E - G . SR - SIM images showing three stages of invading wt parasites . SiR - tubulin staining for microtubules ( in red ) was performed prior to fixation to specifically label microtubules in the parasite . During invasion , the microtubules ( MT ) are deformed at the TJ area ( white arrowhead ) . Accumulation of actin ( green arrow ) at the posterior forms a F - actin meshwork . The nucleus is deformed when it enters the TJ area ( orange arrowhead ) . H - J . In the case of the myoA KO strain , the MTs also deform when passing through the TJ ( white arrowhead ) . However , F - actin is not concentrated at the posterior pole or the TJ , but evenly distributed within the cytosol of the parasite , with some peripheral location . K - M . In case of adfKD , MTs are deformed at the TJ area ( white arrowhead ) . F - actin is accumulated at both ends of the parasite ( green arrowheads ) , with no accumulation at the TJ . Data information : Scale bar represents 5 µm for SR - SIM images White arrow points to direction of invasion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G. SR-SIM images showing three stages of invading wt parasites. SiR-tubulin staining for microtubules (in red) was performed prior to fixation to specifically label microtubules in the parasite. During invasion, the microtubules (MT) are deformed at the TJ area (white arrowhead). Accumulation of actin (green arrow) at the posterior forms a F-actin meshwork. The nucleus is deformed when it enters the TJ area (orange arrowhead). H-J. In the case of the myoA KO strain, the MTs also deform when passing through the TJ (white arrowhead). However, F-actin is not concentrated at the posterior pole or the TJ, but evenly distributed within the cytosol of the parasite, with some peripheral location. K-M. In case of adfKD, MTs are deformed at the TJ area (white arrowhead). F-actin is accumulated at both ends of the parasite (green arrowheads), with no accumulation at the TJ. Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.


A . Different invasion stages of P . falciparum merozoites expressing Cb - EmeraldFP . DAPI labels the nucleus ( DNA ) , Cb - EmeraldFP labels actin filaments ( CB - EME , green ) and anti - RON4 labels the junction ( red ) . Left panel : onset of invasion . Middle panel : A merozoite in the middle of invasion ; note the constriction of the nucleus ( blue ) as it passes through the RON4 junction and F - actin colocalising with it ( green ) . Right panel : A merozoite completing invasion ; note the F - actin ring beyond the RON4 - junction and actin filaments surrounding the nucleus . Data information : Scale bar represents 1 µm for Plasmodium

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Different invasion stages of P. falciparum merozoites expressing Cb-EmeraldFP. DAPI labels the nucleus (DNA), Cb-EmeraldFP labels actin filaments (CB-EME, green) and anti-RON4 labels the junction (red). Left panel: onset of invasion. Middle panel: A merozoite in the middle of invasion; note the constriction of the nucleus (blue) as it passes through the RON4 junction and F-actin colocalising with it (green). Right panel: A merozoite completing invasion; note the F-actin ring beyond the RON4-junction and actin filaments surrounding the nucleus. Data information: Scale bar represents 1 µm for Plasmodium falciparum


B . IFA showing various stages of invasion of erythrocytes by P . knowlesi merozoites . DAPI labels the nucleus ( DNA ) , anti - actin Antibody labels actin filaments ( anti - actin , green ) and the invasion junction is marked with a C - terminally tagged PkRON2 mCherryHA ( red ) . Left panel : a merozoite beginning invasion ; note the formation of an F - actin ring ( green ) behind the PkRON2 junction ( red ) . Middle panel : A merozoite in the middle of the invasion event ; note the constriction of the nucleus ( blue ) as it passes through the PkRON2 junction and F - actin colocalising with it ( green ) . Right panel : A merozoite completing the process of invasion ; note the F - actin ring beyond the PkRON2 - junction and actin filaments colocalising with the nucleus . Data information : Scale bar represents 3 µm for Plasmodium knowlesi .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. IFA showing various stages of invasion of erythrocytes by P. knowlesi merozoites. DAPI labels the nucleus (DNA), anti-actin Antibody labels actin filaments (anti-actin, green) and the invasion junction is marked with a C-terminally tagged PkRON2 mCherryHA (red). Left panel: a merozoite beginning invasion; note the formation of an F-actin ring (green) behind the PkRON2 junction (red). Middle panel: A merozoite in the middle of the invasion event; note the constriction of the nucleus (blue) as it passes through the PkRON2 junction and F-actin colocalising with it (green). Right panel: A merozoite completing the process of invasion; note the F-actin ring beyond the PkRON2-junction and actin filaments colocalising with the nucleus. Data information: Scale bar represents 3 µm for Plasmodium knowlesi.


C . Super - resolution images showing various stages of invasion of erythrocytes by P . falciparum merozoites expressing Cb - EmeraldFP . Left panel : DAPI labels the nucleus ( blue ) , Cb - EmeraldFP labels actin filaments ( CB - EME , green ) and the invasion junction is marked with an anti - RON4 antibody ( red ) . Brightfield images have been marked with red dots depicting the junction , black dots depicting merozoite boundary still outside the erythrocyte and white dots depicting a merozoite that has penetrated the host cell . Top panel shows an attached merozoite beginning the process of invasion , the second panel during the process of invasion and the bottom two panels show merozoites completing the process of invasion . Right panel : Schematic depicting merozoite invasion into red blood cells . Letters depict the corresponding event in the model ( left ) and microscopy pictures ( right ) . White arrows depict actin filaments that colocalise with the nucleus . D . Quantification of Plasmodium falciparum invasion events categorising actin - TJ and actin - nucleus association events . 121 events were counted in total . Data information : Error bars represent standard deviation . Scale bar represents 1 µm for Plasmodium

C. Super-resolution images showing various stages of invasion of erythrocytes by P. falciparum merozoites expressing Cb-EmeraldFP. Left panel: DAPI labels the nucleus (blue), Cb-EmeraldFP labels actin filaments (CB-EME, green) and the invasion junction is marked with an anti-RON4 antibody (red). Brightfield images have been marked with red dots depicting the junction, black dots depicting merozoite boundary still outside the erythrocyte and white dots depicting a merozoite that has penetrated the host cell. Top panel shows an attached merozoite beginning the process of invasion, the second panel during the process of invasion and the bottom two panels show merozoites completing the process of invasion. Right panel: Schematic depicting merozoite invasion into red blood cells. Letters depict the corresponding event in the model (left) and microscopy pictures (right). White arrows depict actin filaments that colocalise with the nucleus. D. Quantification of Plasmodium falciparum invasion events categorising actin-TJ and actin-nucleus association events. 121 events were counted in total. Data information: Error bars represent standard deviation. Scale bar represents 1 µm for Plasmodium falciparum


( H ) - ( J ) mRNA expression of ( H ) U2AF1 , ( I ) SMN2 - FL , or ( J ) SMN2 ∆ 7 in SMA patient - derived fibroblasts ( red bars ) 72 hours post - transfection with SMN2 SSO , scrambled siRNA , or U2AF1 siRNA at the indicated dose . Unaffected , SMA carrier fibroblasts ( blue bars ) treated with scrambled siRNA . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; empirical p - values are determined by a simulation and sampling method The error bars show the Standard Error of Mean ( SEM ) . Data represent 3 biological replicates . Three technical replicates were performed during qPCR for each biological replicate and averaged . Scrambled siRNA condition for each experiment was set to 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)-(J) mRNA expression of (H) U2AF1, (I) SMN2-FL, or (J) SMN2∆7 in SMA patient-derived fibroblasts (red bars) 72 hours post-transfection with SMN2 SSO, scrambled siRNA, or U2AF1 siRNA at the indicated dose. Unaffected, SMA carrier fibroblasts (blue bars) treated with scrambled siRNA. Data information: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; empirical p-values are determined by a simulation and sampling method The error bars show the Standard Error of Mean (SEM). Data represent 3 biological replicates. Three technical replicates were performed during qPCR for each biological replicate and averaged. Scrambled siRNA condition for each experiment was set to 1.


( K ) Representative Western blot . ( L ) - ( M ) Quantification of protein expression of ( L ) U2AF1 or ( M ) SMN normalized to GAPDH ( n = 3 ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; empirical p - values are determined by a simulation and sampling method The error bars show the Standard Error of Mean ( SEM ) . Data represent 3 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot. (L)-(M) Quantification of protein expression of (L) U2AF1 or (M) SMN normalized to GAPDH (n=3). Data information: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; empirical p-values are determined by a simulation and sampling method The error bars show the Standard Error of Mean (SEM). Data represent 3 biological replicates.


Quantitative phosphoproteomics dataset showing that most phosphorylation events induced in rad9Δ cells are dependent on Mec1 . These phosphoproteomic analyses were conducted in the presence of 0 . 02 % MMS for 2 hours .

