C . Transwell migration of primary neutrophils towards conditioned media from HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox in combination with indicated inhibitors for 24 h . Data is presented as individual values with grand mean ( n = 3 biological replicates of conditioned media from GSK ' 872 and NSA - treated wells , n = 6 biological replicates of conditioned media from other conditions ) . Two - way ANOVA and Sidak ' s multiple comparisons test were used to test for statistical differences between indicated conditions . * * * * p < 0 . 0001 ; ns = not significant ( p > 0 . 96 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox in combination with indicated inhibitors for 24 h. Data is presented as individual values with grand mean (n = 3 biological replicates of conditioned media from GSK'872 and NSA-treated wells, n = 6 biological replicates of conditioned media from other conditions). Two-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. **** p < 0.0001; ns = not significant (p > 0.96).


D . CXCL8 concentration in the culture media of HT29 / TO - ZBP1WT cells transfected with siRNA targeting mismatch sequence ( MM ) or RIPK3 and treated with Dox at indicated concentrations for 24 h . Data is presented as mean with S . E . M . ( n = 4 biological replicates ) . Unpaired t - tests were used to test for statistical differences between indicated conditions . * * p = 0 . 0079 . Cell lysates from one biological replicate were analyzed by Western blotting .

O O B-GENEPROD O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O

D. CXCL8 concentration in the culture media of HT29 / TO-ZBP1WT cells transfected with siRNA targeting mismatch sequence (MM) or RIPK3 and treated with Dox at indicated concentrations for 24 h. Data is presented as mean with S.E.M. (n = 4 biological replicates). Unpaired t-tests were used to test for statistical differences between indicated conditions. ** p = 0.0079. Cell lysates from one biological replicate were analyzed by Western blotting.


E . Cytokine concentration in the culture media of HT29 / TO - ZBP1WT or HT29 / RIPK1 - KO clone aA3 / TO - ZBP1WT cells treated with 0 , 50 or 500 ng / mL Dox . data is plotted as mean with S . E . M . ( n = 6 biological replicates ) . Brown - Forsythe and Welch ANOVA tests and Dunnet ' s T3 multiple comparisons test were used to test for statistical differences between indicated conditions . Cell lysates were analyzed by Western blotting for ZBP1 levels . Blots are representative of six biological replicates . ns = not significant ( p > 0 . 2 ) ; * * * p = 0 . 0003 for CXCL8 , p = 0 . 0008 for CXCL1 ; * * * * p < 0 . 0001 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O O O O 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-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O

E. Cytokine concentration in the culture media of HT29 / TO-ZBP1WT or HT29 / RIPK1-KO clone aA3 / TO-ZBP1WT cells treated with 0, 50 or 500 ng/mL Dox. data is plotted as mean with S.E.M. (n = 6 biological replicates). Brown-Forsythe and Welch ANOVA tests and Dunnet's T3 multiple comparisons test were used to test for statistical differences between indicated conditions. Cell lysates were analyzed by Western blotting for ZBP1 levels. Blots are representative of six biological replicates. ns = not significant (p > 0.2); *** p = 0.0003 for CXCL8, p = 0.0008 for CXCL1; **** p < 0.0001.


G , NF - κB activity in HEK293FT cells with and without stable expression of full - length ( FL ) RIPK3 - 2xFV or RIPK3 ∆ C - 2xFV ( ∆ C ) transfected with dual luciferase reporters and ZBP1 or empty vector ( EV ) as indicated . Reporter activities were measured 24 h after transfection . Data is presented as mean with S . E . M ( n = 3 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons test were used to test for statistical differences between indicated conditions . ns = not significant ( p > 0 . 05 ) ; * * * * p < 0 . 0001 . ( G ) Cell lysates were analyzed by Western blotting . Blots are representative of three biological replicates . Asterisk indicates background signals of the antibody .

O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O

G, NF-κB activity in HEK293FT cells with and without stable expression of full-length (FL) RIPK3-2xFV or RIPK3∆C-2xFV (∆C) transfected with dual luciferase reporters and ZBP1 or empty vector (EV) as indicated. Reporter activities were measured 24 h after transfection. Data is presented as mean with S.E.M (n = 3 biological replicates). One-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p > 0.05); **** p<0.0001. (G) Cell lysates were analyzed by Western blotting. Blots are representative of three biological replicates. Asterisk indicates background signals of the antibody.


