B . Internalization ( cytoplasm ) and recycling ( plasma membrane ) of FGF7 - and FGF10 - stimulated endogenous FGFR2 ( green ) for 0 , 15 , 40 , and 120 min . in T47D . TRITC - Tf is a marker of recycling ( red ) . Nuclei are stained in blue . * , cells with receptor recycled to the plasma membrane . Scale bar , 5 μm . C . Red and green pixels overlap fraction representing the co - localization of FGFR2b with the recycling endosomes marker Tf ( above ) and the proportion of green over total pixels representing FGFR2b in the cytoplasm ( below ) upon stimulation with FGF7 ( dark green ) or FGF10 ( burgundy ) for 15 , 40 , or 120 min . . Values represent the median ± SD of at least 3 independent experiments . Representative pictures are shown in B ( cytoplasm ) . * , p value < 0 . 005 ( Student ' s t - test ) . D

O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_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 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 B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Internalization (cytoplasm) and recycling (plasma membrane) of FGF7- and FGF10-stimulated endogenous FGFR2 (green) for 0, 15, 40, and 120 min. in T47D. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm. C. Red and green pixels overlap fraction representing the co-localization of FGFR2b with the recycling endosomes marker Tf (above) and the proportion of green over total pixels representing FGFR2b in the cytoplasm (below) upon stimulation with FGF7 (dark green) or FGF10 (burgundy) for 15, 40, or 120 min.. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in B (cytoplasm). *, p value<0.005 (Student's t-test). D


H . Internalization ( cytoplasm ) and recycling ( plasma membrane ) of FGF10 - stimulated HA - FGFR2b ( green ) transfected in BT549 stimulated for 0 , 40 and 120 min . . Cells were depleted or not of TTP or RCP by a pool of siRNAs . TRITC - Tf is a marker of recycling ( red ) . Nuclei are stained in blue . * , cells with receptor recycled to the plasma membrane . Scale bar , 5 μm .

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

H. Internalization (cytoplasm) and recycling (plasma membrane) of FGF10-stimulated HA-FGFR2b (green) transfected in BT549 stimulated for 0, 40 and 120 min.. Cells were depleted or not of TTP or RCP by a pool of siRNAs. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm.


( J ) control or 40 min . FGF10 - or TGFα - stimulated T47D left untreated or depleted of TTP , followed or not by transfection with siRNA resistant Flag - TTP ( Flag - TTP * ) or depleted of RCP followed or not by transfection with siRNA resistant RCP - GFP ( RCP - GFP * ) were immunoblotted with the indicated antibodies . * * , non - specific band ( J ) .

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

(J) control or 40 min. FGF10- or TGFα-stimulated T47D left untreated or depleted of TTP, followed or not by transfection with siRNA resistant Flag-TTP (Flag-TTP*) or depleted of RCP followed or not by transfection with siRNA resistant RCP-GFP (RCP-GFP*) were immunoblotted with the indicated antibodies. **, non-specific band (J).


F , G Lysates from T47D or BT20 treated with DMSO , PD173074 , AG1478 , or the MEK inhibitors U0126 and MEK162 and stimulated or not with either FGF10 or TGFα ( F ) ; T47D or BT20 treated with DMSO , or the p38 inhibitor BMS582949 and stimulated or not with either FGF10 or TGFα ( G ) were immunoblotted with the indicated antibodies .

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

F, G Lysates from T47D or BT20 treated with DMSO, PD173074, AG1478, or the MEK inhibitors U0126 and MEK162 and stimulated or not with either FGF10 or TGFα (F); T47D or BT20 treated with DMSO, or the p38 inhibitor BMS582949 and stimulated or not with either FGF10 or TGFα (G) were immunoblotted with the indicated antibodies.


Lysates from T47D stimulated or not with FGF10 , EGF for different time periods or treated with PD173074 ( C ) , MEK162 ( D ) , AG1478 ( E ) before stimulation were immunoblotted with the indicated antibodies .

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

Lysates from T47D stimulated or not with FGF10, EGF for different time periods or treated with PD173074 (C), MEK162 (D), AG1478 (E) before stimulation were immunoblotted with the indicated antibodies.


G , H Percentage of EdU incorporation in T47D pre - treated or not with FGF10 for 40 min . and stimulated or not with FGF10 or EGF ( G ) or incubated with MEK162 , PD173074 , or AG1478 before pre - treatment ( H ) . N = 6 . P value = < 0 . 05 * , < 0 . 01 * * , < 0 . 001 * * * ( one - way ANOVA with Tukey test ) .

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

G, H Percentage of EdU incorporation in T47D pre-treated or not with FGF10 for 40 min. and stimulated or not with FGF10 or EGF (G) or incubated with MEK162, PD173074, or AG1478 before pre-treatment (H). N= 6. P value = <0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).


A . Co - localization of FGFR2 ( red ) , EGFR ( green ) , and the recycling marker Tf ( blue ) in T47D stimulated or not with FGF10 or TGFα for 40 min . Scale bar , 5 μm . C . Co - localization of EGFR ( green ) , T693 phosphorylated EGFR ( blue ) , and the recycling marker Tf ( red ) in T47D stimulated or not with FGF10 or TGFα for 40 min . Scale bar , 5 μm .

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

A. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm. C. Co-localization of EGFR (green), T693 phosphorylated EGFR (blue), and the recycling marker Tf (red) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm.


E . Co - localization of FGFR2 ( red ) , EGFR ( green ) , and the recycling marker Tf ( blue ) in T47D depleted of EGFR by siRNA followed by transfection with wt or T693A and stimulated or not with either FGF10 or TGFα for the indicated time periods . Scale bar , 5 μm .

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

E. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D depleted of EGFR by siRNA followed by transfection with wt or T693A and stimulated or not with either FGF10 or TGFα for the indicated time periods. Scale bar, 5 μm.


E , F Lysates from T47D ( E ) and BT20 ( F ) depleted of EGFR , transfected with wt or T693A , and stimulated or not with either FGF10 or TGFα for the indicated time intervals were immunoblotted with the indicated antibodies .

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

E, F Lysates from T47D (E) and BT20 (F) depleted of EGFR, transfected with wt or T693A, and stimulated or not with either FGF10 or TGFα for the indicated time intervals were immunoblotted with the indicated antibodies.


A - D Percentage of EdU incorporation in T47D ( A , C ) or BT20 ( B , D ) depleted of EGFR or not , transfected with wt or T693A , and stimulated or not with FGF10 ( A , B ) or with TGFα ( C , D ) and pre - treated ( C , D ) or not ( A , B ) with FGF10 for 40 min . Values represent the median ± SD of N = 4 . P value = < 0 . 05 * , < 0 . 01 * * , < 0 . 001 * * * ( one - way ANOVA with Tukey test ) .

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

A-D Percentage of EdU incorporation in T47D (A, C) or BT20 (B, D) depleted of EGFR or not, transfected with wt or T693A, and stimulated or not with FGF10 (A, B) or with TGFα (C, D) and pre-treated (C, D) or not (A, B) with FGF10 for 40 min. Values represent the median ± SD of N= 4. P value =<0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).


A ) Seeding efficiency was compared between control non - neurodegenerative and FTLD - TDP patient samples . Cells were transfected using Lipofectamine after mitotic arrest with AraC and were fixed 3 to 6 days post - transfection with SarkoSpin fractions ( Laferrière et al , 2019 ) . Surface masks generated with Imaris show DAPI ( blue ) , nuclear TDP - 43 ( NuclTDP - 43 , cyan ) and neoaggregates ( NeoAgg , orange ) . All images are presented at the same scale , 20 μm as indicated in AraC panel Day 6 . B ) Higher magnification imaging of cells seeded for 6 days revealed presence of large cytoplasmic TDP - 43 - HA aggregates in cells seeded with FTLD - TDP - A and multiple cytoplasmic TDP - 43 - HA aggregates in cells seeded with FTLD - TDP - C . Surface representation generated with Imaris showing DAPI , TDP - 43 - HA ( green ) , phosphorylated TDP - 43 ( pTDP - 43 , magenta ) and neoaggregates ( NeoAgg , orange ) . All images are presented at the same scale , 5 μm as indicated in FTLD - TDP - C top panel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-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 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 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 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 B-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

A) Seeding efficiency was compared between control non-neurodegenerative and FTLD-TDP patient samples. Cells were transfected using Lipofectamine after mitotic arrest with AraC and were fixed 3 to 6 days post-transfection with SarkoSpin fractions (Laferrière et al, 2019). Surface masks generated with Imaris show DAPI (blue), nuclear TDP-43 (NuclTDP-43, cyan) and neoaggregates (NeoAgg, orange). All images are presented at the same scale, 20 μm as indicated in AraC panel Day 6. B) Higher magnification imaging of cells seeded for 6 days revealed presence of large cytoplasmic TDP-43-HA aggregates in cells seeded with FTLD-TDP-A and multiple cytoplasmic TDP-43-HA aggregates in cells seeded with FTLD-TDP-C. Surface representation generated with Imaris showing DAPI , TDP-43-HA (green), phosphorylated TDP-43 (pTDP-43, magenta) and neoaggregates (NeoAgg, orange). All images are presented at the same scale, 5 μm as indicated in FTLD-TDP-C top panel.