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Quantitative phosphoproteomics dataset showing that most phosphorylation events induced in rad9Δ cells are dependent on Mec1. These phosphoproteomic analyses were conducted in the presence of 0.02% MMS for 2 hours.


Immunoblot analysis of Sgs1 N - terminus ( amino acids 1 - 647 ) from cells treated with increasing doses of the DNA alkylating drug MMS in the absence of RAD9 and / or MEC1 . This truncated Sgs1 protein was expressed from its native promoter in this and all subsequent experiments .

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Immunoblot analysis of Sgs1 N-terminus (amino acids 1-647) from cells treated with increasing doses of the DNA alkylating drug MMS in the absence of RAD9 and/or MEC1. This truncated Sgs1 protein was expressed from its native promoter in this and all subsequent experiments.


Rmi1 , a component of the Sgs1 - Top3 - Rmi1 ( STR ) complex , is hyper - phosphorylated on S / T - Q motifs in rad9Δ cells .

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Rmi1, a component of the Sgs1-Top3-Rmi1 (STR) complex, is hyper-phosphorylated on S/T-Q motifs in rad9Δ cells.


Immunoblot showing co - immunoprecipitation of Dpb11 with Sgs1 . Both DPB11 - 3xHA and SGS11 - 647 - 5xFLAG were expressed at their endogenous loci .

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Immunoblot showing co-immunoprecipitation of Dpb11 with Sgs1. Both DPB11-3xHA and SGS11-647-5xFLAG were expressed at their endogenous loci.


Immunoblots showing co - immunoprecipitation between Dpb11 and Sgs1 in the presence of 0 . 04 % MMS in either wildtype , mec1Δ , tel1Δ or mec1Δ tel1Δ cell lines . DPB11 - 3HA was tagged at its endogenous locus and co - immunoprecipitated with SGS11 - 647 - FLAG tagged and truncated at its endogenous locus .

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Immunoblots showing co-immunoprecipitation between Dpb11 and Sgs1 in the presence of 0.04% MMS in either wildtype, mec1Δ, tel1Δ or mec1Δ tel1Δ cell lines. DPB11-3HA was tagged at its endogenous locus and co-immunoprecipitated with SGS11-647-FLAG tagged and truncated at its endogenous locus.


Dilution assay of wild - type or rev3Δ cells expressing BRCT3 / 4 - SGS1 in the presence of 0 . 0025 % MMS .

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Dilution assay of wild-type or rev3Δ cells expressing BRCT3/4-SGS1 in the presence of 0.0025% MMS.


Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3 / 4 fused to wild - type or mutant versions of SGS1 . Mutants include a helicase deficient allele of SGS1 ( SGS1hd ) or an allele lacking the Top3 Interacting Motif ( TIM ; SGS1TIM ) . Error bars represent SEM of at least 3 replicate experiments . P value was calculated with a two - tailed , unpaired t - test . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 . C . Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3 / 4 fused to either SGS1 , TOP3 , or RMI1 . Error bars represent SEM of at least 3 replicate experiments . P value was calculated with a two - tailed , unpaired t - test . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 . D . Measurement of BIR efficiency in cells carrying an empty vector or DPB11BRCT3 / 4 fused to either SGS1 , hBLM , PIF1 , or RRM3 . Error bars represent SEM of at least 3 replicate experiments . P value was calculated with a two - tailed , unpaired t - test . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 .

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

Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3/4 fused to wild-type or mutant versions of SGS1. Mutants include a helicase deficient allele of SGS1 (SGS1hd) or an allele lacking the Top3 Interacting Motif (TIM; SGS1TIM). Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001. C. Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3/4 fused to either SGS1, TOP3, or RMI1. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001. D. Measurement of BIR efficiency in cells carrying an empty vector or DPB11BRCT3/4 fused to either SGS1, hBLM, PIF1, or RRM3. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.


Serial dilution assay of sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH .

Serial dilution assay of sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH.


BIR assay in rad9Δ sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH . Error bars represent SEM of 3 independent experiments . Error bars represent SEM of at least 3 replicate experiments . P value was calculated with a two - tailed , unpaired t - test . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 .

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

BIR assay in rad9Δ sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH. Error bars represent SEM of 3 independent experiments. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.


( H ) Ang - 2 dose and time - dependently activates p - EphrinB , p - TIE2 and p - AKT in HUVEC ; immunoblotting results ; NA3VO4 : sodium orthovanadate .

O O O O O 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 I-CONTROLLED_VAR O

(H) Ang-2 dose and time-dependently activates p-EphrinB, p-TIE2 and p-AKT in HUVEC; immunoblotting results; NA3VO4: sodium orthovanadate.


( I ) Tumor vessels in B16F10 tumors are mostly p - TIE2 ( Tyr992 ) + and pSHP2 ( Tyr542 ) + ; confocal images ( scale bars : 200μm ) .

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

(I) Tumor vessels in B16F10 tumors are mostly p-TIE2 (Tyr992)+ and pSHP2(Tyr542)+; confocal images (scale bars: 200μm).


( A ) Representative tissue immunostaining of human melanoma , colon carcinoma and colon adenoma ; confocal images from serial sections ( scale bars : 50μm tissues ) . Arrowheads point to CD31 + / p - EphrinB + ; CD31 + / p - SHP2 + ; CD31 + / p - Tie2 + and CD31 + / Ang2 + endothelial cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 tissue immunostaining of human melanoma, colon carcinoma and colon adenoma; confocal images from serial sections (scale bars: 50μm tissues). Arrowheads point to CD31+/p-EphrinB+; CD31+/p-SHP2+; CD31+/p-Tie2+ and CD31+/Ang2+ endothelial cells.


( B ) Gene expression in tumor ( black bar ) and control ( white bar ) ; top panel : melanoma ( GSE7553 , primary and metastatic ; tumor n = 54 ; control skin n = 5 ) ; bottom panel : colon cancer ( TCGA , primary and metastatic ; tumor n = 269 ; control n = 41 . * * P < 0 . 01 , * * * * P < 0 . 0001 ; P values from Mann Whitney U - 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 O O O O O O O O O O O O O O

(B) Gene expression in tumor (black bar) and control (white bar); top panel: melanoma (GSE7553, primary and metastatic; tumor n=54; control skin n=5); bottom panel: colon cancer (TCGA, primary and metastatic; tumor n=269; control n=41. **P<0.01, ****P<0.0001; P values from Mann Whitney U-test.


( C ) Impact of high and low ( 75 % quartile ) mRNA levels on the survival probability of patients with primary melanoma ; Kaplan - Meier analysis using TCGA primary melanoma datasets ( n = 103 ) ; P values from Mann Whitney U - test . ( D ) Impact of high and low ( median ) mRNA levels on the survival probability of patients with colon cancer ; Kaplan - Meier analysis using TCGA colon cancer dataset ( n = 262 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Impact of high and low (75% quartile) mRNA levels on the survival probability of patients with primary melanoma; Kaplan-Meier analysis using TCGA primary melanoma datasets (n=103); P values from Mann Whitney U-test. (D) Impact of high and low (median) mRNA levels on the survival probability of patients with colon cancer; Kaplan-Meier analysis using TCGA colon cancer dataset (n=262).


( C , D ) The JAK inhibitor Tofacitinib ( Tofa , 50nM ) enhances the proliferation of HUVEC and BMEC compared to control ( C ) and reduces endogenous p - STAT3 levels in HUVEC and BMEC compared to control ( D ) after 72 - hour culture . SHP099 ( 5μM HUVEC ; 20μM BMEC ) reduces cell proliferation and increases p - STAT3 in HUVEC and BMEC compared to control ; error bars : ± S . D . Results of immunoblotting are representative of 3 independent blots .

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(C,D) The JAK inhibitor Tofacitinib (Tofa, 50nM) enhances the proliferation of HUVEC and BMEC compared to control (C) and reduces endogenous p-STAT3 levels in HUVEC and BMEC compared to control (D) after 72-hour culture. SHP099 (5μM HUVEC; 20μM BMEC) reduces cell proliferation and increases p-STAT3 in HUVEC and BMEC compared to control; error bars: ± S.D. Results of immunoblotting are representative of 3 independent blots.


( E , F ) The MAP kinases inhibitor PD098059 ( PD0980 , 10μM ) and SHP099 ( 5μM HUVEC ; 20μM BMEC ) inhibit HUVEC and BMEC proliferation compared to control ( E ) and reduce p - ERK1 / 2 levels by immunoblotting after 72 - hour culture ( F ) . Results of proliferation in panels C and E ( % of control ) from triplicate cultures are representative of 3 - 4 experiments ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ; two - tailed Student ' s t - test ; error bars : ± S . D . Results of immunoblotting are representative of 3 independent blots .

