E . Livers from sham 48 h following PH were digested to isolate hepatocyte population . extracts were analyzed by Western blot for YAP TAZ expression . β - actin was used as a loading control . Results of a single experiment with n = 2 per group . Each band is representative of an independent .

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E. Livers from sham and 48 h following PH were digested to isolate the hepatocyte population. Protein extracts were analyzed by Western blot for YAP and TAZ expression. β-actin was used as a loading control. Results of a single experiment with n = 2 animals per group. Each band is representative of an independent animal.


F . RT - PCR of isolated from mouse liver for Birc5 , FoxM1B cyclin B1 . Data are presented as log2 fold change were calculated using non - operated mouse liver tissue as a control . Unpaired , two - Student t - was used to determine significance between log2 values of livers 6 , 24 48 h post PH compared to sham operated controls . Log2 values are plotted on a linear scale as mean ± SD .

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F. RT-PCR analysis of RNA isolated from mouse liver for Birc5, FoxM1B and cyclin B1. Data are presented as log2 fold change and were calculated using non-operated mouse liver tissue as a control. Unpaired, two-tailed Student t-test was used to determine the significance between the log2 values of livers 6, 24 and 48 h post PH compared to the sham operated controls. Log2 values are plotted on a linear scale as mean ± SD.


A - C . Representative photomicrograph of young ( 6 - 8 ) aged ( > 12 months ) livers 40 h post PH stained with Ki67 visualized by H E for histology . Sections were scanned using Histech Pannoramic MIDI Scanner Ki67 positive were quantitated with Quant Center 2 . 0 software . A proliferation index ( p . i . ) was calculated on number of Ki67 positive hepatocytes to total hepatocytes livers were classified as ( + ) regenerating , p . i . 10 % or ( - ) non - regenerating , p . i . 10 % . D . Number of young ( 6 - 8 ) aged ( 12 months ) mice with ( - ) non - regenerating ( + ) regenerating or livers on p . i . , 40 h post PH . deemed non - surviving for ethical reasons were euthanized 24 hours post PH . Representative results of a single experiment with n = 9 aged n = 6 young per group .

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A-C. Representative photomicrograph of young (6-8 weeks) and aged (>12 months) livers 40 h post PH stained with Ki67 and visualized by IHC and H E for histology. Sections were scanned using Histech Pannoramic MIDI Scanner and Ki67 positive cells were quantitated with Quant Center 2.0 software. A proliferation index (p.i.) was calculated based on the number of Ki67 positive hepatocytes to total hepatocytes and livers were classified as (+) regenerating, p.i.10% or (-) non-regenerating, p.i.10%.D. Number of young (6-8 weeks) and aged (12 months) mice with (-) non-regenerating and (+) regenerating or livers based on p.i., 40 h post PH. Animals deemed non-surviving for ethical reasons were euthanized 24 hours post PH. Representative results of a single experiment with n=9 aged and n=6 young animals per group.


A . Western blot detection of p - MST1 , MST , p - YAP , YAP TAZ in control 40 h following PH in young aged mice . TBP was used as a loading control . Representative results of a single experiment with n = 6 independent . Each band is from an independent . Western blot ratios are graphically depicted . were normalized to TBP mean value of group is plotted . Unpaired , two - Student t - was used to calculate significance between 0h ( resected liver ) 40h following PH . Error bars represent mean ±

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A. Western blot detection of p-MST1, MST, p-YAP, YAP and TAZ in control and 40 h following PH in young and aged mice. TBP was used as a loading control. Representative results of a single experiment with n = 6 independent animals. Each band is from an independent animal. Western blot quantification ratios are graphically depicted. Proteins levels were normalized to TBP and mean value of the group is plotted. Unpaired, two-tailed Student t-test was used to calculate the significance between 0h (resected liver) and 40h following PH. Error bars represent mean ± SD


B . RT - PCR of isolated from mouse liver for FoxM1B , Birc5 cyclin B1 , Cyclin A2 , Cyclin D1 p21 . RT - PCR data are presented as log2 fold change each value post PH was calculated for each mouse by comparison to its resected liver lobe . Circle , with red outline marks aged non - regenerating livers shown in Figure 2B . Each dot is representing an independent .

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B. RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1, Cyclin A2, Cyclin D1 and p21. RT-PCR data are presented as log2 fold change and each value post PH was calculated for each mouse by comparison to its resected liver lobe. Circle, with red outline marks the aged non-regenerating livers shown in Figure 2B. Each dot is representing an independent animal.


A . Eight old mice were injected with liposomes coupled to scrambled siRNA ( siScr ) or sequences targeting MST1 MST2 ( siMST ) . Each dot is representing an independent . % of Mst1 Mst2 mRNA remaining in liver was tested by RT - PCR , 1 , 3 6 days post injection calculated compared to control non - transfected livers . Unpaired , two - Student t - was used to calculate significance of mRNA remaining in comparison to non - transfected control livers at each time point .

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A. Eight weeks old mice were injected with liposomes coupled to scrambled siRNA (siScr) or sequences targeting MST1 MST2 (siMST). Each dot is representing an independent animal. The % of Mst1 Mst2 mRNA remaining in the liver was tested by RT-PCR, 1, 3 and 6 days post injection and calculated compared to control non-transfected livers. Unpaired, two-tailed Student t-test was used to calculate the significance of mRNA remaining in comparison to non-transfected control livers at each time point.


B . Western blot detection of MST1 , p - LATS1 β - actin 1 , 3 6 days post siRNA injection . A representative from each time point is shown .

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B. Western blot detection of MST1, p-LATS1 and β-actin 1, 3 and 6 days post siRNA injection. A representative animal from each time point is shown.


C . Western blot detection of YAP1 , p - YAP1 , H3 p - H3 , 3 days post siRNA injection . Two representative from each group are shown . samples were run on same blot . blot is split due to non - consecutive loading of samples .

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C. Western blot detection of YAP1, p-YAP1, H3 and p-H3, 3 days post siRNA injection. Two representative animals from each group are shown. All samples were run on the same blot. The blot is split due to non-consecutive loading of the samples.


D . Quantitative RT - PCR of isolated from mouse liver for FoxM1B , Birc5 cyclin B1 , 1 , 3 6 days post injection . Log2 fold change was calculated using non - transfected mouse liver as control . Unpaired two Student t - was used to calculate significance of fold change of in comparison to a panel non - transfected control livers .

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D. Quantitative RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1, 1, 3 and 6 days post injection. Log2 fold change was calculated using non-transfected mouse liver as control. Unpaired two tailed Student t-test was used to calculate the significance of fold change of in comparison to a panel non-transfected control livers.


E . HNF4α Ki67 were detected by positive for both were classified as proliferating hepatocytes . Ki67 positive hepatocytes are indicated with an asterisk Ki67 negative bile / biliary with an arrow in tissue 3 days post siRNA delivery . percentages of positive hepatocytes at day 3 day 6 - post injection are graphed . Two - Student t - was used to calculate significance of of positive hepatocytes in comparison to non - transfected control livers .

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

E. HNF4α and Ki67 were detected by IHC and cells positive for both were classified as proliferating hepatocytes. Ki67 positive hepatocytes are indicated with an asterisk and Ki67 negative bile ducts/biliary cells with an arrow in tissue 3 days post siRNA delivery. The percentages of positive hepatocytes at day 3 and day 6-post injection are graphed. Two-tailed Student t-test was used to calculate the significance of percentage of positive hepatocytes in comparison to non-transfected control livers.


A . Aged mice ( > 12 months ) were injected i . v . with siScr or siMST . Twenty - four h after siRNA injection a 70 % PH was performed remnant / regenerating liver tissue was harvested 40 h later . p . i . of livers was calculated by of Ki67 positive hepatocytes on of positive liver were classified ( - ) non regenerating or ( + ) regenerating . Non - surviving were sacrificed before 40 h end time point . " n " indicates number of per group . B . Ki67 immunostaining of regenerating aged mice livers , 40 h post PH . Sections were scanned using Histotech Pannoramic MIDI Scanner quantitated with Quant Center 2 . 0 software . p . i . values are provided on image .

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A. Aged mice (>12 months) were injected i.v. with siScr or siMST. Twenty-four h after siRNA injection a 70% PH was performed and remnant/regenerating liver tissue was harvested 40 h later. p.i. of the livers was calculated by IHC staining of Ki67 positive hepatocytes and based on the percentage of positive cells liver were classified (-) non regenerating or (+) regenerating. Non-surviving animals were sacrificed before the 40 h end time point. "n" indicates the number of animals per group.B. Ki67 immunostaining of the regenerating aged mice livers, 40 h post PH. Sections were scanned using Histotech Pannoramic MIDI Scanner and quantitated with Quant Center 2.0 software. p.i. values are provided on the image.


C . Representative photomicrograph of an aged liver treated with siMST stained with H & amp ; amp ; E by for CK19 Ki67 . Arrows are indicating regions with no signs of reaction or oval expansion .

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C. Representative photomicrograph of an aged liver treated with siMST and stained with H&amp;E and by IHC for CK19 and Ki67. Arrows are indicating ductal regions with no signs of reaction or oval cell expansion.


