Specificity of the C - terminal Tap - 1 segment . Killing assay in which the lack of tap - 1 in V52 is complemented by empty vector ( control ) or one of two different alleles of tap - 1 . The ability to kill in a TseL - dependent manner is indicated by the logarithm of surviving C6706ΔtsiV1 .

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

Specificity of the C-terminal Tap-1 segment. Killing assay in which the lack of tap-1 in V52 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to kill in a TseL-dependent manner is indicated by the logarithm of surviving C6706ΔtsiV1.


Specificity of N - terminal segment of Tap - 1 . Killing assay in which the lack of tap - 1 in 1587 is complemented by empty vector ( control ) or one of two different alleles of tap - 1 . The ability to facilitate killing by the effector A55 _ 1502 is indicated by the logarithm of surviving 1587ΔA55 _ 1501 - 03 .

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

Specificity of N-terminal segment of Tap-1. Killing assay in which the lack of tap-1 in 1587 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to facilitate killing by the effector A55_1502 is indicated by the logarithm of surviving 1587ΔA55_1501-03.


Representative western - blots of the cortical ( Ctx ) or hippocampal ( Hip ) extracts from mice at the indicated time after seizure activity in pilocarpine ( A , B ) models . ( C , F ) Statistics of Gli1 or Shh expression levels shown in A , B or D , E respectively . n = 8 - 14 mice in C and n = 8 - 23 mice in F .

O O 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

Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in pilocarpine (A,B) models. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F.


Representative western - blots of the cortical ( Ctx ) or hippocampal ( Hip ) extracts from mice at the indicated time after seizure activity in kindling ( D , E ) models . For D and E , samples obtained from mice evoked with a single kindling stimulation to induce seizure activity as evidenced in EEG . ( C , F ) Statistics of Gli1 or Shh expression levels shown in A , B or D , E respectively . n = 8 - 14 mice in C and n = 8 - 23 mice in F .

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

Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in kindling (D,E) models. For D and E, samples obtained from mice evoked with a single kindling stimulation to induce seizure activity as evidenced in EEG. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F.


( G ) Shh levels assayed by ELISA from mouse cortex and hippocampus at the indicated time after the initiation of status epilepticus ( SE ) induced by pilocarpine ( n = 7 - 10 ) .

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(G) Shh levels assayed by ELISA from mouse cortex and hippocampus at the indicated time after the initiation of status epilepticus (SE) induced by pilocarpine (n=7-10).


( H , I ) Shh levels assayed by ELISA in the medium of slices ( H , n = 9 ) or hippocampal neurons ( I , n = 6 ) incubated with picrotoxin ( Pic ) or Mg2 + - free ( 0Mg ) for the indicated times .

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

(H,I) Shh levels assayed by ELISA in the medium of slices (H, n=9) or hippocampal neurons (I, n=6) incubated with picrotoxin (Pic) or Mg2+-free (0Mg) for the indicated times.


( J ) Representative western - blots and statistics of Gli1 expression levels from hippocampal neurons incubated with 0Mg for the indicated times ( n = 13 - 19 ) . a - tubulin ( a - Tub ) , a loading control . Data were mean + SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Control ( Ctrl ) with student ' s t test .

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(J) Representative western-blots and statistics of Gli1 expression levels from hippocampal neurons incubated with 0Mg for the indicated times (n=13-19). a-tubulin (a-Tub), a loading control. Data were mean + SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Control (Ctrl) with student's t test.


( A - C ) Whole - cell recordings of spontaneous epileptiform activity from cultured hippocampal neurons . A , representative traces showing the neuronal activity under the indicated treatments . Ctrl : extracellular solution . Cyclo : cyclopamine . B , the expanded view of a single burst ( arrow ) from A ; C , statistics of the percentage of neurons showing epileptiform activity ( left , n = 6 - 7 ) and the burst frequency ( right , n = 18 - 19 ) shown in A . Cells were incubated in the presence of Cyclo in extracellular solution for 30 min , then transferred to 0Mg plus Cyclo ( 0Mg + Cyclo ) and incubated for another 30 min .

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

(A-C) Whole-cell recordings of spontaneous epileptiform activity from cultured hippocampal neurons. A, representative traces showing the neuronal activity under the indicated treatments. Ctrl: extracellular solution. Cyclo: cyclopamine. B, the expanded view of a single burst (arrow) from A; C, statistics of the percentage of neurons showing epileptiform activity (left, n=6-7) and the burst frequency (right, n=18-19) shown in A. Cells were incubated in the presence of Cyclo in extracellular solution for 30 min, then transferred to 0Mg plus Cyclo (0Mg+Cyclo) and incubated for another 30 min.


( D , E ) The effects of Sant - 1 , robotnikinin ( Robot ) or 5E1 on the percentage of neurons showing epileptiform activity ( D , n = 6 - 10 ) and the burst frequency ( E , n = 15 - 22 ) . ( F - H ) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices . F , representative traces under the indicated treatments . Ctrl : artificial cerebrospinal fluid ( aCSF ) .

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

(D,E) The effects of Sant-1, robotnikinin (Robot) or 5E1 on the percentage of neurons showing epileptiform activity (D, n=6-10) and the burst frequency (E, n=15-22). (F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF).


( F - H ) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices . F , representative traces under the indicated treatments . Ctrl : artificial cerebrospinal fluid ( aCSF ) . G , the expanded view of a single burst ( arrow ) in F . Slices were incubated in the presence of vehicle or Cyclo in aCSF for 30 min , then transferred to picrotoxin in aCSF ( Pic ) or with Cyclo ( Pic + Cyclo ) and recorded for 30 min . H , statistics of the spontaneous burst frequency of F from 10 slices ( 7 rats ) .

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

(F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF). G, the expanded view of a single burst (arrow) in F. Slices were incubated in the presence of vehicle or Cyclo in aCSF for 30 min, then transferred to picrotoxin in aCSF (Pic) or with Cyclo (Pic+Cyclo) and recorded for 30 min. H, statistics of the spontaneous burst frequency of F from 10 slices (7 rats).


( I , J ) Left are representative traces showing the D - CPP - sensitive currents recorded from cultured hippocampal neurons ( I ) or slices ( J ) incubated in 0Mg or Pic , respectively , with or without Cyclo . Black lines : application of D - CPP . Right are statistics of D - CPP - sensitive current density from 18 - 26 cells ( I ) or 16 - 18 slices ( 15 rats ) ( J ) . Unless stated , 10 µM cyclopamine was used . Data were mean + SEM . * p < 0 . 05 , * * < 0 . 01 , * * * p < 0 . 001 vs . Ctrl ; # p < 0 . 05 , # # < 0 . 01 , # # # p < 0 . 001 vs . 0Mg or Pic ; & amp ; amp ; p < 0 . 05 vs . 0Mg + IgG . C - E used one - way ANOVA . H - J used student ' s t test .

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

(I,J) Left are representative traces showing the D-CPP-sensitive currents recorded from cultured hippocampal neurons (I) or slices (J) incubated in 0Mg or Pic, respectively, with or without Cyclo. Black lines: application of D-CPP. Right are statistics of D-CPP-sensitive current density from 18-26 cells (I) or 16-18 slices (15 rats) (J). Unless stated, 10 µM cyclopamine was used. Data were mean + SEM. *p<0.05,**<0.01,***p<0.001 vs. Ctrl; #p<0.05,##<0.01,###p<0.001 vs. 0Mg or Pic; &amp;p<0.05 vs. 0Mg + IgG. C-E used one-way ANOVA. H-J used student's t test.


( A ) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents . n = 4 - 6 .

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

(A) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents. n=4-6.


( B ) The paired - pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle ( Ctrl ) or Shh ( 13 - 14 slices , 6 rats ) . Insets : representative traces recorded in response to paired pulse stimuli with different intervals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The paired-pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle (Ctrl) or Shh (13-14 slices, 6 rats). Insets: representative traces recorded in response to paired pulse stimuli with different intervals.


Statistics of 3H - glutamate uptake by neurons treated with indicated agents ( C , n = 10 )

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Statistics of 3H-glutamate uptake by neurons treated with indicated agents (C, n=10)


Statistics of 3H - glutamate uptake by neurons transfected with two lentivirus - based RNAi against EAAC1 ( R2 and R3 ) or nonsense RNAi ( Non ) in response to vehicle ( Ctrl ) or Shh ( D , n = 3 - 6 ) . No difference between R2 or R3 in Ctrl and those in Shh . Inset of ( D ) : representative western - blots for EAAC1 .

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

Statistics of 3H-glutamate uptake by neurons transfected with two lentivirus-based RNAi against EAAC1 (R2 and R3) or nonsense RNAi (Non) in response to vehicle (Ctrl) or Shh (D, n=3-6). No difference between R2 or R3 in Ctrl and those in Shh. Inset of (D): representative western-blots for EAAC1.