A . Western blot analysis of anti - FLAG ( ZBP1 ) immunoprecipitation from HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16 h . Blots are representative of three biological replicates . Immunoprecipitated material was treated or not with USP21 ( 1 μM ) for deubiquitination . Input loaded was 5 % for ZBP1 and 1 % for co - immunoprecipitants . Asterisk indicates antibody heavy chain signal in IP samples .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O

A. Western blot analysis of anti-FLAG (ZBP1) immunoprecipitation from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16 h. Blots are representative of three biological replicates. Immunoprecipitated material was treated or not with USP21 (1 μM) for deubiquitination. Input loaded was 5% for ZBP1 and 1% for co-immunoprecipitants. Asterisk indicates antibody heavy chain signal in IP samples.


B . Enrichment of Ub - conjugates by GST - 1xUBA for analysis of ubiquitination status of RIPK1 , ZBP1 and RIPK3 in HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16h . Blots are representative of at least three biological replicates . After enrichment , samples were treated or not with USP21 ( 1 μM ) for deubiquitination . Asterisk indicates unspecific bands . Arrows indicate RIPK3 signal .

O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O

B. Enrichment of Ub-conjugates by GST-1xUBA for analysis of ubiquitination status of RIPK1, ZBP1 and RIPK3 in HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16h. Blots are representative of at least three biological replicates. After enrichment, samples were treated or not with USP21 (1 μM) for deubiquitination. Asterisk indicates unspecific bands. Arrows indicate RIPK3 signal.


C . Enrichment of Ub - conjugates for analysis of the ubiquitination status of RIPK1 and ZBP1 using GST - 1xUBA or linkage - specific SUBs in HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16 h .

O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O

C. Enrichment of Ub-conjugates for analysis of the ubiquitination status of RIPK1 and ZBP1 using GST-1xUBA or linkage-specific SUBs in HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16 h.


D . Enrichment of Ub - conjugates by GST - 1xUBA for analysis of ubiquitination status of RIPK1 and ZBP1 in HT29 , HT29 / TO - ZBP1WT and HT29 / RIPK1 - KO aA3 / TO - ZBP1WT cells treated with 500 ng / mL Dox for 16 h . Blots are representative of four biological replicates . Asterisk indicates antibody background signal .

O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O

D. Enrichment of Ub-conjugates by GST-1xUBA for analysis of ubiquitination status of RIPK1 and ZBP1 in HT29, HT29 / TO-ZBP1WT and HT29 / RIPK1-KO aA3 / TO-ZBP1WT cells treated with 500 ng/mL Dox for 16 h. Blots are representative of four biological replicates. Asterisk indicates antibody background signal.


E . NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with dual luciferase reporters , ZBP1 or EV , and GFP ( Ctrl ) or GFP - tagged SUBs as indicated . Luciferase reporter activities were measured 24 h after transfection and normalized to GFP + EV - transfected condition . Data is plotted as mean with S . E . M . ( n = 4 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons tests were used to test for statistical differences between the indicated condition and ZBP1 / GFP - transfected condition . * * * * p < 0 . 0001 . Cell lysates were analyzed by Western blotting to determine expression of ZBP1 , GFP or GFP - tagged SUBs and Actin . Blots are representative of three biological replicates .

O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O

E. NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and GFP (Ctrl) or GFP-tagged SUBs as indicated. Luciferase reporter activities were measured 24 h after transfection and normalized to GFP+EV-transfected condition. Data is plotted as mean with S.E.M. (n = 4 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between the indicated condition and ZBP1/GFP-transfected condition. **** p < 0.0001. Cell lysates were analyzed by Western blotting to determine expression of ZBP1, GFP or GFP-tagged SUBs and Actin. Blots are representative of three biological replicates.


F , NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with dual luciferase reporters , ZBP1 or EV , and variants of OTULIN . Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1 / EV - transfected cells . Data is plotted as mean with S . E . M . Multiple Welch t - tests were used to test for statistical differences between the indicated condition and the ZBP1 / EV - transfected condition . ( F ) n = 4 biological replicates . * * p = 0 . 0012 , ns = not significant ( p = 0 . 2342 ) . Cell lysates were analyzed by Western blotting to determine expression of ZBP1 , or OTULIN , and Actin . Blots are representative of ( F ) four biological replicates .