D . Quantitative RT - PCR of isolated from mouse liver for FoxM1B , Birc5 cyclin B1 at 0 40 h post PH . Log2 fold change was calculated using non - transfected / non - resected mouse liver as a control . Representative results of a single experiment with n = 6 per group are shown . Paired , two - Student t - was used to calculate significant change in signals between liver tissues at time of resection ( 0h ) compared to 40 h post PH .

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D. Quantitative RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1 at 0 and 40 h post PH. Log2 fold change was calculated using non-transfected/non-resected mouse liver as a control. Representative results of a single experiment with n = 6 animals per group are shown. Paired, two-tailed Student t-test was used to calculate the significant change in signals between liver tissues at the time of resection (0h) compared to 40 h post PH.


E . of Ki67 positive hepatocytes in aged aged + siMST 40 hours post resection . Representative results of a single experiment with n = 6 independent aged for group are shown . Two - Student t - was used to calculate significance of of positive hepatocytes of siMST treated compared to non - treated controls .

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E. Percentage of Ki67 positive hepatocytes in aged and aged + siMST 40 hours post resection. Representative results of a single experiment with n = 6 independent aged animals for group are shown. Two-tailed Student t-test was used to calculate the significance of percentage of positive hepatocytes of siMST treated animals compared to non-treated controls.


F . Endpoint liver was taken of remnant lobe expressed as a to total . Representative results of 1experiment with n = 6 independent for group expect for control group were n = 6 are shown . Estimated liver - to - before PH in mice = 3 . 85 ± 0 . 05 ( SD ) . Unpaired , two - Student t - was used to calculate significance of each aged group in comparison to control young group .

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F. Endpoint liver weight was taken of the remnant lobe and expressed as a percentage to total body weight. Representative results of 1experiment with n = 6 independent animals for group expect for control group were n = 6 are shown. Estimated liver-to-body weight ratio before PH in mice = 3.85 ± 0.05 (SD). Unpaired, two-tailed Student t-test was used to calculate the significance of each aged group in comparison to the control young group.


G . Hepatocyte geometric diameter was calculated ( see material methods ) . Representative results of 1 experiment with n = 3 independent per group . Unpaired , two - Student t - was used to calculate significant change in hepatocyte in liver tissue with without siMST 40 h post PH .

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G. Hepatocyte geometric diameter was calculated (see material and methods). Representative results of 1 experiment with n = 3 independent animals per group. Unpaired, two-tailed Student t-test was used to calculate the significant change in hepatocyte size in liver tissue with and without siMST 40 h post PH.


( B ) of SARS - CoV - 2 infection . After siRNA knockdown of each target , viral infection was quantified as mean log2 fold - change ( log2FC ) of of SARS - CoV - 2 + relative to mean of scrambled non - targeting siRNAs ( " SCRAMBLED " , green dots ; accordingly mean log2FC value of scrambled non - targeting siRNAs was normalized to zero ) . Predicted positive targets ( " TARGETING " , grey dots ) , predicted negative targets ( " NEG " , blue dots ) positive controls ( " POS " , red dots , including ACE2 TMPRSS2 ) are shown . Wilcoxon rank - sum P value comparing predicted positive targets to scrambled non - targeting siRNAs is given .

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(B) Quantification of SARS-CoV-2 infection. After the siRNA knockdown of each target, viral infection was quantified as mean log2 fold-change (log2FC) of the percentage of SARS-CoV-2+ cells relative to the mean of scrambled non-targeting siRNAs ("SCRAMBLED", green dots; accordingly the mean log2FC value of scrambled non-targeting siRNAs was normalized to zero). Predicted positive targets ("TARGETING", grey dots), predicted negative targets ("NEG", blue dots) and positive controls ("POS", red dots, including ACE2 and TMPRSS2) are all shown. Wilcoxon rank-sum test P value comparing the predicted positive targets to scrambled non-targeting siRNAs is given.


( C ) of number after each target knockdown , calculated as mean fraction of DAPI + relative to scrambled non - targeting siRNAs ( i . e . latter was normalized to 1 ) . Colors of dots P value are interpreted in same way as in ( B ) .

(C) Quantification of cell number after each target knockdown, calculated as the mean fraction of DAPI+ cells relative to the scrambled non-targeting siRNAs (i.e. the latter was normalized to 1). Colors of dots and P value are interpreted in the same way as in (B).


( D ) Representative from siRNA showing SARS - CoV - 2 infection ( green channel , top row ) , number ( blue channel , middle row ) merged ( bottom row ) . Results for scrambled non - targeting siRNA as negative control ( left column ) , knockdown of three predicted top metabolic targets ( PIKFYVE , SLC16A10 , PIP5K1C , middle columns ) , knockdown of positive control ( ACE2 , right column ) are shown . Scale bar = 10 μm .

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(D) Representative fluorescence images from the siRNA assay showing SARS-CoV-2 infection (green channel, top row), cell number (blue channel, middle row) and merged (bottom row). Results for scrambled non-targeting siRNA as negative control (left column), knockdown of three predicted top metabolic targets (PIKFYVE, SLC16A10, and PIP5K1C, middle columns), and knockdown of positive control (ACE2, right column) are shown. Scale bar=10 μm.


( A ) PCA plot of gene expression profiles for Vero E6 samples from experimental groups : control ( no or remdesivir treatment ) , ( SARS - CoV - 2 - infected ) , + remdesivir ( SARS - CoV - 2 - infected treated by remdesivir ) , remdesivir ( remdesivir treatment alone without ) . There are 3 replicates in each group .

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(A) PCA plot of the gene expression profiles for the Vero E6 samples from all experimental groups: control (no virus or remdesivir treatment), virus (SARS-CoV-2-infected), virus+remdesivir (SARS-CoV-2-infected treated by remdesivir), remdesivir (remdesivir treatment alone without virus). There are 3 replicates in each group.


( C ) PCA plot of average metabolic flux profile computed using iMAT algorithm ( Shlomi et al . 2008 ; Methods ) representative of each of experimental groups , labels are same as ( A ) .

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(C) PCA plot of the average metabolic flux profile computed using the iMAT algorithm (Shlomi et al. 2008; Methods) representative of each of the experimental groups, the labels are the same as (A).


( B ) Representative leak - subtracted ClC - 3 , ClC - 4 ClC - 5 whole - currents measured with ( upper row ) or without ( lower row ) 5 mM ATP added to pipette solution . Currents were elicited every 10 s by voltage steps from 0 mV to + 140 mV , followed by a step to - 100 mV to maximize off - gating currents visible as sharp peaks at end of pulses . recording started shortly ( 10s - 30s ) after obtaining a whole - configuration . first last recorded currents in a series are depicted in green red , respectively . NMDG - solutions were used to characterize ClC - 4 .

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(B) Representative leak-subtracted ClC-3, ClC-4 and ClC-5 whole-cell currents measured with (upper row) or without (lower row) 5 mM ATP added to the pipette solution. Currents were elicited every 10 s by voltage steps from 0 mV to +140 mV, followed by a step to -100 mV to maximize the off-gating currents visible as sharp peaks at the end of the pulses. The recording started shortly (10s - 30s) after obtaining a whole-cell configuration. The first and last recorded currents in a series are depicted in green and red, respectively. NMDG-based solutions were used to characterize ClC-4.


( C ) Effects of various adenine nucleotides or their washout on ionic transport of ClC - 3 , ClC - 4 ClC - 5 . current amplitudes were measured at end of pulses from experiments as shown in ( B ) normalized to initial current amplitudes obtained after establishing whole - configuration . Some of experimental data points have been omitted for clarity . Monoexponential fits to data are depicted as red lines . Asterisks indicate statistical significances evaluated by two - sample t - of residual currents determined at end of each 10 - minute measurement ( * , p < 0 . 05 ; * * * , p < 0 . 001 , n . s . , not significant ; n = 5 - 11 ) .

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(C) Effects of various adenine nucleotides or their washout on the ionic transport of ClC-3, ClC-4 and ClC-5. The current amplitudes were measured at the end of the test pulses from experiments as shown in (B) and normalized to the initial current amplitudes obtained after establishing the whole-cell configuration. Some of the experimental data points have been omitted for clarity. Monoexponential fits to the data are depicted as red lines. Asterisks indicate statistical significances evaluated by two-sample t-test analysis of the residual currents determined at the end of each 10-minute measurement (*, p < 0.05; ***, p < 0.001, n.s., not significant; n= 5-11).


( D ) , Left panel : residual currents determined at end of 10 - minute measurements as shown in ( C ) . ( D ) , Right panel : time constants ( τ ) of current rundown obtained by fitting monoexponential functions to measurements as depicted in ( C ) , ( n = 5 - 11 ) . Asterisks indicate statistical significances evaluated using two - sample t - ( * , p < 0 . 05 ; * * * , p < 0 . 001 , n . s . , not significant ) .

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(D), Left panel: residual currents determined at the end of 10-minute measurements as shown in (C). (D), Right panel: time constants (τ) of the current rundown obtained by fitting monoexponential functions to individual measurements as depicted in (C), (n= 5-11). Asterisks indicate statistical significances evaluated using two-sample t-test analysis (*, p < 0.05; ***, p < 0.001, n.s., not significant).