Aspartate ( Asp ) - evoked currents at - 70 mV from hippocampal neurons transfected with RNAi or Non ( E , n = 17 - 23 ) or treated with indicated agents ( F , n = 16 - 18 ) .

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Aspartate (Asp)-evoked currents at -70 mV from hippocampal neurons transfected with RNAi or Non (E, n=17-23) or treated with indicated agents (F, n=16-18).


( G ) Upper , representative western - blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors ( Ctrl ) , constitutively active form of Smoothened ( SmoA1 ) , EAAC1 , or EAAC1 plus SmoA1 . Lower , 3H - glutamate uptake by cells transfected with the indicated vectors . n = 9 .

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(G) Upper, representative western-blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors (Ctrl), constitutively active form of Smoothened (SmoA1), EAAC1, or EAAC1 plus SmoA1. Lower, 3H-glutamate uptake by cells transfected with the indicated vectors. n=9.


( H , I ) Shh effects on 3H - glutamate uptake ( H , n = 6 ) or Asp - evoked currents ( I , n = 17 - 23 ) of neurons with or without pertussis toxin ( PTX ) pretreatment . For E , F , I , the uppers : representative traces . Shh : 500 ng / mL . Data were mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Ctrl or Non ( Ctrl ) with student ' s t test .

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

(H,I) Shh effects on 3H-glutamate uptake (H, n=6) or Asp-evoked currents (I, n=17-23) of neurons with or without pertussis toxin (PTX) pretreatment. For E,F,I, the uppers: representative traces. Shh: 500 ng/mL. Data were mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl or Non (Ctrl) with student's t test.


Effects of Cyclo ( A - C , n = 14 ) on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In A - F , Ctrl are vehicle - treated mice .

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Effects of Cyclo (A-C, n=14) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice.


Effects of 5E1 ( D - F , n = 16 - 20 ) , on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In A - F , Ctrl are vehicle - treated mice .

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

Effects of 5E1 (D-F, n=16-20), on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice.


Effects of ablation of Smo in CaMKIIα - positive neurons ( G - I ) , on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In G - I , Ctrl : Smofl / fl induced by tamoxifen ; CaMK : Smofl / flCaMKIIα - CreERT2 induced by tamoxifen , n = 14 - 20 .

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

Effects of ablation of Smo in CaMKIIα-positive neurons (G-I), on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In G-I, Ctrl: Smofl/fl induced by tamoxifen; CaMK: Smofl/flCaMKIIα-CreERT2 induced by tamoxifen, n=14-20.


Effects of ablation of Smo in Aldh1l1 - positive astrocytes ( J - L ) on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In J - L , Ctrl : Smo + / fl ; Aldh1l1 : Smo + / flAldh1l1 - Cre , n = 19 - 20 .

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

Effects of ablation of Smo in Aldh1l1-positive astrocytes (J-L) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In J-L, Ctrl: Smo+/fl; Aldh1l1: Smo+/flAldh1l1-Cre, n=19-20.


( M ) Frequency of spontaneous seizures ( class 4 - 5 ) in mice administrated with Cyclo or vehicle ( Ctrl ) at 4th , 6th and 8th week after pilocarpine SE induction . n = 22 - 23 .

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(M) Frequency of spontaneous seizures (class 4-5) in mice administrated with Cyclo or vehicle (Ctrl) at 4th, 6th and 8th week after pilocarpine SE induction. n=22-23.


( N ) Percentage of mice with class 5 seizures within 8 weeks after pilocarpine SE induction . n = 22 - 23 . Cyclo : 10 mg / kg . 5E1 : 900 ng / mouse . Data were mean ± SEM from at least three independent experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Ctrl with student ' s t test .

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(N) Percentage of mice with class 5 seizures within 8 weeks after pilocarpine SE induction. n=22-23. Cyclo: 10 mg/kg. 5E1: 900 ng/mouse. Data were mean ± SEM from at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl with student's t test.


( B ) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit . For quantification , TAG levels were normalized to protein . Error bars represent ± SEM . * * * : P < 0 . 001 ; NS : non - significant .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O 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) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. Error bars represent ± SEM. ***: P < 0.001; NS: non-significant.


( G ) Quantification of the lipid droplet size in ( C and F ) . ND : normal diet ; HFD : high - fat diet . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison test . Each point represents data from one fat body , and at least 30 cells were analyzed for each fat body . Error bars represent ± SEM . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

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(G) Quantification of the lipid droplet size in (C and F). ND: normal diet; HFD: high-fat diet. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were analyzed for each fat body. Error bars represent ±SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


( B 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets and DAPI ( blue ) for nuclei . Scale bars represent 25 µm . ( B ) RDH / CG2064 RNAi suppresses the enlarged lipid droplet phenotype in CSN2 RNAi under DGAT1 overexpression ( ppl > DGAT1 ) or high - fat diet conditions . ( C ) Quantification of the lipid droplet size in ( B ) . HFD : high - fat diet . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison test . Each point represents data from one fat body , and at least 30 cells were examined in each fat body . Error bars represent ± SEM . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (B) RDH/CG2064 RNAi suppresses the enlarged lipid droplet phenotype in CSN2 RNAi under DGAT1 overexpression (ppl>DGAT1) or high-fat diet conditions. (C) Quantification of the lipid droplet size in (B). HFD: high-fat diet. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


, F 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets and DAPI ( blue ) for nuclei . Scale bars represent 25 µm . ( F ) RDH / CG2064 overexpression increases lipid droplet size in ppl > DGAT1 larvae . ( G ) Quantification of the lipid droplet size in ( F ) . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison test . Each point represents data from one fat body , and at least 30 cells were examined in each fat body . Error bars represent ± SEM . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (F) RDH/CG2064 overexpression increases lipid droplet size in ppl>DGAT1 larvae. (G) Quantification of the lipid droplet size in (F). data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets and DAPI ( blue ) for nuclei . Scale bars represent 25 µm . ( H ) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit . For quantification , TAG levels were normalized to protein . ( I ) RDH / CG2064 and CG2065 deletion does not affect lipid droplet size under normal or high - fat diet conditions . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison test . Each point represents data from one fat body , and at least 30 cells were examined in each fat body . Error bars represent ± SEM . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

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

3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (H) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. (I) RDH/CG2064 and CG2065 deletion does not affect lipid droplet size under normal or high-fat diet conditions. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


( B ) 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets and DAPI ( blue ) for nuclei . Overexpressing wild - type or site - mutated RDH / CG2064 increases the lipid droplet size in larvae with DGAT1 overexpression ( ppl > DGAT1 ) . Scale bar represents 25 µm .

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(B) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Overexpressing wild-type or site-mutated RDH/CG2064 increases the lipid droplet size in larvae with DGAT1 overexpression (ppl>DGAT1). Scale bar represents 25 µm.


( E ) Immunofluorescence imaging of RDH / CG2064 - GFP , detected with anti - GFP antibody , and the ER protein marker RFP - KDEL in 3rd instar larval fat body . RDH / CG2064 co - localizes with RFP - KDEL . Scale bar represents 10 µm .

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(E) Immunofluorescence imaging of RDH/CG2064-GFP, detected with anti-GFP antibody, and the ER protein marker RFP-KDEL in 3rd instar larval fat body. RDH/CG2064 co-localizes with RFP-KDEL. Scale bar represents 10 µm.


Lysates of 3rd instar larval fat body samples of different genetic backgrounds were immunoprecipitated with anti - Flag antibody and then probed by anti - GFP or anti - Flag antibody in a western blot . Plin2 , but not Plin1 or Bmm , is specifically coimmunoprecipitated with RDH / CG2064 ( A ) .

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

Lysates of 3rd instar larval fat body samples of different genetic backgrounds were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Plin2, but not Plin1 or Bmm, is specifically coimmunoprecipitated with RDH/CG2064 (A).


( E ) Lysates of Drosophila S2 cells expressing full - length Plin1 or Plin1 with a C - terminal deletion ( Plin1ΔC ) were immunoprecipitated with anti - Flag antibody and then probed by anti - GFP or anti - Flag antibody in a western blot . Plin1ΔC coimmunoprecipitated with RDH / CG2064 .

(E) Lysates of Drosophila S2 cells expressing full-length Plin1 or Plin1 with a C-terminal deletion (Plin1ΔC) were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Plin1ΔC coimmunoprecipitated with RDH/CG2064.


( F ) 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets and DAPI ( blue ) for nuclei . Under high - fat conditions , RDH / CG2064 overexpression increases the lipid droplet size , and Plin2 RNAi suppresses the enlargement induced by RDH / CG2064 overexpression . Scale bar represents 25 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Under high-fat conditions, RDH/CG2064 overexpression increases the lipid droplet size, and Plin2 RNAi suppresses the enlargement induced by RDH/CG2064 overexpression. Scale bar represents 25 µm.