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

(E,F) The MAP kinases inhibitor PD098059 (PD0980, 10μM) and SHP099 (5μM HUVEC; 20μM BMEC) inhibit HUVEC and BMEC proliferation compared to control (E) and reduce p-ERK1/2 levels by immunoblotting after 72-hour culture (F). Results of proliferation in panels C and E (% of control) from triplicate cultures are representative of 3-4 experiments; *P<0.05, **P<0.01 ***P<0.001; two-tailed Student's t-test; error bars: ± S.D. Results of immunoblotting are representative of 3 independent blots.


( G , H ) The ERK1 / 2 inhibitors AZD6244 and Tamnitinib dose - dependently reduce B16F10 cell proliferation ( G ) ; representative experiment of 3 independent experiments ; the results reflect the means of 5 replicate / condition ; error bars : ± S . D . ; and levels of endogenous p - STAT3 and p - ERK in B16F10 cells ( representative of 3 blots ) ( H ) .

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

(G,H) The ERK1/2 inhibitors AZD6244 and Tamnitinib dose-dependently reduce B16F10 cell proliferation (G); representative experiment of 3 independent experiments; the results reflect the means of 5 replicate/condition; error bars: ± S.D.; and levels of endogenous p-STAT3 and p-ERK in B16F10 cells (representative of 3 blots) (H).


( A - C ) SHP099 ( A , 75mg / kg ; B , C : 100mg / kg ) reduces B16F10 tumor growth in syngeneic mice . Panels depict tumor growth from initiation of treatment to endpoint ( left ) and tumor weight ( right ) at endpoint ; no . mice / group : 6 - 11 control ; 7 - 9 SHP099 .

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(A-C) SHP099 (A, 75mg/kg; B, C: 100mg/kg) reduces B16F10 tumor growth in syngeneic mice. Panels depict tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; no. mice/group: 6-11 control; 7-9 SHP099.


( E ) " Vascular sleeves " ( Collagen IV + CD31 - ) identified in a representative SHP099 - treated tumor . Representative confocal images from 6 tumors ( scale bar : 50μm ) . ( F ) Reduced CD31 + vascular area in SHP099 - treated tumors compared to controls ( n = 6 / group ) ; quantification by ImageJ . ( G ) The ratio of Collagen IV + / CD31 + area is increased in SHP099 - treated tumors compared to controls ( n = 6 / group ) ; quantification by ImageJ ; Coll : collagen . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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 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

(E) "Vascular sleeves" (Collagen IV+ CD31-) identified in a representative SHP099-treated tumor. Representative confocal images from 6 tumors (scale bar: 50μm). (F) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ. (G) The ratio of Collagen IV+/CD31+ area is increased in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ; Coll: collagen. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( H ) Cleaved caspase - 3 detection in a CD31 + vessel ( representative of 6 SHP099 - treated tumors ) ; arrowheads point to cleaved caspase - 3 + CD31 + cells . Cleaved caspase - 3 + tumor cell is marked by * . Confocal images ( scale bar : 10μm ) . ( I ) Increased cleaved caspase - 3 fluorescence intensity in CD31 + cells from SHP099 - treated tumors compared to controls ( n = 3 / group ) ; at least 100 cells counted / sample . ( J ) Ki67 + proliferating cells in representative of 3 SHP099 - treated and 3 control tumors ; CD31 identifies the vessels ; DAPI identifies cell nuclei . Confocal images ( scale bar : 50μm ) . ( K ) Quantification of proliferating cells within SHP099 and control ( n = 4 / group ) tumors by ImageJ . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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 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 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 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

(H) Cleaved caspase-3 detection in a CD31+ vessel (representative of 6 SHP099-treated tumors); arrowheads point to cleaved caspase-3+CD31+ cells. Cleaved caspase-3+ tumor cell is marked by *. Confocal images (scale bar: 10μm). (I) Increased cleaved caspase-3 fluorescence intensity in CD31+ cells from SHP099-treated tumors compared to controls (n=3/group); at least 100 cells counted/sample. (J) Ki67+ proliferating cells in representative of 3 SHP099-treated and 3 control tumors; CD31 identifies the vessels; DAPI identifies cell nuclei. Confocal images (scale bar: 50μm). (K) Quantification of proliferating cells within SHP099 and control (n=4/group) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( L ) Hypoxic tumor identified Hypoxyprobe ( green ) representative of 3 SHP099 - treated tumors ; confocal images ( scale bar : 100μm ) , left ; quantification of hypoxic areas in control ( n = 3 ) and SHP099 - treated ( n = 3 ) tumors by ImageJ . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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 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

(L) Hypoxic tumor identified Hypoxyprobe (green) representative of 3 SHP099-treated tumors; confocal images (scale bar: 100μm), left; quantification of hypoxic areas in control (n=3) and SHP099-treated (n=3) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( M ) Representative control ( of 5 ) and SHP099 - treated ( of 5 ) B16F10 tumor sections through the maximum diameter ; H & E staining ( scale bar : 10mm ) . ( N ) Cleaved caspase - 3 + fluorescence intensity in SHP099 and control tumors ( n = 3 / group ) . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative control (of 5) and SHP099-treated (of 5) B16F10 tumor sections through the maximum diameter; H&E staining (scale bar: 10mm). (N) Cleaved caspase-3+ fluorescence intensity in SHP099 and control tumors (n=3/group). Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( B ) Extravasation of FITC - dextran ( green ) in tumors established in NSG mice treated with SHP099 ; scale bar ( 50μm ) . Representative images from 3 mice / group . ( C ) Reduced CD31 + vascular area in SHP099 - treated tumors compared to controls ( n = 5 / group ) ; ImageJ quantification . P values from two - tailed Student ' s t - test ; * P < 0 . 05 . Error bars : ± S . D .

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 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) Extravasation of FITC-dextran (green) in tumors established in NSG mice treated with SHP099; scale bar (50μm). Representative images from 3 mice/group. (C) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=5/group); ImageJ quantification. P values from two-tailed Student's t-test; *P<0.05. Error bars: ± S.D.


E ) SHP099 reduces weight of control ( p - LKO vector ) and SHP2 - depleted ( shSHP2 ) B16F10 tumors compared to untreated controls : p - LKO untreated ( n = 5 ) , p - LKO treated with SHP099 ( n = 9 ) , shSHP2 untreated ( n = 5 ) , and shSHP2 treated with SHP099 ( n = 13 ) . P values by analysis of variance with Dunnett ' s multiple comparison test ; * * * P < 0 . 001 . ( F ) Persistent depletion of SHP2 in tumors induced by shSHP2 - B16F10 cells ( n = 5 ) compared to control tumors induced by p - LKO - B16F10 ( n = 5 ) ; tumors removed from mice 18 days after cell inoculation .

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

E) SHP099 reduces weight of control (p-LKO vector) and SHP2-depleted (shSHP2) B16F10 tumors compared to untreated controls: p-LKO untreated (n=5), p-LKO treated with SHP099 (n=9), shSHP2 untreated (n=5), and shSHP2 treated with SHP099 (n=13). P values by analysis of variance with Dunnett's multiple comparison test; ***P<0.001. (F) Persistent depletion of SHP2 in tumors induced by shSHP2-B16F10 cells (n=5) compared to control tumors induced by p-LKO-B16F10 (n=5); tumors removed from mice 18 days after cell inoculation.


( N ) Representative popliteal lymph nodes from control and SHP099 - treated mice with microscopic evidence of metastases ; H & E stain ; scale bars 500 μm ( low magnification ) 20 μm ( high magnification ) .

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

(N) Representative popliteal lymph nodes from control and SHP099-treated mice with microscopic evidence of metastases; H&E stain; scale bars 500 μm (low magnification) 20 μm (high magnification).


( C , D ) p - TIE2 in tumor vessels from control and AMG386 - treated mice ; representative confocal images ; arrowheads point to CD31 + / p - TIE2 - endothelial cells ( scale bars : 50μm ) ( C ) , and quantification ( D ) of p - TIE - 2 / CD31 + in control ( n = 3 ) and AMG386 treated ( n = 3 ) tumors ( 1 , 200 CD31 + cells / group ) . P values from two - tailed Student ' s t - test ; * * * P < 0 . 001 . ( E , F ) p - EphrinB ( E ) in representative tumor vessels from control ( n = 3 ) and AMG386 - treated ( n = 3 ) tumors ; confocal images ( E ) , ( scale bar : 50μm ) and quantification of p - EphrinB / CD31 + ( F ) ; P values from two - tailed Student ' s t - test ; * * * P < 0 . 001 .

