F . WT MEFs were untreated , treated with 10 μg / ml FAC for 12h or treated with 10 μg / ml FAC , 10 μM E64d 10 μg / ml pepstatin A ( pep A ) for 12h . were immunostained with anti - NCOA4 anti - LAMP1 . Scale bar , 10 μm . Quantitative data are shown as means ± SD of three biological replicates . At least 30 were quantified in each replicate . P = 0 . 033 ( Welch Two Sample t - ) .

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F. WT MEFs were untreated, treated with 10 μg/ml FAC for 12h or treated with 10 μg/ml FAC, 10 μM E64d and 10 μg/ml pepstatin A (pep A) for 12h. The cells were immunostained with anti-NCOA4 and anti-LAMP1. Scale bar, 10 μm. Quantitative data are shown as the means ± SD of three biological replicates. At least 30 cells were quantified in each replicate. P=0.033 (Welch Two Sample t-test).


G . NCOA4 were photobleached in MEFs stably expressing NCOA4 - GFP after treatment with 10 μg / ml FAC for 12 h , then recovery was monitored . p62 were photobleached in MEFs stably expressing GFP - human p62 , then recovery was monitored . Representative are shown . Time 0 indicates start of recovery after photobleaching . Scale bars , 2 μm . H . Quantitative data of recovery in ( G ) shown as means ± SD . 26 dots ( NCOA4 ) or 25 dots ( p62 ) were quantified from three biological replicates .

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G. NCOA4 puncta were photobleached in MEFs stably expressing NCOA4-GFP after treatment with 10 μg/ml FAC for 12 h, and then fluorescent recovery was monitored. p62 puncta were photobleached in MEFs stably expressing GFP-human p62, and then fluorescent recovery was monitored. Representative images are shown. Time 0 indicates the start of recovery after photobleaching. Scale bars, 2 μm. H. Quantitative data of fluorescence recovery in (G) shown as means ± SD. 26 dots (NCOA4) or 25 dots (p62) were quantified from three biological replicates.


I . Time - lapse imaging of MEFs stably expressing NCOA4 - GFP . Representative fusion fission are shown . Red arrowheads indicate fission event . Scale bars , 5 μm .

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I. Time-lapse imaging of MEFs stably expressing NCOA4-GFP. Representative fusion and fission images are shown. Red arrowheads indicate the fission event. Scale bars, 5 μm.


B . NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg / ml FAC for 12 h then fixed for imaging . were immunostained with anti - myc antibody . Representative are shown . Scale bar , 10 μm .

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B. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h and then fixed for imaging. Cells were immunostained with anti-myc antibody. Representative images are shown. Scale bar, 10 μm.


C . Number of per in ( B ) are shown as means ± of at least 40 in each condition from two biological replicates .

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C. Number of puncta per cell in (B) are shown as the means ± SEM of at least 40 cells in each condition from two biological replicates.


D . NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg / ml FAC for 12 h , fractionated , analyzed by immunoblotting with indicated antibodies .

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D. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h, fractionated, and analyzed by immunoblotting with the indicated antibodies.


E . DISOPRED3 disorder score of human NCOA4 .

E. DISOPRED3 disorder score of human NCOA4.


F . WT MEFs stably expressing NCOA4 IDR ( aa 167 - 334 ) fused with tandem FKBP were treated with 10 μg / ml FAC or 20 μM Dfo for 12 h then cultured with 0 . 1 nM FKBP ligand ( AP20187 ) for 6h . were fixed for imaging immunostained with anti - myc to analyze of fusion . Scale bar , 10μm . G . Number of in ( F ) are shown as means ± of at least 35 in each condition from two biological replicates . P < 0 . 0001 ( Dfo + ligand vs FAC + ligand ) P = 0 . 048 ( FAC vs FAC + ligand ) ( Kruskal - Wallis ANOVA with Dunn ' s multiple comparison ) . P values were adjusted using Bonferroni method .

O O O O O O B-MEASURED_VAR 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 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

F. WT MEFs stably expressing NCOA4 IDR (aa 167-334) fused with tandem FKBP were treated with 10 μg/ml FAC or 20 μM Dfo for 12 h and then cultured with 0.1 nM FKBP ligand (AP20187) for 6h. The cells were fixed for imaging and immunostained with anti-myc to analyze puncta of the fusion protein. Scale bar, 10μm. G. Number of puncta in (F) are shown as means ± SEM of at least 35 cells in each condition from two biological replicates. P<0.0001 (Dfo+ligand vs FAC+ligand) and P=0.048 (FAC vs FAC+ligand) (Kruskal-Wallis ANOVA with Dunn's multiple comparison test). P values were adjusted using the Bonferroni method.


H . Coomassie - stained SDS - PAGE gel of purified . I . amount of co - purifying iron in purified in ( H ) was measured by inductively coupled plasma mass spectrometry ( ICP - MS ) . Data are shown as means ± SD of three biological replicates . P = 0 . 032 ( Welch Two Sample t - ) .

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H. Coomassie-stained SDS-PAGE gel of purified proteins. I. The amount of co-purifying iron in purified proteins in (H) was measured by inductively coupled plasma mass spectrometry (ICP-MS). Data are shown as means ± SD of three biological replicates. P=0.032 (Welch Two Sample t-test).


A . Purified NCOA4 were incubated with FeSO4 for 1 h under aerobic conditions fractionated . Samples were subjected to SDS - PAGE stained with Coomassie Brilliant Blue . B . Quantitative results in ( A ) are shown as means ± of three biological replicates .

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A. Purified NCOA4 proteins were incubated with FeSO4 for 1 h under aerobic conditions and fractionated. Samples were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. B. Quantitative results in (A) are shown as means ± SEM of three biological replicates.


F . Electron micrograph of NCOA4 KO MEFs reconstituted with myc - hNCOA4 . Cryosections were labeled with anti - myc antibody . Scale bar , 200nm . G . of diameter of NCOA4 condensates . Red shows mean . dot plot represents diameter of 36 condensates from two biological replicates .

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F. Electron micrograph of NCOA4 KO MEFs reconstituted with myc-hNCOA4. Cryosections were labeled with anti-myc antibody. Scale bar, 200nm. G. Quantification of the diameter of NCOA4 condensates. Red line shows the mean. The dot plot represents the diameter of 36 condensates from two biological replicates.


A . MEFs were treated with 10 μg / ml FAC for indicated times harvested . lysates were fractionated analyzed by SDS - PAGE with indicated antibodies . Asterisks indicate a non - specific band . TfR1 ; Transferrin receptor

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A. MEFs were treated with 10 μg/ml FAC for the indicated times and harvested. Cell lysates were fractionated and analyzed by SDS-PAGE with the indicated antibodies. Asterisks indicate a non-specific band. TfR1; Transferrin receptor 1


B . MEFs were cultured for 12 h in medium containing 10 μg / ml FAC supplemented with DMSO or 10 μM E64d 10 μg / ml pepstatin A ( pepA ) . Samples were analyzed by immunoblotting with indicated antibodies . black arrowhead indicates an intact ferritin band , arrowhead indicates a partial degradation band of ferritin .

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B. MEFs were cultured for 12 h in medium containing 10 μg/ml FAC supplemented with DMSO or 10 μM E64d and 10 μg/ml pepstatin A (pepA). Samples were analyzed by immunoblotting with the indicated antibodies. The black arrowhead indicates an intact ferritin band, and the white arrowhead indicates a partial degradation band of ferritin.


C . NCOA4 KO MEFs reconstituted with hNCOA4 - myc were cultured with 10 μg / ml FAC for indicated times fixed for imaging . were immunostained with ferritin myc antibodies . Scale bar , 10μm .

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C. NCOA4 KO MEFs reconstituted with hNCOA4-myc were cultured with 10 μg/ml FAC for the indicated times and fixed for imaging. Cells were immunostained with ferritin and myc antibodies. Scale bar, 10μm.


D . Myc ( NCOA4 ) were extracted , average signal intensities of myc ferritin in in ( C ) were plotted .

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D. Myc (NCOA4) puncta were extracted, and average signal intensities of myc and ferritin in the puncta in (C) were plotted.


E . MEFs expressing hNCOA4 - TurboID were treated with 10 μg / ml FAC for 6 h or 24 h then cultured with DMSO or 50 μM biotin for 30 min . were lysed with Triton buffer , lysates were pulled down by streptavidin beads . Inputs pulldown samples were analyzed with immunoblotting by indicated antibodies .

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E. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 6 h or 24 h and then cultured with DMSO or 50 μM biotin for 30 min. Cells were lysed with Triton buffer, and lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed with immunoblotting by indicated antibodies.


F . WT MEFs pretreated with 10 μg / ml FAC for 6 h or 24 h were chased with 20 μg / ml CHX analyzed by immunoblotting with indicated antibodies .

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F. WT MEFs pretreated with 10 μg/ml FAC for 6 h or 24 h were chased with 20 μg/ml CHX and analyzed by immunoblotting with the indicated antibodies.


