C . ) Live epifluorescence microscopy of PfMev that are expressing api - SFG ( green ) P . falciparum DPCK - mCherry ( red ) with DAPI marking nuclei ( blue ) . borders of red blood are marked with dotted blue lines , respectively . Microscopy represent fields that are 10µm long by 10µm wide .

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C.) Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG (green) and the P. falciparum DPCK-mCherry protein (red) with DAPI marking the nuclei (blue). The borders of the red blood cell and parasite are marked with dotted blue and white lines, respectively. Microscopy images represent fields that are 10µm long by 10µm wide.


B . ) Live epifluorescence microscopy of PfMev that are expressing api - SFG ( green ) api - EcDPCK - mCherry ( red ) with DAPI marking nuclei ( blue ) . Data information : microscopy represent fields that are 10µm long by 10µm wide .

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B.) Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.


D . ) Live epifluorescence microscopy of PfMev api - EcDPCK - mCherry Δdpck that is expressing api - SFG ( green ) api - EcDPCK - mCherry ( red ) with DAPI marking nuclei ( blue ) . E . ) Live epifluorescence microscopy of PfMev api - EcDPCK - mCherry Δdpck after treatment with 1x azithromycin for 7 days under supplementation with 50μM mevalonate . This expresses api - SFG ( green ) api - EcDPCK - mCherry ( red ) with DAPI marking nuclei ( blue ) . Data information : microscopy represent fields that are 10µm long by 10µm wide .

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

D.) Live epifluorescence microscopy of the PfMev api-EcDPCK-mCherry Δdpck parasite line that is expressing api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). E.) Live epifluorescence microscopy of the PfMev api-EcDPCK-mCherry Δdpck line after treatment with 1x azithromycin for 7 days under supplementation with 50μM mevalonate. This parasite line expresses api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.


F . ) Attempted PCR detection of ldh , sufB , cox1 genes from ( N ) , apicoplast ( A ) , mitochondrial ( M ) genomes , respectively . We failed to amplify sufB from azithromycin - treated ( + Azith ) PfMev api - EcDPCK - mCherry Δdpck , indicating loss of apicoplast organellar genome .

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

F.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We failed to amplify sufB from the azithromycin-treated (+ Azith) PfMev api-EcDPCK-mCherry Δdpck parasites, indicating the loss of the apicoplast organellar genome.


B . ) Live epifluorescence microscopy of PfMev EcDPCK - mCherry . This expresses api - SFG ( green ) EcDPCK - mCherry ( red ) with DAPI marking nuclei ( blue ) . D . ) Live epifluorescence microscopy of PfMev EcDPCK - mCherry Δdpck . This expresses api - SFG ( green ) EcDPCK - mCherry ( red ) with DAPI marking nuclei ( blue ) . Data information : microscopy represent fields that are 10µm long by 10µm wide .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.) Live epifluorescence microscopy of the PfMev EcDPCK-mCherry line. This parasite line expresses api-SFG (green) and EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). D.) Live epifluorescence microscopy of the PfMev EcDPCK-mCherry Δdpck line. This parasite line expresses api-SFG (green) and EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.


E . ) Attempted PCR detection of ldh , sufB , cox1 genes from ( N ) , apicoplast ( A ) , mitochondrial ( M ) genomes , respectively . We were successful in amplifying sufB from PfMev EcDPCK - mCherry Δdpck grown under continuous supplementation with 50μM mevalonate , indicating retention of apicoplast organellar genome .

E.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We were successful in amplifying sufB from the PfMev EcDPCK-mCherry Δdpck parasite line grown under continuous supplementation with 50μM mevalonate, indicating retention of the apicoplast organellar genome.


C . ) Live epifluorescence microscopy of PfMev CLD - EcDPCK - mCherry - apt Δdpck . This expresses api - SFG ( green ) CLD - EcDPCK - mCherry ( red ) with DAPI marking nuclei ( blue ) . This was grown in presence of 0 . 5μM aTc . D . ) PfMev CLD - EcDPCK - mCherry - apt Δdpck grown in absence of aTc for 48 hours . brightness of red image was increased to demonstrate lack of mCherry signal associated with . E . ) PfMev CLD - EcDPCK - mCherry - apt Δdpck grown in presence of 0 . 5μM aTc , in presence of 0 . 5μM Shield1 ( Shld ) for 48 hours . F . ) PfMev CLD - EcDPCK - mCherry - apt Δdpck grown in absence of aTc , in presence of 0 . 5μM Shield1 ( Shld ) for 48 hours . brightness of red image was increased to demonstrate lack of mCherry signal associated with . Data information : microscopy represent fields that are 10µm long by 10µm wide .

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

C.) Live epifluorescence microscopy of the PfMev CLD-EcDPCK-mCherry-apt Δdpck line. This parasite line expresses api-SFG (green) and CLD-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). This parasite line was grown in the presence of 0.5μM aTc. D.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the absence of aTc for 48 hours. The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite. E.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the presence of 0.5μM aTc, and in the presence of 0.5μM Shield1 (Shld) for 48 hours. F.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the absence of aTc, and in the presence of 0.5μM Shield1 (Shld) for 48 hours. The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite. Data information: All microscopy images represent fields that are 10µm long by 10µm wide.


G . ) Growth curve of PfMev CLD - EcDPCK - mCherry - apt Δdpck grown either in presence of 0 . 5μM aTc absence 0 . 5μM Shield1 ( permissive condition , blue ) , or absence of 0 . 5μM aTc presence of 0 . 5μM Shield1 ( non - permissive condition , red ) . Pantothenate was supplied at 50nM in both conditions cultures were diluted 1 : 5 on day 4 . Treatment under non - permissive conditions resulted in reduced growth beginning at day 1 ( two - way ANOVA , followed by Bonferroni ' s correction ; * , P < 0 . 05 ) . Error bars represent standard error of mean from two independent experiments , each conducted in quadruplicate .

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

G.) Growth curve of the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown either in the presence of 0.5μM aTc and the absence 0.5μM Shield1 (permissive condition, blue), or the absence of 0.5μM aTc and the presence of 0.5μM Shield1 (non-permissive condition, red). Pantothenate was supplied at 50nM in both conditions and the cultures were diluted 1:5 on day 4. Treatment under non-permissive conditions resulted in reduced parasite growth beginning at day 1 (two-way ANOVA, followed by Bonferroni's correction; *, P < 0.05). Error bars represent the standard error of the mean from two independent experiments, each conducted in quadruplicate.


A . ) Attempted PCR detection of ldh , sufB , cox1 genes from ( N ) , apicoplast ( A ) , mitochondrial ( M ) genomes , respectively . We were unsuccessful in amplifying sufB from PfMev CLD - EcDPCK - mCherry - apt Δdpck after treatment with 100nM azithromycin ( Azith ) in presence of 50μM mevalonate , indicating disruption of apicoplast organelle .

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

A.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We were unsuccessful in amplifying sufB from the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line after treatment with 100nM azithromycin (Azith) in the presence of 50μM mevalonate, indicating the disruption of the apicoplast organelle.


B . ) Live epifluorescence microscopy of apicoplast - negative PfMev CLD - EcDPCK - mCherry - apt Δdpck post treatment with 100nM azithromycin in presence of 50μM mevalonate . This expresses api - SFG ( green ) CLD - EcDPCK - mCherry construct ( red ) with DAPI marking nuclei ( blue ) . represent fields that are 10µm long by 10µm wide .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.) Live epifluorescence microscopy of the apicoplast-negative PfMev CLD-EcDPCK-mCherry-apt Δdpck line post treatment with 100nM azithromycin in the presence of 50μM mevalonate. This parasite line expresses api-SFG (green) and the CLD-EcDPCK-mCherry construct (red) with DAPI marking the nuclei (blue). Images represent fields that are 10µm long by 10µm wide.


