( C ) Localization of Ykt6 relative to different organelle marker proteins during nitrogen starvation . Cells expressing GFP - tagged Ykt6 under the control of the GAL1 promoter and tdTomato - tagged Snx41 ( endosomes ) , Sec63 ( ER ) , Vac8 ( vacuole ) , or Mnn9 ( Golgi ) were were grown in SGC - LEU , then shifted to SG - N for 2 h , and analyzed by fluorescence microscopy . Single imaging planes are shown . Scale bar , 5 µm . Arrows indicate Ykt6 dots that colocalize with marker proteins .

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(C) Localization of Ykt6 relative to different organelle marker proteins during nitrogen starvation. Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter and tdTomato-tagged Snx41 (endosomes), Sec63 (ER), Vac8 (vacuole), or Mnn9 (Golgi) were were grown in SGC-LEU, then shifted to SG-N for 2 h, and analyzed by fluorescence microscopy. Single imaging planes are shown. Scale bar, 5 µm. Arrows indicate Ykt6 dots that colocalize with marker proteins.


Localization of Ykt6 in multiple atg mutants . ( A ) Wild - type or the respective mutant cells expressing mCherry - tagged Ape1 and GFP - tagged Ykt6 were grown in SGC to allow for Ykt6 expression from the GAL1 - promoter and analyzed Arrows in the images indicate dots of Ykt6 colocalizing with Ape1 ( A )

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Localization of Ykt6 in multiple atg mutants. (A) Wild-type or the respective mutant cells expressing mCherry-tagged Ape1 and GFP-tagged Ykt6 were grown in SGC to allow for Ykt6 expression from the GAL1-promoter and analyzed Arrows in the images indicate dots of Ykt6 colocalizing with Ape1 (A)


( B ) Quantification of colocalization of Ykt6 relative to Ape1 as a PAS marker . Cells ( n ≥ 250 ) and Ape1 dots ( n ≥ 70 ) were quantified .

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(B) Quantification of colocalization of Ykt6 relative to Ape1 as a PAS marker. Cells (n≥250) and Ape1 dots (n≥70) were quantified.


( C ) Localization of Atg9 relative to Ykt6 . Cells expressing GFP - tagged Ykt6 and mScarlet - tagged Atg9 were grown and analyzed The left is the quantification of colocalization of Ykt6 relative to Atg9 . Cells ( n ≥ 200 ) and Atg9 dots ( n ≥ 80 ) were quantified . Arrows in the images indicate dots of Ykt6 colocalizing with Atg9 ( C )

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(C) Localization of Atg9 relative to Ykt6. Cells expressing GFP-tagged Ykt6 and mScarlet-tagged Atg9 were grown and analyzed The left is the quantification of colocalization of Ykt6 relative to Atg9. Cells (n≥200) and Atg9 dots (n≥80) were quantified. Arrows in the images indicate dots of Ykt6 colocalizing with Atg9 (C)


Ykt6 localizes to the isolation membrane during starvation . ( D ) Cells expressing mCherry - tagged Atg8 and GFP - Ykt6 carrying the Ape1 encoding plasmid pRS315 - CUP1pr - BFP - APE1 were grown in SGC medium containing CuSO4 and starved for 1 h , before to be analyzed by microscopy . Scale bar , 5 µm . ( E ) Volume rendering of LLSM image from a single time point after 3D deconvolution from 3D view . Cells expressing mCherry - tagged Atg8 and GFP - Ykt6 carrying plasmid pRS315 - CUP1pr - BFP - APE1 , cells , images were analyzed by Imaris . Scale bar , 5 µm . Arrows in the images indicate dots of Ykt6 colocalizing with Atg8 ( D , E ) .

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Ykt6 localizes to the isolation membrane during starvation. (D) Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying the Ape1 encoding plasmid pRS315-CUP1pr-BFP-APE1 were grown in SGC medium containing CuSO4 and starved for 1 h, before to be analyzed by microscopy. Scale bar, 5 µm. (E) Volume rendering of LLSM image from a single time point after 3D deconvolution from 3D view. Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying plasmid pRS315-CUP1pr-BFP-APE1, cells , images were analyzed by Imaris. Scale bar, 5 µm. Arrows in the images indicate dots of Ykt6 colocalizing with Atg8 (D,E).


( F ) The only partial time points ( 90 s to 600 s ) of images were assembled , and analyzed by Imaris and image J . Scale bar , 5 µm . 500 - 1000 cells were analyzed in each independent experiment . The 3D stacks were cropped by Imaris and different channels were split by Image J .

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(F) The only partial time points (90 s to 600 s) of images were assembled, and analyzed by Imaris and image J. Scale bar, 5 µm. 500-1000 cells were analyzed in each independent experiment. The 3D stacks were cropped by Imaris and different channels were split by Image J.


( A ) Effect of ykt6 and dsl1 complex ts mutants on autophagosome - vacuole fusion . Cells carrying a CEN plasmid expressing GFP - Atg8 were grown at 24 ° C in SGC and then shifted to SG - N for 2 h at 24 ° C or 37 ° C . Vacuoles were stained by FM4 - 64 and analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar of inset is 0 . 6 µm .

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(A) Effect of ykt6 and dsl1 complex ts mutants on autophagosome-vacuole fusion. Cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 24°C in SGC and then shifted to SG-N for 2 h at 24°C or 37°C. Vacuoles were stained by FM4-64 and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset is 0.6 µm.


( B ) Analysis of autophagy over time . Cells were grown at 24 ° C and shifted to starvation medium at 24 ° C or 37 ° C for the indicated time periods . The cells were harvested and proteins were extracted from cells by TCA precipitation . The extracted proteins were analyzed by SDS - PAGE and Western blotting against GFP .

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(B) Analysis of autophagy over time. Cells were grown at 24°C and shifted to starvation medium at 24°C or 37°C for the indicated time periods. The cells were harvested and proteins were extracted from cells by TCA precipitation. The extracted proteins were analyzed by SDS-PAGE and Western blotting against GFP.


( C ) Protease protection assay of ykt6 - 11 and dsl3 mutants . Lysates of atg1 ∆ , ypt7 ∆ , ykt6ts and dsl3ts cells carrying a CEN plasmid expressing GFP - Atg8 were grown at 37 ° C and subjected to the protease ( PK ) -

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(C) Protease protection assay of ykt6-11 and dsl3 mutants. Lysates of atg1∆, ypt7∆, ykt6ts and dsl3ts cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 37°C and subjected to the protease (PK)-protection


( D ) Localization of Atg8 relative to Atg1 during nitrogen starvation condition in sec12 - 1 ts mutant . Cells carrying a CEN plasmid expressing GFP - Atg8 and encoding 3xmCherry - tagged Atg1 were grown at 24 ° C in SDC and then shifted to SD - N for 2 h at 24 ° C or 37 ° C , and analyzed by fluorescence microscopy . Individual slices are shown . Scale bar , 5 µm . Scale bar of inset is 0 . 6 µm . Arrows in images indicate dots colocalization of Atg1 with Atg8 ( D )

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(D) Localization of Atg8 relative to Atg1 during nitrogen starvation condition in sec12-1 ts mutant. Cells carrying a CEN plasmid expressing GFP-Atg8 and encoding 3xmCherry-tagged Atg1 were grown at 24°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Arrows in images indicate dots colocalization of Atg1 with Atg8 (D)


( E ) Localization of Ykt6 relative to Dsl1 during growth and nitrogen starvation conditions . Cells expressing GFP - tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry - tagged Dsl1 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Individual slices are shown . Scale bar , 5 µm . ( F ) Percentage of Ykt6 puncta co - localizing with Dsl1 . The data was quantified from ( E ) . Error bars represent standard deviation of three independent experiments . Cells ( n ≥ 250 ) and Dsl1 dots ( n ≥ 50 ) were quantified . Arrows in images indicate dots colocalization of Ykt6 with Dsl1 ( E ) .

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(E) Localization of Ykt6 relative to Dsl1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Dsl1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. (F) Percentage of Ykt6 puncta co-localizing with Dsl1. The data was quantified from (E). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Dsl1 dots (n≥50) were quantified. Arrows in images indicate dots colocalization of Ykt6 with Dsl1 (E).


( G ) Localization of Dsl3 relative to Atg8 during growth and nitrogen starvation . Cells expressing GFP - tagged Dsl3 and mCherry - tagged Atg8 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Scale bar , 5 µm .