( B ) Voltage dependence of WT ClC - 5 off - gating charge normalized to ionic current at + 165 mV in absence or presence of adenine nucleotides ( n = 7 - 12 ) . Error bars represent . Red lines depict Boltzmann fits to data ; fit parameters are provided in Appendix Table S1 .

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(B) Voltage dependence of the WT ClC-5 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=7-12). Error bars represent SEM. Red lines depict Boltzmann fits to the data; fit parameters are provided in Appendix Table S1.


( C ) Voltage dependence of WT ClC - 3 off - gating charge normalized to ionic current at + 165 mV in absence or presence of adenine nucleotides ( n = 4 - 5 , Error bars represent . ) . Red lines represent Boltzmann fits to

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(C) Voltage dependence of the WT ClC-3 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=4-5, Error bars represent SEM.). Red lines represent Boltzmann fits to the data


( A ) Representative whole - recordings of expressing E211G ClC - 5 upon voltage pulses to + 140 mV from a - 60 - mV holding potential applied in 10 - s intervals starting shortly after obtaining whole - configuration with ( left ) or without ( right ) 5 mM ATP in pipette solution . Leak subtraction was not applied . Asymmetric [ Cl - ] was used ( 110 mM internal NaCl substituted by TrisSO4 ) to achieve a stable negative reversal potential of < - 50 mV allowing to recognize recordings with high unspecific leak current contamination .

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(A) Representative whole-cell recordings of cells expressing E211G ClC-5 upon voltage pulses to +140 mV from a -60-mV holding potential applied in 10-s intervals starting shortly after obtaining the whole-cell configuration with (left) or without (right) 5 mM ATP in the pipette solution. Leak subtraction was not applied. Asymmetric [Cl-] was used (110 mM internal NaCl substituted by TrisSO4) to achieve a stable negative reversal potential of <-50 mV allowing to recognize recordings with high unspecific leak current contamination.


( B ) Normalized time course of steady - state E211G ClC - 5 current amplitudes measured with ( n = 5 ) or without ( n = 4 ) internal ATP . Error bars show . Red represents a monoexponential fit to data with a time constant of 108 ± 8 s ( ) ; currents measured with ATP - free pipette solution declined to 57 . 9 ± 2 . 6 % ( ) of initial amplitude .

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(B) Normalized time course of the steady-state E211G ClC-5 current amplitudes measured with (n=5) or without (n=4) internal ATP. Error bars show SEM. Red line represents a monoexponential fit to the data with a time constant of 108±8 s (SEM); currents measured with ATP-free pipette solution declined to 57.9±2.6% (SEM) of the initial amplitude.


( A ) Effects of different adenine nucleotides or their washout on ClC - 5 current amplitudes ( n = 4 - 7 ) . data were normalized to initial current amplitude determined shortly after establishing whole - configuration . Some of data points are omitted for clarity . For comparison , ADP data set from Figure 1 is also depicted . Red lines indicate monoexponential fits to averaged data .

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(A) Effects of different adenine nucleotides or their washout on the ClC-5 current amplitudes (n=4-7). The data were normalized to the initial current amplitude determined shortly after establishing whole-cell configuration. Some of the data points are omitted for clarity. For comparison, the ADP data set from Figure 1 is also depicted. Red lines indicate monoexponential fits to the averaged data.


( B ) Averaged residual currents determined after 600 - s dialysis as shown in ( A ) , normalized to current at start of measurements ( n = 4 - 7 ) . Asterisks indicate statistically significant differences from a two - sample t - with p < 0 . 001 ; n . d . - not defined .

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(B) Averaged residual currents determined after 600-s cell dialysis as shown in (A), and normalized to the current at the start of the measurements (n=4-7). Asterisks indicate statistically significant differences from a two-sample t-test analysis with p < 0.001; n.d. - not defined.


Averaged rundown time constants obtained from fits with monoexponential decay functions as shown in ( A ) .

Averaged rundown time constants obtained from fits with monoexponential decay functions as shown in (A).


A - D Appearance of autophagic structures ( marked with GFP - Atg8a ) during wound closure in epidermis of control third instar larvae after epidermal knockdown of autophagy pathway components Atg1 , Atg5 , Atg6 , Atg7 or Atg12 . constructs are expressed in epidermis under control of A58 - Gal4 driver . ( A ) Time points from movies of wounded epidermis . wounds have closed by 2 h in cases . ( B ) Higher magnification of areas marked by magenta boxes at t = 120 min . ( C ) of appearance of GFP - Atg8a in imaged ( 10000 µm2 ) 3 control larvae , shown for each larva . ( D ) of number of GFP - Atg8a in different genetic conditions measured in an of 10000 µm2 at time of wound closure ; n = 4 - 10 larvae each genotype , for detail see Data .

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A-D Appearance of autophagic structures (marked with GFP-Atg8a) during wound closure in the epidermis of control third instar larvae and after epidermal knockdown of the autophagy pathway components Atg1, Atg5, Atg6, Atg7 or Atg12. All constructs are expressed in the epidermis under the control of the A58-Gal4 driver. (A) Time points from movies of wounded epidermis. The wounds have closed by 2 h in all cases. (B) Higher magnification of the areas marked by magenta boxes at t=120 min. (C) Quantification of the appearance of GFP-Atg8a puncta in the imaged area (10000 µm2) 3 control larvae, shown for each individual larva. (D) Quantification of number of GFP-Atg8a puncta in different genetic conditions measured in an area of 10000 µm2 at the time of wound closure; n=4-10 larvae each genotype, for the detail see Data analysis.


Control of epidermal autophagy by TOR signalling . ( A ) Epidermis of third instar larvae expressing autophagosome marker GFP - Atg8a together with constructs for up - or down - regulating autophagy pathway in epidermis . Healthy epidermis contains few autophagosomes , but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes . ( B ) Higher magnification of areas marked by magenta boxes in ( A ) .

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Control of epidermal autophagy by TOR signalling. (A) Epidermis of third instar larvae expressing the autophagosome marker GFP-Atg8a together with constructs for up- or down-regulating the autophagy pathway in the epidermis. Healthy epidermis contains few autophagosomes, but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes. (B) Higher magnification of the areas marked by magenta boxes in (A).


Control of epidermal autophagy by TOR signalling . ( C ) of Atg8a in an of 10000 µm2 , n = 6 - 8 larvae each genotype . We assumed unequal sample unequal variances calculations were performed . Values are presented as box plots . Box plot elements are : center , median ; box limits , upper lower quartiles ; whiskers , 1 . 5x interquartile range ; points , outliers .

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Control of epidermal autophagy by TOR signalling. (C) Quantification of Atg8a puncta in an area of 10000 µm2, n=6-8 larvae each genotype. We assumed unequal sample size and unequal variances and calculations were performed. Values are presented as box plots. Box plot elements are: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers.


A - D loss in photobleaching ( FLIP ) to free GFP motility within epidermis . A small ( magenta circle ; 179 or 1098 µm2 ) was - illuminated for indicated times ( 3 or 20 min ) in control or Atg1 - expressing epidermis also expressing free GFP . ( A ) Snapshots before bleaching at 3 points of recovery . ( B ) Kymographs along broken in ( A ) during recovery . ( C , D ) of recovery after bleaching , shown separately for 3 20 min bleaching protocols .

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A-D Fluorescence loss in photobleaching (FLIP) to test free cytoplasmic GFP motility within the epidermis. A small area (magenta circle; 179 or 1098 µm2) was laser-illuminated for the indicated times (3 or 20 min) in control or Atg1-expressing epidermis also expressing free GFP. (A) Snapshots before bleaching and at 3 points of recovery. (B) Kymographs along the broken line in (A) during recovery. (C, D) Quantification of fluorescence recovery after bleaching, shown separately for 3 and 20 min bleaching protocols.


B - D Atg1 - induced syncytium formation is abolished when Atg5 function is downregulated ( compare to rows 2 3 in Fig . 5A , B ) ; same representation as shown in Fig . 5A - D .

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B-D Atg1-induced syncytium formation is abolished when Atg5 function is downregulated (compare to rows 2 and 3 in Fig. 5A, B); same representation as shown in Fig. 5A-D.


C , D Accumulation of neutral lipid in cytoplasm under conditions of uncontrolled autophagy . ( C ) Fixed epidermis from larvae expressing indicated constructs was stained with BodiPy 493 / 503 to mark lipid accumulations . ( D ) of number of lipid in an epidermal of 10000 µm2 in indicated conditions . A - D , n = 7 - 20 larvae each genotype .

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C, D Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy. (C) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493/503 to mark lipid accumulations. (D) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions. A-D, n=7-20 larvae each genotype.


