. RPE cells maintain strong expression of VEGF-A (β-gal+ cells [white arrow]) in the retina with progressive age. Immunofluorescence for β-gal in the retina of a 24-months old VEGF-Ahypermouse is shown. Ocular macrophages (F4/80+ cells [yellow arrow]) show no strong β-galactosidase expression, while some immunolabeling for β-galactosidase is detected in retinal cells of the inner nuclear layer (orange arrow) of the retina. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer. Scale bar 50 μm.


H . VEGF - A continues to be highly expressed in secondary nuclear fiber cells in the aged lens ( shown is a lens of a 24 - months old VEGF - Ahyper mouse ; nuclear β - galactosidasestaining ( arrow ) reflects cellular VEGF - A expression ) . Cor : lenscortex ; Cap : lens capsule . DAPI labels nuclei ( blue ) . Scale bar 25 μm .

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H. VEGF-A continues to be highly expressed in secondary nuclear fiber cells in the aged lens (shown is a lens of a 24-months old VEGF-Ahyper mouse; nuclear β-galactosidasestaining (arrow) reflects cellular VEGF-A expression). Cor: lenscortex; Cap: lens capsule. DAPI labels nuclei (blue). Scale bar 25 μm.


A . Left graph : VEGF - Aprotein levels in lenses of VEGF - Ahypermice ( KI ) are increased ~ 3 - fold throughout life ( measured at 2 - and at 8 . 5 - months of age ; n = 7 mice / group , 2 independent experiments ) . Graph shows mean ± SD . Right graph : VEGF - AmRNA levels are similarly increased in lenses of adult VEGF - Ahypermice ( 9 - months old ; n = 7 mice / group , 3 independent experiments ) . Graph shows mean ± SEM . * * P - value : 0 . 0095 .

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A. Left graph: VEGF-Aprotein levels in lenses of VEGF-Ahypermice (KI) are increased ~3-fold throughout life (measured at 2- and at 8.5-months of age; n=7 mice/group, 2 independent experiments). Graph shows mean ± SD. Right graph: VEGF-AmRNA levels are similarly increased in lenses of adult VEGF-Ahypermice (9-months old; n=7 mice/group, 3 independent experiments). Graph shows mean ± SEM. **P-value: 0.0095.


B . VEGF - A isoforms ( mainly VEGF - A120 and VEGF - A164 ) and the VEGF - A receptor Flk1 are expressed in the adult lens . Flt1 expression was not detected in the lens . N = 3 mice / group .

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B. VEGF-A isoforms (mainly VEGF-A120 and VEGF-A164) and the VEGF-A receptor Flk1 are expressed in the adult lens. Flt1 expression was not detected in the lens. N=3 mice/group.


C . Increased serum levels of the lipid peroxidation marker 4 - HNE ( a marker of increased oxidative damage ) in aged , but not in young VEGF - Ahyper mice . * P - value : 0 . 0325 . N = 7 mice / group ( 2 independent experiments ) . Graph shows mean ± SEM .

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C. Increased serum levels of the lipid peroxidation marker 4-HNE (a marker of increased oxidative damage) in aged, but not in young VEGF-Ahyper mice. *P-value: 0.0325. N=7 mice/group (2 independent experiments). Graph shows mean ± SEM.


A . Increased ERK1 / 2 phosphorylation is observed in lenses from VEGF - Ahyper mice ( KI ) with increased VEGF - A levels already prior to cataract formation ( 11 - months old mice with no cataracts ( 0 cataract ) ) , which is maintained in lenses with mature cataracts ( lens of a 26 - months old VEGF - Ahyper mouse with + 3 cataract ) . Quantification of ERK1 / 2 phosphorylation in 11 - months old lenses ( n = 3 / group ) . Graph shows mean ± SEM . * * * P - value : 0 . 0005 .

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A. Increased ERK1/2 phosphorylation is observed in lenses from VEGF-Ahyper mice (KI) with increased VEGF-A levels already prior to cataract formation (11-months old mice with no cataracts (0 cataract)), which is maintained in lenses with mature cataracts (lens of a 26-months old VEGF-Ahyper mouse with +3 cataract). Quantification of ERK1/2 phosphorylation in 11-months old lenses (n=3/group). Graph shows mean ± SEM. ***P-value: 0.0005.