O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O

F, NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and variants of OTULIN. Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1/EV-transfected cells. Data is plotted as mean with S.E.M. Multiple Welch t-tests were used to test for statistical differences between the indicated condition and the ZBP1/EV-transfected condition. (F) n = 4 biological replicates. ** p = 0.0012, ns = not significant (p = 0.2342). Cell lysates were analyzed by Western blotting to determine expression of ZBP1, or OTULIN, and Actin. Blots are representative of (F) four biological replicates.


G . NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with dual luciferase reporters , ZBP1 or EV , and variants of CYLD Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1 / EV - transfected cells . Data is plotted as mean with S . E . M . Multiple Welch t - tests were used to test for statistical differences between the indicated condition and the ZBP1 / EV - transfected condition . ( G ) n = 3 biological replicates . * p = 0 . 0137 ; ns = not significant ( p = 0 . 8374 ) . Cell lysates were analyzed by Western blotting to determine expression of ZBP1 , CYLD and Actin . Blots are representative of ( G ) three biological replicates .

O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O

G. NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and variants of CYLD Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1/EV-transfected cells. Data is plotted as mean with S.E.M. Multiple Welch t-tests were used to test for statistical differences between the indicated condition and the ZBP1/EV-transfected condition. (G) n = 3 biological replicates. * p = 0.0137; ns = not significant (p = 0.8374). Cell lysates were analyzed by Western blotting to determine expression of ZBP1, CYLD and Actin. Blots are representative of (G) three biological replicates.


A . NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with siRNA targeting HOIP ( siHOIP ) or a mismatch sequence ( siMM ) , and 48 h later transfected with dual luciferase reporters and ZBP1 or GFP ( control ) . Luciferase reporter activities were measured 24 h after transfection and normalized to the siMM + ZBP1 samples . Data is plotted as mean with S . E . M . ( n = 4 biological replicates ) . A Welch ' s t - test was used to test for the statistical difference between indicated conditions . * * * * p < 0 . 0001 . HOIP knockdown and ZBP1 expression levels were analyzed by Western blotting . Blots are representative of three biological replicates .

O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

A. NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with siRNA targeting HOIP (siHOIP) or a mismatch sequence (siMM), and 48 h later transfected with dual luciferase reporters and ZBP1 or GFP (control). Luciferase reporter activities were measured 24 h after transfection and normalized to the siMM+ZBP1 samples. Data is plotted as mean with S.E.M. (n = 4 biological replicates). A Welch's t-test was used to test for the statistical difference between indicated conditions. **** p < 0.0001. HOIP knockdown and ZBP1 expression levels were analyzed by Western blotting. Blots are representative of three biological replicates.


D - F . Western blot analysis of anti - FLAG ( ZBP1 ) immunoprecipitation from HT29 or HT29 / TO - ZBP1WT or HT29 / RIPK1 - KO clone aA3 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16 h . Asterisk indicates antibody heavy chain signal in IP samples . Blots are representative of at least ( D ) two and ( E - F ) three biological replicates .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

D-F. Western blot analysis of anti-FLAG (ZBP1) immunoprecipitation from HT29 or HT29 / TO-ZBP1WT or HT29 / RIPK1-KO clone aA3 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16 h. Asterisk indicates antibody heavy chain signal in IP samples. Blots are representative of at least (D) two and (E-F) three biological replicates.


B . Western blot analysis of HCT116 / RIPK3 - 2xFV cells ( FL and ∆ C ) treated with 100 nM dimerizer . Blots are representative of two biological replicates .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O

B. Western blot analysis of HCT116 / RIPK3-2xFV cells (FL and ∆C) treated with 100 nM dimerizer. Blots are representative of two biological replicates.


D . CXCL8 concentration in the culture media of HCT116 / RIPK3 - 2xFV cells ( WT and RHIMmut ) treated with 100 nM dimerizer for 24 h . In RIPK3 - RHIMmut three key residues , VQV , of the RHIM region was changed to AAAA . Data is plotted as mean with S . E . M . ( n = 5 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons tests were used to test for statistical differences between indicated time points . * * p = 0 . 0011 ; ns = not significant ( p = 0 . 9301 ) . Inset : Western blot analysis to determine RIPK3 expression levels in one biological replicate . Line indicates that image was cut and spliced to remove non - relevant lanes from the scanned blot .

