[ a ] Representative images of indirect immunofluorescence of DIV6 hippocampal neurons cultured from P1 WT and Xrcc1Nes - Cre mouse pups , immunostained for ADP - ribose ( red ) , NeuN to identify neurons ( green ) , and counterstained with DAPI ( blue ) . Cells were pretreated with PARP inhibitor ( 10 µM ) or vehicle for 2 h prior to fixation , with PARG inhibitor ( 10 μM ) additionally present for the final hour . Scale bar 10 µm . [ b ] Histogram of mean ( ± SEM ) relative pan - ADP - ribose fluorescence in NeuN - positive hippocampal neurons pretreated with PARP inhibitor ( 5 µM ) or vehicle for 5 h prior to fixation , with PARG inhibitor ( 10 μM ) additionally present for the final hour . Neurons were cultured from WT ( n = 6 mice , > 180 cells per condition ) , Xrcc1Nes - Cre ( n = 6 , > 180 ) , Parp1 + / - / Xrcc1Nes - Cre ( n = 3 , > 90 ) , and Parp1 - / - / Xrcc1Nes - Cre ( n = 3 , > 90 ) . * indicates significant differences from WT ( Kruskal - Wallis ANOVA , p = 0 . 0013 and Dunn ' s post - hoc tests ) .

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[a] Representative images of indirect immunofluorescence of DIV6 hippocampal neurons cultured from P1 WT and Xrcc1Nes-Cre mouse pups, immunostained for ADP-ribose (red), NeuN to identify neurons (green), and counterstained with DAPI (blue). Cells were pretreated with PARP inhibitor (10 µM) or vehicle for 2 h prior to fixation, with PARG inhibitor (10 μM) additionally present for the final hour. Scale bar 10 µm. [b] Histogram of mean (± SEM) relative pan-ADP-ribose fluorescence in NeuN-positive hippocampal neurons pretreated with PARP inhibitor (5 µM) or vehicle for 5 h prior to fixation, with PARG inhibitor (10 μM) additionally present for the final hour. Neurons were cultured from WT (n = 6 mice, > 180 cells per condition), Xrcc1Nes-Cre (n = 6, > 180), Parp1+/-/Xrcc1Nes-Cre (n = 3, > 90), and Parp1-/-/Xrcc1Nes-Cre (n = 3, > 90). * indicates significant differences from WT (Kruskal-Wallis ANOVA, p = 0.0013 and Dunn's post-hoc tests).


Summary histogram of the mean ( ± SEM ) of the integrated fluorescence responses for all individual synapses for each condition . Synapse number is the same as in ( D - F ) . Responses are significantly higher in Xrcc1Nes - Cre synapses versus both WT and Parp1 - / - / Xrcc1Nes - Cre ( one - way ANOVA , * p < 0 . 0033 , and pairwise student ' s t - tests ) .

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Summary histogram of the mean (± SEM) of the integrated fluorescence responses for all individual synapses for each condition. Synapse number is the same as in (D-F). Responses are significantly higher in Xrcc1Nes-Cre synapses versus both WT and Parp1-/-/Xrcc1Nes-Cre (one-way ANOVA, *p < 0.0033, and pairwise student's t-tests).


B , Immunolocalization of TSN2 - TAPa and GFP - TAPa fusion proteins in root cells of 5 - d - old seedlings . The seedlings were grown under no stress ( NS ) conditions ( 23ºC ) or incubated for 40 min at 39ºC ( HS ) and then immunostained with α - Myc . Scale bars = 10 μm .

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B, Immunolocalization of TSN2-TAPa and GFP-TAPa fusion proteins in root cells of 5-d-old seedlings. The seedlings were grown under no stress (NS) conditions (23ºC) or incubated for 40 min at 39ºC (HS) and then immunostained with α-Myc. Scale bars = 10 μm.


B , Co - localization of RFP - TSN2 ( red ) with GFP - ADH2 ( negative control ) and GFP - UBP1 ( positive control ) in N . benthamiana protoplasts incubated under 23 ° C ( NS ) or at 39ºC for 40 min ( HS ) . Scale bars = 5 μm .

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B, Co-localization of RFP-TSN2 (red) with GFP-ADH2 (negative control) and GFP-UBP1 (positive control) in N. benthamiana protoplasts incubated under 23°C (NS) or at 39ºC for 40 min (HS). Scale bars = 5 μm.


C , Pearson and Spearman coefficients ( rp and rs , respectively ) of co - localization ( PSC ) of RFP - TSN2 with individual GFP - tagged TSN - interacting proteins listed in A and with both negative and positive control proteins ( denoted by red arrowheads ) under HS . Data represent means ± SD of at least 5 replicate measurements from three independent experiments .

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C, Pearson and Spearman coefficients (rp and rs, respectively) of co-localization (PSC) of RFP-TSN2 with individual GFP-tagged TSN-interacting proteins listed in A and with both negative and positive control proteins (denoted by red arrowheads) under HS. Data represent means ± SD of at least 5 replicate measurements from three independent experiments.


D , BiFC between cYFP - TSN2 and nYFP - TSN - interacting proteins in N . benthamiana leaf cells or protoplasts after HS ( 39ºC for 40 min ) . BiFC analysis of cYFP - TSN2 and nYFP - TSN - interacting proteins ( TIPs ) with empty vectors ( EV ) encoding nYFP and cYFP , respectively was used as a negative control . Only one representative example of BiFC between cYFP and nYFP - TIP is shown . Scale bars = 5 μm .

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D, BiFC between cYFP-TSN2 and nYFP-TSN-interacting proteins in N. benthamiana leaf cells or protoplasts after HS (39ºC for 40 min). BiFC analysis of cYFP-TSN2 and nYFP-TSN-interacting proteins (TIPs) with empty vectors (EV) encoding nYFP and cYFP, respectively was used as a negative control. Only one representative example of BiFC between cYFP and nYFP-TIP is shown. Scale bars = 5 μm.


D , BiFC between cYFP - TSN2 ( full - length ) , cYFP - SN or cYFP - Tudor and nYFP - TSN - interacting proteins in N . benthamiana protoplasts after HS ( 39ºC for 40 min ) . Scale bars = 10 μm . Boxplots show quantification of the reconstituted YFP signal . AU , arbitrary units . Upper and lower box boundaries represent the first and third quantiles , respectively , horizontal lines mark the median , and whiskers mark the highest and lowest values . Three independent experiments , each containing seven individual measurements , were performed . * * * P < 0 . 001 vs Tudor ( one - way ANOVA ) .

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D, BiFC between cYFP-TSN2 (full-length), cYFP-SN or cYFP-Tudor and nYFP-TSN-interacting proteins in N. benthamiana protoplasts after HS (39ºC for 40 min). Scale bars = 10 μm. Boxplots show quantification of the reconstituted YFP signal. AU, arbitrary units. Upper and lower box boundaries represent the first and third quantiles, respectively, horizontal lines mark the median, and whiskers mark the highest and lowest values. Three independent experiments, each containing seven individual measurements, were performed. ***P < 0.001 vs Tudor (one-way ANOVA).


B , Co - immunoprecipitation ( Co - Ip ) of the two TSN isoforms and RH12 in protein extracts prepared from N . benthamiana leaves agro - infiltrated with GFP - TSN1 or GFP - TSN2 and Myc - RH12 . Free GFP was used as a negative control . Input and Co - Ip fractions were analyzed by immunoblotting using α - Myc and α - GFP .

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B, Co-immunoprecipitation (Co-Ip) of the two TSN isoforms and RH12 in protein extracts prepared from N. benthamiana leaves agro-infiltrated with GFP-TSN1 or GFP-TSN2 and Myc-RH12. Free GFP was used as a negative control. Input and Co-Ip fractions were analyzed by immunoblotting using α-Myc and α-GFP.


C , Localization of RH12 in root cells of 5 - d - old Arabidopsis seedlings expressing GFP - RH12 under control of the native promoter . The seedlings were grown under 23 ° C ( NS ) or incubated at 39 ° C for 60 min ( HS ) . Scale bars = 10 μm .

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C, Localization of RH12 in root cells of 5-d-old Arabidopsis seedlings expressing GFP-RH12 under control of the native promoter. The seedlings were grown under 23°C (NS) or incubated at 39°C for 60 min (HS). Scale bars = 10 μm.


B , Co - immunoprecipitation ( Co - Ip ) of TSN and RBP47 in protein extracts prepared from 10 - d - old Arabidopsis seedlings expressing Pro35S : GFP - RBP47 and grown under no stress ( NS ) , HS ( 39ºC for 60 min ) or salt ( NaCl ) stress ( 150 mM NaCl for 60 min ) conditions . The GFP - expressing line was used as a negative control . Endogenous TSN ( 107 kD ) was detected in total fractions ( Input ) and fractions co - immunoprecipitated ( Co - Ip ) with RBP47 but not with free GFP in all three conditions . Input and Co - Ip fractions were analyzed by immunoblotting using α - TSN and α - GFP .

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B, Co-immunoprecipitation (Co-Ip) of TSN and RBP47 in protein extracts prepared from 10-d-old Arabidopsis seedlings expressing Pro35S:GFP-RBP47 and grown under no stress (NS), HS (39ºC for 60 min) or salt (NaCl) stress (150 mM NaCl for 60 min) conditions. The GFP-expressing line was used as a negative control. Endogenous TSN (107 kD) was detected in total fractions (Input) and fractions co-immunoprecipitated (Co-Ip) with RBP47 but not with free GFP in all three conditions. Input and Co-Ip fractions were analyzed by immunoblotting using α-TSN and α-GFP.


