I - L . Hep2 ( I ) , Huh - 7 ( J ) , MRC - 5 ( K ) , undifferentiated HTBE ( L ) were treated infected with VSV - GFP Data information : Total Integrated Green Intensities were determined using Incucyte live - system after 24h Data represent means standard deviations from n = 3 biological experiments ( data points shown ) . Statistical significance was determined by 1 - way ANOVA on log - transformed values ( * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 ) . , dotted lines are a visual guide for maximum minimum replication in control in absence presence of IFN - α2 , respectively . Numbers above IFN - α2 - treated bars indicate their approximate difference to respective untreated conditions .

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I-L. Hep2 (I), Huh-7 (J), MRC-5 (K), and undifferentiated HTBE (L) were treated and infected with VSV-GFP Data information: Total Integrated Green Fluorescent Intensities were determined using the Incucyte live-cell analysis system after 24h Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed intensity values (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001). , dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN-α2, respectively. Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions.


C . Impact of dBRD9 - A pre - treatment on induction of ISGs identified in ( B ) . Colors indicate IFN - induced transcripts significantly affected ( blue ) or unaffected ( gray ) by dBRD9 - A pretreatment ( FC > 2 , p - value ≤ 0 . 001 ) .

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C. Impact of dBRD9-A pre-treatment on the induction of ISGs identified in (B). Colors indicate IFN-induced transcripts significantly affected (blue) or unaffected (gray) by dBRD9-A pretreatment (FC>2, p-value ≤ 0.001).


F . RT - qPCR confirmation of transcriptomics results for selected transcripts ( MX1 , MX2 , IFITM1 , ISG15 IFIT1 ) in A549 treated as outlined in ( A ) . GAPDH transcript were used for normalization . Data represent means standard deviations from n = 3 biological experiments ( data points shown ) . Statistical significance was determined by unpaired 2 - t - on ΔCt values ( * p - value < 0 . 05 ; n . s . not significant ) .

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F. RT-qPCR confirmation of transcriptomics results for selected transcripts (MX1, MX2, IFITM1, ISG15 and IFIT1) in A549 cells treated as outlined in (A). GAPDH transcript levels were used for normalization. Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by unpaired 2-tailed t-test on ΔCt values (*p-value < 0.05; n.s. not significant).


G . Western blot of CTRL # 3 or BRD9 - KO # 3 stably transduced with lentiviruses expressing empty vector ( EV ) , or wt chimeric

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G. Western blot analysis of CTRL#3 or BRD9-KO#3 cells stably transduced with lentiviruses expressing empty vector (EV), or wt and chimeric constructs


H . Western blot of input lysates anti - BRD9 immunoprecipitations ( IPs ) from described in ( G ) expressing wt BRD9 or chimeric BRD7DUF . indicated were detected with specific antibodies . Data are representative of two biological replicates .

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H. Western blot analysis of input lysates and anti-BRD9 immunoprecipitations (IPs) from cells described in (G) expressing wt BRD9 or the chimeric BRD7DUF. The indicated proteins were detected with specific antibodies. Data are representative of two biological replicates.


I . various CTRL # 3 or BRD9 - KO # 3 described in ( G ) were stimulated infected as described in ( B ) . Data information : data represent means standard deviations from n = 3 biological experiments ( data points shown ) . Numbers above IFN - α2 - treated bars indicate their approximate difference to respective untreated conditions . Dotted lines are a visual guide for minimum replication in control in presence of IFN - α2 . Statistical significance was determined by 1 - way ANOVA on log - transformed values ( * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 ; n . s . not significant ) .

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I. The various CTRL#3 or BRD9-KO#3 cells described in (G) were stimulated and infected as described in (B). Data information: data represent means and standard deviations from n=3 biological experiments (individual data points shown). Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions. Dotted lines are a visual guide for minimum virus replication in control cells in the presence of IFN-α2. Statistical significance was determined by 1-way ANOVA on log-transformed intensity values (***p-value < 0.001; ****p-value < 0.0001; n.s. not significant).


F . Western blot of samples from independent small - scale streptavidin purifications ( termed ' Eluates ' ) following proximity labeling approach outlined in ( B ) . indicated were detected with specific antibodies . M indicates marker lane . Data are representative of at least two biological replicates .