O O B-GENEPROD O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD 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-EXP_ASSAY O

D. CXCL8 concentration in the culture media of HCT116 / RIPK3-2xFV cells (WT and RHIMmut) treated with 100 nM dimerizer for 24 h. In RIPK3-RHIMmut three key residues, VQV, of the RHIM region was changed to AAAA. Data is plotted as mean with S.E.M. (n = 5 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated time points. ** p = 0.0011; ns = not significant (p = 0.9301). Inset: Western blot analysis to determine RIPK3 expression levels in one biological replicate. Line indicates that image was cut and spliced to remove non-relevant lanes from the scanned blot.


H . Relative mRNA levels of CXCL8 in HCT116 / RIPK3 - 2xFV cells pretreated with 100 nM CpA for 0 . 5 h before treatment with 0 or 100 nM dimerizer for 3 h . Data is presented as mean with S . E . M . ( n = 3 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons tests were used to test for statistical differences between indicated conditions and DMSO + dimerizer - treated condition . * * * * p < 0 . 0001 . ( H ) Cell lysates were analyzed by Western blotting for cIAP1 levels . Blots are representative of two biological replicates .

O O O O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O O O O O O O

H. Relative mRNA levels of CXCL8 in HCT116 / RIPK3-2xFV cells pretreated with 100 nM CpA for 0.5 h before treatment with 0 or 100 nM dimerizer for 3 h. Data is presented as mean with S.E.M. (n = 3 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated conditions and DMSO+dimerizer-treated condition. **** p < 0.0001. (H) Cell lysates were analyzed by Western blotting for cIAP1 levels. Blots are representative of two biological replicates.


J . Relative CXCL8 mRNA levels in WT or HOIP - knockout HCT116 / RIPK3 - 2xFV cells treated with 0 or 100 nM dimerizer for 3 h . Data is plotted as mean with S . E . M . ( n = 3 biological replicates ) . A Welch ' s t - test was used to test for the statistical difference as indicated . * * * p = 0 . 0010 . Cell lysates were loaded for Western blot analysis .

O O O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O

J. Relative CXCL8 mRNA levels in WT or HOIP-knockout HCT116 / RIPK3-2xFV cells treated with 0 or 100 nM dimerizer for 3 h. Data is plotted as mean with S.E.M. (n = 3 biological replicates). A Welch's t-test was used to test for the statistical difference as indicated. *** p = 0.0010. Cell lysates were loaded for Western blot analysis.


K . Relative CXCL8 mRNA levels in HCT116 / Tet - On - GFP - K63 - SUB / RIPK3 - 2xFV , HCT116 / Tet - On - GFP - M1 - SUB / RIPK3 - 2xFV and HCT116 / Tet - On - GFP / RIPK3 - 2xFV cells treated with 0 or 100 ng / mL Dox for 48 h before stimulated with 0 or 100 nM dimerizer for 3 h . Data is presented as mean with S . E . M . ( n = 4 biological replicates ) . Brown - Forsythe and Welch ANOVA tests and Dunnet ' s T3 multiple comparisons test were used to test for statistical differences between indicated conditions . ns = not significant ( p = 0 . 9997 ) ; * * p = 0 . 0030 ; * p = 0 . 0366 . Cells were analyzed by Western blotting for the inducible - expression levels of GFP ( control ) and GFP - SUBs . Blots are representative of three biological replicates .

O O O B-GENEPROD O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O

K. Relative CXCL8 mRNA levels in HCT116 / Tet-On-GFP-K63-SUB / RIPK3-2xFV, HCT116 / Tet-On-GFP-M1-SUB / RIPK3-2xFV and HCT116 / Tet-On-GFP / RIPK3-2xFV cells treated with 0 or 100 ng/mL Dox for 48 h before stimulated with 0 or 100 nM dimerizer for 3 h. Data is presented as mean with S.E.M. (n = 4 biological replicates). Brown-Forsythe and Welch ANOVA tests and Dunnet's T3 multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p = 0.9997); ** p = 0.0030; * p = 0.0366. Cells were analyzed by Western blotting for the inducible-expression levels of GFP (control) and GFP-SUBs. Blots are representative of three biological replicates.


M . Relative CXCL8 mRNA levels in HCT116 / RIPK3 - 2xFV cells stably knocked down against mismatch ( shMM ) or two different sites of caspase - 8 ( shCASP8 - A and shCASP8 - B ) , pretreated with DMSO , 10 µM Nec1s , 10 µM GSK ' 872 or 1 µM NSA for 1h followed by treatment with 0 or 100 nM dimerizer for 3h . Data is plotted as mean with S . E . M . ( n = 3 biological replicates ) . Two - way ANOVA and Tukey ' s multiple comparison tests were used to test for statistical differences between indicated conditions . * * * p = 0 . 0001 ; * * p = 0 . 0035 . Cell lysates from one biological replicate were analyzed by Western blotting to determine caspase - 8 levels .