B ) Bar graph showing the seeding effect at day 5 and day 6 post - seeding in untreated , temperature denaturation or autoclaving denaturation conditions . While seeding in untreated condition showed a significant increase in neoaggregates ( One - way ANOVA , with post - hoc Fisher ' s LSD test , Day 5 : F = ( 5 , 12 ) = 6 . 713 , p = 0 . 0033 , non - neurodegenerative control vs FTLD - TDP - A p = 0 . 0016 , Day 6 : F ( 5 , 12 ) = 4 . 271 , p = 0 . 0183 , non - neurodegenerative control vs FTLD - TDP - A p = 0 . 0397 ) , this effect was abolished in denaturing conditions ( N = 3 biological replicates for non - neurodegenerative control , FTLD - TDP - A and FTLD - TDP - C , bar graph showing mean ± SEM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graph showing the seeding effect at day 5 and day 6 post-seeding in untreated, temperature denaturation or autoclaving denaturation conditions. While seeding in untreated condition showed a significant increase in neoaggregates (One-way ANOVA, with post-hoc Fisher's LSD test, Day 5: F=(5,12)=6.713, p=0.0033, non-neurodegenerative control vs FTLD-TDP-A p=0.0016, Day 6: F(5,12)=4.271, p=0.0183, non-neurodegenerative control vs FTLD-TDP-A p=0.0397), this effect was abolished in denaturing conditions (N=3 biological replicates for non-neurodegenerative control, FTLD-TDP-A and FTLD-TDP-C, bar graph showing mean ± SEM).


C ) TOP : Aggregation kinetics of full length ( fl ) recombinant human TDP - 43 ( rec - hTDP - 43 ) measured as an increase in absorbance . The addition of TEV protease induces the cleavage of the MBP tag and subsequent aggregation of rec - hTDP - 43 . Rec - hTDP - 43 aggregates used for seeding were collected 18 hours after the addition of TEV protease . BOTTOM LEFT : rec - hTDP - 43 aggregates stained by Thioflavin T ( ThT ) . BOTTOM RIGHT : Transmission Electron Microscopy ( TEM ) image of the rec - hTDP - 43 aggregates . All images are presented at the same scale of 1 μm .

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

C) TOP: Aggregation kinetics of full length (fl) recombinant human TDP-43 (rec-hTDP-43) measured as an increase in absorbance. The addition of TEV protease induces the cleavage of the MBP tag and subsequent aggregation of rec-hTDP-43. Rec-hTDP-43 aggregates used for seeding were collected 18 hours after the addition of TEV protease. BOTTOM LEFT: rec-hTDP-43 aggregates stained by Thioflavin T (ThT). BOTTOM RIGHT: Transmission Electron Microscopy (TEM) image of the rec-hTDP-43 aggregates. All images are presented at the same scale of 1 μm.


E ) Quantification of neoaggregates ( left y - axis ) and pTDP - 43 ( right y - axis ) at day 5 and day 6 post - transfection for increasing concentrations of rec - hTDP - 43 . 2 - way ANOVA repeated measure , with post - hoc Fisher ' s LSD , F ( 7 , 80 ) = 5 . 886 , p < 0 . 0001 , Day 5 : AraC vs 50 ng p = 0 . 0497 , AraC vs 500 ng p = 0 . 0002 , AraC vs 5 µg p = 0 . 0161 . 50 ng vs 100 ng p = 0 . 0208 , 50 ng vs 500 ng p = 0 . 0169 , 50 ng vs 2 . 5 µg p = 0 . 0272 , 100 ng vs 1 µg p < 0 . 0001 , 100 ng vs 5 µg p = 0 . 006 , 250 ng vs 1 μg p = 0 . 0005 , 250 ng vs 5 µg p = 0 . 034 , 500 ng vs 1 µg p < 0 . 0001 , 500 ng vs 5 µg p = 0 . 0047 , 1 µg vs 2 . 5 µg p < 0 . 0001 , 2 . 5 µg vs 5 µg p = 0 . 0081 , Day 6 : AraC vs 250 ng p = 0 . 0385 , AraC vs 1 µg p = 0 . 0006 , 50 ng vs 1 µg p = 0 . 0016 , 100 ng vs 1 µg p = 0 . 0101 , 100 ng vs 5 µg p = 0 . 0173 , 250 ng vs 5 µg p = 0 . 0006 , 500 ng vs 1 µg p = 0 . 0099 , 500 ng vs 5 µg p = 0 . 0176 , 1 µg vs 2 . 5 µg p = 0 . 0144 , 1 µg vs 5 µg p < 0 . 0001 , 2 . 5 µg vs 5 µg p = 0 . 0121 . To reduce complexity , only comparisons with AraC are marked in the graph . N = 6 biological replicates for each concentration ; bar graph showing mean ± SEM .

O O O O O O O O O O O O O B-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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of neoaggregates (left y-axis) and pTDP-43 (right y-axis) at day 5 and day 6 post-transfection for increasing concentrations of rec-hTDP-43. 2-way ANOVA repeated measure, with post-hoc Fisher's LSD, F(7,80)=5.886, p<0.0001, Day 5: AraC vs 50 ng p=0.0497, AraC vs 500 ng p=0.0002, AraC vs 5 µg p=0.0161. 50 ng vs 100 ng p=0.0208, 50 ng vs 500 ng p=0.0169, 50 ng vs 2.5 µg p=0.0272, 100 ng vs 1 µg p<0.0001, 100 ng vs 5 µg p=0.006, 250 ng vs 1 μg p=0.0005, 250 ng vs 5 µg p=0.034, 500 ng vs 1 µg p<0.0001, 500 ng vs 5 µg p=0.0047, 1 µg vs 2.5 µg p<0.0001, 2.5 µg vs 5 µg p=0.0081, Day 6: AraC vs 250 ng p=0.0385, AraC vs 1 µg p=0.0006, 50 ng vs 1 µg p=0.0016, 100 ng vs 1 µg p=0.0101, 100 ng vs 5 µg p=0.0173, 250 ng vs 5 µg p=0.0006, 500 ng vs 1 µg p=0.0099, 500 ng vs 5 µg p=0.0176, 1 µg vs 2.5 µg p=0.0144, 1 µg vs 5 µg p<0.0001, 2.5 µg vs 5 µg p=0.0121. To reduce complexity, only comparisons with AraC are marked in the graph. N=6 biological replicates for each concentration; bar graph showing mean ± SEM.


B ) Bar graphs representing the number of NeoAggpS403 only ( bars filled in light orange ) , NeoAggpS409 only ( bars filled in light purple ) and double phosphorylated NeoAggpS403 - 409 ( unfilled bars ) for each condition . Individual patients are labelled in different colored dots ( n = 3 biological replicates for each condition , graph bar showing mean ± SEM ) . Two - way ANOVA repeated measures , with post - hoc Fisher ' s LSD test , F ( 24 . 54 ) = 3 . 799 , p < 0 . 0001 , Day 4 : non - neurodegenerative control pS403 / 404 vs pS409 / 410 , p < 0 . 0001 , non - neurodegenerative control pS403 / 404 vs bi - phosphorylated , p < 0 . 0001 , FTLD - TDP - A pS403 / 404 vs pS409 / 410 , p < 0 . 0001 , FTLD - TDP - A pS403 / 404 vs bi - phosphorylated , p < 0 . 0001 FTLD - TDP - C pS403 / 404 vs pS409 / 410 , p < 0 . 0001 , FTLD - TDP - C pS403 / 404 vs bi - phosphorylated , p < 0 . 0001 . Day 5 : non - neurodegenerative control pS403 / 404 vs bi - phosphorylated , p = 0 . 0219 , FTLD - TDP - A pS403 / 404 vs double , p = 0 . 0650 , FTLD - TDP - C pS403 / 404 vs double , p = 0 . 0157 . Day 6 : Control bi - phosphorylated vs FTLD - TDP - A bi - phosphorylated , p = 0 . 5096 , control bi - phosphorylated vs FTLD - TDP - C , p = 0 . 1010 . Non - neurodegenerative control pS403 / 404 : day 3 vs 4 , p = 0 . 0559 , day 3 vs 6 , p = 0 . 0369 . FTLD - TDP - A pS404 / 404 , day 3 vs 4 , p = 0 . 0075 , day 4 vs 5 , p = 0 . 0714 . FTLD - TDP - C pS403 - 404 , day3 vs 4 , P = 0 . 0759 , day 4 vs 6 , 9 = 0 . 0305 . FTLD - TDP - A bi - phosphorylated : day 3 vs 5 , P = 0 . 0577 , day 3 vs 6 , p = 0 . 0609 . FTLD - TDP - C bi - phosphorylatede : day 3 vs 4 , p = 0 . 0522 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graphs representing the number of NeoAggpS403 only (bars filled in light orange), NeoAggpS409 only (bars filled in light purple) and double phosphorylated NeoAggpS403-409 (unfilled bars) for each condition. Individual patients are labelled in different colored dots (n=3 biological replicates for each condition, graph bar showing mean ± SEM). Two-way ANOVA repeated measures, with post-hoc Fisher's LSD test, F(24.54)=3.799, p<0.0001, Day 4: non-neurodegenerative control pS403/404 vs pS409/410, p<0.0001, non-neurodegenerative control pS403/404 vs bi-phosphorylated, p<0.0001, FTLD-TDP-A pS403/404 vs pS409/410, p<0.0001, FTLD-TDP-A pS403/404 vs bi-phosphorylated, p<0.0001 FTLD-TDP-C pS403/404 vs pS409/410, p<0.0001, FTLD-TDP-C pS403/404 vs bi-phosphorylated, p<0.0001. Day 5: non-neurodegenerative control pS403/404 vs bi-phosphorylated, p=0.0219, FTLD-TDP-A pS403/404 vs double, p=0.0650, FTLD-TDP-C pS403/404 vs double, p=0.0157. Day 6: Control bi-phosphorylated vs FTLD-TDP-A bi-phosphorylated, p=0.5096, control bi-phosphorylated vs FTLD-TDP-C, p=0.1010. Non-neurodegenerative control pS403/404: day 3 vs 4, p=0.0559, day 3 vs 6, p=0.0369. FTLD-TDP-A pS404/404, day 3 vs 4, p=0.0075, day 4 vs 5, p=0.0714. FTLD-TDP-C pS403-404, day3 vs 4, P=0.0759, day 4 vs 6, 9=0.0305. FTLD-TDP-A bi-phosphorylated: day 3 vs 5, P=0.0577, day 3 vs 6, p=0.0609. FTLD-TDP-C bi-phosphorylatede: day 3 vs 4, p=0.0522.