C , Localization of GFP - tagged proteins in root cells of 5 - d - old Arabidopsis seedlings expressing Pro35S : GFP - RBP47 , Pro35S : GFP - UBP1 , Pro35S : GFP - TCTP and ProUBQ : GFP - SnRK1α2 . The seedlings were grown under 23 ° C ( NS ) , incubated at 39 ° C for 60 min ( HS ) or treated with 200 mM NaCl at 23 ° C for 60 min ( NaCl ) . Scale bars = 10

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C, Localization of GFP-tagged proteins in root cells of 5-d-old Arabidopsis seedlings expressing Pro35S:GFP-RBP47, Pro35S:GFP-UBP1, Pro35S:GFP-TCTP and ProUBQ:GFP-SnRK1α2. The seedlings were grown under 23°C (NS), incubated at 39°C for 60 min (HS) or treated with 200 mM NaCl at 23°C for 60 min (NaCl). Scale bars = 10 μm


D , Localization of GFP - SnRK1α1 and GFP - SnRK1α2 in root cells of 5 - d - old Arabidopsis WT and tsn1 tsn2 seedlings grown under 23 ° C ( NS ) or incubated at 39 ° C for 60 min ( HS ) . Insets show enlarged areas inside dashed rectangles . Scale bars = 10 μm .

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D, Localization of GFP-SnRK1α1 and GFP-SnRK1α2 in root cells of 5-d-old Arabidopsis WT and tsn1 tsn2 seedlings grown under 23°C (NS) or incubated at 39°C for 60 min (HS). Insets show enlarged areas inside dashed rectangles. Scale bars = 10 μm.


J , Colocalization of GFP - SnRK1α1 , GFP - SnRK1α1CD , or GFP - SnRK1α1RD with RFP - RBP47 in N . benthamiana protoplasts subjected to HS ( 40 min at 39 ° C ) . For colocalization analysis under NS conditions see Fig . EV5B . For CHX treatment , protoplasts were incubated with 200 ng μL - 1 CHX for 30 min at 23ºC before HS . GFP and RFP fusion proteins were expressed under the control of the UBQ and 35S promoter , respectively . Scale bars = 5 μm .

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J, Colocalization of GFP-SnRK1α1, GFP-SnRK1α1CD, or GFP-SnRK1α1RD with RFP-RBP47 in N. benthamiana protoplasts subjected to HS (40 min at 39°C). For colocalization analysis under NS conditions see Fig. EV5B. For CHX treatment, protoplasts were incubated with 200 ng μL-1 CHX for 30 min at 23ºC before HS. GFP and RFP fusion proteins were expressed under the control of the UBQ and 35S promoter, respectively. Scale bars = 5 μm.


C , Localization of GFP - SnRK1α1 and GFP - SnRK1α2 in root cells of 5 - d - old Arabidopsis WT seedlings incubated at 39 ° C and imaged at the indicated time points . Scale bars = 10 μm . D , Quantification of GFP - SnRK1α1 and GFP - SnRK1α2 foci in the experiment shown in C . Data represent means ± SD of at least 16 replicate measurements . The experiment was repeated three times with similar results . * * * P < 0 . 05 ( two - tailed t - test ) .

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C, Localization of GFP-SnRK1α1 and GFP-SnRK1α2 in root cells of 5-d-old Arabidopsis WT seedlings incubated at 39°C and imaged at the indicated time points. Scale bars = 10 μm. D, Quantification of GFP-SnRK1α1 and GFP-SnRK1α2 foci in the experiment shown in C. Data represent means ± SD of at least 16 replicate measurements. The experiment was repeated three times with similar results. ***P < 0.05 (two-tailed t-test).


E , F , Immunoblot analysis with indicated antibodies of protein extracts prepared from root tips of 10 - d - old Arabidopsis tsn1 tsn2 ( E ) or tsn1 tsn2 expressing ProTSN2 : GFP - TSN2 ( F ) heat - stressed seedlings ( 39ºC ) 0 , 20 , 40 and 60 min after the onset of HS . The charts show SnRK1 activity , expressed as the ratio of phosphorylated to total SnRK1 protein . The data represent mean ratios of integrated band intensities ( for both isoforms ) normalized to 0 min ± SD from at least four different experiments . P values denote statistically significant differences for comparisons to 0 min ( two - tailed t - test ) .

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E, F, Immunoblot analysis with indicated antibodies of protein extracts prepared from root tips of 10-d-old Arabidopsis tsn1 tsn2 (E) or tsn1 tsn2 expressing ProTSN2:GFP-TSN2 (F) heat-stressed seedlings (39ºC) 0, 20, 40 and 60 min after the onset of HS. The charts show SnRK1 activity, expressed as the ratio of phosphorylated to total SnRK1 protein. The data represent mean ratios of integrated band intensities (for both isoforms) normalized to 0 min ± SD from at least four different experiments. P values denote statistically significant differences for comparisons to 0 min (two-tailed t-test).


G , Expression levels of DIN2 and DIN6 in Arabidopsis WT , tsn1 tsn2 , tsn1 tsn2 ; TSN2 and snrk1α1 - / - snrk1α2 - / + 10 - d - old heat - stressed seedlings relative to unstressed controls . For CHX treatment , the WT seedlings were pre - treated with 200 ng μL - 1 CHX for 30 min before HS . Upper and lower box boundaries represent the first and third quantiles , respectively . Horizontal lines mark the median of five replicate measurements , and whiskers mark the highest and lowest values . Means with different letters are significantly different at P < 0 . 05 ( one - way ANOVA ) .

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G, Expression levels of DIN2 and DIN6 in Arabidopsis WT, tsn1 tsn2, tsn1 tsn2;TSN2 and snrk1α1-/- snrk1α2-/+ 10-d-old heat-stressed seedlings relative to unstressed controls. For CHX treatment, the WT seedlings were pre-treated with 200 ng μL-1 CHX for 30 min before HS. Upper and lower box boundaries represent the first and third quantiles, respectively. Horizontal lines mark the median of five replicate measurements, and whiskers mark the highest and lowest values. Means with different letters are significantly different at P < 0.05 (one-way ANOVA).


( B ) Representative fluorescent images ( red ) showing that mouse primary cortical neurons were transduced with shRNA - control or shATIC # 6 at DIV 3 and then transfected with MYC - LRRK2 - WT and DsRed at a plasmid ratio of 10 : 1 at DIV 5 . Neuronal viability was analyzed at 72 - hour post - transfection with non - viable neurons exhibiting no obvious neurite process ( arrow ) . Scale bars , 100 μm . ( C ) Quantification of neuronal viability . Bars indicate the viability ( n > 100 ) for each transfection condition expressed as a percent of control neurons ( DsRed with pcDNA3 . 1 empty vector ) . Data represent the mean ± SEM from three independent experiments with n > 100 cells quantified in each experiment . * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA followed by a Tukey ' s post hoc test .

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(B) Representative fluorescent images (red) showing that mouse primary cortical neurons were transduced with shRNA-control or shATIC #6 at DIV 3 and then transfected with MYC-LRRK2-WT and DsRed at a plasmid ratio of 10:1 at DIV 5. Neuronal viability was analyzed at 72-hour post-transfection with non-viable neurons exhibiting no obvious neurite process (arrow). Scale bars, 100 μm. (C) Quantification of neuronal viability. Bars indicate the viability (n > 100) for each transfection condition expressed as a percent of control neurons (DsRed with pcDNA3.1 empty vector). Data represent the mean ± SEM from three independent experiments with n > 100 cells quantified in each experiment. **p < 0.01, ***p < 0.001 by one-way ANOVA followed by a Tukey's post hoc test.


( D and E ) Endogenous LRRK2 levels in ATIC overexpression cells . SH - SY5Y cells were transfected with V5 - ATIC or empty vector . After 48 hours , LRRK2 expression levels were analyzed by Western blot . Images were quantified by ImageJ software . Data are mean ± SEM , n = 3 independent experiments . * * * p < 0 . 001 by Student ' s t tests .

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(D and E) Endogenous LRRK2 levels in ATIC overexpression cells. SH-SY5Y cells were transfected with V5-ATIC or empty vector. After 48 hours, LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. ***p < 0.001 by Student's t tests.


( H and I ) Endogenous LRRK2 levels in ATIC KO cells upon LV - flag - ATIC expressing . SH - SY5Y WT or ATIC KO cells were infected with lentiviruses carrying Flag - ATIC or empty vector . After 48 hours , LRRK2 expression levels were analyzed by Western blot . Images were quantified by ImageJ software . Data are the means ± SEM , n = 3 independent experiments . * * * p < 0 . 001 by one - way ANOVA followed by a Tukey ' s post hoc test .

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(H and I) Endogenous LRRK2 levels in ATIC KO cells upon LV-flag-ATIC expressing. SH-SY5Y WT or ATIC KO cells were infected with lentiviruses carrying Flag-ATIC or empty vector. After 48 hours, LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are the means ± SEM, n=3 independent experiments. ***p < 0.001 by one-way ANOVA followed by a Tukey's post hoc test.


( A and B ) Overexpressed LRRK2 levels in HEK 293T cells upon AICAr treatment . HEK293 cells were transfected with MYC - LRRK2 and treated with or without 1mM AICAr for 48 hours . LRRK2 levels were analyzed by Western blot .

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(A and B) Overexpressed LRRK2 levels in HEK 293T cells upon AICAr treatment. HEK293 cells were transfected with MYC-LRRK2 and treated with or without 1mM AICAr for 48 hours. LRRK2 levels were analyzed by Western blot.