B . - C . Cataracts with opacification of the lens ( B . , black arrow ) and extruded nuclei ( C . , white arrow ) are observed with progressive age in VEGF - Ahyper mice ( here a representative 30 - months old lens with a cataract is shown ) .

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B.-C. Cataracts with opacification of the lens (B., black arrow) and extruded nuclei (C., white arrow) are observed with progressive age in VEGF-Ahyper mice (here a representative 30-months old lens with a cataract is shown).


D . - E . Histology of lens from b . - c . shows extrusion of sclerotic nucleus ( white arrows in C . and D . ) and massive degenerative changes of the lens cortex with vacuolization ( black arrows in D . and E . ) . Scale bar 500 μm in ( D . ) and 100 μm in ( E . ) .

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D.-E. Histology of lens from b.-c. shows extrusion of sclerotic nucleus (white arrows in C. and D.) and massive degenerative changes of the lens cortex with vacuolization (black arrows in D. and E.). Scale bar 500 μm in (D.) and 100 μm in (E.).


F . - G . Representative histological images of 21 - months old eyes from WT and VEGF - Ahyper mice . The sclerotic nucleus ( red arrow in G . ) was extruded and the lenscortex and capsule are separated from the nucleus ( white arrow in G . ) . The retina in aged VEGF - Ahyper mice is atrophic and shows degenerative changes ( yellow arrow in G . ) , while the retina ( * ) and lens ( * * ) appears normal in age - matched WT littermate mice . Scale bars 200 μm . DAPI ( blue , nuclei ) ; phalloidin ( green ) .

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F.-G. Representative histological images of 21-months old eyes from WT and VEGF-Ahyper mice. The sclerotic nucleus (red arrow in G.) was extruded and the lenscortex and capsule are separated from the nucleus (white arrow in G.). The retina in aged VEGF-Ahyper mice is atrophic and shows degenerative changes (yellow arrow in G.), while the retina (*) and lens (**) appears normal in age-matched WT littermate mice. Scale bars 200 μm. DAPI (blue, nuclei); phalloidin (green).


A . lens progress with age in VEGF - Ahypermice . lens were graded from + 1 ( mild ) , + 2 ( moderate ) , to + 3 ( mature cataract with fully opacified lens ) . While only a small subset of > 18 months - old WT mice have mature ( + 3 ) cataracts , the majority of VEGF - Ahypermice of this age group has mature cataracts . Percentile ( % ) of mice with graded cataracts is indicated in each age group . 0 = no cataract . Absolute mouse numbers of each group are shown in Appendix Figure S1 . B . Targeting NLRP3 inhibits normal age - dependent cataract formation , and delays ( but does not prevent ) VEGF - A - induced cataract formation in VEGF - Ahypermice . Percentile ( % ) of mice with graded cataracts is indicated in each age group . 0 = no cataract . Absolute mouse numbers of each group are shown in Appendix Figure S1 .

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A. lens progress with age in VEGF-Ahypermice. lens were graded from +1 (mild), +2 (moderate), to +3 (mature cataract with fully opacified lens). While only a small subset of >18 months-old WT mice have mature (+3) cataracts, the majority of VEGF-Ahypermice of this age group has mature cataracts. Percentile (%) of mice with graded cataracts is indicated in each age group. 0 = no cataract. Absolute mouse numbers of each group are shown in Appendix Figure S1.B. Targeting NLRP3 inhibits normal age-dependent cataract formation, and delays (but does not prevent) VEGF-A-induced cataract formation in VEGF-Ahypermice. Percentile (%) of mice with graded cataracts is indicated in each age group. 0 = no cataract. Absolute mouse numbers of each group are shown in Appendix Figure S1.


C . Expression of IL - 1β in lenses of 21 - months old VEGF - Ahyper mice with + 3 cataracts is increased compared to lenses from age - matched WT mice with + 3 cataracts , but not in lenses from 11 - months old mouse groups with no cataracts . * P - value : 0 . 0259 . N = 7 / group ( 3 independent experiments ) .

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C. Expression of IL-1β in lenses of 21-months old VEGF-Ahyper mice with +3 cataracts is increased compared to lenses from age-matched WT mice with +3 cataracts, but not in lenses from 11-months old mouse groups with no cataracts. *P-value: 0.0259. N=7/group (3 independent experiments).