( A ) Wild ‐ type rho0 harbouring prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) were exposed to ‐ acid or nitrogen starvation ( ‐ N ) medium supplemented with indicated carbon sources . were analysed at indicated time points by whole extraction western blot using α ‐ GFP α ‐ Cdc11 antibodies . of ATG8 induction is shown in lower left panel . Total GFP signals ( GFP ‐ Atg8 free GFP ) were quantified normalized to Cdc11 signals . Normalized values at 0 h were set as one relative changes are shown after 6 h starvation . of autophagic flux is shown in lower right panel as of free GFP to total GFP signals ( GFP ‐ Atg8 free GFP ) after 6 h starvation . means s . d . of four ( n = 4 ) independent experiments are indicated .

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(A) Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid or nitrogen starvation (‐N) medium supplemented with indicated carbon sources. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐GFP and α‐Cdc11 antibodies. Quantification of ATG8 induction is shown in the lower left panel. Total GFP signals (GFP‐Atg8 and free GFP) were quantified and normalized to Cdc11 signals. Normalized values at 0 h were set as one and relative changes are shown after 6 h starvation. Quantification of autophagic flux is shown in the lower right panel as ratio of free GFP to total GFP signals (GFP‐Atg8 and free GFP) after 6 h starvation. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) microscopical . Wild ‐ type , rho0 , Δatg7 expressing prATG8 ‐ GFP ‐ ATG8 were grown as described in ( A ) exposed to ‐ acid ( left panel , galactose ) or nitrogen starvation ( right panel , glucose ( ‐ N ) ) for 6 h . Vacuoles were visualized by over night FM4 ‐ 64 ( 1 μM ) ( red ) . Arrowhead indicates a punctate GFP ‐ Atg8 structure . Transmission microscopy were superimposed to visualize boundaries . localization of GFP signal was analysed in at least 150 ( n ⩾ 150 ) for each condition . Scale bars represent 1 . 5 μm .

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(B) Fluorescence microscopical analysis. Wild‐type, rho0, and Δatg7 cells expressing prATG8‐GFP‐ATG8 were grown as described in (A) and exposed to amino‐acid (left panel, galactose) or nitrogen starvation (right panel, glucose (‐N)) for 6 h. Vacuoles were visualized by over night FM4‐64 (1 μM) staining (red). Arrowhead indicates a punctate GFP‐Atg8 structure. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Cellular localization of GFP signal was analysed in at least 150 cells (n⩾150) for each strain and condition. Scale bars represent 1.5 μm.


Autophagic response in with compromised respiratory chain III , IV , or V activity during ‐ acid starvation . Wild ‐ type , rho0 , atg7 mutants that are selectively inhibited in biogenesis of respiratory chain III ( CIII : cbs1 ) , IV ( CIV : mss51 , pet111 , pet122 ) , or F1Fo ‐ ATP synthase ( V ; CV : atp10 ) were exposed to ‐ acidstarvation medium supplemented with acetate ( A ) or galactose ( B ) . When indicated , wild ‐ type were exposed to antimycin A ( AA ) or oligomycin ( O ) during ‐ acid starvation period . strains expressed prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) . Samples were analysed as described in Figure 1A . means s . d . of five ( n = 5 ) independent experiments are indicated .

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Autophagic response in cells with compromised respiratory chain complex III, IV, or V activity during amino‐acid starvation. Wild‐type, rho0, atg7 cells and mutants that are selectively inhibited in the biogenesis of respiratory chain complex III (CIII: cbs1), complex IV (CIV: mss51, pet111, pet122), or the F1Fo‐ATP synthase (complex V; CV: atp10) were exposed to amino‐acidstarvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. All strains expressed prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels). Samples were analysed as described in Figure 1A. The means and s.d. of five (n=5) independent experiments are indicated.


( A , B ) Wild ‐ type , rho0 , Δatg7 were exposed to ‐ acid starvation medium supplemented with acetate ( A ) or galactose ( B ) . When indicated , wild ‐ type were exposed to antimycin A ( AA ) or oligomycin ( O ) during ‐ acid starvation period . ATP from total ( upper panels ) or isolated mitochondria ( lower panels ) were determined at indicated time points .

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(A, B) Wild‐type, rho0, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. ATP and protein from total cells (upper panels) or isolated mitochondria (lower panels) were determined at indicated time points.


( C ) Wild ‐ type Δatg7 were exposed to ‐ acid starvation medium supplemented with acetate or galactose for 3 h . Wild ‐ type were exposed to antimycin A ( AA ) , oligomycin ( O ) , or CCCP ( 50 μM ) during ‐ acid starvation period when indicated . were treated with mitochondrial potential ‐ dependent dye DiOC6 ( 3 ) examined by microscopy . Average pixel intensities for at least 10 mitochondrial tubules ( n = 10 ) in 5 representative were determined for each condition . Values for wild ‐ type mitochondria in presence of acetate were defined as 100 % relative values were calculated accordingly . means s . d . are shown .

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(C) Wild‐type and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate or galactose for 3 h. Wild‐type cells were exposed to antimycin A (AA), oligomycin (O), or CCCP (50 μM) during the amino‐acid starvation period when indicated. Cells were treated with the mitochondrial membrane potential‐dependent dye DiOC6(3) and examined by fluorescence microscopy. Average pixel intensities for at least 10 mitochondrial tubules (n=10) in 5 representative cells were determined for each condition. Values for wild‐type mitochondria in the presence of acetate were defined as 100% and relative values were calculated accordingly. The means and s.d. are shown.


( A ) Wild ‐ type , rho0 , Δnpr2 , Δnpr2 rho0 expressing prATG8 ‐ GFP ‐ ATG8 ( upper panel ) or prATG8 ‐ GFP ( lower panel ) were exposed to ‐ acid starvation medium supplemented with galactose in absence or presence of rapamycin . Samples were analysed as described in Figure 1A . means s . d . of four ( n = 4 ) independent experiments are indicated .

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(A) Wild‐type, rho0, Δnpr2, and Δnpr2 rho0 cells expressing prATG8‐GFP‐ATG8 (upper panel) or prATG8‐GFP (lower panel) were exposed to amino‐acid starvation medium supplemented with galactose in the absence or presence of rapamycin. Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) Wild ‐ type , rho0 , Δnpr2 , Δatg7 expressing prNPR1 ‐ NPR1 ‐ HA were exposed to ‐ acid starvation medium supplemented with galactose in absence ( upper panels ) or presence ( lower panels ) of rapamycin . hyperphosphorylated ( Npr1 ‐ P ) dephosphorylated ( Npr1 ) forms of Npr1 are indicated . were analysed at indicated time points by whole extraction western blot using α ‐ HA α ‐ Cdc11 antibodies .

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(B) Wild‐type, rho0, Δnpr2, and Δatg7 cells expressing prNPR1‐NPR1‐HA were exposed to amino‐acid starvation medium supplemented with galactose in the absence (upper panels) or presence (lower panels) of rapamycin. The hyperphosphorylated (Npr1‐P) and dephosphorylated (Npr1) forms of Npr1 are indicated. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐HA and α‐Cdc11 antibodies.


( C ) Wild ‐ type , rho0 , Δnpr2 , Δatg7 were exposed to ‐ acid starvation medium supplemented with galactose . Additionally , wild ‐ type were treated with rapamycin during starvation ( left panel ) . Phosphorylated ( Atg13 ‐ P ) dephosphorylated ( Atg13 ) Atg13 was monitored at indicated time points by whole extraction western blot using α ‐ Atg13 α ‐ Cdc11 antibodies .

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(C) Wild‐type, rho0, Δnpr2, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with galactose. Additionally, wild‐type cells were treated with rapamycin during starvation (left panel). Phosphorylated (Atg13‐P) and dephosphorylated (Atg13) Atg13 was monitored at indicated time points by whole cell extraction and western blot analysis using α‐Atg13 and α‐Cdc11 antibodies.


( A ) PKA ‐ dependent regulation of autophagic response under ‐ acid starvation . Wild ‐ type , rho0 , pka , ras2G19V ‐ expressing harbouring prATG8 ‐ GFP ‐ ATG8 ( upper panels ) or prATG8 ‐ GFP ( lower panels ) were exposed to ‐ acid starvation medium supplemented with galactose . PKA activity in pka was inhibited by addition of 1NM ‐ PP1 ( PP1 ; 1 μg / ml ) . Samples were analysed as described in Figure 1A ; autophagic flux was determined after 3 6 h .

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(A) PKA‐dependent regulation of the autophagic response under amino‐acid starvation. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose. PKA activity in pka was inhibited by addition of 1NM‐PP1 (PP1; 1 μg/ml). Samples were analysed as described in Figure 1A; autophagic flux was determined after 3 and 6 h.


( B ) In vivo activity of PKA . Wild ‐ type , rho0 , pka , ras2G19V ‐ expressing harbouring 6xMYC ‐ cki12 − 200 ( S125 / 130A ) ( Cki1 ) were grown in galactose medium . When indicated , wild ‐ type were grown in galactose medium in presence of antimycin A ( AA ) or oligomycin ( O ) for 6 h . PKA ‐ dependent phosphorylation of Cki1 was analysed by whole extraction western blot using a α ‐ Myc antibody ( upper panels ) . of phosphorylated ( Cki1 ‐ P ) non ‐ phosphorylated ( Cki1 ) forms of Cki1 relative to wild ‐ type ( wt = 1 ) ( lower panels ) .