( G - N ) LRRK2 expression levels in primary neurons ( G and H ) , microglia ( I and J ) , astrocytes ( K and L ) and human fibroblasts ( M and N ) upon AICAr treatment . Mouse primary cortical neurons , microglia , astrocytes or human fibroblasts from a health control were treated with or without 1mM AICAr for 48 hours . LRRK2 expression levels were analyzed by Western blot .

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(G-N) LRRK2 expression levels in primary neurons (G and H), microglia (I and J), astrocytes (K and L) and human fibroblasts (M and N) upon AICAr treatment. Mouse primary cortical neurons, microglia, astrocytes or human fibroblasts from a health control were treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot.


( Q and R ) LRRK2 expression levels in mouse striatum upon AICAr treatment . AICAr ( 72μg / day / animal ) or saline ( used as a control ) was injected into mouse striatum via osmotic pumps . One day after injection , striatal tissues near the injection sites were collected and LRRK2 expression levels were analyzed by Western blot . Images were quantified by ImageJ software . Data are mean ± SEM from total six mice ( three male mice and three female mice ) . * * * * p < 0 . 0001 by Student ' s t tests .

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(Q and R) LRRK2 expression levels in mouse striatum upon AICAr treatment. AICAr (72μg/day/animal) or saline (used as a control) was injected into mouse striatum via osmotic pumps. One day after injection, striatal tissues near the injection sites were collected and LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM from total six mice (three male mice and three female mice). ****p < 0.0001 by Student's t tests.


( S ) Representative fluorescent images ( green ) showing that mouse primary cortical neurons were co - transfected with MYC - LRRK2 WT or G2019S ( GS ) , and eGFP at a plasmid ratio of 10 : 1 at DIV 5 , and treated with or without 1mM AICAr . Neuronal viability was analyzed at 48 - hour post - transfection with non - viable neurons exhibiting no obvious neurite process ( arrow ) . Scale bars , 100 μm . ( T ) Quantification of neuronal viability . Bars indicate the viability ( n > 100 ) for each transfection condition expressed as a percent of control neurons ( eGFP with pcDNA3 . 1 empty vector ) . Data represent the mean ± SEM from three independent experiments with n > 100 cells quantified in each experiment . * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by one - way ANOVA followed by a Tukey ' s post hoc test .

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 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

(S) Representative fluorescent images (green) showing that mouse primary cortical neurons were co-transfected with MYC-LRRK2 WT or G2019S (GS), and eGFP at a plasmid ratio of 10:1 at DIV 5, and treated with or without 1mM AICAr. Neuronal viability was analyzed at 48-hour post-transfection with non-viable neurons exhibiting no obvious neurite process (arrow). Scale bars, 100 μm. (T) Quantification of neuronal viability. Bars indicate the viability (n > 100) for each transfection condition expressed as a percent of control neurons (eGFP with pcDNA3.1 empty vector). Data represent the mean ± SEM from three independent experiments with n > 100 cells quantified in each experiment. *p < 0.05, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by a Tukey's post hoc test.


( A and B ) LRRK2 protein levels upon treatment of AICAr together with a proteasome inhibitor MG132 or an autophagy inhibitor chloroquine ( CQ ) . SH - SY5Y cells were treated with or without 1mM AICAr , 0 . 35μM MG132 , and 5μM CQ as showed in ( A ) for 48 hours . LRRK2 expression levels were analyzed by Western blot . Images were quantified by ImageJ software . Data are mean ± SEM , n = 3 independent experiments . * * * p < 0 . 001 , * * * * p < 0 . 0001 by one - way ANOVA followed by a Tukey ' s post hoc test .

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

(A and B) LRRK2 protein levels upon treatment of AICAr together with a proteasome inhibitor MG132 or an autophagy inhibitor chloroquine (CQ). SH-SY5Y cells were treated with or without 1mM AICAr, 0.35μM MG132, and 5μM CQ as showed in (A) for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by a Tukey's post hoc test.


( C ) LRRK2 and GBA mRNA levels upon AICAr treatment . SH - SY5Y cells were treated with or without 2mM AICAr for 24 hours . Total RNA was extracted . LRRK2 and GBA mRNA levels were analyzed by real - time PCR . Data are mean ± SEM , n = 4 independent experiments . * * p < 0 . 01 by Student ' s t tests . ns : not statistically significant . ( D ) LRRK2 mRNA level upon AICAr treatment in the presence of actinomycin D . SH - SY5Y cells were treated with or without 2mM AICAr for 24 hours and in the presence of 10μg / ml actinomycin D . Total RNA was extracted . LRRK2 mRNA levels were analyzed by real - time PCR . Data are mean ± SEM , n = 4 independent experiments . * * p < 0 . 01 , * * * * p < 0 . 0001 by two - way ANOVA followed by Sidak ' s multiple comparison test .

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

(C) LRRK2 and GBA mRNA levels upon AICAr treatment. SH-SY5Y cells were treated with or without 2mM AICAr for 24 hours. Total RNA was extracted. LRRK2 and GBA mRNA levels were analyzed by real-time PCR. Data are mean ± SEM, n=4 independent experiments. **p < 0.01 by Student's t tests. ns: not statistically significant. (D) LRRK2 mRNA level upon AICAr treatment in the presence of actinomycin D. SH-SY5Y cells were treated with or without 2mM AICAr for 24 hours and in the presence of 10μg/ml actinomycin D. Total RNA was extracted. LRRK2 mRNA levels were analyzed by real-time PCR. Data are mean ± SEM, n=4 independent experiments. **p < 0.01, ****p < 0.0001 by two-way ANOVA followed by Sidak's multiple comparison test.


( E and F ) LRRK2 expression levels from native LRRK2 cDNA and synthesized LRRK2 cDNA with synonymous codons upon AICAr treatment . HEK 293T cells were transfected with Flag - LRRK2 with native cDNA or with synthesized cDNA , and treated with or without 1mM AICAr for 48 hours . LRRK2 expression levels were analyzed by Western blot . Images were quantified by ImageJ software . Data are mean ± SEM , n = 3 independent experiments . * * * p < 0 . 001 by Student ' s t tests .

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

(E and F) LRRK2 expression levels from native LRRK2 cDNA and synthesized LRRK2 cDNA with synonymous codons upon AICAr treatment. HEK 293T cells were transfected with Flag-LRRK2 with native cDNA or with synthesized cDNA, and treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. ***p < 0.001 by Student's t tests.


( I and J ) The specific region ( 2221 - 3927bp ) of the synthesized Flag - LRRK2 cDNA was replaced with the native sequence and the response of this chimeric LRRK2 cDNA expression to AICAr treatment was analyzed by Western blot . Images were quantified by ImageJ software . Data are mean ± SEM , n = 3 independent experiments . * * * * p < 0 . 0001 by Student ' s t tests .

O O O O O O O O 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-MEASURED_VAR 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

(I and J) The specific region (2221-3927bp) of the synthesized Flag-LRRK2 cDNA was replaced with the native sequence and the response of this chimeric LRRK2 cDNA expression to AICAr treatment was analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. ****p < 0.0001 by Student's t tests.


( B LRRK2 protein levels in AUF1 knockout ( KO ) cells upon AICAr treatment . SH - SY5Y WT and AUF1 KO cells were treated with or without 1mM AICAr for 48 hours . LRRK2 expression levels were analyzed by Western blot .

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(B LRRK2 protein levels in AUF1 knockout (KO) cells upon AICAr treatment. SH-SY5Y WT and AUF1 KO cells were treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot.


( E and F ) LRRK2 protein levels in AUF1 KO cells with AUF1 expressing back upon AICAr treatment . SH - SY5Y WT and KO cells were transfected AUF1 - GFP or GFP and selected with 4μg / ml blasticidin . The resulting stable cell lines were treated with or without 1mM AICAr for 48 hours . LRRK2 expression levels were analyzed by Western blot .

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

(E and F) LRRK2 protein levels in AUF1 KO cells with AUF1 expressing back upon AICAr treatment. SH-SY5Y WT and KO cells were transfected AUF1-GFP or GFP and selected with 4μg/ml blasticidin. The resulting stable cell lines were treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot.


( G and H ) Overexpressed LRRK2 levels in AUF1 KO cells with AUF1 expressing back upon AICAr treatment . SH - SY5Y WT and AUF1 KO cells were transfected AUF1 - GFP or GFP and selected with 4μg / ml blasticidin . The resulting cell lines were transfected with a construct with native LRRK2 cDNA or synthesized LRRK2 cDNA with synonymous codons and treated with or without 1mM AICAr for 48 hours . LRRK2 expression levels were analyzed by Western blot . Images were quantified by ImageJ software .

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

(G and H) Overexpressed LRRK2 levels in AUF1 KO cells with AUF1 expressing back upon AICAr treatment. SH-SY5Y WT and AUF1 KO cells were transfected AUF1-GFP or GFP and selected with 4μg/ml blasticidin. The resulting cell lines were transfected with a construct with native LRRK2 cDNA or synthesized LRRK2 cDNA with synonymous codons and treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software.


( A and B ) AUF1 binds to LRRK2 mRNA upon AICAr treatment . SH - SY5Y WT and AUF1 KO cells were treated with or without 1mM AICAr for 24 hours . RNA and protein complexes were immunoprecipitated using AUF1 antibody . The LRRK2 mRNA levels were analyzed by semiquantitative reverse transcription ( RT ) - PCR . 18S ribosomal RNA ( rRNA ) was as a loading control . Images were quantified by ImageJ software . Data are mean ± SEM , n = 3 independent experiments . * p < 0 . 05 by Student ' s t tests .

