B RT - qPCR results showing the relative expression level changes ( MII / GV ) of indicated transcripts . Fully grown GV oocytes of Pabpn1l - / - mice were microinjected with mRNAs encoding PABPN1L or BTG4 and were released from meiotic arrest at 12 h after microinjection . Total RNA was extracted from 10 oocytes in each sample . Error bars , S . E . M . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 by two - tailed Student ' s t - test compared to the first column . ns : non - significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RT-qPCR results showing the relative expression level changes (MII/GV) of indicated transcripts. Fully grown GV oocytes of Pabpn1l-/- mice were microinjected with mRNAs encoding PABPN1L or BTG4 and were released from meiotic arrest at 12 h after microinjection. Total RNA was extracted from 10 oocytes in each sample. Error bars, S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test compared to the first column. ns: non-significant.


C Western blot results showing the expression of exogenous PABPN1L ( full length , RRM - deleted , or R171A mutants ) and BTG4 in Pabpn1l - / - oocytes at the MII stage . The total protein from 100 oocytes is loaded in each lane . pERK1 / 2 is blotted to indicate the developmental stages . DDB1 is blotted as a loading control . At least three independent experiments were done with consistent results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot results showing the expression of exogenous PABPN1L (full length, RRM-deleted, or R171A mutants) and BTG4 in Pabpn1l-/- oocytes at the MII stage. The total protein from 100 oocytes is loaded in each lane. pERK1/2 is blotted to indicate the developmental stages. DDB1 is blotted as a loading control. At least three independent experiments were done with consistent results.


Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly ( A ) variants ( n = 6 ) . The RNA substrate comprised 20 non - poly ( A ) nucleotides followed by a 10 / 20 / 30 / 60 - adenosine poly ( A ) tail . PABPN1L - bound substrates were prepared with 2 / 1 / 0 . 5 / 0 . 2 / 0 . 1 RNA molecules per PABPN1L . The midpoint of the unfolding curve was determined as the melting temperature ( Tm ) .

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

Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly(A) variants (n = 6). The RNA substrate comprised 20 non-poly(A) nucleotides followed by a 10/20/30/60-adenosine poly(A) tail. PABPN1L-bound substrates were prepared with 2/1/0.5/0.2/0.1 RNA molecules per PABPN1L. The midpoint of the unfolding curve was determined as the melting temperature (Tm).


D RNA immunoprecipitation results showing the interaction of PABPN1L with indicated transcripts . Fully grown GV oocytes of WT mice were microinjected with mRNAs encoding PABPN1L ( WT , RRM - deleted , or R171A mutant ) , and were released from meiotic arrest at 12 h after microinjection . Total RNAs were extracted from 350 MI stage oocytes in each sample . RNAs recovered from the immunoprecipitants were subjected to RT - qPCR of the indicated transcripts . Fold change values of both Input and IP samples were normalized by RIP results of the FLAG - PABPN1L microinjection group . Error bars , S . E . M . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 by two - tailed Student ' s t - test comparing with RNA levels pulled - down by PABPN1L WT in the second column .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RNA immunoprecipitation results showing the interaction of PABPN1L with indicated transcripts. Fully grown GV oocytes of WT mice were microinjected with mRNAs encoding PABPN1L (WT, RRM-deleted, or R171A mutant), and were released from meiotic arrest at 12 h after microinjection. Total RNAs were extracted from 350 MI stage oocytes in each sample. RNAs recovered from the immunoprecipitants were subjected to RT-qPCR of the indicated transcripts. Fold change values of both Input and IP samples were normalized by RIP results of the FLAG-PABPN1L microinjection group. Error bars, S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test comparing with RNA levels pulled-down by PABPN1L WT in the second column.


A Western blot results showing the levels of the indicated proteins in WT and Pabpn1l - / - oocytes . Total protein from 100 oocytes is loaded in each lane . DDB1 is blotted as a loading 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

A Western blot results showing the levels of the indicated proteins in WT and Pabpn1l-/- oocytes. Total protein from 100 oocytes is loaded in each lane. DDB1 is blotted as a loading control.


B HeLa cells were transfected with plasmids expressing HA - BTG4 and FLAG - PABPN1L for 12 h and then treated by cycloheximide ( CHX , 10 μM ) . Cells were harvested at the indicated time points for western blots . DDB1 is a loading control .

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

B HeLa cells were transfected with plasmids expressing HA-BTG4 and FLAG-PABPN1L for 12 h and then treated by cycloheximide (CHX, 10 μM). Cells were harvested at the indicated time points for western blots. DDB1 is a loading control.


C Co - IP and western blot results showing the interaction of βTrCP1 with BTG4 in the presence or absence of PABPN1L .

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

C Co-IP and western blot results showing the interaction of βTrCP1 with BTG4 in the presence or absence of PABPN1L.


D Co - IP and western blot results showing BTG4 polyubiquitination . HeLa cells transfected with plasmids encoding the indicated proteins were lysed and subjected to IP with an anti - HA affinity gel . Input cell lysates and precipitates are immunoblotted with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O 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 Co-IP and western blot results showing BTG4 polyubiquitination. HeLa cells transfected with plasmids encoding the indicated proteins were lysed and subjected to IP with an anti-HA affinity gel. Input cell lysates and precipitates are immunoblotted with the indicated antibodies.


E Western blot results showing endogenous BTG4 levels in WT and Pabpn1l - / - oocytes at the MII stage . FLAG - PABPN1L was expressed in Pabpn1l - / - oocytes by mRNA microinjected at the GV stage . Total proteins from 100 oocytes are loaded in each lane . ERK1 / 2 is blotted as a loading control .

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

E Western blot results showing endogenous BTG4 levels in WT and Pabpn1l-/- oocytes at the MII stage. FLAG-PABPN1L was expressed in Pabpn1l-/- oocytes by mRNA microinjected at the GV stage. Total proteins from 100 oocytes are loaded in each lane. ERK1/2 is blotted as a loading control.


B : Hippocampal neurons transduced with GFP - TDP - 25 constructs ( DIV5 + 8 ) or GFP - TDP - 43 ( DIV5 + 4 due to higher toxicity ) were sequentially extracted with RIPA buffer followed by 2 % SDS buffer and analyzed via immuoblotting . Left panel shows representative immunoblot for GFP and loading control Calnexin . Right panel shows quantification of the ratio of the respective GFP densiometric signal in SDS extract to RIPA extract . Barplots showing mean ± SD from n = 3 independent experiments . TDP - 43 ( 0 . 039 ± 0 . 029 , mean ± CI ) vs TDP - 25 wt ( 1 . 22 ± 0 . 296 ) vs TDP - 25 mut ( 0 . 928 ± 0 . 564 ) : F ( 2 , 6 ) = 51 . 4 , * * * p = 0 . 000168 , η² = 0 . 94 . TDP - 43 vs TDP - 25 wt : * * * p = 1 . 67 * 10 - 4 , TDP - 43 vs TDP - 25 mut : p = 7 . 983 * 10 - 4 , TDP - 25 wt vs TDP - 25 mut : p = 0 . 1182 ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Hippocampal neurons transduced with GFP-TDP-25 constructs (DIV5+8) or GFP-TDP-43 (DIV5+4 due to higher toxicity) were sequentially extracted with RIPA buffer followed by 2% SDS buffer and analyzed via immuoblotting. Left panel shows representative immunoblot for GFP and loading control Calnexin. Right panel shows quantification of the ratio of the respective GFP densiometric signal in SDS extract to RIPA extract. Barplots showing mean ± SD from n=3 independent experiments. TDP-43 (0.039 ± 0.029, mean ± CI) vs TDP-25 wt (1.22 ± 0.296) vs TDP-25 mut (0.928 ± 0.564): F(2,6)=51.4, ***p=0.000168, η²=0.94. TDP-43 vs TDP-25 wt: ***p=1.67*10-4, TDP-43 vs TDP-25 mut: p=7.983*10-4, TDP-25 wt vs TDP-25 mut: p=0.1182,


( A - D ) Primary rat hippocampal neurons were transduced with GFP - TDP - 25 variants , GFP - NPM1 or Htt97Q - GFP and analyzed by fluorescence recovery after photobleaching ( FRAP ) at DIV5 + 8 , except Htt97Q - GFP which was analyzed at DIV5 + 6 to avoid excessive toxicity . Example images with indicated bleach regions ( dashed circles ) are shown ( A , C ) . Scale bar = 1 . 5 µm . Resulting normalized FRAP curves ( relative fluorescence intensity over time ) with values representing means ± SD are shown in ( B , D ) . ( B ) Comparison of the averaged recovery fraction of the last six timepoints obtained from three independent experiments . GFP - NPM1 ( 0 . 982 ± 0 . 019 , mean ± CI , n = 37 cells ) vs GFP - TDP - 25 wt ( 0 . 097 ± 0 . 013 , n = 35 cells ) vs GFP - TDP - 25 mut ( 0 . 085 ± 0 . 006 , n = 38 cells ) : H ( 1 ) = 74 . 134 , df = 2 , * * * p < 2 . 2 * 10 - 16 , η² [ H ] = 0 . 674 , Kruskal - Wallis Test . GFP - NPM1 vs GFP - TDP - 25 wt : * * * p < 2 * 10 - 16 , GFP - NPM1 vs GFP - TDP - 25 mut : p < 2 * 10 - 16 , GFP - TDP - 25 wt vs GFP - TDP - 25 mut : p = 0 . 11 , Pairwise Wilcoxon Rank Sum Tests with Benjamini - Hochberg correction . In ( D ) , the recovery fraction averaged at > 20 min timepoints was compared from four independent experiments . Htt97Q - GFP ( 0 . 094 ± 0 . 017 , n = 25 cells ) vs GFP - TDP - 25 wt ( 0 . 312 ± 0 . 041 , n = 23 cells ) vs GFP - TDP - 25 mut ( 0 . 271 ± 0 . 029 , n = 24 cells ) : H ( 1 ) = 46 . 376 , df = 2 , * * * p = 8 . 501 * 10 - 11 , η² [ H ] = 0 . 643 , Kruskal - Wallis Test . Htt97Q - GFP vs GFP - TDP - 25 wt * * * p = 1 . 9 * 10 - 11 , Htt97Q - GFP vs GFP - TDP - 25 mut * * * p = 4 . 3 * 10 - 12 , GFP - TDP - 25 wt vs GFP - TDP - 25 mut : p = 0 . 12 , Pairwise Wilcoxon Rank Sum Tests with Benjamini - Hochberg correction .