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F. Western blot analysis of samples from independent small-scale streptavidin purifications (termed 'Eluates') following the proximity labeling approach outlined in (B). The indicated proteins were detected with specific antibodies. M indicates the marker lane. Data are representative of at least two biological replicates.


siRNA depletion of COPI increases autophagy . ( A ) siRNA depletion of β ′ - , β - , α - COPI ( top ) Sec23A B ( bottom ) in 293 / GFP - LC3 for 48 h . Decreased of corresponding subunits is shown by immunoblots .

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siRNA depletion of COPI increases autophagy. (A) siRNA depletion of β′-, β-, and α-COPI (top) and Sec23A and B (bottom) in 293/GFP-LC3 cells for 48 h. Decreased protein levels of the corresponding subunits is shown by immunoblots.


( B ) Loss of β ′ - , α - , β - COP but not subunits of COPII ( Sec 23A B ) increase GFP - LC3 - II . In A B , tubulin was loading control .

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(B) Loss of β′-, α-, and β-COP but not subunits of COPII (Sec 23A and B) increase GFP-LC3-II. In A and B, tubulin was the loading control.


( C ) GFP - LC3 - positive vesicles were detectable in 293 / GFP - LC3 after siRNA depletion of β ′ - , α - , β - COP but only basal of GFP - LC3 - positive vesicles are observed in control siRNA - depleted .

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(C) GFP-LC3-positive vesicles were detectable in 293/GFP-LC3 cells after siRNA depletion of β′-, α-, and β-COP but only basal levels of GFP-LC3-positive vesicles are observed in control siRNA-depleted cells.


Time course of COPI depletion shows that Golgi disperses before AVs are formed , but Golgi dispersal does not cause AV formation . ( A ) COPI subunits were depleted in 293 / GFP - LC3 analyzed 24 , 36 , 48 h after addition of siRNA . Immunoblotting for β ′ - α - COP confirms loss of COP subunits .

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Time course of COPI depletion shows that Golgi disperses before AVs are formed, but Golgi dispersal does not cause AV formation. (A) COPI subunits were depleted in 293/GFP-LC3 cells and analyzed 24, 36, and 48 h after addition of siRNA. Immunoblotting for β′- and α-COP confirms loss of COP subunits.


( B ) GFP - LC3 - I - II were monitored by immunoblots in parallel lysates using anti - GFP antibodies at indicated times .

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(B) GFP-LC3-I and -II were monitored by immunoblots in parallel lysates using anti-GFP antibodies at the indicated times.


( C ) After 24 h siRNA treatment , depletion of β ′ - COP caused morphological changes a reduction in perinuclear population of GM130 . Box indicates enlarged .

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(C) After 24 h siRNA treatment, depletion of β′-COP caused morphological changes and a reduction in perinuclear population of GM130. Box indicates enlarged area.


( D ) Rab1a / b was depleted using siRNAs for 48 h . Lysates were probed with anti - Rab1 to confirm depletion .

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(D) Rab1a/b was depleted using siRNAs for 48 h. Lysates were probed with anti-Rab1 to confirm depletion.


( E ) β ′ - COP or Rab1a / b were depleted as in D , analyzed by indirect immunofluorescence using anti - GM130 GFP . In β ′ - COP panel , asterisk indicates that did not show an accumulation of GFP - LC3 - positive AVs .

(E) β′-COP or Rab1a/b were depleted as in D, and analyzed by indirect immunofluorescence using anti-GM130 and GFP fluorescence. In β′-COP panel, the asterisk indicates cells that did not show an accumulation of GFP-LC3-positive AVs.


AV formation after siRNA depletion of COPI requires Atg5 Atg7 . 293 / GFP - LC3 were incubated with siRNA for β ′ - , α - COP , or control siRNA alone or combined with siRNA against Atg5 or Atg7 for 48 h , then ( A ) lysed analyzed by immunoblot for Atg5 , Atg7 , GFP - LC3 , tubulin ( loading control ) ,

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AV formation after siRNA depletion of COPI requires Atg5 and Atg7. 293/GFP-LC3 cells were incubated with siRNA for β′-, α-COP, or control siRNA alone or combined with siRNA against Atg5 or Atg7 for 48 h, then (A) lysed and analyzed by immunoblot for Atg5, Atg7, GFP-LC3, and tubulin (loading control),


( B ) fixed for immunofluorescence using anti - ERGIC - 53 antibodies , or GFP . In A , asterisk indicates nonspecific band ; in B C , asterisk indicates that did not show a fragmented ERGIC - 53 .