G . NCOA4 KO MEFs reconstituted with myc - hNCOA4 were pretreated with 10 μg / ml FAC then cultured with 20 μg / ml CHX or 200 nM bafilomycin A1 ( BafA ) . were fixed for imaging immunostained using myc LAMP1 antibodies . Scale bar , 10μm .

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G. NCOA4 KO MEFs reconstituted with myc-hNCOA4 were pretreated with 10 μg/ml FAC and then cultured with 20 μg/ml CHX or 200 nM bafilomycin A1 (BafA). Cells were fixed for imaging and immunostained using myc and LAMP1 antibodies. Scale bar, 10μm.


A . WT , ATG7 KO , TAX1BP1 KO , FIP200KO MEFs were treated or not treated with 10 μg / ml FAC for 12 h . lysates were fractionated analyzed by immunoblotting with indicated antibodies .

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A. WT, ATG7 KO, TAX1BP1 KO, and FIP200KO MEFs were treated or not treated with 10 μg/ml FAC for 12 h. Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.


C . HEK293T were transfected with GFP - NCOA4 indicated FLAG - His - TAX1BP1 variants . Soluble lysates extracted from transfected were immunoprecipitated with anti - FLAG antibody . Inputs immunoprecipitated samples were analyzed by immunoblotting with indicated antibodies .

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C. HEK293T cells were transfected with GFP-NCOA4 and the indicated FLAG-His-TAX1BP1 variants. Soluble lysates extracted from transfected cells were immunoprecipitated with anti-FLAG antibody. Inputs and immunoprecipitated samples were analyzed by immunoblotting with the indicated antibodies.


WT MEFs TAX1BP1 KO MEFs reconstituted with indicated TAX1BP1 variants were untreated or treated with 10 μg / ml FAC for 12 h . ( D , F ) lysates were fractionated analyzed by immunoblotting with indicated antibodies .

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WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were untreated or treated with 10 μg/ml FAC for 12 h. (D, F) Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.


WT MEFs TAX1BP1 KO MEFs reconstituted with indicated TAX1BP1 variants were treated with 10 μg / ml FAC for 12 h . ( E ) were fixed for imaging immunostained to analyze ferritin localization . Scale bar , 10μm .

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WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were treated with 10 μg/ml FAC for 12 h. (E) Cells were fixed for imaging and immunostained to analyze ferritin localization. Scale bar, 10μm.


A . MEFs were pretreated with 10 μg / ml FAC for 12 h then cultured with 100 μM Dfo for indicated time . were lysed with SDS - containing sample buffer or Triton buffer . Lysates were analyzed by immunoblotting with indicated antibodies .

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A. MEFs were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo for the indicated time. Cells were lysed with SDS-containing sample buffer or Triton buffer. Lysates were analyzed by immunoblotting with the indicated antibodies.


B . MEFs were treated with 20 μM Dfo for 12 h , 10 μg / ml FAC for 12h , or 100 μM Dfo for 6 h after treatment with 10 μg / ml FAC for 12 h . Triton - soluble lysates were subjected to glycerol gradient ultracentrifugation .

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B. MEFs were treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12h, or 100 μM Dfo for 6 h after treatment with 10 μg/ml FAC for 12 h. Triton-soluble lysates were subjected to glycerol gradient ultracentrifugation analysis.


C . WT TAX1BP1 KO MEFs were pretreated with 25 μg / ml FAC for 16 h then cultured with 100 μM Dfo . Lysates were fractionated then analyzed by immunoblotting with indicated antibodies . D . Soluble FTL bands in ( C ) were quantified by densitometry . Data are shown as means ± SD of three biological replicates .

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C. WT and TAX1BP1 KO MEFs were pretreated with 25 μg/ml FAC for 16 h and then cultured with 100 μM Dfo. Lysates were fractionated and then analyzed by immunoblotting with the indicated antibodies. D. Soluble FTL bands in (C) were quantified by densitometry. Data are shown as the means ± SD of three biological replicates.


E . TAX1BP1 KO MEFs ( sg1 clone ) stably expressing FLAG - OsTIR1 ( F74G ) FLAG - mAID - mTAX1BP1 were pretreated with 10 μg / ml FAC for 12 h , cultured with DMSO or 1 μM 5 - Ph - IAA for 2 h , then treated with 100 μM Dfo . Soluble lysates were analyzed by immunoblotting with indicated antibodies . 5 - Ph - IAA , a derivative of Auxin ; mAID , mini auxin - inducible

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E. TAX1BP1 KO MEFs (sg1 clone) stably expressing FLAG-OsTIR1 (F74G) and FLAG-mAID-mTAX1BP1 were pretreated with 10 μg/ml FAC for 12 h, cultured with DMSO or 1 μM 5-Ph-IAA for 2 h, and then treated with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies. 5-Ph-IAA, a derivative of Auxin; mAID, mini auxin-inducible degron


F . TAX1BP1 KO MEFs ( clone sg1 ) in which ATG7 was knocked down were pretreated with 10 μg / ml FAC for 12 h then cultured with 100 μM Dfo . Soluble lysates were analyzed by immunoblotting with indicated antibodies .

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F. TAX1BP1 KO MEFs (clone sg1) in which ATG7 was knocked down were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.


G . TAX1BP1 KO MEFs ( clone sg1 ) stably expressing myc - hNCOA4 pretreated with 25 μg / ml FAC for 16 h were cultured with 100 μM Dfo , soluble lysates were analyzed by immunoblotting with indicated antibodies .

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G. TAX1BP1 KO MEFs (clone sg1) stably expressing myc-hNCOA4 pretreated with 25 μg/ml FAC for 16 h were cultured with 100 μM Dfo, and soluble lysates were analyzed by immunoblotting with the indicated antibodies.


H , I . NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg / ml FAC for 12 h then chased with ( H ) 20 μg / ml CHX or ( I ) 100 μM Dfo for indicated times . Soluble lysates were analyzed by immunoblotting with indicated antibodies .

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H, I. NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg/ml FAC for 12 h and then chased with (H) 20 μg/ml CHX or (I) 100 μM Dfo for the indicated times. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.


J . MEFs expressing hNCOA4 - TurboID were treated with 10 μg / ml FAC for 3 h ( indicated as F ) or 100 μM DFO for 3 h after treatment with 10 μg / ml FAC for 12 h ( indicated as D ) , then cultured with DMSO or 50 μM biotin for 30 min . Triton - soluble lysates were pulled down by streptavidin beads . Inputs pulldown samples were analyzed by immunoblotting with indicated antibodies . black arrowheads indicate hNCOA4 - TurboID endogenous NCOA4 , respectively .

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J. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 3 h (indicated as F) or 100 μM DFO for 3 h after treatment with 10 μg/ml FAC for 12 h (indicated as D), and then cultured with DMSO or 50 μM biotin for 30 min. Triton-soluble cell lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed by immunoblotting with the indicated antibodies. The black and white arrowheads indicate hNCOA4-TurboID and endogenous NCOA4, respectively.


A . U2OS were either mock or treated with 10 μM XL413 for 30 minutes , at which point 4 mM HU was added further incubated for indicated times . Whole extracts were then analysed by western blotting with indicated antibodies . Reduction of phosphorylation of Ser40 / 41 on MCM2 is indicative of CDC7 inhibition by XL413 . Representative of two independent experiments .

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A. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells further incubated for the indicated times. Whole cell extracts were then analysed by western blotting with the indicated antibodies. Reduction of phosphorylation of Ser40/41 on MCM2 is indicative of CDC7 inhibition by XL413. Representative of two independent experiments.


B . U2OS were either mock treated or treated with 10 μM XL413 , 4 mM HU or both for 24 hours before performing neutral comet assays . Representative of comets are shown . Scale bar = 100 μM . In dot plots ~ 800 comets per each condition were analysed , means are indicated with a red their value shown above plot . Data are from two independent experiments .

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B. U2OS cells were either mock treated or treated with 10 μM XL413, 4 mM HU or both for 24 hours before performing neutral comet assays. Representative images of comets are shown. Scale bar = 100 μM. In the dot plots ~800 comets per each condition were analysed, means are indicated with a red line and their value shown above the plot. Data are from two independent experiments.


C . U2OS were either untreated or treated with 10 μM XL413 or with 5 µM AZD6738 alone or in combination , where indicated , after 30 minutes 4 mM HU was added for a further 2 hours . Whole extracts were analysed by western blotting with indicated antibodies . Total stain ( TPS ) as a loading control . Representative of two independent experiments .

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C. U2OS cells were either untreated or treated with 10 μM XL413 or with 5 µM AZD6738 alone or in combination and, where indicated, after 30 minutes 4 mM HU was added for a further 2 hours. Whole cell extracts were analysed by western blotting with the indicated antibodies. Total protein stain (TPS) as a loading control. Representative of two independent experiments.