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

(A-D) Primary rat hippocampal neurons were transduced with GFP-TDP-25 variants, GFP-NPM1 or Htt97Q-GFP and analyzed by fluorescence recovery after photobleaching (FRAP) at DIV5+8, except Htt97Q-GFP which was analyzed at DIV5+6 to avoid excessive toxicity. Example images with indicated bleach regions (dashed circles) are shown (A, C). Scale bar = 1.5 µm. Resulting normalized FRAP curves (relative fluorescence intensity over time) with values representing means ± SD are shown in (B, D). (B) Comparison of the averaged recovery fraction of the last six timepoints obtained from three independent experiments. GFP-NPM1 (0.982 ± 0.019, mean ± CI, n=37 cells) vs GFP-TDP-25 wt (0.097 ± 0.013, n=35 cells) vs GFP-TDP-25 mut (0.085 ± 0.006, n=38 cells): H(1)=74.134, df=2, ***p<2.2*10-16, η²[H]=0.674, Kruskal-Wallis Test. GFP-NPM1 vs GFP-TDP-25 wt: ***p<2*10-16, GFP-NPM1 vs GFP-TDP-25 mut: p<2*10-16, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.11, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction. In (D), the recovery fraction averaged at >20 min timepoints was compared from four independent experiments. Htt97Q-GFP (0.094 ± 0.017, n=25 cells) vs GFP-TDP-25 wt (0.312 ± 0.041, n=23 cells) vs GFP-TDP-25 mut (0.271 ± 0.029, n=24 cells): H(1)=46.376, df=2, ***p=8.501*10-11, η²[H]=0.643, Kruskal-Wallis Test. Htt97Q-GFP vs GFP-TDP-25 wt ***p=1.9*10-11, Htt97Q-GFP vs GFP-TDP-25 mut ***p=4.3*10-12, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.12, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction.


E : Immunofluorescence of hippocampal neurons transduced with GFP , GFP - TDP - 25 wild - type or mutant and treated with MG132 ( 10 µM , 14h , DIV5 + 8 ) . Representative images of GFP - TDP - 25 inclusions and diffuse cytoplasmic staining . Automated quantification of GFP intensity normalized to the number of DAPI stained nuclei ( omitted here for clarity ) in MG132 treated cells compared to DMSO control . Counterstain to label the neuronal cytoskeleton ( MAP2 ) . Scale bar = 10 µm . Box plot as in Fig . 3C . GFP ( 1 . 23 ± 0 . 393 , n = 5 independent experiments ) vs GFP - TDP - 25 wt ( 2 . 11 ± 0 . 804 , n = 5 ) vs GFP - TDP - 25 mut ( 2 . 36 ± 1 . 16 , n = 5 ) : H ( 1 ) = 7 . 34 , df = 2 , p = 0 . 02548 , η² [ H ] = 0 . 445 , Kruskal - Wallis test . GFP vs GFP - TDP - 25 wt : * p = 0 . 048 , GFP vs GFP - TDP - 25 mut : * p = 0 . 048 , GFP - TDP - 25 wt vs GFP - TDP - 25 mut : p = 0 . 690 , Pairwise Wilcoxon Rank Sum Tests with Benjamini - Hochberg correction .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Immunofluorescence of hippocampal neurons transduced with GFP, GFP-TDP-25 wild-type or mutant and treated with MG132 (10 µM, 14h, DIV5+8). Representative images of GFP-TDP-25 inclusions and diffuse cytoplasmic staining. Automated quantification of GFP intensity normalized to the number of DAPI stained nuclei (omitted here for clarity) in MG132 treated cells compared to DMSO control. Counterstain to label the neuronal cytoskeleton (MAP2). Scale bar = 10 µm. Box plot as in Fig. 3C. GFP (1.23 ± 0.393, n=5 independent experiments) vs GFP-TDP-25 wt (2.11 ± 0.804, n=5) vs GFP-TDP-25 mut (2.36 ± 1.16, n=5): H(1)=7.34, df=2, p=0.02548, η²[H]=0.445, Kruskal-Wallis test. GFP vs GFP-TDP-25 wt: *p=0.048, GFP vs GFP-TDP-25 mut: *p=0.048, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.690, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction.


( C ) Representative FACS plot showing loss of hCD2 marker expression of Ch14 ICL MEFs after treatment with 4 - hydroxy tamoxifen ( 4OHT ) for 14 days to induce Cre , compared to Ethanol treated control cells ( See also Appendix figure S1A ) .

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

(C) Representative FACS plot showing loss of hCD2 marker expression of Ch14 ICL MEFs after treatment with 4-hydroxy tamoxifen (4OHT) for 14 days to induce Cre, compared to Ethanol treated control cells (See also Appendix figure S1A).


( D ) Shallow whole genome sequencing ( copy number profiles ) of large T antigen immortalized , MEFs after exposure to Cre recombinase and sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosomes 12 , 14 and 9 and 10 , without any in vitro culturing , post FACS sort .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Shallow whole genome sequencing (copy number profiles) of large T antigen immortalized, MEFs after exposure to Cre recombinase and sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10, without any in vitro culturing, post FACS sort.


( A ) Shallow whole genome sequencing ( copy number profiles ) of large T antigen immortalized , early passage MEFs after exposure to Cre recombinase and sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosomes 12 , 14 and 9 and 10 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Shallow whole genome sequencing (copy number profiles) of large T antigen immortalized, early passage MEFs after exposure to Cre recombinase and sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10.


( B ) Tumor growth curve after early passage ICL cells were injected into flanks of athymic nude mice ( n = 5 per group and error bars denote SEM , p < 0 . 05 for Ch9 and Ch14 ) .

O O O O O O O O O O O O O O O O O O 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) Tumor growth curve after early passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SEM, p<0.05 for Ch9 and Ch14).


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( A ) Ch12 Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (A) Ch12 Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( B ) Ch14 . Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (B) Ch14. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( C ) Ch9 Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (C) Ch9 Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Representative karyotypes of late passage , large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control ( hCD2 plus ) and ICL ( hCD2 Minus ) cells for chromosome lines ( D ) Ch10 . Boxed panels denote targeted chromosome loss ; arrows and asterisks denote shared and unshared , non - targeted chromosomal copy number variations respectively .

Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (D) Ch10. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch12 ( A ) . All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

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

Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch12 (A). All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch10 ( B ) All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

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

Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch10 (B) All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch14 ( C ) All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch14 (C) All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


Karyotypic analysis of later passage , control and ICL of a replicate ( # 1 ) of Ch9 ( D ) lines . All chromosomes are shown for 20 metaphases . Target chromosomes are boxed in red .

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

Karyotypic analysis of later passage, control and ICL of a replicate (#1) of Ch9 (D) lines. All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.


( A ) Tumor growth curve after later passage ICL cells were injected into flanks of athymic nude mice ( n = 5 per group and error bars denote SD , p < 0 . 0005 for Ch9 , p < 0 . 05 for Ch10 and p < 0 . 005 for Ch14 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tumor growth curve after later passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SD, p<0.0005 for Ch9, p<0.05 for Ch10 and p<0.005 for Ch14).


( B ) Tumors sections were stained with H & E to assess tumor histology ( scale bars denote 5mm for the inset and 200μm for the zoomed image ) Arrows in the ICL tumors sections indicate regions where tumors have invaded into the adjacent tissue .

(B) Tumors sections were stained with H&E to assess tumor histology (scale bars denote 5mm for the inset and 200μm for the zoomed image) Arrows in the ICL tumors sections indicate regions where tumors have invaded into the adjacent tissue.


( C ) Tumor growth curve after later passage ICL cells were injected into flanks of NOD / SCID mice ( n = 5 per group and error bars denote SD p < 0 . 0005 for Ch10 and p < 0 . 005 for Ch14 ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Tumor growth curve after later passage ICL cells were injected into flanks of NOD/SCID mice (n = 5 per group and error bars denote SD p<0.0005 for Ch10 and p<0.005 for Ch14).


( A ) Growth curve of the later passage ICL lines Ch10 and Ch14 under adherent in vitro culture conditions ( n = 3 for each data set and error bars denote SD , p < 0 . 005 for Ch10 and p < 0 . 0001 for Ch14 ) . ( See also Appendix Figure S3A )

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(A) Growth curve of the later passage ICL lines Ch10 and Ch14 under adherent in vitro culture conditions (n=3 for each data set and error bars denote SD, p<0.005 for Ch10 and p<0.0001 for Ch14). (See also Appendix Figure S3A)


( B ) Colony formation in ICL and control MEFs . 5000 cells were seeded in 10 cm plates and stained with Methylene blue after three days . Representative colony formation images in Ch10 and Ch14 are shown ( left ) and quantification of colonies formed ( right ) in the 4 ICL lines ( n = 3 for each data set and error bars denote SD , p < 0 . 005 for Ch10 , Ch12 and Ch14 and ns for Ch9 ) .