( A , B Images of Plin2 - GFP in 3rd instar larval fat body cells of different genotypes . Scale bar represents 25 µm . ( A ) CSN2 RNAi or RDH / CG2064 overexpression reduces the peripheral localization of Plin2 - GFP and increases the localization of Plin2 - GFP on small peri - nuclear lipid droplets ( boxed ) . DAPI ( blue ) stains nuclei . White arrows indicate peripheral lipid droplet . Red arrows indicate medial lipid droplets . ( B ) The peripheral localization of Plin2 - GFP is greatly reduced in fat body cells from larvae with CSN2 RNAi or RDH / CG2064 overexpression . Images were taken by focusing on the periphery of fat cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Images of Plin2-GFP in 3rd instar larval fat body cells of different genotypes. Scale bar represents 25 µm. (A) CSN2 RNAi or RDH/CG2064 overexpression reduces the peripheral localization of Plin2-GFP and increases the localization of Plin2-GFP on small peri-nuclear lipid droplets (boxed). DAPI (blue) stains nuclei. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets. (B) The peripheral localization of Plin2-GFP is greatly reduced in fat body cells from larvae with CSN2 RNAi or RDH/CG2064 overexpression. Images were taken by focusing on the periphery of fat cells.


C , Images of Plin2 - GFP in 3rd instar larval fat body cells of different genotypes . Scale bar represents 25 µm . ( C ) In fat body cells from ppl > DGAT1 larvae with CSN2 RNAi or RDH / CG2064 overexpression , the localization of Plin2 - GFP is greatly reduced at the periphery and is dramatically increased on large medial lipid droplets . White arrows indicate peripheral lipid droplet . Red arrows indicate medial lipid droplets . ( D ) Fluorescence intensity line plots of the images in ( C ) . GFP intensity along the line across a lipid droplet was measured by ImageJ . In ppl > DGAT1 larvae with CSN2 RNAi or RDH / CG2064 overexpression , Plin2 - GFP localization on large medial lipid droplets is represented by two peaks . More than 30 lipid droplets were measured for each genotype . The plot shown is a typical image curve for the indicated genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Images of Plin2-GFP in 3rd instar larval fat body cells of different genotypes. Scale bar represents 25 µm. (C) In fat body cells from ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression, the localization of Plin2-GFP is greatly reduced at the periphery and is dramatically increased on large medial lipid droplets. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets. (D) Fluorescence intensity line plots of the images in (C). GFP intensity along the line across a lipid droplet was measured by ImageJ. In ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression, Plin2-GFP localization on large medial lipid droplets is represented by two peaks. More than 30 lipid droplets were measured for each genotype. The plot shown is a typical image curve for the indicated genotype.


( G ) The peripheral lipid droplets in larval fat body cells from different genetic backgrounds were stained by BODIPY ( green ) . CSN3 RNAi or RDH / CG2064 overexpression reduces the number of peripheral lipid droplets in both wild - type and ppl > DGAT1 larvae .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 peripheral lipid droplets in larval fat body cells from different genetic backgrounds were stained by BODIPY (green). CSN3 RNAi or RDH/CG2064 overexpression reduces the number of peripheral lipid droplets in both wild-type and ppl>DGAT1 larvae.


( A , Confocal images of Bmm - GFP ( green ) in 3rd instar larval fat bodies of different genotypes . DAPI ( blue ) stains nuclei . Scale bar represents 25 µm . ( A ) Knocking down CSN2 or overexpressing RDH / CG2064 in animals with DGAT1 overexpression ( ppl > DGAT1 ) decreases the level and lipid droplet localization of Bmm - GFP . ( B ) The GFP intensity along the line across a lipid droplet in ( A ) was measured by ImageJ . The lipid droplet localization of Bmm - GFP , represented by two peaks , is clearly visible in fat cells from ppl > DGAT1 larvae , but it is lost in fat cells from ppl > DGAT1 larvae with CSN2 RNAi or overexpression of RDH / CG2064 . More than 30 lipid droplets of each genotype were measured . One typical image curve is shown for each genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Confocal images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (A) Knocking down CSN2 or overexpressing RDH/CG2064 in animals with DGAT1 overexpression (ppl>DGAT1) decreases the level and lipid droplet localization of Bmm-GFP. (B) The GFP intensity along the line across a lipid droplet in (A) was measured by ImageJ. The lipid droplet localization of Bmm-GFP, represented by two peaks, is clearly visible in fat cells from ppl>DGAT1 larvae, but it is lost in fat cells from ppl>DGAT1 larvae with CSN2 RNAi or overexpression of RDH/CG2064. More than 30 lipid droplets of each genotype were measured. One typical image curve is shown for each genotype.


F ) Confocal images of Bmm - GFP ( green ) in 3rd instar larval fat bodies of different genotypes . DAPI ( blue ) stains nuclei . Scale bar represents 25 µm . ( F ) Overexpression of Plin2 decreases the level and lipid droplet localization of Bmm - GFP in fat cells from larvae with DGAT1 overexpression . ( G ) GFP intensity along the line across a lipid droplet from ( F ) was measured by ImageJ . The lipid droplet localization of Bmm - GFP , represented by two peaks , was evident in fat cells from ppl > DGAT1 larvae , but is lost in ppl > DGAT1 larvae with overexpression of Plin2 . More than 30 lipid droplets of each genotype were measured . One typical image curve is shown for each genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (F) Overexpression of Plin2 decreases the level and lipid droplet localization of Bmm-GFP in fat cells from larvae with DGAT1 overexpression. (G) GFP intensity along the line across a lipid droplet from (F) was measured by ImageJ. The lipid droplet localization of Bmm-GFP, represented by two peaks, was evident in fat cells from ppl>DGAT1 larvae, but is lost in ppl>DGAT1 larvae with overexpression of Plin2. More than 30 lipid droplets of each genotype were measured. One typical image curve is shown for each genotype.


( H ) 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets and DAPI ( blue ) for nuclei . bmm RNAi increases the lipid droplet size in fat cells from larvae with DGAT1 overexpression . Scale bar represents 25 µm .

O O O O O O O O O O O O O O O O O O O O O O O O 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) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. bmm RNAi increases the lipid droplet size in fat cells from larvae with DGAT1 overexpression. Scale bar represents 25 µm.


( A ) S2 cells expressing RDH / CH2064 - GFP were treated with the protein synthesis inhibitor cycloheximide ( CHX ) for the indicated time ( h : hours ) . The level of RDH / CH2064 - GFP was determined by western blotting using anti - GFP antibody . Tubulin was used as loading control . ( B ) Quantification of the relative RDH / CH2064 - GFP protein levels in ( A ) . Error bars represent ± SEM .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) S2 cells expressing RDH/CH2064-GFP were treated with the protein synthesis inhibitor cycloheximide (CHX) for the indicated time (h: hours). The level of RDH/CH2064-GFP was determined by western blotting using anti-GFP antibody. Tubulin was used as loading control. (B) Quantification of the relative RDH/CH2064-GFP protein levels in (A). Error bars represent ± SEM.


( C ) S2 cells expressing Bmm - GFP were treated with the protein synthesis inhibitor cycloheximide ( CHX ) for the indicated time ( h : hours ) . The level of Bmm - GFP was determined by western blotting using anti - GFP antibody . Tubulin was used as loading control . ( D ) Quantification of the relative Bmm - GFP protein levels in ( C ) . Error bars represent ± SEM .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) S2 cells expressing Bmm-GFP were treated with the protein synthesis inhibitor cycloheximide (CHX) for the indicated time (h: hours). The level of Bmm-GFP was determined by western blotting using anti-GFP antibody. Tubulin was used as loading control. (D) Quantification of the relative Bmm-GFP protein levels in (C). Error bars represent ± SEM.


( F ) Oil red staining of 3rd instar larval oenocytes in different genetic backgrounds under fed or starvation conditions .

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

(F) Oil red staining of 3rd instar larval oenocytes in different genetic backgrounds under fed or starvation conditions.


G - I ) Experimental validation of collective survival . All populations grew to high cell densities in the absence of antibiotics ( Blue curves ) . In the presence of antibiotics , only populations with initial densities greater than NCT grew . NCT was increased with the concentration of antibiotics . Engineered bacteria with the QS - BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS - CAT circuits . Carbenicillin was used for the BlaM and QS - BlaM circuits ; chloramphenicol for the QS - CAT circuit . Each error bar represents the standard deviation from triplicate measurements .

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

G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.


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


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


( G - I ) Experimental validation of collective survival . All populations grew to high cell densities in the absence of antibiotics ( Blue curves ) . In the presence of antibiotics , only populations with initial densities greater than NCT grew . NCT was increased with the concentration of antibiotics . Engineered bacteria with the QS - BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS - CAT circuits . Carbenicillin was used for the BlaM and QS - BlaM circuits ; chloramphenicol for the QS - CAT circuit . Each error bar represents the standard deviation from triplicate measurements .