D . U2OS were either untreated or treated with 4 mM HU for 5 hours in presence or absence of 10 μM XL413 which had either been added to 30 minutes prior ( pre - treatment ) or at same time ( co - treatment ) to addition of HU . Whole extracts were analysed by western blotting with indicated antibodies total stain ( TPS ) is displayed as a loading control . Representative of two independent experiments .

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D. U2OS cells were either untreated or treated with 4 mM HU for 5 hours in the presence or absence of 10 μM XL413 which had either been added to cells 30 minutes prior (pre-treatment) or at the same time (co-treatment) to the addition of HU. Whole cell extracts were analysed by western blotting with the indicated antibodies and total protein stain (TPS) is displayed as a loading control. Representative of two independent experiments.


B . CSK soluble - enriched fractions were analysed by western blotting with indicated antibodies . Representative of two independent experiments .

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B. CSK soluble and chromatin-enriched protein fractions were analysed by western blotting with the indicated antibodies. Representative of two independent experiments.


C - D . Flow cytometry to assess of pS139 Histone H2AX ( c ) . Histograms show mono - parametric of against pS139 Histone H2AX . Histograms are overlaid to appreciate changes in pS139 Histone H2AX upon treatment ( Red lines ) relative to appropriate experimental baseline controls ( Grey lines ) . Data are representative of two independent experiments .

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C-D. Flow cytometry analysis to assess levels of pS139 Histone H2AX (c). Histograms show the mono-parametric analysis of cell count against pS139 Histone H2AX intensity. Histograms are overlaid to appreciate changes in pS139 Histone H2AX intensity upon treatment (Red lines) relative to appropriate experimental baseline controls (Grey lines). Data are representative of two independent experiments.


A . U2OS were labelled with EdU for 30 minutes , then EdU was washed were further incubated for either 1 or 2 hours in presence of thymidine . At indicated time points were fixed binding to EdU - labelled captured by mediated Pull down technique ( DmChP ) . Graphical experimental outline is shown above by western blot of relevant in both input captured material .

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A. U2OS cells were labelled with EdU for 30 minutes, then EdU was washed and cells were further incubated for either 1 or 2 hours in presence of thymidine. At the indicated time points cells were fixed and proteins binding to EdU-labelled DNA captured by DNA mediated chromatin Pull down technique (DmChP). Graphical experimental outline is shown above the analysis by western blot of relevant proteins in both input and captured material.


B . U2OS were labelled with EdU for 30 minutes then treated with 4 mM HU for 2 hours in presence or absence of 10 μM XL413 . binding to EdU - labelled captured by DmChP were then analysed by western blot as above .

B. U2OS cells were labelled with EdU for 30 minutes and then treated with 4 mM HU for 2 hours in the presence or absence of 10 μM XL413. Proteins binding to EdU-labelled DNA captured by DmChP were then analysed by western blot as above.


C . As in panel B but incubation with HU was extended to 24 hours . Black arrow indicates MRE11 electrophoretic - mobility shift .

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C. As in panel B but incubation with HU was extended to 24 hours. Black arrow indicates MRE11 electrophoretic-mobility shift.


D . U2OS were either mock or treated with 10 μM XL413 for 30 minutes , at which point 4 mM HU was added incubated for further 24 hours . Extracts prepared from HU treated were then incubated in presence or absence of λ - phosphatase . were analysed by western blotting with anti - MRE11 antibodies . Total stain ( TPS ) is used as a loading control . Black arrow indicates MRE11 electrophoretic - mobility shift .

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D. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells incubated for further 24 hours. Extracts prepared from HU treated cells were then incubated in presence or absence of λ-phosphatase. Proteins were analysed by western blotting with anti-MRE11 antibodies. Total protein stain (TPS) is used as a loading control. Black arrow indicates MRE11 electrophoretic-mobility shift.


E . U2OS were treated with 4 mM HU in presence or absence of 10 μM XL413 for 24 hours . PCNA ( green ) MRE11 ( red ) were detected by immunofluorescence . Inset I - II represent enlargements of selected region of merged .

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E. U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 hours. PCNA (green) and MRE11 (red) were detected by immunofluorescence. Inset I-II represent enlargements of selected region of the merged images.


F . of PCNA MRE11 colocalization was assessed with ImageJ in ~ 70 randomly selected for each condition from four biological replicates expressed as Pearson ' s correlation coefficient . Error bars represent . Statistical significance was assessed by student t ( * p ˂ 0 .

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F. Quantification of PCNA and MRE11 colocalization was assessed with ImageJ in ~70 randomly selected cells for each condition from four biological replicates and expressed as Pearson's correlation coefficient. Error bars represent SEM. Statistical significance was assessed by student t test (*p˂0.05


A . MCF10A were either mock or treated with 10 μM XL413 labelled with IdU ( red ) for 30 minutes at which point 4 mM HU was added for 2 hours . HU was then washed off labelled with CldU ( green ) in continued presence or absence of XL413 . A set of representative fibres from each condition is shown of IdU ( red ) only tracts is plotted . At least 200 replication forks were analysed for each condition . Error bars represent from three biological repeats statistical significance was assessed by student t ( * * p ˂ 0 .

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A. MCF10A cells were either mock or treated with 10 μM XL413 and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from three biological repeats and statistical significance was assessed by the student t test (** p˂0.01


B . U2OS were either mock or treated with 10 μM XL413 , 50 μM Mirin or both , labelled with IdU ( red ) for 30 minutes at which point 4 mM HU was added for 2 hours . HU was then washed off labelled with CldU ( green ) in continued presence or absence of XL413 / or Mirin . A set of representative fibres from each condition is shown of IdU ( red ) only tracts is plotted . At least 200 replication forks were analysed for each condition . Error bars represent from four biological repeats statistical significance was assessed by student t ( * p < 0 . 05 , * * p ˂ 0 .

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B. U2OS cells were either mock or treated with 10 μM XL413, 50 μM Mirin or both, and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413 and/or Mirin. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from four biological repeats and statistical significance was assessed by the student t test (*p<0.05, ** p˂0.01


C . U2OS were either mock or pretreated for indicated times with 10 μM XL413 , 50 μM Mirin or both , labelled with CldU ( red ) . Then 50 nM CPT was added labelled with of IdU ( green ) for further 30 minutes . A set of representative fibres from each condition is shown IdU / CIdU tract is plotted . box extends from 25th to 75th percentile with in box representing median . Whiskers indicate 10 - 90 percentiles with data outside this range for outliers being plotted as dots . At least 100 replication forks were analyzed for each condition . P values were calculated using one way ANOVA Kruskal - Wallis Dunn ' s multiple comparison post ( ns , not significant , * * * * p < 0 . 0001 ) are related to experiment shown . Similar results were obtained in a second independent experiment .

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C. U2OS cells were either mock or pretreated for the indicated times with 10 μM XL413, 50 μM Mirin or both, and labelled with CldU (red). Then 50 nM CPT was added and cells labelled with of IdU (green) for further 30 minutes. A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment.


D . U2OS were transfected with control ( siLuc ) or MRE11 targeting siRNA ( siMRE11 ) . After 48 hours were either mock or pretreated with 10 μM XL413 labelled with CldU ( red ) for 30 minutes . Where indicated 50 nM CPT was added then were labelled with IdU ( green ) . A set of representative fibres from each condition is shown IdU / CIdU tract is plotted . box extends from 25th to 75th percentile with in box representing median . Whiskers indicate 10 - 90 percentileswith data outside this range for outliers being plotted as dots . At least 100 replication forks were analyzed for each condition . P values were calculated using one way ANOVA Kruskal - Wallis Dunn ' s multiple comparison post ( ns , not significant , * * * * p < 0 . 0001 ) are related to experiment shown . Similar results were obtained in a second independent experiment .

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D. U2OS cells were transfected with control (siLuc) or MRE11 targeting siRNA (siMRE11). After 48 hours cells were either mock or pretreated with 10 μM XL413 and labelled with CldU (red) for 30 minutes. Where indicated 50 nM CPT was added and then cells were labelled with IdU (green). A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentileswith data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment.


B . Representative western blot of indicated obtained from whole extracts . pS40 / 41 on MCM2 indicates effective CDC7 inhibition by XL413 .

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B. Representative western blot of indicated proteins obtained from whole cell extracts. pS40/41 on MCM2 indicates effective CDC7 inhibition by XL413.


C . Ratios of IdU / CldU tracts is plotted . box extends from 25th to 75th percentile with in box representing median . Whiskers indicate 10 - 90 percentiles with data outside this range for outliers being plotted as dots . Mean values of IdU / CldU ratios are indicated above plot . At least 100 replication forks were analyzed for each condition .

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C. Ratios of IdU/CldU tracts length is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. Mean values of IdU/CldU ratios are indicated above the plot. At least 100 replication forks were analyzed for each condition.


D . mean values of IdU / CldU tract ratios from three independent experiments are plotted , with standard deviation ( blue lines ) mean ( red ) . Statistical : student t - ; ns , not significant ; * * P < 0 . 01 , * * * P < 0 .