(B) fixed for immunofluorescence analysis using anti-ERGIC-53 antibodies, or GFP fluorescence. In A, the asterisk indicates nonspecific band; in B and C, asterisk indicates cells that did not show a fragmented ERGIC-53.


Correlative - electron microscopy of GFP - LC3 demonstrates they are AVs . ( A ) Phase , ( B ) , ( C ) of same field of 293 / GFP - LC3 48 h after siRNA depletion of α - COP . In B image was inverted to allow better visualization of GFP - LC3 - positive structures . ( D ) Higher magnification of shown with asterisk in A C . Top region of of interest is enlarged in E to show two AVs structures indicated by arrows in B . ( F G ) Higher magnification of AVs shown by arrows in E .

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Correlative light-electron microscopy analysis of GFP-LC3 puncta demonstrates they are AVs. (A) Phase, (B) fluorescent, and (C) TEM of the same field of 293/GFP-LC3 cells 48 h after siRNA depletion of α-COP. In B the image was inverted to allow better visualization of the GFP-LC3-positive structures. (D) Higher magnification of cell shown with asterisk in A and C. Top region of cell of interest is enlarged in E to show the two AVs structures indicated by arrows in B. (F and G) Higher magnification of AVs shown by arrows in E.


Ub p62 accumulate in α - β ′ - COP - depleted 293 / GFP - LC3 . ( A ) 293 / GFP - LC3 were treated with control or β ′ - COP siRNA for 48 h , then fixed labeled with anti - p62 anti - ubiquitin antibodies for colocalization with GFP - LC3 .

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Ub and p62 accumulate in α- and β′-COP-depleted 293/GFP-LC3 cells. (A) 293/GFP-LC3 cells were treated with control or β′-COP siRNA for 48 h, then fixed and labeled with anti-p62 and anti-ubiquitin antibodies for colocalization with GFP-LC3.


( B ) HEK293 treated as in A labeled with anti - ubiquitin anti - p62 antibodies .

(B) HEK293 cells treated as in A and labeled with anti-ubiquitin and anti-p62 antibodies.


( C ) Cryosections from β ′ - COP - depleted were labeled with anti - Ub antibodies followed by 10 - nm A - gold . AV , autophagosomes .

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(C) Cryosections from β′-COP-depleted cells were labeled with anti-Ub antibodies followed by 10-nm protein A-gold. AV, autophagosomes.


AVs in COPI - depleted are not degradative . ( A ) Long - lived degradation was assessed 72 h after transfection with control or β ′ - COP siRNA in 293 / GFP - LC3 . were either starved of for 2 h ( St ) or incubated in fresh growth medium ( Fed ) for 2 h . amount of degradation is presented as mean ± .

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AVs in COPI-depleted cells are not degradative. (A) Long-lived protein degradation was assessed 72 h after transfection with control or β′-COP siRNA in 293/GFP-LC3 cells. Cells were either starved of amino acids for 2 h (St) or incubated in fresh growth medium (Fed) for 2 h. The amount of protein degradation is presented as mean ± SEM.


( B ) siControl - treated β ′ - α - COP were depleted for 48 h , then incubated with Lysotracker red ( 50 nM ) for 30 min before fixation . Merged image shows very little colocalization of GFP - LC3 AV - positive structures . Asterisk shows enlarged in

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(B) siControl-treated cells and β′- and α-COP were depleted for 48 h, then incubated with Lysotracker red (50 nM) for 30 min before fixation. Merged image shows very little colocalization of GFP-LC3 AV-positive structures. Asterisk shows cell enlarged in inset


( C ) After β ′ - α - COP siRNA depletion , Magic Red - RR2 ( MR ) was used to detect active cathepsin B in which were fed or starved ( St ) for 2 h . of MR labeling was measured in live by flow cytometry .