D . VEGF - Ahyper mice have decreased levels of the major antioxidant in the lens , reduced glutathione ( μmol / gm lens weight ) , prior to cataract formation . Lenses with 0 to + 1 cataracts from 11 - months old mice were used for measurements . * * P - value : 0 . 0097 . N = 7 / group ( 2 independent experiments ) .

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D. VEGF-Ahyper mice have decreased levels of the major antioxidant in the lens, reduced glutathione (μmol/gm lens weight), prior to cataract formation. Lenses with 0 to +1 cataracts from 11-months old mice were used for measurements. ** P-value: 0.0097. N=7/group (2 independent experiments).


E - F . Overexpression of NADPH oxidase gp91phox [ * p - value : 0 . 0435 ] ( E . ) and of the antioxidant enzymes SOD1 [ * p - value : 0 . 0330 ] , catalase ( CAT ) [ * p - value : 0 . 0448 ] and GPx - 1 [ * p - value : 0 . 0443 ] ( F . ) in 21 - months old lenses of VEGF - Ahyper mice with cataracts ( + 3 ) , but not in 11 - months old lenses with no cataracts . N = 7 / group ( 3 independent experiments ) .

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E-F. Overexpression of NADPH oxidase gp91phox [*p-value: 0.0435] (E.) and of the antioxidant enzymes SOD1 [*p-value: 0.0330], catalase (CAT) [*p-value: 0.0448] and GPx-1 [*p-value: 0.0443] (F.) in 21-months old lenses of VEGF-Ahyper mice with cataracts (+3), but not in 11-months old lenses with no cataracts. N=7/group (3 independent experiments).


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A - B . Choroidalflat mount staining of a white VEGF - Ahypermouse shows choroidalvessels ( green [ fluorescein - conjugated isolectin B4 ] ) from which neovessels ( strong red [ CD31 ] ) originate and that are covered by a RPEcell layer ( white [ phalloidin ] ) , which separates the CNV lesion from the photoreceptors . White arrows demarcate CNV lesion . Yellow arrows show round autofluorescent deposits . Scale bars 100 μm .

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A-B. Choroidalflat mount staining of a white VEGF-Ahypermouse shows choroidalvessels (green [fluorescein-conjugated isolectin B4]) from which neovessels (strong red [CD31]) originate and that are covered by a RPEcell layer (white [phalloidin]), which separates the CNV lesion from the photoreceptors. White arrows demarcate CNV lesion. Yellow arrows show round autofluorescent deposits. Scale bars 100 μm.


A - B . Choroidalflat mount staining of a white VEGF - A hypermouse shows choroidal vessels ( green [ fluorescein - conjugated isolectin B4 ] ) from which neovessels ( strong red [ CD31 ] ) originate and that are covered by a RPEcell layer ( white [ phalloidin ] ) , which separates the CNV lesion from the photoreceptors . White arrows demarcate CNV lesion . Yellow arrows show round autofluorescent deposits . Scale bars 100 μm .

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A-B. Choroidalflat mount staining of a white VEGF-A hypermouse shows choroidal vessels (green [fluorescein-conjugated isolectin B4]) from which neovessels (strong red [CD31]) originate and that are covered by a RPEcell layer (white [phalloidin]), which separates the CNV lesion from the photoreceptors. White arrows demarcate CNV lesion. Yellow arrows show round autofluorescent deposits. Scale bars 100 μm.


C . Section through a CNV lesion in a VEGF - Ahyper mouse . Bruch ' s membrane location is indictaed by yellow arrows . Choroidal neovessels ( white arrows ) are covered by a layer of RPE cells ( black arrows ) . Chor : choroid ; ONL : outer nuclear layer ; PR : photoreceptors . Scale bar 20 μm . A . - C . 21 - months old VEGF - Ahyper mice .

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C. Section through a CNV lesion in a VEGF-Ahyper mouse. Bruch's membrane location is indictaed by yellow arrows. Choroidal neovessels (white arrows) are covered by a layer of RPE cells (black arrows). Chor: choroid; ONL: outer nuclear layer; PR: photoreceptors. Scale bar 20 μm. A.-C. 21-months old VEGF-Ahyper mice.