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(B) Colony formation in ICL and control MEFs. 5000 cells were seeded in 10 cm plates and stained with Methylene blue after three days. Representative colony formation images in Ch10 and Ch14 are shown (left) and quantification of colonies formed (right) in the 4 ICL lines (n=3 for each data set and error bars denote SD, p<0.005 for Ch10, Ch12 and Ch14 and ns for Ch9).


( C ) Quantification of growth in ultra - low adherent , sphere forming conditions for the 4 ICL lines ( n = 3 for Ch10 and Ch14 , n = 6 for Ch9 and Ch12 and error bars denote SD , p < 0 . 0001 for Ch9 , p < 0 . 001 for Ch10 and p < 0 . 05 for Ch12 and Ch14 ) .

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(C) Quantification of growth in ultra-low adherent, sphere forming conditions for the 4 ICL lines (n=3 for Ch10 and Ch14, n=6 for Ch9 and Ch12 and error bars denote SD, p<0.0001 for Ch9, p<0.001 for Ch10 and p<0.05 for Ch12 and Ch14).


( D ) Analysis of metabolites ( glucose consumed , lactate produced and glutamine consumed ) for the ICL lines . Spent media was analyzed after each ICL & control line was grown in culture for three days . Media in identical culture conditions , but with no plated cells , was used as a baseline for all samples .

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(D) Analysis of metabolites (glucose consumed, lactate produced and glutamine consumed) for the ICL lines. Spent media was analyzed after each ICL & control line was grown in culture for three days. Media in identical culture conditions, but with no plated cells, was used as a baseline for all samples.


( A ) Flow cytometry analysis of tail blood from Ch10 / + ( control ) at 30 days post tamoxifen administration , Ch10 / + ; Id1 Cre / + at 30 days post tamoxifen administration , and Ch10 / + ; Id1Cre / + at 80 days post tamoxifen administration .

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(A) Flow cytometry analysis of tail blood from Ch10/+ (control) at 30 days post tamoxifen administration, Ch10/+; Id1 Cre/+ at 30 days post tamoxifen administration, and Ch10/+; Id1Cre/+ at 80 days post tamoxifen administration.


( B ) Analysis from the Mitelman database for the fraction of human solid tumors that have lost ( top ) or gained ( bottom ) individual chromosomes , that have modal chromosome number > 2N or ~ 2N ( top , p < 0 . 0001 for chromosome 1 - 22 , X and p < 0 . 05 for chromosome Y ; bottom , p < 0 . 0001 for all chromosomes ) .

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(B) Analysis from the Mitelman database for the fraction of human solid tumors that have lost (top) or gained (bottom) individual chromosomes, that have modal chromosome number >2N or ~2N (top, p<0.0001 for chromosome 1-22, X and p<0.05 for chromosome Y; bottom, p<0.0001 for all chromosomes).


( C ) Analysis from the Mitelman database for the fraction of human solid tumors that have lost or gained any chromosome , that have modal chromosome number > 2N or ~ 2N ( p < 0 . 0001 for both modal number > 2N and ~ 2N ) .

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(C) Analysis from the Mitelman database for the fraction of human solid tumors that have lost or gained any chromosome, that have modal chromosome number >2N or ~2N (p<0.0001 for both modal number >2N and ~2N).


( A ) Chromosomal instability readouts , of later passage lines , assessed by average chromosomes loss per metaphase , average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes ( n = 20 for each dataset and error bars denote SD ) .

(A) Chromosomal instability readouts, of later passage lines, assessed by average chromosomes loss per metaphase, average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes (n=20 for each dataset and error bars denote SD).


( B ) Determination of number of γH2AX foci in ICL and control nuclei and quantification ( scale bars denote 10μm , p < 0 . 0001 for Ch9 , Ch10 and Ch12 , p < 0 . 05 for Ch14 ; n = 25 for each dataset and error bars denote SD ) . ( See also Figures S5A and S5B )

(B) Determination of number of γH2AX foci in ICL and control nuclei and quantification (scale bars denote 10μm, p<0.0001 for Ch9, Ch10 and Ch12, p<0.05 for Ch14; n=25 for each dataset and error bars denote SD). (See also Figures S5A and S5B)


( C ) Number of γH2AX positive cells in formalin fixed ICL and control tumor sections by immunofluorescence staining and quantification ( Scale bars denote 20μm , p < 0 . 0001 for Ch10 and p < 0 . 05 for Ch14 ) .

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(C) Number of γH2AX positive cells in formalin fixed ICL and control tumor sections by immunofluorescence staining and quantification (Scale bars denote 20μm, p<0.0001 for Ch10 and p<0.05 for Ch14).


Defects in midbrain - type dopamine ( mDA ) neuron - specific developmental factor expressions in the ventral midbrain ( VM ) of Lin28a / b cKO embryos . Embryonic VM sections from 3 WT and cKO animals each were immunoflorescent stained for the midbrain - specific markers in parallel and mean fluoresecent intensities ( MFI ) were measured . n = 20 cells for each group . * * * p < 0 . 0001 , t - test . Reduced mDA neurons in the embryonic VM ( E12 . 5 ) and adult SN at 2 - 7 months ( young age ) and at 14 - 18 months ( old age ) of the Lin28a / b cKO mice . * p < 0 . 05 , * * p < 0 . 005 , n = 4 each wild - type ( WT ) and cKO embryos , and 9 ( WT ) , 8 ( cKO ) in adult mice . Enhanced DA transporter ( DAT ) + and TH + fiber intensities of Lin28a / b cKO adult mice ( age : 4 - 7 months ) . Striatal sections of WT and cKO mice ( 3 animals for each group ) were subjected to immunofluorescent staining against DAT and TH in parallel , and MFI was measured from 101 microscopic fields of dorsolateral striatum .

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Defects in midbrain-type dopamine (mDA) neuron-specific developmental factor expressions in the ventral midbrain (VM) of Lin28a/b cKO embryos. Embryonic VM sections from 3 WT and cKO animals each were immunoflorescent stained for the midbrain-specific markers in parallel and mean fluoresecent intensities (MFI) were measured. n = 20 cells for each group. *** p<0.0001, t-test. Reduced mDA neurons in the embryonic VM (E12.5) and adult SN at 2-7 months (young age) and at 14-18 months (old age) of the Lin28a/b cKO mice. *p<0.05, **p<0.005, n=4 each wild-type (WT) and cKO embryos, and 9 (WT), 8 (cKO) in adult mice. Enhanced DA transporter (DAT)+ and TH+ fiber intensities of Lin28a/b cKO adult mice (age: 4-7 months). Striatal sections of WT and cKO mice (3 animals for each group) were subjected to immunofluorescent staining against DAT and TH in parallel, and MFI was measured from 101 microscopic fields of dorsolateral striatum.


DA uptakes assessed by nanoScanPET / MRI analysis using [ 18F ] FP - CIT . n = 9 ( WT ) , 8 ( cKO ) . * p < 0 . 05 , * * * p < 0 . 001 .

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DA uptakes assessed by nanoScanPET/MRI analysis using [18F]FP-CIT. n=9 (WT), 8 (cKO). *p<0.05, ***p<0.001.


Enhanced susceptibility of Lin28a / b cKO mice to the Parkinsonian toxin MPTP . WT and cKO mice were injected with MPTP ( 20mg / kg , i . p ) for 5 days and behaviors of the animals were assessed for 1 month after the MPTP

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Enhanced susceptibility of Lin28a/b cKO mice to the Parkinsonian toxin MPTP. WT and cKO mice were injected with MPTP (20mg/kg, i.p) for 5 days and behaviors of the animals were assessed for 1 month after the MPTP injection


Enhanced susceptibility of Lin28a / b cKO mice to the Parkinsonian toxin MPTP . The mouse brains at 1 month after initial MPTP exposure were subjected to immunohistochemical analyses for TH + mDA neuron counts at SN ( L ) and DAT MFI at striatum ( M ) .

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Enhanced susceptibility of Lin28a/b cKO mice to the Parkinsonian toxin MPTP. The mouse brains at 1 month after initial MPTP exposure were subjected to immunohistochemical analyses for TH+ mDA neuron counts at SN (L) and DAT MFI at striatum (M).


Cell growth and population doubling levels ( PDLs ) during passaging . In ( B ) , dots represent individual PDLs , boxes and bars represent mean ± SE of five PDL values of + / + and + / m lines . * Significantly different from control with p < 0 . 05 , ANOVA . Phase - contrast ( left ) , alkaline phosphatase - stained ( middle ) , and immunocytochemical images stained with pluripotent cell markers ( right ) . Images were taken at cell passage P8 ( + / + , + / m ) or P6 ( m / m ) . Scale bars , 100 µm .

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Cell growth and population doubling levels (PDLs) during passaging. In (B), dots represent individual PDLs, boxes and bars represent mean ± SE of five PDL values of +/+ and +/m lines. *Significantly different from control with p<0.05, ANOVA. Phase-contrast (left), alkaline phosphatase-stained (middle), and immunocytochemical images stained with pluripotent cell markers (right). Images were taken at cell passage P8 (+/+, +/m) or P6 (m/m). Scale bars, 100 µm.


Unsupervised hierarchical clustering heatmap of the gene expression ( ratios of mutant / WT ) categorized in " neurodegenerative disorders " ( ' Parkinson ' s disease ' , ' Alzheimer ' s disease ' , and ' Huntington ' s disease ' ) .

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Unsupervised hierarchical clustering heatmap of the gene expression (ratios of mutant/WT) categorized in "neurodegenerative disorders" ('Parkinson's disease', 'Alzheimer's disease', and 'Huntington's disease').