(C) After β′- and α-COP siRNA depletion, Magic Red-RR2 (MR) was used to detect active cathepsin B in cells which were fed or starved (St) for 2 h. The intensity of MR labeling was measured in live cells by flow cytometry.


GFP - LC3 AVs are LAMP2 positive . ( A ) 293 / GFP - LC3 were incubated with control or β ′ - or α - COP siRNA for 48 h , fixed labeled with anti - LAMP2 antibodies , followed by Alexa 555 anti - mouse antibodies . After COPI depletion many GFP - LC3 - positive AVs are positive for LAMP2 . Asterisk indicates enlarged in inset .

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GFP-LC3 AVs are LAMP2 positive. (A) 293/GFP-LC3 cells were incubated with control or β′- or α-COP siRNA for 48 h, fixed and labeled with anti-LAMP2 antibodies, followed by Alexa 555 anti-mouse antibodies. After COPI depletion many GFP-LC3-positive AVs are positive for LAMP2. Asterisk indicates cell enlarged in inset.


( B ) Cryosections of β ′ - COP - depleted labeled with anti - GFP ( left ) or anti - GFP anti - LAMP2 ( right ) followed by A - gold as indicated . AV , autophagosomes , M , mitochondria .

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(B) Cryosections of β′-COP-depleted cells labeled with anti-GFP (left) or anti-GFP and anti-LAMP2 (right) followed by protein A-gold as indicated. AV, autophagosomes, M, mitochondria.


GFP - LC3 - positive AVs colocalize TGN46 early endocytotic markers after COPI depletion . ( A ) 293 / GFP - LC3 were incubated with siRNA for β ′ - COP or control siRNA ( Cont ) for 48 h , processed for immunofluorescence , labeled with antibodies for TGN46 .

GFP-LC3-positive AVs colocalize TGN46 and early endocytotic markers after COPI depletion. (A) 293/GFP-LC3 cells were incubated with siRNA for β′-COP or control siRNA (Cont) for 48 h, processed for immunofluorescence, and labeled with antibodies for TGN46.


( B ) siRNA - treated were fixed labeled , or starved for 2 h ( St ) , labeled with antibodies to EEA1 . (

(B) siRNA-treated cells were fixed and labeled, or starved for 2 h (St), and labeled with antibodies to EEA1. (


( C ) Control - starved HEK293 were labeled for endogenous LC3 EEA1 by double labeling after methanol fixation .

(C) Control-starved HEK293 cells were labeled for endogenous LC3 and EEA1 by double labeling after methanol fixation.


( D ) After siRNA treatment , rhodamine - labeled dextran was internalized for 20 min , followed by a 40 - min chase after which 293 / GFP - LC3 were fixed analyzed by confocal microscopy .

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(D) After siRNA treatment, rhodamine-labeled dextran was internalized for 20 min, followed by a 40-min chase after which the 293/GFP-LC3 cells were fixed and analyzed by confocal microscopy.


Transferrin EGF trafficking is inhibited by COPI depletion . 293 / GFP - LC3 were treated with β ′ - COP siRNA or control siRNA . After 48 h were incubated with Alexa 555 transferrin ( A ) for 2 min , ( B ) 30 min , or ( C ) 30 min followed by a 30 - min chase , then washed fixed analyzed by confocal microscopy .

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Transferrin and EGF trafficking is inhibited by COPI depletion. 293/GFP-LC3 cells were treated with β′-COP siRNA or control siRNA. After 48 h cells were incubated with Alexa 555 transferrin (A) for 2 min, (B) 30 min, or (C) 30 min followed by a 30-min chase, then washed and fixed and analyzed by confocal microscopy.


( D E ) 125I - EGF was added to HeLa after 48 h of siRNA depletion of β ′ - COP , or control siRNA for 10 min . were then washed as described in Materials methods , chased for 30 , 60 , 120 , 180 min ( including 10 - min incubation ) . ( D ) amount of 125I - EGF internalized was shown as a of total 125I - EGF taken up in 10 min .

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(D and E) 125I-EGF was added to HeLa cells after 48 h of siRNA depletion of β′-COP, or control siRNA for 10 min. The cells were then washed as described in the Materials and methods, and chased for 30, 60, 120, and 180 min (including 10-min incubation). (D) The amount of 125I-EGF internalized was shown as a percentage of the total 125I-EGF taken up in 10 min.


