Alteration of CY side chain ( Cpd B ) caused loss of HDAC inhibitory activity and resulted in reduced ability to induce DNA damage . Cells were treated for 12 h . Cells treated with 200 μM Cpd B and 20 μM CY exhibited similar levels of γ - H2AX . Actin was used as a loading control .

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Alteration of CY side chain (Cpd B) caused loss of HDAC inhibitory activity and resulted in reduced ability to induce DNA damage. Cells were treated for 12 h. Cells treated with 200 μM Cpd B and 20 μM CY exhibited similar levels of γ-H2AX. Actin was used as a loading control.


Summary of lethal dose 50 ( LD50 ) of bendamustine , CY , Cpd A ( HDAC inhibition only ) , and Cpd B ( nitrogen mustard only ) against 11 human cancer cell lines .

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Summary of lethal dose 50 (LD50) of bendamustine, CY, Cpd A (HDAC inhibition only), and Cpd B (nitrogen mustard only) against 11 human cancer cell lines.


Cpd A enhances the anticancer activity of bendamustine . H1650 cells were treated with various doses of bendamustine with or without 40 μM Cpd A ( nontoxic dose , see Fig1C and Supplementary Fig S2 ) for 48 h and subjected to MTT viability assays .

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Cpd A enhances the anticancer activity of bendamustine. H1650 cells were treated with various doses of bendamustine with or without 40 μM Cpd A (nontoxic dose, see Fig1C and Supplementary Fig S2) for 48 h and subjected to MTT viability assays.


CY , but not bendamustine treatment , led to suppression of CBP , TIP60 , MORF , and MSL1 in H1650 cells . Cells were treated with DMSO , bendamustine ( 350 μM ) , CY ( 15 μM ) , or SAHA ( 10 μM ) for 6 h , and mRNA was extracted for qPCR analysis . Data represent mean ± SEM from three independent experiments , and statistical significance was determined by unpaired two - tailed t - test . * * * P < 0 . 01 .

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CY, but not bendamustine treatment, led to suppression of CBP, TIP60, MORF, and MSL1 in H1650 cells. Cells were treated with DMSO, bendamustine (350 μM), CY (15 μM), or SAHA (10 μM) for 6 h, and mRNA was extracted for qPCR analysis. Data represent mean ± SEM from three independent experiments, and statistical significance was determined by unpaired two-tailed t-test. ***P < 0.01.


Drug treatment up to 10 h at the concentrations indicated in ( A ) did not cause apoptosis as judged by PARP cleavage . Actin was used as a loading control .

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Drug treatment up to 10 h at the concentrations indicated in (A) did not cause apoptosis as judged by PARP cleavage. Actin was used as a loading control.


ShRNA - mediated suppression of CBP , TIP60 , MORF , and MSL1 at mRNA level . Data represent mean ± SEM from three independent experiments , and statistical significance was determined by unpaired two - tailed t - test . * P < 0 . 1 , * * P < 0 . 05 , * * * P < 0 . 01 .

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ShRNA-mediated suppression of CBP, TIP60, MORF, and MSL1 at mRNA level. Data represent mean ± SEM from three independent experiments, and statistical significance was determined by unpaired two-tailed t-test. *P < 0.1, **P < 0.05, ***P < 0.01.


Suppression of CBP , TIP60 , MORF , and MSL1 sensitized cells to bendamustine . Y - axis represents relative resistance calculated from results of GFP competition assays , and statistical significance was determined by unpaired two - tailed t - test . Data represent mean ± SEM from two independent experiments . * * P < 0 . 05 , * * * P < 0 . 01 .

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Suppression of CBP, TIP60, MORF, and MSL1 sensitized cells to bendamustine. Y-axis represents relative resistance calculated from results of GFP competition assays, and statistical significance was determined by unpaired two-tailed t-test. Data represent mean ± SEM from two independent experiments. **P < 0.05, ***P < 0.01.


A Determination of maximally tolerated dose ( MTD ) of CY at 60 mg / kg . Other drugs were used at MTD according to the literature . For each drug , n = 5 and all mice survived treatment . Data represent mean ± SEM .

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A Determination of maximally tolerated dose (MTD) of CY at 60 mg/kg. Other drugs were used at MTD according to the literature. For each drug, n = 5 and all mice survived treatment. Data represent mean ± SEM.


B CY exhibited enhanced activity against BCR - ABL Arf − / − murine ALL cells in vitro .

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B CY exhibited enhanced activity against BCR-ABL Arf−/− murine ALL cells in vitro.


C , D CY showed superior antitumor activity compared with bendamustine , SAHA , and other chemotherapeutic drugs in mice transplanted with BCR - ABL ALL cells . P - values of CY versus other drugs : NT ( P = 5 . 02E - 06 ) ( C ) , SAHA ( P = 4 . 45E - 06 ) , Bend ( P = 5 . 18E - 06 ) , NT ( P = 1 . 20E - 05 ) ( D ) , VCR ( P = 1 . 13E - 05 ) , Dox ( P = 8 . 08E - 06 ) , AraC ( P = 7 . 62E - 06 ) , and CTX ( P = 1 . 13E - 05 ) . Survival statistical analysis was done with the Mantel - Cox ( log - rank ) test of GraphPad Prism .

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C, D CY showed superior antitumor activity compared with bendamustine, SAHA, and other chemotherapeutic drugs in mice transplanted with BCR-ABL ALL cells. P-values of CY versus other drugs: NT (P = 5.02E-06) (C), SAHA (P = 4.45E-06), Bend (P = 5.18E-06), NT (P = 1.20E-05) (D), VCR (P = 1.13E-05), Dox (P = 8.08E-06), AraC (P = 7.62E-06), and CTX (P = 1.13E-05). Survival statistical analysis was done with the Mantel-Cox (log-rank) test of GraphPad Prism.


E Therapeutic effects of CY ( weekly dose ) and targeted drugs PP242 ( daily dose ) and dasatinib ( daily dose ) in mice transplanted with BCR - ABL ALL cells . P - values of CY versus other drugs : NT ( P = 3 . 94E - 05 ) , PP242 ( P = 1 . 20E - 05 ) , and dasatinib ( P = 0 . 99 ) . Survival statistical analysis was done with the Mantel - Cox ( log - rank ) test of GraphPad Prism .

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E Therapeutic effects of CY (weekly dose) and targeted drugs PP242 (daily dose) and dasatinib (daily dose) in mice transplanted with BCR-ABL ALL cells. P-values of CY versus other drugs: NT (P = 3.94E-05), PP242 (P = 1.20E-05), and dasatinib (P = 0.99). Survival statistical analysis was done with the Mantel-Cox (log-rank) test of GraphPad Prism.


F , G Antitumor activity of CY and bendamustine in nude mice models . Mice were inoculated with human NSCLC cell line H460 . When tumor volume reached 200 mm3 , mice were treated with single dose of bendamustine ( 40 mg / kg , MTD ) or CY ( 20 , 40 , 60 mg / kg ) . In ( G ) , tumors were dissected out after 15 days posttreatment .

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F, G Antitumor activity of CY and bendamustine in nude mice models. Mice were inoculated with human NSCLC cell line H460. When tumor volume reached 200 mm3, mice were treated with single dose of bendamustine (40 mg/kg, MTD) or CY (20, 40, 60 mg/kg). In (G), tumors were dissected out after 15 days posttreatment.


Dose curve of CY , fludarabine , and bendamustine using fresh CLL samples . Data represent mean ± SD .

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Dose curve of CY, fludarabine, and bendamustine using fresh CLL samples. Data represent mean ± SD.


CY preferentially killed CLL cells over healthy B cells . Data represent mean ±

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CY preferentially killed CLL cells over healthy B cells. Data represent mean ± SD


Toxicity of CY190602 in patients harboring deletion of chromosome 17p ( including p53 locus ) compared to patients without del17p . Viability of samples was normalized to background apoptosis for individual patient samples . For all experiments , cell viability was determined after 72 h of drug treatment . Data represent mean ± SD .