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D. The mean values of IdU/CldU tract length ratios from three independent experiments are plotted, with the standard deviation (blue lines) and mean (red line). Statistical analysis: student t-test; ns, not significant; **P<0.01, ***P < 0.001


E . Electron of representative replication forks from U2OS A regressed arm is labelled : P = parental strand ; D = daughter strand ; R = reversed fork ; four - way junction at reversed fork is magnified in inset . red arrows indicates single stranded at fork . Scale bar is 200 nm ( = 460 bp ) , 100 nm ( = 230 bp ) in inset .

E. Electron micrographs of representative replication forks from U2OS cells A regressed arm is labelled: P=parental strand; D=daughter strand; and R=reversed fork; the four-way junction at the reversed fork is magnified in the inset. The red arrows indicates single stranded DNA at the fork. Scale bar is 200 nm ( = 460 bp), 100 nm ( = 230 bp) in the inset.


F . Frequency of reversed replication forks isolated from mock - depleted ( siLuc ) BRCA2 - depleted ( siBRCA2 ) U2OS upon 5 hour treatment with 4 mM HU in presence absence of 10 μM XL413 . number of replication intermediates analyzed is indicated in parentheses . Similar results were obtained in two independent experiments ( Appendix

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F. Frequency of reversed replication forks isolated from mock-depleted (siLuc) and BRCA2-depleted (siBRCA2) U2OS cells upon 5 hour treatment with 4 mM HU in the presence and absence of 10 μM XL413. The number of replication intermediates analyzed is indicated in parentheses. Similar results were obtained in two independent experiments (Appendix Table


G . Amount of ssDNA at junction ( Red arrows in Figure 4D ) in siLuc siBRCA2 U2OS treated with 4 mM of HU for 5 hours in presence or absence of XL413 . N indicates number of forks observed , only molecules with detectable ssDNA stretches are included in . lines show mean of ssDNA regions at fork value is displayed above

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G. Amount of ssDNA length at the junction (Red arrows in Figure 4D) in siLuc and siBRCA2 U2OS cells treated with 4 mM of HU for 5 hours in the presence or absence of XL413. N indicates the number of forks observed, and only molecules with detectable ssDNA stretches are included in the analysis. The lines show the mean length of ssDNA regions at the fork and the value is displayed above the plot


A . Representative metaphase spread from BRCA2 depleted U2OS treated with HU . Scale bar = 5 μM . Arrows indicate chromosomes enlarged in insets , two of which show chromatid breaks ( *

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A. Representative metaphase spread from BRCA2 depleted U2OS cell treated with HU. Scale bar = 5 μM. Arrows indicate the chromosomes enlarged in the insets, two of which show chromatid breaks (*


B - C . graphs show average number of chromatid breaks per spread . In each experiment 30 chromosome spreads for each condition were analysed three independent experiments were performed . Error bars represent . Statistical significance assessed by student t ( * * p ˂ 0 . 01 , * * * p < 0 .

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B-C. The graphs show the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (** p˂0.01, *** p<0.001


D - E . U2OS AS - CDC7 were transfected with control ( siCon ) or BRCA2 ( siBRCA2 ) targeting siRNAs . 48 hours post transfection were either mock - treated or treated with 10 μM XL413 or 10 μM 3MB - PP1 respectively for 24 hours with Nocodazole added for last 16 hours of experiment . graph shows average number of chromatid breaks per spread . In each experiment 30 chromosome spreads for each condition were analysed three independent experiments were performed . Error bars represent . Statistical significance assessed by student t ( * p < 0 . 05 , * * * p < 0 .

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D-E. U2OS and AS-CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2) targeting siRNAs. 48 hours post transfection cells were either mock-treated or treated with 10 μM XL413 or 10 μM 3MB-PP1 respectively for 24 hours with Nocodazole added for the last 16 hours of the experiment. The graph shows the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (*p<0.05, *** p<0.001


B ) VEGF neutralizing antibody ( B20 ) failed to suppress BCE growth induced by LN - 229 CM , n = 3 . Data information : Bars error bars represent mean ± SD . experiments were carried out in three independent studies . Two - way ANOVA was used as statistical . ns , not statistically significant .

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B) VEGF neutralizing antibody (B20) failed to suppress BCE cell growth induced by LN-229 CM, n=3. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. ns, not statistically significant.


G ) LIF VEGF synergistically stimulated BCE growth . proliferation was analyzed after 6 days using alamar blue as described in Materials Methods , n = 3 . Data information : Bars error bars represent mean ± SD . experiments were carried out in three independent studies . Two - way ANOVA was used as statistical . ns , not statistically significant .

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G) LIF and VEGF synergistically stimulated BCE cell growth. Cell proliferation was analyzed after 6 days using alamar blue as described in Materials and Methods, n=3. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. ns, not statistically significant.


A ) JAK inhibitor baricitinib ( Ba ) blocked LIF - induced STAT3 phosphorylation . BCE were pre - incubated with DMSO , baricitinib ( 2 μM ) , cobimetinib ( Co ) ( 150 nM ) or BEZ235 ( BE ) ( 5 nM ) for 1 hour were then treated with vehicle or LIF ( 10 ng / ml , Sigma ) for 15 minutes . Ctrl , no pre - incubation with inhibitors .

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A) The JAK inhibitor baricitinib (Ba) blocked LIF-induced STAT3 phosphorylation. BCE cells were pre-incubated with DMSO, baricitinib (2 μM), cobimetinib (Co) (150 nM) or BEZ235 (BE) (5 nM) for 1 hour and were then treated with vehicle or LIF (10 ng/ml, Sigma) for 15 minutes. Ctrl, no pre-incubation with inhibitors.


C ) D ) STAT3 knockdown in BCE . BCE were transfected with siNegative siRNAs targeting STAT3 . qRT - PCR were performed to examine STAT3 mRNA . STAT3 level in siNegative was set as 1 . Data from three independent experiments were averaged are presented in C . In D , transfected with siRNAs were treated with LIF ( 10 ng / ml ) or vehicle for 15 minutes . Whole - lysates were subjected to Western blotting with indicated antibodies . Data information : Bars error bars represent mean ± SD . experiments were carried out in three independent studies . Two - way ANOVA was used as statistical .

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C) and D) STAT3 knockdown in BCE cells. BCE cells were transfected with siNegative and siRNAs targeting STAT3. qRT-PCR were performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and are presented in C. In D, cells transfected with siRNAs were treated with LIF (10 ng/ml) or vehicle for 15 minutes. Whole-cell lysates were subjected to Western blotting with the indicated antibodies. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


E ) LIF - induced BCE growth was abolished by STAT3 knockdown . BCE with STAT3 knockdown were cultured with LIF ( 10 ng / ml , Sigma ) or vehicle . proliferation was analyzed after 3 days . reading at 590 nm for each vehicle group was set as 1 , n = 3 . siNegative , negative control siRNA not targeting any known genes . Data information : Bars error bars represent mean ± SD . experiments were carried out in three independent studies . Two - way ANOVA was used as statistical .

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E) LIF-induced BCE cell growth was abolished by STAT3 knockdown. BCE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) or vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading at 590 nm for each vehicle group was set as 1, n=3. siNegative, negative control siRNA not targeting any known genes. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


B ) . JAK inhibitor baricitinib blocked activation of STAT3 by LIF . BAE pre - incubated with DMSO inhibitors for 1 hour were treated with vehicle LIF ( 10 ng / ml ) for 15 minutes . Whole - lysates were subjected to Western blotting with indicated antibodies . Ctrl , no pre - incubation with inhibitors ; Ba , baricitinib ( 2 μM ) ; Co , cobimetinib ( 150 nM ) ; BE , BEZ235 ( 5 nM ) .

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B). JAK inhibitor baricitinib blocked activation of STAT3 by LIF. BAE cells pre-incubated with DMSO and inhibitors for 1 hour were treated with vehicle and LIF (10 ng/ml) for 15 minutes. Whole-cell lysates were subjected to Western blotting with indicated antibodies. Ctrl, no pre-incubation with inhibitors; Ba, baricitinib (2 μM); Co, cobimetinib (150 nM); BE, BEZ235 (5 nM).


E ) . Knockdown of STAT3 in BAE . BAE were transfected with siRNAs targeting STAT3 . In E , transfected with siRNAs were treated with LIF ( 10 ng / ml , Sigma ) vehicle for 15 minutes . Whole - lysates were subjected to Western blotting with indicated antibodies .

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E). Knockdown of STAT3 in BAE cells. BAE cells were transfected with siRNAs targeting STAT3. In E, cells transfected with siRNAs were treated with LIF (10 ng/ml, Sigma) and vehicle for 15 minutes. Whole-cell lysates were subjected to Western blotting with indicated antibodies.