( E ) amount of degraded 125I - EGF was determined as a of total radioactivity present after TCA precipitation over total 125I - EGF internalized . experiment was performed three times in duplicate data shown is mean ± . significance was determined by Student ' s t . * , P ≤ 0 . 05 .

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(E) The amount of degraded 125I-EGF was determined as a percentage of the total radioactivity present after TCA precipitation over the total 125I-EGF internalized. The experiment was performed three times in duplicate and the data shown is the mean ± SEM. The significance was determined by Student's t test. *, P ≤ 0.05.


B . Western blot of dose - dependent expression of ZBP1 in HT29 / TO - ZBP1WT HT29 / TO - ZBP1Zα1α2mut treated with indicated concentrations of Dox for 24 h . Blots are representative of three biological repeats .

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B. Western blot analysis of the dose-dependent expression of ZBP1 in HT29 / TO-ZBP1WT and HT29 / TO-ZBP1Zα1α2mut cells treated with indicated concentrations of Dox for 24 h. Blots are representative of three biological repeats.


I . Western blot of from same wells as used in ( H ) for ZBP1 expression , representative of four biological replicates .

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I. Western blot analysis of cells from the same wells as used in (H) for ZBP1 expression levels, representative of four biological replicates.


L . Transwell migration of neutrophils towards conditioned media from HT29 / TO - ZBP1WT treated with DMSO or 500 ng / mL Dox for 24 hours , or HT29 chemotaxis buffer ( control buffer ) containing equal volume amount of DMSO or Dox as conditioned media . Data is presented as values with grand mean of migrated induced by control buffer containing DMSO or Dox ( n = 10 biological replicates ) , or conditioned media from DMSO - treated ( n = 11 biological replicates ) or Dox - treated ( n = 12 biological replicates ) , where number of biological replicates is defined by total number of independent cultures tested on neutrophils from four donors . An unpaired t - was used to for statistical differences between indicated conditions . * * * * p < 0 . 0001 .

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L. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with DMSO or 500 ng/mL Dox for 24 hours, or HT29 chemotaxis buffer (control buffer) containing equal volume and amount of DMSO or Dox as the conditioned media. Data is presented as individual values with grand mean of the migrated percentage induced by control buffer containing DMSO or Dox (n = 10 biological replicates), or conditioned media from DMSO-treated cells (n = 11 biological replicates) or Dox-treated cells (n = 12 biological replicates), where the number of biological replicates is defined by the total number of independent cell cultures tested on primary neutrophils from four donors. An unpaired t-test was used to test for statistical differences between indicated conditions. **** p < 0.0001.


M . Transwell migration of neutrophils towards conditioned media from HT29 / TO - ZBP1WT treated with 0 or 500 ng / mL Dox in combination with DMSO or 20 µM zVAD + 10 μM Nec1s for 24 h . Data is presented as values with mean S . E . M . ( n = 3 biological replicates of conditioned media ) . One - way ANOVA Sidak ' s multiple comparisons were used to for statistical differences between indicated conditions . ns = not significant ( p = 0 . 4129 ) ; * * * p = 0 . 0004 .

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M. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox in combination with DMSO or 20 µM zVAD + 10 μM Nec1s for 24 h. Data is presented as individual values with mean and S.E.M. (n = 3 biological replicates of conditioned media). One-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p = 0.4129); *** p = 0.0004.


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

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

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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 in culture media of HT29 / TO - ZBP1WT or HT29 / RIPK1 - KO clone aA3 / TO - ZBP1WT treated with 0 , 50 or 500 ng / mL Dox . data is plotted as mean with S . E . M . ( n = 6 biological replicates ) . Brown - Forsythe Welch ANOVA Dunnet ' s T3 multiple comparisons were used to for statistical differences between indicated conditions . lysates were analyzed by Western blotting for ZBP1 . 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 .