C . ) Growth curve of PfMev CLD - EcDPCK - mCherry - apt Δdpck containing a disrupted apicoplast grown either in presence of 0 . 5μM aTc absence 0 . 5μM Shield1 ( permissive condition , blue ) , or absence of 0 . 5μM aTc presence of 0 . 5μM Shield1 ( non - permissive condition , red ) . Pantothenate ( 50nM ) mevalonate ( 50μM ) were supplied in both conditions cultures were diluted 1 : 5 on day 4 . Treatment under non - permissive conditions resulted in reduced growth beginning at day 2 ( two - way ANOVA , followed by Bonferroni ' s correction ; * * , P < 0 . 01 ) . Error bars represent standard error of mean from two independent experiments , each conducted in quadruplicate .

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

C.) Growth curve of the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line containing a disrupted apicoplast grown either in the presence of 0.5μM aTc and the absence 0.5μM Shield1 (permissive condition, blue), or the absence of 0.5μM aTc and the presence of 0.5μM Shield1 (non-permissive condition, red). Pantothenate (50nM) and mevalonate (50μM) were supplied in both conditions and the cultures were diluted 1:5 on day 4. Treatment under non-permissive conditions resulted in reduced parasite growth beginning at day 2 (two-way ANOVA, followed by Bonferroni's correction; **, P < 0.01). Error bars represent the standard error of the mean from two independent experiments, each conducted in quadruplicate.


a . Fold change in mRNA expression of members of PI3K / AKT / Foxo pathway in IFN + as compared to IFN - Tregs in microarray .

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a. Fold change in mRNA expression of members of the PI3K/AKT/Foxo pathway in IFN+ as compared to IFN- Tregs in the microarray analysis.


b . mRNA expression of members of PI3K / AKT / Foxo signaling pathway in sorted IFN + IFN - Tregs .

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b. mRNA expression of members of the PI3K/AKT/Foxo signaling pathway in sorted IFN+ and IFN- Tregs.


c . Sorted CD4 + CD25highCD127 - Tregs were stimulated in presence of IL - 2 ( ) or IL - 2 + IL - 12 ( black dots ) was isolated every 24 hours for 4 days . mRNA expression of members of PI3K / AKT / Foxo signaling pathway .

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c. Sorted CD4+CD25highCD127- Tregs were stimulated in the presence of IL-2 (white) or IL-2+IL-12 (black dots) and RNA was isolated every 24 hours for 4 days. mRNA expression of members of the PI3K/AKT/Foxo signaling pathway.


d . Representative example of expression of AKT1 , AKT2 , AKT3 , PTEN FOXO3 on sorted CD4 + CD25highCD127 - Tregs stimulated in presence of IL - 2 ( grey ) or IL - 2 + IL - 12 ( black ) day 2 for AKT1 , AKT2 , AKT3 PTEN day 3 for FOXO3 . Grey histogram represents isotype . e . Statistical of AKT1 , AKT2 , AKT3 , FOXO3 PTEN gMFI of n = 6 experiments performed . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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d. Representative example of the protein expression of AKT1, AKT2, AKT3, PTEN and FOXO3 on sorted CD4+CD25highCD127- Tregs stimulated in the presence of IL-2 (grey line) or IL-2+IL-12 (black line) and day 2 for AKT1, AKT2, AKT3 and PTEN and day 3 for FOXO3. Grey histogram represents the isotype. e. Statistical analysis of AKT1, AKT2, AKT3, FOXO3 and PTEN gMFI of n=6 experiments performed. * p<0.05, ** p<0.005, *** p<0.0005.


Sorted CD4 + CD25hiCD127 - Tregs were stimulated with anti - CD3 , anti - CD28 IL - 2 in presence or absence of IL - 12 . a . Tregs were stimulated for 50 minutes with IL - 12 or vehicle . Histograms show a representative example of n = 8 independent experiments performed of expression of phosphorylated molecules at 30 minutes after stimulation for pAKT pFoxo1 , at 40 minutes for pFoxo3 in vehicle - ( grey open histogram ) or IL - 12 - treated ( open histogram ) Tregs . Grey histograms represent isotype . time points selected were on kinetic experiments performed for a total of 50

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Sorted CD4+CD25hiCD127- Tregs were stimulated with anti-CD3, anti-CD28 and IL-2 in the presence or absence of IL-12. a. Tregs were stimulated for 50 minutes with IL-12 or vehicle. Histograms show a representative example of n=8 independent experiments performed of the expression of phosphorylated molecules at 30 minutes after stimulation for pAKT and pFoxo1, and at 40 minutes for pFoxo3 in vehicle- (grey open histogram) or IL-12-treated (dark open histogram) Tregs. Grey histograms represent the isotype. The time points selected were based on kinetic experiments performed for a total of 50 minutes


Sorted CD4 + CD25hiCD127 - Tregs were stimulated with anti - CD3 , anti - CD28 IL - 2 in presence or absence of IL - 12 . b . Tregs were stimulated in presence ( black ) or absence ( grey ) of IL - 12 expression of phosphorylated molecules was examined 16 hours later . c . gMFI of pAKT , pFoxo1 pFoxo3 of n = 6 independent experiments performed .

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

Sorted CD4+CD25hiCD127- Tregs were stimulated with anti-CD3, anti-CD28 and IL-2 in the presence or absence of IL-12. b. Tregs were stimulated in the presence (black) or absence (grey) of IL-12 and the expression of phosphorylated molecules was examined 16 hours later. c. gMFI of pAKT, pFoxo1 and pFoxo3 of n=6 independent experiments performed.


d . Foxo1 Foxo3 transcriptional targets expression on sorted CD4 + CD25hiCD127 - Tregs stimulated in presence of vehicle or IL - 12 at day 2 after stimulation ( n = 8 ) . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

d. Foxo1 and Foxo3 transcriptional targets expression on sorted CD4+CD25hiCD127-Tregs stimulated in the presence of vehicle or IL-12 at day 2 after stimulation (n=8). *p < 0.05, **p < 0.005, ***p < 0.0005.


a . Sorted CD4 + CD25hiCD127 - Tregs were stimulated in presence of vehicle ( dots ) or either SF1670 ( first row ) , SC - 79 ( second row ) , 740 Y - P ( third row ) or AS1842856 ( fourth row , black dots ) for 3 days IFNG TBX21 gene expression was examined every 24 hours . Statistical of n = 4 independent experiments performed .

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a. Sorted CD4+CD25hiCD127- Tregs were stimulated in the presence of vehicle (white dots) or either SF1670 (first row), SC-79 (second row), 740 Y-P (third row) or AS1842856 (fourth row, black dots) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=4 independent experiments performed.


b . Representative example of frequency of IFN + Foxp3 + in Tregs stimulated in presence of vehicle , SF1670 , SC - 79 , 740 Y - P or AS1842856 for 3 days after 4 hours of stimulation with PMA ionomycin . Numbers in brackets represent total numbers c . Statistical of frequency of IFN + Foxp3 + in Tregs stimulated in presence of vehicle , SF1670 , SC - 79 , 740 Y - P or AS1842856 of n = 5 experiments performed .

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

b. Representative example of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle, SF1670, SC-79, 740 Y-P or AS1842856 for 3 days after 4 hours of stimulation with PMA and ionomycin. Numbers in brackets represent total cell numbers c. Statistical analysis of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle, SF1670, SC-79, 740 Y-P or AS1842856 of n=5 experiments performed.


d . Proliferation of CFSE - labeled responder T alone ( left histogram ) or co - cultured with Tregs that have been pre - incubated with either vehicle , SF1670 , SC - 79 or 740 Y - P at day 4 after co - culture at a 1 : 2 Treg : Tresp . e . Statistical of of suppression at different Treg : Tresp ratios of n = 6 experiments performed .

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d. Proliferation of CFSE-labeled responder T cells alone (left histogram) or co-cultured with Tregs that have been pre-incubated with either vehicle, SF1670, SC-79 or 740 Y-P at day 4 after co-culture at a 1:2 Treg:Tresp ratio. e. Statistical analysis of the percentage of suppression at different Treg:Tresp ratios of n=6 experiments performed.


a . Sorted CD4 + CD25hiCD127 - were pre - incubated with increasing concentrations of AS1842856 , washed co - cultured with CFSE - labeled responder T . Representative histograms of proliferation of Tresp alone ( left ) or in co - culture with Tregs treated with either vehicle or different concentrations of AS1842856 measured at day 4 at a 1 : 2 Treg : Tresp . b . Statistical of suppression of proliferation at different Treg : Tresp ratios ( n = 6 ) on Tregs pretreated with vehicle ( dots ) or 10 nM ( black dots , left diagram ) or 50 nM AS1842856 ( black dots , right diagram ) .