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

(G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.


Configuration of the two - compartment model . The microenvironment of microbial swarmbot is separated from that of the chamber by its permeable hydrogel shell ( orange ) , resulting in two separate compartments , defined as swarmbot and environment ( chamber ) . Four major components involved in the population dynamics are explored in the model , including cell density ( n ) , nutrient ( s ) , carbenicillin ( a ) and BlaM concentration ( b ) . Each component has a different transport rate , depending on the molecule size . Simulated microbial swarmbot dynamics under a static condition . a = 0 . 4 , a microbial swarmbot with a low n0 cannot initiate growth ( red lines ) but one with a high n0 leads to growth both in the swarmbot and , after a delay , in the chamber ( green lines ) . While ( C ) swarmbots with both low and high initial densities lead to eventual full growth in the chamber in the absence of the antibiotic ( a = 0 ) . Solid lines : cell density inside swarmbots ; dashed lines : cell density in the chambers . ( D ) A microfluidic device for controlling and monitoring population dynamics of microbial swarmbot . The device consists of an input channel leading to a culturing chamber and a trapping slot . The chamber is used for culturing engineered bacterial populations both inside and outside the swarmbot , while the trapping slot is for trapping the swarmbot due to its smaller size but allowing the medium to flow through . Input flow patterns ( static or pulsing ) are controlled by a programmable syringe pump . ( E ) Experimental validation of model prediction . At 100µg / ml carbenicillin , a microbial swarmbot with a high initial density led to robust growth in the capsule and , after a delay , in the chamber ( green lines ) . While a swarmbot with a low initial cell density did not exhibit growth either in the swarmbot or in the chamber at the same carbenicillin concentration ( red lines ) . ( F ) In contrast , both the swarmbot and culturing chamber reached high cell densities in absence of carbenicillin , regardless of the initial cell densities .

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

Configuration of the two-compartment model. The microenvironment of microbial swarmbot is separated from that of the chamber by its permeable hydrogel shell (orange), resulting in two separate compartments, defined as swarmbot and environment (chamber). Four major components involved in the population dynamics are explored in the model, including cell density (n), nutrient (s), carbenicillin (a) and BlaM concentration (b). Each component has a different transport rate, depending on the molecule size.Simulated microbial swarmbot dynamics under a static condition. a = 0.4, a microbial swarmbot with a low n0 cannot initiate growth (red lines) but one with a high n0 leads to growth both in the swarmbot and, after a delay, in the chamber (green lines). While (C) swarmbots with both low and high initial densities lead to eventual full growth in the chamber in the absence of the antibiotic (a = 0). Solid lines: cell density inside swarmbots; dashed lines: cell density in the chambers.(D) A microfluidic device for controlling and monitoring population dynamics of microbial swarmbot. The device consists of an input channel leading to a culturing chamber and a trapping slot. The chamber is used for culturing engineered bacterial populations both inside and outside the swarmbot, while the trapping slot is for trapping the swarmbot due to its smaller size but allowing the medium to flow through. Input flow patterns (static or pulsing) are controlled by a programmable syringe pump.(E) Experimental validation of model prediction. At 100µg/ml carbenicillin, a microbial swarmbot with a high initial density led to robust growth in the capsule and, after a delay, in the chamber (green lines). While a swarmbot with a low initial cell density did not exhibit growth either in the swarmbot or in the chamber at the same carbenicillin concentration (red lines). (F) In contrast, both the swarmbot and culturing chamber reached high cell densities in absence of carbenicillin, regardless of the initial cell densities.


( G ) Time - lapse images of microbial swarmbot dynamics . The population initiated growth inside the swarmbot and subsequently in the chamber at a high initial density . But no growth occurred for a swarmbot with a low initial density at the same antibiotic concentration ( rightmost ) . The concentration of carbenicillin was 100 µg / ml and the scale bar represents 200 µm . ( H ) The control strain lacking BlaM circuit . The population could not survive at the same concentration of carbenicillin at the high initial density .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time-lapse images of microbial swarmbot dynamics. The population initiated growth inside the swarmbot and subsequently in the chamber at a high initial density. But no growth occurred for a swarmbot with a low initial density at the same antibiotic concentration (rightmost). The concentration of carbenicillin was 100 µg/ml and the scale bar represents 200 µm.(H) The control strain lacking BlaM circuit. The population could not survive at the same concentration of carbenicillin at the high initial density.


Time courses of swarmbots treated with varying concentrations of carbenicillin and glucose . Solid lines indicate cell densities inside swarmbots ; dotted lines indicate those in the chamber . Safeguard was established for lower nutrient levels ( 0 . 004 % or 0 . 04 % glucose ) along with carbenicillin treatment . At a high nutrient level ( 0 . 4 % glucose ) , the safeguard was lost due to either growth in the chamber ( panels 3 and 6 ) or gradual killing of the population inside swarmbot ( solid line in panel 9 ) . These results are qualitatively consistent with the model predictions ( Fig 4 ) .

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 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

Time courses of swarmbots treated with varying concentrations of carbenicillin and glucose. Solid lines indicate cell densities inside swarmbots; dotted lines indicate those in the chamber. Safeguard was established for lower nutrient levels (0.004% or 0.04% glucose) along with carbenicillin treatment. At a high nutrient level (0.4% glucose), the safeguard was lost due to either growth in the chamber (panels 3 and 6) or gradual killing of the population inside swarmbot (solid line in panel 9). These results are qualitatively consistent with the model predictions (Fig 4).


Snapshots of safeguard performance at the end time point of ( A ) . The red arrow in panel 6 indicates significant growth in the chamber ; the dimmer fluorescence of the swarmbot in panel 9 indicates killing of the population .

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

Snapshots of safeguard performance at the end time point of (A). The red arrow in panel 6 indicates significant growth in the chamber; the dimmer fluorescence of the swarmbot in panel 9 indicates killing of the population.


Figure 6 . Experimental control of microbial swarmbot safeguard by other circuits . Demonstration of modular safeguard performance of swarmbots with different collective survival systems . For the swarmbots containing cells carrying the QS - CAT circuit , pulsing flow of medium containing 100µg / ml chloramphenicol established safeguard control but not for the static condition . For the swarmbots with the QS - BlaM circuit , the static condition of 100 µg / ml carbenicillin was sufficient for safeguard control . For all the conditions , safeguard did not occur in the absence of antibiotic ( top panels from each condition ) . All the images were from the swarmbots cultured at 37 ˚ C for 16 hours . The scale bar is 250 µm .

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

Figure 6. Experimental control of microbial swarmbot safeguard by other circuits.Demonstration of modular safeguard performance of swarmbots with different collective survival systems. For the swarmbots containing cells carrying the QS-CAT circuit, pulsing flow of medium containing 100µg/ml chloramphenicol established safeguard control but not for the static condition. For the swarmbots with the QS-BlaM circuit, the static condition of 100 µg/ml carbenicillin was sufficient for safeguard control. For all the conditions, safeguard did not occur in the absence of antibiotic (top panels from each condition). All the images were from the swarmbots cultured at 37˚C for 16 hours. The scale bar is 250 µm.


( A ) Transfer of K18 - ATTO 594 fibrils from donor cells to H2B - GFP - expressing acceptor cells . Representative confocal images of each population are in the upper panels , below are pictures after 24 hours of co - culture of the two populations ( left ) , and of acceptor cells treated with conditioned medium from donor cells for 24 hours ( SN , right ) . In white is the cell membrane labelling with WGA ( wheat germ agglutinin ) coupled to Alexa 647 . The arrows point to acceptor cells containing fibrils , scale bars are 10 μm .

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(A) Transfer of K18-ATTO 594 fibrils from donor cells to H2B-GFP-expressing acceptor cells. Representative confocal images of each population are in the upper panels, below are pictures after 24 hours of co-culture of the two populations (left), and of acceptor cells treated with conditioned medium from donor cells for 24 hours (SN, right). In white is the cell membrane labelling with WGA (wheat germ agglutinin) coupled to Alexa 647. The arrows point to acceptor cells containing fibrils, scale bars are 10 μm.


( B ) Quantification by flow cytometry of the percentage of K18 - ATTO 594 - positive acceptor cells after co - culturing donor and acceptor cells ( total ) , or culturing acceptor cells with donor - conditioned medium for 24 hours ( secretion ) . The total transfer is arbitrarily set at 100 % , and cell - cell contact transfer is calculated by subtracting secretion transfer from total transfer . Data represent the means ( + SD ) of 4 independent experiments , with statistical analysis by two - tailed unpaired t - test ( * * * * P = 4 . 64E - 08 ) .