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(G) Localization of Dsl3 relative to Atg8 during growth and nitrogen starvation. Cells expressing GFP-tagged Dsl3 and mCherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Scale bar, 5 µm.


( A ) Effect of Dsl and COPII ts mutants on Ykt6 localization to autophagosomes . Wild - type and selected ts strains ( sec12 - 1 , tip20 - 5 and dsl3 - 2 ) expressing GFP - tagged Ykt6 and mCherry - tagged Atg8 were grown at 24 ° C in SGC for 8 h and then shifted to SG - N for 2 h at 37 ° C . Vacuoles were stained by CMAC and analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar in inset , 0 . 7 µm . Arrows indicate dot colocalization of Ykt6 and Atg8 ( A )

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(A) Effect of Dsl and COPII ts mutants on Ykt6 localization to autophagosomes. Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. Vacuoles were stained by CMAC and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar in inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 and Atg8 (A)


Wild - type and selected ts strains ( sec12 - 1 , tip20 - 5 and dsl3 - 2 ) expressing GFP - tagged Ykt6 and mCherry - tagged Atg8 were grown at 24 ° C in SGC for 8 h and then shifted to SG - N for 2 h at 37 ° C . ( B ) Percentage of Ykt6 puncta co - localizing with Atg8 . Cells ( n ≥ 150 ) and Atg8 dots ( n ≥ 100 ) were quantified .

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Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. (B) Percentage of Ykt6 puncta co-localizing with Atg8. Cells (n≥150) and Atg8 dots (n≥100) were quantified.


( C ) Localization of Ykt6 relative to Sec63 and Sec13 during growth and nitrogen starvation conditions . tip20 - 5 cells expressing GFP - tagged Ykt6 and 3xmCherry - tagged Sec63 or Sec13 were grown at 24 ° C in SGC for 8 h and then shifted to SG - N for 2 h at 24 ° C or 37 ° C , and analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar of inset , 0 . 7 µm . Arrows indicate dot colocalization of Ykt6 with Sec63 ( C ) .

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(C) Localization of Ykt6 relative to Sec63 and Sec13 during growth and nitrogen starvation conditions. tip20-5 cells expressing GFP-tagged Ykt6 and 3xmCherry-tagged Sec63 or Sec13 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 with Sec63 (C).


( D ) Effect of dsl mutation on autophagosome - vacuole fusion in vitro . The vam3 ∆ or dsl ts strain expressing Atg9 - 3xFLAG and GFP - Atg8 were starved for 3 h at 30 ° C before purifying autophagosomes , which was sufficient to induce the ts phenotype . Vacuoles were isolated from pep4 ∆ cells expressing Vac8 - 3xmCherry and then incubated with autophagosomes at 26 ° C for 1 h with or without ATP ( E ) Quantification of ( D ) . Error bars represent standard deviation of 3 independent experiments . Fluorescence intensity of GFP - Atg8 in the vacuolar lumen was quantified by image J using the ROI ( region of interest ) manager tool . Vacuoles ( n ≥ 40 ) for each experiment were quantified .

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(D) Effect of dsl mutation on autophagosome-vacuole fusion in vitro. The vam3∆ or dsl ts strain expressing Atg9-3xFLAG and GFP-Atg8 were starved for 3 h at 30°C before purifying autophagosomes, which was sufficient to induce the ts phenotype. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.


( A - B ) Quantification of size and number of autophagosomes in the indicated ts mutant strains . Error bars represent standard deviation of 3 independent experiments . GFP - tagged Atg8 in the different mutants were grown at 23 ° C ( ts mutant ) or 30 ° C ( atg1 ∆ ) in SDC and then shifted to SD - N for 2 h at 30 ° C ( atg1 ∆ ) or 37 ° C ( ts mutant ) , and analyzed by fluorescence microscopy . Scale bar , 1 µm .

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(A-B) Quantification of size and number of autophagosomes in the indicated ts mutant strains. Error bars represent standard deviation of 3 independent experiments. GFP-tagged Atg8 in the different mutants were grown at 23°C (ts mutant) or 30°C (atg1∆) in SDC and then shifted to SD-N for 2 h at 30°C (atg1∆) or 37°C (ts mutant), and analyzed by fluorescence microscopy. Scale bar, 1 µm.


( C ) Localization of Atg8 relative to Atg9 during nitrogen starvation . dsl3 cells expressing mCherry - tagged Atg9 and carrying a CEN plasmid expressing GFP - Atg8 were grown at 23 ° C in SDC and then shifted to SD - N for 2 h at 24 ° C or 37 ° C . Cells were analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar for inset is 0 . 6 µm .

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(C) Localization of Atg8 relative to Atg9 during nitrogen starvation. dsl3 cells expressing mCherry-tagged Atg9 and carrying a CEN plasmid expressing GFP-Atg8 were grown at 23°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.


( D ) Atg9 localization in dsl3 cells during starvation . dsl3 ts cells expressing mCherry - tagged Atg9 , and carrying a CEN plasmid expressing GFP - Atg8 and plasmid pRS315 - CUP1pr - BFP - APE1 were grown in SDC medium containing CuSO4 to induce formation of the giant Ape1 structure , and starved for 1 h . Cells were analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar for inset is 0 . 6 µm .

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(D) Atg9 localization in dsl3 cells during starvation. dsl3 ts cells expressing mCherry-tagged Atg9, and carrying a CEN plasmid expressing GFP-Atg8 and plasmid pRS315-CUP1pr-BFP-APE1 were grown in SDC medium containing CuSO4 to induce formation of the giant Ape1 structure, and starved for 1 h. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.


( B ) Localization of Ykt6 relative to Atg1 during growth and nitrogen starvation conditions . Cells expressing GFP - tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry - tagged Atg1 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Individual slices are shown . Scale bar , 5 µm . Scale bar of inset is 0 . 6 µm . Right , percentage of Ykt6 puncta co - localizing with Atg1 . Error bars represent standard deviation of three independent experiments . Cells ( n ≥ 200 ) and Atg1 dots ( n ≥ 100 ) were quantified . Arrows indicate Ykt6 positive dots colocalizing with Atg1 ( B )

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(B) Localization of Ykt6 relative to Atg1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Atg1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Right, percentage of Ykt6 puncta co-localizing with Atg1. Error bars represent standard deviation of three independent experiments. Cells (n≥200) and Atg1 dots (n≥100) were quantified. Arrows indicate Ykt6 positive dots colocalizing with Atg1 (B)


( C ) In vitro phosphorylation of purified Ykt6 by the Atg1 kinase complex . Purified Ykt6 was incubated with the Atg1 - Atg13 complex in the presence or absence of ATP , and Ykt6 band was cut out and analyzed by mass spectrometry .

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(C) In vitro phosphorylation of purified Ykt6 by the Atg1 kinase complex. Purified Ykt6 was incubated with the Atg1-Atg13 complex in the presence or absence of ATP, and Ykt6 band was cut out and analyzed by mass spectrometry.


( D ) Localization of Ykt6 relative to Atg8 under growth and nitrogen starvation conditions . Wild - type and atg1D211A cells expressing GFP - tagged Ykt6 under the control of the PHO5 promoter and genomically Cherry - tagged Atg8 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Individual focal planes are shown . Scale bar , 5 µm . ( E ) Percentage of Ykt6 puncta co - localizing with Atg8 . Numbers in the column indicates the percentage . Cells ( n ≥ 300 ) and Atg8 dots ( n ≥ 60 ) were quantified . Arrows indicate Ykt6 positive dots colocalizing with Atg8 ( D ) .

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(D) Localization of Ykt6 relative to Atg8 under growth and nitrogen starvation conditions. Wild-type and atg1D211A cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically Cherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual focal planes are shown. Scale bar, 5 µm. (E) Percentage of Ykt6 puncta co-localizing with Atg8. Numbers in the column indicates the percentage. Cells (n≥300) and Atg8 dots (n≥60) were quantified. Arrows indicate Ykt6 positive dots colocalizing with Atg8 (D).


( A ) Growth test of Ykt6 wild - type and mutants . ykt6 ∆ cells carrying a URA3 plasmid coding for wild - type Ykt6 and pRS413 - GAL1pr - YKT6 - GFP ( wild - type or mutant ) were spotted as serial dilutions on SGC - HIS to select for the mutant plasmid or 5 - Fluoroorotic acid ( 5 - FOA ) to force the loss of the plasmid coding for wild - type Ykt6 .