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M. Relative CXCL8 mRNA levels in HCT116 / RIPK3-2xFV cells stably knocked down against mismatch (shMM) or two different sites of caspase-8 (shCASP8-A and shCASP8-B), pretreated with DMSO, 10 µM Nec1s, 10 µM GSK'872 or 1 µM NSA for 1h followed by treatment with 0 or 100 nM dimerizer for 3h. Data is plotted as mean with S.E.M. (n = 3 biological replicates). Two-way ANOVA and Tukey's multiple comparison tests were used to test for statistical differences between indicated conditions. *** p = 0.0001; ** p = 0.0035. Cell lysates from one biological replicate were analyzed by Western blotting to determine caspase-8 levels.


A . Western blot analysis of ZBP1 , RIPK1 , RIPK3 , MLKL levels in Calu - 3 cell lines with stable expression of shRNAs targeting ZBP1 or a mismatch sequence ( shMM ) , untreated ( NT ) or treated with 10 ng / mL IFNβ for 48 h . Blots are representative of two biological replicates .

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A. Western blot analysis of ZBP1, RIPK1, RIPK3, MLKL levels in Calu-3 cell lines with stable expression of shRNAs targeting ZBP1 or a mismatch sequence (shMM), untreated (NT) or treated with 10 ng/mL IFNβ for 48 h. Blots are representative of two biological replicates.


( A ) Localization of Ape1 relative to Ykt6 and Sec63 during nitrogen starvation . Cells expressing GFP - tagged Ykt6 under the control of the GAL1 promoter and tdTomato - tagged Sec63 were transformed with a centromeric plasmid expressing BFP - Ape1 . Cells were grown in SGC - LEU , shifted to SG - N for the indicated time , and analyzed by fluorescence microscopy . Images are shown as single planes . Scale bar , 5 µm . Arrows indicate Ykt6 dots colocalizing with Ape1 . ( B ) Percentage of Ykt6 puncta simultaneously co - localizing with both Ape1 and Sec63 . The data were quantified from ( A ) . Error bars represent standard deviation of three independent experiments . Cells ( n ≥ 250 ) and Ape1 dots ( n ≥ 50 ) were quantified .

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(A) Localization of Ape1 relative to Ykt6 and Sec63 during nitrogen starvation. Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter and tdTomato-tagged Sec63 were transformed with a centromeric plasmid expressing BFP-Ape1. Cells were grown in SGC-LEU, shifted to SG-N for the indicated time, and analyzed by fluorescence microscopy. Images are shown as single planes. Scale bar, 5 µm. Arrows indicate Ykt6 dots colocalizing with Ape1. (B) Percentage of Ykt6 puncta simultaneously co-localizing with both Ape1 and Sec63. The data were quantified from (A). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Ape1 dots (n≥50) were quantified.


( 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 .

O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O

(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 .

O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O

(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 )

O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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-GENEPROD B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(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 ) .

O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O

(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 .

O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

(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 .

O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O

(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

O O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

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

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY

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

O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O 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-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O

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

O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

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 /

O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O 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-GENEPROD O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O B-DISEASE O O O O O 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 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 ) .

O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O B-TISSUE O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE O O O O B-DISEASE O O O

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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-DISEASE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE O O O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O O O O B-DISEASE 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-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE 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-GENEPROD O B-TISSUE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O B-GENEPROD O O O B-TISSUE B-DISEASE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O 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 B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE 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 B-DISEASE O O O O O O O O O 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-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE O O O B-DISEASE O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE O O O O B-DISEASE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O B-GENEPROD O O O B-TISSUE B-DISEASE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O 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 ) .

B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM 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 .

B-TISSUE I-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O

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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM 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 B-EXP_ASSAY B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O 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-DISEASE O B-EXP_ASSAY O 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-EXP_ASSAY B-TISSUE O O B-GENEPROD O O O O O O B-ORGANISM 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 B-EXP_ASSAY B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O 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-DISEASE O B-EXP_ASSAY 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-GENEPROD O O O O O O B-ORGANISM 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.