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(B) Quantification by flow cytometry of the percentage of K18-ATTO 594-positive acceptor cells after co-culturing donor and acceptor cells (total), or culturing acceptor cells with donor-conditioned medium for 24 hours (secretion). The total transfer is arbitrarily set at 100%, and cell-cell contact transfer is calculated by subtracting secretion transfer from total transfer. Data represent the means (+SD) of 4 independent experiments, with statistical analysis by two-tailed unpaired t-test (****P = 4.64E-08).


( C ) Quantification by flow cytometry of the relative percentage of K18 - ATTO 594 - positive acceptor cells upon treatment with CK666 during the co - culture . Left , data represent the means ( 132 % ) + SD , normalized to non - treated co - culture arbitrarily set at 100 % , of 3 independent experiments , with statistical analysis by two - tailed unpaired t - test ( * P = 0 . 015 ) . The hatched area of each bar represents the part due to secretion ( respectively 4 . 8 and 4 . 3 % in the absence and presence of CK666 ) . Right is the same analysis when the cells were cultured in sparse conditions , not allowing cell - to - cell contacts ( mean = 47 . 7 % , 3 independent experiments , * P = 0 . 020 ) .

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 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

(C) Quantification by flow cytometry of the relative percentage of K18-ATTO 594-positive acceptor cells upon treatment with CK666 during the co-culture. Left, data represent the means (132%) +SD, normalized to non-treated co-culture arbitrarily set at 100%, of 3 independent experiments, with statistical analysis by two-tailed unpaired t-test (*P = 0.015). The hatched area of each bar represents the part due to secretion (respectively 4.8 and 4.3% in the absence and presence of CK666). Right is the same analysis when the cells were cultured in sparse conditions, not allowing cell-to-cell contacts (mean= 47.7%, 3 independent experiments, *P = 0.020).


( D ) Representative confocal images ( 40x ) of CAD cells treated with 1 µM K18 - ATTO 594 fibrils , 24h after fibrils addition , fixed and stained with WGA - Alexa 488 ( green ) and DAPI ( blue in the merge panels ) . Bottom panels are a bottom slice corresponding to the substrate - attached surface of cells ( z3 ) , upper panels correspond to slice 10 of the same picture , ( z10 ) , not attached to the substrate ) . On the right and below z10 pictures , are the orthogonal views ( xz and yz ) of the same region covering 27 slices over 11 μm in total . The arrows point to red fibrils into a WGA - positive TNT . Scale bars are 10 μm .

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(D) Representative confocal images (40x) of CAD cells treated with 1 µM K18-ATTO 594 fibrils, 24h after fibrils addition, fixed and stained with WGA-Alexa 488 (green) and DAPI (blue in the merge panels). Bottom panels are a bottom slice corresponding to the substrate-attached surface of cells (z3), upper panels correspond to slice 10 of the same picture, (z10), not attached to the substrate). On the right and below z10 pictures, are the orthogonal views (xz and yz) of the same region covering 27 slices over 11 μm in total. The arrows point to red fibrils into a WGA-positive TNT. Scale bars are 10 μm.


( A ) Representative confocal images of DS1 cells ( expressing soluble Tau RD - YFP ) , non - treated and treated with increasing concentrations of non - labelled K18 fibrils , fixed after two days and stained with WGA - Alexa 594 ( red ) . The arrows point to examples of aggregate - containing cells , scale bars are 40 μm . On the right is the quantification of the mean percentage + SEM of cells where green aggregates were detected ( the total number of cells analyzed over 3 independent experiments were 1188 for non - treated , 1552 for 0 . 3 μM , and 1506 for 1 μM ) , with statistical analysis by one - way ANOVA and Bonferroni post hoc test ( * * * * P = 6 . 20E - 06 and 6 . 74E - 05 for 0 to 0 . 3 and 0 to 1 comparisons respectively ) .

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(A) Representative confocal images of DS1 cells (expressing soluble Tau RD-YFP), non-treated and treated with increasing concentrations of non-labelled K18 fibrils, fixed after two days and stained with WGA-Alexa 594 (red). The arrows point to examples of aggregate-containing cells, scale bars are 40 μm. On the right is the quantification of the mean percentage +SEM of cells where green aggregates were detected (the total number of cells analyzed over 3 independent experiments were 1188 for non-treated, 1552 for 0.3 μM, and 1506 for 1 μM), with statistical analysis by one-way ANOVA and Bonferroni post hoc test (****P = 6.20E-06 and 6.74E-05 for 0 to 0.3 and 0 to 1 comparisons respectively).


( B ) Representative confocal images ( 40x ) of RD - YFP SH cells , non - treated and treated with 1 μM K18 - ATTO 594 fibrils , 48h after fibrils addition . Right insets are threefold enlargements of the boxed region of treated cells , merge and single channel pictures ; white arrows point to partial colocalization . Scale bars are 10 μm , 2 μm in the insets . On the right is the quantification of the mean percentage ( 24 % ) + SD of cells where green aggregates were detected ( 2874 treated cells analyzed over 6 independent experiments ) , with statistical analysis by two - tailed unpaired t - test ( * * * * P = 3 . 75E - 07 ) .

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(B) Representative confocal images (40x) of RD-YFP SH cells, non-treated and treated with 1 μM K18-ATTO 594 fibrils, 48h after fibrils addition. Right insets are threefold enlargements of the boxed region of treated cells, merge and single channel pictures; white arrows point to partial colocalization. Scale bars are 10 μm, 2 μm in the insets. On the right is the quantification of the mean percentage (24%) +SD of cells where green aggregates were detected (2874 treated cells analyzed over 6 independent experiments), with statistical analysis by two-tailed unpaired t-test (****P = 3.75E-07).


( C ) Relative proportion of DS9 cells expressing aggregates of Tau RD - YFP over a 50 - hour period monitored by IncuCyte . DS9 cells were left untreated , the graph represents means of 3 independent experiments ± SD .

(C) Relative proportion of DS9 cells expressing aggregates of Tau RD-YFP over a 50-hour period monitored by IncuCyte. DS9 cells were left untreated, the graph represents means of 3 independent experiments ±SD.


( D ) Monitoring the conversion of DS1 cells into inclusion - containing cells over a 70 - hour period upon treatment with K18 fibrils . Cells were plated , treated or not with K18 - ATTO 594 fibrils at 1 μM ( time 0 ) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change . The scatter plot represents means of 5 independent experiments ( ± SD for green and black curves ) .

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(D) Monitoring the conversion of DS1 cells into inclusion-containing cells over a 70-hour period upon treatment with K18 fibrils. Cells were plated, treated or not with K18-ATTO 594 fibrils at 1 μM (time 0) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change. The scatter plot represents means of 5 independent experiments (± SD for green and black curves).


Cells were plated , treated or not with K18 - ATTO 594 fibrils at 1 μM ( time 0 ) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change . ( E ) Monitoring the conversion of RD - YFP - expressing SH - SY5Y cells into inclusion - containing cells over a 70 - hour period upon treatment with K18 fibrils . the scatter plot shows means of 3 independent experiments ± SD for the green and black curves .

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Cells were plated, treated or not with K18-ATTO 594 fibrils at 1 μM (time 0) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change. (E) Monitoring the conversion of RD-YFP-expressing SH-SY5Y cells into inclusion-containing cells over a 70-hour period upon treatment with K18 fibrils. the scatter plot shows means of 3 independent experiments ±SD for the green and black curves.


Cells were plated , treated or not with K18 - ATTO 594 fibrils at 1 μM ( time 0 ) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change . ( F ) Monitoring the conversion of RD - YFP - expressing SH - SY5Y cells into inclusion - containing cells over a 70 - hour period upon treatment with a cortex crude extracts from a AD - deceased patient . , the scatter plot shows means of 2 independent experiments ± SD for the green curve . IncuCyte ( 20x objective ) was set to acquire images every 30 minutes , phase - contrast and green channel ( Excitation 440 - 480 nm , 400 ms ) were acquired ( 9 images / well ) . Analysis was performed with IncuCyte software to give the relative proportion of cells being converted to inclusion - containing cells ( the end point of treated cells was arbitrarily set at 100 % , black curve : non - treated cells , green curve : treated cells ) .

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Cells were plated, treated or not with K18-ATTO 594 fibrils at 1 μM (time 0) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change. (F) Monitoring the conversion of RD-YFP-expressing SH-SY5Y cells into inclusion-containing cells over a 70-hour period upon treatment with a cortex crude extracts from a AD-deceased patient. , the scatter plot shows means of 2 independent experiments ±SD for the green curve. IncuCyte (20x objective) was set to acquire images every 30 minutes, phase-contrast and green channel (Excitation 440-480 nm, 400 ms) were acquired (9 images/well). Analysis was performed with IncuCyte software to give the relative proportion of cells being converted to inclusion-containing cells (the end point of treated cells was arbitrarily set at 100%, black curve: non-treated cells, green curve: treated cells).