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a. Sorted CD4+CD25hiCD127- were pre-incubated with increasing concentrations of AS1842856, washed and co-cultured with CFSE-labeled responder T cells. Representative histograms of proliferation of Tresp alone (left) or in co-culture with Tregs treated with either vehicle or different concentrations of AS1842856 measured at day 4 at a 1:2 Treg:Tresp ratio. b. Statistical analysis of the suppression of proliferation at different Treg:Tresp ratios (n=6) on Tregs pretreated with vehicle (white dots) or 10 nM (black dots, left diagram) or 50 nM AS1842856 (black dots, right diagram).


c . Representative dot plots of frequency of IFN + Foxp3 + Tregs in co - cultures in a at a 1 : 2 Treg : Tresp after adding GolgiStop for last 6 hours .

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c. Representative dot plots of the frequency of IFN+Foxp3+ Tregs in the co-cultures in a at a 1:2 Treg:Tresp ratio after adding GolgiStop for the last 6 hours.


d . Statistical of frequency of IFN + Foxp3 + Tregs in co - cultures in a of n = 6 experiments performed .

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d. Statistical analysis of the frequency of IFN+Foxp3+ Tregs in the co-cultures in a of n=6 experiments performed.


e . Correlation between frequency of IFN + Foxp3 + Tregs in co - cultures of suppression in Tregs pre - incubated with different doses of AS1842856 at a 1 : 4 Treg : Tresp .

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e. Correlation between frequency of IFN+Foxp3+ Tregs in co-cultures and percentage of suppression in Tregs pre-incubated with different doses of AS1842856 at a 1:4 Treg:Tresp ratio.


f . IFNG TBX21 gene expression on Tregs transduced with a Foxo1 or Foxo3 shRNA as compared to NT - shRNA control , measured at day 6 after 4 hours of stimulation with PMA ionomycin .

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f. IFNG and TBX21 gene expression on Tregs transduced with a Foxo1 or Foxo3 shRNA as compared to the NT-shRNA control, measured at day 6 after 4 hours of stimulation with PMA and ionomycin.


g . of suppression of CFSE - labeled Tresp proliferation co - cultured with Tregs transduced with a non - target ( dots ) , Foxo1 ( left diagram , black dots ) or Foxo3 shRNA ( right diagram , black dots ) at different Treg : Tresp ratios ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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g. Percentage of suppression of CFSE-labeled Tresp proliferation co-cultured with Tregs transduced with a non-target (white dots), Foxo1 (left diagram, black dots) or Foxo3 shRNA (right diagram, black dots) at different Treg:Tresp ratios (n=4). *p < 0.05, **p < 0.005, ***p < 0.0005.


Sorted Tregs were stimulated with anti - CD3 , anti - CD28 , IL - 2 shRNA specific for AKT1 , AKT2 , AKT3 , or non - target control resorted on GFP expression at day 5 . a . IFNG , TBX21 , IL10 , FOXP3 gene expression after sort at day 5 ( n = 5 ) .

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Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. a. IFNG, TBX21, IL10, FOXP3 gene expression after sort at day 5 (n=5).


Sorted Tregs were stimulated with anti - CD3 , anti - CD28 , IL - 2 shRNA specific for AKT1 , AKT2 , AKT3 , or non - target control resorted on GFP expression at day 5 . b . Representative example of on resorted Tregs stimulated with PMA ionomicin for 4 hours at day 5 ( n = 4 ) .

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Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. b. Representative example of staining on resorted Tregs stimulated with PMA and ionomicin for 4 hours at day 5 (n=4).


Sorted Tregs were stimulated with anti - CD3 , anti - CD28 , IL - 2 shRNA specific for AKT1 , AKT2 , AKT3 , or non - target control resorted on GFP expression at day 5 . c . Statistical of frequency of IFN + Foxp3 + Tregs after AKT silencing ( n = 4 ) .

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Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. c. Statistical analysis of the frequency of IFN+Foxp3+ Tregs after AKT silencing (n=4).


Sorted Tregs were stimulated with anti - CD3 , anti - CD28 , IL - 2 shRNA specific for AKT1 , AKT2 , AKT3 , or non - target control resorted on GFP expression at day 5 . d . shRNA transduced were co - cultured with CFSE - labeled Tresp Tresp proliferation was measured after 4 days . e . Statistical of of suppression by AKT1 - , AKT2 - AKT3 - silenced Tregs as compared to NT - transduced of n = 6 experiments performed . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. d. shRNA transduced cells were co-cultured with CFSE-labeled Tresp and Tresp proliferation was measured after 4 days. e. Statistical analysis of the percentage of suppression by AKT1-, AKT2- and AKT3-silenced Tregs as compared to NT-transduced cells of n=6 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.


a . Sorted CD4 + CD25hiCD127 - Tregs were stimulated in presence of vehicle ( squares ) IL - 12 either alone ( circles ) or in presence of LY294002 or MK2206 ( black circles ) for 3 days IFNG TBX21 gene expression was examined every 24 hours . Statistical of n = 8 independent experiments performed .

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a. Sorted CD4+CD25hiCD127- Tregs were stimulated in the presence of vehicle (white squares) and IL-12 either alone (white circles) or in the presence of LY294002 or MK2206 (black circles) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=8 independent experiments performed.


b . Representative example of frequency of IFN + Foxp3 + in Tregs stimulated in presence of vehicle or IL - 12 either alone or in presence of LY294002 or MK2206 for 3 days after a 4 hour stimulation with PMA ionomycin .

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b. Representative example of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle or IL-12 either alone or in the presence of LY294002 or MK2206 for 3 days after a 4 hour stimulation with PMA and ionomycin.


c . Statistical of frequency of IFN + Foxp3 + in Tregs stimulated as in c of n = 8 experiments performed .

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c. Statistical analysis of the frequency of IFN+Foxp3+ cells in Tregs stimulated as in c of n=8 experiments performed.


d . Proliferation measured at day 4 of CFSE - labeled responder T alone ( left histogram ) or co - cultured with Tregs that had been pre - incubated with either vehicle or IL - 12 alone or with LY294002 or MK2206 at a 1 : 2 Treg : Tresp . e . Statistical of of suppression as in d at different Treg : Tresp ratios of n = 6 experiments performed . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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d. Proliferation measured at day 4 of CFSE-labeled responder T cells alone (left histogram) or co-cultured with Tregs that had been pre-incubated with either vehicle or IL-12 alone or with LY294002 or MK2206 at a 1:2 Treg:Tresp ratio. e. Statistical analysis of the percentage of suppression as in d at different Treg:Tresp ratios of n=6 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.


a . mRNA expression on ex vivo isolated Tregs from MS healthy controls ( n = 15 ) .

a. mRNA expression on ex vivo isolated Tregs from MS patients and healthy controls (n=15).


b . Representative example of expression of AKT3 , Foxo3 PTEN on ex vivo sorted Tregs from MS ( black open histogram ) HC ( grey open histogram ) . Grey histograms represent isotype . c . Ex vivo expression of pAKT , pFoxo1 pFoxo3 in Tregs from RRMS ( black open histogram ) healthy controls ( open histograms ) . Grey histograms represent isotype .

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b. Representative example of the expression of AKT3, Foxo3 and PTEN on ex vivo sorted Tregs from MS patients (black open histogram) and HC (grey open histogram). Grey histograms represent isotype. c. Ex vivo expression of pAKT, pFoxo1 and pFoxo3 in Tregs from RRMS patients (black open histogram) and healthy controls (white open histograms). Grey histograms represent isotype.


d . Representative example of frequency of IFN + Foxp3 + Tregs isolated ex vivo from RRMS ( lower row ) healthy controls ( HC , upper row ) stimulated in presence of vehicle ( left histogram ) , LY294002 ( middle histogram ) or MK2206 ( right histogram ) for 3 days . was performed after a 4 - hour stimulation with PMA ionomycin . e . Statistical of frequency of IFN + Foxp3 + Tregs in HC ( bars ) RRMS ( black bars ) stimulated in presence of either vehicle , LY294002 or MK2206 ( n = 10 ) .