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

(A and B) AUF1 binds to LRRK2 mRNA upon AICAr treatment. SH-SY5Y WT and AUF1 KO cells were treated with or without 1mM AICAr for 24 hours. RNA and protein complexes were immunoprecipitated using AUF1 antibody. The LRRK2 mRNA levels were analyzed by semiquantitative reverse transcription (RT)-PCR. 18S ribosomal RNA (rRNA) was as a loading control. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. *p < 0.05 by Student's t tests.


( D ) Direct interaction of LRRK2 mRNA and four AUF1 isoforms . Biotin - labelled 62 - nt LRRK2 RNA probes from native LRRK2 cDNA or synthesized LRRK2 cDNA were incubated with purified AUF1 isoforms in an RNA electrophoretic mobility shift assay ( EMSA ) . Free probes and AUF1 bound probes were detected using a chemiluminescent RNA EMSA kit .

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(D) Direct interaction of LRRK2 mRNA and four AUF1 isoforms. Biotin-labelled 62-nt LRRK2 RNA probes from native LRRK2 cDNA or synthesized LRRK2 cDNA were incubated with purified AUF1 isoforms in an RNA electrophoretic mobility shift assay (EMSA). Free probes and AUF1 bound probes were detected using a chemiluminescent RNA EMSA kit.


( A and B ) DCP1A and DCP2 expression levels upon AICAr treatment . SH - SY5Y cells were treated with or without 1mM AICAr for 48 hours . DCP1A and DCP2 expression levels were analyzed by Western blot . Images were quantified by ImageJ software . Data are mean ± SEM , n = 3 independent experiments . * * p < 0 . 01 by Student ' s t tests .

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

(A and B) DCP1A and DCP2 expression levels upon AICAr treatment. SH-SY5Y cells were treated with or without 1mM AICAr for 48 hours. DCP1A and DCP2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. **p < 0.01 by Student's t tests.


( E ) LRRK2 mRNA decapping by DCP1A and DCP2 . Recombinant DCP1A and DCP2 proteins were purified from HEK 293T cells and incubated with [ α - 32P ] cap - labeled LRRK2 RNA . LRRK2 mRNA decapping was analyzed by one - dimensional thin layer chromatography . The decapping activity is demonstrated by the increased m7GDP levels .

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

(E) LRRK2 mRNA decapping by DCP1A and DCP2. Recombinant DCP1A and DCP2 proteins were purified from HEK 293T cells and incubated with [α-32P]cap-labeled LRRK2 RNA. LRRK2 mRNA decapping was analyzed by one-dimensional thin layer chromatography. The decapping activity is demonstrated by the increased m7GDP levels.


( A and B ) LRRK2 protein levels upon the treatment of AICAr , AICAr plus an adenosine transporter ( AT ) inhibitor ABT702 or an adenosine kinase ( ADK ) inhibitors NBT - 1 . SH - SY5Y cells were treated with or without 1mM AICAr , AICAr plus 1μM ABT - 702 or 50μM NBT - 1 for 48 hours .

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(A and B) LRRK2 protein levels upon the treatment of AICAr, AICAr plus an adenosine transporter (AT) inhibitor ABT702 or an adenosine kinase (ADK) inhibitors NBT-1. SH-SY5Y cells were treated with or without 1mM AICAr, AICAr plus 1μM ABT-702 or 50μM NBT-1 for 48 hours.


( C and D ) LRRK2 protein levels upon the treatment of AICAr or plus AMPK direct activator A769662 . SH - SY5Y cells were treated with or without 1mM AICAr , 10 , 20 , or 50 μM A769662 for 48 hours .

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(C and D) LRRK2 protein levels upon the treatment of AICAr or plus AMPK direct activator A769662. SH-SY5Y cells were treated with or without 1mM AICAr, 10, 20, or 50 μM A769662 for 48 hours.


( E and F ) LRRK2 protein levels upon the treatment of 1mM AICAr in WT and AMPK KO SH - SY5Ycells for 48 hours . LRRK2 expression levels were analyzed by Western blot . Images were quantified by ImageJ software .

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

(E and F) LRRK2 protein levels upon the treatment of 1mM AICAr in WT and AMPK KO SH-SY5Ycells for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software.


( B ) Representative confocal images ( GFP ) of dopamine neurons in each cluster from 9 - week - old WT and LRRK2 - G2019S transgenic flies with or without 1mM AICAr treatment after posteclosion . Scale bars , 25 μm .

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(B) Representative confocal images (GFP) of dopamine neurons in each cluster from 9-week-old WT and LRRK2-G2019S transgenic flies with or without 1mM AICAr treatment after posteclosion. Scale bars, 25 μm.


( C - E ) Quantification of dopamine neurons per DA cluster in 9 - week - old WT and LRRK2 - G2019S transgenic flies with or without 1mM AICAr treatment after posteclosion . ( F ) Total numbers of dopamine neurons in four major DA clusters of 9 - week - old WT and LRRK2 - G2019S transgenic flies with or without AICAr treatment after posteclosion . Data are mean ± SEM , n = 12 flies per group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by one - way ANOVA followed by a Tukey ' s post hoc test .

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(C-E) Quantification of dopamine neurons per DA cluster in 9-week-old WT and LRRK2-G2019S transgenic flies with or without 1mM AICAr treatment after posteclosion. (F) Total numbers of dopamine neurons in four major DA clusters of 9-week-old WT and LRRK2-G2019S transgenic flies with or without AICAr treatment after posteclosion. Data are mean ± SEM, n=12 flies per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by a Tukey's post hoc test.


( A ) Representative images of DAneuron staining . WT and LRRK - KO mice were injected with 1ul LPS ( 5ug / animal ) or phosphate buffered saline ( PBS ) into the substantia nigra pars compacta ( SNpc ) and administrated with AICAr ( 2 . 4μg / day / animal ) or saline to same stereotaxic coordinates using osmotic pumps . After 21 days of injection , DA neurons were stained by anti - tyrosine hydroxylase ( TH ) antibody . Scale bars , 500 μm .

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(A) Representative images of DAneuron staining. WT and LRRK-KO mice were injected with 1ul LPS (5ug/animal) or phosphate buffered saline (PBS) into the substantia nigra pars compacta (SNpc) and administrated with AICAr (2.4μg/day/animal) or saline to same stereotaxic coordinates using osmotic pumps. After 21 days of injection, DA neurons were stained by anti-tyrosine hydroxylase (TH) antibody. Scale bars, 500 μm.


( B ) Quantification of TH positive neurons in substantia nigra pars compacta ( SNpc ) using an unbiased stereological method with stereo investigator software from 7 - 8 animals per group . ( C ) Quantification of Nissl - positive neurons in SNpc using an unbiased stereological method with stereo investigator software from 7 - 8 animals per group .

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(B) Quantification of TH positive neurons in substantia nigra pars compacta (SNpc) using an unbiased stereological method with stereo investigator software from 7-8 animals per group. (C) Quantification of Nissl-positive neurons in SNpc using an unbiased stereological method with stereo investigator software from 7-8 animals per group.


( E ) CD68 levels in WT and LRRK - KO mice injected with 1μl LPS ( 5μg / animal ) or PBS into striatum followed by administration of AICAr ( 2 . 4μg / day / animal ) or saline to same stereotaxic coordinates using osmotic pumps . After 21 days of injection , LRRK2 and CD68 expression in striatal tissues near the needle sites was analyzed by Western blot . ( F ) Quantification of images from ( E ) using ImageJ software , n = 3 animals per group .

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(E) CD68 levels in WT and LRRK-KO mice injected with 1μl LPS (5μg/animal) or PBS into striatum followed by administration of AICAr (2.4μg/day/animal) or saline to same stereotaxic coordinates using osmotic pumps. After 21 days of injection, LRRK2 and CD68 expression in striatal tissues near the needle sites was analyzed by Western blot. (F) Quantification of images from (E) using ImageJ software, n=3 animals per group.


qRT - PCR for INHBA relative to RPL27 using RNA from SCC13 cells transduced with a lentiviral vector allowing expression of INHBA in a doxycycline ( DOX ) - inducible manner ( SCC13 Act clone 1 and 2 ) or empty vector ( EV ) ( N = 3 ) . SCC13 Act and EV cell lines were generated from lentivirally transduced cells upon single cell clonal expansion .

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qRT-PCR for INHBA relative to RPL27 using RNA from SCC13 cells transduced with a lentiviral vector allowing expression of INHBA in a doxycycline (DOX)-inducible manner (SCC13 Act clone 1 and 2) or empty vector (EV) (N=3). SCC13 Act and EV cell lines were generated from lentivirally transduced cells upon single cell clonal expansion.


Western blot of cell lysate and conditioned media ( CM ; bottom ) of transduced SCC13 cells for the activin βA subunit and GAPDH ( loading control for cell lysate ) under reducing conditions . The higher molecular weight of recombinant activin βA results from the HA - epitope tag . Ponceau S staining of the membrane was used as a loading control for the CM .

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Western blot of cell lysate and conditioned media (CM; bottom) of transduced SCC13 cells for the activin βA subunit and GAPDH (loading control for cell lysate) under reducing conditions. The higher molecular weight of recombinant activin βA results from the HA-epitope tag. Ponceau S staining of the membrane was used as a loading control for the CM.