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 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 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

(C,D) p-TIE2 in tumor vessels from control and AMG386-treated mice; representative confocal images; arrowheads point to CD31+/p-TIE2- endothelial cells (scale bars: 50μm) (C), and quantification (D) of p-TIE-2/CD31+ in control (n=3) and AMG386 treated (n=3) tumors (1,200 CD31+ cells/group). P values from two-tailed Student's t-test; ***P<0.001. (E,F) p-EphrinB (E) in representative tumor vessels from control (n=3) and AMG386-treated (n=3) tumors; confocal images (E), (scale bar: 50μm) and quantification of p-EphrinB/CD31+ (F); P values from two-tailed Student's t-test; ***P<0.001.


( G ) Vessel perfusion visualized by FITC - dextran ( green ) ; CD31 ( red ) identifies the endothelium ; DAPI ( blue ) identifies the nuclei ; representative tumor confocal images ( scale bar : 50μm ) of 3 mice / group ; arrow heads point to FITC - dextran extravasation ; asterisks to extravasated erythrocytes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Vessel perfusion visualized by FITC-dextran (green); CD31 (red) identifies the endothelium; DAPI (blue) identifies the nuclei; representative tumor confocal images (scale bar:50μm) of 3 mice/group; arrow heads point to FITC-dextran extravasation; asterisks to extravasated erythrocytes.


( H ) Quantification of cleaved caspase - 3 in the tumor vasculature ( CD31 + cells ) ; n = 3 tumors / group ; number of CD31 + cells counted : control ( n = 311 ) ; SHP099 ( n = 425 ) ; AMG386 ( n = 451 ) ; combined SHP099 + AMG386 ( n = 484 ) . Error bars : ± S . D . ; P values by analysis of variance with Dunnett ' s multiple comparison test ; * * P < 0 . 01 , * * * P < 0 . 001 .

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

(H) Quantification of cleaved caspase-3 in the tumor vasculature (CD31+ cells); n=3 tumors/group; number of CD31+ cells counted: control (n=311); SHP099 (n=425); AMG386 (n=451); combined SHP099+AMG386 (n=484). Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.


( A - D ) Nuclear p - STAT3 ( A , B ) and p - ERK1 / 2 ( C , D ) in tumor CD31 + endothelial cells of mice treated for 14 days with SHP099 ( 200 mg / kg ) , AMG386 ( 5 . 6mg / kg ) or the combination of SHP099 ( 200 mg / kg ) + AMG386 ( 5 . 6mg / kg ) . Representative ( 3 tumors / group ) immunostaining ( A , C ) and quantification of staining intensity / CD31 + cell ( B , D ) ; horizontal line : mean intensity / group ; n = 3 tumors / group ; number of CD31 + cells counted in B : control ( n = 180 ) , SHP099 ( n = 308 ) , AMG386 ( n = 300 ) , combined SHP099 + AMG386 ( n = 322 ) ; number of CD31 + cells counted in D : control ( n = 1 , 733 ) , SHP099 ( n = 821 ) , AMG386 ( n = 1 , 399 ) , combined SHP099 + AMG386 ( n = 783 ) . Scale bars : A , 50μm ; C , 10μm .

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

(A-D) Nuclear p-STAT3 (A,B) and p-ERK1/2 (C,D) in tumor CD31+ endothelial cells of mice treated for 14 days with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) immunostaining (A,C) and quantification of staining intensity/CD31+ cell (B, D); horizontal line: mean intensity/group; n=3 tumors/group; number of CD31+ cells counted in B: control (n=180), SHP099 (n=308), AMG386 (n=300), combined SHP099+AMG386 (n=322); number of CD31+ cells counted in D: control (n=1,733), SHP099 (n=821), AMG386 (n=1,399), combined SHP099+AMG386 (n=783). Scale bars: A, 50μm; C, 10μm.


FOXO1 detection in tumor CD31 + endothelial cells after treatment with SHP099 ( 200 mg / kg ) , AMG386 ( 5 . 6mg / kg ) or the combination of SHP099 ( 200 mg / kg ) + AMG386 ( 5 . 6mg / kg ) . Representative ( 3 tumors / group ) confocal

O O O O O 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 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

FOXO1 detection in tumor CD31+ endothelial cells after treatment with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) confocal images


( C ) Vessel perfusion visualized by FITC - dextran ( green ) ; CD31 identifies endothelial cells ( red ) ; DAPI ( blue ) identifies the nuclei ; representative ( of 3 mice / group ) confocal images ( scale bar : 50μm ) ; arrow heads point to FITC - dextran extravasation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Vessel perfusion visualized by FITC-dextran (green); CD31 identifies endothelial cells (red); DAPI (blue) identifies the nuclei; representative (of 3 mice/group) confocal images (scale bar: 50μm); arrow heads point to FITC-dextran extravasation.


( H ) Representative images depicting Ki67 + cells in control and treated MC38 tumors ; representative images ( scale bar : 50μm ) of 4 tumors / 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

(H) Representative images depicting Ki67+ cells in control and treated MC38 tumors; representative images (scale bar: 50μm) of 4 tumors/group.


d ) representative images of Ki67 and p16Ink4a immunofluorescence and SA - β - Gal staining ; e ) % of Ki67 positive cells ( 28 days after co - culture ) ; f ) % of p16INK4A positive cells ( 28days after co - culture ) ; g ) % of SA - β - Gal positive cells ( 28 days after co - culture ) ; h ) Western blotting for p21 and quantification ; i ) MitoSOX fluorescence ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Ki67 and p16Ink4a immunofluorescence and SA-β-Gal staining; e) % of Ki67 positive cells (28 days after co-culture); f) % of p16INK4A positive cells (28days after co-culture); g) % of SA-β-Gal positive cells (28 days after co-culture); h) Western blotting for p21 and quantification; i) MitoSOX fluorescence;


h ) Representative Immuno - FISH micrographs ( using telomere ‐ specific ( CCCTAA ) peptide nucleic acid probe and anti - 53BP1 antibody ) 20 days following co - culture ; i ) quantification of mean TAF and % TAF in MRC5 fibroblasts 8 and 20 days following neutrophil co - culture . Data are mean ± S . E . M . of 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

h) Representative Immuno-FISH micrographs (using telomere‐specific (CCCTAA) peptide nucleic acid probe and anti-53BP1 antibody) 20 days following co-culture; i) quantification of mean TAF and % TAF in MRC5 fibroblasts 8 and 20 days following neutrophil co-culture. Data are mean±S.E.M. of 3 independent experiments;


J - L ) mRNA expression of p21 ( j ) , IL - 6 ( k ) and IL - 8 ( l ) 20 days after neutrophil co - culture ; Data are mean ± S . E . M . of 2 - 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

J-L) mRNA expression of p21 (j), IL-6 (k) and IL-8 (l) 20 days after neutrophil co-culture; Data are mean±S.E.M. of 2-3 independent experiments.


f ) mean number of TAF in hepatocytes ; g ) % of TAF positive hepatocytes ; h ) number of p21 positive hepatocytes per field ; i ) number of p16Ink4a positive hepatocytes per field .

O O O O O O O O O O O O O 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) mean number of TAF in hepatocytes; g) % of TAF positive hepatocytes; h) number of p21 positive hepatocytes per field; i) number of p16Ink4a positive hepatocytes per field.


e ) Representative micrographs of neutrophil marker Ly6G and senescent - associated marker p21 ( 96h ) ; f ) number of Ly6G positive cells per field ( 96h ) ; g ) % of TAF positive hepatocytes ( 96h ) ; h ) mean number of TAF in hepatocytes ( 96h ) ; i ) total number of γH2A . X foci in hepatocytes ( 96h ) ; j ) Lactate dehydrogenase ( LD ) activity ( 96h ) ; k ) p21 positive cells per field ( 96h ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative micrographs of neutrophil marker Ly6G and senescent-associated marker p21 (96h); f) number of Ly6G positive cells per field (96h); g) % of TAF positive hepatocytes (96h); h) mean number of TAF in hepatocytes (96h); i) total number of γH2A.X foci in hepatocytes (96h); j) Lactate dehydrogenase (LD) activity (96h); k) p21 positive cells per field (96h).


m ) Representative immunoFISH ( Telomere - FISH and 8 - oxodG ) ; n ) mean number of 8 - oxodG co - localizing with telomeres in hepatocytes ; o ) Mean telomere FISH intensity in hepatocytes .