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Toxicity of CY190602 in patients harboring deletion of chromosome 17p (including p53 locus) compared to patients without del17p. Viability of samples was normalized to background apoptosis for individual patient samples. For all experiments, cell viability was determined after 72 h of drug treatment. Data represent mean ± SD.


( a - d ) Rotenone ( Rot ) increased the number of GFP - LC3 puncta and the level of co - localization with mitochondria ( arrows ) in SH - SY5Y cells ( a - c ; 1 μM ) and primary cortical neurons ( d ; 250 nM ) , quantified in Fig . 3f and Supplementary Fig . S1b , c . Veh , vehicle .

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(a-d) Rotenone (Rot) increased the number of GFP-LC3 puncta and the level of co-localization with mitochondria (arrows) in SH-SY5Y cells (a-c; 1 μM) and primary cortical neurons (d; 250 nM), quantified in Fig. 3f and Supplementary Fig. S1b,c. Veh, vehicle.


( e , f ) Rotenone increased delivery of MitoTracker Green - stained mitochondria ( mt ) to LysoTracker Red ( LTR ) - stained lysosomes , inhibited by siRNA knockdown of ATG7 or LC3 in SH - SY5Y cells . Ctrl , control . Right : RNAi knockdown .

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(e,f) Rotenone increased delivery of MitoTracker Green-stained mitochondria (mt) to LysoTracker Red (LTR)-stained lysosomes, inhibited by siRNA knockdown of ATG7 or LC3 in SH-SY5Y cells. Ctrl, control. Right: RNAi knockdown.


( g ) Rotenone decreased IMM ( COXIV ) , OMM ( TOM40 ) and matrix ( MnSOD ) protein expression levels in primary neurons , reversed by bafilomycin ( Baf ) inhibition of autolysosomal degradation , quantified in Supplementary Fig . S1e . Mean ± s . d . of n = 7 independent experiments for b , c , and n = 3 independent experiments for f , g ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle control ; † P0 . 05 versus Rot / control siRNA . Scale bars , 10 μm . See Supplementary Fig . S7 for uncropped blots .

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(g) Rotenone decreased IMM (COXIV), OMM (TOM40) and matrix (MnSOD) protein expression levels in primary neurons, reversed by bafilomycin (Baf) inhibition of autolysosomal degradation, quantified in Supplementary Fig. S1e. Mean ± s.d. of n = 7 independent experiments for b,c, and n = 3 independent experiments for f,g (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control; †P0.05 versus Rot/control siRNA. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.


( a , b ) IMM and OMM fractions isolated from primary neurons following the indicated treatments were lipid extracted for LC - MS analysis . ( a ) MS spectra demonstrated increased cardiolipin ( CL ) content of the OMM after rotenone ( Rot ) treatment , with diversification of the cluster distribution to the 7 clusters exhibited by the IMM . Veh , vehicle . ( b ) Pie charts showing the CL distribution between IMM and OMM fractions from toxin - treated neurons , normalized to mitochondrial lipid phosphorus .

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(a,b) IMM and OMM fractions isolated from primary neurons following the indicated treatments were lipid extracted for LC-MS analysis. (a) MS spectra demonstrated increased cardiolipin (CL) content of the OMM after rotenone (Rot) treatment, with diversification of the cluster distribution to the 7 clusters exhibited by the IMM. Veh, vehicle. (b) Pie charts showing the CL distribution between IMM and OMM fractions from toxin-treated neurons, normalized to mitochondrial lipid phosphorus.


( c , d ) Treatment with rotenone caused significant increases in PLA2 - hydrolysable ( surface accessible ) CL assessed by LC - MS in primary cortical neurons ( c ; 250 nM ) and SH - SY5Y cells ( d ; 1 μM ) .

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(c,d) Treatment with rotenone caused significant increases in PLA2-hydrolysable (surface accessible) CL assessed by LC-MS in primary cortical neurons (c; 250 nM) and SH-SY5Y cells (d; 1 μM).


( e , f ) Treatment with 6 - OHDA and CCCP increased surface exposure of CL probed with annexin V in SH - SY5Y ( e , 120 μM ) and parkin - expressing HeLa cells ( f , 20 μM ) , respectively . Mean ± s . d . of n = 3 independent experiments for c - f ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle control .

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(e,f) Treatment with 6-OHDA and CCCP increased surface exposure of CL probed with annexin V in SH-SY5Y (e, 120 μM) and parkin-expressing HeLa cells (f, 20 μM), respectively. Mean ± s.d. of n = 3 independent experiments for c-f (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control.


( a - e ) SH - SY5Y cells expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , and treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( a ) Anionic phospholipids exposed to the outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647 - annexin V binding .

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(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (a) Anionic phospholipids exposed to the outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647-annexin V binding.


( a - e ) SH - SY5Y cells expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , and treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( b - d ) PLS3 knockdown had no effect on rotenone - induced autophagy ( b ) , but decreased mitophagy in SH - SY5Y cells treated with rotenone ( c ; 1 μM ) or 6 - OHDA ( d ; 120 μM ) .

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(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (b-d) PLS3 knockdown had no effect on rotenone-induced autophagy (b), but decreased mitophagy in SH-SY5Y cells treated with rotenone (c; 1 μM) or 6-OHDA (d; 120 μM).


( a - e ) SH - SY5Y cells expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , and treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( d , e ) The effects of PLS3 siRNA were recapitulated using a second siRNA # 433 ( d , Supplementary Fig . S3b , g ) , and reversed by transfection with an RNAi - resistant mouse PLS3 vector ( e ) . Inset : PLS3 overexpression .

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(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (d,e) The effects of PLS3 siRNA were recapitulated using a second siRNA #433 (d, Supplementary Fig. S3b,g), and reversed by transfection with an RNAi-resistant mouse PLS3 vector (e). Inset: PLS3 overexpression.


( f - i ) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone ( 250 nM ) and analysed for autophagy ( Supplementary Fig . S1b ) or mitophagy by either co - localization analysis ( f )

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(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f)


( f - i ) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone ( 250 nM ) and analysed for autophagy ( Supplementary Fig . S1b ) or mitophagy by either co - localization analysis ( f ) , or with mitochondrial protein immunoblotting ( g ) and densitometry ( h , i and Supplementary Fig . S4c ) . Knockdown efficiencies are shown in Supplementary Figs S3 and S4 . Mean ± s . d . of n = 4 independent experiments for b , c , and n = 3 independent experiments for a , d - f , h - i ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle ; † P0 . 05 versus toxin - treated control siRNA ; * * P0 . 05 versus Rot / PLS3 siRNA . See Supplementary Fig . S7 for uncropped blots .

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(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f), or with mitochondrial protein immunoblotting (g) and densitometry (h,i and Supplementary Fig. S4c). Knockdown efficiencies are shown in Supplementary Figs S3 and S4. Mean ± s.d. of n = 4 independent experiments for b,c, and n = 3 independent experiments for a,d-f,h-i (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle; †P0.05 versus toxin-treated control siRNA; **P0.05 versus Rot/PLS3 siRNA. See Supplementary Fig. S7 for uncropped blots.


( a ) Western blot analysis showed beclin 1 cleavage products ( Mr49 K ) were not observed in primary neurons exposed to mitophagy - inducing sublethal concentrations of rotenone ( 250 nM × 2 h : Rot low ) or STS ( 100 nM × 2 h ) . However , beclin 1 was cleaved on exposure to a lethal dose of rotenone ( 1 mM × 24 h : Rot high ) . Veh , vehicle ; CTD , C - terminal domain .

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(a) Western blot analysis showed beclin 1 cleavage products (Mr49 K) were not observed in primary neurons exposed to mitophagy-inducing sublethal concentrations of rotenone (250 nM×2 h: Rot low) or STS (100 nM×2 h). However, beclin 1 was cleaved on exposure to a lethal dose of rotenone (1 mM×24 h: Rot high). Veh, vehicle; CTD, C-terminal domain.


( b , c ) Likewise , SH - SY5Y cells did not show beclin 1 cleavage on exposure to rotenone ( b ) or 6 - OHDA ( c ; duplicate lanes ) . Data are representative of 2 - 3 independent experiments per toxin .