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d. Representative example of the frequency of IFN+Foxp3+ Tregs isolated ex vivo from RRMS patients (lower row) and healthy controls (HC, upper row) and stimulated in the presence of vehicle (left histogram), LY294002 (middle histogram) or MK2206 (right histogram) for 3 days. Staining was performed after a 4-hour stimulation with PMA and ionomycin. e. Statistical analysis of the frequency of IFN+Foxp3+ Tregs in HC (white bars) and RRMS patients (black bars) stimulated in the presence of either vehicle, LY294002 or MK2206 (n=10).


f . Representative example of proliferation of Tresp from HC ( upper row ) RRMS ( lower row ) either alone ( first column ) or in co - culture with Tregs that had been previously incubated with either vehicle , LY294002 or MK2206 for one hour washed to remove drug traces . g . Statistical of of suppression as in h at different Treg : Tresp ratios of n = 8 experiments performed . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 .

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f. Representative example of the proliferation of Tresp cells from HC (upper row) and RRMS patients (lower row) either alone (first column) or in co-culture with Tregs that had been previously incubated with either vehicle, LY294002 or MK2206 for one hour and washed to remove drug traces. g. Statistical analysis of the percentage of suppression as in h at different Treg:Tresp ratios of n=8 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.


B Representative examples of accessibility gene expression of KRT5 KRT10 , two key epidermal differentiation - related genes , in main keratinocyte populations .

B Representative examples of chromatin accessibility and gene expression of KRT5 and KRT10, two key epidermal differentiation-related genes, in the main keratinocyte populations.


C Co - accessibility at human epidermal differentiation ( EDC ) in undifferentiated ( cluster 2 ) differentiated ( cluster 3 ) keratinocytes . Only connections ( arcs ) with a co - accessibility score above 0 . 25 are plotted . Grey boxes below tracks represent scATAC - seq peaks .

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C Co-accessibility at the human epidermal differentiation complex (EDC) in undifferentiated (cluster 2) and differentiated (cluster 3) keratinocytes. Only connections (arcs) with a co-accessibility score above 0.25 are plotted. Grey boxes below tracks represent scATAC-seq peaks.


D Left : displaying differentially accessible peaks between undifferentiated ( cluster 2 ) differentiated ( cluster 3 ) keratinocytes . Number of Tn5 insertion sites in each region were scaled by row . Right : motifs for top six overrepresented transcription factor ( TF ) motifs in undifferentiated ( cluster 2 ) differentiated ( cluster 3 ) keratinocyte - specific accessible peaks .

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D Left: Heatmap displaying the differentially accessible peaks between undifferentiated (cluster 2) and differentiated (cluster 3) keratinocytes. Number of Tn5 insertion sites in each region were scaled by row. Right: DNA sequence motifs for the top six overrepresented transcription factor (TF) motifs in undifferentiated (cluster 2) and differentiated (cluster 3) keratinocyte-specific accessible peaks.


E Heatmaps displaying predicted top five transcription factors in each cluster using chromVAR motif activity ( left ) gene expression ( right ) . F chromVAR deviations ( in quantiles ) gene expression for representative enriched TF projected onto joint UMAP plot of keratinocytes from single - multi - omics dataset .

E Heatmaps displaying the predicted top five transcription factors in each cell cluster using chromVAR motif activity (left) and gene expression (right). F chromVAR deviations (in quantiles) and gene expression for representative enriched TF projected onto the joint UMAP plot of keratinocytes from the single-cell multi-omics dataset.


G showing top five active regulons in each cluster , on expression data of multi - omics dataset . TF regulons also identified by other approaches are highlighted .

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G Heatmap showing the top five active regulons in each cell cluster, based on the expression data of the multi-omics dataset. TF regulons also identified by other approaches are highlighted.


A Average histone modification profiles of undifferentiated differentiated keratinocyte - specific peaks using previously published data generated on NHEK ( ENCODE ) . normalized signal of H3K27ac , H3K4me1 / me2 / me3 H3K27me3 were measured in a window of ± 10 , 000 base - pair ( bp ) .

A Average histone modification profiles of undifferentiated and differentiated keratinocyte-specific peaks using previously published data generated on NHEK cells (ENCODE). The normalized signal of H3K27ac, H3K4me1/me2/me3 and H3K27me3 were measured in a window of ±10,000 base-pair (bp).


B Fractions of CpGs located within epigenomic substructures for 4 , 351 InfiniumEPIC CpG probes found within undifferentiated differentiated keratinocyte - specific peaks .

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B Fractions of CpGs located within epigenomic substructures for the 4,351 InfiniumEPIC CpG probes found within undifferentiated and differentiated keratinocyte-specific peaks.


C Unsupervised hierarchical clustering of 12 healthy , 20 AK 35 cSCC epidermal samples on methylation status at undifferentiated differentiated keratinocyte - specific peaks . Each row represents average methylation value of CpGs contained in a particular peak .

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C Unsupervised hierarchical clustering of 12 healthy, 20 AK and 35 cSCC epidermal samples based on the methylation status at undifferentiated and differentiated keratinocyte-specific peaks. Each row represents the average methylation value of all CpGs contained in a particular peak.


B , C Average methylation in 15 states defined by ChromHMM in NHEK for ( B ) both EpSC Keratinocytes from sci - MET single - dataset , or for ( C ) EpSC - like keratinocyte - like AK / cSCC from EPIC dataset . ChromHMM state 1 : active promoter ; state 2 : weak promoter ; state 3 : inactive / poised promoter ; state 4 : strong enhancer ; state 5 : strong enhancer ; state 6 : weak / poised enhancer ; state 7 : weak / poised enhancer ; state 8 : insulator ; state 9 : transcriptional transition ; state 10 : transcriptional elongation ; state 11 : weak transcription ; state 12 : polycomb - repressed ; state 13 : heterochromatin / low signal ; state 14 : repetitive / copy number variation ; state 15 : repetitive / copy number variation . AK : actinic keratosis , cSCC : cutaneous squamous carcinoma . Data information : Statistical analyses in B C were performed using a Wilcoxon Rank Sum comparing average methylation values in genomic states 4 5 between EpSC ( n = 6 ) keratinocytes ( n = 548 ) ( B ) or EpSC - like ( n = 22 ) keratinocyte - like ( n = 33 ) AK / cSCC samples ( C ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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B, C Average DNA methylation in the 15 chromatin states defined by ChromHMM in NHEK for (B) both EpSC and Keratinocytes from the sci-MET single-cell dataset, or for (C) EpSC-like and keratinocyte-like AK/cSCC from the EPIC dataset. ChromHMM state 1: active promoter; state 2: weak promoter; state 3: inactive/poised promoter; state 4: strong enhancer; state 5: strong enhancer; state 6: weak/poised enhancer; state 7: weak/poised enhancer; state 8: insulator; state 9: transcriptional transition; state 10: transcriptional elongation; state 11: weak transcription; state 12: polycomb-repressed; state 13: heterochromatin/low signal; state 14: repetitive/copy number variation; state 15: repetitive/copy number variation. AK: actinic keratosis, cSCC: cutaneous squamous cell carcinoma. Data information: Statistical analyses in B and C were performed using a Wilcoxon Rank Sum test comparing the average methylation values in genomic states 4 and 5 between EpSC (n= 6) and keratinocytes (n= 548) (B) or EpSC-like (n= 22) and keratinocyte-like (n= 33) AK/cSCC samples (C). *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001.


D Heatmaps displaying gene expression ( upper ) imputed promoter methylation ( lower ) reference matrices for keratinocyte populations involved in terminal differentiation , calculated by EpiSCORE . of each gene in reference matrix is also depicted .

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D Heatmaps displaying the gene expression (upper) and the imputed promoter DNA methylation (lower) reference matrices for keratinocyte populations involved in terminal differentiation, calculated by EpiSCORE. The weight of each gene in the reference DNA matrix is also depicted.