C ) Bar graph representing the percentage of NeoAggpS403 / 404 bearing double phosphorylation ( left side , bars filled in light orange ) and percentage of NeoAggpS409 bearing double phosphorylation ( right side , bars filled in light purple ) . Each individual patient is labelled in different colored dots . N = 3 biological replicates per condition , graph bar showing mean ± SEM . Two - way ANOVA repeated measures , with post - hoc Fisher ' s LSD test , pS403 / 404 also double positive : F ( 3 , 6 ) = 6 . 567 , p = 0 . 0253 : FTLD - TDP - A : Day 3 vs 5 , p = 0 . 0491 , day 4 vs 6 , p = 0 . 0321 , FTLD - TDP - C : ay 4 vs 6 , p = 0 . 037 . pS409 / 419 also double positive : F ( 3 , 6 ) = 1 . 246 , p = 0 . 3731 , FTLD - TDP - C : Day 4 vs 5 , p = 0 . 0144 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graph representing the percentage of NeoAggpS403/404 bearing double phosphorylation (left side, bars filled in light orange) and percentage of NeoAggpS409 bearing double phosphorylation (right side, bars filled in light purple). Each individual patient is labelled in different colored dots. N=3 biological replicates per condition, graph bar showing mean ± SEM. Two-way ANOVA repeated measures, with post-hoc Fisher's LSD test, pS403/404 also double positive: F(3,6)=6.567, p=0.0253: FTLD-TDP-A: Day 3 vs 5, p=0.0491, day 4 vs 6, p=0.0321, FTLD-TDP-C: ay 4 vs 6, p=0.037. pS409/419 also double positive: F(3,6)=1.246, p=0.3731, FTLD-TDP-C: Day 4 vs 5, p=0.0144.


E ) Overexpression of GFP - TDP - 43 variants with different mutations that either block ( CC and AA mutation , red labels ) or mimic phosphorylation ( DD and EE , green label ) at S403 / 404 or S409 / 410 in motor neuron - like NSC - 34 cells . Western blot showed a specific signal for total TDP - 43 ( top panel ) , TDP - 43pS403 / 404 ( middle panel ) and TDP - 43p409 / 410 ( lower panel ) and load control SOD1 ( bottom panel ) .

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E) Overexpression of GFP-TDP-43 variants with different mutations that either block (CC and AA mutation, red labels) or mimic phosphorylation (DD and EE, green label) at S403/404 or S409/410 in motor neuron-like NSC-34 cells. Western blot showed a specific signal for total TDP-43 (top panel), TDP-43pS403/404 (middle panel) and TDP-43p409/410 (lower panel) and load control SOD1 (bottom panel).


A - C ) dSTORM images of cells seeded for 6 days with non - neurodegenerative control ( non - ND control ) , FTLD - TDP - A and FTLD - TDP - C seeds extracted from post - mortem brain tissue . Staining shows localization of TDP - 43 ( HA tag , cyan ) along with LaminB1 ( yellow , A ) , nucleopore complexes ( nucleopores , yellow , B ) and nucleolus ( yellow , C ) . Cells seeded with FTLD - TDP seeds show coaggregation of nuclear membrane proteins upon neoaggregate formation . All images are presented at the same scale , 2 μm as indicated in the non - neurodegenerative control top panels . D ) dSTORM images of cells containing aggregates after 6 days of seeding with SarkoSpin fractions of postmortem brain from patients with FTLD - TDP - A or FTLD - TDP - C . Red arrows point to cytoplasmic aggregates . Cells were labelled with TDP - 43 ( HA - tag , cyan ) and phosphorylated TDP - 43 pS403 / 404 ( pTDP - 43 , magenta ) . All images are presented at the same scale , 2 μm as indicated in the FTLD - TDP - A top panel .

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

A-C) dSTORM images of cells seeded for 6 days with non-neurodegenerative control (non-ND control), FTLD-TDP-A and FTLD-TDP-C seeds extracted from post-mortem brain tissue. Staining shows localization of TDP-43 (HA tag, cyan) along with LaminB1 (yellow, A), nucleopore complexes (nucleopores, yellow, B) and nucleolus (yellow, C). Cells seeded with FTLD-TDP seeds show coaggregation of nuclear membrane proteins upon neoaggregate formation. All images are presented at the same scale, 2 μm as indicated in the non-neurodegenerative control top panels. D) dSTORM images of cells containing aggregates after 6 days of seeding with SarkoSpin fractions of postmortem brain from patients with FTLD-TDP-A or FTLD-TDP-C. Red arrows point to cytoplasmic aggregates. Cells were labelled with TDP-43 (HA-tag, cyan) and phosphorylated TDP-43 pS403/404 (pTDP-43, magenta). All images are presented at the same scale, 2 μm as indicated in the FTLD-TDP-A top panel.


Light microscopy images ( LM ) showing the HA - immunoreactive inclusions ( green color , black arrowheads ) identified in ultrathin sections from resin - embedded cell monolayers . The LM images were overlaid with low magnification EM images ( EM / LM overlay ) to localize the same region within the cells for EM imaging at higher magnification ( dotted black square , EM ) . A ) HA - immunopositive regions within cells seeded with FTLD - TDPA patient material showed accumulated filaments ( examples indicated with white arrowheads ) . B ) HA - immunopositive regions within cells seeded with FTLD - TDP - C patient material showed aggregated amorphous material . Scale bars LM and EM / LM overlay , 10 µm ; EM , 500

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

Light microscopy images (LM) showing the HA-immunoreactive inclusions (green color, black arrowheads) identified in ultrathin sections from resin-embedded cell monolayers. The LM images were overlaid with low magnification EM images (EM/LM overlay) to localize the same region within the cells for EM imaging at higher magnification (dotted black square, EM). A) HA-immunopositive regions within cells seeded with FTLD-TDPA patient material showed accumulated filaments (examples indicated with white arrowheads). B) HA-immunopositive regions within cells seeded with FTLD-TDP-C patient material showed aggregated amorphous material. Scale bars LM and EM/LM overlay, 10 µm; EM, 500 nm


A ) 2D TEM ( first column ) shows an example of the overall morphology of neuronal cytoplasmic inclusions from FTLD - TDP - A localized in postmortem human brain by correlative light and electron microscopy ( also shown in Fig . EV4D ) . 2D projections of the central slices of reconstructed 3D tomograms ( middle column ) taken at higher magnification within the aggregate show a dense accumulation of filaments seemingly enclosed by a boundary membrane ( white arrowheads ) . The positions of filaments selected for subtomogram analysis are shown within a single orthoslice from the same tomogram ( colored , dots last column ) . B ) STED imaging of nuclear structures ( left column ) and neuronal cytoplasmic inclusions found in a postmortem tissue of a patient with FTLD - TDP - A . LaminB1 ( green ) was used as a nuclear membrane marker along with TDP - 43 ( cyan ) and TDP - 43pS403 / 404 ( magenta ) .