( A ) RD - YFP SH cells were challenged with non - labelled K18 fibrils , incubated 2 days later with Bafilomycin A1 ( second line of panels ) , Bortezomide ( bottom panels ) or left non - treated ( NT , upper panels ) for 4 hours before fixation , saponin - permeabilization and staining with antibody recognizing p62 ( red ) and WGA ( white in left panels ) . Images are confocal pictures after deconvolution ( 63x , zoom 1 . 8 , px size = 60 nm , slice of 0 . 43 μm ) representative of three independent experiments . Insets are threefold enlargements of the boxed regions of second column ; white arrows point to co - labelling ; scale bars are 10 μm , 2 μm in insets .

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(A) RD-YFP SH cells were challenged with non-labelled K18 fibrils, incubated 2 days later with Bafilomycin A1 (second line of panels), Bortezomide (bottom panels) or left non-treated (NT, upper panels) for 4 hours before fixation, saponin-permeabilization and staining with antibody recognizing p62 (red) and WGA (white in left panels). Images are confocal pictures after deconvolution (63x, zoom 1.8, px size = 60 nm, slice of 0.43 μm) representative of three independent experiments. Insets are threefold enlargements of the boxed regions of second column; white arrows point to co-labelling; scale bars are 10 μm, 2 μm in insets.


( B ) Quantification of colocalization of RD - YFP material with p62 - positive structures ( left graph ) or LAMP1 - positive vesicles ( right ) upon K18 fibril - induced aggregation . Confocal pictures were analyzed in 3D with Imaris software from pictures obtained as in A , the graphs represent the mean percentage ( + SEM ) of green material overlapping with p62 or LAMP1 : aggregates if cells have been converted ( agg ) or soluble material otherwise . Below each graph , the corresponding Pearson correlation coefficient ( PCC ) is indicated . The number of cells analyzed over 2 independent experiments is 40 , 51 , 63 , 34 for each respective condition for p62 , 36 , 59 , 44 , 27 for LAMP1 .

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(B) Quantification of colocalization of RD-YFP material with p62-positive structures (left graph) or LAMP1-positive vesicles (right) upon K18 fibril-induced aggregation. Confocal pictures were analyzed in 3D with Imaris software from pictures obtained as in A, the graphs represent the mean percentage (+ SEM) of green material overlapping with p62 or LAMP1: aggregates if cells have been converted (agg) or soluble material otherwise. Below each graph, the corresponding Pearson correlation coefficient (PCC) is indicated. The number of cells analyzed over 2 independent experiments is 40, 51, 63, 34 for each respective condition for p62, 36, 59, 44, 27 for LAMP1.


( A ) RD - YFP SH cells were challenged with non - labelled K18 fibrils , incubated 2 days later with Bafilomycin A1 ( second line of panels ) , Bortezomide ( bottom panels ) or left non - treated ( NT , upper panels ) for 4 hours before fixation , saponin - permeabilization and staining with antibody and WGA ( white in left panels ) . Images are confocal pictures after deconvolution ( 63x , zoom 1 . 8 , px size = 60 nm , slice of 0 . 43 μm ) representative of three independent experiments . Insets are threefold enlargements of the boxed regions of second column ; white arrows point to co - labelling ; scale bars are 10 μm , 2 μm in insets . anti - LAMP1 antibody was

(A) RD-YFP SH cells were challenged with non-labelled K18 fibrils, incubated 2 days later with Bafilomycin A1 (second line of panels), Bortezomide (bottom panels) or left non-treated (NT, upper panels) for 4 hours before fixation, saponin-permeabilization and staining with antibody and WGA (white in left panels). Images are confocal pictures after deconvolution (63x, zoom 1.8, px size = 60 nm, slice of 0.43 μm) representative of three independent experiments. Insets are threefold enlargements of the boxed regions of second column; white arrows point to co-labelling; scale bars are 10 μm, 2 μm in insets. anti-LAMP1 antibody was used


( D ) RD - YFP SH cells were challenged with AD - derived brain extracts , trypsinized and replated 3 days later for an additional 24h before fixation , saponin - permeabilization and staining with antibody recognizing p62 or LAMP1 ( in red ) . Images are confocal pictures ( 63x , zoom 1 . 6 , px size = 60 nm , slice of 0 . 43 μm ) representative of two independent experiments . Insets are threefold enlargements of the boxed regions ; white arrows point to co - labelling ; scale bars are 10 μm , 2 μm in the insets .

(D) RD-YFP SH cells were challenged with AD-derived brain extracts, trypsinized and replated 3 days later for an additional 24h before fixation, saponin-permeabilization and staining with antibody recognizing p62 or LAMP1 (in red). Images are confocal pictures (63x, zoom 1.6, px size = 60 nm, slice of 0.43 μm) representative of two independent experiments. Insets are threefold enlargements of the boxed regions; white arrows point to co-labelling; scale bars are 10 μm, 2 μm in the insets.


( E ) Quantification of colocalization of RD - YFP material with p62 - positive structures ( left graph ) or LAMP1 - positive vesicles ( right ) upon AD extract - induced aggregation . Confocal pictures were analyzed in 3D with Imaris software from pictures obtained as in D , the graphs represent the mean percentage ( + SEM ) of green material overlapping with p62 or LAMP1 : aggregates if cells have been converted ( agg ) or soluble material otherwise . Below each graph , the corresponding Pearson correlation coefficient ( PCC ) is indicated . The number of cells analyzed over 2 independent experiments is 21 ( sol ) , 27 ( agg ) for p62 , 12 ( sol ) , 17 ( agg ) for LAMP1 .

(E) Quantification of colocalization of RD-YFP material with p62-positive structures (left graph) or LAMP1-positive vesicles (right) upon AD extract-induced aggregation. Confocal pictures were analyzed in 3D with Imaris software from pictures obtained as in D, the graphs represent the mean percentage (+ SEM) of green material overlapping with p62 or LAMP1: aggregates if cells have been converted (agg) or soluble material otherwise. Below each graph, the corresponding Pearson correlation coefficient (PCC) is indicated. The number of cells analyzed over 2 independent experiments is 21 (sol), 27 (agg) for p62, 12 (sol), 17 (agg) for LAMP1.


( F ) Confocal pictures of RD - YFP cells , treated with Bortezomide phalloidin - Texas red was incubated together with the secondary Alexa 647 antibody . Upper panels are the bottom slice corresponding to the substrate - attached surface of cells ( z1 ) , below are projections of slices 6 to 9 of the same pictures ( z6 - z9 , not attached to the substrate ) . Below is a two - fold magnification of the area framed in the merged projection , showing a TNT . White arrows point to overlapping between RD - YFP aggregates and p62 , and arrowheads to aggregates not colocalizing with p62 inside the TNT ; scale bars are 10 μm , 5 μm in the insets . Bottom is a 3D reconstruction ( using Imaris software ) of the same region .

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(F) Confocal pictures of RD-YFP cells, treated with Bortezomide phalloidin-Texas red was incubated together with the secondary Alexa 647 antibody. Upper panels are the bottom slice corresponding to the substrate-attached surface of cells (z1), below are projections of slices 6 to 9 of the same pictures (z6-z9, not attached to the substrate). Below is a two-fold magnification of the area framed in the merged projection, showing a TNT. White arrows point to overlapping between RD-YFP aggregates and p62, and arrowheads to aggregates not colocalizing with p62 inside the TNT; scale bars are 10 μm, 5 μm in the insets. Bottom is a 3D reconstruction (using Imaris software) of the same region.


( A ) Below the schematic representation of the experiment are representative confocal images of two - day co - cultures of RD - YFP SH cells ( acceptor cells ) with K18 - ATTO 594 fibrils - treated SH - SY5Y cells ( donor cells , upper panel ) . Bottom panels are acceptor cells cultured with the supernatant of K18 - ATTO 594 fibrils - treated SH - SY5Y cells ( donor SN ) . WGA ( white ) labels cell membrane and DAPI the nuclei in the merge panels . Arrows point to cells with green and red aggregates , scale bars are 20 μm . ( B ) Quantification of seeding after transfer in RD - YFP SH cells in the experiments described in ( A ) , depending on the condition ( donor cells or donor SN ) . Analysis was performed using ICY software , data represent the number of aggregate - containing cells over the total number of green cells ± SD ( the total number of RD - YFP cells analyzed over 3 independent experiments were 1011 for co - culture and 1265 for SN ) with statistical analysis by two - tailed unpaired t - test ( * * * * P = 4 . 18E - 07 ) .