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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 with without stable expression of full - ( FL ) RIPK3 - 2xFV or RIPK3 ∆ C - 2xFV ( ∆ C ) transfected with dual luciferase reporters 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 Sidak ' s multiple comparisons were used to for statistical differences between indicated conditions . ns = not significant ( p > 0 . 05 ) ; * * * * p < 0 . 0001 . ( G ) lysates were analyzed by Western blotting . Blots are representative of three biological replicates . Asterisk indicates background signals of antibody .

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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 of anti - FLAG ( ZBP1 ) immunoprecipitation from HT29 / TO - ZBP1WT 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 1 % for co - immunoprecipitants . Asterisk indicates antibody heavy chain signal in IP samples .

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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 of ubiquitination status of RIPK1 , ZBP1 RIPK3 in HT29 / TO - ZBP1WT 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 .

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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 of ubiquitination status of RIPK1 ZBP1 using GST - 1xUBA or linkage - specific SUBs in HT29 / TO - ZBP1WT treated with 0 or 500 ng / mL Dox for 16 h .

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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 of ubiquitination status of RIPK1 ZBP1 in HT29 , HT29 / TO - ZBP1WT HT29 / RIPK1 - KO aA3 / TO - ZBP1WT treated with 500 ng / mL Dox for 16 h . Blots are representative of four biological replicates . Asterisk indicates antibody background signal .

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

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

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

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

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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 of anti - FLAG ( ZBP1 ) immunoprecipitation from HT29 or HT29 / TO - ZBP1WT or HT29 / RIPK1 - KO clone aA3 / TO - ZBP1WT 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 ( E - F ) three biological replicates .

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

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

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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 of CXCL8 in HCT116 / RIPK3 - 2xFV 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 Sidak ' s multiple comparisons were used to for statistical differences between indicated conditions DMSO + dimerizer - treated condition . * * * * p < 0 . 0001 . ( H ) lysates were analyzed by Western blotting for cIAP1 . Blots are representative of two biological replicates .

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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 in WT or HOIP - knockout HCT116 / RIPK3 - 2xFV 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 - was used to for statistical difference as indicated . * * * p = 0 . 0010 . lysates were loaded for Western blot .

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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 in HCT116 / Tet - On - GFP - K63 - SUB / RIPK3 - 2xFV , HCT116 / Tet - On - GFP - M1 - SUB / RIPK3 - 2xFV HCT116 / Tet - On - GFP / RIPK3 - 2xFV 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 Welch ANOVA Dunnet ' s T3 multiple comparisons were used to for statistical differences between indicated conditions . ns = not significant ( p = 0 . 9997 ) ; * * p = 0 . 0030 ; * p = 0 . 0366 . were analyzed by Western blotting for inducible - expression of GFP ( control ) GFP - SUBs . Blots are representative of three biological replicates .

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

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 in HCT116 / RIPK3 - 2xFV stably knocked down against mismatch ( shMM ) or two different sites of caspase - 8 ( shCASP8 - A 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 Tukey ' s multiple comparison were used to for statistical differences between indicated conditions . * * * p = 0 . 0001 ; * * p = 0 . 0035 . lysates from one biological replicate were analyzed by Western blotting to determine caspase - 8 .

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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 of ZBP1 , RIPK1 , RIPK3 , MLKL in Calu - 3 lines with stable expression of shRNAs targeting ZBP1 or a mismatch ( 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 Sec63 during nitrogen starvation . expressing GFP - tagged Ykt6 under control of GAL1 promoter tdTomato - tagged Sec63 were transformed with a centromeric plasmid expressing BFP - Ape1 . were grown in SGC - LEU , shifted to SG - N for indicated time , analyzed by microscopy . are shown as single planes . Scale bar , 5 µm . Arrows indicate Ykt6 dots colocalizing with Ape1 . ( B ) of Ykt6 simultaneously co - localizing with both Ape1 Sec63 . data were quantified from ( A ) . Error bars represent standard deviation of three independent experiments . ( n ≥ 250 ) 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 during nitrogen starvation . expressing GFP - tagged Ykt6 under control of GAL1 promoter 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 , analyzed by microscopy . Single imaging planes are shown . Scale bar , 5 µm . Arrows indicate Ykt6 dots that colocalize with marker .