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(A) Growth test of Ykt6 wild-type and mutants. ykt6∆ cells carrying a URA3 plasmid coding for wild-type Ykt6 and pRS413-GAL1pr-YKT6-GFP (wild-type or mutant) were spotted as serial dilutions on SGC-HIS to select for the mutant plasmid or 5-Fluoroorotic acid (5-FOA) to force the loss of the plasmid coding for wild-type Ykt6.


( B ) Effect of Ykt6 phospho - mutants on autophagy . ykt6 ∆ cells expressing wild - type Ykt6 plus a pRS413 plasmid encoding GAL1pr - YKT6 ( wild - type or mutants with S to A mutations [ SA ; S182 , S183A ] , or S to D mutations [ SD ; S182D , S183D ] ) , and a plasmid expressing GFP - Atg8 were grown in SGC and then shifted to SG - N for 2 h . Cells were analyzed by fluorescence microscopy . Individual slices are shown . Scale bar , 5 µm . ( C ) Quantification of Atg8 dots per cell from images in ( B ) . Cells ( n ≥ 100 ) and Atg8 dots ( n ≥ 50 ) were quantified .

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(B) Effect of Ykt6 phospho-mutants on autophagy. ykt6∆ cells expressing wild-type Ykt6 plus a pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutants with S to A mutations [SA; S182, S183A], or S to D mutations [SD; S182D, S183D]), and a plasmid expressing GFP-Atg8 were grown in SGC and then shifted to SG-N for 2 h. Cells were analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. (C) Quantification of Atg8 dots per cell from images in (B). Cells (n≥100) and Atg8 dots (n≥50) were quantified.


( D ) Effect of Atg1 - Atg13 subcomplex on autophagosome - vacuole fusion in vitro . The vam3 ∆ strain carrying pRS413 plasmid encoding GAL1pr - YKT6 ( wild - type or mutant ) , and expressing Atg9 - 3xFLAG and GFP - Atg8 , were starved for 3 h at 30 ° C before purifying autophagosomes . Vacuoles were isolated from pep4 ∆ cells expressing Vac8 - 3xmCherry and then incubated with autophagosomes in the presence or absence of 50 nM Atg1 - Atg13 subcomplex at 26 ° C for 1 h with or without ATP ( E ) Quantification of ( D ) . Error bars represent standard deviation of 3 independent experiments . Fluorescence intensity of GFP - Atg8 in the vacuolar lumen was quantified by image J using the ROI ( region of interest ) manager tool . Vacuoles ( n ≥ 40 ) for each experiment were quantified .

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(D) Effect of Atg1-Atg13 subcomplex on autophagosome-vacuole fusion in vitro. The vam3∆ strain carrying pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutant), and expressing Atg9-3xFLAG and GFP-Atg8, were starved for 3 h at 30°C before purifying autophagosomes. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes in the presence or absence of 50 nM Atg1-Atg13 subcomplex at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.


After 2 hour starvation , E . coli cultures were pulse fed 10 µM glucose at varying frequencies using the spin flask and plate reader systems , and optical density ( OD ) was measured over time ( inset example figures ) . Grey dots are OD measurements and the black lines are an empirical fit ( see Methods ) . For separate experiments ( n = 18 ) , the lag time is plotted against the frequency , represented as the time - integrated ( TI ) feedrate f ( mmol glucose / g dry cell weight / hour ) . An empirical fit ( grey solid line , see Methods ) was used to separate the lag ( non - dividing ) and dividing

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After 2 hour starvation, E. coli cultures were pulse fed 10 µM glucose at varying frequencies using the spin flask and plate reader systems, and optical density (OD) was measured over time (inset example figures). Grey dots are OD measurements and the black lines are an empirical fit (see Methods). For separate experiments (n = 18), the lag time is plotted against the frequency, represented as the time-integrated (TI) feedrate f (mmol glucose/g dry cell weight/hour). An empirical fit (grey solid line, see Methods) was used to separate the lag (non-dividing) and dividing phases


Normalized absolute cell counts versus time show linear increases after lag time for exemplary feedrates . Data are mean ± standard error of technical replicates ( n = 2 - 3 ) . Lag predicted from the empirical fit is indicated by vertical dotted

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Normalized absolute cell counts versus time show linear increases after lag time for exemplary feedrates. Data are mean ± standard error of technical replicates (n = 2-3). Lag predicted from the empirical fit is indicated by vertical dotted lines


The average size of cells decreased after lag . Micrographs and cell length distributions ( n > 400 per distribution ) are shown for specific time points , with f = 0 . 6 mmol / g /

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The average size of cells decreased after lag. Micrographs and cell length distributions (n > 400 per distribution) are shown for specific time points, with f = 0.6 mmol/g/h


Immobilized cells in the microfluidic experiment divided after a lag time that decreased with increasing glucose pulse frequency . The labeled times indicate the period , time between pulses , for a given

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Immobilized cells in the microfluidic experiment divided after a lag time that decreased with increasing glucose pulse frequency. The labeled times indicate the period, time between pulses, for a given experiment


Time course of the distribution of cellular DNA content . Sampled cells were stained with SYBR Green I and measured with flow cytometry over the course of a pulsing experiment ( f = 0 . 6 mmol / g / h ) The DNA content distribution over time is shown on the left side , and three specific time points are shown on the right . Within the first time point ( t = 0 ) , the highest distribution is taken to be high DNA content ( 2N ) , and the distribution at half of the 2N average was taken ( 1N ) as medium DNA . DNA distributions were separated into medium ( 1N ) and high DNA ( 2N ) . Distribution - specific estimated counts ( see Methods ) over time ( f = 0 . 6 mmol / g / h ) suggested that net division from high to medium DNA cells can explain the increase in cell counts and OD

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Time course of the distribution of cellular DNA content. Sampled cells were stained with SYBR Green I and measured with flow cytometry over the course of a pulsing experiment (f = 0.6 mmol/g/h) The DNA content distribution over time is shown on the left side, and three specific time points are shown on the right. Within the first time point (t = 0), the highest distribution is taken to be high DNA content (2N), and the distribution at half of the 2N average was taken (1N) as medium DNA. DNA distributions were separated into medium (1N) and high DNA (2N). Distribution-specific estimated counts (see Methods) over time (f = 0.6 mmol/g/h) suggested that net division from high to medium DNA cells can explain the increase in cell counts and OD increase


The spin flask system for glucose pulsing was connected to a real - time metabolomics platform . Traces of exemplary ions are shown that correspond to hexose phosphate , guanine , phenylalanine , and hypoxanthine for pulsing at non - division supporting frequencies of 0 . 06 , 0 . 12 , and 0 . 18 mmol / g / h . The TI feedrate is abbreviated as f ( units : mmol glucose / g dry cell weight / hour ) . Glucose pulses are indicated by the grey bars , and the pink region shows a no pulse control . Dots are ion intensity measurements . Solid lines are a moving average filter of the measured ion intensity . For clarity , dots are not shown for f = 0 mmol / g / h condition A metabolic scheme describing the propagation of fed glucose . Pulsed glucose is hypothesized to pass through central carbon metabolism and then be converted to downstream pathways including amino acid synthesis and nucleic acid synthesis . For nucleic acid synthesis , glucose is converted to the intermediate PRPP , which then can combine with nitrogen bases to form nucleotides for nucleic acid synthesis . Different pathways can be blocked with

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The spin flask system for glucose pulsing was connected to a real-time metabolomics platform. Traces of exemplary ions are shown that correspond to hexose phosphate, guanine, phenylalanine, and hypoxanthine for pulsing at non-division supporting frequencies of 0.06, 0.12, and 0.18 mmol/g/h. The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Glucose pulses are indicated by the grey bars, and the pink region shows a no pulse control. Dots are ion intensity measurements. Solid lines are a moving average filter of the measured ion intensity. For clarity, dots are not shown for f = 0 mmol/g/h condition A metabolic scheme describing the propagation of fed glucose. Pulsed glucose is hypothesized to pass through central carbon metabolism and then be converted to downstream pathways including amino acid synthesis and nucleic acid synthesis. For nucleic acid synthesis, glucose is converted to the intermediate PRPP, which then can combine with nitrogen bases to form nucleotides for nucleic acid synthesis. Different pathways can be blocked with antibiotics