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(B)In vivo activity of PKA. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring 6xMYC‐cki12−200(S125/130A) (Cki1) were grown in galactose medium. When indicated, wild‐type cells were grown in galactose medium in the presence of antimycin A (AA) or oligomycin (O) for 6 h. PKA‐dependent phosphorylation of Cki1 was analysed by whole cell extraction and western blot analysis using a α‐Myc antibody (upper panels). Ratio of phosphorylated (Cki1‐P) and non‐phosphorylated (Cki1) forms of Cki1 relative to wild‐type cells (wt=1) (lower panels).


( C ) PKA inhibition restores autophagic flux , but not ATG8 induction in presence of mitochondrial dysfunction . Wild ‐ type , rho0 , pka , pka rho0 expressing prATG8 ‐ GFP ‐ ATG8 were treated as described in ( A ) . Samples were analysed as described in Figure 1A . means s . d . of four ( n = 4 ) independent experiments are indicated in ( A - C ) .

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(C) PKA inhibition restores autophagic flux, but not ATG8 induction in the presence of mitochondrial dysfunction. Wild‐type, rho0, pka, and pka rho0 cells expressing prATG8‐GFP‐ATG8 were treated as described in (A). Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated in (A-C).


( A ) ATG8 induction is independent of Atg1 - Atg13 autophagic flux . Wild ‐ type , rho0 , Δatg1 , Δatg7 , Δatg9 , Δatg11 harbouring prATG8 ‐ GFP ‐ ATG8 were analysed as described in Figure 1A . means s . d . of four ( n = 4 ) independent experiments are indicated .

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(A)ATG8 induction is independent of the Atg1-Atg13 complex and autophagic flux. Wild‐type, rho0, Δatg1, Δatg7, Δatg9, and Δatg11 cells harbouring prATG8‐GFP‐ATG8 were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated.


( B ) Atg1 Atg13 recruitment to PAS depends on mitochondrial function . Wild ‐ type rho0 harbouring prATG8 ‐ GFP ‐ ATG8 prATG1 ‐ ATG1 ‐ mCherry ( upper panels ) or prATG13 ‐ ATG13 ‐ mCherry ( lower panels ) were exposed to ‐ acid starvation medium supplemented with galactose for 3 h . Wild ‐ type were treated with antimycin A ( AA 30 ′ ) after 2 . 5 h of starvation for 30 min or with oligomycin ( O ) for 3 h of starvation . Arrowheads indicate position of GFP ‐ Atg8 . Transmission microscopy were superimposed to visualize boundaries . Scale bar represents 1 . 5 μm .

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(B) Atg1 and Atg13 recruitment to the PAS depends on mitochondrial function. Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 and prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose for 3 h. Wild‐type cells were treated with antimycin A (AA 30′) after 2.5 h of starvation for 30 min or with oligomycin (O) for 3 h of starvation. Arrowheads indicate the position of GFP‐Atg8 puncta. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Scale bar represents 1.5 μm.


( C ) Steady ‐ state of Atg1 ‐ Atg13 ‐ mCherry during ‐ acid starvation . Wild ‐ type , rho0 , Δatg7 expressing prATG1 ‐ ATG1 ‐ mCherry ( upper panels ) or prATG13 ‐ ATG13 ‐ mCherry ( lower panels ) were exposed treated as described in ( B ) analysed by whole extraction western blot using α ‐ dsRed α ‐ Cdc11 antibodies .

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(C) Steady‐state levels of Atg1‐ and Atg13‐mCherry during amino‐acid starvation. Wild‐type, rho0, and Δatg7 cells expressing prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed and treated as described in (B) and analysed by whole cell extraction and western blot analysis using α‐dsRed and α‐Cdc11 antibodies.


C , Growth curves for E . coli 113 / 3 in defined media in presence or absence of B12 ( o , r espectively ) . When challenged with 40 nM isolated R - domain ( ) , growth was largely impaired whereas growth in presence of 40 nM ColE9 W39A ( ▫ ) a pproached that of no colicin control , consistent with OmpF threading causing disengagement of ColE9 from BtuB . Error bars represent standard deviation across three biological replicates .

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C, Growth curves for E. coli 113/3 in defined media and in the presence or absence of B12 ( and o, respectively). When challenged with 40 nM isolated R-domain ( ), cell growth was largely impaired whereas growth in the presence of 40 nM ColE9 W39A (▫) approached that of the no colicin control, consistent with OmpF threading causing the disengagement of ColE9 from BtuB. Error bars represent the standard deviation across three biological replicates.


ColE9 * denotes ColE9 H551A , an active site mutation that inactivates toxin . A , Widefield microscopy of E . coli JM83 labelled with ColE9 * K469CAF647 ( 1 . 5 µM ) Hoechst stain ( 20 µM ) for 30 min at 37 ° C with or without trypsin treatment with or without prior treatment with CCCP . Each panel shows same in DIC ( grey ) , Hoechst stain ( blue ) ColE9 * K469CAF647 ( red ) . Overlays of Hoechst ColE9 * K469CAF647 are also shown . ColE9 * K469CAF647 remains bound to OM in presence of CCCP but this signal is lost on treatment with trypsin . Trypsin treatment in absence of CCCP yields some - associated ColE9 * K469CAF647 that likely represents internalised molecules ( see text ) . Scale bar , 1 µm .

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ColE9* denotes ColE9 H551A, an active site mutation that inactivates the toxin. A, Widefield fluorescence microscopy images of E. coli JM83 cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst DNA stain (20 µM) for 30 min at 37 °C with or without trypsin treatment with or without prior treatment with CCCP. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and ColE9* K469CAF647 fluorescence (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. ColE9* K469CAF647 remains bound to the OM in the presence of CCCP but this signal is lost on treatment with trypsin. Trypsin treatment in the absence of CCCP yields some cell-associated ColE9* K469CAF647 fluorescence that likely represents internalised molecules (see text). Scale bar, 1 µm.


C , Box whisker plots showing mean pixel intensities for ColE9 * K469CAF647 per measured for indicated condition used , whiskers represent minimum maximum mean pixel , box shows 1st 3rd quartile with median shown as a . From left - to - right : E . coli JM83 ; E . coli btuB deletion showing loss of ColE9 * K469CAF647 - associated ; E . coli JM83 in presence of CCCP ; E . coli JM83 following trypsin treatment showing significant ColE9 * K469CAF647 remains associated with indicative of import ; E . coli JM83 treated with CCCP trypsin showing complete loss of internalised ColE9 * K469CAF647 . n , number of used , typically from 2 - 4 biological replicates . * * * * , indicates a t value < 0 . 05 in a student T - as a statistically significant result , ns indicates no significant difference as determined by student T - .

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C, Box and whisker plots showing mean pixel intensities for ColE9* K469CAF647 fluorescence per cell measured for the indicated cells and condition used, whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. From left-to-right: E. coli JM83 cells; E. coli btuB deletion strain showing loss of all ColE9* K469CAF647 cell-associated fluorescence; E. coli JM83 cells in the presence of CCCP; E. coli JM83 cells following trypsin treatment showing significant ColE9* K469CAF647 fluorescence remains associated with cells indicative of import; E. coli JM83 cells treated with CCCP and trypsin showing the complete loss of internalised ColE9* K469CAF647 fluorescence. n, number of cells used, typically from 2-4 biological replicates. ****, indicates a t value <0.05 in a student T-test as a statistically significant result, ns indicates no significant difference as determined by student T-test.


B , Overnight plate assays comparing cytotoxic activity of wild - type ColE9 with ColE9 K469C labelled with AF488 , AF568 or AF647 against a lawn of E . coli with either OmpF ( JW2203 ) or OmpC ( JW0912 ) in outer ( shown as + OmpF + OmpC , respectively ) . Each plate was spotted with a serial dilution of colicin ( 10 µM - 2 . 4 pM , 4 - fold dilution per spot ) . Wild - type ColE9 was more active against E . coli with OmpF in outer than OmpC . Labelling ColE9 K469C with fluorophores reduced colicin activity in with OmpF in outer but largely abolished colicin activity in with OmpC .

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B, Overnight plate assays comparing the cytotoxic activity of wild-type ColE9 with ColE9 K469C labelled with AF488, AF568 or AF647 against a lawn of E. coli with either OmpF (JW2203) or OmpC (JW0912) in the outer membrane (shown as +OmpF and +OmpC, respectively). Each plate was spotted with a serial dilution of the colicin (10 µM - 2.4 pM, 4-fold dilution per spot). Wild-type ColE9 was more active against E. coli cells with OmpF in the outer membrane than OmpC. Labelling ColE9 K469C with fluorophores reduced colicin activity in cells with OmpF in the outer membrane but largely abolished colicin activity in cells with OmpC.