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

A) 2D TEM (first column) shows an example of the overall morphology of neuronal cytoplasmic inclusions from FTLD-TDP-A localized in postmortem human brain by correlative light and electron microscopy (also shown in Fig. EV4D). 2D projections of the central slices of reconstructed 3D tomograms (middle column) taken at higher magnification within the aggregate show a dense accumulation of filaments seemingly enclosed by a boundary membrane (white arrowheads). The positions of filaments selected for subtomogram analysis are shown within a single orthoslice from the same tomogram (colored, dots last column). B) STED imaging of nuclear structures (left column) and neuronal cytoplasmic inclusions found in a postmortem tissue of a patient with FTLD-TDP-A. LaminB1 (green) was used as a nuclear membrane marker along with TDP-43 (cyan) and TDP-43pS403/404 (magenta).


C ) Confocal imaging of dystrophic neurites found in a postmortem tissue of a patient with FTLD - TDP - C . Dystrophic neurites were identified using TDP - 43 ( cyan ) , and TDP - 43pS403 / 404 ( magenta ) . Neurite markers were used to visualize neuronal projections ( MAP2 for dendrites , PNF for axons ; yellow and orange , respectively ) . D ) 2D TEM ( first row ) of dystrophic neurites from FTLD - TDP - C , localized in postmortem human brain by correlative light and electron microscopy ( also shown in Fig . EV5D ) . 2D projections of the central slices of reconstructed 3D tomograms ( middle row ) taken at higher magnification from regions within each aggregate show a sparse arrangement of filaments which are interspersed with vesicles ( white asterisk ) . A similar arrangement of sparse filaments and vesicles is seen in a cross - section of a dystrophic neurite from FTLD - TDP - C ( cross - section , also shown in Fig . EV5G ) . The positions of filaments selected for subtomogram analysis are shown within a single orthoslice from the same tomogram ( colored dots , last row ) .

O O O 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal imaging of dystrophic neurites found in a postmortem tissue of a patient with FTLD-TDP-C. Dystrophic neurites were identified using TDP-43 (cyan), and TDP-43pS403/404 (magenta). Neurite markers were used to visualize neuronal projections (MAP2 for dendrites, PNF for axons; yellow and orange, respectively). D) 2D TEM (first row) of dystrophic neurites from FTLD-TDP-C, localized in postmortem human brain by correlative light and electron microscopy (also shown in Fig. EV5D). 2D projections of the central slices of reconstructed 3D tomograms (middle row) taken at higher magnification from regions within each aggregate show a sparse arrangement of filaments which are interspersed with vesicles (white asterisk). A similar arrangement of sparse filaments and vesicles is seen in a cross-section of a dystrophic neurite from FTLD-TDP-C (cross-section, also shown in Fig. EV5G). The positions of filaments selected for subtomogram analysis are shown within a single orthoslice from the same tomogram (colored dots, last row).


( A - C ) Atg7 + / + and Atg7 - / - MEFs stably expressing GFP ‐ Gal3 ( A , C ) or GFP ‐ Ub ( B ) were treated with 1000 μM LLOMe or 250 μg / ml silica for 3 h . Cells were subjected to immunocytochemistry using the following antibodies : anti ‐ LC3 and anti ‐ Lamp1 ( A ) , anti ‐ p62 and anti ‐ Lamp1 ( B ) , or anti ‐ FK2 and anti ‐ p62 ( C ) . Bars : 10 μm .

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(A-C) Atg7+/+ and Atg7-/- MEFs stably expressing GFP‐Gal3 (A, C) or GFP‐Ub (B) were treated with 1000 μM LLOMe or 250 μg/ml silica for 3 h. Cells were subjected to immunocytochemistry using the following antibodies: anti‐LC3 and anti‐Lamp1 (A), anti‐p62 and anti‐Lamp1 (B), or anti‐FK2 and anti‐p62 (C). Bars: 10 μm.


( A , B ) NIH3T3 cells stably expressing GFP ‐ Gal3 and either empty vector ( control ) or mStrawberry ‐ Atg4BC74A ( Atg4B mutant ) were treated with 1000 μM LLOMe for 1 h . After LLOMe washout , cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI ( blue ) ( A ) . The number of GFP ‐ Gal3 or Lamp1 puncta per cell was quantified using G ‐ Count ( see also Supplementary Figure S4A and B ) . Then , the percent of GFP ‐ Gal3 ‐ positive Lamp1 puncta was determined ( B ) . The data represent means ± s . d . At least 70 cells were counted ( n = 3 ) . Bars : 20 μm . Source data for this figure is available on the online supplementary information page .

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(A, B) NIH3T3 cells stably expressing GFP‐Gal3 and either empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP‐Gal3 or Lamp1 puncta per cell was quantified using G‐Count (see also Supplementary Figure S4A and B). Then, the percent of GFP‐Gal3‐positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.


( C - E ) HeLa cells transfected with tfGal3 and either One ‐ STrEP ‐ FLAG ‐ tagged Atg4BC74A ( Atg4B mutant ) or empty vector ( control ) were observed by confocal microscopy after treatment as shown in Figure 2A and B . The number of GFP ± RFP + or GFP + RFP + puncta per cell was quantified using G ‐ Count ( D ) . Then , the percent of GFP + RFP + tfGal3 puncta was calculated ( E ) . The data represent means ± s . d . At least 30 cells were counted ( n = 3 ) . Bar : 10 μm .

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(C-E) HeLa cells transfected with tfGal3 and either One‐STrEP‐FLAG‐tagged Atg4BC74A (Atg4B mutant) or empty vector (control) were observed by confocal microscopy after treatment as shown in Figure 2A and B. The number of GFP±RFP+ or GFP+RFP+ puncta per cell was quantified using G‐Count (D). Then, the percent of GFP+RFP+tfGal3 puncta was calculated (E). The data represent means±s.d. At least 30 cells were counted (n=3). Bar: 10 μm.


( F - H ) NIH3T3 cells stably expressing empty vector ( control ) or mStrawberry ‐ Atg4BC74A ( Atg4B mutant ) were treated with 1000 μM LLOMe for 1 h . After LLOMe washout , cells were cultured in the presence or absence of both 10 μg / ml E64d and Pepstatin A , fixed at the indicated time points , and subjected to immunocytochemistry for Gal3 ( green ) and DAPI ( blue ) ( F ) . The number of endogenous Gal3 puncta per control ( G ) or Atg4B ‐ mutant ( H ) cells was quantified by G ‐ Count . The data represent means ± s . d . At least 50 cells were counted ( n = 3 ) . Bars : 20 μm . Source data for this figure is available on the online supplementary information page .

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(F-H) NIH3T3 cells stably expressing empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were cultured in the presence or absence of both 10 μg/ml E64d and Pepstatin A, fixed at the indicated time points, and subjected to immunocytochemistry for Gal3 (green) and DAPI (blue) (F). The number of endogenous Gal3 puncta per control (G) or Atg4B‐mutant (H) cells was quantified by G‐Count. The data represent means±s.d. At least 50 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.


( A - F ) HeLa cells stably expressing GFP ‐ LC3 were transfected with mStrawberry ‐ Gal3 , and treated with 1000 μM LLOMe for 1 h . Then , cells were fixed and observed by confocal microscopy ( A ) . The same specimens were further examined by transmission electron microscopy ( B - F ) . Green : LC3 ; magenta : Gal3 ; blue : DAPI .

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(A-F) HeLa cells stably expressing GFP‐LC3 were transfected with mStrawberry‐Gal3, and treated with 1000 μM LLOMe for 1 h. Then, cells were fixed and observed by confocal microscopy (A). The same specimens were further examined by transmission electron microscopy (B-F). Green: LC3; magenta: Gal3; blue: DAPI.


( G - I ) NIH3T3 cells stably expressing CFP ‐ Gal3 and YFP ‐ LC3 , and either empty vector ( control ) ( G , H ) or mStrawberry ‐ Atg4BC74A ( I ) were treated with 1000 μM LLOMe for 2 h , fixed , and observed by confocal microscopy . The electron micrographs were taken in the same sample field as the transmission electron microscope . Green : LC3 ; blue : Gal3 and DAPI ; black arrow : single membrane ; white arrow : autophagosome ; white arrowhead : ER membrane .

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(G-I) NIH3T3 cells stably expressing CFP‐Gal3 and YFP‐LC3, and either empty vector (control) (G, H) or mStrawberry‐Atg4BC74A (I) were treated with 1000 μM LLOMe for 2 h, fixed, and observed by confocal microscopy. The electron micrographs were taken in the same sample field as the transmission electron microscope. Green: LC3; blue: Gal3 and DAPI; black arrow: single membrane; white arrow: autophagosome; white arrowhead: ER membrane.


( A ) Plasmacreatinine and ureanitrogen in Atg5F / F and Atg5F / F ; KAPmice treated with vehicle or UA + OA . The values displayed represent means ± s . e . Statistically significant differences ( * P0 . 05 ) are indicated . F / F : Atg5F / Fmice ; F / F ; KAP : Atg5F / F ; KAPmice .

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(A) Plasmacreatinine and ureanitrogen in Atg5F/F and Atg5F/F;KAPmice treated with vehicle or UA+OA. The values displayed represent means±s.e. Statistically significant differences (*P0.05) are indicated. F/F: Atg5F/Fmice; F/F;KAP: Atg5F/F;KAPmice.