Influence of antibiotics that inhibit macromolecular synthesis at the non - division TI feedrate of 0 . 18 mmol / g / h . Antibiotics were added one minute after the second pulse ( yellow region ) . Four different ions are shown corresponding to glutamate , phenylalanine , guanine , and thymine . Chloramphenicol ( blue ) inhibits protein biosynthesis , rifamycin ( orange ) inhibits RNA polymerase , and azidothymidine ( AZT ; red ) inhibits DNA

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Influence of antibiotics that inhibit macromolecular synthesis at the non-division TI feedrate of 0.18 mmol/g/h. Antibiotics were added one minute after the second pulse (yellow region). Four different ions are shown corresponding to glutamate, phenylalanine, guanine, and thymine. Chloramphenicol (blue) inhibits protein biosynthesis, rifamycin (orange) inhibits RNA polymerase, and azidothymidine (AZT; red) inhibits DNA synthesis


Percentage of labeled threonine and deoxyribose from protein and DNA hydrolysate shows de novo protein and DNA synthesis in non - dividing cells . After 6 hours of pulsing uniformly labeled 13C - glucose , cultures were lysed , and their macromolecules were washed free of latent metabolites and hydrolyzed to monomers . Labeling data are presented as the mean ± standard error of independent biological replicates ( n = 3 , all pairwise P < 0 . 02 as determined by one sided Student " s t -

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Percentage of labeled threonine and deoxyribose from protein and DNA hydrolysate shows de novo protein and DNA synthesis in non-dividing cells. After 6 hours of pulsing uniformly labeled 13C-glucose, cultures were lysed, and their macromolecules were washed free of latent metabolites and hydrolyzed to monomers. Labeling data are presented as the mean ± standard error of independent biological replicates (n = 3, all pairwise P < 0.02 as determined by one sided Student"s t-


Pulsing experiment was repeated in the presence of protease inhibitor ( PI ) that reduced the lag time for a given TI feedrate ( f = 0 . 28 mmol / g / h ) . The TI feedrate is abbreviated as f ( units : mmol glucose / g dry cell weight / hour ) . Wild - type lag ( from Figure 2A empirical fit ) is indicated by the dotted grey

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Pulsing experiment was repeated in the presence of protease inhibitor (PI) that reduced the lag time for a given TI feedrate (f = 0.28 mmol/g/h). The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Wild-type lag (from Figure 2A empirical fit) is indicated by the dotted grey line


Cells expressing a nearly functional FtsZ - mVenus fluorescent fusion as their sole copy of ftsZ were monitored after transition into carbon starvation . Two low motile MG1655 strains , one ( wild - type ) containing clpX and one without ( ΔclpX ) , were grown in M9 glucose to steady - state . At time zero , M9 media without glucose was flushed through device . Cells rapidly ceased elongation as measured by phase contrast imaging at 2 minute intervals . Fluorescent images were taken every hour throughout to measure both total Ftsz - mVenus per cell and localization patterns Thin individual lines show the total fluorescent per cell in a single lineage ( wild - type in solid blue , n = 154 . ΔclpX in dotted red , n = 144 ) . Thick solid lines are the time average across individual lineages for each subset ( wild - type in blue , ΔclpX in red ) . After starvation , cells containing clpX degrade FtsZ faster than cells which do not . Fluorescent signals are normalized to the average value for their respective subset at time 0 Distributions of cellular fluorescence for each subset at times corresponding to the top panel . Cellular fluorescence varies widely as total FtsZ per cell is a function of cell size ; a typical size distribution is shown at left . FtsZ concentration is roughly constant at steady - stat Student " s t - test P value shown for when distributions differ with significance level α = 0 . 01 . Representative images from cells in a single lineage with and without clpX . Wild - type strains may contain the characteristic FtsZ ring at midcell after shift down but it dissipates after several hours . ΔclpX strains display an abnormal FtsZ localization pattern even in steady - state . After shift down , the FtsZ may disassemble and reform along the cell body in distinct puncta . Image timing corresponds to the abscissa in the

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Cells expressing a nearly functional FtsZ-mVenus fluorescent fusion as their sole copy of ftsZ were monitored after transition into carbon starvation. Two low motile MG1655 strains, one (wild-type) containing clpX and one without (ΔclpX), were grown in M9 glucose to steady-state. At time zero, M9 media without glucose was flushed through device. Cells rapidly ceased elongation as measured by phase contrast imaging at 2 minute intervals. Fluorescent images were taken every hour throughout to measure both total Ftsz-mVenus per cell and localization patterns Thin individual lines show the total fluorescent per cell in a single lineage (wild-type in solid blue, n = 154. ΔclpX in dotted red, n = 144). Thick solid lines are the time average across individual lineages for each subset (wild-type in blue, ΔclpX in red). After starvation, cells containing clpX degrade FtsZ faster than cells which do not. Fluorescent signals are normalized to the average value for their respective subset at time 0 Distributions of cellular fluorescence for each subset at times corresponding to the top panel. Cellular fluorescence varies widely as total FtsZ per cell is a function of cell size; a typical size distribution is shown at left. FtsZ concentration is roughly constant at steady-stat Student"s t-test P value shown for when distributions differ with significance level α = 0.01. Representative images from cells in a single lineage with and without clpX. Wild-type strains may contain the characteristic FtsZ ring at midcell after shift down but it dissipates after several hours. ΔclpX strains display an abnormal FtsZ localization pattern even in steady-state. After shift down, the FtsZ may disassemble and reform along the cell body in distinct puncta. Image timing corresponds to the abscissa in the top


Genetically induced titration of PdhR in a pdhR mutant reintroduced lag commensurate with expression level for a given TI feedrate ( f = 0 . 38 mmol / g / h ) . Induction level corresponds to the amount of doxycycline ( max - 50 ng / uL , half - 10 ng / uL , and zero 0 ng / uL ) added at the onset of starvation . The TI feedrate is abbreviated as f ( units : mmol glucose / g dry cell weight / hour ) . Wild - type lag ( from Figure 2A empirical fit ) is indicated by the dotted grey line . Lag time reduced with synthesis levels of titrated FtsZ in the wild - type strain at f = 0 . 31 - 0 . 32 mmol / g / h . Lag time increased with titrated synthesis of ClpX in wild - type cells at f = 0 . 43 - 0 . 45 mmol / g /

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Genetically induced titration of PdhR in a pdhR mutant reintroduced lag commensurate with expression level for a given TI feedrate (f = 0.38 mmol/g/h). Induction level corresponds to the amount of doxycycline (max - 50 ng/uL, half - 10 ng/uL, and zero 0 ng/uL) added at the onset of starvation. The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Wild-type lag (from Figure 2A empirical fit) is indicated by the dotted grey line. Lag time reduced with synthesis levels of titrated FtsZ in the wild-type strain at f = 0.31-0.32 mmol/g/h. Lag time increased with titrated synthesis of ClpX in wild-type cells at f = 0.43-0.45 mmol/g/h


E Quantification of mean fluorescence intensity ( MFI ) for CD80 , CD86 , CD206 , CD163 , CCR2 , and MHC - Ⅱ in TAMEMs and typical macrophages subtypes via flow cytometry ( * P < 0 . 05 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; n = 6 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using ordinary one - way ANOVA with Tukey ' s multiple comparison test in ( E ) in Graph Pad Prism 8 .

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E Quantification of mean fluorescence intensity (MFI) for CD80, CD86, CD206, CD163, CCR2, and MHC-Ⅱ in TAMEMs and typical macrophages subtypes via flow cytometry (*P < 0.05, ***P < 0.001, and ****P < 0.0001; n=6, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (E) in Graph Pad Prism 8.


F Representative immunofluorescent ( IF ) staining images for CD86 and CD206 in TAMEMs and typical macrophages subtypes ( scale bar : 10 μm ; n = 6 , biological replicates ) .

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F Representative immunofluorescent (IF) staining images for CD86 and CD206 in TAMEMs and typical macrophages subtypes (scale bar: 10 μm; n=6, biological replicates).


A Quantification of concentrations of 22 soluble proteins from the cytokine and angiogenesis array analysis ( * P < 0 . 05 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; ns : not significan ; n = 3 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using two - way ANOVA with Tukey ' s multiple comparison test in ( A , in Graph Pad Prism 8 .

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A Quantification of concentrations of 22 soluble proteins from the cytokine and angiogenesis array analysis (*P < 0.05, ***P < 0.001, and ****P < 0.0001; ns: not significan; n=3, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in (A, in Graph Pad Prism 8.