Neural differentiation of hESCs was induced on a feeder layer of MS5 stromal cells . Representative colonies differentiated from the WT ( + / + , c42 ) and homozygous mutant ( m / m , c38 ) hESCs are shown ( left ) . Neural induction efficiencies were estimated by % neuroepithelial colonies out of total colonies ( n = 5 ~ 7 dishes for each group ) . Neuroepithelial cell colonies were identified by neuroepithelial cell structure ( neural rosettes ) and by staining of markers specific for primitive neuroepithelial cells ( PLZF , N - CADHERIN , and SOX2 ) and rostral embryonic brain ( OTX2 ) . Scale bars , 100 µm . Ventral midbrain ( VM ) patterning was assessed during 24 days of + / + and + / m hESC differentiation ( chemically defined protocol ) . Each dot represents mRNA expression detected by qPCR ; boxes and bars represent means and SEs of + / + and + / m lines . The gene expression levels of + / + and + / m at day 24 are compared in the last graph ( n = 4 , * p < 0 . 05 or * * p < 0 . 001 , t - test ) .

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Neural differentiation of hESCs was induced on a feeder layer of MS5 stromal cells. Representative colonies differentiated from the WT (+/+, c42) and homozygous mutant (m/m, c38) hESCs are shown (left). Neural induction efficiencies were estimated by % neuroepithelial colonies out of total colonies (n=5~7 dishes for each group). Neuroepithelial cell colonies were identified by neuroepithelial cell structure (neural rosettes) and by staining of markers specific for primitive neuroepithelial cells (PLZF, N-CADHERIN, and SOX2) and rostral embryonic brain (OTX2). Scale bars, 100 µm. Ventral midbrain (VM) patterning was assessed during 24 days of +/+ and +/m hESC differentiation (chemically defined protocol). Each dot represents mRNA expression detected by qPCR; boxes and bars represent means and SEs of +/+ and +/m lines. The gene expression levels of +/+ and +/m at day 24 are compared in the last graph (n=4, *p<0.05 or **p<0.001, t-test).


Neural stem cell ( NSC ) expansion estimated by cell growth curve and PDL during NSC passages ( n = 12 , * p < 0 . 001 , t - test ) . NSCs were derived from neuroepithelial cell colonies ( shown in ( A ) ) .

Neural stem cell (NSC) expansion estimated by cell growth curve and PDL during NSC passages (n=12, *p<0.001, t-test). NSCs were derived from neuroepithelial cell colonies (shown in (A)).


The + / + ( c42 , c44 ) and + / m ( c27 , c43 ) cells which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . General cell survival ( calcein - AM ) / death ( EthD - 1 ) . ( B ) Cleaved caspase - 3 + apoptotic cell death . ( C ) Cellular senescence ( β - gal staining ) . ( D ) DNA damage estimated by γH2AX staining . Cells with > 4 γH2AX foci in the nucleus were counted . ( E ) Mitochondrial ROS ( MitoSox ) . ( F ) Mitochondrial membrane potential ( MitoID ) .

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The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. General cell survival (calcein-AM)/death (EthD-1). (B) Cleaved caspase-3+ apoptotic cell death. (C) Cellular senescence (β-gal staining). (D) DNA damage estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted. (E) Mitochondrial ROS (MitoSox). (F) Mitochondrial membrane potential (MitoID).


The + / + ( c42 , c44 ) and + / m ( c27 , c43 ) cells which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . Seahorse test . Terminal differentiation of neural stem cells ( NSCs ) was induced and the oxygen consumption rate ( OCR ) and extracellular acidification rate ( ECAR ) were analyzed on the NSCs at differentiation day 4 . p < 0 . 001 * , n = 7 ( WT ) , 10 ( mutant ) , t - test .

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The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Seahorse test. Terminal differentiation of neural stem cells (NSCs) was induced and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed on the NSCs at differentiation day 4. p<0.001*, n=7 (WT), 10 (mutant), t-test.


The + / + ( c42 , c44 ) and + / m ( c27 , c43 ) cells which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . Western blot analyses for ER stress marker proteins .

The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Western blot analyses for ER stress marker proteins.


The + / + ( c42 , c44 ) and + / m ( c27 , c43 ) cells which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . Resistance of mDA neurons against mitochondrial toxins . Differentiated mDA neurons were exposed to the toxins indicated for 1 h , and viable TH + mDA neurons were counted . Data are expressed as % TH + cells relative to untreated . Three cultures were analyzed for each cell line . N = 3 independent experiments , * Significance at p < 0 . 05 . Scale bars , 100 µm .

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The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Resistance of mDA neurons against mitochondrial toxins. Differentiated mDA neurons were exposed to the toxins indicated for 1 h, and viable TH+ mDA neurons were counted. Data are expressed as % TH+ cells relative to untreated. Three cultures were analyzed for each cell line. N=3 independent experiments, *Significance at p<0.05. Scale bars, 100 µm.


Representative in vitro phase - contrast ( A ) , TH / DAPI or TH ( insets ) ( B ) , and TH / SYNAPSIN - stained ( C ) images in the cultures terminally differentiated from mutant ( c13 , c19 ) and corrected hiPSCs ( c22 , c59 ) .

Representative in vitro phase-contrast (A), TH/DAPI or TH (insets) (B), and TH/SYNAPSIN-stained (C) images in the cultures terminally differentiated from mutant (c13, c19) and corrected hiPSCs (c22, c59).


DA neuron differentiation of DA neurons were assessed by DA neuronal yields


DA neuron morphological of DA neurons were assessed by TH + fiber

DA neuron morphological of DA neurons were assessed by TH+ fiber lengths


DA neuron , synaptical of DA neurons were assessed by synaptic puncta densities


DA neuron differentiation functional maturation of DA neurons were assessed by presynaptic DA neuron releases DA levels were measured in the media conditioned in the differentiated cultures for 24 h ( D15 ) and evoked by KCl - mediated depolarization for 30 min .

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DA neuron differentiation functional maturation of DA neurons were assessed by presynaptic DA neuron releases DA levels were measured in the media conditioned in the differentiated cultures for 24 h (D15) and evoked by KCl-mediated depolarization for 30 min.


DNA breaks estimated by γH2AX staining . Cells with > 4 γH2AX foci in the nucleus were counted .

DNA breaks estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted.


SA - β - gal staining was performed for cellular senescence analysis .

SA-β-gal staining was performed for cellular senescence analysis.


Mitochondrial ROS ( MitoSox ) were analyzed after terminal differentiation .

Mitochondrial ROS (MitoSox) were analyzed after terminal differentiation.


mitochondrial membrane potential ( MitoID ) were analyzed after terminal differentiation .

mitochondrial membrane potential (MitoID) were analyzed after terminal differentiation.


Accumulation of α - synuclein oligomers analyzed by western blotting . The mutant ( c13 ) and corrected ( c22 ) neural stem cells ( NSCs ) were transduced with α - synuclein - expressing lentiviruses . At 6 days after differentiation , cells were extracted and fractionated to Triton X - 100 ( 1 % ) - soluble and insoluble fractions . Shown is the representative WB image of three independent experiments . α - Synuclein oligomers are indicated with a bracket , while monomer bands are marked with an arrowhead .

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Accumulation of α-synuclein oligomers analyzed by western blotting. The mutant (c13) and corrected (c22) neural stem cells (NSCs) were transduced with α-synuclein-expressing lentiviruses. At 6 days after differentiation, cells were extracted and fractionated to Triton X-100 (1%)-soluble and insoluble fractions. Shown is the representative WB image of three independent experiments. α-Synuclein oligomers are indicated with a bracket, while monomer bands are marked with an arrowhead.


Amphetamine - induced rotation scores . The ipsilateral net rotation values of individual animals transplanted with differentiated mutant ( n = 7 ) and corrected ( n = 6 ) pt - hiPSCs are depicted . Significant differences from the mutant cell - transplanted at each post - transplantation time point are at * p < 0 . 05 or * * p < 0 . 001 . Cell engraftments measured by the integrated fluorescent intensities in the animals grafted with DiR - labeled pt and corrected hiPSCs . * p < 0 . 05 .

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Amphetamine-induced rotation scores. The ipsilateral net rotation values of individual animals transplanted with differentiated mutant (n=7) and corrected (n=6) pt-hiPSCs are depicted. Significant differences from the mutant cell-transplanted at each post-transplantation time point are at *p<0.05 or **p<0.001. Cell engraftments measured by the integrated fluorescent intensities in the animals grafted with DiR-labeled pt and corrected hiPSCs. *p<0.05.


Overviews of hNCAM + / TH + cell grafts at 8 weeks post - transplantation . Insets , TH + DA neuron morphology in grafts shown with high magnification .

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Overviews of hNCAM+/TH+ cell grafts at 8 weeks post-transplantation. Insets, TH+ DA neuron morphology in grafts shown with high magnification.


Co - expression of the mDA neuron - specific FOXA2 , LMX1A , NURR1 , DAT in TH + DA cells generated by the corrected pt - hiPSC transplantation .

Co-expression of the mDA neuron-specific FOXA2, LMX1A, NURR1, DAT in TH+ DA cells generated by the corrected pt-hiPSC transplantation.


Cell engraftments measured by the integrated fluorescent intensities in the animals grafted with DiR - labeled pt and corrected hiPSCs . * p < 0 . 05 . Total number of TH + cells in the graft ( TH + numbers / mm3 ) per animal .

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Cell engraftments measured by the integrated fluorescent intensities in the animals grafted with DiR-labeled pt and corrected hiPSCs. *p<0.05. Total number of TH+ cells in the graft (TH+ numbers/mm3) per animal.