F ) . LIF - induced BAE growth inhibition was abolished by knockdown of STAT3 . BAE with STAT3 knockdown were cultured with LIF ( 10 ng / ml , Sigma ) vehicle . proliferation was analyzed after 3 days . reading for each vehicle group was set as 1 , n = 3 . Data information : Bars error bars represent mean ± SD . experiments were carried out in three independent studies . siNegative , negative control siRNA not targeting any known genes . Two - way ANOVA was used as statistical .

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F). LIF-induced BAE cell growth inhibition was abolished by knockdown of STAT3. BAE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) and vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading for each vehicle group was set as 1, n=3. Data information: Bars and error bars represent mean±SD. All experiments were carried out in three independent studies. siNegative, negative control siRNA not targeting any known genes. Two-way ANOVA was used as statistical test.


C , D ) OCT - A imaging of LIF - treated mouse retina . Adult mice were intravitreally injected with 1 μl of LIF ( 50 ng , Sigma ) or vehicle solution ( PBS ) . Retinal OCT - A were obtained 7 days after injection representative are shown . Blood vessel density was determined as of vessel - covered / total surface using ImageJ software shown in D ) , n = 7 - 8 . Data information : Bars error bars represent mean ± . experiments were carried out in three independent studies . Two - way ANOVA was used as statistical .

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C, D) OCT-A imaging of LIF-treated mouse retina. Adult mice were intravitreally injected with 1 μl of LIF (50 ng, Sigma) or vehicle solution (PBS). Retinal OCT-A images were obtained 7 days after the injection and representative images are shown. Blood vessel density was determined as percentage of vessel-covered area/total area surface using ImageJ software and shown in D), n=7-8. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


E ) . STAT3 was phosphorylated in retinal ECs after LIF treatment . 50 ng of our in - house LIF with low - endotoxin was injected . Phospho - Stat3 was examined on cryosection of retina after 2 hour by . Magnification in dashed box is shown in lower panel . n = 5 . Scale bar = 100 µm .

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E). STAT3 was phosphorylated in retinal ECs after LIF treatment. 50 ng of our in-house LIF with low-endotoxin levels was injected. Phospho-Stat3 was examined on cryosection of retina after 2 hour by IHC. Magnification in the dashed box is shown in the lower panel. n = 5. Scale bar = 100 µm.


F ) . Five - day old neonatal mice were intravitreally injected with LIF ( 50 ng ) or vehicle solution ( PBS ) . After 3 days , retinas were subjected to IF with Dyight - 488 - labeled lectin . Representative for similar ocular loci of lectin - labeled using ImageJ software were shown , n = 4 . Scale bar = 100 µm . Data information : Bars error bars represent mean ± . experiments were carried out in three independent studies . Two - way ANOVA was used as statistical .

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F). Five-day old neonatal mice were intravitreally injected with LIF (50 ng) or vehicle solution (PBS). After 3 days, retinas were subjected to IF staining with Dyight-488-labeled lectin. Representative images for similar ocular loci and quantification of lectin-labeled area using ImageJ software were shown, n=4. Scale bar = 100 µm. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


A ) . LIF enhanced - induced CNV . Intravitreal administration of LIF ( 10 ng 100 ng , Sigma ) OSM ( 10 ng ) after - induced CNV . PBS was used as vehicle control . OCT - A imaging immunostaining of choroid flatmounts was carried out 10 days after induction . of CNV is shown . Dashed circles indicate CNV in OCT - A . n = 5 . Dot plot shows data points from three independent experiments . Scale bar = 100 µm .

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A). LIF enhanced laser-induced CNV. Intravitreal administration of LIF (10 ng and 100 ng, Sigma) and OSM (10 ng) after laser-induced CNV. PBS was used as vehicle control. OCT-A imaging and immunostaining of choroid flatmounts was carried out 10 days after laser induction. Quantification of CNV area is shown. Dashed line circles indicate the CNV in OCT-A images. n = 5. Dot plot shows all data points from three independent experiments. Scale bar = 100 µm.


D ) . Avascular in choroid in sodium iodate model was determined quantified using Image J . E ) . Thickness of retina in H & E was quantified bar graph was shown using ImageJ . Data information : Bars error bars represent mean ± . experiments were carried out in three independent studies . Two - way ANOVA was used as statistical .

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D). Avascular area in choroid in sodium iodate model was determined and quantified using Image J. E). Thickness of retina in H&E staining was quantified and bar graph was shown using ImageJ. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.


( G ) Human choroidal EC ( HCEC ) mouse retinal EC ( MREC ) were treated with LIF 10 ng , LIF 100 ng for 30 min . ( PBS was used as control ) . STAT3 phosphorylation was detected by western blot .

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(G) Human choroidal EC (HCEC) and mouse retinal EC (MREC) were treated with LIF 10 ng, LIF 100 ng for 30 min. (PBS was used as control). STAT3 phosphorylation was detected by western blot.


( H ) H & E of LIFR CD31 in mouse eye sections . magnified of dashed boxes , a b , are shown on right panel . Box a shows LIFR in retina box b showed LIFR in RPE / choroid layer .

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(H) H&E staining of LIFR and CD31 in mouse eye sections. The magnified images of the dashed line boxes, a and b, are shown on the right panel. Box a shows LIFR staining in retina and box b showed LIFR staining in RPE/choroid layer.


( I ) Whole mount of LIFR CD31 in mouse retina . for CD31 ( green ) LIFR ( red ) is shown . arrows indicate co - of CD31 LIFR on retinal ECs .

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(I) Whole mount staining of LIFR and CD31 in mouse retina. The fluorescent staining for CD31 (green) and LIFR (red) is shown. White arrows indicate the co-staining of CD31 and LIFR on retinal ECs.


Violin plots of log scale normalized expression of LIF receptor subunits , IL6ST ( A ) , LIFR ( B ) , LIF ( C ) , IL6R ( D ) in types in human choroid Note wide distribution of IL6ST ( gp130 ) , common subunit to receptors of IL - 6 family ( A ) . In contrast , LIFR is strongly expressed in ECs to a lesser extent in fibroblasts pericytes ( B ) . LIF is expressed in fibroblasts , macrophages / monocytes pericytes minimally in ECs ( C ) . specific IL - 6 receptor ( IL6R ) is highly expressed in macrophage / monocytes ( D ) , while LIFR has little expression in these ( B ) .

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Violin plots of the log scale normalized expression of LIF receptor subunits, IL6ST (A), LIFR (B), LIF (C), IL6R (D) in all cell types in the human choroid Note the wide distribution of IL6ST (gp130), the common subunit to all receptors of the IL-6 family (A). In contrast, LIFR is strongly expressed in ECs and to a lesser extent in fibroblasts and pericytes (B). LIF is expressed in fibroblasts, macrophages/monocytes and pericytes and minimally in ECs (C). The specific IL-6 receptor (IL6R) is highly expressed in macrophage/monocytes (D), while LIFR has little expression in these cells (B).


Violin plots of log scale normalized expression of LIF receptor subunits KDR ( E ) in types in human choroid KDR shows , as expected , high expression selectively in EC ( E ) .

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Violin plots of the log scale normalized expression of LIF receptor subunits KDR (E) in all cell types in the human choroid KDR shows, as expected, high expression selectively in EC (E).


A , A targeted MAPPIT screen identifies several DUBs as putative RAS interactors . A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait . pSEL ( + 2L ) - HRAS G12V was expressed in HEK293T together with indicated prey . BRAF served as a positive control . Each measurement was done in triplicate . results are expressed as a mean of normalized luciferase activity ( leptin - treated vs leptin - untreated ) . overall mean value + 2 s . d . served as a threshold .

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A, A targeted MAPPIT screen identifies several DUBs as putative RAS interactors. A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait. pSEL(+2L)-HRAS G12V was expressed in HEK293T cells together with the indicated prey. BRAF served as a positive control. Each measurement was done in triplicate. The results are expressed as a mean of normalized luciferase activity (leptin-treated cells vs leptin-untreated cells). The overall mean value + 2 s.d. served as a threshold.


B , MAPPIT confirms interaction between OTUB1 RAS . pSEL ( + 2L ) vectors coding RAS were expressed in HEK293T together with indicated prey . Empty vector two random baits , MAL eDHFR , were used as negative controls . REM2 EFHA1 preys that bind to bait receptor itself was used to evaluate expression of RAS baits . results are expressed as a mean of normalized luciferase activity ± s . e . m ( leptin - treated vs leptin - untreated ) , n = 3 .

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B, MAPPIT assay confirms the interaction between OTUB1 and RAS proteins. pSEL(+2L) vectors coding RAS proteins were expressed in HEK293T cells together with the indicated prey. Empty vector and two random baits, MAL and eDHFR, were used as negative controls. REM2 and EFHA1 preys that bind to the bait receptor itself was used to evaluate expression of the RAS baits. The results are expressed as a mean of normalized luciferase activity ± s.e.m (leptin-treated cells vs leptin-untreated cells), n=3.