( B , C ) PAS ‐ stained renal cortical region of Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA ( n = 4 - 7 ) . Bars : 40 μm ( B ) . Kidney injury score of Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA ( C ) .

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(B, C) PAS‐stained renal cortical region of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (n=4-7). Bars: 40 μm (B). Kidney injury score of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (C).


( D ) Kidney cortexes from Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA were subjected to immunohistocheminal analysis of Lamp1 and LC3 . Green : LC3 ; magenta : Lamp1 ; blue : DAPI . F / F : Atg5F / F mice ; F / F ; KAP : Atg5F / F ; KAP mice . Bar : 5 μm .

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(D) Kidney cortexes from Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA were subjected to immunohistocheminal analysis of Lamp1 and LC3. Green: LC3; magenta: Lamp1; blue: DAPI. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. Bar: 5 μm.


( E ) Electron micrographs of kidney proximal tubules in Atg5 F / F and Atg5F / F ; KAP mice treated with UA + OA . Yellow arrowhead : autophagosome ; asterisk : lysosome ; Mt : mitochondrion . Bar : 1 μm .

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(E) Electron micrographs of kidney proximal tubules in Atg5 F/F and Atg5F/F;KAP mice treated with UA+OA. Yellow arrowhead: autophagosome; asterisk: lysosome; Mt: mitochondrion. Bar: 1 μm.


Immunofluorescence microscopy analysis reveals highly similar and mostly nuclear localization of the SMARCB1 wild type and the R377C mutant , visualized by anti - HA staining ( green ) . Phalloidin and DAPI staining was used to visualize the actin cytoskeleton and the nucleus , respectively . A novel MS - microscopy approach was used to further define the molecular context of the proteins . This analysis identified possible chromosomal , endosomal and membrane localization of the proteins . The possible endosomal localization is in agreement with the anti - HA immunofluorescence microscopy results . ( key : the scale bar for immunofluorescence images is 10 µm , and the color gradient on the MS - microscopy indicates the localization scores calculated by the MS - microscopy tool .

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Immunofluorescence microscopy analysis reveals highly similar and mostly nuclear localization of the SMARCB1 wild type and the R377C mutant, visualized by anti-HA staining (green). Phalloidin and DAPI staining was used to visualize the actin cytoskeleton and the nucleus, respectively. A novel MS-microscopy approach was used to further define the molecular context of the proteins. This analysis identified possible chromosomal, endosomal and membrane localization of the proteins. The possible endosomal localization is in agreement with the anti-HA immunofluorescence microscopy results. (key: the scale bar for immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-microscopy tool.


The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the SMARCB1 R377C mutation compared with the wild type SMARCB1 or the vector control . The error bars designate the standard deviation ( SD )

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The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the SMARCB1 R377C mutation compared with the wild type SMARCB1 or the vector control. The error bars designate the standard deviation (SD)


Immunofluorescence microscopy analysis of the STK38L wild type and the R105W mutant display no clear difference in localization . Both the wild type and the R105W mutant display nuclear , cytoplasmic and plasma membrane localization . However , the MS - microscopy analysis suggests a more prominent endosomal localization with the R105W mutant . ( key : the scale bar for the immunofluorescence images is 10 µm , and the color gradient on the MS - microscopy indicates the localization scores calculated by the MS - Microscopy tool .

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Immunofluorescence microscopy analysis of the STK38L wild type and the R105W mutant display no clear difference in localization. Both the wild type and the R105W mutant display nuclear, cytoplasmic and plasma membrane localization. However, the MS-microscopy analysis suggests a more prominent endosomal localization with the R105W mutant. (key: the scale bar for the immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-Microscopy tool.


The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the expression of the wild type STK38L , whereas the R105W mutant only slightly increased the number of colonies . The error bars designate the standard deviation ( SD )

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The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the expression of the wild type STK38L, whereas the R105W mutant only slightly increased the number of colonies. The error bars designate the standard deviation (SD)


Each mitochondrial protein is colored according to ( A ) its fold change calculated by standard differential expression using the limma package ( Ritchie et al , 2015 ; Phipson et al , 2016 ) ; ( B ) its CNV value ( 5 proteins with the highest CNV value and 5 proteins with the lowest CNV values are highlighted and annotated in boxes ) ; and ( C ) the absolute difference between the two

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Each mitochondrial protein is colored according to (A) its fold change calculated by standard differential expression using the limma package (Ritchie et al, 2015; Phipson et al, 2016); (B) its CNV value (5 proteins with the highest CNV value and 5 proteins with the lowest CNV values are highlighted and annotated in boxes); and (C) the absolute difference between the two values


Analysis of the abundance distribution for ( A ) cytoplasmic , ( B ) mitochondrial , and ( C ) extracellular proteins across 5 age groups in C . elegans ( Walther et al , 2015 ) . Each age group was compared against a pool reference sample using SILAC ( Ong et al , 2002 ) . Distribution of protein fold changes for the different time points are shown as density plots and as as boxplots ( inset ) ; below the boxplots , asterisks mark the time points that show significantly different distribution of fold changes compared to the first time point ( Mann - Whitney test p < 0 . 01 )

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Analysis of the abundance distribution for (A) cytoplasmic, (B) mitochondrial, and (C) extracellular proteins across 5 age groups in C.elegans (Walther et al, 2015). Each age group was compared against a pool reference sample using SILAC (Ong et al, 2002). Distribution of protein fold changes for the different time points are shown as density plots and as as boxplots (inset); below the boxplots, asterisks mark the time points that show significantly different distribution of fold changes compared to the first time point (Mann-Whitney test p<0.01)


Abundance variation of complex members for ( D ) three mitochondrial complexes ( respiratory complex I , mitochondrial ribosome and mitochondrial import / export proteins ) . For each complex , the abundance variation of each member ( colored in grey ) over the 5 age groups is reported with the Δ SILAC ratio ( difference between the experimental SILAC ratio of a time point and the SILAC ratio at day 1 , left ) and the CNV value ( right ) . For each age group , the box plot ( colored as the corresponding time - point in the panels A - B - C ) represents the variance between the abundances of the complex members . Significant changes ( Mann - Whitney test p < 0 . 05 ) are marked with a red

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Abundance variation of complex members for (D) three mitochondrial complexes (respiratory complex I, mitochondrial ribosome and mitochondrial import/export proteins). For each complex, the abundance variation of each member (colored in grey) over the 5 age groups is reported with the Δ SILAC ratio (difference between the experimental SILAC ratio of a time point and the SILAC ratio at day 1, left) and the CNV value (right). For each age group, the box plot (colored as the corresponding time-point in the panels A-B-C) represents the variance between the abundances of the complex members. Significant changes (Mann-Whitney test p<0.05) are marked with a red star


( E ) Variation of the mitochondrial proteome over the lifespan of C . elegans ( day 1 and day 22 ) . Respiratory chain complex I and mitochondrial ribosomal proteins are highlighted in green and purple , respectively ( selected proteins , with extreme CNV values , are indicated )

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(E) Variation of the mitochondrial proteome over the lifespan of C.elegans (day 1 and day 22). Respiratory chain complex I and mitochondrial ribosomal proteins are highlighted in green and purple, respectively (selected proteins, with extreme CNV values, are indicated)


( F ) Variation of the extracellular proteome abundance over the lifespan of C . elegans ( day 1 and day 22 ) . Four proteins have been selected ( highlighted with a colored circle : purple for NAS - 34 , green for Y45F10C . 4 , blue for Vitellogenin - 1 , black for Y45F10C . 2 ) for their slower or accelerated increase of abundance compared to the rest of extracellular proteins . The variation of the selected proteins ( highlighted with the same colors ) throughout the worm lifespan is represented in the middle box , with the average of the extracellular proteome colored in yellow . Related to Figure EV5 and Dataset

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(F) Variation of the extracellular proteome abundance over the lifespan of C.elegans (day 1 and day 22). Four proteins have been selected (highlighted with a colored circle: purple for NAS-34, green for Y45F10C.4, blue for Vitellogenin-1, black for Y45F10C.2) for their slower or accelerated increase of abundance compared to the rest of extracellular proteins. The variation of the selected proteins (highlighted with the same colors) throughout the worm lifespan is represented in the middle box, with the average of the extracellular proteome colored in yellow. Related to Figure EV5 and Dataset EV6


B MAPKKK5 - C interacts with PBL27 in yeast two - hybrid experiments . The growth of yeast colonies on plates ( - ULWH ) lacking uracil ( U ) , leucine ( L ) , tryptophan ( W ) , and histidine ( H ) with 2 mM 3 - aminotriazole ( 3 - AT ) indicates a positive interaction .

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B MAPKKK5-C interacts with PBL27 in yeast two-hybrid experiments. The growth of yeast colonies on plates (-ULWH) lacking uracil (U), leucine (L), tryptophan (W), and histidine (H) with 2 mM 3-aminotriazole (3-AT) indicates a positive interaction.