Representative pictures of 5 week - old tumors ( indicated by arrows ) formed in NOD / SCID mice upon intradermal injection of SCC13 Act ( clone 2 ) or SCC13 EV cells and tumor volume at different time points of tumor development . N = 3 - 6 tumors .

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Representative pictures of 5 week-old tumors (indicated by arrows) formed in NOD/SCID mice upon intradermal injection of SCC13 Act (clone 2) or SCC13 EV cells and tumor volume at different time points of tumor development. N=3-6 tumors.


Representative images of H & E stainings of tumors formed by SCC13 EV and Act cells ( clone 2 ) at day 35 ( asterisk indicates cartilage , arrow indicates site of invasion ) . An inset panel indicates the area where tumor cells invade into the cartilage . Tumor sections were evaluated for invasion through the basement membrane . N = 6 ( EV ) , N = 6 Act ( 3 tumors from clone 1 and 3 tumors from clone 2 ) . Tumors from Act mice were pooled in the graph ) .

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Representative images of H&E stainings of tumors formed by SCC13 EV and Act cells (clone 2) at day 35 (asterisk indicates cartilage, arrow indicates site of invasion). An inset panel indicates the area where tumor cells invade into the cartilage. Tumor sections were evaluated for invasion through the basement membrane. N=6 (EV), N=6 Act (3 tumors from clone 1 and 3 tumors from clone 2). Tumors from Act mice were pooled in the graph).


Tumor weight at endpoint ( 5 weeks after injection ) . N = 3 - 6 tumors .

Tumor weight at endpoint (5 weeks after injection). N=3-6 tumors.


Herovici staining and quantification of percentage of collagen - positive ( dark blue ) area per field of view . N = 3 tumors , n = 3 - 4 histological sections .

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Herovici staining and quantification of percentage of collagen-positive (dark blue) area per field of view. N=3 tumors, n=3-4 histological sections.


Representative images of sections from tumors formed by SCC13 EV and SCC13 Act ( clone 2 ) stained for Ki67 ( red ) , PDGFR - α ( green ) and counterstained with Hoechst ( blue ) . Quantification of Ki67 / PDGFR - α positive cells is shown in the graph . N = 3 tumors , n = 3 - 4 histological sections .

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Representative images of sections from tumors formed by SCC13 EV and SCC13 Act (clone 2) stained for Ki67 (red), PDGFR-α (green) and counterstained with Hoechst (blue). Quantification of Ki67/PDGFR-α positive cells is shown in the graph. N=3 tumors, n=3-4 histological sections.


Representative images of sections from 3D cultures of primary human skin fibroblasts with either SCC13 EV or SCC13 Act ( clones 1 and 2 ) cells stained with either H & E or Herovici .

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Representative images of sections from 3D cultures of primary human skin fibroblasts with either SCC13 EV or SCC13 Act (clones 1 and 2) cells stained with either H&E or Herovici.


Quantification of Ki67 - positive cells in sections from SCC13 EV and SCC13 Act ( clone 2 ) organotypic cultures ( co - stained with E - cadherin to indicate epithelial cells ) . N = 3 independent cultures , n = 3 histological sections .

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Quantification of Ki67-positive cells in sections from SCC13 EV and SCC13 Act (clone 2) organotypic cultures (co-stained with E-cadherin to indicate epithelial cells). N=3 independent cultures, n=3 histological sections.


Representative images of sections from SCC13 EV and SCC13 Act ( clone 2 ) organotypic cultures stained for K14 ( red ) and collagen IV ( green ) combined with Hoechst staining ( blue ) and quantification of the K14 - positive area below the basement membrane ( BM ) . N = 3 independent cultures , n = 4 histological sections . The area indicated with a white rectangle in the left picture is shown at higher magnification in the right picture .

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Representative images of sections from SCC13 EV and SCC13 Act (clone 2) organotypic cultures stained for K14 (red) and collagen IV (green) combined with Hoechst staining (blue) and quantification of the K14-positive area below the basement membrane (BM). N=3 independent cultures, n=4 histological sections. The area indicated with a white rectangle in the left picture is shown at higher magnification in the right picture.


Primary murine dermal fibroblasts , which had been treated with recombinant activin A at different concentrations , were analyzed for BrdU incorporation . N = 3 - 4 .

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Primary murine dermal fibroblasts, which had been treated with recombinant activin A at different concentrations, were analyzed for BrdU incorporation. N=3-4.


Representative images of collagen gels and quantification of the extent of gel contraction ( percentage of initial gel size ) in the presence or absence of activin A ( 20 ng / ml ) and / or TGF - β1 ( 1 ng / ml ) . N = 3 - 4 .

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Representative images of collagen gels and quantification of the extent of gel contraction (percentage of initial gel size) in the presence or absence of activin A (20 ng/ml) and/or TGF-β1 (1 ng/ml). N=3-4.


qRT - PCR and Western blot analyses of ACTA2 / αSMA expression using total RNA / lysates of fibroblasts treated with activin A or TGF - β1 for 6 h . N = 3 . The membrane was re - probed with GAPDH and mDia2 antibodies .

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qRT-PCR and Western blot analyses of ACTA2/αSMA expression using total RNA/lysates of fibroblasts treated with activin A or TGF-β1 for 6 h. N=3. The membrane was re-probed with GAPDH and mDia2 antibodies.


Representative images of fibroblasts treated with activin A , TGF - β1 or vehicle ( control ) as in ( C ) , stained for αSMA ( green ) , counterstained with rhodamine - coupled phalloidin ( red ) and Hoechst ( blue ) . Bar graph shows percentage of fibroblasts with stress fibers . N = 3 .

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Representative images of fibroblasts treated with activin A, TGF-β1 or vehicle (control) as in (C), stained for αSMA (green), counterstained with rhodamine-coupled phalloidin (red) and Hoechst (blue). Bar graph shows percentage of fibroblasts with stress fibers. N=3.


epresentative images of control and activin A - treated fibroblasts stained with rhodamine - coupled phalloidin ; scatter plot shows quantification of filopodium length in control , activin A - and TGF - β1 - treated fibroblasts . N = 27 - 70 filopodia .

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epresentative images of control and activin A-treated fibroblasts stained with rhodamine-coupled phalloidin; scatter plot shows quantification of filopodium length in control, activin A- and TGF-β1-treated fibroblasts. N=27-70 filopodia.


Scratch assay of fibroblasts treated with activin A or vehicle for 24 h . N = 3 . The area covered by cells in the original scratch was quantified . Migration in one control culture was set to 1 .

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Scratch assay of fibroblasts treated with activin A or vehicle for 24 h. N=3. The area covered by cells in the original scratch was quantified. Migration in one control culture was set to 1.


Chemotactic migration of primary mouse fibroblasts from wt mice for 24 h in a transwell assay towards increasing concentrations of activin A or vehicle . N = 3 .

Chemotactic migration of primary mouse fibroblasts from wt mice for 24 h in a transwell assay towards increasing concentrations of activin A or vehicle. N=3.


qRT - PCR analysis of CAF marker genes relative to Rps29 using RNA from fibroblasts treated with activin A for 6 h . N = 3 .

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qRT-PCR analysis of CAF marker genes relative to Rps29 using RNA from fibroblasts treated with activin A for 6 h. N=3.


qRT - PCR for INHBA relative to RPL27 using RNA from primary human fibroblasts transduced with a lentiviral vector allowing expression of INHBA in an inducible manner after treatment with DOX for 24 h ( Fb Act , clones 1 and 2 ) or with empty vector ( Fb EV ) ( N = 3 ) . Fb Act and Fb EV cultures were generated from lentivirally - transduced cells upon clonal expansion of resistant single cells .

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qRT-PCR for INHBA relative to RPL27 using RNA from primary human fibroblasts transduced with a lentiviral vector allowing expression of INHBA in an inducible manner after treatment with DOX for 24 h (Fb Act, clones 1 and 2) or with empty vector (Fb EV) (N=3). Fb Act and Fb EV cultures were generated from lentivirally-transduced cells upon clonal expansion of resistant single cells.


Western blot of conditioned media ( CM ) of transduced fibroblasts showing mature activin βA . Ponceau S staining of the membrane was used as a loading control .

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Western blot of conditioned media (CM) of transduced fibroblasts showing mature activin βA. Ponceau S staining of the membrane was used as a loading control.


Chemotactic migration of SCC13 cells for 24 h in a transwell assay containing CM from Fb Act ( clone 2 ) or Fb EV . N = 4 . Migration in one control culture was set to 1 .

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Chemotactic migration of SCC13 cells for 24 h in a transwell assay containing CM from Fb Act (clone 2) or Fb EV. N=4. Migration in one control culture was set to 1.


Relative colony area of SCC13 cells upon culture in CM from Fb Act ( clone 2 ) or EV . N = 3 .

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Relative colony area of SCC13 cells upon culture in CM from Fb Act (clone 2) or EV. N=3.


Representative immunofluorescence images of de - cellularized matrix from Fb Act ( clone 2 ) and Fb EV stained for fibronectin or collagen I .

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Representative immunofluorescence images of de-cellularized matrix from Fb Act (clone 2) and Fb EV stained for fibronectin or collagen I.


Relative colony area of SCC13 cells plated on either de - cellularized matrix from Fb Act ( clone 2 ) or Fb EV . N = 3 .

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Relative colony area of SCC13 cells plated on either de-cellularized matrix from Fb Act (clone 2) or Fb EV. N=3.


Migration of SCC13 cells plated on either de - cellularized matrix from Fb Act ( clone 2 ) or Fb EV . N = 6 . The experimental setup is shown schematically above the graph .