C Upper : Heatmaps displaying unsupervised clustering of 67 AK / cSCC healthy controls on methylation patterns of MIR200C / 141 cluster MIR205 gene . Lower : Bar plots showing of methylation values at CpGs located at promoter regions in each - of - origin - subclass . For MIR200C / 141 cluster , probes located at regulatory CpG island are depicted with green arrows . For MIR205 gene , probes shown in are part of promoter region were used for . Data information : AK : actinic keratosis , cSCC : cutaneous squamous carcinoma .

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C Upper: Heatmaps displaying the unsupervised clustering of 67 AK/cSCC and healthy controls based on the methylation patterns of the MIR200C/141 cluster and the MIR205 gene. Lower: Bar plots showing the quantification of the methylation values at CpGs located at promoter regions in each cell-of-origin-based subclass. For the MIR200C/141 cluster, probes located at the regulatory CpG island are depicted with green arrows. For the MIR205 gene, all probes shown in the heatmap are part of the promoter region and were used for quantification. Data information: AK: actinic keratosis, cSCC: cutaneous squamous cell carcinoma.


D Left : Representative of EpSC - like ( n = 5 ) Keratinocyte - like ( n = 5 ) cSCC tumors stained for EMT - marker ZEB2 ( green ) keratinocyte marker TP63 ( red ) . Nuclei were counterstained with DAPI . are shown at 40x original magnification . Scale bar , 50 µm . Right : of ZEB2 - positive ( TP63 - positive ) in three independent regions per sample . Each dot represents a region with at least 500 ( TP63 - positive ) counted . Data information : Statistical in D was performed using an unpaired two - sided t - ( EpSC - like : n = 5 samples , 15 technical replicates ; keratinocyte - like : n = 5 samples , 15 technical replicates ) . Central bar represents mean error bars represent standard deviation . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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D Left: Representative images of EpSC-like (n=5) and Keratinocyte-like (n=5) cSCC tumors stained for the EMT-marker ZEB2 (green) and the keratinocyte marker TP63 (red). Nuclei were counterstained with DAPI. Images are shown at 40x original magnification. Scale bar, 50 µm. Right: Quantification of ZEB2-positive tumor cells (TP63-positive) in three independent regions per sample. Each dot represents a region with at least 500 tumor cells (TP63-positive) counted. Data information: Statistical analysis in D was performed using an unpaired two-sided t-test (EpSC-like: n= 5 samples, 15 technical replicates; keratinocyte-like: n= 5 samples, 15 technical replicates). Central bar represents the mean and error bars represent the standard deviation. *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001.


( D , E ) Representative examples of dose - response curves time courses . Lines through dose - response curves are fits to Michaelis - Menten equation ; time courses are fitted to linear equations . Shown are means standard errors over at least three independent repeats .

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(D, E) Representative examples of dose-response curves and time courses. Lines through the dose-response curves are fits to the Michaelis-Menten equation; time courses are fitted to linear equations. Shown are means and standard errors over at least three independent repeats.


( F ) Fold increase ( positive values ) or decrease ( negative values ) in initial uptake rates of variants relative to WT GltPh . Circles are averages of technical triplicates . Bars are means over independent repeats ( as indicated ) . dashed marks a four - fold increase in initial rate .

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(F) Fold increase (positive values) or decrease (negative values) in the initial uptake rates of the variants relative to WT GltPh. Circles are averages of technical triplicates. Bars are means over independent repeats (as indicated). The dashed line marks a four-fold increase in the initial rate.


( A , B ) Schematic representations of experimental conditions are shown on far left . Data are shown for WT GltPh select gain - of - function mutants , as indicated above panels . Transition density plots ( top of each panel ) show frequency of transitions between EFRET values . number of trajectories analyzed ( N ) population - wide mean frequency of transitions is shown on panels . Representative 50 s sections of single - molecule EFRET trajectories ( middle ) with raw data in black idealizations in brown . Scale bar conformational states corresponding to low intermediate EFRET values are shown to left of panels . Dwell - time distributions for OFS in blue IFS in red ( bottom ) . Mean dwell times are shown on panels in corresponding colors . Black lines represent photobleaching survival plots normalized from 1 to 0 . Shown are means standard errors over at least three independent repeats .

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(A, B) Schematic representations of the experimental conditions are shown on the far left. Data are shown for WT GltPh and select gain-of-function mutants, as indicated above the panels. Transition density plots (top of each panel) show the frequency of transitions between the EFRET values. The number of trajectories analyzed (N) and the population-wide mean frequency of transitions is shown on the panels. Representative 50 s sections of single-molecule EFRET trajectories (middle) with raw data in black and idealizations in brown. Scale bar and the conformational states corresponding to the low and intermediate EFRET values are shown to the left of the panels. Dwell-time distributions for the OFS in blue and the IFS in red (bottom). Mean dwell times are shown on the panels in corresponding colors. Black lines represent photobleaching survival plots normalized from 1 to 0. Shown are means and standard errors over at least three independent repeats.


( A ) Changes in initial rates of substrate uptake ( gray bars ) , transport domain dynamics ( black bars ) , l - Asp dissociation constant ( bars ) of mutant GltPh variants relative to WT transporter . transition frequencies were measured under non - equilibrium transport conditions , frequencies obtained in presence of d , l - TBOA were subtracted from data . Error bars represent standard errors for at least three independent measurements .

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(A) Changes in the initial rates of substrate uptake (gray bars), transport domain dynamics (black bars), and l-Asp dissociation constant (white bars) of the mutant GltPh variants relative to the WT transporter. The transition frequencies were measured under non-equilibrium transport conditions, and frequencies obtained in the presence of d,l-TBOA were subtracted from the data. Error bars represent standard errors for at least three independent measurements.


( A ) Dwell - time distributions of OFS ( blue ) IFS ( red ) for WT ( top ) K290A ( bottom ) GltPh observed under equilibrium conditions in saturating Na + / l - Asp ( left ) . Lines are fits to three exponentials . Data are averages standard errors of at least three independent measurements .

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(A) Dwell-time distributions of the OFS (blue) and the IFS (red) for WT (top) and K290A (bottom) GltPh observed under equilibrium conditions in saturating Na+/l-Asp (left). Lines are fits to three exponentials. Data are averages and standard errors of at least three independent measurements.


( B ) Representative EFRET trajectories of WT GltPh showing different transition frequencies . Raw data are in black , idealizations are in brown . Scalebar is above trajectories .

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(B) Representative EFRET trajectories of WT GltPh showing different transition frequencies. Raw data are in black, and idealizations are in brown. Scalebar is above the trajectories.


( C ) Distribution of WT ( top ) K290A GltPh ( bottom ) molecules with different mean transition frequencies ( bars ) . N is number of trajectories longer than 90 s used in . stacked black bars are fractions of trajectories without transitions . Pink bars show expected binomial distribution if trajectories shared mean transition frequency of 0 . 04 s - 1 for WT 0 . 34 s - 1 for K290A . Data are averages standard errors of at least three independent measurements .

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(C) Distribution of the WT (top) and the K290A GltPh (bottom) molecules with different mean transition frequencies (white bars). N is the number of trajectories longer than 90 s used in the analysis. The stacked black bars are fractions of trajectories without transitions. Pink bars show the expected binomial distribution if all trajectories shared the mean transition frequency of 0.04 s-1 for the WT and 0.34 s-1 for the K290A. Data are averages and standard errors of at least three independent measurements.


( D ) 2D - histograms of consecutive dwell lengths in OFS ( top ) IFS ( bottom ) . From left to right : calculated distribution of dwell times randomly selected from distributions in panel A ; measured distribution for WT GltPh K290A mutant .

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(D) 2D-histograms of the consecutive dwell lengths in the OFS (top) and the IFS (bottom). From left to right: calculated distribution of dwell times randomly selected from the distributions in panel A; measured distribution for WT GltPh and the K290A mutant.


( E ) Representative trajectories of WT ( left ) K290A ( right ) GltPh molecules showing switching between dynamic modes . Raw data are in black idealizations are in brown ( top ) . Black grey bars under trajectories indicate apparent slower faster dynamic modes . Survival plots for OFS IFS ( middle ) . Solid lines are fits to a single ( IFS ) double ( OFS ) exponentials . Dashed lines ( OFS only ) are rejected fits to single exponentials . Autocorrelation plots of sequential dwell durations ( bottom ) .