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(b,c) Likewise, SH-SY5Y cells did not show beclin 1 cleavage on exposure to rotenone (b) or 6-OHDA (c; duplicate lanes). Data are representative of 2-3 independent experiments per toxin.


( d ) Cell lysates from stable PINK1 - Flag - expressing SH - SY5Y cells were treated with 6 - OHDA ( 120 μM ) , rotenone ( 1 μM ) or FCCP ( 2 μM ) , and analysed by Flag immunoblot for PINK1 - Flag levels .

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(d) Cell lysates from stable PINK1-Flag-expressing SH-SY5Y cells were treated with 6-OHDA (120 μM), rotenone (1 μM) or FCCP (2 μM), and analysed by Flag immunoblot for PINK1-Flag levels.


( e ) SH - SY5Y cells transfected with HA - parkin were treated with the indicated toxins , immunolabelled for HA ( green ) and mitochondrial p60 antigen ( red ) and analysed for HA - parkin redistribution to mitochondria . FCCP elicited translocation of parkin to mitochondria ( arrows ) , which was not seen with the other mitophagy stimuli .

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(e) SH-SY5Y cells transfected with HA-parkin were treated with the indicated toxins, immunolabelled for HA (green) and mitochondrial p60 antigen (red) and analysed for HA-parkin redistribution to mitochondria. FCCP elicited translocation of parkin to mitochondria (arrows), which was not seen with the other mitophagy stimuli.


( f ) Cytosolic and mitochondrial pellets were prepared from SH - SY5Y cells treated with vehicle ( V ) , 6 - OHDA ( O , 120 μM ) or rotenone ( R , 1 μM ) . Gels were loaded with cytosolic or mitochondrial proteins , and immunoblotted for p62 / SQSMT1 , and the indicated fractionation markers .

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(f) Cytosolic and mitochondrial pellets were prepared from SH-SY5Y cells treated with vehicle (V), 6-OHDA (O, 120 μM) or rotenone (R, 1 μM). Gels were loaded with cytosolic or mitochondrial proteins, and immunoblotted for p62/SQSMT1, and the indicated fractionation markers.


( g , h ) Confocal analysis of p62 / SQSMT1 distribution in SH - SY5Y cells ( g ) and in primary cortical neurons ( h ) that were co - transfected with mCherry - tagged p62 / SQSMT1 and mitochondrially targeted GFP . Note recruitment of p62 to large mitochondrial aggregates ( g , yellow , arrows ) in FCCP - treated cells , which was not observed in control or rotenone - treated cells . Scale bars , 10 μm . See Supplementary Fig . S7 for uncropped blots .

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

(g,h) Confocal analysis of p62/SQSMT1 distribution in SH-SY5Y cells (g) and in primary cortical neurons (h) that were co-transfected with mCherry-tagged p62/SQSMT1 and mitochondrially targeted GFP. Note recruitment of p62 to large mitochondrial aggregates (g, yellow, arrows) in FCCP-treated cells, which was not observed in control or rotenone-treated cells. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.


( a ) Recombinant LC3 incubated with tetraoleoyl - CL ( TOCL ) or dioleoylphosphatidylcholine ( DOPC ) liposomes at the indicated molar ratios was analysed by blue native PAGE ; liposome binding impaired the gel entry of proteins .

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

(a) Recombinant LC3 incubated with tetraoleoyl-CL (TOCL) or dioleoylphosphatidylcholine (DOPC) liposomes at the indicated molar ratios was analysed by blue native PAGE; liposome binding impaired the gel entry of proteins.


( b ) Titration of phospholipids ( for example , TOCL ) to LC3 was performed to evaluate the ratio that prevents 50 % of the LC3 from entering the gels ( IC50 ) as an index of relative affinity . Bottom left inset : representative gel for TOCL / LC3 = 12 ; top right inset : comparison of IC50 values for TOCL versus dioleoyl - phosphatidic acid ( DOPA ) and tetralinoleoyl - CL ( TLCL ) versus monolyso - trilinoleoyl - CL ( lyso - CL ) . The IC50 for DOPG was > 15 . * P0 . 05 versus TOCL ; † P0 . 05 versus TLCL .

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

(b) Titration of phospholipids (for example, TOCL) to LC3 was performed to evaluate the ratio that prevents 50% of the LC3 from entering the gels (IC50) as an index of relative affinity. Bottom left inset: representative gel for TOCL/LC3 = 12; top right inset: comparison of IC50 values for TOCL versus dioleoyl-phosphatidic acid (DOPA) and tetralinoleoyl-CL (TLCL) versus monolyso-trilinoleoyl-CL (lyso-CL). The IC50 for DOPG was >15. *P0.05 versus TOCL; †P0.05 versus TLCL.


( f - j ) The arrowhead in e shows the position of the N - terminal truncation used to create GFP - LC3 deletion mutants , which were analysed for GFP - LC3 puncta formation ( f , g , i ) and participation in mitophagy ( h , j ) in response to the indicated stimuli . Rot , rotenone ; Veh , vehicle .

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

(f-j) The arrowhead in e shows the position of the N-terminal truncation used to create GFP-LC3 deletion mutants, which were analysed for GFP-LC3 puncta formation (f,g,i) and participation in mitophagy (h,j) in response to the indicated stimuli. Rot, rotenone; Veh, vehicle.


( e , k ) Dual point mutations were prepared on the basis of predicted CL contact residues ( e ) , and analysed for participation in mitophagy elicited by Rot or 6 - OHDA ( k ) . Inset : expression of GFP - LC3 plasmids ( uncropped blots in Supplementary Fig . S7 ) . UT , untransfected . Mean ± s . d . of n = 3 independent experiments for f - h , and n = 4 independent experiments for i , j ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus respective vehicle . † P0 . 05 versus toxin - treated wild type ( WT ) .

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

(e,k) Dual point mutations were prepared on the basis of predicted CL contact residues (e), and analysed for participation in mitophagy elicited by Rot or 6-OHDA (k). Inset: expression of GFP-LC3 plasmids (uncropped blots in Supplementary Fig. S7). UT, untransfected. Mean ± s.d. of n = 3 independent experiments for f-h, and n = 4 independent experiments for i,j (see Supplementary Table S4 for statistics source data); *P0.05 versus respective vehicle. †P0.05 versus toxin-treated wild type (WT).


D ) HEK293 cells were genetically engineered to knockout RIG - I , MDA5 , or both , and subsequently subjected to retroviral transduction to stably express FLAG - LGP2 or an empty vector ( EV ) . Correct gene editing and intact type I IFN responsiveness was validated by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 2 ) . * , non - specific band .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells were genetically engineered to knockout RIG-I, MDA5, or both, and subsequently subjected to retroviral transduction to stably express FLAG-LGP2 or an empty vector (EV). Correct gene editing and intact type I IFN responsiveness was validated by SDS-PAGE and immunoblotting using the indicated antibodies (n=2). *, non-specific band.


E ) Cells generated in ( D ) were transfected with siCtrl or siADAR1 . The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post - transfection as in ( B ) . Data are means ± s . d . from a representative of four biological replicate 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

E) Cells generated in (D) were transfected with siCtrl or siADAR1. The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post-transfection as in (B). Data are means ± s.d. from a representative of four biological replicate experiments.


F ) Cells generated in ( D ) were transfected with siCtrl ( siC ) or siADAR1 ( siA ) . Protein lysates were prepared 78h post - transfection , followed by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O 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) Cells generated in (D) were transfected with siCtrl (siC) or siADAR1 (siA). Protein lysates were prepared 78h post-transfection, followed by SDS-PAGE and immunoblotting using the indicated antibodies (n=2).