LIN28A protein stability assays . HEK293 cells were transfected with pFLAG - WT - LIN28A and pFLAG - LIN28A R192G . Protein samples were prepared during 24 h of cycloheximide ( 50 μg / ml ) treatment . Representative western blot data for LIN28A protein level changes normalized to β - actin protein levels ( upper ) or with normalization based on initial ( 0 h ) LIN28A protein levels ( lower ) . Band intensities were quantified using ImageJ , and normalized values are depicted ( lower graph ) ( n = 5 ) .

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LIN28A protein stability assays. HEK293 cells were transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G. Protein samples were prepared during 24 h of cycloheximide (50 μg/ml) treatment. Representative western blot data for LIN28A protein level changes normalized to β-actin protein levels (upper) or with normalization based on initial (0 h) LIN28A protein levels (lower). Band intensities were quantified using ImageJ, and normalized values are depicted (lower graph) (n=5).


The proteasome inhibitor MG132 treatment increases LIN28A ( R192G ) protein level . After serum starvation for 3 hours , HEK293T cells expressing LIN28A ( R912G ) were treated with or without MG132 ( 0 . 1 - 10 uM ) for 30 minutes , and then followed by WB analyses .

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The proteasome inhibitor MG132 treatment increases LIN28A(R192G) protein level. After serum starvation for 3 hours, HEK293T cells expressing LIN28A(R912G) were treated with or without MG132 (0.1-10 uM) for 30 minutes, and then followed by WB analyses.


LC - MS / MS analysis . Immunoprecipitated FLAG - LIN28A WT was loaded on an SDS gel ( left upper ) . Bold red sequences indicate peptides matching LIN28A ( left lower ) . Right , LC - MS / MS spectra of WT LIN28A .

LC-MS/MS analysis. Immunoprecipitated FLAG-LIN28A WT was loaded on an SDS gel (left upper). Bold red sequences indicate peptides matching LIN28A (left lower). Right, LC-MS/MS spectra of WT LIN28A.


Cytoplasmic localization of WT LIN28A and LIN28A ( R192G ) proteins . HeLa cells transfected with pFLAG - WT - LIN28 and pFLAG - LIN28A ( R192G ) were stained with FLAG antibody and counterstained with DAPI .

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Cytoplasmic localization of WT LIN28A and LIN28A(R192G) proteins. HeLa cells transfected with pFLAG-WT-LIN28 and pFLAG-LIN28A(R192G) were stained with FLAG antibody and counterstained with DAPI.


LET - 7 microRNA regulatory activity of LIN28A . HeLa cells were transfected with plasmids expressing FLAG - tagged WT - LIN28A , LIN28AR192G , or mock pFLAG vector ( control ) , and Let - 7a , b , and c levels were quantified by RT - qPCR . The level of RNA was normalized to that of U6 snRNA . p < 0 . 05 , n = 26 from 6 independent experiments , ns = not significant , ANOVA .

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LET-7 microRNA regulatory activity of LIN28A. HeLa cells were transfected with plasmids expressing FLAG-tagged WT- LIN28A, LIN28AR192G, or mock pFLAG vector (control), and Let-7a, b, and c levels were quantified by RT-qPCR. The level of RNA was normalized to that of U6 snRNA. p<0.05, n=26 from 6 independent experiments, ns = not significant, ANOVA.


Affinity of LIN28A protein binding to its cognate mRNA 3 ′ UTR region . Upper : schematic for pmirGLO - LIN28A used in this assay , a bicistronic Firefly / Renilla luciferase ( FLuc / RLuc ) plasmid containing the part of the LIN28A 3 ′ UTR containing the putative LIN28A protein binding sites ( black dots ) Wilbert et al . , 2012 ( ) . Lower : HeLa cells were transiently co - transfected with pmirGLO - LIN28A 3 ′ UTR along with pFLAG - WT - LIN28A , pFLAG - LIN28A R192G , or mock pFLAG control . LIN28A protein - mRNA binding activities were determined by relative luciferase activity levels of FLuc normalized to those of RLuc . * p < 0 . 05 , n = 18 from 6 independent experiments .

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Affinity of LIN28A protein binding to its cognate mRNA 3′UTR region. Upper: schematic for pmirGLO-LIN28A used in this assay, a bicistronic Firefly/Renilla luciferase (FLuc/RLuc) plasmid containing the part of the LIN28A 3′UTR containing the putative LIN28A protein binding sites (black dots)Wilbert et al., 2012(). Lower: HeLa cells were transiently co-transfected with pmirGLO-LIN28A 3′UTR along with pFLAG-WT-LIN28A, pFLAG-LIN28A R192G, or mock pFLAG control. LIN28A protein-mRNA binding activities were determined by relative luciferase activity levels of FLuc normalized to those of RLuc. *p<0.05, n=18 from 6 independent experiments.


LIN28A - RHA co - IP assays in HEK293 cells transfected with pFLAG - WT - LIN28A and pFLAG - LIN28A R192G .

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LIN28A-RHA co-IP assays in HEK293 cells transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G.


A . Whole mount in situ hybridization ( WISH ) of evi1at 20 ( left ) and 32 ( middle ) hpf . evi1expression is visible in various structures of the brain , neuronal structures , the posterior pronephric duct ( ppnd ) and the branchial arches ( ba ) , as well as in the VDA ( ventral dorsal aorta ) region . Additionally , evi1co - localizes with the endothelial marker flk1 ( right ) .

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A. Whole mount in situ hybridization (WISH) of evi1at 20 (left) and 32 (middle) hpf. evi1expression is visible in various structures of the brain, neuronal structures, the posterior pronephric duct (ppnd) and the branchial arches (ba), as well as in the VDA (ventral dorsal aorta) region. Additionally, evi1co-localizes with the endothelial marker flk1 (right).


B , C . WISH of runx1 / c - myb ; in HSPCs ( B ) , mpo in neutrophils ( left ) and l - plastin ( right ) in monocytes / macrophages ( C ) at 36 hpf in control ( upper ) and evi1 MO ( lower ) injected embryos . D - G . WISH of globinin erythrocytes of 6 dpf old embryos ( D ) , of rag - 1in the thymus of 5 dpf embryos ( E ; red asterisk ) , of cd41at 52 hpf ( F ) , and gata2bat 32 hpf ( G ) for both control ( upper ) and evi1 morpholino ( lower ) injected embryos . H . Quantitation of results is shown for each gene , displaying the percentages of embryos with normal vs . changed expression in each condition . A Fisher ´ s exact test was applied to calculate statistical significance . P < 0 . 001 ( * * * ) .

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B, C. WISH of runx1/c-myb ;in HSPCs (B), mpo in neutrophils (left) and l-plastin (right) in monocytes/macrophages (C) at 36 hpf in control (upper) and evi1 MO (lower) injected embryos.D-G. WISH of globinin erythrocytes of 6 dpf old embryos (D), of rag-1in the thymus of 5 dpf embryos (E; red asterisk), of cd41at 52 hpf (F), and gata2bat 32 hpf (G) for both control (upper) and evi1 morpholino (lower) injected embryos.H. Quantitation of results is shown for each gene, displaying the percentages of embryos with normal vs. changed expression in each condition. A Fisher´s exact test was applied to calculate statistical significance. P < 0.001 (***).


I . WISH of runx1 / c - myb in HSPCs of uninjected control and UAS : mEvi1 plasmid DNA injected Tg ( - 1 . 5hsp70l : Gal4 ) embryos with heat - shock induction performed at 14 hpf . J . WISH of runx1 / c - myb in HSPCs of uninjected control and UAS : mEvi1 mRNA in Tg ( fli . 1 : Gal4FF ; UAS : RFP ) embryos , leading to endothelial - specific evi1 overexpression . K . Graph displays quantitation of results from I and J using the same method as above .

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I. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 plasmid DNA injected Tg(-1.5hsp70l:Gal4) embryos with heat-shock induction performed at 14 hpf.J. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 mRNA in Tg(fli.1:Gal4FF;UAS:RFP) embryos, leading to endothelial-specific evi1 overexpression.K. Graph displays quantitation of results from I and J using the same method as above.


A . WISH of notch1b ( upper ) and dll4 ( lower ) in both control ( left ) and evi1 MO injected embryos ( right ) .

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A. WISH of notch1b(upper) and dll4(lower) in both control (left) and evi1 MO injected embryos (right).


B . WISH of notch1b in uninjected ( left ) and UAS : mEvi1 plasmid DNA injected ( right ) Tg ( fli . 1 : Gal4FF ; UAS : RFP ) embryos resulting in endothelial - specific evi1 induction .

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B. WISH of notch1b in uninjected (left) and UAS:mEvi1 plasmid DNA injected (right) Tg(fli.1:Gal4FF;UAS:RFP) embryos resulting in endothelial-specific evi1 induction.


A . Confocal time - lapse live imaging was performed in Tg ( c - myb : GFP ; kdrl : mKate - CAAX ) embryos from 28 to 42 hpf ( Movie EV 1 ) . Shown are three representative time - points in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible , indicated by the white arrowheads . For each time - point merged images are shown . White arrowheads denote double - positive cells . B . Cell counts of total numbers of kdrl + cmyb1 + cells " in view " , dividing and respectively emerging HSPCs . n = 10 movies from n = 3 biological replicates were analyzed . A non - parametric Mann - Whitney test was used to test for statistical significance and error bars are shown as ± s . d .

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A. Confocal time-lapse live imaging was performed in Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos from 28 to 42 hpf (Movie EV 1). Shown are three representative time-points in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible, indicated by the white arrowheads. For each time-point merged images are shown. White arrowheads denote double-positive cells.B. Cell counts of total numbers of kdrl+cmyb1+ cells "in view", dividing and respectively emerging HSPCs. n=10 movies from n=3 biological replicates were analyzed. A non-parametric Mann-Whitney test was used to test for statistical significance and error bars are shown as ± s.d.