C , Widefield microscopy of E . coli JW2203 ( + OmpF ) JW0912 ( + OmpC ) labelled with ColE9 * K469CAF647 ( 1 . 5 µM ) Hoechst stain ( 20 µM ) for 30 min at 37 ° C with or without trypsin treatment . Each panel shows same in DIC ( grey ) , Hoechst stain ( blue ) of ColE9 * K469CAF647 ( red ) . Overlays of Hoechst ColE9 * K469CAF647 are also shown . Data show that following trypsin - treatment , significant ColE9 * K469CAF647 remains associated with ompF - expressing E . coli whereas little or no remains associated with ompC - expressing E . coli . Scale bar , 1 µm . D , Box whisker plots of E . coli JW2203 E . coli JW0912 labelled with ColE9 * K469CAF647 with without trypsin treatment : whiskers represent minimum maximum mean pixel , box shows 1st 3rd quartile with median shown as a . Microscopy data were collected as in c . mean pixel of ColE9 * K469CAF647 per was measured for each condition . n , number of , typically from 3 or 4 biological replicates . Data show that ColE9 * K469CAF647 translocates across OM through OmpF that this is impeded when E . coli has OmpC in outer .

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C, Widefield fluorescence microscopy images of E. coli JW2203 (+OmpF) and JW0912 (+OmpC) cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst stain (20 µM) for 30 min at 37 °C with or without trypsin treatment. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and fluorescence of ColE9* K469CAF647 (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. Data show that following trypsin-treatment, significant ColE9* K469CAF647 fluorescence remains associated with ompF-expressing E. coli whereas little or no fluorescence remains associated with ompC-expressing E. coli. Scale bar, 1 µm. D, Box and whisker plots of E. coli JW2203 and E. coli JW0912 cells labelled with ColE9* K469CAF647 with and without trypsin treatment: whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. Microscopy data were collected as in c. The mean pixel intensity of ColE9* K469CAF647 per cell was measured for each cell condition. n, number of cells, typically from 3 or 4 biological replicates. Data show that ColE9* K469CAF647 translocates across the OM through OmpF and that this is impeded when E. coli has OmpC in the outer membrane.


C . TIRF microscopy revealed time courses of CAR - GFP microcluster formation as CAR T spread on CD19 ICAM - 1 - coated SLBs .

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C. TIRF microscopy revealed time courses of the CAR-GFP microcluster formation as CAR T cells spread on the CD19 and ICAM-1-coated SLBs.


D . Enrichment of phosphor - tyrosine ( pY ) at CAR microclusters . CAR T were fixed 20 min after being plated on SLB , followed by with an anti - pan phosphor - tyrosine antibody . No CD19 was included in control experiment ( only ICAM - 1 ) . Scale bar : 2 μm .

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D. Enrichment of phosphor-tyrosine (pY) at CAR microclusters. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-pan phosphor-tyrosine antibody. No CD19 was included in the control experiment (only ICAM-1). Scale bar: 2 μm.


T expressing GFP - CAR were plated on CD19 ICAM - 1 coated SLBs . TIRF microscopy was implemented to visualize CAR localization . A . CAR microclusters underwent centripetal movement to form cSMAC ( upper panel ) or remained separated ( lower panel ) . B . of categories regarding formation of CAR cSMAC . Unstable cSMACs are defined by a cSMAC duration below 12 min . Data are shown as mean ± SD . n = 3 independent experiments . 37 , 38 , 47 were quantified in experiments . C . of time from landing to start of retrograde flow . Data are shown as mean ± SD . n = 33 ( stable ) , 31 ( unstable ) , 10 ( kinapse ) , 41 ( no cSMAC ) . Results are pooled from three independent experiments .

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T cells expressing GFP-CAR were plated on CD19 and ICAM-1 coated SLBs. TIRF microscopy was implemented to visualize CAR localization. A. CAR microclusters underwent centripetal movement to form cSMAC (upper panel) or remained separated (lower panel). B. Quantification of the categories regarding the formation of CAR cSMAC. Unstable cSMACs are defined by a cSMAC duration below 12 min. Data are shown as mean ± SD. n = 3 independent experiments. 37, 38, and 47 cells were quantified in individual experiments. C. Quantification of time from cell landing to the start of retrograde flow. Data are shown as mean ± SD. n = 33 (stable), 31 (unstable), 10 (kinapse), and 41 (no cSMAC) cells. Results are pooled from three independent experiments.


D . Localization of CAR CD45 in immunological synapse . CAR T were fixed 20 min after being plated on SLB , followed by with an anti - CD45 antibody . E . scan of CAR CD45 across long axis of immunological synapse . Shown are normalized .

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D. Localization of CAR and CD45 in the immunological synapse. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-CD45 antibody. E. Line scan of CAR and CD45 across the long axis of the immunological synapse. Shown are the normalized fluorescence intensity.


A . WT or LAT - deficient Jurkat ( Jcam2 . 5 ) expressing CAR - GFP were stimulated on SLB - coated with OKT3 ( anti - TCR antibody ) or CD19 ( CAR antigen ) ; in both conditions , lipid bilayers were also coated with ICAM - 1 to facilitate attachment . TIRF microscopy revealed clustering of Ax647 - labeled streptavidin - Biotin - OKT3 or CD19 , which serves as a probe for TCR or CAR respectively . Scale bar , 2 µm . B . of clustering as normalized variance . n = 100 . Shown are mean ± SD . Statistical : unpaired two - t - . * * * * p < 0 . 0001 . * * 0 . 005 < p < 0 . 05 .

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A. WT or LAT-deficient Jurkat cells (Jcam2.5) expressing CAR-GFP were stimulated on SLB-coated with OKT3 (anti-TCR antibody) or CD19 (CAR antigen); in both conditions, the lipid bilayers were also coated with ICAM-1 to facilitate cell attachment. TIRF microscopy revealed clustering of Ax647-labeled streptavidin-Biotin-OKT3 or CD19, which serves as a probe for TCR or CAR respectively. Scale bar, 2 µm. B. Quantification of clustering as normalized variance. n = 100 cells. Shown are the mean ± SD. Statistical test: unpaired two-tailed t-test. **** p < 0.0001. ** 0.005 < p < 0.05.


B . WT or LAT - deficient ( Jcam2 . 5 ) expressing 3rd or 1st generation CAR were stimulated on supported lipid bilayers coated with CD19 ICAM - 1 . TIRF microscopy revealed clustering of CAR Ax647 - labeled streptavidin - Biotin - CD19 . Scale bar , 2 µm . C . of clustering level of Ax647 - labeled streptavidin - Biotin - CD19 . Shown are means ± SD . Clustering is quantified as normalized variance . N = 50 for CAR 3rd WT 1st WT , 63 for CAR 3rd lat - , 51 for CAR 1st lat - . * * : 0 . 0005 < p < 0 . 005 . Statistical : unpaired two - t - .

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B. WT or LAT-deficient (Jcam2.5) cells expressing the 3rd or 1st generation CAR were stimulated on supported lipid bilayers coated with CD19 and ICAM-1. TIRF microscopy revealed clustering of CAR and Ax647-labeled streptavidin-Biotin-CD19. Scale bar, 2 µm. C. Quantification of clustering level of Ax647-labeled streptavidin-Biotin-CD19. Shown are means ± SD. Clustering is quantified as normalized variance. N = 50 cells for CAR 3rd WT and 1st WT, 63 cells for CAR 3rd lat-, and 51 cells for CAR 1st lat-. **: 0.0005 < p < 0.005. Statistical test: unpaired two-tailed t-test.


CAR ( 3rd generation ) T were plated on supported lipid bilayers coated with CD19 ICAM - 1 imaged by TIRF microscopy . A . Gads was recruited to CAR microclusters in both wild - type LAT - deficient ( Jcam2 . 5 ) . n = 33 or 34 . Shown are means ± SE . n . s . , p = 0 . 58 by unpaired two - t - . As a control , Gads is not recruited to LAT - deficient activated by OKT3 .

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CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. A. Gads was recruited to CAR microclusters in both the wild-type and LAT-deficient (Jcam2.5) cells. n = 33 or 34 cells. Shown are the means ± SE. n.s., p = 0.58 by unpaired two-tailed t-test. As a control, Gads is not recruited to LAT-deficient cells activated by OKT3.


CAR ( 3rd generation ) T were plated on supported lipid bilayers coated with CD19 ICAM - 1 imaged by TIRF microscopy . B . Phosphorylated SLP76 , recognized by an anti - SLP76 pY145 antibody , was enriched in CAR microclusters in both wild - type LAT - deficient . In contrast , phosphorylated SLP76 was not enriched in TCR microclusters in LAT - deficient . n = 50 . Shown are means ± SE . n . s . , p = 0 . 95 by unpaired two - t - .

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CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. B. Phosphorylated SLP76, recognized by an anti-SLP76 pY145 antibody, was enriched in CAR microclusters in both the wild-type and LAT-deficient cells. In contrast, phosphorylated SLP76 was not enriched in TCR microclusters in LAT-deficient cells. n = 50 cells. Shown are the means ± SE. n.s., p = 0.95 by unpaired two-tailed t-test.


CAR ( 3rd generation ) T were plated on supported lipid bilayers coated with CD19 ICAM - 1 imaged by TIRF microscopy . C . Actin remodeling was induced in LAT - deficient by CD19 - , but not OKT3 - coated SLB . No ICAM - 1 was supplemented to SLB in this experiment to rule out costimulation of actin via LFA - 1 / ICAM - 1 pathway . Actin network was revealed by F - tractin - mScarlet . n = 6 . Shown are means ± SD . n . s . , p = 0 . 68 , 0 . 98 by unpaired two - t - .