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

m) Representative immunoFISH (Telomere-FISH and 8-oxodG); n) mean number of 8-oxodG co-localizing with telomeres in hepatocytes; o) Mean telomere FISH intensity in hepatocytes.


a ) RNA - sequencing data showing expression of genes associated with neutrophil recruitment during liver aging ;

a) RNA-sequencing data showing expression of genes associated with neutrophil recruitment during liver aging;


i ) Comparison between p16Ink4a mRNA levels of young ( 3 months old ) and old INK - ATTAC mice ( 28 - 29 month old ) treated with vehicle or AP20187 . Data are mean ± SEM of n = 5 - 8 mice per age group ; j ) % of TAF positive hepatocytes in old INK - ATTAC mice ( 28 - 29 months old ) treated with vehicle or AP20187 . Data are mean ± SEM of n = 5 - 6 mice per group ; k ) Liver neutrophils ( Ly6G ) in old INK - ATTAC mice ( 28 - 29 months old ) treated with vehicle or AP20187 . Data are mean ± SEM of n = 5 - 7 mice per 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 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 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

i) Comparison between p16Ink4a mRNA levels of young (3 months old) and old INK-ATTAC mice (28-29 month old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-8 mice per age group; j) % of TAF positive hepatocytes in old INK-ATTAC mice (28-29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-6 mice per group; k) Liver neutrophils (Ly6G) in old INK-ATTAC mice (28-29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-7 mice per group;


p ) Quantification of liver neutrophils ( Ly6G ) in mice injected with vehicle or Cxcl1 . Data are mean ± SEM of n = 7 - 10 mice per group ; q ) Quantification of the % of TAF positive hepatocytes in mice injected with vehicle or Cxcl1 . Data are mean ± SEM of n = 6 - 7 mice per 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 O O O O O O O O O O O O O O O O O O O O O O

p) Quantification of liver neutrophils (Ly6G) in mice injected with vehicle or Cxcl1. Data are mean ± SEM of n = 7-10 mice per group ; q) Quantification of the % of TAF positive hepatocytes in mice injected with vehicle or Cxcl1. Data are mean ± SEM of n = 6-7 mice per group;


Primary BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with

Primary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SM


Primary BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with TAK1i for 6 or 4 h 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

Primary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with TAK1i for 6 or 4 h respectively.


Time - lapse confocal images ( hour : min ) of BMDMs stimulated with recombinant murine TNF ( 100 ng / ml ) and SM ( 250 nM ) stained with propidium iodide ( red ) for 6 h . Black arrowheads indicate membrane ballooning while white arrowheads indicate apoptotic bodies .

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

Time-lapse confocal images (hour:min) of BMDMs stimulated with recombinant murine TNF (100 ng/ml) and SM (250 nM) stained with propidium iodide (red) for 6 h. Black arrowheads indicate membrane ballooning while white arrowheads indicate apoptotic bodies.


immortalized BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with

immortalized BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SM


immortalized BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with TAK1i for 6 or 4 h respectively .

immortalized BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with TAK1i for 6 or 4 h respectively.


BMDMs were stimulated with TNF ( 100 ng / ml ) in combination with TAK1i ( 125 nM ) for the indicated time points . LDH

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

BMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. LDH release


BMDMs were stimulated with TNF ( 100 ng / ml ) in combination with TAK1i ( 125 nM ) for the indicated time points . mixed supernatant and cell extracts were analysed .

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

BMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. mixed supernatant and cell extracts were analysed.


BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h in the presence or absence of KCl ( 50 mM ) . Where indicated , cells were pre - incubated with MCC950 ( 10 μM ) 20 - 30 min prior to TNF / TAK1i stimulation .

O O 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

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h in the presence or absence of KCl (50 mM). Where indicated, cells were pre-incubated with MCC950 (10 μM) 20-30 min prior to TNF/TAK1i stimulation.


BMDMs were costimulated with TNF ( 100 ng / ml ) and SM ( 250 nM ; 6 h ) and mixed supernatant and cell extracts were analysed by

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

BMDMs were costimulated with TNF (100 ng/ml) and SM (250 nM; 6 h) and mixed supernatant and cell extracts were analysed by immunoblot


BMDMs were costimulated with TNF ( 100 ng / ml ) and SM LDH release in the cell culture supernatant were quantified at the indicated time points .

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

BMDMs were costimulated with TNF (100 ng/ml) and SM LDH release in the cell culture supernatant were quantified at the indicated time points.


BMDMs were costimulated with TNF ( 100 ng / ml ) and

BMDMs were costimulated with TNF (100 ng/ml) and SM


HEK 293T cells were transfected with doxycycline - inducible DmrB - caspase - 8 and the indicated GSDMD constructs . Cells were stimulated with doxycycline ( 10 μg / ml ) for 18 h to induce DmrB - caspase - 8 expression and exposed to B / B homodimerizer ( 12 . 5 nM ) for another 2 h to activate caspase - 8 . Mixed supernatant and extracts were analysed by immunoblot .

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

HEK 293T cells were transfected with doxycycline-inducible DmrB-caspase-8 and the indicated GSDMD constructs. Cells were stimulated with doxycycline (10 μg/ml) for 18 h to induce DmrB-caspase-8 expression and exposed to B/B homodimerizer (12.5 nM) for another 2 h to activate caspase-8. Mixed supernatant and extracts were analysed by immunoblot.


Immortalized Gsdmd - / - BDMDs expressing GSDMDWT and GSDMDD88A were costimulated with TNF ( 100 ng / ml ) and SM for 6 h and LDH release was quantified .

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

Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were costimulated with TNF (100 ng/ml) and SM for 6 h and LDH release was quantified.


Immortalized Gsdmd - / - BDMDs expressing GSDMDWT and GSDMDD88A were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) and stimulated with LCL161 ( 1 μM ) for 24 h and LDH release was quantified .

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

Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with LCL161 (1 μM) for 24 h and LDH release was quantified.


BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i for 4 h and ( D ) mixed supernatant and extracts were analysed by

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

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h and (D) mixed supernatant and extracts were analysed by immunoblot


BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i for 4 h and LDH release was quantified at the indicated time points .

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h and LDH release was quantified at the indicated time points.


BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h and LDH release was quantified Where indicated , cells were treated with the inhibitors Nec - 1s ( 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and LDH release was quantified Where indicated, cells were treated with the inhibitors Nec-1s (50 μM)


BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h and LDH release was

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

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and LDH release was quantified


BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h and mixed supernatant and extracts were analysed by immunoblot . Where indicated , cells were treated with the inhibitors MCC950 ( 10 μM ) , GSK ' 872 ( 1 μM ) , 20 - 30 min prior to cell stimulation . KCl ( 50 mM ) was added together with TNF and TAK1i . To induce necroptosis , BMDMs were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) and Q - VD - OPh ( 10 μM ) was added at the last 20 - 30 min of priming and stimulated with SM ( 500 nM ) for 4 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 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 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

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors MCC950 (10 μM), GSK'872 (1 μM), 20-30 min prior to cell stimulation. KCl (50 mM) was added together with TNF and TAK1i. To induce necroptosis, BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and Q-VD-OPh (10 μM) was added at the last 20-30 min of priming and stimulated with SM (500 nM) for 4 h.


BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h and mixed supernatant and extracts were analysed by immunoblot . Where indicated , cells were treated with the inhibitors GSK ' 872 ( 1 μM ) , 20 - 30 min prior to cell stimulation .

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

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors GSK'872 (1 μM), 20-30 min prior to cell stimulation.


BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h and mixed supernatant and extracts were analysed by immunoblot . Where indicated , cells were treated with the inhibitors probenecid ( 1 mM ) 20 - 30 min prior to cell stimulation . To activate the NLRP3 inflammasome , BMDM were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 4 h and stimulated with nigericin ( 10 μM ) for 1 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 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 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

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors probenecid (1 mM) 20-30 min prior to cell stimulation. To activate the NLRP3 inflammasome, BMDM were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 4 h and stimulated with nigericin (10 μM) for 1 h.


BMDMs were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) and stimulated with SM ( 0 . 5 μM ) for a further 4 h . Probenecid ( 1 mM ) was added 20 - 30 min prior to cell stimulation and mixed supernatant and extracts were analysed by immunoblot .

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

BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with SM (0.5 μM) for a further 4 h. Probenecid (1 mM) was added 20-30 min prior to cell stimulation and mixed supernatant and extracts were analysed by immunoblot.


BMDMs were stimulated with TNF ( 100 ng / ml ) and SM ( 0 . 5 μM ) for 6 h and ( G ) mixed supernatant and extracts were analysed by

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

BMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h and (G) mixed supernatant and extracts were analysed by immunoblot


BMDMs were stimulated with TNF ( 100 ng / ml ) and SM ( 0 . 5 μM ) for 6 h and LDH release was quantified .

BMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h and LDH release was quantified.


BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT - 737 ( 0 . 5 μM ) and LDH release was quantified at 6 h .

O O O O O O O O B-CONTROLLED_VAR 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

BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT-737 (0.5 μM) and LDH release was quantified at 6 h.