G ) HEK293 WT cells and FLAG - LGP2 - expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence . Cells were fixed , permeabilized and stained 72h post - transfection with anti - FLAG ( red ) and anti - IRF3 ( green ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar is 50 μm . Total nuclei ( > 450 nuclei per experimental condition ) and IRF3 - positive nuclei were counted using semi - automated software analysis and plotted as percentage IRF3 - positive nuclei of total nuclei per field of view ( a representative of three biological replicate experiments is quantified ) . The boxplot indicates the interquartile range as a box , the median as a central line , and the whiskers extend from the minimum to the maximum value . Statistical analyses were performed using unpaired two - tailed Mann - Whitney U tests . ns , not significant ; * * * * , p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 WT cells and FLAG-LGP2-expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence. Cells were fixed, permeabilized and stained 72h post-transfection with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>450 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of three biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analyses were performed using unpaired two-tailed Mann-Whitney U tests. ns, not significant; ****, p<0.0001.


A ) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR / Cas9 . Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness , respectively . Protein lysates were analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR/Cas9. Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness, respectively. Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3).


B ) ADAR1 - knockout HEK293 cells ( clone 1 ) were co - transfected with an empty vector ( EV ) or a FLAG - LGP2 - encoding vector ( LGP2 ) combined with a vector encoding GFP - tagged ADAR1 p110 or p150 . Cells were harvested 72h post - transfection and the type I IFN response was monitored by RT - qPCR analysis of IFN - β and IFIT1 expression , normalized to ACTB . Data are means ± s . d . from a representative of four biological replicate experiments .

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

B) ADAR1-knockout HEK293 cells (clone 1) were co-transfected with an empty vector (EV) or a FLAG-LGP2-encoding vector (LGP2) combined with a vector encoding GFP-tagged ADAR1 p110 or p150. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 expression, normalized to ACTB. Data are means ± s.d. from a representative of four biological replicate experiments.


C ) ADAR1 - knockout cells ( clone 1 ) were transfected as in ( B ) . Protein lysates were analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 4 ) .

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

C) ADAR1-knockout cells (clone 1) were transfected as in (B). Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=4).


A ) ADAR1 - knockout HEK293 cells ( clone 1 ) were modified with a lentiviral - based inducible system to express FLAG - LGP2 WT or a FLAG - LGP2 RNA binding mutant ( K138E / R490E / K634E , denoted as " KRK mutant " ) in a doxycycline - regulated manner . Cells were treated 72h with doxycycline ( dox ) . Protein lysates were analyzed by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 3 ) . iEV = inducible empty vector ; iLGP2 = inducible LGP2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ADAR1-knockout HEK293 cells (clone 1) were modified with a lentiviral-based inducible system to express FLAG-LGP2 WT or a FLAG-LGP2 RNA binding mutant (K138E/R490E/K634E, denoted as "KRK mutant") in a doxycycline-regulated manner. Cells were treated 72h with doxycycline (dox). Protein lysates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies (n=3). iEV = inducible empty vector; iLGP2 = inducible LGP2.


C ) Cells generated in ( A ) were plated on coverslips and treated with or without doxycycline for 72h . Cells were fixed , permeabilized and stained with anti - FLAG ( red ) and anti - IRF3 ( green ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar is 50 μm . Total nuclei ( > 500 nuclei per experimental condition ) and IRF3 - positive nuclei were counted using semi - automated software analysis and plotted as percentage IRF3 - positive nuclei of total nuclei per field of view ( a representative of two biological replicate experiments is quantified ) . The boxplot indicates the interquartile range as a box , the median as a central line , and the whiskers extend from the minimum to the maximum value . Statistical analysis was performed using a Kruskal - Wallis test with a Dunn ' s post hoc test for multiple comparisons . ns , not significant ; * * * * , p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells generated in (A) were plated on coverslips and treated with or without doxycycline for 72h. Cells were fixed, permeabilized and stained with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>500 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of two biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analysis was performed using a Kruskal-Wallis test with a Dunn's post hoc test for multiple comparisons. ns, not significant; ****, p<0.0001.


E ) MDA5 - knockout HEK293 cells , were transfected with an ADAR1 - targeting siRNA ( siADAR1 ) or a control siRNA ( siCtrl ) and 8h later with an empty vector ( EV ) or a vector encoding the indicated WT , truncation , or point mutant ( s ) of MDA5 . Cells were harvested 72h post - siRNA transfection and the type I IFN response was monitored by RT - qPCR analysis of IFIT1 and ISG15 transcript expression , normalized to ACTB . Data are means ± s . d . from a representative of two biological replicate experiments .

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

E) MDA5-knockout HEK293 cells, were transfected with an ADAR1-targeting siRNA (siADAR1) or a control siRNA (siCtrl) and 8h later with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of MDA5. Cells were harvested 72h post-siRNA transfection and the type I IFN response was monitored by RT-qPCR analysis of IFIT1 and ISG15 transcript expression, normalized to ACTB. Data are means ± s.d. from a representative of two biological replicate experiments.


B ) HEK293 WT or ADAR1 - knockout cells ( clone 1 ) were transiently transfected with an empty vector ( EV ) or a vector encoding the indicated WT , truncation , or point mutant ( s ) of LGP2 . Cells were harvested 72h post - transfection and the type I IFN response was monitored by RT - qPCR analysis of IFN - β and IFIT1 transcript expression , normalized to ACTB . Data are means ± s . d . from a representative of three biological replicate experiments .

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

B) HEK293 WT or ADAR1-knockout cells (clone 1) were transiently transfected with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of LGP2. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 transcript expression, normalized to ACTB. Data are means ± s.d. from a representative of three biological replicate experiments.


C ) Cells were treated as in ( B ) . Protein lysates were prepared and analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 3 ) . The dotted line indicates the juxtaposition of two non - adjacent lanes .

C) Cells were treated as in (B). Protein lysates were prepared and analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3). The dotted line indicates the juxtaposition of two non-adjacent lanes.


A ) Kaplan - Meier plots showing overall survival of ADARlow and ADARhigh patients stratified by DHX58 levels in bladder cancer ( BLCA ; n = 407 ) , breast cancer ( BRCA ; n = 1099 ) and sarcoma ( SARC ; n = 263 ) TCGA datasets . Median cut - offs were used for patient stratification and logrank test P values are 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

A) Kaplan-Meier plots showing overall survival of ADARlow and ADARhigh patients stratified by DHX58 levels in bladder cancer (BLCA; n=407), breast cancer (BRCA; n=1099) and sarcoma (SARC; n=263) TCGA datasets. Median cut-offs were used for patient stratification and logrank test P values are shown.


B ) CAL27 cells transduced with doxycycline - inducible shRNAs targeting ADAR1 or GFP ( negative control ) were treated with doxycycline and transfected with two independent siRNAs targeting LGP2 ( siLGP2 # 1 or # 2 ) or a control siRNA ( siCtrl ) . To determine cell confluency at 120h post - transfection , cells were fixed , stained with Crystal Violet , and imaged . Images of a representative experiment are shown ( n = 3 ) . C ) Crystal Violet was extracted from stained cells ( B ) and the dye intensity was quantified using a colorimetric assay ( OD590 ) . OD590 values of doxycycline - treated cells were normalized to the OD 590 values of untreated cells . Quantification of data from three independent experiments is shown as mean ± s . d . . Statistical analysis was performed using ordinary two - way ANOVA with Tukey ' s post hoc test . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CAL27 cells transduced with doxycycline-inducible shRNAs targeting ADAR1 or GFP (negative control) were treated with doxycycline and transfected with two independent siRNAs targeting LGP2 (siLGP2 #1 or #2) or a control siRNA (siCtrl). To determine cell confluency at 120h post-transfection, cells were fixed, stained with Crystal Violet, and imaged. Images of a representative experiment are shown (n=3). C) Crystal Violet was extracted from stained cells (B) and the dye intensity was quantified using a colorimetric assay (OD590). OD590 values of doxycycline-treated cells were normalized to the OD590 values of untreated cells. Quantification of data from three independent experiments is shown as mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001.