C . Gene expression analysis of endothelial ( kdrl , flt1 , dab2 , cdh5 ) and blood specific genes ( c - myb , CD45 , CD41 , runx1 ) in sorted kdrl + c - myb + cells from 34 hpf Tg ( c - myb : GFP ; kdrl : mKate - CAAX ) embryos . Cells were isolated from 15 - 25 pooled embryos per sample . Three biological experiments were performed with one representative shown . Error bars indicate s . d . of three technical replicates for each representative experiment .

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C. Gene expression analysis of endothelial (kdrl, flt1, dab2, cdh5) and blood specific genes (c-myb, CD45, CD41, runx1) in sorted kdrl+c-myb+ cells from 34 hpf Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos. Cells were isolated from 15-25 pooled embryos per sample. Three biological experiments were performed with one representative shown. Error bars indicate s.d.
of three technical replicates for each representative experiment.


A . WISH for runx1 / c - mybin 36 hpf evi1 morphant ( left ) and evi1 morphant transgenic Tg ( 5xUAS - E1b : 6xMYC - notch1a ; - 1 . 5hsp70l : Gal4 ) embryos ( right ) with global heat - shock ( HS ) induction at 20 hpf . B . WISH for runx1 / c - myb in 36 hpf evi1 morphant ( left ) and evi1 morphant transgenic Tg ( 5xUAS - E1b : 6xMYC - notch1a ; cdh5 : gal4ff ) embryos with endothelial - specific NICD induction ( right ) . C . WISH for runx1 / c - myb in 36 hpf evi1 morphant ( left ) and evi1 morphant transgenic Tg ( Hsp70l : vegfaa ; myl7 : eGFP ) embryos ( right ) with global heat - shock induction at 20 hpf . D . WISH for runx1 / c - mybin 32 hpf evi1 MO injected embryos ( left ) and embryos injected with both evi1 MO and capped gata2 mRNA ( right ) . E . Quantitation of all rescue experiments . Shown are the percentages of embryos displaying normal or changed runx1 / c - myb expression . A Fisher ´ s exact test was applied to calculate statistical significance . ( n . s . , not significant ; p < 0 . 001 ( * * * ) ; p < 0 . 01 ( * * ) ) .

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A. WISH for runx1/c-mybin 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right) with global heat-shock (HS) induction at 20 hpf.B. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;cdh5:gal4ff) embryos with endothelial-specific NICD induction (right).C. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(Hsp70l:vegfaa;myl7:eGFP) embryos (right) with global heat-shock induction at 20 hpf.D. WISH for runx1/c-mybin 32 hpf evi1 MO injected embryos (left) and embryos injected with both evi1 MO and capped gata2 mRNA (right).E. Quantitation of all rescue experiments. Shown are the percentages of embryos displaying normal or changed runx1/c-myb expression. A Fisher´s exact test was applied to calculate statistical significance. (n.s., not significant; p < 0.001 (***); p<0.01 (**)).


A . Confocal time - lapse live imaging was performed in Tg ( c - myb : GFP ; fli . 1 : UAS ; Gal4 : RFP ) evi1 morphant embryos ( top , Movie EV 2 ) or in Tg ( c - myb : GFP ; fli . 1 : UAS ; Gal4 : RFP ) crossed to Tg ( 5xUAS - E1b : 6xMYC - notch1a ) embryos prior to MO injection ( bottom , Movie EV 3 ) from 28 to 42 hpf . Shown are three representative time - points , in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible . For each time - point merged images are shown . NICD induction controlled by endothelial fli . 1 ( lower panel ) can rescue the evi1 morphant phenotype and increases the number of HSPCs cells emerging from the VDA ( arrows ) . B . Cell counts of emerging HSPCs in control and evi1 morphant fish with or without endothelial NICD induction . At least n = 5 movies from n = 3 biological replicates were analyzed . A non - parametric Mann - Whitney test was used to test for statistical significance and error bars are shown as ± s . d . ( n . s . , not significant , p < 0 . 05 ( * ) ) .

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A. Confocal time-lapse live imaging was performed in Tg(c-myb:GFP;fli.1:UAS;Gal4:RFP) evi1 morphant embryos (top, Movie EV 2) or in Tg(c-myb:GFP;fli.1:UAS;Gal4:RFP) crossed to Tg(5xUAS-E1b:6xMYC-notch1a) embryos prior to MO injection (bottom, Movie EV 3) from 28 to 42 hpf. Shown are three representative time-points, in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible. For each time-point merged images are shown. NICD induction controlled by endothelial fli.1 (lower panel) can rescue the evi1 morphant phenotype and increases the number of HSPCs cells emerging from the VDA (arrows).B. Cell counts of emerging HSPCs in control and evi1 morphant fish with or without endothelial NICD induction. At least n=5 movies from n=3 biological replicates were analyzed. A non-parametric Mann-Whitney test was used to test for statistical significance and error bars are shown as ± s.d. (n.s., not significant, p<0.05 (*)).


A . WISH of dll4 ( upper ) and notch1b ( lower ) in control injected embryos ( left ) , evi1 morphants ( middle ) and evi1 morphant transgenic Tg ( Hsp70l : vegfaa ; myl7 : eGFP ) embryos at 32 hpf after heat - shock induction at 20 hpf ( right ) .

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A. WISH of dll4(upper) and notch1b(lower) in control injected embryos (left), evi1 morphants (middle) and evi1 morphant transgenic Tg(Hsp70l:vegfaa;myl7:eGFP) embryos at 32 hpf after heat-shock induction at 20 hpf (right).


B , C . WISH of runx1 / c - myb ( B ) or notch1b ( C ) after treatment with DMSO ( left ) , the VEGF receptor inhibitor SU5461 ( middle ) and respectively SU5461 after injection of UAS : mEvi1 ( inducing endothelial evi1 expression ) in Tg ( fli . 1 : Gal4FF ; UAS : RFP ) embryos ( right ) .

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B, C. WISH of runx1/c-myb(B) or notch1b(C) after treatment with DMSO (left), the VEGF receptor inhibitor SU5461 (middle) and respectively SU5461 after injection of UAS:mEvi1 (inducing endothelial evi1 expression) in Tg(fli.1:Gal4FF;UAS:RFP) embryos (right).


A . Expression of runx1 / c - myb in the VDA after treatment with DMSO vehicle control ( left ) , the PI3K / AKT inhibitor Wortmannin ( WM , middle ) and after joined treatment with WM and enforced NICD expression via HS at 14 hpf in Tg ( 5xUAS - E1b : 6xMYC - notch1a ; - 1 . 5hsp70l : Gal4 ) embryos ( right ) . Graph displays quantitation of results . Shown are the percentages of embryos with normal or decreased runx1 / c - mybexpression for each condition .

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A. Expression of runx1/c-myb in the VDA after treatment with DMSO vehicle control (left), the PI3K/AKT inhibitor Wortmannin (WM, middle) and after joined treatment with WM and enforced NICD expression via HS at 14 hpf in Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased runx1/c-mybexpression for each condition.


B . Expression of notch1b after treatment with DMSO vehicle control ( left ) or Wortmannin ( WM , middle ) . Graph displays quantitation of results . Shown are the percentages of embryos with normal or decreased notch1b expression for each condition .

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B. Expression of notch1b after treatment with DMSO vehicle control (left) or Wortmannin (WM, middle). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased notch1b expression for each condition.


C . WISH for runx1 / c - myb in control injected embryos ( upper left ) , evi1 morphants ( upper right ) or respectively evi1 MO with myr - AKT injected embryos with HS at 14 hpf ( lower right ) and evi1 MO with UAS - myr - AKT injected transgenic Tg ( fli . 1 : Gal4FFubs3 ; UAS : RFP ) rk embryos . D . Quantitation of results from ( C ) . Shown are the percentages of embryos with normal or decreased runx1 / c - myb expression for each condition .

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C. WISH for runx1/c-myb in control injected embryos (upper left), evi1 morphants (upper right) or respectively evi1 MO with myr-AKT injected embryos with HS at 14 hpf (lower right) and evi1 MO with UAS-myr-AKT injected transgenic Tg(fli.1:Gal4FFubs3; UAS:RFP)rk embryos.D. Quantitation of results from (C). Shown are the percentages of embryos with normal or decreased runx1/c-myb expression for each condition.


E . WISH for notch1b in evi1 Morpholino injected ( left ) and evi1 plus UAS : myr - AKT injected transgenic Tg ( fli . 1 : Gal4FFubs3 ; UAS : RFP ) rk embryos ( right ) . Graph displays quantitation of results . Shown are the percentages of embryos with normal or decreased notch1b expression for each condition .

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E. WISH for notch1b in evi1 Morpholino injected (left) and evi1 plus UAS:myr-AKT injected transgenic Tg(fli.1:Gal4FFubs3; UAS:RFP)rk embryos (right). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased notch1b expression for each condition.


A - D Cells were infected at the indicated MOI . Viral replication ( Left ) and release ( Right ) were assessed by flow cytometry and RT - qPCR . A ) Caco2 / TC7 cells ( MOI 0 . 01 ) B ) Calu - 3 cells ( MOI 0 . 001 ) C ) Vero cells ( MOI 0 , 01 ) D ) primary human airway epithelial cells ( HAEC ) virus release ( Right ) and infectious virus release ( Left ) ( MOI 0 . 01 ) . Data are mean ± SD of at least 3 independent experiments . Statistical analysis : Mixed - effect analysis or Two ‐ way ANOVA compared to D614G reference , ns : non ‐ significant , * P < 0 . 05 , * * * * P < 0 . 0001 .