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(A) Below the schematic representation of the experiment are representative confocal images of two-day co-cultures of RD-YFP SH cells (acceptor cells) with K18-ATTO 594 fibrils-treated SH-SY5Y cells (donor cells, upper panel). Bottom panels are acceptor cells cultured with the supernatant of K18-ATTO 594 fibrils-treated SH-SY5Y cells (donor SN). WGA (white) labels cell membrane and DAPI the nuclei in the merge panels. Arrows point to cells with green and red aggregates, scale bars are 20 μm. (B) Quantification of seeding after transfer in RD-YFP SH cells in the experiments described in (A), depending on the condition (donor cells or donor SN). Analysis was performed using ICY software, data represent the number of aggregate-containing cells over the total number of green cells ±SD (the total number of RD-YFP cells analyzed over 3 independent experiments were 1011 for co-culture and 1265 for SN) with statistical analysis by two-tailed unpaired t-test (****P =4.18E-07).


( C ) Below the schematic representation of the experiment are representative confocal images showing maximal intensity projections of 6 z - slices ( covering 2 μm of thickness ) of 24 - hour co - culture of RD - YFP SH cells ( acceptor cells ) with RD - YFP SH cells expressing nls - Red and challenged with K18 - ATTO 594 fibrils ( donor cells ) . WGA ( white ) labels cell membrane and DAPI the nuclei in the merge panel . Stars label donor cells containing green and red aggregates , arrows indicate an acceptor cell with green and red aggregates , and the arrowheads show an acceptor cell with green aggregates but devoid of red fibrils . Below are two - fold enlargement of the respective framed cells . The apparent discontinuity in the picture corresponds to the boundary between two adjacent tiles . Scale bars are 10 μm , 5 μm in the enlargements .

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(C) Below the schematic representation of the experiment are representative confocal images showing maximal intensity projections of 6 z-slices (covering 2 μm of thickness) of 24-hour co-culture of RD-YFP SH cells (acceptor cells) with RD-YFP SH cells expressing nls-Red and challenged with K18-ATTO 594 fibrils (donor cells). WGA (white) labels cell membrane and DAPI the nuclei in the merge panel. Stars label donor cells containing green and red aggregates, arrows indicate an acceptor cell with green and red aggregates, and the arrowheads show an acceptor cell with green aggregates but devoid of red fibrils. Below are two-fold enlargement of the respective framed cells. The apparent discontinuity in the picture corresponds to the boundary between two adjacent tiles. Scale bars are 10 μm, 5 μm in the enlargements.


( D ) Analysis using ICY software of the percentage of cells containing red fibrils among the population of cells containing green aggregates . The graph shows the mean percentages with SEM , respectively 66 . 3 % and 36 . 9 % for donor and acceptor cells , statistical analysis was performed by two - tailed unpaired t - test ( * * * * p = 3 . 65E - 05 ) .

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(D) Analysis using ICY software of the percentage of cells containing red fibrils among the population of cells containing green aggregates. The graph shows the mean percentages with SEM, respectively 66.3% and 36.9% for donor and acceptor cells, statistical analysis was performed by two-tailed unpaired t-test (**** p =3.65E-05).


( E ) Dot plot showing the number of red fibrils per cell with SEM among the population of cells containing green and red aggregates . Each analyzed cell is represented ( note that because of the chosen scale , 6 data points are outside the axis limits ) , the bars indicate the means ± SEM ( respectively 3 . 9 and 1 . 9 for donor and acceptor cells ) , statistical analysis was performed by two - tailed unpaired t - test ( * * p = 0 . 0015 ) .

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(E) Dot plot showing the number of red fibrils per cell with SEM among the population of cells containing green and red aggregates. Each analyzed cell is represented (note that because of the chosen scale, 6 data points are outside the axis limits), the bars indicate the means ± SEM (respectively 3.9 and 1.9 for donor and acceptor cells), statistical analysis was performed by two-tailed unpaired t-test (** p=0.0015).


( F ) Analysis of the number of green dots per cell among the population of cells containing green aggregates . Each analyzed cell is represented on the dot plot , the bars indicate the means ± SEM ( respectively 30 . 2 , 26 . 1 , 32 . 0 , and 32 . 2 ) . Statistical analysis was performed by one - way ANOVA and Tukey post - hoc test and all the pair - wise comparisons were not significant ( n . s . ) .

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(F) Analysis of the number of green dots per cell among the population of cells containing green aggregates. Each analyzed cell is represented on the dot plot, the bars indicate the means ± SEM (respectively 30.2, 26.1, 32.0, and 32.2). Statistical analysis was performed by one-way ANOVA and Tukey post-hoc test and all the pair-wise comparisons were not significant (n.s.).


( A ) Below the schematics of the experiment are representative confocal images of donor neuron - like cells ( CAD cells labelled with Cell Tracker Green ( CTG ) ) and acceptor primary neurons after 24 hours in co - culture . The upper panels show control conditions ( non - challenged CAD cells , CTL ) , and the bottom panels show donor CTG - CADs that were loaded with K18 - ATTO 594 fibrils prior to co - culture with primary cortical neurons . The images are representative Z - stack projections covering the whole cell body of donor and acceptor cells . In the merged images , white show neurons labelled with MAP - 2 , green are CTG - labelled donor CAD cells , red corresponds to K18 - ATTO 594 puncta and nuclei are stained in blue . The single channel pictures are gray scale images . Yellow arrowheads point to K18 puncta inside acceptor cells . MAP - 2 and K18 - ATTO 594 panels are threefold enlargements of the boxed regions in the merged pictures . On the right are the orthogonal views of the same regions covering 14 slices . Scale bars are 20 μm in the merge panels , and 5 μm in the insets and orthogonal views .

(A) Below the schematics of the experiment are representative confocal images of donor neuron-like cells (CAD cells labelled with Cell Tracker Green (CTG)) and acceptor primary neurons after 24 hours in co-culture. The upper panels show control conditions (non-challenged CAD cells, CTL), and the bottom panels show donor CTG-CADs that were loaded with K18-ATTO 594 fibrils prior to co-culture with primary cortical neurons. The images are representative Z-stack projections covering the whole cell body of donor and acceptor cells. In the merged images, white show neurons labelled with MAP-2, green are CTG-labelled donor CAD cells, red corresponds to K18-ATTO 594 puncta and nuclei are stained in blue. The single channel pictures are gray scale images. Yellow arrowheads point to K18 puncta inside acceptor cells. MAP-2 and K18-ATTO 594 panels are threefold enlargements of the boxed regions in the merged pictures. On the right are the orthogonal views of the same regions covering 14 slices. Scale bars are 20 μm in the merge panels, and 5 μm in the insets and orthogonal views.


( B ) Below the schematics of the experiment are representative confocal images of acceptor primary neurons ( labelled by MAP - 2 ) cultured with donor cells , consisting in RD - YFP SH cells , first K18 - challenged for 2 days , therefore expressing RD - YFP aggregates ( upper panels ) or with supernatant ( SN ) of donor cells ( lower panels ) after 24 hours . The single channel pictures are gray scale images . Yellow arrowheads point to Tau RD - YFP puncta inside acceptor cells , red arrowheads point to aggregates inside donor cells . Scale bars are 20 μm .

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(B) Below the schematics of the experiment are representative confocal images of acceptor primary neurons (labelled by MAP-2) cultured with donor cells, consisting in RD-YFP SH cells, first K18-challenged for 2 days, therefore expressing RD-YFP aggregates (upper panels) or with supernatant (SN) of donor cells (lower panels) after 24 hours. The single channel pictures are gray scale images. Yellow arrowheads point to Tau RD-YFP puncta inside acceptor cells, red arrowheads point to aggregates inside donor cells. Scale bars are 20 μm.


( C ) Below the schematics of the co - culture experiment are representative confocal images of neuron - like cells ( CTG - CADs growing on top of organotypic slices ( n = 6 ) , in which the astrocytes were labelled with GFAP . The upper panels show control CTG - CADs and the middle panels show donor CTG - CADs loaded with K18 - ATTO 594 fibrils , co - cultured with an acceptor hippocampal slice . The images are representative Z - stack projections covering the whole cell body of donor cells . In the merged images , white are acceptor astrocytes ( GFAP positive ) , green are donor CTG - CADs , red are K18 - ATTO 594 puncta and nuclei are stained in blue . Insets are threefold enlargements of the boxed region in the merged picture . The orthogonal views of the bottom panels show 25 slices of K18 - ATTO 594 fibrils in donor cells ( 1 ) , or transfer of K18 - ATTO 594 fibrils from donor CTG - CADs to hippocampal astrocytes of the organotypic slice ( 2 to 5 ) . Yellow arrowheads point to Tau puncta inside acceptor astrocytes . Orthogonal views show each of the five K18 - ATTO 594 positive - puncta contained inside acceptor cells as indicated in the inset of the middle panel . Scale bars are 100 μm in the merged panel and 10 μm in the insets .