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CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy. C. Actin remodeling was induced in LAT-deficient cells by CD19-, but not OKT3-coated SLB. No ICAM-1 was supplemented to SLB in this experiment to rule out the costimulation of actin via the LFA-1/ICAM-1 pathway. Actin network was revealed by F-tractin-mScarlet. n = 6 cells. Shown are the means ± SD. n.s., p = 0.68, and 0.98 by unpaired two-tailed t-test.


A . - conjugation . Wild - type or Lat - null ( J . LAT ) Jurkat T expressing CAR - GFP were mixed with Raji expressing mCherry - CAAX at a 1 : 1 for 1 hr before being fixed . Three independent experiments were performed over 90 were scored in each experiment . Scale bar : 5 µm .

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A. Cell-cell conjugation assay. Wild-type or Lat-null (J.LAT) Jurkat T cells expressing CAR-GFP were mixed with Raji cells expressing mCherry-CAAX at a 1:1 ratio for 1 hr before being fixed. Three independent experiments were performed and over 90 cells were scored in each experiment. Scale bar: 5 µm.


B . CAR T were incubated with Raji B at a 1 : 1 for 24 hrs IL - 2 from supernatant were measured by ELISA . C . CAR T were incubated with Raji B at a 1 : 1 fixed at 24hr , stained with an anti - CD69 antibody , analyzed by FACS . CD69 positive were scored within CAR - GFP + population .

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B. CAR T cells were incubated with Raji B cells at a 1:1 ratio for 24 hrs and IL-2 from the supernatant were measured by ELISA. C. CAR T cells were incubated with Raji B cells at a 1:1 ratio and fixed at 24hr, stained with an anti-CD69 antibody, and analyzed by FACS. CD69 positive cells were scored within the CAR-GFP+ population.


A . Western blot showed that LAT is efficiently depleted in human T using CRISPR . Shown are means ± SD . n = 4 independent experiments .

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A. Western blot showed that LAT is efficiently depleted in human primary T cells using CRISPR. Shown are the means ± SD. n = 4 independent experiments.


B . CAR T were stimulated on SLBs coated with biotin - OKT3 ( left ) or biotin - CD19 ( right ) supplemented with ICAM - 1 . TIRF microscopy revealed clustering of Alexa647 - labeled streptavidin - Biotin - OKT3 or CD19 . Scale bar , 5 µm . C . of clustering of OKT3 or CD19 as normalized variance . n > 200 from two independent donors were scored for each condition . Central band : mean ; Box : quartiles ; Whisker : of distribution . * * * * , p < 0 . 0001 ; n . s . p = 0 . 48 .

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B. CAR T cells were stimulated on SLBs coated with biotin-OKT3 (left) or biotin-CD19 (right) supplemented with ICAM-1. TIRF microscopy revealed clustering of Alexa647-labeled streptavidin-Biotin-OKT3 or CD19. Scale bar, 5 µm. C. Quantification of clustering of OKT3 or CD19 as normalized variance. n > 200 cells from two independent donors were scored for each condition. Central band: mean; Box: quartiles; Whisker: rest of the distribution. ****, p < 0.0001; n.s. p = 0.48.


D . Immunofluorescence revealed - localized phosphorylated SLP76 . were plated on supported lipid bilayer coated with ICAM - 1 only ( control ) or ICAM - 1 supplemented with CD19 or OKT3 as indicated . dashed circle indicated boundary . Scale bar , 5 µm . E . of pSLP76 level on . n > 200 were scored , were isolated from two independent donors . Central band : mean ; Box : quartiles ; Whisker : of distribution . * * * * , p < 0 . 0001 ; n . s . p = 0 . 11 .

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D. Immunofluorescence analysis revealed membrane-localized phosphorylated SLP76. Cells were plated on supported lipid bilayer coated with ICAM-1 only (control) or ICAM-1 supplemented with CD19 or OKT3 as indicated. The white dashed circle indicated cell boundary. Scale bar, 5 µm. E. Quantification of pSLP76 level on the membrane. n > 200 cells were scored, and the cells were isolated from two independent donors. Central band: mean; Box: quartiles; Whisker: rest of the distribution. ****, p < 0.0001; n.s. p = 0.11.


F . T were incubated with Raji B at a 4 : 1 IL - 2 production was measured by ELISA at 24 hr . Data were pooled from three donors . * , 0 . 005 < p < 0 . 05 ; * * , 0 . 0005 < p < 0 . 005 . Unpaired two - t - was applied .

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F. T cells were incubated with Raji B cells at a 4:1 ratio and IL-2 production was measured by ELISA at 24 hr. Data were pooled from three donors. *, 0.005 < p < 0.05; **, 0.0005 < p < 0.005. Unpaired two-tailed t-test was applied.


exclusion chromatography ( SEC ) results for MICU2 _ WT a set of mutants in presence of Ca2 + monitored at 280 nm absorption wavelength . exclusion chromatography ( SEC ) results for MICU2 _ WT a set of mutants in presence of EGTA monitored at 280 nm absorption wavelength .

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Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of Ca2+ monitored at 280 nm absorption wavelength. Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength.


MICU2 _ WT ( A ) , MICU2 _ K172A ( B ) were pulled - down by GST - MICU1 both in absence presence of 2 mM Ca2 + . MICU2 _ EF1mut was not pulled - down by GST - MICU1 in presence of 2 mM Ca2 + ( C ) , while MICU2 _ EF2mut was pulled - down by GST - MICU1 both in absence presence of 2 mM Ca2 + ( D ) . MICU2 _ R352A was not pulled - down by GST - MICU1 in absence of 2 mM Ca2 + ( E ) , while MICU2 _ E329A was pulled - down by GST - MICU1 both in absence presence of 2 mM Ca2 + ( F ) . MICU2 _ D185A was partially pulled - down by GST - MICU1 in presence of Ca2 + , while they showed a strong interaction in absence of 2 mM Ca2 + . MICU2 _ E196K was not pulled - down by GST - MICU1 in presence of 2 mM Ca2 + , while a strong interaction was observed in absence of 2 mM Ca2 + .

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MICU2_WT (A), MICU2_K172A (B) were pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+. MICU2_EF1mut was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+ (C), while MICU2_EF2mut was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (D). MICU2_R352A was not pulled-down by GST-MICU1 in the absence of 2 mM Ca2+ (E), while MICU2_E329A was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (F). MICU2_D185A was partially pulled-down by GST-MICU1 in the presence of Ca2+, while they showed a strong interaction in the absence of 2 mM Ca2+. MICU2_E196K was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+, while a strong interaction was observed in the absence of 2 mM Ca2+.


GST - MICU1 _ EF1mut failed to pull down MICU2 _ WT in absence of 2 mM Ca2 + , whereas they displayed a strong interaction in presence of 2 mM Ca2 + . GST - MICU1 _ F383A & H385A failed to pull - down MICU2 _ WT in presence of Ca2 + , whereas they displayed a strong interaction in absence of 2 mM Ca2 + . C GST - MICU1 _ E242K failed to pull - down MICU2 _ WT in absence of Ca2 + , whereas they displayed a strong interaction in presence of 2 mM Ca2 + . GST - MICU1 _ D231A partly pulled - down MICU2 _ WT in absence of Ca2 + , whereas they displayed a strong interaction in presence of 2 mM Ca2 + . GST - MICU1 _ F383A failed to pull down MICU2 _ WT in presence of Ca2 + , whereas they displayed a strong interaction in absence of 2 mM Ca2 + . GST - MICU1 _ H385A exhibited a strong interaction with MICU2 both in absence presence of 2 mM Ca2 + .

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GST-MICU1_EF1mut failed to pull down MICU2_WT in the absence of 2 mM Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A & H385A failed to pull-down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. C GST-MICU1_E242K failed to pull-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_D231A partly pulled-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A failed to pull down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. GST-MICU1_H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2+.


ITC analyses of MICU1 MICU2 binding properties in presence of 2 mM EGTA ( G ) CaCl2 ( H ) .

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ITC analyses of MICU1 and MICU2 binding properties in the presence of 2 mM EGTA (G) and CaCl2 (H).


A . Chemical structure of Nec - 1 . schematic diagram was drawn by S . Lee using ChemDraw Professional 15 . 0 . software .

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A. Chemical structure of Nec-1. The schematic diagram was drawn by S. Lee using ChemDraw Professional 15.0. software.


B , C . MTT cytotoxicity . ( B ) HT22 was treated with various concentrations of Nec - 1 ( 0 - 200 μM ) . Aggregated Aβ ( 1 - 42 ) was used as positive control for induction of neural . data represented mean ± standard deviation of three independent experiments performed in triplicate .

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B, C. MTT cytotoxicity assay. (B) HT22 cell line was treated with various concentrations of Nec-1 (0 - 200 μM). Aggregated Aβ(1-42) was used as the positive control for the induction of neural cell death. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.