BMDMs were stimulated with ABT - 737 ( 0 . 5 μM ) and S63845 ( 0 . 5 μM ) and LDH release was

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 O

BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and LDH release was quantified


BMDMs were stimulated with ABT - 737 ( 0 . 5 μM ) and S63845 ( 0 . 5 μM ) mixed supernatant and extracts were analysed by immunoblot at 6

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 O O O O O O O

BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) mixed supernatant and extracts were analysed by immunoblot at 6 h


BMDMs were stimulated with ABT - 737 ( 0 . 5 μM ) and S63845 ( 0 . 5 μM ) and mixed supernatant and extracts were analysed by immunoblot at 6

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 O O O O O O O O

BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and mixed supernatant and extracts were analysed by immunoblot at 6 h


BMDMs were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 3 h and further stimulated with ABT - 737 ( 1 μM ) and S63845 ( 1 μM ) for 24 h and mixed supernatant and extracts were analysed by

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

BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and mixed supernatant and extracts were analysed by immunoblot


BMDMs were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 3 h and further stimulated with ABT - 737 ( 1 μM ) and S63845 ( 1 μM ) for 24 h and IL - 1β in cell - free supernatant were quantified by

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

BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and IL-1β in cell-free supernatant were quantified by ELISA


A , B MAGE - A3 / 6 protein levels decrease upon EBSS treatment of HeLa cells for the indicated times . Note both bands are MAGE - A3 / 6 . Quantitation represents average of n = 3 with standard deviation 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

A, B MAGE-A3/6 protein levels decrease upon EBSS treatment of HeLa cells for the indicated times. Note both bands are MAGE-A3/6. Quantitation represents average of n=3 with standard deviation shown.


C Quantitative TMT proteomics of A375 cells grown in complete media ( CM ) or EBSS for 3 hrs . Number of proteins affected are shown . Autophagy related proteins are indicated in grey and MAGE - A proteins in blue .

O O O O O O O O O O O O O O O O O O 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 Quantitative TMT proteomics of A375 cells grown in complete media (CM) or EBSS for 3 hrs. Number of proteins affected are shown. Autophagy related proteins are indicated in grey and MAGE-A proteins in blue.


D , E MAGE - A3 / 6 protein levels are actively degraded upon nutrient deprivation . HeLa cells were treated for the indicated times with complete media ( DMEM + 10 % FBS ) or EBSS containing 100 µg / mL translation inhibitor cycloheximide ( CHX ) . Protein degradation rates are shown ( E ) . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 . ns indicates p > 0 . 05 using 2 - way ANOVA .

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D, E MAGE-A3/6 protein levels are actively degraded upon nutrient deprivation. HeLa cells were treated for the indicated times with complete media (DMEM + 10% FBS) or EBSS containing 100 µg/mL translation inhibitor cycloheximide (CHX). Protein degradation rates are shown (E). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01. ns indicates p>0.05 using 2-way ANOVA.


FBS ( 10 % ) , but not non - essential amino acids ( NEAA ; 10 mM ) or glucose ( 4 . 5 g / L ) , rescues MAGE - A3 / 6 protein levels upon EBSS treatment . HeLa cells were treated with EBSS and the indicated supplements for the indicated times . Quantitation represents average of n = 3 with standard deviation shown .

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

FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown.


FBS ( 10 % ) , but not non - essential amino acids ( NEAA ; 10 mM ) or glucose ( 4 . 5 g / L ) , rescues MAGE - A3 / 6 protein levels upon EBSS treatment . HeLa cells were treated with EBSS and the indicated supplements for the indicated times . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA .

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

FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.


C Removal of FBS from normal growth conditions ( DMEM + 10 % FBS ) results in MAGE - A3 / 6 protein degradation in HeLa cells .

O O O O 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 Removal of FBS from normal growth conditions (DMEM+10% FBS) results in MAGE-A3/6 protein degradation in HeLa cells.


D , E MAGE - A3 / 6 protein stability is controlled by a < 3 kDa , heat stable , lipophilic molecule present in FBS . HeLa cells were treated with EBSS alone , or EBSS containing 10 % complete FBS , 10 % charcoal - dextran stripped FBS , 10 % boiled FBS , 10 % dialyzed > 3 kDa FBS , or 10 % dialyzed < 3 kDa FBS for the indicated times . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E MAGE-A3/6 protein stability is controlled by a <3 kDa, heat stable, lipophilic molecule present in FBS. HeLa cells were treated with EBSS alone, or EBSS containing 10% complete FBS, 10% charcoal-dextran stripped FBS, 10% boiled FBS, 10% dialyzed >3 kDa FBS, or 10% dialyzed <3 kDa FBS for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.


F MAGE - A3 / 6 protein instability upon nutrient deprivation of HeLa cells is not altered by EGF 0 . 2 ng / mL , testosterone , or bovine pituitary extract 25 µg / mL .

O O 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

F MAGE-A3/6 protein instability upon nutrient deprivation of HeLa cells is not altered by EGF 0.2 ng/mL, testosterone, or bovine pituitary extract 25 µg/mL.


Insulin partially rescues MAGE - A3 / 6 protein levels upon nutrient deprivation . HeLa cells were treated for the indicated times with EBSS alone or EBSS containing NEAA ( 10 mM ) or insulin ( 10 µg / mL ) . Quantitation represents average of n = 3 with standard deviation shown .

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

Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing NEAA (10 mM) or insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown.


, H Insulin partially rescues MAGE - A3 / 6 protein levels upon nutrient deprivation . HeLa cells were treated for the indicated times with EBSS alone or EBSS containing insulin ( 10 µg / mL ) . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA .

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

, H Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.


A MAGE - A3 / 6 mRNA levels do not change upon nutrient deprivation . RNA was isolated from HeLa cells treated with EBSS for the indicated times . RT - QPCR analysis was performed to detect both MAGE - A3 / 6 with a common primer set . Data was normalized to 18S rRNA levels . Quantitation represents average of n = 3 with standard deviation 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

A MAGE-A3/6 mRNA levels do not change upon nutrient deprivation. RNA was isolated from HeLa cells treated with EBSS for the indicated times. RT-QPCR analysis was performed to detect both MAGE-A3/6 with a common primer set. Data was normalized to 18S rRNA levels. Quantitation represents average of n=3 with standard deviation shown.


B , Proteasome inhibition rescues MAGE - A3 / 6 protein levels upon nutrient deprivation . HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 ( BafA1 , 50 nM ) or proteasome inhibitor MG132 ( 10 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O

B, Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM).


C Proteasome inhibition rescues MAGE - A3 / 6 protein levels upon nutrient deprivation . HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 ( BafA1 , 50 nM ) or proteasome inhibitor MG132 ( 10 µM ) . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA .

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

C Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.


D Nutrient deprivation increases ubiquitination of endogenous MAGE - A3 / 6 . HeLa cells were treated with MG132 ( 10 µM ) in complete media or EBSS for 6 hrs . Cell extracts were incubated with control agarose or agarose - TUBE2 to isolate ubiquitinated proteins . Input and pulldown samples were probed for the indicated proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Nutrient deprivation increases ubiquitination of endogenous MAGE-A3/6. HeLa cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.


E MAGE - A3 / 6 protein instability upon nutrient deprivation is not rescued by knockdown of its associated E3 ubiquitin ligase , TRIM28 . HeLa cells were treated with control or TRIM28 siRNAs for 6 days before incubation in EBSS for the indicated times .

O O O O O O O O O O O O O O O O O O O O O O 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 MAGE-A3/6 protein instability upon nutrient deprivation is not rescued by knockdown of its associated E3 ubiquitin ligase, TRIM28. HeLa cells were treated with control or TRIM28 siRNAs for 6 days before incubation in EBSS for the indicated times.


F , MAGE - A3 / 6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase . HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 ( 1 µM ) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times .

O O O O O O O O O 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

F, MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times.


G MAGE - A3 / 6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase . HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 ( 1 µM ) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA .

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

G MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.


H Degradation of MAGE - A3 / 6 upon nutrient deprivation is dependent on a Cul4 E3 ubiquitin ligase complex . HeLa cells were transfected with vector control or the indicated dominant negative ( D / N ) Cullin constructs for 48 hrs before harvesting cells after 0 or 4 hrs EBSS treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on a Cul4 E3 ubiquitin ligase complex. HeLa cells were transfected with vector control or the indicated dominant negative (D/N) Cullin constructs for 48 hrs before harvesting cells after 0 or 4 hrs EBSS treatment.


I Degradation of MAGE - A3 / 6 upon nutrient deprivation is dependent on Cul4A / B . HeLa cells were transfected with control siRNAs or siRNAs targeting both Cul4A and Cul4B . 72 hrs after treatment , cells were incubated for the indicated times with EBSS before harvesting and immunoblotting . Note , time 0 samples between two siRNA groups were normalized to more readily discern differences in MAGE - A3 / 6 degradation upon nutrient deprivation .

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I Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on Cul4A/B. HeLa cells were transfected with control siRNAs or siRNAs targeting both Cul4A and Cul4B. 72 hrs after treatment, cells were incubated for the indicated times with EBSS before harvesting and immunoblotting. Note, time 0 samples between two siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation.