A-D Cells were infected at the indicated MOI. Viral replication (Left) and release (Right) were assessed by flow cytometry and RT-qPCR. A) Caco2/TC7 cells (MOI 0.01) B) Calu-3 cells (MOI 0.001) C) Vero cells (MOI 0,01) D) primary human airway epithelial cells (HAEC) virus release (Right) and infectious virus release (Left) (MOI 0.01). Data are mean ± SD of at least 3 independent experiments. Statistical analysis: Mixed-effect analysis or Two‐way ANOVA compared to D614G reference, ns: non‐significant, *P < 0. 05, ****P < 0.0001.


( B ) Left Panel : Fusion was quantified by GFP area / number of nuclei and normalized to D614G for U2OS - ACE2 20h post infection at MOI 0 . 001 . Right Panel : Representative images of U2OS - ACE2 20h post infection , GFP - Split ( Green ) and Hoechst ( Blue ) . Top and bottom are the same images with and without Hoechst channel .

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(B) Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for U2OS-ACE2 20h post infection at MOI 0.001. Right Panel: Representative images of U2OS-ACE2 20h post infection, GFP-Split (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel.


( C ) Left Panel : Quantified fusion of Vero cells infected at MOI 0 . 01 . Right Panel : Representative images of Vero cells 48h post infection , GFP - Split ( Green ) and Hoechst ( Blue ) .

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(C) Left Panel: Quantified fusion of Vero cells infected at MOI 0.01. Right Panel: Representative images of Vero cells 48h post infection, GFP-Split (Green) and Hoechst (Blue).


( B ) Left Panel : Fusion was quantified by GFP area / number of nuclei and normalized to D614G for each of the transfected variant S proteins . Right Panel : Representative images of Vero GFP - split cells 18h post - transfection , GFP ( Green ) and Hoechst ( Blue ) . Top and bottom are the same images with and without Hoechst channel . Scale bars : 200 µm .

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(B) Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.


( C ) Left Panel : Quantification of variant S protein mediated fusion in Vero - GFP split cells by video microscopy . Results are mean ± SD from three fields per condition from one representative experiment . Right Panel : Fusion quantification of at least 3 independent video microscopy experiments , 20h post transfection , normalized to D614G .

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(C) Left Panel: Quantification of variant S protein mediated fusion in Vero-GFP split cells by video microscopy. Results are mean ± SD from three fields per condition from one representative experiment. Right Panel: Fusion quantification of at least 3 independent video microscopy experiments, 20h post transfection, normalized to D614G.


( B ) Left Panel : Quantified fusion of the Alpha + E484K variant S protein normalized to D614G S . Right Panel : Representative images of fusion at 20h . Scale bar : 200 µm . Top and bottom are the same images with and without Hoechst channel .

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(B) Left Panel: Quantified fusion of the Alpha + E484K variant S protein normalized to D614G S. Right Panel: Representative images of fusion at 20h. Scale bar: 200 µm. Top and bottom are the same images with and without Hoechst channel.


( B ) Left Panel : EC50 values ( concentration of ACE2 needed for 50 % binding ) for Alpha and associated mutations . Colour code corresponds to location on S protein functional domains and lower EC50 values signifies higher affinity to ACE2 binding . Right Panel : EC50 values for Beta and associated mutations . Data set for N501Y and D614G reference mutations are duplicated between left and right panels as mutations are common to both variants .

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(B) Left Panel: EC50 values (concentration of ACE2 needed for 50% binding) for Alpha and associated mutations. Colour code corresponds to location on S protein functional domains and lower EC50 values signifies higher affinity to ACE2 binding. Right Panel: EC50 values for Beta and associated mutations. Data set for N501Y and D614G reference mutations are duplicated between left and right panels as mutations are common to both variants.


( A ) Vero GFP - split cells were transfected with variant S proteins and imaged 18h post - transfection . Left Panel : Fusion was quantified by GFP area / number of nuclei and normalized to D614G for each of the transfected variant S proteins . Right Panel : Representative images of Vero GFP - split cells 18h post - transfection , GFP ( Green ) and Hoechst ( Blue ) . Top and bottom are the same images with and without Hoechst channel . Scale bars : 200 µm .

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(A) Vero GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.


( D ) Caco2 GFP - split cells were transfected with variant S proteins and imaged 18h post - transfection . Left Panel : Fusion was quantified by GFP area / number of nuclei and normalized to D614G for each of the transfected variant S proteins . Right Panel : Representative images of Caco2 GFP - split cells 18h post - transfection , GFP ( Green ) and Hoechst ( Blue ) . Top and bottom are the same images with and without Hoechst channel . Scale bars : 200

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(D) Caco2 GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Caco2 GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm


dHepaRG - TR - A3A cells were infected with HBV at an MOI of 300 virions / cell . Cells were treated with tetracycline ( Tet ( A3A ) ) or / and 300 U / ml IFNα from day 9 to 18 p . i . cccDNA amplicons were analyzed by 3D ‑

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dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell. Cells were treated with tetracycline (Tet (A3A)) or/and 300 U/ml IFNα from day 9 to 18 p.i. cccDNA amplicons were analyzed by 3D‑PCR


dHepaRG cells were infected with HBV at an MOI of 100 virions / cell and treated 9 days later with 300 U / ml IFNα ( B ) or 200 U / ml IFNγ ( C ) for indicated times ( h : hours ) . Expression of indicated genes was analyzed by qRT - PCR relative to TATA - box binding protein ( TBP ) mRNA ( n = 6 biological replicates of two independent experiments ) .

dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated 9 days later with 300 U/ml IFNα (B) or 200 U/ml IFNγ (C) for indicated times (h: hours). Expression of indicated genes was analyzed by qRT-PCR relative to TATA-box binding protein (TBP) mRNA (n=6 biological replicates of two independent experiments).


Liver tissue samples obtained from HBV - negative patients undergoing metastasis resection ( Control ) , or patients with acute , self - limiting HBV infection or chronic hepatitis B , or chronic coinfection with HBV and hepatitis D virus ( HDV ) were stained for ISG20 by immunohistochemistry . For each clinical entity , tissue sections from three different patients are shown ; scale bar : 50 µm . ISG20 positive area of each sample ( n = 3 biological replicates ) was determined by Tissue IA image analysis software and is given in % of total tissue area scanned .

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Liver tissue samples obtained from HBV-negative patients undergoing metastasis resection (Control), or patients with acute, self-limiting HBV infection or chronic hepatitis B, or chronic coinfection with HBV and hepatitis D virus (HDV) were stained for ISG20 by immunohistochemistry. For each clinical entity, tissue sections from three different patients are shown; scale bar: 50 µm. ISG20 positive area of each sample (n=3 biological replicates) was determined by Tissue IA image analysis software and is given in % of total tissue area scanned.


dHepaRG cells were infected with HBV at an MOI of 100 virions / cell and treated with 600 U / ml IFNα or 400 U / ml IFNγ from day 10 to 14 p . i . and ISG20 was stained by immunofluorescence . Primary human hepatocytes were infected with HBV at an MOI of 100 virions / cell and treated with 500 U / ml IFNα or 200 U / ml IFNγ from day 4 to 6 p . i . and ISG20 was stained by immunofluorescence .

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dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated with 600 U/ml IFNα or 400 U/ml IFNγ from day 10 to 14 p.i. and ISG20 was stained by immunofluorescence. Primary human hepatocytes were infected with HBV at an MOI of 100 virions/cell and treated with 500 U/ml IFNα or 200 U/ml IFNγ from day 4 to 6 p.i. and ISG20 was stained by immunofluorescence.


dHepaRG cells were infected with HBV at an MOI of 500 virions / cell , treated with 600 U / ml IFNα or 400 U / ml IFNγ from day 7 to 9 p . i . and stained by immunofluorescence for ISG20 and PML ( C ) or ISG20 and nucleophosmin ( D ) .

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dHepaRG cells were infected with HBV at an MOI of 500 virions/cell, treated with 600 U/ml IFNα or 400 U/ml IFNγ from day 7 to 9 p.i. and stained by immunofluorescence for ISG20 and PML (C) or ISG20 and nucleophosmin (D).


dHepaRG cells were infected at an MOI of 100 virions / cell and transduced at day 7 p . i . with an adenoviral vector for expression of shRNA targeting ISG20 ( AdV - shISG20 ) . After 2 days , cells were treated with 300 U / ml IFNα or 200 U / ml IFNγ . ISG20 expression was analyzed by western blot after 7 days of IFNα or 12 days of IFNγ treatment , respectively .

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dHepaRG cells were infected at an MOI of 100 virions/cell and transduced at day 7 p.i. with an adenoviral vector for expression of shRNA targeting ISG20 (AdV-shISG20). After 2 days, cells were treated with 300 U/ml IFNα or 200 U/ml IFNγ. ISG20 expression was analyzed by western blot after 7 days of IFNα or 12 days of IFNγ treatment, respectively.


A subsequence of the 3D - PCR amplicon was used as ( - ) ssDNA oligomer , complementary ( + ) ssDNA and a dU - containing ( ‑ ) ssDNA , where all cytosines were replaced by uracils . These oligomers were digested in vitro with recombinant ISG20 with or without EDTA as indicated .

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A subsequence of the 3D-PCR amplicon was used as (-) ssDNA oligomer, complementary (+) ssDNA and a dU-containing (‑) ssDNA, where all cytosines were replaced by uracils. These oligomers were digested in vitro with recombinant ISG20 with or without EDTA as indicated.


dHepaRG - TR - A3A cells were infected with HBV at an MOI of 300 virions / cell and at day 7 p . i . transduced with AdV - ISG20 and treated with tetracycline to induce A3A expression as indicated ( ± Tet ( A3A ) ) for 7 days . Cell lysates were analyzed for cccDNA after T5 digest ( B ) and total intracellular HBV DNA ( C ) by qPCR relative to Prnp ( n = 5 - 6 biological replicates from two independent experiments ) .