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(C) Below the schematics of the co-culture experiment are representative confocal images of neuron-like cells (CTG-CADs growing on top of organotypic slices (n=6), in which the astrocytes were labelled with GFAP. The upper panels show control CTG-CADs and the middle panels show donor CTG-CADs loaded with K18-ATTO 594 fibrils, co-cultured with an acceptor hippocampal slice. The images are representative Z-stack projections covering the whole cell body of donor cells. In the merged images, white are acceptor astrocytes (GFAP positive), green are donor CTG-CADs, red are K18-ATTO 594 puncta and nuclei are stained in blue. Insets are threefold enlargements of the boxed region in the merged picture. The orthogonal views of the bottom panels show 25 slices of K18-ATTO 594 fibrils in donor cells (1), or transfer of K18-ATTO 594 fibrils from donor CTG-CADs to hippocampal astrocytes of the organotypic slice (2 to 5). Yellow arrowheads point to Tau puncta inside acceptor astrocytes. Orthogonal views show each of the five K18-ATTO 594 positive-puncta contained inside acceptor cells as indicated in the inset of the middle panel. Scale bars are 100 μm in the merged panel and 10 μm in the insets.


( C ) Below the schematics of the co - culture experiment are representative confocal images of neuron - like cells ( CTG - CADs as in A ) growing on top of organotypic slices ( n = 6 ) , in which the astrocytes were labelled with The upper panels show control CTG - CADs and the middle panels show donor CTG - CADs loaded with K18 - ATTO 594 fibrils , co - cultured with an acceptor hippocampal slice . The images are representative Z - stack projections covering the whole cell body of donor cells . In the merged images , white are acceptor astrocytes , green are donor CTG - CADs , red are K18 - ATTO 594 puncta and nuclei are stained in blue . Insets are threefold enlargements of the boxed region in the merged picture . of K18 - ATTO 594 fibrils in donor cells ( 1 ) , or transfer of K18 - ATTO 594 fibrils from donor CTG - CADs to hippocampal astrocytes of the organotypic slice ( 2 to 5 ) . Yellow arrowheads point to Tau puncta inside acceptor astrocytes . Orthogonal views show each of the five K18 - ATTO 594 positive - puncta contained inside acceptor cells as indicated in the inset of the middle panel . Scale bars are 100 μm in the merged panel and 10 μm in the insets . the neurons in the slices were labelled with the dendrite marker MAP - 2 and the axonal marker β - III - tubulin ( white ) , and the orthogonal views show 26 slices .

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(C) Below the schematics of the co-culture experiment are representative confocal images of neuron-like cells (CTG-CADs as in A) growing on top of organotypic slices (n=6), in which the astrocytes were labelled with The upper panels show control CTG-CADs and the middle panels show donor CTG-CADs loaded with K18-ATTO 594 fibrils, co-cultured with an acceptor hippocampal slice. The images are representative Z-stack projections covering the whole cell body of donor cells. In the merged images, white are acceptor astrocytes , green are donor CTG-CADs, red are K18-ATTO 594 puncta and nuclei are stained in blue. Insets are threefold enlargements of the boxed region in the merged picture. of K18-ATTO 594 fibrils in donor cells (1), or transfer of K18-ATTO 594 fibrils from donor CTG-CADs to hippocampal astrocytes of the organotypic slice (2 to 5). Yellow arrowheads point to Tau puncta inside acceptor astrocytes. Orthogonal views show each of the five K18-ATTO 594 positive-puncta contained inside acceptor cells as indicated in the inset of the middle panel. Scale bars are 100 μm in the merged panel and 10 μm in the insets. the neurons in the slices were labelled with the dendrite marker MAP-2 and the axonal marker β-III-tubulin (white), and the orthogonal views show 26 slices.


Cells were fixed and the viral bait fused with the V5 - tag was visualized by immunofluorescence staining using Alexa Fluor - 488 labeled anti - V5 immunostaining ( yellow ) , actin with Alexa Fluor594 - labeled phalloidin ( magenta ) , and nuclei were stained with DAPI ( cyan ) ( Scale bar : 10 μm ) .

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Cells were fixed and the viral bait fused with the V5-tag was visualized by immunofluorescence staining using Alexa Fluor-488 labeled anti-V5 immunostaining (yellow), actin with Alexa Fluor594-labeled phalloidin (magenta), and nuclei were stained with DAPI (cyan) (Scale bar: 10 μm).


A For the image - based drug screening , Calu - 1 cells were infected with SARS - CoV - 2 in the presence of drugs with indicated doses for 48 h , immunostained , imaged and analyzed . The example images of mock - infected cells and virus - infected control cells ( no drugs ) . The viral yield in cells was detected with an antibody for the viral N protein ( virus - infected cells ; upper image ) , and nuclear staining ( mock - infected cells ; lower image ) ( Scale bar : 200 μm ) .

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A For the image-based drug screening, Calu-1 cells were infected with SARS-CoV-2 in the presence of drugs with indicated doses for 48 h, immunostained, imaged and analyzed. The example images of mock-infected cells and virus-infected control cells (no drugs). The viral yield in cells was detected with an antibody for the viral N protein (virus-infected cells; upper image), and nuclear staining (mock-infected cells; lower image) (Scale bar: 200 μm).


B The dose - response curves for six drugs with potential antiviral effects . The drug - induced inhibition of virus infection ( count of N protein positive cells ) was quantified using the area under the curve ( AUC ) values .

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B The dose-response curves for six drugs with potential antiviral effects. The drug-induced inhibition of virus infection (count of N protein positive cells) was quantified using the area under the curve (AUC) values.


C The effect of drugs on cell viability in the presence of the virus , e . g . , cytotoxicity was evaluated by the count of nuclei .

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C The effect of drugs on cell viability in the presence of the virus, e.g., cytotoxicity was evaluated by the count of nuclei.


D Inhibition of interactions formed with DDX39B in the presence of MTX using co - IP assay . DDX39B is fused to the C terminus of a V5 tag , and the interaction partner is fused with a Strep - HA tag . The diagram ( upper ) shows the interaction pairs formed with DDX39B used for co - IP assay . Dot - blot results ( lower ) show the interaction pairs ( arrangement as upper panel ) in presence of different concentration of MTX as indicated on the top of the image . The image displayed is representative of 3 replicates ( n = 3 ) .

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D Inhibition of interactions formed with DDX39B in the presence of MTX using co-IP assay. DDX39B is fused to the C terminus of a V5 tag, and the interaction partner is fused with a Strep-HA tag. The diagram (upper) shows the interaction pairs formed with DDX39B used for co-IP assay. Dot-blot results (lower) show the interaction pairs (arrangement as upper panel) in presence of different concentration of MTX as indicated on the top of the image. The image displayed is representative of 3 replicates (n = 3).


E Quantification of DDX39B binding to different interaction partners . Quantification of V5 - tagged DDX39B protein amount was normalized over HA - tagged interaction partner intensity and displayed as a fold change over the ratio of untreated cells . Data that represent the mean of 3 replicates are highlighted in red . A fold change > 0 . 5 is considered significant .

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E Quantification of DDX39B binding to different interaction partners. Quantification of V5-tagged DDX39B protein amount was normalized over HA-tagged interaction partner intensity and displayed as a fold change over the ratio of untreated cells. Data that represent the mean of 3 replicates are highlighted in red. A fold change > 0.5 is considered significant.


( C ) Expression of miRNAs miR - 146a and miR - 505 correlated with overall survival in ovarian cancer patients . The validation cohort ( n = 150 samples ) is shown . See Figure EV1 for the training cohort .

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(C) Expression of miRNAs miR-146a and miR-505 correlated with overall survival in ovarian cancer patients. The validation cohort (n=150 samples) is shown. See Figure EV1 for the training cohort.


( A ) The consequence of miR - 155 and miR - 181b mimics on cell viability , 5 days post transfection , relative to a negative control miRNA mimic is shown across a panel of normal and ovarian cancer cell lines as indicated . Each data point is the mean of n = 3 / cell line . In the screen , miR - 155 was found to reduce cell viability ( Z - score < - 2 ) in one cell line , and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors . miR - 181 was found to reduce viability ( Z - score < - 2 ) in 2 cell lines , and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors . N , normal cell lines ( IHH , HOSE , HBEC3 , HBEC13 , HBEC30 , and HBEC34 ) , EOC , epithelial ovarian cancer cell lines . miR - 155 data points for each cell line are shown in black while miR - 181b data points are displayed in red .

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(A) The consequence of miR-155 and miR-181b mimics on cell viability, 5 days post transfection, relative to a negative control miRNA mimic is shown across a panel of normal and ovarian cancer cell lines as indicated. Each data point is the mean of n=3/cell line. In the screen, miR-155 was found to reduce cell viability (Z-score < -2) in one cell line, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors. miR-181 was found to reduce viability (Z-score < -2) in 2 cell lines, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors. N, normal cell lines (IHH, HOSE, HBEC3, HBEC13, HBEC30, and HBEC34), EOC, epithelial ovarian cancer cell lines. miR-155 data points for each cell line are shown in black while miR-181b data points are displayed in red.