C , NRAS Q61K mutant co - immunoprecipitates with OTUB1 . At 48 hours post - transfection with Flag - tagged NRAS Q61K HA - tagged OTUB1 expression constructs , HA - tagged OTUB1 was immunoprecipitated with anti - HA - agarose followed by immunoblotting using anti - Flag or anti - HA antibodies .

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C, NRAS Q61K mutant co-immunoprecipitates with OTUB1. At 48 hours post-transfection with Flag-tagged NRAS Q61K and HA-tagged OTUB1 expression constructs, HA-tagged OTUB1 was immunoprecipitated with anti-HA-agarose followed by immunoblotting using anti-Flag or anti-HA antibodies.


D , OTUB1 interacts with wt NRAS active NRAS mutant . Flag - tagged NRAS were immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T overexpressing HA - tagged OTUB1 or empty vector ( V ) .

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D, OTUB1 interacts with wt NRAS and active NRAS mutant. Flag-tagged NRAS proteins were immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V).


E , GTP binding does not affect formation between NRAS OTUB1 . Recombinant Flag - tagged NRAS was incubated with lysates derived from HEK293T expressing HA - tagged OTUB1 in excess of GTP - γ - S or GDP , followed by immunoblotting with indicated antibodies .

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E, GTP binding does not affect the complex formation between NRAS and OTUB1. Recombinant Flag-tagged NRAS was incubated with lysates derived from HEK293T cells expressing HA-tagged OTUB1 in the excess of GTP-γ-S or GDP, followed by immunoblotting with the indicated antibodies.


F , OTUB1 interacts with wt KRAS . Flag - tagged KRAS was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T overexpressing HA - tagged OTUB1 or empty vector ( V ) . C , D , E , F , Immunoprecipitates , IP . Whole lysate , WCL .

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F, OTUB1 interacts with wt KRAS. Flag-tagged KRAS was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V). C, D, E, F, Immunoprecipitates, IP. Whole cell lysate, WCL.


G , OTUB1 co - localizes with KRAS at plasmamembrane . At 24 hours after co - transfection with GFP - tagged KRAS HA - tagged OTUB1 , HeLa were immunostained with anti - HA antibody . outlined areas are shown at higher magnification at top of each image . Scale bar , 10 micron .

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G, OTUB1 co-localizes with KRAS at the plasmamembrane. At 24 hours after co-transfection with GFP-tagged KRAS and HA-tagged OTUB1, HeLa cells were immunostained with anti-HA antibody. The outlined areas are shown at higher magnification at the top of each image. Scale bar, 10 micron.


A , Suppression of OTUB1 expression increases NRAS mono - di - ubiquitination . 6xHis - tagged ubiquitin Flag - NRAS were introduced into HEK293T expressing shGFP or shRNAs against OTUB1 . Ubiquitinated NRAS was purified by Co2 + metal affinity chromatography detected by anti - Flag antibody .

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A, Suppression of OTUB1 expression increases NRAS mono- and di-ubiquitination. 6xHis-tagged ubiquitin and Flag-NRAS were introduced into HEK293T cells expressing shGFP or shRNAs against OTUB1. Ubiquitinated NRAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody.


B , C , D , Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination . 6xHis - tagged ubiquitin RAS expression constructs were introduced into HEK293T expressing wt HA - OTUB1 , catalytically dead mutant HA - OTUB1 C91S , or empty vector ( V ) . Ubiquitinated RAS was purified by Co2 + metal affinity chromatography detected by anti - Flag antibody .

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B, C, D, Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination. 6xHis-tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA-OTUB1, the catalytically dead mutant HA-OTUB1 C91S, or empty vector (V). Ubiquitinated RAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody.


E , OTUB1 induces RAS re - localization . Confocal imaging of HeLa expressing indicated constructs . For each sample , z - stacks obtained by scanning sample from apical to basal layer . Step - , 2 micron .

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E, OTUB1 induces membrane RAS re-localization. Confocal imaging of HeLa cells expressing the indicated constructs. For each sample, z-stacks obtained by scanning the sample from the apical to the basal layer. Step-size, 2 micron.


F , RAS distribution expressed as of with specific RAS localization . For of RAS localization , were randomly imaged using IN Analyzer . RAS localization ( > 200 ) was scored as intracellular diffused ( IN ) , mostly at plasmamembrane ( PM ) , or both intracellular plasmamembrane ( PM / IN ) . p - value = 0 . 0005 as determined by Chi2 - , n = 3 . Representative of HeLa expressing GFP - tagged KRAS are shown in Appendix Figure S1 . Pull - down , PD . Whole lysate , WCL . Not modified , n . m .

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F, RAS cellular distribution expressed as percentage of cells with specific RAS localization. For quantification of RAS localization, cells were randomly imaged using IN Cell Analyzer. RAS localization (>200 cells) was scored as intracellular and diffused (IN), mostly at the plasmamembrane (PM), or both intracellular and plasmamembrane (PM/IN). p-value=0.0005 as determined by Chi2-test, n=3. Representative images of HeLa cells expressing GFP-tagged KRAS are shown in Appendix Figure S1. Pull-down, PD. Whole cell lysate, WCL. Not modified, n.m.


A , B , OTUB1 overexpression promotes serum - induced activation of endogenous wt RAS ( A ) or wt NRAS ( B ) . GTP - bound RAS was pulled down from HEK293T expressing HA - tagged OTUB1 or empty vector ( V ) using recombinant RAF1 RBD conjugated to agarose beads . Input was controlled by immunoblotting using anti - panRAS or anti - Flag

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A, B, OTUB1 overexpression promotes serum-induced activation of endogenous wt RAS (A) or wt NRAS (B). GTP-bound RAS was pulled down from HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) using recombinant RAF1 RBD conjugated to agarose beads. Input was controlled by immunoblotting using anti-panRAS or anti-Flag antibodies


C , D OTUB1 enhances serum - induced ERK1 / 2 phosphorylation in endogenous wt RAS ( C ) or wt KRAS ( D ) . of phosphorylated ERK1 / 2 total ERK1 / 2 in HEK293T expressing HA - tagged OTUB1 or empty vector ( V ) were analyzed by Meso Scale . results are expressed as a mean of pERK1 / 2 relative to total ERK1 / 2 ± s . e . m . n = 2 .

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C, D OTUB1 enhances serum-induced ERK1/2 phosphorylation in endogenous wt RAS (C) or wt KRAS (D). Levels of phosphorylated ERK1/2 and total ERK1/2 in HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m. n=2.


. E , F , Overexpression of wt OTUB1 ( E ) or catalytically dead mutant OTUB1 C91S mutant ( F ) promotes serum - induced MAPK activation in expressing wt NRAS . Whole lysates were analyzed by immunoblotting using indicated antibodies .

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. E, F, Overexpression of wt OTUB1 (E) or the catalytically dead mutant OTUB1 C91S mutant (F) promotes serum-induced MAPK activation in cell expressing wt NRAS. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies.


G , H , Overexpression of OTUB1 has no effect on serum - induced MAPK activation in expressing mutant NRAS Q61K or KRAS G12V . Whole lysates were analyzed by immunoblotting using antibodies as indicated . A , B , C , D , E , F , G , H Serum - starved HEK293T expressing indicated constructs were stimulated with 10 % serum for indicated time periods .

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G, H, Overexpression of OTUB1 has no effect on serum-induced MAPK activation in cell expressing mutant NRAS Q61K or KRAS G12V. Whole cell lysates were analyzed by immunoblotting using antibodies as indicated. A,B,C,D,E,F,G,H Serum-starved HEK293T cells expressing the indicated constructs were stimulated with 10% serum for the indicated time periods.


I , Immunoblot of OTUB1 overexpression in immortalized human embryonic kidney epithelial ( HEK ) , expressing empty vector ( V ) , myristoylated AKT1 ( myr - AKT ) , MEK1 D218 , D222 mutant ( MEKDD ) .

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I, Immunoblot of OTUB1 overexpression in immortalized human embryonic kidney epithelial cells (HEK TEST), expressing empty vector (V), myristoylated AKT1 (myr-AKT), MEK1 D218,D222 mutant (MEKDD).


J , K , OTUB1 cooperates with active AKT1 to promote anchorage - independent growth . Representative of soft - agar colonies formed by HEK expressing indicated constructs . number of soft agar colonies formed by expressing OTUB1 compared to expressing an empty vector . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - , n = 2 .

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J, K, OTUB1 cooperates with active AKT1 to promote anchorage-independent growth. Representative images of soft-agar colonies formed by HEK TEST cells expressing the indicated constructs. The number of soft agar colonies formed by cells expressing OTUB1 compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=2.


B , C , OTUB1 overexpression in TCGA lung carcinomas is associated with 11q13 . 1 copy number alteration . Pearson correlation of OTUB1 copy number ( log2 ) with OTUB1 mRNA ( RNAseq normalized read , log2 transformed ) was analyzed .