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(E) Representative trajectories of the WT (left) and the K290A (right) GltPh molecules showing switching between dynamic modes. Raw data are in black and idealizations are in brown (top). Black and grey bars under the trajectories indicate apparent slower and faster dynamic modes. Survival plots for the OFS and the IFS (middle). Solid lines are fits to a single (IFS) and double (OFS) exponentials. Dashed lines (OFS only) are rejected fits to single exponentials. Autocorrelation plots of the sequential dwell durations (bottom).


( A , B ) 2D - histograms of lifetime pairs obtained for trajectories exhibiting single exponential behavior . N is number of traces used . Scale bar shows relative density normalized by number of molecules . Above to right of each panel are stacked histograms of , respectively , OFS IFS lifetimes of analyzed molecules ( open bars ) photobleaching time of molecules showing no transitions ( black bars ) . Data from three independent measurements were combined for presentation . ( C ) mean lifetimes of molecules falling within 30 % of most populated bins of 2D - histograms ( yellow to red ) . open blue bar represents calculated mean OFS lifetime for WT GltPh Shown are means standard errors of mean of at least three independent 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 O O O O O O O O O O O O O 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) 2D-histograms of lifetime pairs obtained for trajectories exhibiting single exponential behavior. N is the number of traces used. Scale bar shows relative density normalized by the number of molecules. Above and to the right of each panel are stacked histograms of, respectively, the OFS and the IFS lifetimes of the analyzed molecules (open bars) and the photobleaching time of molecules showing no transitions (black bars). Data from three independent measurements were combined for presentation. (C) The mean lifetimes of the molecules falling within 30 % of the most populated bins of the 2D-histograms (yellow to red). The open blue bar represents the calculated mean OFS lifetime for WT GltPh Shown are means and standard errors of the mean of at least three independent measurements.


A , B Temporal expression of Snail genes in intestinal tract in ( A ) embryonic E12 . 5 to E18 . 5 ( B ) postnatal P0 to P20 measured by qRT - PCR . Shown are means ± SD .

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

A, B Temporal expression of Snail genes in the intestinal tract in (A) embryonic E12.5 to E18.5 and (B) postnatal P0 to P20 measured by qRT-PCR. Shown are means ± SD.


C , E , G , I Immunohistochemical for Snai1 / 2 ( Abcam ab85931 ) ( C ) E14 . 5midgut , ( E ) E14 . 5hind gut , ( G ) postnatal day 14 SI , ( I ) postnatal day 14 colon . Snai1immunohistochemistry peptide competition controls are shown as inserts ( C ' , E ' , G ' , I ' ) . Scale bars : 20 mm . D , F , H , J , K X ‐ gal for β ‐ galactosidase activity in tissue from Snai2LacZ mice stained in whole mount , sectioned counterstained with fast red ( D ) E14 . 5midgut , ( F ) E14 . 5hind gut , ( H ) postnatal day 14 SI , ( J ) postnatal day 14 colon , ( K ) adult SI . Controls for X ‐ gal are shown as inserts ( D ' , F ' , H ' , J ' ) . L , M , N Double immunostaining of small intestinalcrypts for Snai1 / 2 ( red ) ( M ) Lgr5 ‐ GFP ( green ) ; ( N ) merge shows Snai1 is present in both CBCstem proliferating transit amplifying . Scale bars : 10 mm . O , P Immunohistochemistry for Snai1 using a different antibody ( Genetex GTX125918 ) shows minimal expression of Snai1 in villi . ( P ) Expression of Snai1 is detected at base of crypts ( P ) with strong expression in CBCstem indicated by arrows . Scale bars : 20 mm ( O ) , 10 mm ( P ) . Q Schematic diagram showing Snai1 expression ( red ) within different types in crypt . SI = small intestine . See also Supplementary Fig S1 .

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C, E, G, I Immunohistochemical staining for Snai1/2 (Abcam ab85931) (C) E14.5midgut, (E) E14.5hind gut, (G) postnatal day 14 SI, (I) postnatal day 14 colon. Snai1immunohistochemistry peptide competition controls are shown as inserts (C', E', G', I'). Scale bars: 20 mm.D, F, H, J, K X‐gal staining for β‐galactosidase activity in tissue from Snai2LacZ mice stained in whole mount, sectioned and counterstained with nuclear fast red (D) E14.5midgut, (F) E14.5hind gut, (H) postnatal day 14 SI, (J) postnatal day 14 colon, (K) adult SI. Controls for X‐gal staining are shown as inserts (D', F', H', J').L, M, N Double immunostaining of small intestinalcrypts for Snai1/2 (red) and (M) Lgr5‐GFP (green); (N) merge shows Snai1 is present in both CBCstem cells and proliferating transit amplifying cells. Scale bars: 10 mm.O, P Immunohistochemistry for Snai1 using a different primary antibody (Genetex GTX125918) shows minimal expression of Snai1 in villi. (P) Expression of Snai1 is detected at the base of crypts (P) with strong nuclear expression in CBCstem cells indicated by arrows. Scale bars: 20 mm (O), 10 mm (P).Q Schematic diagram showing Snai1 expression (red) within different cell types in the crypt. SI = small intestine. See also Supplementary Fig S1.


Periodic Acid Schiff ( PAS ) was used to detect goblet in sections of small intestine from control Snai1 KO mice . Graph shows number of goblet per crypt - villus axes ( n = 3 ) . Immunohistochemistry for synaptophysin was used to detect enteroendocrine ( marked by arrows ) . of enteroendocrine per crypt - villus axes in control Snai1 KO tissue revealed a significant increase in number of enteroendocrine following Snai1 loss ( n = 3 , P = 0 . 004 ) . Immunohistochemistry for lysozyme was used to detect Paneth . of Paneth in control Snai1 KO indicates a significant increase in Paneth numbers following knockout of Snai1 ( n = 4 , P = 0 . 002 ) .

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Periodic Acid Schiff (PAS) staining was used to detect goblet cells in sections of small intestine from control and Snai1 KO mice. Graph shows number of goblet cells per crypt-villus axes (n = 3).Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells (marked by arrows). Quantification of enteroendocrine cells per crypt-villus axes in control and Snai1 KO tissue revealed a significant increase in the number of enteroendocrine cells following Snai1 loss (n = 3, P = 0.004).Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cells in control and Snai1 KO indicates a significant increase in Paneth cell numbers following knockout of Snai1 (n = 4, P = 0.002).


Measurement of villus ( μm ) ( n = 3 , P = 0 . 013 ) of enterocytes per crypt - villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss ( n = 3 , P = 0 . 036 ) . Measurement of villus ( μm ) ( n = 3 , P = 0 . 003 ) 8 days after β ‐ NF induction demonstrates a significant shortening of villi . Bars represent mean ± SD . Two ‐ Student ' s t ‐ was used to assess significance . Scale bars : 20 μm .

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Measurement of villus length (μm) (n = 3, P = 0.013) and quantification of enterocytes per crypt-villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss (n = 3, P = 0.036).Measurement of villus length (μm) (n = 3, P = 0.003) 8 days after β‐NF induction demonstrates a significant shortening of villi. Bars represent mean ± SD. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 20 μm.


Sections from control Snai1 KO small intestine were stained for PCNA to detect proliferating ( arrows depicting proliferating CBC at base of crypts ) . of total PCNA ‐ positive per crypt PCNA ‐ positive in base of crypts both revealed a significant reduction in number of proliferating after conditional depletion of Snai1 ( n = 4 , P = 0 . 049 , 0 . 026 ) . Scale bars : 20 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 O O O O O O O O O O O

Sections from control and Snai1 KO small intestine were stained for PCNA to detect proliferating cells (arrows depicting proliferating CBC stem cells at the base of crypts). Quantification of total PCNA‐positive cells per crypt and PCNA‐positive cells in the base of crypts both revealed a significant reduction in the number of proliferating cells after conditional depletion of Snai1 (n = 4, P = 0.049, 0.026). Scale bars: 20 μm.


expression of 2 CBCstem markers Olfm4 Lgr5 were examined by in situ hybridisation in control Snai1KO small intestine . Lgr5 Olfm4in situ hybridisation shows a loss of CBCstem marker expression after loss of Snai1 ( high magnification inserts depict a representative crypt demarcated by a dotted ) . Loss of Snai1 was confirmed in knockout tissue using immunohistochemistry . Scale bars : 20 μm .