RPE - specific overexpression of VEGF - A in VMD2Cre + / WTROSA - STOPfl / fl - VEGF - A164mice ( VEGF - ARPEmice ) results in CNV at sites of VEGF - A overexpression ( Cre + patches ( white nuclei ) ; red arrow ) , while Cre - areas with normal VEGF - A expression show no CNV ( white arrow ) . Neovessels are CD31 + ( red ) . VEGF - A - induced CNV lesions and RPE barrier breakdown in VMD2Cre + / WT ROSA - STOPfl / fl - VEGF - A164mice resemble those seen in VEGF - Ahypermice ( B . - C . , arrows ; phalloidin staining in green ) . Scale bars 100 μm .

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RPE-specific overexpression of VEGF-A in VMD2Cre+/WTROSA-STOPfl/fl-VEGF-A164mice (VEGF-ARPEmice) results in CNV at sites of VEGF-A overexpression (Cre+ patches (white nuclei); red arrow), while Cre- areas with normal VEGF-A expression show no CNV (white arrow). Neovessels are CD31+ (red). VEGF-A-induced CNV lesions and RPE barrier breakdown in VMD2Cre+/WT ROSA-STOPfl/fl-VEGF-A164mice resemble those seen in VEGF-Ahypermice (B.-C., arrows; phalloidin staining in green). Scale bars 100 μm.


D . Genetic inactivation of Flk1 specifically in the RPE in VEGF - Ahypermice ( VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice ) prevents RPE barrier breakdown and CNV lesion formation . Choroidalflat mounts of 5 months old VEGF - Ahypermice and VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice are shown . β - gal shows uniform VEGF - A expression in these mice ( green ) . Crestaining ( red ) shows co - localization of β - gal and Crestaining in the nuclei of RPE cells in VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice ( red areas of staining occur in CNV lesions of VEGF - Ahypermice due to labeling of anti - mouseIgG and not Cre , as the anti - mouseIgG secondary antibodies were used to detect the mouse anti - Cre antibody ) . Phalloidinstaining ( white ) reveals extensive RPE barrier breakdown and CNV lesions ( arrow ) in VEGF - Ahypermice , which is not observed in VMD2Cre + / WTFlk1fl / flVEGF - Ahypermice . Scale bar 100 μm .

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D. Genetic inactivation of Flk1 specifically in the RPE in VEGF-Ahypermice (VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice) prevents RPE barrier breakdown and CNV lesion formation. Choroidalflat mounts of 5 months old VEGF-Ahypermice and VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice are shown. β-gal shows uniform VEGF-A expression in these mice (green). Crestaining (red) shows co-localization of β-gal and Crestaining in the nuclei of RPE cells in VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice (red areas of staining occur in CNV lesions of VEGF-Ahypermice due to labeling of anti-mouseIgG and not Cre, as the anti-mouseIgG secondary antibodies were used to detect the mouse anti-Cre antibody). Phalloidinstaining (white) reveals extensive RPE barrier breakdown and CNV lesions (arrow) in VEGF-Ahypermice, which is not observed in VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice. Scale bar 100 μm.


A . NLRP3 is expressed in RPE cells at sites of CNV lesions ( arrow ; white ) . B . Lack of NLRP3 staining in VEGF - Ahyper / NLRP3 - / - mice ( control ) . Scale bars 100 μm .

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A. NLRP3 is expressed in RPE cells at sites of CNV lesions (arrow; white).B. Lack of NLRP3 staining in VEGF-Ahyper/NLRP3-/- mice (control). Scale bars 100 μm.


C . Expression of NLRP3 [ * p - value : 0 . 0478 ] and IL - 1β [ * p - value : 0 . 0398 ] , but not of IL - 18 [ p - value : 0 . 3529 ] is increased in RPE / choroid lysates from VEGF - Ahyper mice when compared to matched control littermate mice . N = 7 mice / group ( 3 independent experiments ) . Graphs show mean ± SEM .

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C. Expression of NLRP3 [*p-value: 0.0478] and IL-1β [*p-value: 0.0398], but not of IL-18 [p-value: 0.3529] is increased in RPE/choroid lysates from VEGF-Ahyper mice when compared to matched control littermate mice. N=7 mice/group (3 independent experiments). Graphs show mean ± SEM.