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B,C, OTUB1 overexpression in TCGA lung carcinomas is associated with 11q13.1 copy number alteration. Pearson correlation of OTUB1 copy number (log2 ratio) with OTUB1 mRNA levels (RNAseq normalized read counts, log2 transformed) was analyzed.


D , E , F , OTUB1 expression in TCGA lung adenocarcinoma ( LUAD ) lung squamous carcinoma ( SCC ) . were stratified according to their OTUB1 mRNA / or their KRAS status as described in Materials Methods . Box whisker plots represent OTUB1 mRNA expression in TCGA lung carcinoma determined by RNAseq . p - values were determined by two - sided t - . Total number of , n .

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D,E,F, OTUB1 expression in TCGA lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (SCC) patients. Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in Materials and Methods. Box whisker plots represent OTUB1 mRNA expression levels in TCGA lung carcinoma patients determined by RNAseq analysis. p-values were determined by two-sided t-Test. Total number of patients, n.


G , Gain of 11q13 . 1 locus is an early event in lung adenocarcinoma development . TCGA lung adenocarcinoma with diploid or gain of OTUB1 locus were stratified according stages ( T1 - T4 ) . Total numbers of , n . Statistical comparison of sample distributions were compared using Chi - square .

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G, Gain of 11q13.1 locus is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients with diploid or gain of the OTUB1 locus were stratified according tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.


H , OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development . TCGA lung adenocarcinoma were stratified by stages ( T1 - T4 ) OTUB1 expression as described in Materials Methods . Total numbers of , n . Statistical comparison of sample distributions were compared using Chi - square .

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H, OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients were stratified by tumor stages (T1-T4) and OTUB1 expression levels as described in Materials and Methods. Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.


I , KRAS mutation is a late event in lung adenocarcinoma progression . TCGA lung adenocarcinoma with different KRAS mutation status were stratified by stages ( T1 - T4 ) . Total numbers of , n . Statistical comparison of sample distributions were compared using Chi - square .

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I, KRAS mutation is a late event in lung adenocarcinoma progression. TCGA lung adenocarcinoma patients with different KRAS mutation status were stratified by tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.


, Suppression of OTUB1 expression in NSCLC lines by specific shRNAs against OTUB1 was confirmed by immunoblotting using antibody against OTUB1 .

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, Suppression of OTUB1 expression in NSCLC cell lines by specific shRNAs against OTUB1 was confirmed by immunoblotting using antibody against OTUB1.


B , OTUB1 suppression decreases serum - induced ERK1 / 2 phosphorylation . Serum - starved A549 expressing shRNAs against OTUB1 or GFP were stimulated with 10 % serum for indicated time periods . of phosphorylated ERK1 / 2 total ERK1 / 2 were analyzed by Meso Scale . results are expressed as a mean of pERK1 / 2 relative to total ERK1 / 2 ± s . e . m ; p - values were determined by two - sided t - , n = 2 .

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B, OTUB1 suppression decreases serum-induced ERK1/2 phosphorylation. Serum-starved A549 cells expressing shRNAs against OTUB1 or GFP were stimulated with 10% serum for the indicated time periods. Levels of phosphorylated ERK1/2 and total ERK1/2 were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m; p-values were determined by two-sided t-Test, n=2.


C , D , OTUB1 suppression impairs anchorage - independent growth of Western blot cancer lines . Representative of soft - agar colonies formed by Western blot expressing shRNAs against OTUB1 or GFP . number of soft agar colonies formed by expressing shOTUB1 compared to expressing shGFP . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - , n = 3 .

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C, D, OTUB1 suppression impairs the anchorage-independent growth of Western blot cancer cell lines. Representative images of soft-agar colonies formed by Western blot cells expressing shRNAs against OTUB1 or GFP. The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.


C , D , OTUB1 suppression impairs anchorage - independent growth of lung cancer lines . Representative of soft - agar colonies formed by lung expressing shRNAs against OTUB1 or GFP . number of soft agar colonies formed by expressing shOTUB1 compared to expressing shGFP . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - , n = 3 .

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C, D, OTUB1 suppression impairs the anchorage-independent growth of lung cancer cell lines. Representative images of soft-agar colonies formed by lung cells expressing shRNAs against OTUB1 or GFP. The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.


E , Xenograft growth of A549 expressing shRNAs against GFP or OTUB1 subcutaneously injected into nude mice . p - value was determined by 2 - way ANOVA .

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E, Xenograft tumor growth of A549 cells expressing shRNAs against GFP or OTUB1 subcutaneously injected into nude mice. p-value was determined by 2-way ANOVA.


A , OTUB1 overexpression promotes serum - induced MAPK activation in wt KRAS H1993 lung adenocarcinoma .

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A, OTUB1 overexpression promotes serum-induced MAPK activation in wt KRAS H1993 lung adenocarcinoma cells.


B , OTUB1 overexpression does not affect serum - induced MAPK activation in mutant KRAS A549 lung adenocarcinoma . A , B , Serum - starved lung expressing Flag - OTUB1 or an empty vector ( V ) were stimulated with 10 % serum for indicated time periods . Whole lysates were analyzed by immunoblotting using antibodies against pERK1 / 2 , ERK1 / 2 , Flag vinculin .

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B, OTUB1 overexpression does not affect serum-induced MAPK activation in mutant KRAS A549 lung adenocarcinoma cells. A,B, Serum-starved lung cells expressing Flag-OTUB1 or an empty vector (V) were stimulated with 10% serum for the indicated time periods. Whole cell lysates were analyzed by immunoblotting using antibodies against pERK1/2, ERK1/2, Flag and vinculin.


C , Representative immunoblots of Flag - tagged OTUB1 overexpression in lung adenocarcinoma

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C, Representative immunoblots of Flag-tagged OTUB1 overexpression in lung adenocarcinoma cell lines


D , E , OTUB1 overexpression affects anchorage - independent growth of lung adenocarcinoma lines . Representative of soft - agar colonies formed by indicated expressing Flag - OTUB1 or empty vector ( V ) . number of soft agar colonies formed by OTUB1 expressing compared to expressing an empty vector . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - , n = 3 .

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D, E, OTUB1 overexpression affects anchorage-independent growth of lung adenocarcinoma cell lines. Representative images of soft-agar colonies formed by the indicated cells expressing Flag-OTUB1 or empty vector (V). The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.


F , G , Xenograft growth of A549 , H1993 or H1437 expressing Flag - OTUB1 or an empty vector ( V ) subcutaneously injected into nude mice . p - values were determined by 2 - way ANOVA .

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F,G, Xenograft tumor growth of A549, H1993 or H1437 cells expressing Flag-OTUB1 or an empty vector (V) subcutaneously injected into nude mice. p-values were determined by 2-way ANOVA.


A , Representative examples of OTUB1 immunostaining in lung adenocarcinomas . Samples were scored as negative / low , medium or high as described in Materials Methods . Scale bar , 100 micron .

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A, Representative examples of OTUB1 immunostaining in lung adenocarcinomas. Samples were scored as negative/low, medium or high staining as described in Materials and Methods. Scale bar, 100 micron.


B , OTUB1 expression in lung non - small carcinomas with different KRAS mutation status . OTUB1 expression was assessed in TMA samples by .

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B, OTUB1 expression in lung non-small cell carcinomas with different KRAS mutation status. OTUB1 expression was assessed in TMA samples by IHC.


C , OTUB1 up - regulation is an early event in lung adenocarcinoma development . Lung adenocarcinoma expressing different of OTUB1 were stratified by stages ( T1 - T3 ) . Total number of , n .

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C, OTUB1 up-regulation is an early event in lung adenocarcinoma development. Lung adenocarcinoma patients expressing different levels of OTUB1 were stratified by tumor stages (T1-T3). Total number of patients, n.


D , Representative examples of phosphorylated - ERK1 / 2 immunostaining in lung adenocarcinomas . Samples were scored as negative / low , medium or high as described in Material Methods . Scale bar , 100 micron .

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D, Representative examples of phosphorylated-ERK1/2 immunostaining in lung adenocarcinomas. Samples were scored as negative/low, medium or high staining as described in Material and Methods. Scale bar, 100 micron.


E , OTUB1 correlates with increased of phosphorylated - ERK1 / 2 in wt KRAS lung adenocarcinomas . Association between OTUB1 pERK1 / 2 expression was assessed in same samples by .

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E, OTUB1 levels correlates with increased levels of phosphorylated-ERK1/2 in wt KRAS lung adenocarcinomas. Association between OTUB1 and pERK1/2 expression levels was assessed in the same tumor samples by IHC.


F , Ki67 expression in lung adenocarcinomas expressing different of OTUB1 . Ki67 expression was assessed by . of Ki67 - positive was counted , samples were scored as negative / low , < 10 % ; medium , between 10 40 % ; high , > 40 % . See Figure EV4 for representative of Ki67 .