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Migration of SCC13 cells plated on either de-cellularized matrix from Fb Act (clone 2) or Fb EV. N=6. The experimental setup is shown schematically above the graph.


Fb Act ( clone 2 ) or Fb EV cells were cultured together in 2D with SCC13 cells . After 7 day of culture SCC13 cells were identified by K14 and the number of K14 - positive colony area was quantified . N = 6 .

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Fb Act (clone 2) or Fb EV cells were cultured together in 2D with SCC13 cells. After 7 day of culture SCC13 cells were identified by K14 and the number of K14-positive colony area was quantified. N=6.


Quantification of SCC13 tumor spheroid area in single hanging drop including CM from Fb EV or Fb Act ( clone 2 ) ( including 1 % FBS ) , DMEM / 1 % FBS , or DMEM / 1 % FBS plus 20 ng / mL recombinant activin A . The area of the spheroid formed in one Fb EV sample was set to 1 . N = 9 - 10 hanging drops .

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Quantification of SCC13 tumor spheroid area in single hanging drop including CM from Fb EV or Fb Act (clone 2) (including 1% FBS), DMEM/1% FBS, or DMEM/1% FBS plus 20 ng/mL recombinant activin A. The area of the spheroid formed in one Fb EV sample was set to 1. N=9-10 hanging drops.


Representative images and quantification of K14 - stained areas from 3D cultures of SCC13 cells seeded on top of either Fb Act ( clone 2 ) or Fb EV . N = 3 independent cultures , n = 3 histological sections .

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Representative images and quantification of K14-stained areas from 3D cultures of SCC13 cells seeded on top of either Fb Act (clone 2) or Fb EV. N=3 independent cultures, n=3 histological sections.


Representative pictures of tumors formed in NOD / SCID mice 6 - 7 weeks after intradermal injection of SCC13 cells with Fb Act ( clone 2 ) or Fb EV . Tumor volume at different time points of tumor development ( 6 - 7 weeks after injection ) . N = 5 tumors per group .

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Representative pictures of tumors formed in NOD/SCID mice 6-7 weeks after intradermal injection of SCC13 cells with Fb Act (clone 2) or Fb EV. Tumor volume at different time points of tumor development (6-7 weeks after injection). N=5 tumors per group.


mDia2 ( Diap3 ) expression quantified by RNA sequencing of fibroblasts isolated from ear skin . N = 3 pools of 3 - 6 mice .

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mDia2 (Diap3) expression quantified by RNA sequencing of fibroblasts isolated from ear skin. N=3 pools of 3-6 mice.


Primary mouse fibroblasts were treated with activin A or TGF - β1 or vehicle for 6h and analyzed by qRT - PCR ( N = 3 ) and Western blot for mDia2 ( Diap3 ) and mDia2 expression , respectively . The GAPDH loading control is identical to the one in Fig 2C , since the same membrane was probed for mDia2 , αSMA and GAPDH .

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Primary mouse fibroblasts were treated with activin A or TGF-β1 or vehicle for 6h and analyzed by qRT-PCR (N=3) and Western blot for mDia2 (Diap3) and mDia2 expression, respectively. The GAPDH loading control is identical to the one in Fig 2C, since the same membrane was probed for mDia2, αSMA and GAPDH.


Immortalized mouse fibroblasts were transduced with a lentivirus expressing a DOX - inducible dominant - negative ActRIB mutant ( dnActRIB ) or an empty lentivirus ( EV ) , treated with activin A for 2 . 5 h and analyzed by Western blot for mDia2 and GAPDH .

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Immortalized mouse fibroblasts were transduced with a lentivirus expressing a DOX-inducible dominant-negative ActRIB mutant (dnActRIB) or an empty lentivirus (EV), treated with activin A for 2.5 h and analyzed by Western blot for mDia2 and GAPDH.


Representative sections of ear skin tumors formed by SCC13 EV or SCC13 Act cells ( clone 2 ) stained for mDia2 or K14 ( green ) ( top ) , mDia2 ( green ) or MECA32 / LYVE1 ( red , bottom ) and counterstained with Hoechst ( blue ) .

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Representative sections of ear skin tumors formed by SCC13 EV or SCC13 Act cells (clone 2) stained for mDia2 or K14 (green) (top), mDia2 (green) or MECA32/LYVE1 (red, bottom) and counterstained with Hoechst (blue).


qRT - PCR for mDia2 / DIAPH3 relative to RPL27 using RNA from normal human skin ( N = 11 ) , SCCs ( N = 9 ) and BCCs ( N = 7 ) .

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qRT-PCR for mDia2/DIAPH3 relative to RPL27 using RNA from normal human skin (N=11), SCCs (N=9) and BCCs (N=7).


Representative skin / tumor sections from normal human skin , SCC and BCC patients stained for mDia2 / DIAPH3 . Note the strong mDia2 / DIAPH3 - positivity in the tumor stroma . Scale bars : 100 μm . D : Dermis , E : Epidermis : T : Tumor .

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Representative skin/tumor sections from normal human skin, SCC and BCC patients stained for mDia2/DIAPH3. Note the strong mDia2/DIAPH3-positivity in the tumor stroma. Scale bars: 100 μm. D: Dermis, E: Epidermis: T: Tumor.


Expression levels of mDia2 / DIAPH3 in tumor and normal tissues based on TCGA cancer data ( TCGA data version 20141017 , http : / / firebrowse . org / ) . Gene expression levels were presented as normalized log2 RSEM ( RNA - Seq by Expectation - Maximization ) index from the database . BLCA : Bladder Urothelial Carcinoma ( N = 408 & 19 tumor and normal tissue samples , respectively ) , BRCA : Breast Invasive Carcinoma ( N = 1100 & 112 ) , CESC : Cervical SCC ( N = 306 & 3 ) , ESCA : Esophageal SCC ( N = 185 & 11 ) , HNSC : Head - Neck SCC ( N = 522 & 44 ) , LIHC : Liver Hepatocellular Carcinoma ( N = 373 & 47 ) , LUSC : Lung SCC ( N = 501 & 51 ) , PAAD : Pancreatic Adenocarcinoma ( N = 179 & 4 ) , PRAD : Prostate Adenocarcinoma ( N = 496 & 50 ) , READ : Rectum Adenocarcinoma ( N = 167 & 10 ) , STES : Stomach and Esophageal Carcinoma ( N = 600 & 46 ) , UCEC : Uterine Corpus Endometrial Carcinoma ( N = 546 & 35 ) . Box plot shows relative mDia2 / DIAPH3 expression in human tumor ( red ) and normal ( blue ) tissue . Boxes indicate the interquartile range , whiskers indicate data points within 1 . 5 times interquartile range , dots outside the whiskers indicate outliers , and the central line indicates median .

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Expression levels of mDia2/DIAPH3 in tumor and normal tissues based on TCGA cancer data (TCGA data version 20141017, http://firebrowse.org/). Gene expression levels were presented as normalized log2 RSEM (RNA-Seq by Expectation-Maximization) index from the database. BLCA: Bladder Urothelial Carcinoma (N = 408 & 19 tumor and normal tissue samples, respectively), BRCA: Breast Invasive Carcinoma (N = 1100 & 112), CESC: Cervical SCC (N = 306 & 3), ESCA: Esophageal SCC (N = 185 & 11), HNSC: Head-Neck SCC (N = 522 & 44), LIHC: Liver Hepatocellular Carcinoma (N = 373 & 47), LUSC: Lung SCC (N = 501 & 51), PAAD: Pancreatic Adenocarcinoma (N = 179 & 4), PRAD: Prostate Adenocarcinoma (N = 496 & 50), READ: Rectum Adenocarcinoma (N = 167 & 10), STES: Stomach and Esophageal Carcinoma (N = 600 & 46), UCEC: Uterine Corpus Endometrial Carcinoma (N = 546 & 35). Box plot shows relative mDia2/DIAPH3 expression in human tumor (red) and normal (blue) tissue. Boxes indicate the interquartile range, whiskers indicate data points within 1.5 times interquartile range, dots outside the whiskers indicate outliers, and the central line indicates median.


Relative gene expression of mDia2 / DIAPH3 and INHBA in the stroma of intrahepatic cholangiocarcinoma , breast and colorectal cancers vs . stroma of respective normal tissues based on datasets GSE45001 ( D ) ( N = 10 normal tissue & 10 cancer samples , GSE9014 ( E ) ( N = 4 & 13 ) , and GSE35602 ( F ) ( N = 6 & 53 ) , and in CAFs from breast cancer samples vs . normal breast fibroblasts ( GSE29270 dataset ) ( G ) ( N = 5 & 23 ) . NS : Normal stroma , CS : Cancer stroma , NF : Normal fibroblasts , CAF : Cancer - associated fibroblasts ) . The expression values of the datasets were obtained using GEO2R tool in the GEO database .

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Relative gene expression of mDia2/DIAPH3 and INHBA in the stroma of intrahepatic cholangiocarcinoma, breast and colorectal cancers vs. stroma of respective normal tissues based on datasets GSE45001 (D) (N = 10 normal tissue & 10 cancer samples, GSE9014 (E) (N = 4 & 13), and GSE35602 (F) (N = 6 & 53), and in CAFs from breast cancer samples vs. normal breast fibroblasts (GSE29270 dataset) (G) (N = 5 & 23). NS: Normal stroma, CS: Cancer stroma, NF: Normal fibroblasts, CAF: Cancer-associated fibroblasts). The expression values of the datasets were obtained using GEO2R tool in the GEO database.