A Tandem affinity purification of MAGE - A3 from HEK293 cells identified DDB1 , DCAF5 , and DCAF12 as binding partners . Mascot score , number of peptides identified , and percent coverage is shown .

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A Tandem affinity purification of MAGE-A3 from HEK293 cells identified DDB1, DCAF5, and DCAF12 as binding partners. Mascot score, number of peptides identified, and percent coverage is shown.


B MAGE - A3 interacts with DCAF12 , but not DCAF5 . HeLa cells were transfected with the indicated constructs ( Myc - MAGE - A3 and HA - DCAF5 or HA - DCAF12 ) . Two days later cells were treated with complete media or EBSS for 4 hrs . Cells were treated in the presence of 1 µM MLN4924 to stabilize MAGE - A3 . Cell extracts were subjected to anti - Myc IP .

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B MAGE-A3 interacts with DCAF12, but not DCAF5. HeLa cells were transfected with the indicated constructs (Myc-MAGE-A3 and HA-DCAF5 or HA-DCAF12). Two days later cells were treated with complete media or EBSS for 4 hrs. Cells were treated in the presence of 1 µM MLN4924 to stabilize MAGE-A3. Cell extracts were subjected to anti-Myc IP.


C Nutrient deprivation increases endogenous DCAF12 binding to MAGE - A3 . HeLa cells were transfect with Myc - vector or Myc - MAGE - A3 for 48 hrs . Cells were then treated with 10 µM MG132 in complete media , EBSS , or EBSS containing 20 µg / mL insulin for 6 hrs . Cell extracts were subjected to anti - Myc IP .

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C Nutrient deprivation increases endogenous DCAF12 binding to MAGE-A3. HeLa cells were transfect with Myc-vector or Myc-MAGE-A3 for 48 hrs. Cells were then treated with 10 µM MG132 in complete media, EBSS, or EBSS containing 20 µg/mL insulin for 6 hrs. Cell extracts were subjected to anti-Myc IP.


Knockdown of DCAF12 , but not DCAF5 , altered MAGE - A3 / 6 protein levels and partially rescued its instability upon nutrient deprivation . HeLa cells were treated with control , DCAF5 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 ( 1 µM ) .

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Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control, DCAF5 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM).


E Knockdown of DCAF12 , but not DCAF5 , altered MAGE - A3 / 6 protein levels and partially rescued its instability upon nutrient deprivation . HeLa cells were treated with control or DCAF12 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 ( 1 µM ) .

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E Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control or DCAF12 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM).


F Knockdown of DCAF12 with individual siRNAs blocks MAGE - A3 / 6 degradation upon nutrient deprivation . HeLa cells were treated with the indicated siRNAs for 72 hrs before incubation in EBSS for the indicated times . Note , time 0 samples between siRNA groups were normalized to more readily discern differences in MAGE - A3 / 6 degradation upon nutrient deprivation .

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F Knockdown of DCAF12 with individual siRNAs blocks MAGE-A3/6 degradation upon nutrient deprivation. HeLa cells were treated with the indicated siRNAs for 72 hrs before incubation in EBSS for the indicated times. Note, time 0 samples between siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation.


G Ubiquitination of MAGE - A3 / 6 upon nutrient deprivation is dependent on DCAF12 . A375 parental or DCAF12 KO cells were treated with MG132 ( 10 µM ) in complete media or EBSS for 6 hrs . Cell extracts were incubated with control agarose or agarose - TUBE2 to isolate ubiquitinated proteins . Input and pulldown samples were probed for the indicated proteins .

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G Ubiquitination of MAGE-A3/6 upon nutrient deprivation is dependent on DCAF12. A375 parental or DCAF12 KO cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.


A Identification of DCAF12 substrates . Control ( WT ) or DCAF12 knockout ( KO ) A375 cells were subjected to quantitative TMT proteomics to identify potential DCAF12 targets . MAGE - A proteins ( shown in blue ) are stabilized in DCAF12 knockout cells .

A Identification of DCAF12 substrates. Control (WT) or DCAF12 knockout (KO) A375 cells were subjected to quantitative TMT proteomics to identify potential DCAF12 targets. MAGE-A proteins (shown in blue) are stabilized in DCAF12 knockout cells.


B Knockout of DCAF12 rescues degradation of MAGE - A proteins in A375 cells . DCAF12 KO A375 cells were treated with complete media or EBSS before quantitative TMT proteomics . Note MAGE - A genes are not significantly altered by EBSS in DCAF12 KO cells .

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B Knockout of DCAF12 rescues degradation of MAGE-A proteins in A375 cells. DCAF12 KO A375 cells were treated with complete media or EBSS before quantitative TMT proteomics. Note MAGE-A genes are not significantly altered by EBSS in DCAF12 KO cells.


C DCAF12 target proteins are differentially affected by EBSS compared to remainder of the proteome . DCAF12 targets ( n = 33 ) identified show significantly greater decrease upon EBSS treatment compared to non - DCAF12 altered genes ( n = 8243 ) . Average with standard deviation is shown with statistical analysis by student t - test . Asterisks * * * indicate p < 0 . 001 ( student t - test ) .

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C DCAF12 target proteins are differentially affected by EBSS compared to remainder of the proteome. DCAF12 targets (n=33) identified show significantly greater decrease upon EBSS treatment compared to non-DCAF12 altered genes (n=8243). Average with standard deviation is shown with statistical analysis by student t-test. Asterisks *** indicate p<0.001 (student t-test).


E Overexpression of DCAF12 decreases wild - type MAGE - A3 , but not MAGE - A3 degron mutant . The indicated constructs were expressed for 48 hrs in HeLa cells before immunoblotting . The MAGE - A3 degron mutant was created ] , in which DNYNEPKANQ * were added to the extreme C - term to obscure the DCAF12 degron motif .

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E Overexpression of DCAF12 decreases wild-type MAGE-A3, but not MAGE-A3 degron mutant. The indicated constructs were expressed for 48 hrs in HeLa cells before immunoblotting. The MAGE-A3 degron mutant was created ], in which DNYNEPKANQ* were added to the extreme C-term to obscure the DCAF12 degron motif.


F Analysis of proteomics indicates that MAGE - A1 , MAGE - A4 , MAGE - A10 , and MAGE - A11 are not regulated by nutrient deprivation or DCAF12 . Average ( n = 3 ) with standard deviation is shown with statistical analysis by student t - test . Asterisks * * * indicate p < 0 . 001 ( student t - test ) .

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F Analysis of proteomics indicates that MAGE-A1, MAGE-A4, MAGE-A10, and MAGE-A11 are not regulated by nutrient deprivation or DCAF12. Average (n=3) with standard deviation is shown with statistical analysis by student t-test. Asterisks *** indicate p<0.001 (student t-test).


G MAGE - A10 and MAGE - A11 are not altered upon nutrient deprivation or DCAF12 deletion . A375 parental or DCAF12 KO cells were treated with EBSS for the indicated times . Cell lysates were probed for the indicated proteins .

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G MAGE-A10 and MAGE-A11 are not altered upon nutrient deprivation or DCAF12 deletion. A375 parental or DCAF12 KO cells were treated with EBSS for the indicated times. Cell lysates were probed for the indicated proteins.


A - B U2OS GFP - LC3 cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs . Cells were then fixed , imaged , and the number of GFP - LC3 puncta were quantitated per cell . Representative images ( A ) and quantitation of > 50 cells from three independent experiments ( B ) are shown . Scale bar indicates 50 µm . Data information : Asterisks indicate * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using student t - test .

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A-B U2OS GFP-LC3 cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, imaged, and the number of GFP-LC3 puncta were quantitated per cell. Representative images (A) and quantitation of >50 cells from three independent experiments (B) are shown. Scale bar indicates 50 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test.


C - D A375 parental or DCAF12 KO cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs . Cells were then fixed , endogenous LC3 was immunostained , and the number of LC3 puncta were quantitated per cell . Representative images ( C ) and quantitation of > 50 cells from three independent experiments ( D ) are shown . Scale bar indicates 20 µm . Data information : Asterisks indicate * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using student t - test .

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C-D A375 parental or DCAF12 KO cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, endogenous LC3 was immunostained, and the number of LC3 puncta were quantitated per cell. Representative images (C) and quantitation of >50 cells from three independent experiments (D) are shown. Scale bar indicates 20 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test.


E A375 parental or DCAF12 KO cells were treated Cell lysates were collected and immunoblotted for the indicated antibodies to confirm MAGE - A3 / 6 depletion and monitor the autophagy adaptor / substrate p62 / SQSTM1 .

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E A375 parental or DCAF12 KO cells were treated Cell lysates were collected and immunoblotted for the indicated antibodies to confirm MAGE-A3/6 depletion and monitor the autophagy adaptor/substrate p62/SQSTM1.