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dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell and at day 7 p.i. transduced with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. Cell lysates were analyzed for cccDNA after T5 digest (B) and total intracellular HBV DNA (C) by qPCR relative to Prnp (n=5-6 biological replicates from two independent experiments).


dHepaRG - TR - A3A cells were infected with wildtype HBV ( HBVwt ) or HBV ∆ x at an MOI of 300 virions / cell and transduced on day 7 p . i . with AdV - ISG20 and treated with tetracycline to induce A3A expression as indicated ( ± Tet ( A3A ) ) for 7 days . HBeAg ( E ) was measured by ELISA and cccDNA after T5 digest ( F ) by qPCR relative to Prnp ( n = 6 biological replicates from two independent experiments ) .

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dHepaRG-TR-A3A cells were infected with wildtype HBV (HBVwt) or HBV∆x at an MOI of 300 virions/cell and transduced on day 7 p.i. with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. HBeAg (E) was measured by ELISA and cccDNA after T5 digest (F) by qPCR relative to Prnp (n=6 biological replicates from two independent experiments).


A BreastMark microarray platform ; Kaplan - Meier curves of overall survival ( OS ) , demonstrating that high expression of ESRP1 ( red line ) is associated with poor prognosis in ER + breast cancer . A log rank test was used to calculate P = 2 . 8 e - 07 ( n = 934 , number of events = 216 ) .

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A BreastMark microarray platform; Kaplan-Meier curves of overall survival (OS), demonstrating that high expression of ESRP1 (red line) is associated with poor prognosis in ER+ breast cancer. A log rank test was used to calculate P=2.8 e-07 (n= 934, number of events= 216).


B BreastMark microarray platform ; Kaplan - Meier curves of overall survival ( OS ) of ER - breast cancer . A log rank test was used to calculate P = 0 . 4481 ( n = 322 , number of events = 130 ) .

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B BreastMark microarray platform; Kaplan-Meier curves of overall survival (OS) of ER- breast cancer. A log rank test was used to calculate P=0.4481 (n= 322, number of events= 130).


C BreastMark microarray platform ; correlation of ESRP1 expression with overall survival in tamoxifen - treated patients . A log rank test was used to calculate P = 1 . 218 , e - 06 ( n = 210 , number of events = 49 ) .

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C BreastMark microarray platform; correlation of ESRP1 expression with overall survival in tamoxifen-treated patients. A log rank test was used to calculate P=1.218, e-06 (n= 210, number of events= 49).


D BreastMark microarray platform ; correlation of ESRP1 expression with overall survival in chemotherapy - treated patients . A log rank test was used to calculate the P = 0 . 28 ( n = 129 , number of events = 21 )

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D BreastMark microarray platform; correlation of ESRP1 expression with overall survival in chemotherapy-treated patients. A log rank test was used to calculate the P=0.28 (n= 129, number of events= 21)


E The Cancer Genome Atlas Breast Invasive Carcinoma ( TCGA - BRCA ) RNA - seq dataset ; Kaplan - Meier curves of overall survival ( OS ) in ER + breast cancer , demonstrating that high expression of ESRP1 ( red line ) is associated with poor prognosis in ER + . A log rank test was used to calculate P = 0 . 00011 ( n = 656 , number of events = 62 ) .

E The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) RNA-seq dataset; Kaplan-Meier curves of overall survival (OS) in ER+ breast cancer, demonstrating that high expression of ESRP1 (red line) is associated with poor prognosis in ER+. A log rank test was used to calculate P=0.00011 (n= 656, number of events= 62).


F The Cancer Genome Atlas Breast Invasive Carcinoma ( TCGA - BRCA ) RNA - seq dataset ; Kaplan - Meier curves of overall survival ( OS ) in ER - breast cancer . Red - high ESRP1 expression ; Black - low ESRP1 expression . A log rank test was used to calculate P = 0 . 19 ( n = 100 , number of events = 17 ) .

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F The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) RNA-seq dataset; Kaplan-Meier curves of overall survival (OS) in ER- breast cancer. Red-high ESRP1 expression; Black-low ESRP1 expression. A log rank test was used to calculate P=0.19 (n=100, number of events=17).


A RT - qPCR confirmed the stable knockdown of ESRP1 expression in endocrine - resistant cells using a shRNA lentiviral system ( Mission TRC human shRNA constructs - Sigma ) ; 2 - control ( LCC2 cells transduced with pLKO . 1 control vector ) and 2C1 and 2C3 ( LCC2 cells transduced with pLKO . 1 shRNA ESRP1 ) . Clones were selected for expression verification ( clone 1 and clone 3 ) ; 9 - control ( LCC9 cells transduced with pLKO . 1 control vector ) , 9C2 and 9C3 ( LCC9 cells transduced with pLKO . 1 shRNA ESRP1 . Clones were selected for expression verification ( clone 2 and clone 3 ) . * represents P < 0 . 05 ; statistically significant . Data ( mean ± SD ) were calculated using two - way ANOVA based on three independent biological replicates .

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A RT-qPCR confirmed the stable knockdown of ESRP1 expression in endocrine-resistant cells using a shRNA lentiviral system (Mission TRC human shRNA constructs-Sigma); 2-control (LCC2 cells transduced with pLKO.1 control vector) and 2C1 and 2C3 (LCC2 cells transduced with pLKO.1 shRNA ESRP1). Clones were selected for expression verification (clone 1 and clone 3); 9-control (LCC9 cells transduced with pLKO.1 control vector), 9C2 and 9C3 (LCC9 cells transduced with pLKO.1 shRNA ESRP1. Clones were selected for expression verification (clone 2 and clone 3). * represents P <0.05; statistically significant. Data (mean ± SD) were calculated using two-way ANOVA based on three independent biological replicates.


B Western blot analysis using ESRP1 antibody GTX 131373 ( GeneTex ) . GAPDH is used as the reference control .

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B Western blot analysis using ESRP1 antibody GTX 131373 (GeneTex). GAPDH is used as the reference control.


C Relative cell density was determined by crystal violet assay . Cells were treated with TAM ( 10 - 6 M ) or ICI ( 10 - 9 M ) for 6 days in the presence and absence of E2 ( β - estradiol ; 10 - 10 M ) . Data ( mean + SD ) were calculated using two - way ANOVA based on three independent biological replicates .

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C Relative cell density was determined by crystal violet assay. Cells were treated with TAM (10-6 M) or ICI (10-9 M) for 6 days in the presence and absence of E2 (β-estradiol; 10-10 M). Data (mean + SD) were calculated using two-way ANOVA based on three independent biological replicates.


D Cell viability was determined using Cell Biolabs CytoSelect ™ Cell Transformation Assay using 2 - control , 2C3 , 9 - control and 9C2 cells in CSM - charcoal stripped media , E2 ( β - estradiol ; 10 - 10 M ) and 10 % fetal bovine serum . Data ( mean + SD ) were calculated using two - way ANOVA based on three independent biological replicates .

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D Cell viability was determined using Cell Biolabs CytoSelect™ Cell Transformation Assay using 2-control, 2C3, 9-control and 9C2 cells in CSM-charcoal stripped media, E2 (β-estradiol; 10-10 M) and 10% fetal bovine serum. Data (mean + SD) were calculated using two-way ANOVA based on three independent biological replicates.


E Impact of ESRP1 knockdown on in vivo tumor growth . Five million cells ( 2 - control , 2C3 , 9 - control and 9C2 ) were implanted into mammary fat pads of athymic mice ( five mice per group ) in the presence of supplemental estrogen . Tumors were measured weekly using calipers for external measurements . Tumor volume was calculated as L x W2 / 2 , where L is length and W is width ( note the different scale for tumor volumes ) . Data ( mean ± SD ) were calculated using two - way ANOVA ( n = 5 mice per group ) .

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E Impact of ESRP1 knockdown on in vivo tumor growth. Five million cells (2-control, 2C3, 9-control and 9C2) were implanted into mammary fat pads of athymic mice (five mice per group) in the presence of supplemental estrogen. Tumors were measured weekly using calipers for external measurements. Tumor volume was calculated as L x W2/2, where L is length and W is width (note the different scale for tumor volumes). Data (mean ± SD) were calculated using two-way ANOVA (n= 5 mice per group).


A Cell morphology using high - power magnification ( 40x ) of the cultured cell lines under a microscope in LCC2 model - 2 - control and 2C3 cells . Scale bar : 50 µm .

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A Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC2 model-2-control and 2C3 cells. Scale bar: 50 µm.


B Cell morphology using high - power magnification ( 40x ) of the cultured cell lines under a microscope in LCC9 model - 9 - control and 9C2 cells . Scale bar : 50 µm .

B Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC9 model-9-control and 9C2 cells. Scale bar: 50 µm.


C - D Protein expression of key EMT - TFs in ESRP1 knockdown cells ( 2C3 and 9C2 ) compared to endocrine resistant control cell lines ( 2 - control and 9 - control ) using Western blotting analysis .

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C-D Protein expression of key EMT-TFs in ESRP1 knockdown cells (2C3 and 9C2) compared to endocrine resistant control cell lines (2-control and 9-control) using Western blotting analysis.


E Protein expression of key EMT - TFs in ESRP1 knockdown cells ( T - 47D - kESRP1 ) and ESRP1 - overexpressing MCF - 7 cells compared to their control counterparts ( T - 47D - control and MCF - 7 - control , respectively ) .

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E Protein expression of key EMT-TFs in ESRP1 knockdown cells (T-47D-kESRP1) and ESRP1-overexpressing MCF-7 cells compared to their control counterparts (T-47D-control and MCF-7-control, respectively).