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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 respective mutant expressing mCherry - tagged Ape1 GFP - tagged Ykt6 were grown in SGC to allow for Ykt6 expression from GAL1 - promoter analyzed Arrows in 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 ) of colocalization of Ykt6 relative to Ape1 as a PAS marker . ( n ≥ 250 ) 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 . expressing GFP - tagged Ykt6 mScarlet - tagged Atg9 were grown analyzed left is of colocalization of Ykt6 relative to Atg9 . ( n ≥ 200 ) Atg9 dots ( n ≥ 80 ) were quantified . Arrows in 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 isolation during starvation . ( D ) expressing mCherry - tagged Atg8 GFP - Ykt6 carrying Ape1 encoding plasmid pRS315 - CUP1pr - BFP - APE1 were grown in SGC medium containing CuSO4 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 . expressing mCherry - tagged Atg8 GFP - Ykt6 carrying plasmid pRS315 - CUP1pr - BFP - APE1 , , were analyzed by Imaris . Scale bar , 5 µm . Arrows in 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 ) only partial time points ( 90 s to 600 s ) of were assembled , analyzed by Imaris image J . Scale bar , 5 µm . 500 - 1000 were analyzed in each independent experiment . 3D stacks were cropped by Imaris 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 dsl1 ts mutants on autophagosome - vacuole fusion . carrying a CEN plasmid expressing GFP - Atg8 were grown at 24 ° C in SGC then shifted to SG - N for 2 h at 24 ° C or 37 ° C . Vacuoles were stained by FM4 - 64 analyzed by microscopy . Scale bar , 5 µm . Scale bar of inset is 0 . 6 µm .

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

(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 of ykt6 - 11 dsl3 mutants . Lysates of atg1 ∆ , ypt7 ∆ , ykt6ts dsl3ts carrying a CEN plasmid expressing GFP - Atg8 were grown at 37 ° C subjected to 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 . carrying a CEN plasmid expressing GFP - Atg8 encoding 3xmCherry - tagged Atg1 were grown at 24 ° C in SDC then shifted to SD - N for 2 h at 24 ° C or 37 ° C , analyzed by microscopy . slices are shown . Scale bar , 5 µm . Scale bar of inset is 0 . 6 µm . Arrows in 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 nitrogen starvation conditions . expressing GFP - tagged Ykt6 under control of PHO5 promoter genomically 3xmCherry - tagged Dsl1 were grown in SDC or SD - N for 2 h , analyzed by microscopy . slices are shown . Scale bar , 5 µm . ( F ) of Ykt6 co - localizing with Dsl1 . data was quantified from ( E ) . Error bars represent standard deviation of three independent experiments . ( n ≥ 250 ) Dsl1 dots ( n ≥ 50 ) were quantified . Arrows in 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 nitrogen starvation . expressing GFP - tagged Dsl3 mCherry - tagged Atg8 were grown in SDC or SD - N for 2 h , analyzed by 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 COPII ts mutants on Ykt6 localization to autophagosomes . Wild - type selected ts strains ( sec12 - 1 , tip20 - 5 dsl3 - 2 ) expressing GFP - tagged Ykt6 mCherry - tagged Atg8 were grown at 24 ° C in SGC for 8 h then shifted to SG - N for 2 h at 37 ° C . Vacuoles were stained by CMAC analyzed by microscopy . Scale bar , 5 µm . Scale bar in inset , 0 . 7 µm . Arrows indicate dot colocalization of Ykt6 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 selected ts strains ( sec12 - 1 , tip20 - 5 dsl3 - 2 ) expressing GFP - tagged Ykt6 mCherry - tagged Atg8 were grown at 24 ° C in SGC for 8 h then shifted to SG - N for 2 h at 37 ° C . ( B ) of Ykt6 co - localizing with Atg8 . ( n ≥ 150 ) 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 Sec13 during growth nitrogen starvation conditions . tip20 - 5 expressing GFP - tagged Ykt6 3xmCherry - tagged Sec63 or Sec13 were grown at 24 ° C in SGC for 8 h then shifted to SG - N for 2 h at 24 ° C or 37 ° C , analyzed by microscopy . Scale bar , 5 µm . Scale bar of inset , 0 . 7 µm . Arrows indicate dot colocalization of Ykt6 with Sec63 ( C ) .