A ) HT29 cells were treated with or without 300 nM 5 - AZA - CdR for 2 days and subsequently washed and transfected with the indicated siRNAs . Cells were harvested 72h post - transfection and the type I IFN response was analyzed by RT - qPCR analysis of IFN - β , IFIT1 , and ISG15 transcript expression , normalized to ACTB . B ) LIM1215 cells were treated with or without 300 nM 5 - AZA - CdR for 2 days and subsequently washed and transfected with the indicated siRNAs . Cells were harvested 72h post - transfection and the type I IFN response was analyzed as in ( A ) . C ) LIM1215 cells were treated with 250 nM palbociclib or a DMSO control for 7 days . Three days after treatment initiation , cells were transfected with the indicated siRNAs and cultured for an additional 72h . The type I IFN response was monitored as in ( A ) . Data information : Data from three biological independent experiments are shown with mean ± s . d . . Statistical analysis was performed using ordinary two - way ANOVA with Tukey ' s post hoc test . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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

A) HT29 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed by RT-qPCR analysis of IFN-β, IFIT1, and ISG15 transcript expression, normalized to ACTB. B) LIM1215 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed as in (A). C) LIM1215 cells were treated with 250 nM palbociclib or a DMSO control for 7 days. Three days after treatment initiation, cells were transfected with the indicated siRNAs and cultured for an additional 72h. The type I IFN response was monitored as in (A). Data information: Data from three biological independent experiments are shown with mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


Ribosome occupancy map at the LINC00665 locus . MCF10A were either mock - treated or treated with TGF - β and Ribosome profiling on three biological replicates were shown .

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Ribosome occupancy map at the LINC00665 locus. MCF10A were either mock-treated or treated with TGF-β and Ribosome profiling on three biological replicates were shown.


Relative levels of CIP2A - BP were determined by polysome profiling and qRT - PCR in MCF - 10A and TNBC cells either mock treated or treated with TGF - β . Relative levels of LINC00665 were determined by qRT - PCR in MCF - 10A and TNBC cells either mock treated or treated with TGF - β .

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

Relative levels of CIP2A-BP were determined by polysome profiling and qRT-PCR in MCF-10A and TNBC cells either mock treated or treated with TGF-β. Relative levels of LINC00665 were determined by qRT-PCR in MCF-10A and TNBC cells either mock treated or treated with TGF-β.


Upper : putative ORFs in LINC00665 . Lower : The ORFs were constructed into pcDNA3 . 1 vector and transfected to MCF - 10A cells for 24h . The ORFs - His fusion proteins were determined by western blot with anti - His antibody .

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

Upper: putative ORFs in LINC00665. Lower: The ORFs were constructed into pcDNA3.1 vector and transfected to MCF-10A cells for 24h. The ORFs-His fusion proteins were determined by western blot with anti-His antibody.


The indicated constructs were transfected into MCF - 10A cells for 24h , then the GFP fluorescence was detected using fluorescence microscope ( F ) and fusion protein levels were determined by western blot with anti - GFP and anti - CIP2A - BP antibodies

The indicated constructs were transfected into MCF-10A cells for 24h, then the GFP fluorescence was detected using fluorescence microscope (F) and fusion protein levels were determined by western blot with anti-GFP and anti-CIP2A-BP antibodies respectively


Diagram of the His fusion constructs . The start codon ATG of the LINC00665 ORF is mutated to ATT . The indicated constructs were stably expressed in MDA - MB - 231 cells , micropeptide CIP2A - BP was immuno - stained using anti - His ( I ) and anti - CIP2A - BP antibodies ( K ) respectively ; CIP2A - BP - His fusion protein levels were determined by western blotting with anti - His and CIP2A - BP

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

Diagram of the His fusion constructs. The start codon ATG of the LINC00665 ORF is mutated to ATT. The indicated constructs were stably expressed in MDA-MB-231 cells, micropeptide CIP2A-BP was immuno-stained using anti-His (I) and anti-CIP2A-BP antibodies (K) respectively; CIP2A-BP-His fusion protein levels were determined by western blotting with anti-His and CIP2A-BP antibodies


MCF - 10A cells incubated in the absence or presence of puromycin ( 200μM ) were fractionated on sucrose gradients , and distribution of CIP2A - BP transcripts was quantified by qPCR .

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

MCF-10A cells incubated in the absence or presence of puromycin (200μM) were fractionated on sucrose gradients, and distribution of CIP2A-BP transcripts was quantified by qPCR.


Micropeptide CIP2A - BP was immune - stained with anti - CIP2A - BP antibody in MCF - 10A and MDA - MB - 231 cells either mock treated or treated with TGF - β .

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

Micropeptide CIP2A-BP was immune-stained with anti-CIP2A-BP antibody in MCF-10A and MDA-MB-231 cells either mock treated or treated with TGF-β.


Micropeptide CIP2A - BP levels were determined by western blot with anti - CIP2A - BP antibody in MCF - 10A and TNBC cells either mock treated or treated with TGF - β .

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

Micropeptide CIP2A-BP levels were determined by western blot with anti-CIP2A-BP antibody in MCF-10A and TNBC cells either mock treated or treated with TGF-β.


MCF - 10A cells were first transfected with anti - LINC00665 translation - blocking antisense oligo , then micropeptide CIP2A - BP was detected .

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

MCF-10A cells were first transfected with anti-LINC00665 translation-blocking antisense oligo, then micropeptide CIP2A-BP was detected.


Micropeptide CIP2A - BP levels were detected by western blot with anti - CIP2A - BP antibody in the indicated non - metastatic TNBC tissues ( non - metastasis ) and metastatic TNBC tissues ( metastasis ) . Micropeptide CIP2A - BP expression in MCF - 10A cells was used as the positive control .

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

Micropeptide CIP2A-BP levels were detected by western blot with anti-CIP2A-BP antibody in the indicated non-metastatic TNBC tissues (non-metastasis) and metastatic TNBC tissues (metastasis). Micropeptide CIP2A-BP expression in MCF-10A cells was used as the positive control.


Kaplan - Meier overall survival curves for TNBC patients with high or low micropeptide CIP2A - BP expression in Suzhou cohort ( discovery set , n = 112 ) and Guangzhou cohort ( validation set , n = 105 ) .

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

Kaplan-Meier overall survival curves for TNBC patients with high or low micropeptide CIP2A-BP expression in Suzhou cohort (discovery set, n = 112) and Guangzhou cohort (validation set, n = 105).


Migration and invasion of TNBC cells transfected with LINC00665 ORF and control lentiviruses for 48h . Right panel is the quantification of TNBC cells that migrated through the membrane without the Matrigel ( upper ) and invaded through Matrigel - coated membrane ( lower ) .

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

Migration and invasion of TNBC cells transfected with LINC00665 ORF and control lentiviruses for 48h. Right panel is the quantification of TNBC cells that migrated through the membrane without the Matrigel (upper) and invaded through Matrigel-coated membrane (lower).


Migration and invasion of wild - type and CIP2A - BP knockout TNBC cells and CIP2A - BP re - expressed Hs578TKO and MDA - MB - 231KO cells . Right panel is the quantification of cells that migrated through the membrane without the Matrigel ( upper ) and invaded through Matrigel - coated membrane ( lower ) .

Migration and invasion of wild-type and CIP2A-BP knockout TNBC cells and CIP2A-BP re-expressed Hs578TKO and MDA-MB-231KO cells. Right panel is the quantification of cells that migrated through the membrane without the Matrigel (upper) and invaded through Matrigel-coated membrane (lower).


Wound - healing assay on Hs578T and MDA - MB - 231 cells transfected with LINC00665 ORF expressing and control lentiviruses . The wound edges were photographed at the indicated time points after wounding . Right panel is the quantification of the relative wound healing area .

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

Wound-healing assay on Hs578T and MDA-MB-231 cells transfected with LINC00665 ORF expressing and control lentiviruses. The wound edges were photographed at the indicated time points after wounding. Right panel is the quantification of the relative wound healing area.


Wild - type and CIP2A - BP knockout TNBC cells and CIP2A - BP re - expressed Hs578TKO and MDA - MB - 231KO were seeded on cell culture inserts for wound - healing assay . The wound edges were photographed at the indicated time points after wounding . Right panel is the quantification of the relative wound healing area .