C Chitin - induced MAPK activation was analyzed by immunoblots with α - pMAPK .

C Chitin-induced MAPK activation was analyzed by immunoblots with α-pMAPK.


D Complementation of chitin - induced MAPK activation in mapkkk5 mutants by expression of MAPKKK5 - FLAG . MAPK activation was analyzed by immunoblots with α - pMAPK .

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D Complementation of chitin-induced MAPK activation in mapkkk5 mutants by expression of MAPKKK5-FLAG. MAPK activation was analyzed by immunoblots with α-pMAPK.


E Flg22 - induced MAPK activation was analyzed by immunoblots with α - pMAPK . Experiments ( B ) - ( E ) were performed three times with similar results .

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E Flg22-induced MAPK activation was analyzed by immunoblots with α-pMAPK. Experiments (B) - (E) were performed three times with similar results.


A Chitin - induced callose deposition in the mapkkk5 mutants . Seedlings were analyzed at 18 hours after treatment with 10 μM chitin . Representative pictures from the biological replicates are presented . Data are means ± SD from three independent biological replicates , where each biological replicate consists of two technical replicates . The asterisks indicate statistically significant differences from the WT controls by Student ' s t - test ( P < 0 . 05 ) .

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A Chitin-induced callose deposition in the mapkkk5 mutants. Seedlings were analyzed at 18 hours after treatment with 10 μM chitin. Representative pictures from the biological replicates are presented. Data are means ±SD from three independent biological replicates, where each biological replicate consists of two technical replicates. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).


B Leaf discs were treated with 10 μM ( GlcNAc ) 7 in a solution containing 500 μM L - 012 and 10 μg / ml horseradish peroxidase . ROS production was quantified using a luminescence microplate reader . Data are means ± SD calculated using three biological replicates , where each biological replicate consisted of two technical replicates .

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B Leaf discs were treated with 10 μM (GlcNAc)7 in a solution containing 500 μM L-012 and 10 μg/ml horseradish peroxidase. ROS production was quantified using a luminescence microplate reader. Data are means ±SD calculated using three biological replicates, where each biological replicate consisted of two technical replicates.


C mapkkk5 mutations reduce resistance to Alternaria brassiciicola . Lesions sizes were measured 6 days after inoculation . Values are mean ± S . E . , n ≥ 92 . Asterisks indicate significant difference from wild - type controls by Student ' s t - test ( P < 0 . 01 ) .

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C mapkkk5 mutations reduce resistance to Alternaria brassiciicola. Lesions sizes were measured 6 days after inoculation. Values are mean ±S.E., n ≥92. Asterisks indicate significant difference from wild-type controls by Student's t-test (P < 0.01).


D Analysis of chitin - induced transcriptional reprogramming in mapkkk5 - 1 . Total RNA was extracted from seedlings treated with mock or 40 μM chitin for 3 hours and analyzed by RNA - seq . Genes that are significantly induced or suppressed by chitin in the wild type were selected ( q - value < 0 . 05 ; 12 , 992 genes ) . The log2 fold changes of the selected genes compared to mock in the wild type and mapkkk5 - 1 were plotted . Yellow and blue dots indicate genes that show reduced induction or suppression ( 506 genes ) or enhanced induction or suppression ( 151 genes ) in mapkkk5 - 1 compared to the wildtype , respectively . The slope of the linear regression line ( red ) indicates that the overall transcriptional response is weakened in mapkkk5 - 1 compared to the wild type . As a comparison , the line y = x ( black ) is shown . For more details , see the Methods . E A heatmap showing MAPKKK5 - dependent genes . Genes showing reduced induction or suppression in mapkkk5 - 1 compared to the wild type were selected as described in the Methods ( 339 genes ) . The log2 fold changes of the selected genes compared to mock were subjected to hierarchical clustering analysis . Yellow indicates positive values , blue indicates negative values and black indicates zero : see the color scale .

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D Analysis of chitin-induced transcriptional reprogramming in mapkkk5-1. Total RNA was extracted from seedlings treated with mock or 40 μM chitin for 3 hours and analyzed by RNA-seq. Genes that are significantly induced or suppressed by chitin in the wild type were selected (q-value < 0.05; 12,992 genes). The log2 fold changes of the selected genes compared to mock in the wild type and mapkkk5-1 were plotted. Yellow and blue dots indicate genes that show reduced induction or suppression (506 genes) or enhanced induction or suppression (151 genes) in mapkkk5-1 compared to the wildtype, respectively. The slope of the linear regression line (red) indicates that the overall transcriptional response is weakened in mapkkk5-1 compared to the wild type. As a comparison, the line y=x (black) is shown. For more details, see the Methods.E A heatmap showing MAPKKK5-dependent genes. Genes showing reduced induction or suppression in mapkkk5-1 compared to the wild type were selected as described in the Methods (339 genes). The log2 fold changes of the selected genes compared to mock were subjected to hierarchical clustering analysis. Yellow indicates positive values, blue indicates negative values and black indicates zero: see the color scale.


F Expression patterns of representative MAPKKK5 - dependent genes . qRT - PCR analysis of defense - related genes in 8 - day - old seedlings exposed to 40μM ( GlcNAc ) 7 for 3h . Data are shown as the average of three independent biological replicates ± SD . The asterisks indicate statistically significant differences from the WT controls by Student ' s t - test ( P < 0 . 05 ) .

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F Expression patterns of representative MAPKKK5-dependent genes. qRT-PCR analysis of defense-related genes in 8-day-old seedlings exposed to 40μM (GlcNAc)7 for 3h. Data are shown as the average of three independent biological replicates ± SD. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).


A The MAPKKK5 - GFP protein was detected in the membrane fraction . The total ( T ) , soluble ( S ) , and microsomal membrane ( M ) fractions purified from Nb leaves expressing MAPKKK5 - GFP were used for immunoblots with antibodies against GFP , PM - localized aquaporin PIP1 and cytosolic ascorbate peroxidase EAP1 .

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A The MAPKKK5-GFP protein was detected in the membrane fraction. The total (T), soluble (S), and microsomal membrane (M) fractions purified from Nb leaves expressing MAPKKK5-GFP were used for immunoblots with antibodies against GFP, PM-localized aquaporin PIP1 and cytosolic ascorbate peroxidase EAP1.


B Subcellular localization of MAPKKK5 - GFP and GFP in Nb leaves . The results were photographed at 38 hpi . Scale bar = 50µm .

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B Subcellular localization of MAPKKK5-GFP and GFP in Nb leaves. The results were photographed at 38 hpi. Scale bar=50µm.


C Detection of MAPKKK5 - GFP and PBL27 - HA transiently expressed in Nb leaves . Total proteins were purified from Nb leaves at 38 hpi .

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C Detection of MAPKKK5-GFP and PBL27-HA transiently expressed in Nb leaves. Total proteins were purified from Nb leaves at 38 hpi.


D Localization of MAPKKK5K375M - GFP was analyzed by using total ( T ) , soluble ( S ) , and microsomal membrane ( M ) fractions purified from Nb leaves expressing MAPKKK5 K375M -

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D Localization of MAPKKK5K375M-GFP was analyzed by using total (T), soluble (S), and microsomal membrane (M) fractions purified from Nb leaves expressing MAPKKK5 K375M -GFP


E Co - immunoprecipitation assays show that MAPKKK5K375M - GFP forms the complex with PBL27 - HA in Nb leaves . The microsomal fractions purified from Nb leaves co - expressing MAPKKK5K375M - GFP and PBL27 - HA at 48 hpi were used for co - immunoprecipitation with α - GFP .

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E Co-immunoprecipitation assays show that MAPKKK5K375M-GFP forms the complex with PBL27-HA in Nb leaves. The microsomal fractions purified from Nb leaves co-expressing MAPKKK5K375M-GFP and PBL27-HA at 48 hpi were used for co-immunoprecipitation with α-GFP.


F Visualization of the interaction between PBL27 and MAPKKK5K375M by bimolecular fluorescence complementation ( BiFC ) analysis in Nb leaves . The results were photographed at 38 hpi . Venus fluorescence indicates the interaction between PBL27 and MAPKKK5K375M . Scale bar = 50µm .

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F Visualization of the interaction between PBL27 and MAPKKK5K375M by bimolecular fluorescence complementation (BiFC) analysis in Nb leaves. The results were photographed at 38 hpi. Venus fluorescence indicates the interaction between PBL27 and MAPKKK5K375M. Scale bar=50µm.


A Detection of MAPKKK5 - GFP and MAPKKK5K375M - GFP transiently expressed in Arabidopsis protoplasts .

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A Detection of MAPKKK5-GFP and MAPKKK5K375M-GFP transiently expressed in Arabidopsis protoplasts.


B Expression of MAPKKK5 - GFP reduces the level of PBL27 . CBB , Coomassie brilliant blue .

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B Expression of MAPKKK5-GFP reduces the level of PBL27. CBB, Coomassie brilliant blue.


C Arabidopsisprotoplasts expressing PBL27 - HA and MAPKKK5 - GFP were treated with 30 μM MG132 for 3 h and subjected to immunoblots with α - HA and α - GFP .