(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 . vam3 ∆ or dsl ts expressing Atg9 - 3xFLAG GFP - Atg8 were starved for 3 h at 30 ° C before purifying autophagosomes , which was sufficient to induce ts phenotype . Vacuoles were isolated from pep4 ∆ expressing Vac8 - 3xmCherry then incubated with autophagosomes at 26 ° C for 1 h with or without ATP ( E ) of ( D ) . Error bars represent standard deviation of 3 independent experiments . of GFP - Atg8 in vacuolar lumen was quantified by image J using 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 ) of number of autophagosomes in indicated ts mutant strains . Error bars represent standard deviation of 3 independent experiments . GFP - tagged Atg8 in different mutants were grown at 23 ° C ( ts mutant ) or 30 ° C ( atg1 ∆ ) in SDC then shifted to SD - N for 2 h at 30 ° C ( atg1 ∆ ) or 37 ° C ( ts mutant ) , analyzed by 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 expressing mCherry - tagged Atg9 carrying a CEN plasmid expressing GFP - Atg8 were grown at 23 ° C in SDC then shifted to SD - N for 2 h at 24 ° C or 37 ° C . were analyzed by microscopy . Scale bar , 5 µm . Scale bar for inset is 0 . 6 µm .

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


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

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


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

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


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

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


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

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


( A ) Growth of Ykt6 wild - type mutants . ykt6 ∆ carrying a URA3 plasmid coding for wild - type Ykt6 pRS413 - GAL1pr - YKT6 - GFP ( wild - type or mutant ) were spotted as serial dilutions on SGC - HIS to select for mutant plasmid or 5 - Fluoroorotic acid ( 5 - FOA ) to force loss of 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 ∆ 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 ] ) , a plasmid expressing GFP - Atg8 were grown in SGC then shifted to SG - N for 2 h . were analyzed by microscopy . slices are shown . Scale bar , 5 µm . ( C ) of Atg8 dots per from in ( B ) . ( n ≥ 100 ) Atg8 dots ( n ≥ 50 ) were quantified .

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


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

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


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


average of decreased after lag . 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 in microfluidic experiment divided after a lag time that decreased with increasing glucose frequency . labeled times indicate period , time between pulses , for a given

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


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

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


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 , hypoxanthine for pulsing at non - division supporting frequencies of 0 . 06 , 0 . 12 , 0 . 18 mmol / g / h . TI feedrate is abbreviated as f ( units : mmol glucose / g dry / hour ) . Glucose pulses are indicated by grey bars , pink region shows a no control . Dots are ion measurements . Solid lines are a moving average filter of measured ion . For clarity , dots are not shown for f = 0 mmol / g / h condition A metabolic scheme describing propagation of fed glucose . Pulsed glucose is hypothesized to pass through central carbon metabolism then be converted to downstream pathways including acid synthesis nucleic acid synthesis . For nucleic acid synthesis , glucose is converted to intermediate PRPP , which then can combine with nitrogen bases to form nucleotides for nucleic acid synthesis . Different pathways can be blocked with

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


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

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


of labeled threonine deoxyribose from hydrolysate shows de novo synthesis in non - dividing . After 6 hours of pulsing uniformly labeled 13C - glucose , cultures were lysed , their macromolecules were washed free of latent metabolites hydrolyzed to monomers . Labeling data are presented as mean ± standard error of independent biological replicates ( n = 3 , 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 presence of protease inhibitor ( PI ) that reduced lag time for a given TI feedrate ( f = 0 . 28 mmol / g / h ) . TI feedrate is abbreviated as f ( units : mmol glucose / g dry / hour ) . Wild - type lag ( from Figure 2A empirical fit ) is indicated by dotted grey

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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 amount of doxycycline ( max - 50 ng / uL , half - 10 ng / uL , zero 0 ng / uL ) added at onset of starvation . TI feedrate is abbreviated as f ( units : mmol glucose / g dry / hour ) . Wild - type lag ( from Figure 2A empirical fit ) is indicated by dotted grey . Lag time reduced with synthesis of titrated FtsZ in wild - type at f = 0 . 31 - 0 . 32 mmol / g / h . Lag time increased with titrated synthesis of ClpX in wild - type at f = 0 . 43 - 0 . 45 mmol / g /

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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

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

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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).