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The expression of 2 CBCstem cell markers Olfm4 and Lgr5 were examined by in situ hybridisation in control and Snai1KO small intestine. Lgr5 and Olfm4in situ hybridisation staining shows a loss of CBCstem cell marker expression after loss of Snai1 (high magnification inserts depict a representative crypt demarcated by a dotted line). Loss of Snai1 was confirmed in knockout tissue using immunohistochemistry. Scale bars: 20 μm.


of sorted EGFP ‐ positive from Lgr5 ‐ EGFP ‐ IRES ‐ creERT2AhCreSnai1fl / fl Lgr5 ‐ EGFP ‐ IRES ‐ creERT2AhCreSnai1 + / + mice 5 days after βNF induction show an ˜ tenfold reduction in Lgr5GFP ‐ positive CBCstem .

The analysis of sorted and EGFP‐positive cells from Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1fl/fl and Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1+/+mice 5 days after βNF induction show an ˜tenfold reduction in Lgr5GFP‐positive CBCstem cells.


Immunohistochemistry for active caspase ‐ 3 to detect apoptotic revealed a significant increase in number of apoptotic in both crypt ( n = 5 , P = 0 . 0001 ) villus ( n = 5 , P = 0 . 005 ) regions in Snai1KO tissue ( arrows mark active caspase ‐ 3 ‐ positive ) . Scale bars : 20 μm .

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Immunohistochemistry for active caspase‐3 to detect apoptotic cells revealed a significant increase in the number of apoptotic cells in both crypt (n = 5, P = 0.0001) and villus (n = 5, P = 0.005) regions in Snai1KO tissue (arrows mark active caspase‐3‐positive cells). Scale bars: 20 μm.


A lineage tracing strategy was employed to determine fate of CBCcells that lack Snai1 . Tissue from Lgr5 ‐ EGFP ‐ IRES ‐ creERT2Snai1 + / + ( control ) Lgr5 ‐ EGFP ‐ IRES ‐ creERT2Snai1fl / fl ( Snai1KO ) that also contained ROSA26LacZCre reporter allele was stained with X ‐ Gal 5 days after tamoxifen injection . of X ‐ Gal ‐ positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout . Bars represent mean ± SD ( n = 3 , P = 0 . 0001 ) . Scale bars : 0 . 50 mm . Two ‐ Student ' s t ‐ was used to assess significance . See also Supplementary Figure S2 .

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A lineage tracing strategy was employed to determine the fate of CBCcells that lack Snai1. Tissue from Lgr5‐EGFP‐IRES‐creERT2Snai1+/+ (control) and Lgr5‐EGFP‐IRES‐creERT2Snai1fl/fl (Snai1KO) that also contained the ROSA26LacZCre reporter allele was stained with X‐Gal 5 days after tamoxifen injection. Quantification of X‐Gal‐positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells. Bars represent mean ± SD (n = 3, P = 0.0001). Scale bars: 0.50 mm. Two‐tailed Student's t‐test was used to assess significance. See also Supplementary Figure S2.


VillinCreERT2Snai1fl / florganoids treated with tamoxifen show arrested growth after 2 4 days of treatment compared to controls . Scale bars : 50 μm . of viable growth in VillinCreERT2Snai1fl / florganoids treated with tamoxifen compared to controls over 4 days . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 002 .

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VillinCreERT2Snai1fl/florganoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cell growth in VillinCreERT2Snai1fl/florganoids treated with tamoxifen compared to controls over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.


Assessment of apoptosis in control VillinCreERT2 Snai1fl / fl organoids after 4 days of growth . show an overlay of bright ‐ field with propidium iodide Hoechst . of apoptotic organoids . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 00001 . Two ‐ Student ' s t ‐ was used to assess significance . Scale bars : 50 μm . See also Supplementary Figure S3 .

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

Assessment of apoptosis in control and VillinCreERT2 Snai1fl/fl organoids after 4 days of growth. Photographs show an overlay of bright‐field and fluorescent images with propidium iodide and Hoechst staining. Quantification of apoptotic organoids. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.00001. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 50 μm. See also Supplementary Figure S3.


consequences of Snai1 knockout were analysed over a time course . Immunohistochemistry for Snai1 revealed significant loss of expression at day 5 following induction compared to control . epithelium is re ‐ populated by wild ‐ type as shown on day 60 . Scale bars : 20 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

The consequences of Snai1 knockout were analysed over a time course. Immunohistochemistry for Snai1 revealed significant loss of expression at day 5 following induction compared to control. The epithelium is re‐populated by wild‐type cells as shown on day 60. Scale bars: 20 μm.


A PAS was used to detect goblet in small intestine , no differences in numbers were observed in versus control mice ( n = 4 , NS ) .

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A PAS staining was used to detect goblet cells in the small intestine, and no differences in cell numbers were observed in test versus control mice (n = 4, NS).


B Immunohistochemistry for synaptophysin was used to detect enteroendocrine . of number of enteroendocrine per crypt - villus axes revealed a significant decrease in enteroendocrine numbers when Snai1 are elevated ( n = 4 , P = 0 . 003 ) .

B Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells. Quantification of the number of enteroendocrine cells per crypt-villus axes revealed a significant decrease in enteroendocrine cell numbers when Snai1 levels are elevated (n = 4, P = 0.003).


C Immunohistochemistry for lysozyme was used to detect Paneth . of Paneth numbers indicated a significant decrease in Paneth in base of crypts in mice with elevated Snai1 ( n = 5 , P = 0 . 027 ) .

O O O O O O O O O O O O O O O O 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 Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cell numbers indicated a significant decrease in Paneth cells in the base of crypts in mice with elevated Snai1 levels (n = 5, P = 0.027).


D , E of total PCNA ‐ positive per crypt revealed a significant increase when Snai1 were elevated ( n = 4 , P = 0 . 009 ) . This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where of ( E ) PCNA ‐ positive in base of crypts revealed a significant increase in proliferating in crypts with elevated Snai1 compared to control tissue ( n = 4 , P = 0 . 0005 ) . Bars represent mean ± SD .

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

D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD.


F PCNAimmunostaining ( green ) DAPIstaining ( blue ) of VillinCreERT2ROSA26Snai1 compared to control VillinCreERT2 . An expansion of proliferating CBCcells ( indicated by arrows ) is observed at base of crypts . Scale bars : 10 μm .

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F PCNAimmunostaining (green) and DAPIstaining (blue) of VillinCreERT2ROSA26Snai1 compared to control VillinCreERT2 littermates. An expansion of proliferating CBCcells (indicated by arrows) is observed at the base of crypts. Scale bars: 10 μm.


G qRT - PCR of marker expression in small intestine from VillinCreERT2 ROSA26Snai1 compared to control VillinCreER2T . Bars represent mean ± SD . n = 3 experiments per group , Olfm4 P = 0 . 013 , Cdh1 / E ‐ cadherin P = 0 . 003 , Lgr5 P = 0 . 013 .

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G qRT-PCR analysis of stem cell marker expression in the small intestine from VillinCreERT2 ROSA26Snai1 compared to control VillinCreER2T littermates. Bars represent mean ± SD. n = 3 experiments per group, Olfm4 P = 0.013, Cdh1/E‐cadherin P = 0.003, Lgr5 P = 0.013.


H CD44v6 immunostaining of tissue from AhCre control compared to AhCre RosaSnai1 tissue . A significant increase in CD44v6 ‐ positive localised at base of crypts ( depicted by arrows ) was observed . Bars represent mean ± SD . n = 3 mice per group , * P = 0 . 00023 . Two ‐ Student ' s t ‐ was used to assess significance . Scale bars : 10 μm .