B , C . MTT cytotoxicity . ( C ) HT22 , BV2cell lines astrocytes were pre - treated with Nec - 1 ( 50 μM ) 15 minutes prior to application of 10 μM of pre - aggregated Aβ ( 1 - 42 ) for indicated times . proliferation was then measured by MTT . data represented mean ± standard deviation of three independent experiments performed in triplicate .

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B, C. MTT cytotoxicity assay. (C) HT22, BV2cell lines and primary astrocytes were pre-treated with Nec-1 (50 μM) 15 minutes prior to application of 10 μM of pre-aggregated Aβ(1-42) for indicated times. Cell proliferation was then measured by MTT assay. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.


D , E . Neural measured by with Live / Dead Viability / Cytotoxicity Kit ( Molecular Probes ) . Pre - treatment of with 50 μM Nec - 1 preceded application of 10 μM pre - aggregated Aβ ( 1 - 42 ) by 15 minutes . was performed 24 hours after Aβ ( 1 - 42 ) application . ( D ) Representation of stained . Scale bar = 500 μm ( E ) of dead stained by EthD - 1 .

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D, E. Neural cell death levels measured by staining with the Live/Dead Viability/Cytotoxicity Assay Kit (Molecular Probes). Pre-treatment of cells with 50 μM Nec-1 preceded the application of 10 μM pre-aggregated Aβ(1-42) by 15 minutes. The assay was performed 24 hours after Aβ(1-42) application. (D) Representation of the stained cells. Scale bar = 500 μm (E) Quantification of dead cells stained by EthD-1.


F . Internalization of Aβ ( 1 - 42 ) into HT22 BV2 lines in presence or absence of Nec - 1 pre - treatment for indicated periods .

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F. Internalization of Aβ(1-42) into HT22 and BV2 cell lines in the presence or the absence of Nec-1 pre-treatment for indicated periods.


G . expression of indicated in HT22 , BV2 lines , astrocyte stimulated with Aβ ( 1 - 42 ) in presence or absence of Nec - 1 pre - treatment for 24 hours .

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G. Cellular expression levels of indicated proteins in HT22, BV2 cell lines, and primary astrocyte stimulated with Aβ(1-42) aggregates in the presence or the absence of Nec-1 pre-treatment for 24 hours.


A - E . Behavioural of APP / PS1mice . ( B - D ) Y - maze passive avoidance on 7 - month - old APP / PS1mice after Nec - 1 administration for 12 . Average alternation ( % ) for each group ( B ) total entry number into each arm ( C ) on Y - maze . ( D ) Average latency time in seconds for each group on passive avoidance .

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A-E. Behavioural tests of APP/PS1mice. (B-D) Y-maze and passive avoidance tests on 7-month-old APP/PS1mice after Nec-1 administration for 12 weeks. Average alternation (%) for each test group (B) and total entry number into each arm (C) on Y-maze. (D) Average latency time in seconds for each test group on passive avoidance test.


A - E . Behavioural of APP / PS1mice . ( E ) Survival of Wt APP / PS1mice after injection of Nec - 1 ( 6 . 25 mg / kg ) .

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A-E. Behavioural tests of APP/PS1mice. (E) Survival of Wt and APP/PS1mice after injection of Nec-1 (6.25 mg/kg).


F - I . Behavrioural of Aβ ( 1 - 42 ) infusion mice . ( H , I ) Y - maze on Aβ ( 1 - 42 ) infusion mice model injected with Nec - 1 . Average alternation ( % ) for each group ( H ) total entry number into each arm ( I ) on Y - maze .

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F-I. Behavrioural tests of Aβ(1-42) infusion mice. (H, I) Y-maze tests on Aβ(1-42) infusion mice model injected with Nec-1. Average alternation (%) for each test group (H) and total entry number into each arm (I) on Y-maze.


A . Co - immunoprecipitation of RIPK3 with Aβ ( 1 - 42 ) in HT22 , BV2 lines , astrocytes in response to Aβ ( 1 - 42 ) . IP , immunoprecipitation ; IB , immunoblot .

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A. Co-immunoprecipitation of RIPK3 with Aβ(1-42) in HT22, BV2 cell lines, and primary astrocytes in response to Aβ(1-42) aggregates. IP, immunoprecipitation; IB, immunoblot.


B . A close - up view of interactions between Nec - 1 surrounding residues in RIPK1 . Nec - 1 is shown in green . hydrophobic residues around Nec - 1 in RIPK1 kinase domain are shown in sticks . Hydrogen bonds in this other figures are represented by yellow dashed lines . Docking score of Nec - 1 in RIPK1 was - 9 . 550 .

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B. A close-up view of the interactions between Nec-1 and surrounding residues in RIPK1. Nec-1 is shown in green. The hydrophobic residues around Nec-1 in RIPK1 kinase domain are shown in sticks. Hydrogen bonds in this and all other figures are represented by yellow dashed lines. Docking score of Nec-1 in RIPK1 was -9.550.


C . Surface treated view of 12 - mer Aβ fibrils ( 2LMO , VDW radius = 0 . 55 Å ) having hydrophobic pockets plausible for Nec - 1 binding .

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C. Surface treated view of 12-mer Aβ fibrils (2LMO, VDW radius = 0.55 Å) having hydrophobic pockets plausible for Nec-1 binding.


D . Docked structure of 12 - mer Aβ fibrils ( 2LMO ) with 4 molecules of Nec - 1 . Docking scores for four top - ranked Glide docking poses of Nec - 1 in Aβ plaques ; - 8 . 338 , - 4 . 755 , - 8 . 493 , - 6 . 754 in clockwise order starting from top - left corner .

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D. Docked structure of 12-mer Aβ fibrils (2LMO) with 4 molecules of Nec-1. Docking scores for four top-ranked Glide docking poses of Nec-1 in Aβ plaques; -8.338, -4.755, -8.493, and -6.754 in clockwise order starting from the top-left corner.


E . Surface plasmon resonance sensorgrams of Nec - 1 targeting Aβ ( 1 - 42 ) ( left ) , corresponding dissociation fitting curve from saturated region ( right ) under various concentrations of Nec - 1 .

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E. Surface plasmon resonance sensorgrams of Nec-1 targeting Aβ(1-42) aggregates (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.


4 - month - old APP / PS1 ( n = 13 , ) - matched wild - type ( Wt , n = 14 , ) mice were intravenously injected with Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) for 12 . After completion of behavioural , brains of each group of mice were subjected to . A . ThS - stained insoluble Aβplaques in whole brain hippocampal region of each group . Scale bars = 1 mM ( upper ) , 200 μM ( lower ) . B - G . Statistics of ThS - positive Aβplaques in brains of APP / PS1mice . Total numbers areas of ThS - positive Aβplaques in whole brains ( B C ) , cortex ( D E ) hippocampus ( F G ) .

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4-month-old APP/PS1 (n = 13, male) and age-matched wild-type (Wt, n = 14, male) mice were intravenously injected with Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for 12 weeks. After the completion of behavioural tests, the brains of each group of mice were subjected to analysis.A. ThS-stained insoluble Aβplaques in whole brain and the hippocampal region of each group. Scale bars = 1 mM (upper), 200 μM (lower).B-G. Statistics of ThS-positive Aβplaques in the brains of APP/PS1mice. Total numbers and areas of ThS-positive Aβplaques in the whole brains (B and C), cortex (D and E) and hippocampus (F and G).


A . Immunohistochemical of cortical hippocampal regions of brains in wild - type ( Wt ) APP / PS1mice after administration of Nec - 1 ( 6 . 25 mg / kg ) or vehicle ( 2 . 5 % DMSO in PBS ) . Diffuse plaques in brain sections were stained by anti - Aβ antibody ( 6E10 clone , green colour ) anti - GFAP antibody ( red colour ) . Hoechst 33342 ( blue colour ) was applied for counterstaining . Scale bars = 200 μM .

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A. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). Diffuse plaques in the brain sections were stained by anti-Aβ antibody (6E10 clone, green colour) and anti-GFAP antibody (red colour). Hoechst 33342 (blue colour) was applied for nuclear counterstaining. Scale bars = 200 μM.


B - D . Dot blot of total Aβ ( anti - Aβ : 6E10 clone , also recognizes APP ) oligomer ( anti - amyloidogenic oligomer A11 ) in cortical ( B ) hippocampal ( C ) region of indicated groups . ( D ) of total Aβ oligomer in cortical hippocampal region of indicated groups . Relative of each band was determined by densitometry of dot blots ( B C ) using ImageJ software normalized to vehicle - administered APP / PS1mouse group .

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B-D. Dot blot analysis of total Aβ (anti-Aβ: 6E10 clone, also recognizes APP) and protein oligomer (anti-amyloidogenic protein oligomer A11) in the cortical (B) and hippocampal (C) region of indicated groups. (D) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups. Relative intensity of each band was determined by densitometry of dot blots (B and C) using ImageJ software and normalized to vehicle-administered APP/PS1mouse group.


E . Sandwich ELISA of Aβ - soluble - insoluble fractions from cortical hippocampal lysates of APP / PS1mice .

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E. Sandwich ELISA of Aβ-soluble and -insoluble fractions from cortical and hippocampal lysates of APP/PS1mice.