F , G Western Blotting analysis of phosphorylated IκB ( F ) and quantification of gray value ( G ) in fibroblasts ( L929 ) that pre - treated with LPS and then cultured with CMs from TAMEMs and other controls for 24 h ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , and ns : not significant ( P > 0 . 05 ) vs . the LPS - treated group ; n = 3 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using two - way ANOVA with Tukey ' s multiple comparison test in G , in Graph Pad Prism 8 .

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F, G Western Blotting analysis of phosphorylated IκB (F) and quantification of gray value (G) in fibroblasts (L929) that pre-treated with LPS and then cultured with CMs from TAMEMs and other controls for 24 h (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the LPS-treated group; n=3, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in G, in Graph Pad Prism 8.


H Representative images and quantification of the Matrigel - based angiogenesis assay in mouse vascular endothelial cell ( SVEC4 - 10 ) treated with CMs from TAMEMs and other controls , VEGF treatment as a positive control ( * * * * P < 0 . 0001 and ns : not significant ( P > 0 . 05 ) vs . the normal medium group ; scale bar : 100 μm ; n = 6 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using ordinary one - way ANOVA with Tukey ' s multiple comparison test in H ) in Graph Pad Prism 8 .

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H Representative images and quantification of the Matrigel-based angiogenesis assay in mouse vascular endothelial cell (SVEC4-10) treated with CMs from TAMEMs and other controls, VEGF treatment as a positive control (****P < 0.0001 and ns: not significant (P > 0.05) vs. the normal medium group; scale bar: 100 μm; n=6, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in H) in Graph Pad Prism 8.


E Representative images of the wound closure process were captured during the twenty - one - day in vivo experiments ( scale bar : 500 μm ; n = 8 , biological replicates ) .

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E Representative images of the wound closure process were captured during the twenty-one-day in vivo experiments (scale bar: 500 μm; n=8, biological replicates).


G H & E ( day 7 and 14 ) and Masson ' s ( day 14 ) staining for full - thickness skin samples containing the entire wound area ( scale bar : 500 μm ; n = 8 ) , the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 ( scale bar : 100 μm ; n = 8 , biological replicates ) , ( gt : granulation tissue , he : hyperproliferative epithelium , black arrow : wound edges ) .

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G H&E (day 7 and 14) and Masson's (day 14) staining for full-thickness skin samples containing the entire wound area (scale bar: 500 μm; n=8), the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 (scale bar: 100 μm; n=8, biological replicates), (gt: granulation tissue, he: hyperproliferative epithelium, black arrow: wound edges).


H - J Quantitative analysis of regenerated granulation ( H ) on day 7 and Re - epithelialization of wound bed ( I ) and Masson ' s trichrome - stained tissue ( J ) on day 14 ( blue for collagen ) ( * * * P < 0 . 001 , * * * * P < 0 . 0001 , and ns : not significant ( P > 0 . 05 ) vs . the blank control group ; n = 8 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using ordinary one - way ANOVA with Tukey ' s multiple comparison test in ( H , I , J , in Graph Pad Prism 8 .

O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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- J Quantitative analysis of regenerated granulation (H) on day 7 and Re-epithelialization of wound bed (I) and Masson's trichrome-stained tissue (J) on day 14 (blue for collagen) (***P < 0.001, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (H, I, J, in Graph Pad Prism 8.


E IF staining for CD31 ( red ; endothelial marker , for new capillary formation ) at day 7 and α - SMA ( green ; smooth muscle marker , for vascular maturation ) at day 14 , with both the entire wound region and magnified yellow - box zone ( scale bar : 100 μm ; n = 8 , biological replicates ) ( gt : granulation tissue , he : hyperproliferative epithelium ) .

O O O O B-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 O O O O O O O O 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 IF staining for CD31 (red; endothelial marker, for new capillary formation) at day 7 and α-SMA (green; smooth muscle marker, for vascular maturation) at day 14, with both the entire wound region and magnified yellow-box zone (scale bar: 100 μm; n=8, biological replicates) (gt: granulation tissue, he: hyperproliferative epithelium).


F Quantification of the CD31 + vessels at day 7 and CD31 + α - SMA + vessels and CD31 + α - SMA - vessels at day 14 ( * * * * P < 0 . 0001 and ns : not significant ( P > 0 . 05 ) vs . the blank control group ; n = 8 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using ordinary one - way ANOVA with Tukey ' s multiple comparison test in ( F , in Graph Pad Prism 8 .

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

F Quantification of the CD31+ vessels at day 7 and CD31+ α-SMA+ vessels and CD31+ α-SMA- vessels at day 14 (****P < 0.0001 and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (F, in Graph Pad Prism 8.


G Representative multiphoton projection images of blood vessels ( lectin ) in healed skin wound tissue after the administration of different treatments at day 21 post - injury , uninjured skin was analyzed as positive control . Up : the images of the entire sampling area ( yellow circled ) containing the healed wound ( green circled ) ; Down : magnified zone ( blue boxed in the upper images ; scale bar : 100 μm ; n = 3 , biological replicates ) .

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

G Representative multiphoton projection images of blood vessels (lectin) in healed skin wound tissue after the administration of different treatments at day 21 post-injury, uninjured skin was analyzed as positive control. Up: the images of the entire sampling area (yellow circled) containing the healed wound (green circled); Down: magnified zone (blue boxed in the upper images; scale bar: 100 μm; n=3, biological replicates).


G The proliferation rate of L929 cells pretreated with different CMs was analyzed using the CCK - 8 kit ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , and ns : not significant ( P > 0 . 05 ) vs . the normal medium group ; n = 6 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using two - way ANOVA with Tukey ' s multiple comparison test in G ) in Graph Pad Prism 8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The proliferation rate of L929 cells pretreated with different CMs was analyzed using the CCK-8 kit (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the normal medium group; n=6, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in G) in Graph Pad Prism 8.


H Immunofluorescent staining for α - SMA and type I Collagen in L929 cells after 48 h treatment with different CMs ( scale bar : 10 μm ; n = 6 , biological replicates ) .

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

H Immunofluorescent staining for α-SMA and type I Collagen in L929 cells after 48 h treatment with different CMs (scale bar: 10 μm; n=6, biological replicates).


B Gross view of the wound closure process was captured during the twenty - one - day in vivo experiments ( scale bar : 500 μm ; n = 8 , biological replicates ) . C The percentage of wound closure determined at each time point ( * * * * P < 0 . 0001 and ns : not significant ( P > 0 . 05 ) vs . the blank control group ; n = 8 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using two - way ANOVA with Tukey ' s multiple comparison test in ( C ) in Graph Pad Prism 8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Gross view of the wound closure process was captured during the twenty-one-day in vivo experiments (scale bar: 500 μm; n=8, biological replicates). C The percentage of wound closure determined at each time point (****P < 0.0001 and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in (C) in Graph Pad Prism 8.


D Full - thickness skin samples containing entire wound sites were stained with H & E ( day 7 and day 14 ) and Masson ( day 14 ) ( scale bar : 500 μm , n = 8 , biological replicates ) , the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 ( scale bar : 100 μm ; n = 8 , biological replicates ) ( gt : granulation tissue , he : hyperproliferative epithelium , black arrow : wound edges ) .

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

D Full-thickness skin samples containing entire wound sites were stained with H&E (day 7 and day 14) and Masson (day 14) (scale bar: 500 μm, n=8, biological replicates), the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 (scale bar: 100 μm; n=8, biological replicates) (gt: granulation tissue, he: hyperproliferative epithelium, black arrow: wound edges).


F Representative multiphoton projection images of blood vessels ( lectin ) in healed skin wound tissue after the administration of different treatments at day 21 postinjury , uninjured skin was analyzed as positive control ( Up : the images of entire sampling sites ( the yellow circular region ) containing healed wound sites ( the green circular region ) ; Down : the images of higher magnification that indicates an area in the blue boxed region in the upper images ; scale bar : 100 μm ; n = 3 , biological replicates ) .

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

F Representative multiphoton projection images of blood vessels (lectin) in healed skin wound tissue after the administration of different treatments at day 21 postinjury, uninjured skin was analyzed as positive control (Up: the images of entire sampling sites (the yellow circular region) containing healed wound sites (the green circular region); Down: the images of higher magnification that indicates an area in the blue boxed region in the upper images; scale bar: 100 μm; n=3, biological replicates).