A / B . Hybrid Rab7 / Arl8b compartment . A . Representative confocal images of fixed HeLa cells expressing Arl8b - GFP ( green ) , immunolabeled against endogenous Rab7 ( eRab7 , magenta ) . Zoom insets ( 3x ) highlight select regions of colocalization ( white ) , white arrowheads point to vesicles positive for both GTPases . B . Colocalization ( Mander ' s overlap ) of endogenous Rab7 with Arl8b - GFP versus free GFP ( EV ) , n EV = 4 , n Arl8b = 9 images ( 3 ≥ cells per image ) analysed from 2 independent experiments . Significance : 2 - tailed student ' s t - test , * p < 0 . 05 .

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A/B. Hybrid Rab7/Arl8b compartment. A. Representative confocal images of fixed HeLa cells expressing Arl8b-GFP (green), immunolabeled against endogenous Rab7 (eRab7, magenta). Zoom insets (3x) highlight select regions of colocalization (white), white arrowheads point to vesicles positive for both GTPases. B. Colocalization (Mander's overlap) of endogenous Rab7 with Arl8b-GFP versus free GFP (EV), n EV=4, n Arl8b=9 images (3≥ cells per image) analysed from 2 independent experiments. Significance: 2-tailed student's t-test, * p<0.05.


C - F . Analysis of Arl8b compartment organization and dynamics as a function of Rab7 activity status . C . Left and middle panels : representative confocal images of live HeLa cells expressing mCherry - Rab7 or its mutants Q67L and T22N ( white ) , together with Arl8b - GFP ( green ) , taken at the start of time - lapse ( t0 ) . Right panels : tracks followed by Arl8b - positive null during the time - lapse lasting 50 s recorded at 0 . 5 s per frame , with highest displacement rates for each track depicted on a rainbow colour scale ( blue : immobile ; red : maximum mobility per time interval ) . Zoom insets ( 2 . 8x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions D . Plot of Arl8b - positive pixel distribution expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , numbers of ( pixels ) plotted given above each scatter , n = 7 cells analysed per condition from 2 independent experiments . E / F . Quantification of mean Arl8b vesicle displacement and maximum speed , respectively , n Rab7 = 21 , n QL = 25 , n TN = 24 images ( 3 ≥ cells per image ) analysed from 2 independent experiments .

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C-F. Analysis of Arl8b compartment organization and dynamics as a function of Rab7 activity status. C. Left and middle panels: representative confocal images of live HeLa cells expressing mCherry-Rab7 or its mutants Q67L and T22N (white), together with Arl8b-GFP (green), taken at the start of time-lapse (t0). Right panels: tracks followed by Arl8b-positive null during the time-lapse lasting 50 s recorded at 0.5 s per frame, with highest displacement rates for each track depicted on a rainbow colour scale (blue: immobile; red: maximum mobility per time interval). Zoom insets (2.8x) highlight select peripheral (PP) and perinuclear (PN) cell regions D. Plot of Arl8b-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), numbers of (pixels) plotted given above each scatter, n=7 cells analysed per condition from 2 independent experiments. E/F. Quantification of mean Arl8b vesicle displacement and maximum speed, respectively, n Rab7=21, n QL=25, n TN=24 images (3≥ cells per image) analysed from 2 independent experiments.


Effects of select transport route activation on the spatial organization of late compartments . A . Representative confocal images of fixed HeLa cells ectopically expressing Arl8b - GFP or Arl8b - Myc ( green ) , in combination with HA - RILP ( cyan ) and / or Myc - SKIP , PLEKHM1 - FLAG , or GFP - FYCO1 ( red ) , immunolabeled against endogenous Rab7 ( eRab7 , magenta ) and the indicated epitope tags . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets ( 3 . 5x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions , scale bar : 10µm . B . Colocalization ( Mander ' s overlap ) between the indicated pairs of proteins , n ≥ 3 images ( 3 ≥ cells per image ) analysed per condition from 2 independent experiments .

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Effects of select transport route activation on the spatial organization of late compartments. A. Representative confocal images of fixed HeLa cells ectopically expressing Arl8b-GFP or Arl8b-Myc (green), in combination with HA-RILP (cyan) and/or Myc-SKIP, PLEKHM1-FLAG, or GFP-FYCO1 (red), immunolabeled against endogenous Rab7 (eRab7, magenta) and the indicated epitope tags. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bar: 10µm. B. Colocalization (Mander's overlap) between the indicated pairs of proteins, n≥3 images (3≥ cells per image) analysed per condition from 2 independent experiments.


C . Plot of eRab7 - or Arl8b - GFP / Myc - Arl8b - positive pixel distribution in response to the indicated effector perturbations , expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , number of ( pixels ) plotted given above each scatter , n ≥ 6 cells per condition analysed from 2 independent experiments .

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C. Plot of eRab7- or Arl8b-GFP / Myc-Arl8b-positive pixel distribution in response to the indicated effector perturbations, expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥6 cells per condition analysed from 2 independent experiments.


D . Colocalization ( Mander ' s overlap ) between Arl8b and Rab7 as a function of the indicated effector perturbations , n ≥ 5 images ( 3 ≥ cells per image ) analysed per condition from 2 independent experiments .

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D. Colocalization (Mander's overlap) between Arl8b and Rab7 as a function of the indicated effector perturbations, n≥5 images (3≥ cells per image) analysed per condition from 2 independent experiments.


A - D . Correlative light and electron microscopy ( CLEM ) on A . untransfected ( WT ) HeLa cells harbouring endogenous CD63 labeled with GFP as compared to those ectopically expressing B . HA - RILP , C . HA - SKIP or D . both HA - RILP and HA - SKIP . Left panels : wide field fluorescence images of fixed cells showing endogenous GFP - CD63 ( green ) and SiR - lysosome - stained endosomes and / or lysosomes ( red ) . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets highlight regions selected for EM imaging . Middle panels : overview electron micrographs of perinuclear ( PN ) and peripheral ( PP ) cell regions selected for further analysis . Various endolysosomal subtypes are designated by arrowheads : MVBs ( white ) , lysosomes ( black ) , endolysosomes ( yellow ) and abnormal / enlarged endolysosomes ( red ) . Right panels : graphical representations of endosomal distribution under the indicated conditions based on ultrastructural characterization .

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A-D. Correlative light and electron microscopy (CLEM) on A. untransfected (WT) HeLa cells harbouring endogenous CD63 labeled with GFP as compared to those ectopically expressing B. HA-RILP, C. HA-SKIP or D. both HA-RILP and HA-SKIP. Left panels: wide field fluorescence images of fixed cells showing endogenous GFP-CD63 (green) and SiR-lysosome-stained endosomes and/or lysosomes (red). Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets highlight regions selected for EM imaging. Middle panels: overview electron micrographs of perinuclear (PN) and peripheral (PP) cell regions selected for further analysis. Various endolysosomal subtypes are designated by arrowheads: MVBs (white), lysosomes (black), endolysosomes (yellow) and abnormal/enlarged endolysosomes (red). Right panels: graphical representations of endosomal distribution under the indicated conditions based on ultrastructural characterization.


A . Electron micrograph of sections immunolabelled against RFP - SKIP ( 15nm gold ) and GFP - Rab7 ( 10nm gold ) . Arrowheads and zoom inset ( 1 . 75x ) highlight presence of RFP - SKIP and GFP - Rab7 on the same endosomal membrane , scale bar : 200nm .

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A. Electron micrograph of sections immunolabelled against RFP-SKIP (15nm gold) and GFP-Rab7 (10nm gold). Arrowheads and zoom inset (1.75x) highlight presence of RFP-SKIP and GFP-Rab7 on the same endosomal membrane, scale bar: 200nm.


B . Co - immunoprecipitations ( Co - IP ) of HA - RILP and RFP - SKIP with GFP - Rab7 ( R7 ) versus its mutants Q67L ( QL ) and T22N ( TN ) from HEK293T cells using GFP - trap beads . Representative immunoblots against GFP , HA and RFP are shown , EV : empty vector , IP : immunoprecipitation , TL : total lysate ( see also Fig EV1E ) . C . Quantification of interaction between SKIP and Rab7 mutants expressed as fraction Co - IP relative to wild type Rab7 , n = 3 independent experiments .

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B. Co-immunoprecipitations (Co-IP) of HA-RILP and RFP-SKIP with GFP-Rab7 (R7) versus its mutants Q67L (QL) and T22N (TN) from HEK293T cells using GFP-trap beads. Representative immunoblots against GFP, HA and RFP are shown, EV: empty vector, IP: immunoprecipitation, TL: total lysate (see also Fig EV1E). C. Quantification of interaction between SKIP and Rab7 mutants expressed as fraction Co-IP relative to wild type Rab7, n=3 independent experiments.