Kaplan - Meier survival curves based on datasets from GEO , EGA and TCGA , demonstrating that high expression of both mDia2 / DIAPH3 and INHBA in patients with liver , stomach and breast cancer correlates with poor overall survival . Data and statistical analyses were collected from KMPlotter .

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Kaplan-Meier survival curves based on datasets from GEO, EGA and TCGA, demonstrating that high expression of both mDia2/DIAPH3 and INHBA in patients with liver, stomach and breast cancer correlates with poor overall survival. Data and statistical analyses were collected from KMPlotter.


Immortalized mouse fibroblasts were transduced with a lentiviral vector expressing different mDia2 - targeting shRNAs ( shmDia2 # 1 - 3 ) or empty vector ( sh - EV ) . The knock - down was confirmed by qRT - PCR ( N = 3 ) , Western blot and immunofluorescence .

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Immortalized mouse fibroblasts were transduced with a lentiviral vector expressing different mDia2-targeting shRNAs (shmDia2 #1-3) or empty vector (sh-EV). The knock-down was confirmed by qRT-PCR (N=3), Western blot and immunofluorescence.


Ki67 staining of mDia2 knock - down and control mouse fibroblasts . N = 3 .

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Ki67 staining of mDia2 knock-down and control mouse fibroblasts. N=3.


migration in scratch assays of mDia2 knock - down and control mouse fibroblasts . N = 3 .

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migration in scratch assays of mDia2 knock-down and control mouse fibroblasts. N=3.


qRT - PCR for mDia1 , mDia3 , Fmn1 and Fmn2 and several CAF genes relative to Rps29 using RNA from control ( sh - EV ) and mDia2 knock - down ( sh - mDia2 ) fibroblasts . N = 3 .

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qRT-PCR for mDia1, mDia3, Fmn1 and Fmn2 and several CAF genes relative to Rps29 using RNA from control (sh-EV) and mDia2 knock-down (sh-mDia2) fibroblasts. N=3.


Representative images of mDia2 knockdown and control mouse fibroblasts stained for p53 ( left ) , and quantification of cells showing accumulation of p53 in the nucleus ( right ) . N = 3 .

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Representative images of mDia2 knockdown and control mouse fibroblasts stained for p53 (left), and quantification of cells showing accumulation of p53 in the nucleus (right). N=3.


Representative Western blots for p53 , vinculin and histone H1 using nuclear and cytoplasmic fractions and total lysate of mDia2 knock - down ( F ) or activin A - treated fibroblasts ( 6h , 20 ng / ml ) ( G ) and control mouse fibroblasts and quantification of the nuclear p53 / histone H1 ratio . Vinculin and histone H1 were used as loading controls for the cytoplasmic and nuclear fractions , respectively . N = 6 .

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

Representative Western blots for p53, vinculin and histone H1 using nuclear and cytoplasmic fractions and total lysate of mDia2 knock-down (F) or activin A-treated fibroblasts (6h, 20 ng/ml) (G) and control mouse fibroblasts and quantification of the nuclear p53/histone H1 ratio. Vinculin and histone H1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. N=6.


Representative Western blots for p53 , mDia2 and GAPDH using total lysates of mDia2 knock - down and control mouse fibroblasts treated for 24 h with pifithrin - α ( PFT - α ) ( 15 μM ) .

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

Representative Western blots for p53, mDia2 and GAPDH using total lysates of mDia2 knock-down and control mouse fibroblasts treated for 24 h with pifithrin-α (PFT-α) (15 μM).


Representative p53 immunofluorescence images of mDia2 knock - down mouse fibroblasts treated with PFT - α or vehicle ( DMSO ) ( top ) and quantification of cells showing accumulation of p53 in the nucleus ( bottom ) . N = 3 .

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

Representative p53 immunofluorescence images of mDia2 knock-down mouse fibroblasts treated with PFT-α or vehicle (DMSO) (top) and quantification of cells showing accumulation of p53 in the nucleus (bottom). N=3.


qRT - PCR for mDia2 , Postn , Mmp13 , Fn1 , Spp1 and Acta2 relative to Rps29 using RNA from mDia2 knock - down and control fibroblasts treated with PFT - α or vehicle . N = 3 .

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

qRT-PCR for mDia2, Postn, Mmp13, Fn1, Spp1 and Acta2 relative to Rps29 using RNA from mDia2 knock-down and control fibroblasts treated with PFT-α or vehicle. N=3.


Colony size of SCC13 Act cells ( clone 2 ) after co - culture with either immortalized mDia2 knockdown ( sh - mDia2 ) or control ( sh - EV ) mouse fibroblasts . N = 4 .

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

Colony size of SCC13 Act cells (clone 2) after co-culture with either immortalized mDia2 knockdown (sh-mDia2) or control (sh-EV) mouse fibroblasts. N=4.


Representative pictures of 5 - week - old tumors formed upon intradermal co - injection of SCC13 Act cells ( clone 2 ) with sh - EV or sh - mDia2 knock - down immortalized mouse fibroblasts . Graph shows tumor volume at different timepoints of tumor development . N = 3 - 5 tumors per group .

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

Representative pictures of 5-week-old tumors formed upon intradermal co-injection of SCC13 Act cells (clone 2) with sh-EV or sh-mDia2 knock-down immortalized mouse fibroblasts. Graph shows tumor volume at different timepoints of tumor development. N=3-5 tumors per group.


Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 Act ( clone 2 ) co - injected with sh - EV or sh - mDia2 fibroblasts , stained for E - cadherin ( green ) and periostin or p53 ( red ) , counterstained with Hoechst ( blue ) .

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

Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 Act (clone 2) co-injected with sh-EV or sh-mDia2 fibroblasts, stained for E-cadherin (green) and periostin or p53 (red), counterstained with Hoechst (blue).


Tumor volume at different time points after co - injection of SCC13 cells with EV or dnActRIB fibroblasts . N = 5 tumors .

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

Tumor volume at different time points after co-injection of SCC13 cells with EV or dnActRIB fibroblasts. N=5 tumors.


Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 cells co - injected with EV or dnActRIB fibroblasts stained for K14 ( left ) or PDGFRα ( right ) ( green ) and mDia2 ( red ) , counterstained with Hoechst ( blue ) . Bar graph shows the percentage of mDia2 - positive area . N = 3 .

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

Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 cells co-injected with EV or dnActRIB fibroblasts stained for K14 (left) or PDGFRα (right) (green) and mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3.


SCC13 EV or SCC13 Fst cells were injected into the ear skin of NOD / SCID mice and tumor weight was determined 12 weeks after injection . N = 9 - 10 tumors .

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

SCC13 EV or SCC13 Fst cells were injected into the ear skin of NOD/SCID mice and tumor weight was determined 12 weeks after injection. N=9-10 tumors.


Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 EV or SSC13 Fst cells stained for K14 or mDia2 ( red ) , counterstained with Hoechst ( blue ) . Bar graph shows the percentage of mDia2 - positive area . N = 3 .

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

Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 EV or SSC13 Fst cells stained for K14 or mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3.


Tumors formed by SCC13 Act cells were treated ex vivo for 6h with follistatin ( FST ) ( 50 ng / ml ) and / or SMIFH2 ( 10 μM ) , or with activin A ( 20 ng / ml ) and stained for Ki67 . N = 3 tumor explants , n = 4 histological sections .

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

Tumors formed by SCC13 Act cells were treated ex vivo for 6h with follistatin (FST) (50 ng/ml) and/or SMIFH2 (10 μM), or with activin A (20 ng/ml) and stained for Ki67. N=3 tumor explants, n=4 histological sections.


Representative images of spheroids formed by SCC13 Act cells labeled with PKH67 ( green ) with primary human fibroblasts embedded in collagen gels and graph showing increase in the spheroid area between day 0 and day 3 . Spheroids were either treated with FST ( 50 ng / ml ) or SMIFH2 ( 10 μM ) or left untreated . N = 6 - 8 tumor spheroids .

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

Representative images of spheroids formed by SCC13 Act cells labeled with PKH67 (green) with primary human fibroblasts embedded in collagen gels and graph showing increase in the spheroid area between day 0 and day 3. Spheroids were either treated with FST (50 ng/ml) or SMIFH2 (10 μM) or left untreated. N=6-8 tumor spheroids.


Increase in A431 tumor volume ( mm3 ) compared to day 0 ( time when the tumors were first injected with FST , approximately 3 - 4 weeks after injection of the cells ) upon treatment of the tumors with FST ( 100 ng per injection ) or vehicle ( PBS ) . N = 3 - 5 tumors per group . Representative immunofluorescence images the FST or vehicle - treated tumors , stained for mDia2 ( green ) and K14 ( red ) , counterstained with Hoechst ( blue ) .

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

Increase in A431 tumor volume (mm3) compared to day 0 (time when the tumors were first injected with FST, approximately 3-4 weeks after injection of the cells) upon treatment of the tumors with FST (100 ng per injection) or vehicle (PBS). N=3-5 tumors per group. Representative immunofluorescence images the FST or vehicle-treated tumors, stained for mDia2 (green) and K14 (red), counterstained with Hoechst (blue).


Representative flow cytometric analysis of BAL from Csf2ra - / - recipients 8 weeks after neonatal i . n . transfer of YS EMP from CD45 . 1 + E10 . 5 embryos , 8 - week old un - transferred Csf2ra - / - , and WT ( CD45 . 2 ) mice . Pre - gated viable CD45 + singlets .