Western blot analysis of total brain lysates of wild type , Tmem106b - / - , Grn - / - and Grn - / - / Tmem106b - / - mice using antibody 6F2 to detect TMEM106B . ( 4 . 5 - month - old mice with the given genotype ; n = 3 biological replicates per genotype ) .

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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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

Western blot analysis of total brain lysates of wild type, Tmem106b-/-, Grn-/- and Grn-/-/Tmem106b-/- mice using antibody 6F2 to detect TMEM106B. (4.5-month-old mice with the given genotype; n = 3 biological replicates per genotype).


Hind limb clasping test wild type , Tmem106b - / - , Grn - / - , Grn + / - / Tmem106b - / - and Grn - / - / Tmem106b - / - mice . Representative pictures are shown of 3 mice analyzed for each genotype at 3 months of age .

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Hind limb clasping test wild type, Tmem106b-/-, Grn-/-, Grn+/-/Tmem106b-/- and Grn-/-/Tmem106b-/- mice. Representative pictures are shown of 3 mice analyzed for each genotype at 3 months of age.


Rotarod performance of wild type , Tmem106b - / - , Grn - / - , Grn + / - / Tmem106b - / - and double knockout animals at 3 to 4 months of age . Number of animals used for analysis , 10 wild type , 12 Tmem106b - / - , 5 Grn - / - , 7 Grn + / - / Tmem106b - / - , 3 Grn - / - / Tmem106b - / - mice .

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

Rotarod performance of wild type, Tmem106b-/-, Grn-/-, Grn+/- /Tmem106b-/- and double knockout animals at 3 to 4 months of age. Number of animals used for analysis, 10 wild type, 12 Tmem106b-/-, 5 Grn-/-, 7 Grn+/-/Tmem106b-/-, 3 Grn-/-/Tmem106b-/- mice.


Representative picture of a 4 . 5 - month - old double knockout ( Grn - / - / Tmem106b - / - ) mouse after manual flip .

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Representative picture of a 4.5-month-old double knockout (Grn-/-/Tmem106b-/- ) mouse after manual flip.


Volcano plot of differentially expressed brain mRNAs of 4 . 5 - month - old mice ( n = 3 biological replicates per genotype ) detected by the Neuropathology panel of NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for all volcano plots . Gliosis and Myelination related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( A ) Comparison of differentially expressed brain mRNAs from Tmem106b - / - and wild type mice . From 680 detected genes 21 are significantly upregulated , while 10 are significantly reduced .

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

Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (A) Comparison of differentially expressed brain mRNAs from Tmem106b-/- and wild type mice. From 680 detected genes 21 are significantly upregulated, while 10 are significantly reduced.


Volcano plot of differentially expressed brain mRNAs of 4 . 5 - month - old mice ( n = 3 biological replicates per genotype ) detected by the Neuropathology panel of NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for all volcano plots . Gliosis and Myelination related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( B ) Comparison of Grn - / - and wild type mice . From 680 detected genes 12 are significantly upregulated , while 6 are significantly reduced .

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

Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (B) Comparison of Grn-/- and wild type mice. From 680 detected genes 12 are significantly upregulated, while 6 are significantly reduced.


Volcano plot of differentially expressed brain mRNAs of 4 . 5 - month - old mice ( n = 3 biological replicates per genotype ) detected by the Neuropathology panel of NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for all volcano plots . Gliosis and Myelination related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( C ) Volcano plot for Grn - / - / Tmem106b - / - and wild type mice . From 680 detected genes 79 are significantly upregulated , while 24 are significantly reduced .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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

Volcano plot of differentially expressed brain mRNAs of 4.5-month-old mice (n = 3 biological replicates per genotype) detected by the Neuropathology panel of NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for all volcano plots. Gliosis and Myelination related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (C) Volcano plot for Grn-/-/Tmem106b-/- and wild type mice. From 680 detected genes 79 are significantly upregulated, while 24 are significantly reduced.


D . Display of differently expressed genes overlapping between the analyzed genotypes from ( A - C ) . Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20 % change in at least one mouse model . Note that 9 genes are significantly altered in all 3 genotypes in comparison to the wild type . 7 genes are exclusively overlapping between the Grn - / - and the Grn - / - / Tmem106b - / - mice . Tmem106b - / - and Grn - / - / Tmem106b - / - mice share 15 significantly altered genes . Tmem106b - / - and Grn - / - mice share one significant altered gene . Data represent the mean ± SD .

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

D. Display of differently expressed genes overlapping between the analyzed genotypes from (A-C). Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20% change in at least one mouse model. Note that 9 genes are significantly altered in all 3 genotypes in comparison to the wild type. 7 genes are exclusively overlapping between the Grn-/- and the Grn-/-/Tmem106b-/- mice. Tmem106b-/- and Grn-/-/Tmem106b-/- mice share 15 significantly altered genes. Tmem106b-/- and Grn-/- mice share one significant altered gene. Data represent the mean ± SD.


Analysis of brain mRNA expression of 4 . 5 - month - old mice ( 5 wild type 4 Tmem106b - / - mice ) detected by the Neuroinflammation panel by NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for in all volcano plots . Gliosis related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( A ) Comparison of differentially expressed mRNA of Tmem106b - / - mice in comparison to wild type mice . From 622 detected genes 21 are significantly upregulated , while 12 are significantly reduced .

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 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

Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type 4 Tmem106b-/- mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (A) Comparison of differentially expressed mRNA of Tmem106b-/- mice in comparison to wild type mice. From 622 detected genes 21 are significantly upregulated, while 12 are significantly reduced.


Analysis of brain mRNA expression of 4 . 5 - month - old mice ( 5 wild type , 4 Grn - / mice ) detected by the Neuroinflammation panel by NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for in all volcano plots . Gliosis related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( B ) Comparison of differentially expressed mRNA in Grn - / - and wild type mice . From 622 detected genes 32 are significantly upregulated , while only 1 is significantly reduced .

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 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

Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type, 4 Grn-/ mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (B) Comparison of differentially expressed mRNA in Grn-/- and wild type mice. From 622 detected genes 32 are significantly upregulated, while only 1 is significantly reduced.


Analysis of brain mRNA expression of 4 . 5 - month - old mice ( 5 wild type 5 Grn - / - / Tmem106b - / - mice ) detected by the Neuroinflammation panel by NanoString . Expression changes were only taken into consideration when the extent of change was above 20 % as indicated by vertical lines for in all volcano plots . Gliosis related genes are highlighted if changes are above 20 % . Fold changes are displayed after log2 transformation . ( C ) Volcano plot of differentially expressed brain mRNAs from Grn - / - / Tmem106b - / - mice in comparison to wild type mice . From 622 detected genes 110 are significantly upregulated , while 16 are significantly reduced .

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-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 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

Analysis of brain mRNA expression of 4.5-month-old mice (5 wild type 5 Grn-/-/Tmem106b-/- mice) detected by the Neuroinflammation panel by NanoString. Expression changes were only taken into consideration when the extent of change was above 20% as indicated by vertical lines for in all volcano plots. Gliosis related genes are highlighted if changes are above 20%. Fold changes are displayed after log2 transformation. (C) Volcano plot of differentially expressed brain mRNAs from Grn-/-/Tmem106b-/- mice in comparison to wild type mice. From 622 detected genes 110 are significantly upregulated, while 16 are significantly reduced.


D . Display of differently expressed genes overlapping between the analyzed genotypes from ( A - C ) . Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20 % change in at least one mouse model . Note that 19 genes are significantly altered in all 3 genotypes in comparison to the wild type . 17 genes are exclusively overlapping between the Grn - / - and the Grn - / - / Tmem106b - / - mice . Tmem106b - / - and Grn - / - / Tmem106b - / - mice share 17 significantly altered genes . Data represent the mean ± SD .

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

D. Display of differently expressed genes overlapping between the analyzed genotypes from (A-C). Overlap was considered if a gene was significantly changed in the respective mouse models with more than 20% change in at least one mouse model. Note that 19 genes are significantly altered in all 3 genotypes in comparison to the wild type. 17 genes are exclusively overlapping between the Grn-/- and the Grn-/-/Tmem106b-/- mice. Tmem106b-/- and Grn-/-/Tmem106b-/- mice share 17 significantly altered genes. Data represent the mean ± SD.