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F, Ki67 expression in lung adenocarcinomas expressing different levels of OTUB1. Ki67 expression was assessed by IHC. The percentage of Ki67-positive cells was counted, and the samples were scored as negative/low, <10%; medium, between 10 and 40%; high, > 40%. See Figure EV4 for representative images of Ki67 staining.


G , Wt KRAS lung adenocarcinoma TCGA with medium OTUB1 expression have poorer overall survival .

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G, Wt KRAS lung adenocarcinoma TCGA patients with medium OTUB1 expression have poorer overall survival.


H , OTUB1 expression are not associated with survival rate of KRAS - mutant lung adenocarcinoma TCGA . G , H , were stratified according to their OTUB1 mRNA / or their KRAS status as described in Materials Methods . Overall survival of lung adenocarcinoma expressing different of OTUB1 mRNA as measured by Kaplan - Meier curves . p - values were determined by Log Rank .

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H, OTUB1 expression levels are not associated with survival rate of KRAS-mutant lung adenocarcinoma TCGA patients. G,H, Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in Materials and Methods. Overall survival of lung adenocarcinoma patients expressing different levels of OTUB1 mRNA as measured by Kaplan-Meier curves. p-values were determined by Log Rank Test.


( C ) Representative flow cytometry results showing CD71 , a surface marker , can be efficiently knocked - down in majority of organoid after 5 days of Dox TMP treatment . N = 3 different organoid lines 3 different gRNAs for CD71 were used . Mean ± is labelled .

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(C) Representative flow cytometry results showing CD71, a cell surface marker, can be efficiently knocked-down in the majority of organoid cells after 5 days of Dox and TMP treatment. N = 3 different organoid lines and 3 different gRNAs for CD71 were used. Mean ± SEM is labelled.


( A ) Human lung tip organoids faithfully express in vivo tip progenitor markers , SOX2 SOX9 . Upper panel : SOX2 / SOX9 dual expression in human lungs . Lower panel : SOX2 / SOX9 in human lung tip organoids . SOX9 green , SOX2 red , E - Cadherin . Data information : Scale bars denote 50 μm (

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(A) Human fetal lung tip organoids faithfully express in vivo tip progenitor markers, SOX2 and SOX9. Upper panel: SOX2/SOX9 dual expression in human fetal lungs. Lower panel: SOX2/SOX9 in human fetal lung tip organoids. SOX9 green, SOX2 red, E-Cadherin white. Data information: Scale bars denote 50 μm (A


( B ) qRT - PCR showing that SOX9 gRNAs effectively knocked down SOX9 transcript using inducible CRISPRi after 5 days of Dox TMP treatment . 3 different organoid lines 2 different SOX9 gRNAs were used . Error bars : mean ± . Two - sided Student ' s t - with equal variance * * * p < 0 . 001 .

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(B) qRT-PCR showing that SOX9 gRNAs effectively knocked down SOX9 transcript levels using inducible CRISPRi after 5 days of Dox and TMP treatment. 3 different organoid lines and 2 different SOX9 gRNAs were used. Error bars: mean ± SEM. Two-sided Student's t-test with equal variance ***p < 0.001.


( D ) CD44 expression in SOX9 + tip progenitor . SOX9 green , SOX2 red , CD44 . Data information : Scale bars = 50

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(D) CD44 protein expression in SOX9+ tip progenitor cells. SOX9 green, SOX2 red, CD44 white. Data information: Scale bars = 50 μm


( E ) LGR5 mRNA expression enrichment in SOX9 expressing tip progenitor . SOX9 red , LGR5 cyan . ( F ) Tip progenitor are of high WNT signalling activity . NOTUM yellow , SOX9 red . Data information : Scale bars = 50

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(E) LGR5 mRNA expression enrichment in SOX9 expressing tip progenitor cells. SOX9 red, LGR5 cyan. (F) Tip progenitor cells are of high WNT signalling activity. NOTUM yellow, SOX9 red. Data information: Scale bars = 50 μm


( H ) qRT - PCR showing rescue of SOX9 transcription after constitutively activated β - catenin overexpression in organoids cultured without WNT activators . N = 3 different organoid lines . Error bars : mean ± .

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(H) qRT-PCR showing rescue of SOX9 transcription after constitutively activated β-catenin overexpression in organoids cultured without WNT activators. N = 3 different organoid lines. Error bars: mean ± SEM.


( I ) WB showing SOX9 was rescued after constitutively activated β - catenin overexpression in organoids cultured without WNT activators .

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(I) WB showing SOX9 protein was rescued after constitutively activated β-catenin overexpression in organoids cultured without WNT activators.


( A , B ) HCR showing ETV5 ( A ) ETV4 ( B ) expression in SOX9 expressing tip progenitors . ETV4 / 5 red , SOX9 cyan . Data information : Scale bars denote 50 μm ( A , B )

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(A,B) HCR images showing ETV5 (A) and ETV4 (B) expression in SOX9 expressing tip progenitors. ETV4/5 red, SOX9 cyan. Data information: Scale bars denote 50 μm (A, B)


( C ) qRT - PCR showing ETV4 ETV5 dual knockdown in tip organoids . N = 3 different organoid lines 2 different ETV4 ETV5 gRNA combinations were used . Error bars : mean ± . Two paired t - : * * p < 0 . 01 ; * * * p < 0 . 001 .

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(C) qRT-PCR showing ETV4 and ETV5 dual knockdown in tip organoids. N =3 different organoid lines and 2 different ETV4 and ETV5 gRNA combinations were used. Error bars: mean ± SEM. Two tailed paired t-test: **p < 0.01; ***p < 0.001.


( F ) qRT - PCR of ETV4 , ETV5 SOX9 expression level in organoids in self - renewing medium self - renewing medium without RTK ligands . N = 3 different organoid lines used . Error bars : mean ± . Two - paired T - : * * p < 0 . 01 ; n . s . non - significant .

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(F) qRT-PCR of ETV4, ETV5 and SOX9 expression level in organoids in self-renewing medium and self-renewing medium without RTK ligands. N = 3 different organoid lines used. Error bars: mean ± SEM. Two-tailed paired T-test: **p < 0.01; n.s. non-significant.


Immunoblots qRT - PCRs showing that expression of miR - 203 , miR - 200 , E - cadherin are increased after ZEB1 knockdown in Panc1 , MDA - MB - 231 . Vice versa , drug - resistant clones of BxPC3 , H358 , DU - 145 show increased expression of ZEB1 decreased expression of miRNAs E - cadherin . n = 3 , mean ± , except for H358 ( data from microarray ) . Unpaired Student ' s t - .

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Immunoblots and qRT-PCRs showing that expression levels of miR-203, miR-200, and E-cadherin are increased after ZEB1 knockdown in Panc1, MDA-MB-231. Vice versa, the drug-resistant clones of BxPC3, H358, and DU-145 show increased expression of ZEB1 and decreased expression of the miRNAs and E-cadherin. n = 3, mean ± SEM, except for H358 (data from microarray). Unpaired Student's t-test.


Lentiviral overexpression of miR - 200c miR - 203 in Panc1 hPaca1 induces sensitivity to gemcitabine treatment as measured by MTT . For changes in EC50 values , see Table1 . n = 3 , mean ± , Dunnett ' s multiple comparisons ( P - values in graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

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Lentiviral overexpression of miR-200c and miR-203 in Panc1 and hPaca1 induces sensitivity to gemcitabine treatment as measured by MTT assay. For the changes in EC50 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


Overexpression of miR - 203 decreases expression of anti - apoptotic factor survivin sensitizes to gemcitabine - triggered apoptosis as evaluated by caspase - 3 in Western blot immunofluorescence . Panc1 hPaca1 were treated with 50 5 nM gemcitabine , respectively , for 48 h . Scale bar 20 μm .

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Overexpression of miR-203 decreases expression of the anti-apoptotic factor survivin and sensitizes to gemcitabine-triggered apoptosis as evaluated by cleaved caspase-3 in Western blot and immunofluorescence. Panc1 and hPaca1 were treated with 50 and 5 nM gemcitabine, respectively, for 48 h. Scale bar 20 μm.


MTT showing increase in gemcitabine resistance after inhibition of endogenous miRNAs in hPaca2 by specific antagomirs against miR - 203 or miR - 200 members . For changes in EC80 values , see Table1 . n = 3 , mean ± , Dunnett ' s multiple comparisons ( P - values in graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

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MTT assay showing increase in gemcitabine resistance after inhibition of endogenous miRNAs in hPaca2 by specific antagomirs against miR-203 or all miR-200 members. For the changes in EC80 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


Overexpression of miR - 203 shows reduced numbers of CD24 / CD44 double - positive cancer population as determined by FACS . arrow indicates reduction in CD24 high subpopulation reduction in CD133 by miR - 203 overexpression in hPaca1 .

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Overexpression of miR-203 shows reduced numbers of the CD24/CD44 double-positive cancer stem cell population as determined by FACS analysis. The arrow indicates the reduction in the CD24 high subpopulation and reduction in CD133 by miR-203 overexpression in hPaca1 cells.