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C Arabidopsisprotoplasts expressing PBL27-HA and MAPKKK5-GFP were treated with 30 μM MG132 for 3 h and subjected to immunoblots with α-HA and α-GFP.


D The co - immunoprecipitation assay shows that PBL27 forms the complex with MAPKKK5 in Arabidopsis protoplasts . Total proteins purified from protoplasts co - expressing PBL27 and MAPKKK5 were subjected to immunoprecipitation with α - HA .

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D The co-immunoprecipitation assay shows that PBL27 forms the complex with MAPKKK5 in Arabidopsis protoplasts. Total proteins purified from protoplasts co-expressing PBL27 and MAPKKK5 were subjected to immunoprecipitation with α-HA.


E Visualization of the interaction between PBL27 and MAPKKK5K375M by BiFC analysis in Arabidopsis protoplasts . FLS - associated RLCK BIK1 was also used for this assay . Venus fluorescence indicates interaction between PBL27 and MAPKKK5K375M . Scale bar = 10µm .

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E Visualization of the interaction between PBL27 and MAPKKK5K375M by BiFC analysis in Arabidopsis protoplasts. FLS-associated RLCK BIK1 was also used for this assay. Venus fluorescence indicates interaction between PBL27 and MAPKKK5K375M. Scale bar=10µm.


F The disassociation of PBL27 - MAPKKK5 interaction after chitin treatment in Arabidopsis protoplasts . Protoplasts were co - transfected with MAPKKK5K375M - GFP and PBL27 - HA , stimulated with or without 10μM chitin or 1 μM flg22 for 10 min and subjected to the co - immunoprecipitation assay . Experiments ( A ) - ( F ) were performed three times with similar results .

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F The disassociation of PBL27-MAPKKK5 interaction after chitin treatment in Arabidopsis protoplasts. Protoplasts were co-transfected with MAPKKK5K375M-GFP and PBL27-HA, stimulated with or without 10μM chitin or 1 μM flg22 for 10 min and subjected to the co-immunoprecipitation assay. Experiments (A) - (F) were performed three times with similar results.


A PBL27 phosphorylates MAPKKK5 - C in vitro . The in vitro phosphorylation reaction was carried out using [ 32P ] γ - ATP , and the phosphorylated proteins were detected by autoradiography . CBB , Coomassie brilliant blue .

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A PBL27 phosphorylates MAPKKK5-C in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. CBB, Coomassie brilliant blue.


B BIK1 does not phosphorylate MAPKKK5 - C in vitro . RBOHD was used as a positive control . Asterisk indicates artificial bands .

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B BIK1 does not phosphorylate MAPKKK5-C in vitro. RBOHD was used as a positive control. Asterisk indicates artificial bands.


D The S622A mutation strongly reduces phosphorylation by PBL27 . Each of phosphorylation sites was substituted by alanine , and subjected to the in vitro phosphorylation assays .

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D The S622A mutation strongly reduces phosphorylation by PBL27. Each of phosphorylation sites was substituted by alanine, and subjected to the in vitro phosphorylation assays.


E PBL27 does not phosphorylate MAPKKK5 - C6xAin vitro . The in vitro phosphorylation reaction was carried out using [ 32P ] γ - ATP , and the phosphorylated proteins were detected by autoradiography .

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E PBL27 does not phosphorylate MAPKKK5-C6xAin vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography.


F PBL27 interacts with MAPKKK5 - C6xA in yeast two - hybrid experiments . The growth of yeast colonies on plates ( - ULWH ) lacking uracil ( U ) , leucine ( L ) , tryptophan ( W ) , and histidine ( H ) with 10 mM 3 - AT indicated a positive interaction .

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F PBL27 interacts with MAPKKK5-C6xA in yeast two-hybrid experiments. The growth of yeast colonies on plates (-ULWH) lacking uracil (U), leucine (L), tryptophan (W), and histidine (H) with 10 mM 3-AT indicated a positive interaction.


A Chitin - induced MAPK activation in a mapkkk5 mutant ( Line 1 ) expressing MAPKKK56xA . MAPK activity was detected using immunoblots with α - pMAPK .

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A Chitin-induced MAPK activation in a mapkkk5 mutant (Line 1) expressing MAPKKK56xA. MAPK activity was detected using immunoblots with α-pMAPK.


B Chitin - induced callose deposition in the MAPKKK56xA plant ( Line 1 ) . Seedlings were analyzed at 18 hours after treatment with 10 μM chitin . Data are means ± SD from three independent biological replicates , where each biological replicate consists of two technical replicates . The asterisks indicate statistically significant differences from the WT controls by Student ' s t - test ( P < 0 . 05 ) .

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B Chitin-induced callose deposition in the MAPKKK56xA plant (Line 1). Seedlings were analyzed at 18 hours after treatment with 10 μM chitin. Data are means ±SD from three independent biological replicates, where each biological replicate consists of two technical replicates. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).


C Detection of MAPKKK5K375M - GFP and MAPKKK5K375M , 6xA - GFP transiently expressed in Arabidopsis protoplasts .

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C Detection of MAPKKK5K375M -GFP and MAPKKK5K375M,6xA-GFP transiently expressed in Arabidopsis protoplasts.


D Visualization of interaction between PBL27 and MAPKKK5K375M , 6xA by BiFC analysis in Arabidopsis protoplasts . Venus fluorescence indicates interaction between PBL27 and MAPKKK5K375M , 6xA . Scale bar = 10µm .

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D Visualization of interaction between PBL27 and MAPKKK5K375M, 6xA by BiFC analysis in Arabidopsis protoplasts. Venus fluorescence indicates interaction between PBL27 and MAPKKK5K375M, 6xA. Scale bar=10µm.


A PBL27 does not interact with MAPKKK5 - KD : C in a yeast two - hybrid assay .

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A PBL27 does not interact with MAPKKK5-KD:C in a yeast two-hybrid assay.


B Addition of CERK1 to an in vitro kinase assay enhances phosphorylation of MAPKKK5 - KDK375M : C by PBL27 . Asterisks indicate artificial bands derived from CERK1 : IC .

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B Addition of CERK1 to an in vitro kinase assay enhances phosphorylation of MAPKKK5-KDK375M:C by PBL27. Asterisks indicate artificial bands derived from CERK1:IC.


C CERK1 does not phosphorylate the C - terminal domain of MAPKKK5 in vitro . The in vitro kinase assay was performed with recombinant proteins of GST - PBL27 , MAPKKK5 - C and the intracellular kinase domain of CERK1 ( GST - CERK1 : IC ) . The protein loading control was shown by staining with Coomassie Brilliant Blue . The in vitro phosphorylation reaction was carried out using [ 32P ] γ - ATP , and the phosphorylated proteins were detected by autoradiography . Asterisks indicate artificial bands .

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C CERK1 does not phosphorylate the C-terminal domain of MAPKKK5 in vitro. The in vitro kinase assay was performed with recombinant proteins of GST-PBL27, MAPKKK5-C and the intracellular kinase domain of CERK1 (GST-CERK1:IC). The protein loading control was shown by staining with Coomassie Brilliant Blue. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. Asterisks indicate artificial bands.


D The alanine substitution mutations at the residues conserved in the activation loop of reduces the phosphorylation of PBL27 by CERK1 : IC . The in vitro kinase assay was carried out with recombinant proteins of GST - PBL27K112E , GST - PBL27K112E , 3xA and CERK1 - IC .

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D The alanine substitution mutations at the residues conserved in the activation loop of reduces the phosphorylation of PBL27 by CERK1:IC. The in vitro kinase assay was carried out with recombinant proteins of GST-PBL27K112E , GST-PBL27K112E, 3xA and CERK1-IC.


E The kinase activity of GST - PBL273xA was analyzed by the in vitro kinase assay using [ 32P ] γ - ATP .

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E The kinase activity of GST-PBL273xA was analyzed by the in vitro kinase assay using [32P]γ-ATP.


A Visualization of interaction between MAPKKK5K375M and MKK1 / MKK2 / MKK4 / MKK5 by BiFC analysis in Arabidopsis protoplasts . Venus fluorescence indicates interaction . Scale bar = 10µm .

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A Visualization of interaction between MAPKKK5K375M and MKK1 / MKK2 / MKK4 / MKK5 by BiFC analysis in Arabidopsis protoplasts. Venus fluorescence indicates interaction. Scale bar=10µm.


B MAPKKK5 - KD phosphorylates MKK4 and MKK5 in vitro . The in vitro phosphorylation reaction was carried out using [ 32P ] γ - ATP , and the phosphorylated proteins were detected by autoradiography .

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B MAPKKK5-KD phosphorylates MKK4 and MKK5 in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography.


C MAPKKK5 - KD does not phosphorylate a T224A / S230A mutant of MKK4 or a T215A / S221A mutant of MKK5 . The above experiments were performed three times with similar results .

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C MAPKKK5-KD does not phosphorylate a T224A/S230A mutant of MKK4 or a T215A/S221A mutant of MKK5. The above experiments were performed three times with similar results.