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

Representative flow cytometric analysis of BAL from Csf2ra-/- recipients 8 weeks after neonatal i.n. transfer of YS EMP from CD45.1+ E10.5 embryos, 8-week old un-transferred Csf2ra-/-, and WT (CD45.2) mice. Pre-gated viable CD45+ singlets.


Numbers of EMP effectively transferred in neonates ( white circle ) and numbers of EMP - derived AM ( red circles ) in BAL and lung of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) .

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

Numbers of EMP effectively transferred in neonates (white circle) and numbers of EMP-derived AM (red circles) in BAL and lung of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice).


Numbers of EMP - derived AM in the BAL ( left ) and lung ( right ) ( C ) in the BAL of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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

Numbers of EMP-derived AM in the BAL (left) and lung (right) (C) in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


total protein in the BAL of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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

total protein in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


total eFluor780 + events ( E ) in the BAL of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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

total eFluor780+ events (E) in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) in the BAL and lung from Csf2ra - / - recipients 8 weeks after neonatal transfer of 1 : 1 : 1 mixture with E17 . 5 fetal liver ( FeLi ) monocytes ( Mo ) ( CD45 . 1 + ) , E14 . 5 FeLi Mo ( CD45 . 1 + CD45 . 2 + ) , and E10 . 5 YS EMP ( CD45 . 2 + ) ( F ) or E17 . 5 fetal lung ( FeLu ) Mo ( CD45 . 1 + ) , E14 . 5 FeLu Mo ( CD45 . 1 + CD45 . 2 + ) , and E10 . 5 YS EMP ( CD45 . 2 + ) ( G ) ( n = 3 mice ) .

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

Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) in the BAL and lung from Csf2ra-/- recipients 8 weeks after neonatal transfer of 1:1:1 mixture with E17.5 fetal liver (FeLi) monocytes (Mo) (CD45.1+), E14.5 FeLi Mo (CD45.1+CD45.2+), and E10.5 YS EMP (CD45.2+) (F) or E17.5 fetal lung (FeLu) Mo (CD45.1+), E14.5 FeLu Mo (CD45.1+CD45.2+), and E10.5 YS EMP (CD45.2+) (G) (n = 3 mice).


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) in the BAL ( top ) and lung ( bottom ) from Csf2ra - / - recipients 10 weeks after neonatal transfer of 1 : 1 mixture with CD45 . 1 + E14 . 5 FeLi Mo and CD45 . 2 + E14 . 5 FeLi pMΦ ( Group 1 ) or CD45 . 1 + E14 . 5 FeLi pMΦ and CD45 . 2 + FeLi Mo ( Group 2 ) ( n = 3 mice ) . The expansion fold of donor cells in Csf2ra - / - recipients 10 weeks after neonatal transfer of cells described above in A ( n = 3 mice ) .

O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O 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 flow cytometric analysis (left) and percentage of donor-derived AM (right) in the BAL (top) and lung (bottom) from Csf2ra-/- recipients 10 weeks after neonatal transfer of 1:1 mixture with CD45.1+ E14.5 FeLi Mo and CD45.2+ E14.5 FeLi pMΦ (Group 1) or CD45.1+ E14.5 FeLi pMΦ and CD45.2+ FeLi Mo (Group 2) (n = 3 mice). The expansion fold of donor cells in Csf2ra-/- recipients 10 weeks after neonatal transfer of cells described above in A (n = 3 mice).


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) of BAL and lung from Csf2ra - / - recipients 8 weeks after neonatal transfer of 1 : 1 : 1 mixture with CD45 . 1 + E17 . 5 FeLi pMΦ , CD45 . 1 + CD45 . 2 + E14 . 5 FeLi pMΦ , and CD45 . 2 + E10 . 5 YS pMΦ ( n = 3 mice ) . Total numbers of donor - derived AM in the BAL and lung of adult Csf2ra - / - recipients 8 weeks after transfer of cells described above in C ( n = 3 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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

Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) of BAL and lung from Csf2ra-/- recipients 8 weeks after neonatal transfer of 1:1:1 mixture with CD45.1+ E17.5 FeLi pMΦ, CD45.1+CD45.2+ E14.5 FeLi pMΦ, and CD45.2+ E10.5 YS pMΦ (n = 3 mice). Total numbers of donor-derived AM in the BAL and lung of adult Csf2ra-/- recipients 8 weeks after transfer of cells described above in C (n = 3 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) of BAL and lung from Csf2ra - / - recipients 8 weeks after neonatal transfer of 1 : 1 : 1 mixture with CD45 . 1 + E17 . 5 FeLu pMΦ , CD45 . 1 + CD45 . 2 + E14 . 5 FeLu pMΦ , and CD45 . 2 + E10 . 5 YS pMΦ ( n = 3 mice ) . Total numbers of donor - derived AM in the BAL and lung of adult Csf2ra - / - recipients 8 weeks after transfer of cells described above in E ( n = 3 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) of BAL and lung from Csf2ra-/- recipients 8 weeks after neonatal transfer of 1:1:1 mixture with CD45.1+ E17.5 FeLu pMΦ, CD45.1+CD45.2+ E14.5 FeLu pMΦ, and CD45.2+ E10.5 YS pMΦ (n = 3 mice). Total numbers of donor-derived AM in the BAL and lung of adult Csf2ra-/- recipients 8 weeks after transfer of cells described above in E (n = 3 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) in the BAL and lung from Csf2ra - / - recipients 7 weeks after neonatal transfer of 1 : 1 mixture with CD45 . 1 + E14 . 5 FeLi Mo from WT embryos and CD45 . 2 + E14 . 5 FeLi pMΦ from Myb - / - ( G ) or Myb + / + ( H ) embryos ( n = 3 mice ) .

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

Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) in the BAL and lung from Csf2ra-/- recipients 7 weeks after neonatal transfer of 1:1 mixture with CD45.1+ E14.5 FeLi Mo from WT embryos and CD45.2+ E14.5 FeLi pMΦ from Myb-/- (G) or Myb+/+ (H) embryos (n = 3 mice).


Representative flow cytometric analysis of BAL from Csf2ra - / - recipients 7 weeks after neonatal i . n . transfer of pMΦ or fetal Mo from CD45 . 1 + E14 . 5 fetal livers , 7 - week old un - transferred Csf2ra - / - , WT ( CD45 . 2 ) mice . Pre - gated viable CD45 + singlets .

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

Representative flow cytometric analysis of BAL from Csf2ra-/- recipients 7 weeks after neonatal i.n. transfer of pMΦ or fetal Mo from CD45.1+ E14.5 fetal livers, 7-week old un-transferred Csf2ra-/-, WT (CD45.2) mice. Pre-gated viable CD45+ singlets.


Numbers of donor - derived AM in the BAL ( left ) and lung ( right ) of adult Csf2ra - / - recipients 7 weeks ( B ) or 1 year ( C ) after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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

Numbers of donor-derived AM in the BAL (left) and lung (right) of adult Csf2ra-/- recipients 7 weeks (B) or 1 year (C) after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


Total protein in the BAL of adult Csf2ra - / - recipients 7 weeks after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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

Total protein in the BAL of adult Csf2ra-/- recipients 7 weeks after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


total eFluor780 + events in the BAL of adult Csf2ra - / - recipients 7 weeks after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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

total eFluor780+ events in the BAL of adult Csf2ra-/- recipients 7 weeks after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


Total protein in the BAL of adult Csf2ra - / - recipients 1 year ( F , G ) after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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

Total protein in the BAL of adult Csf2ra-/- recipients 1 year (F, G) after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


total eFluor780 + events in the BAL of adult Csf2ra - / - recipients 1 year ( F , G ) after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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 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

total eFluor780+ events in the BAL of adult Csf2ra-/- recipients 1 year (F, G) after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


Body weight measurement of influenza virus ( 20 pfu of PR8 strain ) - infected Csf2ra - / - recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14 . 5 fetal livers ( n = 7 mice from each transferred group ) , 24 - week old un - transferred Csf2ra - / - ( n = 5 ) , and WT ( n = 8 ) .

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

Body weight measurement of influenza virus (20 pfu of PR8 strain)-infected Csf2ra-/- recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14.5 fetal livers (n = 7 mice from each transferred group), 24-week old un-transferred Csf2ra-/- (n = 5), and WT (n = 8).


temperature ( B ) measurement of influenza virus ( 20 pfu of PR8 strain ) - infected Csf2ra - / - recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14 . 5 fetal livers ( n = 7 mice from each transferred group ) , 24 - week old un - transferred Csf2ra - / - ( n = 5 ) , and WT ( n = 8 ) .

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

temperature (B) measurement of influenza virus (20 pfu of PR8 strain)-infected Csf2ra-/- recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14.5 fetal livers (n = 7 mice from each transferred group), 24-week old un-transferred Csf2ra-/- (n = 5), and WT (n = 8).


Oxygen ( O2 ) saturation in influenza - infected mice described in A , B 7 days post - infection ( p . i . ) ( n = 5 mice from Csf2ra - / - group ; n = 7 mice from each transferred group ; n = 8 from WT group ) .

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

Oxygen (O2) saturation in influenza-infected mice described in A, B 7 days post-infection (p.i.) (n = 5 mice from Csf2ra-/- group; n = 7 mice from each transferred group; n=8 from WT group).


Survival curve of influenza - infected mice described in A , B ( n = 5 mice from Csf2ra - / - group ; n = 7 mice from each transferred group ; n = 8 from WT group ) .

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

Survival curve of influenza-infected mice described in A, B (n = 5 mice from Csf2ra-/- group; n = 7 mice from each transferred group; n=8 from WT group).