Western blot analysis of GFAP and IBA1 in total brain lysates from 4 . 5 - month - old mice with the given genotype . Quantification of A . Protein expression was normalized to levels in wild type animals . Data represent the mean ± SD .

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

Western blot analysis of GFAP and IBA1 in total brain lysates from 4.5-month-old mice with the given genotype. Quantification of A. Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.


Expression of GFAP in sagittal brain sections . Representative images of indicated brain regions ( CTX = Cortex ; TH = Thalamus ; CBX = Cerebellum ; MY = Medulla ) . Expression of IBA1 in sagittal brain sections . Representative images of indicated brain regions ( CTX = Cortex ; TH = Thalamus ; CBX = Cerebellum ; MY = Medulla ) . Scale bar indicates 100 µm .

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Expression of GFAP in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; MY = Medulla). Expression of IBA1 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; MY = Medulla). Scale bar indicates 100 µm.


In vivo 18F - GE180 TSPO μPET imaging . Two female Grn - / - / Tmem106b - / - and five female wild type mice underwent longitudinal TSPO μPET at 2 . 0 and 3 . 5 months of age . All analyses were performed by PMOD ( V3 . 5 , PMOD technologies ) . Normalization of injected activity was performed by the previously validated myocardium correction method .

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In vivo 18F-GE180 TSPO μPET imaging. Two female Grn-/-/Tmem106b-/- and five female wild type mice underwent longitudinal TSPO μPET at 2.0 and 3.5 months of age. All analyses were performed by PMOD (V3.5, PMOD technologies). Normalization of injected activity was performed by the previously validated myocardium correction method.


Western blot analysis of CD68 , CLEC7A , TREM2 and ApoE in total brain lysates from 4 . 5 - month - old mice with the given genotype . Asterisk indicate unspecific band . ( n = 3 biological replicates per genotype ) . Quantification of A . Protein expression was normalized to levels in wild type animals .

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Western blot analysis of CD68, CLEC7A, TREM2 and ApoE in total brain lysates from 4.5-month-old mice with the given genotype. Asterisk indicate unspecific band. (n = 3 biological replicates per genotype). Quantification of A. Protein expression was normalized to levels in wild type animals.


Expression of CD68 and the microglial marker IBA1 in sagittal brain sections . Representative images of indicated brain regions ( TH = Thalamus ) . Expression of CLEC7A and the microglial marker IBA1 in sagittal brain sections . Representative images of indicated brain regions ( MY = medulla ) . Expression of TREM2 and the microglial marker IBA1 in sagittal brain sections . Representative images of indicated brain regions ( MY = medulla ) . Scale bar indicates 25 µm .

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Expression of CD68 and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (TH = Thalamus). Expression of CLEC7A and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (MY = medulla). Expression of TREM2 and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (MY = medulla). Scale bar indicates 25 µm.


Western blot analysis of cathepsin ( Cat ) D , B , and L in total brain lysates from 4 . 5 - month - old mice with the indicated genotype . Different maturation variants are indicated ( p = preform , sc = single chain , hc = heavy chain ) ( n = 3 biological replicates per genotype ) . Quantification of proteolytically generated variants of cathepsin D , B , and L normalized to levels in wild type animals from ( A ) .

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Western blot analysis of cathepsin (Cat) D, B, and L in total brain lysates from 4.5-month-old mice with the indicated genotype. Different maturation variants are indicated (p = preform, sc = single chain, hc = heavy chain) (n = 3 biological replicates per genotype). Quantification of proteolytically generated variants of cathepsin D, B, and L normalized to levels in wild type animals from (A).


Catalytic activity of cathepsin D , B , and L . Total brain lysates from 4 . 5 - month - old mice were incubated with quenched fluorogenic substrate for Cat D , Cat B and Cat L . The linear increase of fluorescence signal was measured and then normalized to wildtype . Data represent the mean normalized activity ± SD . ( n = 3 biological replicates per genotype ) .

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Catalytic activity of cathepsin D, B, and L. Total brain lysates from 4.5-month-old mice were incubated with quenched fluorogenic substrate for Cat D, Cat B and Cat L. The linear increase of fluorescence signal was measured and then normalized to wildtype. Data represent the mean normalized activity ± SD. (n = 3 biological replicates per genotype).


Immunofluorescence analysis of lipofuscin ( red ) and IBA1 ( green ) in sagittal brain sections . Representative images of the Thalamus ( TH ) . Scale bar indicates 25 µm .

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Immunofluorescence analysis of lipofuscin (red) and IBA1 (green) in sagittal brain sections. Representative images of the Thalamus (TH). Scale bar indicates 25 µm.


Western blot analysis of ubiquitin and the autophagy marker LC3 in RIPA , and p62 in RIPA and urea total brain lysates from 4 . 5 - month - old mice with the indicated genotype . Calnexin used as a loading control . ( n = 3 biological replicates per genotype ) . Quantification of B . Protein expression was normalized to levels in wild type animals . Data represent the mean ± SD .

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Western blot analysis of ubiquitin and the autophagy marker LC3 in RIPA, and p62 in RIPA and urea total brain lysates from 4.5-month-old mice with the indicated genotype. Calnexin used as a loading control. (n = 3 biological replicates per genotype). Quantification of B. Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.


Immunofluorescence analysis of p62 in sagittal brain sections . Representative images of indicated brain regions ( CTX = Cortex ; TH = Thalamus ; CBX = Cerebellum ; Medulla = MY ) . Scale bar indicates 100 µm .

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Immunofluorescence analysis of p62 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; Medulla = MY). Scale bar indicates 100 µm.


Localization of p62 mainly in NeuN positive cells in the cortex ( CTX ) of 4 . 5 - month - old mice . Minor overlap of aggregates with microglial marker IBA1 . Scale bar indicates 15 µm .

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Localization of p62 mainly in NeuN positive cells in the cortex (CTX) of 4.5-month-old mice. Minor overlap of aggregates with microglial marker IBA1. Scale bar indicates 15 µm.


Complete overlap of co - staining of p62 with NeuN in the cortex ( CTX ) and not with the reactive astrocycte marker GFAP . Scale bar indicates 15 µm .

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Complete overlap of co-staining of p62 with NeuN in the cortex (CTX) and not with the reactive astrocycte marker GFAP. Scale bar indicates 15 µm.


Immunofluorescence analysis of TDP - 43 in sagittal brain sections . Representative images of indicated brain regions ( CTX = Cortex ; MB = Midbrain ) . White arrow heads indicate TDP - 43 aggregation . Scale bar indicates 100 µm . Zoom in on TDP - 43 aggregation in double knockout mouse brain sections of the midbrain , scale bar zoom in indicates 50 μm . ( Animals 4 . 5 months of age ) .

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Immunofluorescence analysis of TDP-43 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; MB = Midbrain). White arrow heads indicate TDP-43 aggregation. Scale bar indicates 100 µm. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain, scale bar zoom in indicates 50 μm. (Animals 4.5 months of age).


Immunofluorescence analysis of total TDP - 43 and phosphorylated TDP - 43 ( p - TDP - 43 ) in sagittal brain sections by confocal microscopy . Representative images of one confocal layer in the midbrain . Zoom in on TDP - 43 aggregation in double knockout mouse brain sections of the midbrain . Scale bar indicates 25 µm . ( Animals 4 . 5 months of age ) .

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Immunofluorescence analysis of total TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sagittal brain sections by confocal microscopy. Representative images of one confocal layer in the midbrain. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain. Scale bar indicates 25 µm. (Animals 4.5 months of age).


Western blot analysis of TDP - 43 and phosphorylated TDP - 43 ( p - TDP - 43 ) in sequential high salt ( HS ) / RIPA / Urea fractionations of total brain lysates from 4 . 5 - month - old mice with the indicated genotype . Analysis of C - terminal fragments of TDP - 43 ( CTF ) in RIPA fraction . p - TDP - 43 analyzed in Urea fraction . Quantification of ( C ) Protein expression was normalized to levels in wild type animals . Data represent the mean ± SD .

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Western blot analysis of TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sequential high salt (HS)/RIPA/Urea fractionations of total brain lysates from 4.5-month-old mice with the indicated genotype. Analysis of C-terminal fragments of TDP-43 (CTF) in RIPA fraction. p-TDP-43 analyzed in Urea fraction. Quantification of (C) Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.