Body weights of WT and Rcan1 - KO mice fed normal chow diet ( NC ) for 20 weeks starting at 8 - 10 weeks of age ( male , n = 15 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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Body weights of WT and Rcan1-KO mice fed normal chow diet (NC) for 20 weeks starting at 8-10 weeks of age (male, n=15 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


Body weights of WT and Rcan1 - KO mice fed a high - fat diet ( HFD ) for 20 weeks starting at 8 - 10 weeks of age ( male , n = 15 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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Body weights of WT and Rcan1-KO mice fed a high-fat diet (HFD) for 20 weeks starting at 8-10 weeks of age (male, n=15 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


C Food consumption per gram of body weight gained ( g / g ∆ BW ) over the first 15 weeks on NC or HFD , starting at 8 weeks of age ( male , n = 9 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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C Food consumption per gram of body weight gained (g/g ∆BW) over the first 15 weeks on NC or HFD, starting at 8 weeks of age (male, n=9 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


Cholesterol remaining in the feces of WT and Rcan1 - KO mice fed a HFD , measured as mMol per mg of feces ( males , n = 4 over 3 days ) . Cholesterol content of NC and the HFD chow ( input ) were measured for comparison . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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Cholesterol remaining in the feces of WT and Rcan1-KO mice fed a HFD, measured as mMol per mg of feces (males, n=4 over 3 days). Cholesterol content of NC and the HFD chow (input) were measured for comparison. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


triglyceride remaining in the feces of WT and Rcan1 - KO mice fed a HFD , measured as mMol per mg of feces ( males , n = 4 over 3 days ) . triglyceride content of NC and the HFD chow ( input ) were measured for comparison . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

triglyceride remaining in the feces of WT and Rcan1-KO mice fed a HFD, measured as mMol per mg of feces (males, n=4 over 3 days). triglyceride content of NC and the HFD chow (input) were measured for comparison. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


MRI measures of total body weight , percentage lean mass , and percent fat mass prior to and after 6 weeks on the HFD ( males , n = 5 ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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MRI measures of total body weight, percentage lean mass, and percent fat mass prior to and after 6 weeks on the HFD (males, n=5). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


MRI image of WT and KO males following 8 weeks on a HFD . Yellow delineates visceral WAT . Pink delineates subcutaneous WAT . Quantification graphed on the right . Data information : values shown are mean ± SEM * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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MRI image of WT and KO males following 8 weeks on a HFD. Yellow delineates visceral WAT. Pink delineates subcutaneous WAT. Quantification graphed on the right. Data information: values shown are mean ±SEM * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


Representative H & E staining of gWAT from WT and KO males after 25 weeks on NC or HFD . Scale bar = 100 μm . K Quantification of cross - sectional area ( CSA ) of adipocytes in K ( n = 3 animals , 3 images / each , 75 - 100 adipocytes ) . Scale bar = 100 μm . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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Representative H&E staining of gWAT from WT and KO males after 25 weeks on NC or HFD. Scale bar = 100 μm. K Quantification of cross-sectional area (CSA) of adipocytes in K (n=3 animals, 3 images/each, 75-100 adipocytes). Scale bar = 100 μm. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


L Transcript levels for inflammatory markers Monocyte chemoattractant protein - 1 ( Mcp - 1 ) , macrophage antigen F4 / 80 ( F480 ) , tumor necrosis factor - alpha ( Tnfα ) , and tumor necrosis factor - beta ( Tnfβ ) in gWAT of WT and KO after 25 weeks on NC or HFD ( males , n = 4 - 5 ) . Transcript levels were normalized to 18S Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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L Transcript levels for inflammatory markers Monocyte chemoattractant protein-1 (Mcp-1), macrophage antigen F4/80 (F480), tumor necrosis factor-alpha (Tnfα), and tumor necrosis factor-beta (Tnfβ) in gWAT of WT and KO after 25 weeks on NC or HFD (males, n=4-5). Transcript levels were normalized to 18S Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


A , B Oil Red O staining of livers from WT and KO males after 25 weeks on NC or HFD , with quantification of relative stain intensity ( n = 3 , 3 images each ) . Scale bar = 100 μm . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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A, B Oil Red O staining of livers from WT and KO males after 25 weeks on NC or HFD, with quantification of relative stain intensity (n=3, 3 images each). Scale bar = 100 μm. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


C Transcript levels for genes involved in lipid metabolism and gluconeogenesis in livers of WT and KO mice after 25 weeks on NC or HFD . Stearoyl - Coenzyme A desaturase ( Scd1 ) , fatty acid transporter ( CD36 ) , glycerol - 3 - phosphate acyltransferase ( Gpat ) , carnitine palmitoyltransferase I ( Cpt1 ) , peroxisome proliferator - activated gamma ( PPARγ ) , pyruvate dehydrogenase acetyl - transferase ( Pdk4 ) , PPARγ coactivator 1 alpha ( PGC1α ) , diacylglycerol O - acyltransferase ( Dgat ) , and facilitated glucose transporter ( Glut4 ) . ( males , n = 4 - 5 ) . Transcript levels were normalized to 18S . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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C Transcript levels for genes involved in lipid metabolism and gluconeogenesis in livers of WT and KO mice after 25 weeks on NC or HFD. Stearoyl-Coenzyme A desaturase (Scd1), fatty acid transporter (CD36), glycerol-3-phosphate acyltransferase (Gpat), carnitine palmitoyltransferase I (Cpt1), peroxisome proliferator-activated gamma (PPARγ), pyruvate dehydrogenase acetyl-transferase (Pdk4), PPARγ coactivator 1 alpha (PGC1α), diacylglycerol O-acyltransferase (Dgat), and facilitated glucose transporter (Glut4). (males, n=4-5). Transcript levels were normalized to 18S. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


D - F Serum levels of free fatty acids ( FAA ) , insulin , and leptin in WT and KO after 25 weeks on NC or HFD ( males , n = 8 - 10 ) . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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D-F Serum levels of free fatty acids (FAA), insulin, and leptin in WT and KO after 25 weeks on NC or HFD (males, n=8-10). Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


Glucose tolerance test ( GTT ) after 25 weeks on NC or HFD . GTT was performed following an overnight fast Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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Glucose tolerance test (GTT) after 25 weeks on NC or HFD. GTT was performed following an overnight fast Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


Insulin tolerance test ( ITT ) after 25 weeks on NC or HFD . , ITT following a three hour fast ( males , n = 6 ) . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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Insulin tolerance test (ITT) after 25 weeks on NC or HFD. , ITT following a three hour fast (males, n=6). Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


Oxygen consumption ( VO2 ) of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

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Oxygen consumption (VO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


carbon dioxide production ( VCO2 ) of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

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carbon dioxide production (VCO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


respiratory exchange ratio VCO2 / VO2 ( RER ) of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

respiratory exchange ratio VCO2/VO2 (RER) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


physical activity of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

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

physical activity of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


Transcript levels for Rcan1 . 1 and Rcan1 . 4 during differentiation of 3T3 - L1 adipocytes , measured at 0 , 1 , 2 , 3 , 4 , 6 and 24 hours following the addition MDI as well as at 3 , 7 and 10 days as indicated ( n = 3 ) . * = p < 0 . 05 ; * * = p < 0 . 01 , student ' s t - test , unpaired , vs 0 time point . Data information : Transcript levels were normalized to 18S . All values shown are mean ±

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

Transcript levels for Rcan1.1 and Rcan1.4 during differentiation of 3T3-L1 adipocytes, measured at 0, 1, 2, 3, 4, 6 and 24 hours following the addition MDI as well as at 3, 7 and 10 days as indicated (n=3). * = p < 0.05; ** = p < 0.01, student's t-test, unpaired, vs 0 time point. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


B Western blot for RCAN1 . 1 , RCAN1 . 4 and tubulin ( TUB ) in whole cell extracts of 3T3 - L1 adipocytes transfected with a control si - RNA or ones targeting either Rcan1 . 4 or Rcan1 . 1 ( 20 μg per lane ) .

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

B Western blot for RCAN1.1, RCAN1.4 and tubulin (TUB) in whole cell extracts of 3T3-L1 adipocytes transfected with a control si-RNA or ones targeting either Rcan1.4 or Rcan1.1 (20 μg per lane).


C Representative images of 3T3 - L1 adipocytes transfected with the indicated si - RNAs prior to differentiation and stained with Oil Red O on day 10 . Scale bar = 100 μm .

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C Representative images of 3T3-L1 adipocytes transfected with the indicated si-RNAs prior to differentiation and stained with Oil Red O on day 10. Scale bar = 100 μm.


D Western blot for adiponectin ( ADPOQ ) released into the media during differentiation by 3T3 - L1 adipocytes transfected with the indicated si - RNAs . Ponceau stained image provides loading control .

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D Western blot for adiponectin (ADPOQ) released into the media during differentiation by 3T3-L1 adipocytes transfected with the indicated si-RNAs. Ponceau stained image provides loading control.