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

Wild-type and CIP2A-BP knockout TNBC cells and CIP2A-BP re-expressed Hs578TKO and MDA-MB-231KO were seeded on cell culture inserts for wound-healing assay. The wound edges were photographed at the indicated time points after wounding. Right panel is the quantification of the relative wound healing area.


Macroscopic and histological analysis of the lungs of nude mice injected with the indicated Hs578T ( G ) and MDA - MB - 231 cells ( H ) ( 1 . 0 × 105 - 106 cells / mouse ) via tail vein ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases .

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

Macroscopic and histological analysis of the lungs of nude mice injected with the indicated Hs578T (G) and MDA-MB-231 cells (H) (1.0×105-106 cells/mouse) via tail vein; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.


Immunoblotting analysis of expression of the indicated proteins in TNBC cells cultured with or without TGF - β .

Immunoblotting analysis of expression of the indicated proteins in TNBC cells cultured with or without TGF-β.


Chromatin immunoprecipitation showing Smad4 occupancy at the 4E - BP1 locus in TNBC cells . Co - precipitated DNA was analyzed for amplicons A - E by qPCR . Values represent the enrichment of bound protein fractions relative to input .

Chromatin immunoprecipitation showing Smad4 occupancy at the 4E-BP1 locus in TNBC cells. Co-precipitated DNA was analyzed for amplicons A-E by qPCR. Values represent the enrichment of bound protein fractions relative to input.


Luciferase reporter assay was performed in Hs578T ( F ) and MDA - MB - 231 ( G ) cells following co - transfection with wildtype or mutant 4E - BP1 promoter fragment and Smad4 over - expressed , or knocked down and respective controls for 36h and treatment with TGF - β for 24h . The reporter constructs were expressing the luciferase gene under 4E - BP1 promoter segment or 4E - BP1 promoter deleted 0 to + 200 region .

Luciferase reporter assay was performed in Hs578T (F) and MDA-MB-231 (G) cells following co-transfection with wildtype or mutant 4E-BP1 promoter fragment and Smad4 over-expressed, or knocked down and respective controls for 36h and treatment with TGF-β for 24h. The reporter constructs were expressing the luciferase gene under 4E-BP1 promoter segment or 4E-BP1 promoter deleted 0 to +200 region.


Regulation of 4E - BP1 expression and binding to eIF4E by TGF - β . TNBC cells were treated with TGF - β and equal amounts of proteins were subjected to immunoprecipitation and / or examined by immunoblotting analysis .

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

Regulation of 4E-BP1 expression and binding to eIF4E by TGF-β. TNBC cells were treated with TGF-β and equal amounts of proteins were subjected to immunoprecipitation and/or examined by immunoblotting analysis.


Immunoblotting analysis of expression of micropeptide CIP2A - BP and β - actin in TNBC cells cultured with or without TGF - β .

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

Immunoblotting analysis of expression of micropeptide CIP2A-BP and β-actin in TNBC cells cultured with or without TGF-β.


Smad4 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF - β . The indicated proteins were determined by Immunoblotting analysis .

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

Smad4 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF-β. The indicated proteins were determined by Immunoblotting analysis.


TNBC cells were pretreated with specific antagonist against Smad3 ( SIS3 , 3μM ) for 1h and then cultured with or without TGF - β . The indicated proteins were determined by immunoblotting analysis .

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

TNBC cells were pretreated with specific antagonist against Smad3 (SIS3, 3μM) for 1h and then cultured with or without TGF-β. The indicated proteins were determined by immunoblotting analysis.


4E - BP1 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF - β . The indicated proteins were determined by immunoblotting analysis .

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

4E-BP1 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF-β. The indicated proteins were determined by immunoblotting analysis.


LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 cells . Whole - cell lysates were subjected to co - immunoprecipitation using anti - His antibody and immunoblot analysis with CIP2A antibody to verify that CIP2A bound to CIP2A - BP .

LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation using anti-His antibody and immunoblot analysis with CIP2A antibody to verify that CIP2A bound to CIP2A-BP.


LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 cells . Whole - cell lysates were subjected to co - immunoprecipitation by CIP2A antibody and immunoblot analysis with CIP2A - BP antibodies to verify that CIP2A - BP bound to CIP2A .

LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation by CIP2A antibody and immunoblot analysis with CIP2A-BP antibodies to verify that CIP2A-BP bound to CIP2A.


Endogenous CIP2A - BP and CIP2A were co - immunoprecipitated in MCF - 10A cell line .

Endogenous CIP2A-BP and CIP2A were co-immunoprecipitated in MCF-10A cell line.


LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 cells . Whole - cell lysates were treated with 30 mg / mL RNase A for 30 min , the indicated complexes were co - immunoprecipitated by anti - His or anti - CIP2A antibody , and CIP2A or micropeptide CIP2A - BP were detected .

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

LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were treated with 30 mg/mL RNase A for 30 min, the indicated complexes were co-immunoprecipitated by anti-His or anti-CIP2A antibody, and CIP2A or micropeptide CIP2A-BP were detected.


Upper : A diagram of the domain structure of full length CIP2A protein . Red : RIP = Required for Interaction with PPP2R5C ; Orange : RH = Required for Homodimerization ; Blue , CC = Coiled coil . Lower : Co - immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A - BP with CIP2A via the N - terminus domain of CIP2A . Upper : A diagram of the domain structure of CIP2A protein fragment ( 1 - 245 ) . Red : RIP = Required for Interaction with PPP2R5C . Lower : Co - immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A - BP with CIP2A via the N - terminus domain of CIP2A ( amino acid 159 - 245 ) .

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

Upper: A diagram of the domain structure of full length CIP2A protein. Red: RIP=Required for Interaction with PPP2R5C; Orange: RH=Required for Homodimerization; Blue, CC=Coiled coil. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A. Upper: A diagram of the domain structure of CIP2A protein fragment (1-245). Red: RIP=Required for Interaction with PPP2R5C. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A (amino acid 159-245).


Co - immunoprecipitation assays showed that the interaction between CIP2A and B56γ in TNBC cells was affected by CIP2A - BP expression .

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

Co-immunoprecipitation assays showed that the interaction between CIP2A and B56γ in TNBC cells was affected by CIP2A-BP expression.


The expression levels of the indicated proteins were detected by immunoblotting analysis in CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) and respective controls of TNBC 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

The expression levels of the indicated proteins were detected by immunoblotting analysis in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective controls of TNBC cells.


PP2A activity was measured using a protein phosphatase assay kit . PP2A activity assay was performed to evaluate PP2A activity in CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) and respective control TNBC 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

PP2A activity was measured using a protein phosphatase assay kit. PP2A activity assay was performed to evaluate PP2A activity in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control TNBC cells.


Immunohistochemistry ( IHC ) staining of p - AKT from TNBC primary tumor ( n = 20 ) with high or low micropeptide CIP2A - BP expression . Lower panels show higher - magnification images of insets in upper panels . Right panel is the quantification of IHC staining p - AKT .

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

Immunohistochemistry (IHC) staining of p-AKT from TNBC primary tumor (n = 20) with high or low micropeptide CIP2A-BP expression. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT.


MDA - MB - 231 cells were transfected with anti - CIP2A siRNAs . Whole - cell lysates of these cells were subjected to immunoblot analysis with CIP2A , CIP2A - BP , PP2Ac , p - AKT , AKT and β - actin antibodies .

MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. Whole-cell lysates of these cells were subjected to immunoblot analysis with CIP2A, CIP2A-BP, PP2Ac, p-AKT, AKT and β-actin antibodies.


CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) and respective control MDA - MB - 231 cells were transfected with anti - CIP2A siRNAs . PP2A activity assay was performed to evaluate PP2A activity in the indicated 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

CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. PP2A activity assay was performed to evaluate PP2A activity in the indicated cells.


The indicated cells were pretreated with specific antagonist against AKT ( MK - 2206 ， 3μM ) for 1h . Then whole lysates of these cells were subjected to immunoblot analysis .

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

The indicated cells were pretreated with specific antagonist against AKT (MK-2206，3μM) for 1h. Then whole lysates of these cells were subjected to immunoblot analysis.