, B . survival of Ssrp1fl / fl ; CreERT2 + / + mice of different ages ( weeks ) after the start of tamoxifen ( TMX ) treatment . B . Kaplan - Meier survival curves . Log - rank test p = 0 . 032 for the oldest male age group versus all other male groups ; p = 0 . 013 for all females versus all males ; for all other comparisons p > 0 . 05 . n = 6 - 10 mice .

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, B. survival of Ssrp1fl/fl; CreERT2+/+ mice of different ages (weeks) after the start of tamoxifen (TMX) treatment. B. Kaplan-Meier survival curves. Log-rank test p = 0.032 for the oldest male age group versus all other male groups; p = 0.013 for all females versus all males; for all other comparisons p > 0.05. n = 6-10 mice.


C . Normalized blood cell counts in Ssrp1fl / fl ; CreERT2 + / + mice treated with vehicle or tamoxifen on day 7 after start of treatment . Mean ± SD , * indicates p < 0 . 05 ( unpaired t - test ) . n = 3 mice .

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C. Normalized blood cell counts in Ssrp1fl/fl; CreERT2+/+ mice treated with vehicle or tamoxifen on day 7 after start of treatment. Mean ± SD, * indicates p < 0.05 (unpaired t-test). n = 3 mice.


A . H & E staining of mouse organ sections . Scale bars : spleen , colon 100 μM , bone marrow - 50 μM .

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A. H&E staining of mouse organ sections. Scale bars: spleen, colon 100 μM, bone marrow - 50 μM.


A , B . Effect of tamoxifen on replication in the spleen ( A ) and intestine ( B ) of Ssrp1fl / fl ; CreERT2 + / + mice . EdU was given to mice three days after the end of tamoxifen treatment ( 1 hour before tissue collection ) . Organ sections were stained for total DNA ( Hoechst ) , EdU , and SSRP1 . Scale bars - 100 μM . C . Quantitation of EdU staining . Mean number of EdU positive cells per field of view + / - SD . n > 5 fields of view per mouse . 3 mice were analyzed . D . Quantitation of cleaved caspase - 3 staining . Mean number of positive cells per fields of view + / - SD , n > 5 fields of view per mouse Two male and female mice were analyzed . Data information : P - values for C and D were calculated using the unpaired t - test .

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A, B. Effect of tamoxifen on replication in the spleen (A) and intestine (B) of Ssrp1fl/fl; CreERT2+/+ mice. EdU was given to mice three days after the end of tamoxifen treatment (1 hour before tissue collection). Organ sections were stained for total DNA (Hoechst), EdU, and SSRP1. Scale bars - 100 μM. C. Quantitation of EdU staining. Mean number of EdU positive cells per field of view +/- SD. n > 5 fields of view per mouse. 3 mice were analyzed. D. Quantitation of cleaved caspase-3 staining. Mean number of positive cells per fields of view +/- SD, n > 5 fields of view per mouse Two male and female mice were analyzed. Data information: P-values for C and D were calculated using the unpaired t-test.


D . Fold - change in the proportion of cells in each cluster shown in panel A in samples from tamoxifen - treated versus vehicle - treated mice . Cell types are identified by color as in panel B .

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D. Fold-change in the proportion of cells in each cluster shown in panel A in samples from tamoxifen-treated versus vehicle-treated mice. Cell types are identified by color as in panel B.


D . UMAP plots with color - coded intestinal stem cells identified by transcriptional signatures of intestinal stem cells or by expression of Lgr5 or Gkn3 genes .

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D. UMAP plots with color-coded intestinal stem cells identified by transcriptional signatures of intestinal stem cells or by expression of Lgr5 or Gkn3 genes.


A , B . Effect of 4 - OHT administration on colony formation by bone marrow cells of different genotypes . A . Photographs of colonies from bone marrow cells kept in culture for seven days in the presence or absence of 4 - OHT . B . Quantitation of bone marrow colony assay . Mean colony number per plate ± SD , n = 3 plates per mouse . P values < 0 . 05 are shown ( unpaired t - test ) . 2 mice of each genotype were analyzed . C , D . Growth of intestinal organoids in the presence or absence of 4 - OHT . C . Photographs of small intestine organoids on different days after plating . D . Number of organoids in the presence and absence of 4 - OHT . Mean fold change between number of organoids in control and 4 - OHT treated wells ± SD , n = 4 wells per mouse . P < 0 . 05 are shown ( unpaired t - test ) . Organoids were isolated from one mouse of each genotype in two independent experiments .

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A, B. Effect of 4-OHT administration on colony formation by bone marrow cells of different genotypes. A. Photographs of colonies from bone marrow cells kept in culture for seven days in the presence or absence of 4-OHT. B. Quantitation of bone marrow colony assay. Mean colony number per plate ± SD, n = 3 plates per mouse. P values < 0.05 are shown (unpaired t-test). 2 mice of each genotype were analyzed. C, D. Growth of intestinal organoids in the presence or absence of 4-OHT. C. Photographs of small intestine organoids on different days after plating. D. Number of organoids in the presence and absence of 4-OHT. Mean fold change between number of organoids in control and 4-OHT treated wells ± SD, n = 4 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from one mouse of each genotype in two independent experiments.


F , G . Effect of 4 - OHT administration on intestinal organoids of different genotypes kept in the presence ( undifferentiated ) or absence ( differentiated ) of R - spondin - 1 . F . The viability of organoids assessed by the resorufin assay . Mean ± SD , n = 3 wells per mouse . P < 0 . 05 are shown ( unpaired t - test ) . Organoids were isolated from two mice of each genotype . G . IF images of intestinal organoids from Ssrp1fl / fl ; CreERT2 + / + mice kept in the presence or absence of R - Spondin - 1 . The organoids were stained with antibodies to LGR5 and SSRP1 and counterstained with Hoechst ( DNA ) .

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F, G. Effect of 4-OHT administration on intestinal organoids of different genotypes kept in the presence (undifferentiated) or absence (differentiated) of R-spondin-1. F. The viability of organoids assessed by the resorufin assay. Mean ± SD, n = 3 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from two mice of each genotype. G. IF images of intestinal organoids from Ssrp1fl/fl; CreERT2+/+ mice kept in the presence or absence of R-Spondin-1. The organoids were stained with antibodies to LGR5 and SSRP1 and counterstained with Hoechst (DNA).


Effect of 4 - OHT administration on MSCs and their differentiated counterparts . H . Methylene blue viability assay for undifferentiated MSCs or MSCs differentiated into osteocytes or adipocytes for 21 days and then treated with vehicle or 4 - OHT . Mean ± SD , n = 3 plates per mouse . P < 0 . 05 are shown ( unpaired t - test ) . MSCs were isolated from two mice of each genotype .

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Effect of 4-OHT administration on MSCs and their differentiated counterparts. H. Methylene blue viability assay for undifferentiated MSCs or MSCs differentiated into osteocytes or adipocytes for 21 days and then treated with vehicle or 4-OHT. Mean ± SD, n = 3 plates per mouse. P < 0.05 are shown (unpaired t-test). MSCs were isolated from two mice of each genotype.


A . EU incorporation measured by flow cytometry . Histograms of EU fluorescent signals from two biological replicates for each condition ( except no EU ) . B . SSRP1 staining of the cells in A .

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A. EU incorporation measured by flow cytometry. Histograms of EU fluorescent signals from two biological replicates for each condition (except no EU). B. SSRP1 staining of the cells in A.


G . Box plots of mean read density ( y - axis ) at the gene body ( bins 21 - 100 out of 100 between TSS - TES ) for all genes and genes expressed at different levels . * * * - p < 1e - 10 ( paired t - test ) . n = 2 biological replicates . Central band is mean , box is interquartile range , whiskers are minimum and maximum .

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G. Box plots of mean read density (y-axis) at the gene body (bins 21-100 out of 100 between TSS - TES) for all genes and genes expressed at different levels. *** - p < 1e-10 (paired t-test). n = 2 biological replicates. Central band is mean, box is interquartile range, whiskers are minimum and maximum.


H . Comparison of Hoechst 33342 binding to live control and 4 - OHT - treated MSCs from different genotypes . Data are presented as the median fluorescence intensity ± SD , n = 4 biological replicates . P < 0 . 05 is shown ( unpaired t - test ) .

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H. Comparison of Hoechst 33342 binding to live control and 4-OHT-treated MSCs from different genotypes. Data are presented as the median fluorescence intensity ± SD, n = 4 biological replicates. P < 0.05 is shown (unpaired t-test).