Macroscopic and histological analysis of the lungs of 140 days old MMTV - PyMT mice ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases .

Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.


Immunohistochemistry ( IHC ) staining of p - AKT of mammary tumors from MMTV - PyMT ; CIP2A - BP + / + or MMTV - PyMT ; CIP2A - BP - / - mice . Lower panels show higher - magnification images of insets in upper panels . Right panel is the quantification of IHC staining p - AKT .

Immunohistochemistry (IHC) staining of p-AKT of mammary tumors from MMTV-PyMT;CIP2A-BP+/+ or MMTV-PyMT;CIP2A-BP-/- mice. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT .


MMTV - PyMT mice were injected with CIP2A - BP or svCIP2A - BP via mammary fat pad . Macroscopic and histological analysis of the lungs of 140 days old MMTV - PyMT mice ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases .

MMTV-PyMT mice were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad. Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.


Representative IHC images of p - AKT of mammary tumors from MMTV - PyMT mice that were injected with CIP2A - BP or svCIP2A - BP via mammary fat pad ; Lower panels show higher - magnification images of insets in upper panels . Right panel is the quantification of IHC staining p - AKT .

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

Representative IHC images of p-AKT of mammary tumors from MMTV-PyMT mice that were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad; Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT.


Kaplan - Meier survival curves of nude mice transplanted with MDA - MB - 231 cells and injected with CIP2A - BP or svCIP2A - BP ( 10 mice per group ) . The horizontal line indicates the time after the mice were injected with the cells via the tail vein .

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

Kaplan-Meier survival curves of nude mice transplanted with MDA-MB-231 cells and injected with CIP2A-BP or svCIP2A-BP (10 mice per group). The horizontal line indicates the time after the mice were injected with the cells via the tail vein.


Macroscopic and histological analysis of the lungs of nude mice injected with MDA - MB - 231 cells and CIP2A - BP or svCIP2A - BP via tail vein ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases .

Macroscopic and histological analysis of the lungs of nude mice injected with MDA-MB-231 cells and CIP2A-BP or svCIP2A-BP via tail vein; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.


Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM . Scale bars , 5μm . Data are representative of n = 3 biological replicates . ( C ) Western blot images ( top ) and TRF1 protein levels ( bottom ) of h676 cells treated with the indicated compounds for 24h at 1 μM . Data are representative of n = 3 ( PLKi , HSP90i and RTKi ) and n = 4 ( AuroraI , mTOR , CDKi , Docetaxel , Gemcitabine , ERKi , MEKi ) 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O

Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM. Scale bars, 5μm. Data are representative of n=3 biological replicates. (C) Western blot images (top) and TRF1 protein levels (bottom) of h676 cells treated with the indicated compounds for 24h at 1 μM. Data are representative of n=3 (PLKi, HSP90i and RTKi) and n=4 (AuroraI, mTOR, CDKi, Docetaxel, Gemcitabine, ERKi, MEKi) biological replicates.


Representative images ( left ) and percentage ( right ) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci ( TIFs ) upon treatment of CHA9 - 3 lung cancer cells with the indicated compounds . White arrowheads point to colocalization of γH2AX and RAP1 . Scale bars , 5μm . Data are representative of n = 6 ( DMSO ) and n = 3 ( mTORi , PI3Ki , RTKi , MEKi , ERKi , HSPO90i , CDKi , Docetaxel ) biological

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

Representative images (left) and percentage (right) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci (TIFs) upon treatment of CHA9-3 lung cancer cells with the indicated compounds. White arrowheads point to colocalization of γH2AX and RAP1. Scale bars, 5μm. Data are representative of n=6 (DMSO) and n=3 (mTORi, PI3Ki, RTKi, MEKi, ERKi, HSPO90i, CDKi, Docetaxel) biological replicates


Representative images ( left ) and percentage ( right ) of γH2AX - positive cells per field in DMSO or compound treated patient - derived h676 GSCs . Scale bars , 50μm . Data are representative of 6 ( DMSO ) and 3 ( mTORi , Docetaxel , ERKi , MEKi , RTKi , HSP90i , Gemcitabine , CDKi ) biological

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

Representative images (left) and percentage (right) of γH2AX-positive cells per field in DMSO or compound treated patient-derived h676 GSCs. Scale bars, 50μm. Data are representative of 6 (DMSO) and 3 (mTORi, Docetaxel, ERKi, MEKi, RTKi, HSP90i, Gemcitabine, CDKi) biological replicates


Quantification of multitelomeric signals ( MTS ) in h676 GSC metaphases upon treatment with the indicated compounds . Representative images of the qFISH in the metaphases ( left ) . Multitelomeric signals are indicated by arrowheads . Scale bars , 1μm . Data are representative of n = 31 ( DMSO ) , n = 18 ( mTORi ) , n = 11 ( MEKi ) and n = 24 ( RTKi ) biological replicates . Data are represented as mean ± SEM . Significant differences using unpaired t ‐ test are indicated by * 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

Quantification of multitelomeric signals (MTS) in h676 GSC metaphases upon treatment with the indicated compounds. Representative images of the qFISH in the metaphases (left). Multitelomeric signals are indicated by arrowheads. Scale bars, 1μm. Data are representative of n=31 (DMSO), n=18 (mTORi), n=11 (MEKi) and n=24 (RTKi) biological replicates. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.


Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO .

Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.


1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography .

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

1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.


1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human b - Raf kinase ( WT or V600E ) in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography .

1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human b-Raf kinase (WT or V600E) in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.


1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of mouse MEK1 kinase in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography .

1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse MEK1 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.


1 μM of GST - TRF1 and 0 . 2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors .

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

1 μM of GST-TRF1 and 0.2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors.


2 μM of GST - TRF1 and 0 . 1 μM of human b - Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib .

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

2 μM of GST-TRF1 and 0.1 μM of human b-Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib.


1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human mTOR

1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase


1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human mTOR kinase in the presence of the mTOR inhibitors rapamycin and

O O O 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

1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase in the presence of the mTOR inhibitors rapamycin and Ku0063794


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

Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus ERK2


Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus bRAFWT or bRAFV600E


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

Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus mTOR


Representative image ( down ) and quantification ( up ) of in vitro phosphorylation assays with the indicated GST - TRF1 wild - type or mutated forms in the presence of mouse ERK2 kinase .

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

Representative image (down) and quantification (up) of in vitro phosphorylation assays with the indicated GST-TRF1 wild-type or mutated forms in the presence of mouse ERK2 kinase.


Western blot images of Trf1lox / lox MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed by Cre recombinase transduction .

Western blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction.


Quantification of eGFP - TRF1 inhibition in Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 15 biological

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

Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=15 biological replicates


Growth curves of Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 5 biological

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Growth curves of Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicates


Western blot images of Trf1lox / lox MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed by Cre recombinase transduction . Quantification of eGFP - TRF1 inhibition in Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 5 biological

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Western blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction. Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicates


Western blot images of p53 - / - MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed treatment with ERKi . Data are representative of n = 2 biological

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Western blot images of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed treatment with ERKi. Data are representative of n=2 biological replicates


Representative images ( above ) and percentage ( bottom ) of Telomeric and 53BP1 colocalizing foci ( TIFs ) per cells of p53 - / - MEFS with or without overexpression of eGFP - Trf1 WT and the indicated mutants upon treatment with the ERKi . White arrowheads : colocalization of telomeric and 53BP1 . Scale bars , 10μm .

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Representative images (above) and percentage (bottom) of Telomeric and 53BP1 colocalizing foci (TIFs) per cells of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT and the indicated mutants upon treatment with the ERKi. White arrowheads: colocalization of telomeric and 53BP1. Scale bars, 10μm.


Western blot image ( above ) and quantification ( bottom ) of TRF1 protein levels upon genetic depletion of ERK1 / 2 in p53 - / - MEF line . Data are representative of n = 3 independent experiments .

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Western blot image (above) and quantification (bottom) of TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line. Data are representative of n=3 independent experiments.


Representative images ( above ) and quantification ( bottom ) of TRF1 telomeric foci in ERK1 / 2 RNA interfered p53 - / - MEFs . Scale bars , 5μm . Data are representative of n = 2 independent experiments .

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Representative images (above) and quantification (bottom) of TRF1 telomeric foci in ERK1/2 RNA interfered p53-/- MEFs. Scale bars, 5μm. Data are representative of n=2 independent experiments.


Western blot image of eGFP - tagged and endogenous TRF1 protein levels upon genetic depletion of ERK1 / 2 in p53 - / - MEF line .

Western blot image of eGFP-tagged and endogenous TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line.


Representative images ( above ) and percentage ( bottom ) of telomeric and 53BP1 colocalizing foci ( TIFs ) per cell in p53 - / - MEF with or without overexpression of eGFP - Trf1 WT and indicated mutants upon genetic depletion of ERK1 / 2 . White arrowheads : colocalization of telomeric and 53BP1 . Scale bars , 10μm .

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Representative images (above) and percentage (bottom) of telomeric and 53BP1 colocalizing foci (TIFs) per cell in p53-/- MEF with or without overexpression of eGFP-Trf1 WT and indicated mutants upon genetic depletion of ERK1/2. White arrowheads: colocalization of telomeric and 53BP1.Scale bars, 10μm.


Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates .

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Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.


Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of n = 3 ( RTKi in G ( DMSO in

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Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of n=3 (RTKi in G (DMSO in G


Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSO in and ERKi in H )

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Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in and ERKi in H)


Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSO in ( PI3Ki

Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in (PI3Ki in


Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of (

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Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO


Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of Docetaxel (

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Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of Docetaxel (DMSO


Longitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in A : vehicle n = 16 , PI3Ki n = 8 , ERKi n = 16 , Combination n = 8 Pvalues represent the mean of all the time

Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in A: vehicle n=16, PI3Ki n=8, ERKi n=16, Combination n=8 Pvalues represent the mean of all the time points


Longitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in B : Vehicle n = 16 , PI3Ki n = 16 , MEKi n = 8 , Combination n = 8 Pvalues represent the mean of all the time

Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in B: Vehicle n=16, PI3Ki n=16, MEKi n=8, Combination n=8 Pvalues represent the mean of all the time points


Longitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in C : Vehicle n = 8 , PI3Ki n = 16 , Docetaxel n = 16 , Combination n = 8 Pvalues represent the mean of all the time

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Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in C: Vehicle n=8, PI3Ki n=16, Docetaxel n=16, Combination n=8 Pvalues represent the mean of all the time points


Longitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in D : Vehicle n = 16 , PI3K n = 16 , Gemcitabine n = 4 , Combination n = 4 . Pvalues represent the mean of all the time

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Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in D: Vehicle n=16, PI3K n=16, Gemcitabine n=4, Combination n=4. Pvalues represent the mean of all the time points


Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors : in E : vehicle n = 15 , PI3Ki n = 10 , ERKi n = 8 , Combination n =

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Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors: in E: vehicle n=15, PI3Ki n=10, ERKi n=8, Combination n=2


Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors in F : Vehicle n = 15 , PI3Ki n = 10 , MEKi n = 8 , Combination n =

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Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors in F: Vehicle n=15, PI3Ki n=10, MEKi n=8, Combination n=6


Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors ; in G : Vehicle n = 15 , PI3Ki n = 10 , Docetaxel n = 8 , Combination n =

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Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors ; in G: Vehicle n=15, PI3Ki n=10, Docetaxel n=8, Combination n=4


Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors ; in H : Vehicle n = 15 , PI3K n = 10 , Gemcitabine n = 8 , Combination n = 6 .

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Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors ; in H: Vehicle n=15, PI3K n=10, Gemcitabine n=8, Combination n=6.


EdU - labeled viral DNA forms large foci in ThP1 cells infected by HIV - 1 . EdU images are shown in red , DAPI images in blue . ( A ) Left : nucleus of an infected cell at 48 h p . i . displays three large and bright EdU foci ( colored arrows ) , as well as small and dim punctae ( grey arrows ) . Center : Magnified views have colored borders matching the colors of the arrows . 3D images show that EdU foci are located within the nucleoplasm ( Movie EV1 , Appendix Fig . S2 ) . Right : nucleus of an uninfected cell displays no EdU signal . ( B ) Graph shows EdU intensity profiles along lines shown in A , with colors matching the corresponding arrows .

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EdU-labeled viral DNA forms large foci in ThP1 cells infected by HIV-1. EdU images are shown in red, DAPI images in blue. (A) Left: nucleus of an infected cell at 48 h p.i. displays three large and bright EdU foci (colored arrows), as well as small and dim punctae (grey arrows). Center: Magnified views have colored borders matching the colors of the arrows. 3D images show that EdU foci are located within the nucleoplasm (Movie EV1, Appendix Fig. S2). Right: nucleus of an uninfected cell displays no EdU signal. (B) Graph shows EdU intensity profiles along lines shown in A, with colors matching the corresponding arrows.


( C ) Dual - color image of an infected cell with EdU ( red ) and immunolabeled lamin ( grey ) . Curves to the right plot the intensity profile of EdU and lamin along the dashed blue line . The EdU intensity peaks do not coincide with lamin enrichment . See also Fig . EV1A , B .

(C) Dual-color image of an infected cell with EdU (red) and immunolabeled lamin (grey). Curves to the right plot the intensity profile of EdU and lamin along the dashed blue line. The EdU intensity peaks do not coincide with lamin enrichment. See also Fig. EV1A,B.


( D ) Multi - color images of an infected cell showing EdU ( red ) with CA ( yellow ) and integrase ( green ) . The colocalization of nuclear EdU foci with CA and IN ( arrows ) confirms that EdU specifically labels viral DNA and shows the presence of these proteins in vDNA foci . See also Appendix Fig . S3 . ( E , F ) vDNA foci are much larger than the predicted size of single viral genomes .

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(D) Multi-color images of an infected cell showing EdU (red) with CA (yellow) and integrase (green). The colocalization of nuclear EdU foci with CA and IN (arrows) confirms that EdU specifically labels viral DNA and shows the presence of these proteins in vDNA foci. See also Appendix Fig. S3. (E,F) vDNA foci are much larger than the predicted size of single viral genomes.


( E ) Left image shows an observed , EdU - labeled vDNA focus . Right : simulations of a single linear or circular 10 Kb long chromatinized DNA polymer chain and corresponding predicted images in diffraction - limited ( ~ 300 nm resolution ) microscopy . ( F ) Boxplots show the distribution of sizes ( FWHM , full width at half maximum ) of n = 40 measured vDNA foci ( for MOI 100 ) compared to the sizes predicted for linear ( left ) and circular ( right ) chains based on n = 100 simulated configurations each . Blue lines in boxes define medians , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . All differences are highly significant ( Wilcoxon test data vs . model : * * * p ≈ 3 ⋅ 10 − 20 , circular vs . linear model : * * * p ≈ 10 − 17 ) .

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(E) Left image shows an observed, EdU-labeled vDNA focus. Right: simulations of a single linear or circular 10 Kb long chromatinized DNA polymer chain and corresponding predicted images in diffraction-limited (~300 nm resolution) microscopy. (F) Boxplots show the distribution of sizes (FWHM, full width at half maximum) of n=40 measured vDNA foci (for MOI 100) compared to the sizes predicted for linear (left) and circular (right) chains based on n=100 simulated configurations each. Blue lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. All differences are highly significant (Wilcoxon test data vs. model: ***p ≈ 3 ⋅ 10 − 20, circular vs. linear model: ***p ≈ 10 − 17).


( G ) Images of vDNA foci ( arrows ) in ThP1 cells infected with multiplicities of infection ( MOI ) 10 , 50 and 100 . Images for MOI 10 are shown at two different contrast levels to reveal dimmer spots . Scale bars , 3 µm .

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(G) Images of vDNA foci (arrows) in ThP1 cells infected with multiplicities of infection (MOI) 10, 50 and 100. Images for MOI 10 are shown at two different contrast levels to reveal dimmer spots. Scale bars, 3 µm.