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H CD44v6 immunostaining of tissue from AhCre control compared to AhCre RosaSnai1 tissue. A significant increase in CD44v6‐positive cells localised at the base of crypts (depicted by arrows) was observed. Bars represent mean ± SD. n = 3 mice per group, *P = 0.00023. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 10 μm.


VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 4 days of treatment compared to controls . Scale bars : 50 μm . of viable in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 002 .

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VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cells in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.


Assessment of apoptosis in control VillinCreERT2 RosaSnai1 organoids after 4 days of growth . show an overlay of bright ‐ field , propidium iodide Hoechst . Scale bars : 50 μm . of apoptotic organoids with without Wnt3A treatment shown as mean ± SD . n = 3 experiments per group , * * P = 0 . 00003 . Two ‐ Student ' s t ‐ was used to assess significance .

Assessment of apoptosis in control and VillinCreERT2 RosaSnai1 organoids after 4 days of growth. Photographs show an overlay of bright‐field, propidium iodide and Hoechst fluorescence. Scale bars: 50 μm.Quantification of apoptotic organoids with and without Wnt3A treatment shown as mean ± SD. n = 3 experiments per group, **P = 0.00003. Two‐tailed Student's t‐test was used to assess significance.


A - C Droplet digital PCR of Bmf , Bbc3 / Puma Bcl2l 11 / Bim after conditional Snai1 loss in in vitro organoid culture . VillinCreERT2 Snai1 + / + ( control ) VillinCreERT2 Snai1fl / fl SI organoids were treated for 24 h with tamoxifen . No differences in gene expression were observed ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C Droplet digital PCR analysis of Bmf, Bbc3/Puma and Bcl2l 11/Bim after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 h with tamoxifen. No differences in gene expression were observed (n = 3).


D qRT - PCR of Serinc3 downregulation after conditional Snai1 loss in in vitro organoid culture . VillinCreERT2 Snai1 + / + ( control ) VillinCreERT2 Snai1fl / fl SI organoids were treated for 24 ( P = 0 . 0001 ) 72 h ( P = 0 . 006 ) with tamoxifen . Bars represent mean ± SD . n = 3 mice per group .

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D qRT-PCR analysis of Serinc3 downregulation after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 (P = 0.0001) and 72 h (P = 0.006) with tamoxifen. Bars represent mean ± SD. n = 3 mice per group.


E SW480 , which transiently express GFP ‐ SNAI1 , were used to detect direct binding of SNAI1 to promoters of known target genes CDH1 , IL8 a novel target SERINC3 revealed by microarray . An unrelated region was used as a negative control . Anti ‐ GFP antibody was used to immunoprecipitate GFP ‐ tagged SNAI1 , while IgG control antibody was used as a negative control . results are given as relative enrichment compared to input material are representative of three independent experiments .

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E SW480 cells, which transiently express GFP‐SNAI1, were used to detect direct binding of SNAI1 to the promoters of known target genes CDH1, IL8 and a novel target SERINC3 revealed by microarray analysis. An unrelated DNA region was used as a negative control. Anti‐GFP antibody was used to immunoprecipitate GFP‐tagged SNAI1, while IgG control antibody was used as a negative control. The results are given as relative enrichment compared to input material and are representative of three independent experiments.


Down regulation of SREBP target genes in Cideb - deficient liver . Microarray of fatty / cholesterol synthesis genes that were down - regulated in Cideb - / - mice Scap liver - specific knockout mice , comparing to respective WT controls ( WT VS Cideb - / - , WT VS L - Scap - / - , n = 2 per group ) . expression level of each gene was linearly normalized to range from - 1 to 1 before plotting .

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

Down regulation of SREBP target genes in Cideb-deficient liver. Microarray heatmap of fatty acids/cholesterol synthesis genes that were down-regulated in Cideb-/- mice and Scap liver-specific knockout mice, comparing to the respective WT controls (WT VS Cideb-/-, WT VS L-Scap-/-, n = 2 per group). The expression level of each gene was linearly normalized to range from -1 to 1 before plotting.


Decreased SREBP processing maturation in Cideb deficient liver . Immune blotting ( IB ) of full , precursor SREBP - 1 / 2 ( SREBP FL ) , active SREBP - 1 / 2 ( SREBP N ) from WT Cideb - / - mice liver under ad - lib ( ND ) , fasting 12h ( F ) , refeeding ( R ) conditions . Mice were refed with high - carbohydrate low - fat diet for 12h after 12h fasting .

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Decreased SREBP processing and maturation in Cideb deficient liver. Immune blotting (IB) of the full length, precursor SREBP-1/2 (SREBP FL) and the cleaved, active SREBP-1/2 (SREBP N) from WT and Cideb-/- mice liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting.


Defective SREBP transcriptional response to refeeding in Cideb - deficient liver . Transcript of lipogenic cholesterol metabolism genes determined by qPCR from WT Cideb - / - liver under ad - lib ( ND ) , fasting 12h ( F ) , refeeding ( R ) conditions . Mice were refed with high - carbohydrate low - fat diet for 12h after 12h fasting ( n = 3 per group ) .

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Defective SREBP transcriptional response to refeeding in Cideb-deficient liver. Transcript levels of lipogenic and cholesterol metabolism genes determined by qPCR from WT and Cideb-/- liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting (n = 3 per group).


Re - introduction of Cideb rescues SREBP processing in Cideb - / - liver . WT Cideb - / - mice were injected with AAV - GFP or AAV - Cideb , sacrificed for IB with indicated antibodies 20 days after injection .

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Re-introduction of Cideb rescues SREBP processing in Cideb-/- liver. WT and Cideb-/- mice were injected with AAV-GFP or AAV-Cideb, and sacrificed for IB with the indicated antibodies 20 days after the injection.


Re - introduction of Cideb rescues expression of SREBP target genes in Cideb - / - liver . Transcript of lipogenic genes determined by qPCR from WT Cideb - / - liver . WT Cideb - / - mice were injected with AAV - GFP or AAV - Cideb , sacrificed for qPCR 20 days after injection ( n = 3 per group ) .

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Re-introduction of Cideb rescues the expression of SREBP target genes in Cideb-/- liver. Transcript levels of lipogenic genes determined by qPCR from WT and Cideb-/- liver. WT and Cideb-/- mice were injected with AAV-GFP or AAV-Cideb, and sacrificed for qPCR 20 days after the injection (n = 3 per group).


Liver of WT Cideb - / - liver under chow diet HFLF diet . H / E , oil red - O , immunohistochemistry ultra - structure ( Electron Microscope ) were performed . Scale bar represented 50 µm in upper three rows of , 2 µm in bottom row of . LD : lipid droplet , ER : endoplasmic reticulum , M : mitochondrial , N : nucleus .

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Liver morphology of WT and Cideb-/- liver under chow diet and HFLF diet. H/E staining, oil red-O staining, immunohistochemistry and ultra-structure (Electron Microscope) were performed. Scale bar represented 50 µm in the upper three rows of images, and 2 µm in the bottom row of images. LD: lipid droplet, ER: endoplasmic reticulum, M: mitochondrial, N: nucleus.


Total liver TAG ( B ) of WT Cideb - / - mice under chow diet HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

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Total liver TAG levels (B) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Liver cholesterol ester ( C ) of WT Cideb - / - mice under chow diet HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

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Liver cholesterol ester levels (C) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


D Loss of Cideb inhibits diet - induced SREBP activation . IB of hepatic SREBP processing lipogenic enzyme ( Fasn , Scd1 ) of WT Cideb - / - mice under chow diet HFLF diet .

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D Loss of Cideb inhibits diet-induced SREBP activation. IB of hepatic SREBP processing and lipogenic enzyme levels (Fasn, Scd1) of WT and Cideb-/- mice under chow diet and HFLF diet.


E Serum AST ALT of WT Cideb - / - mice under chow diet HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

E Serum AST and ALT levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


F Cideb deficiency alleviates whole inflammation response . Serum TNFα , MCP1 IL6 of WT Cideb - / - mice under chow diet HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

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F Cideb deficiency alleviates the whole body inflammation response. Serum TNFα, MCP1 and IL6 levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Serum fed glucose ( G ) of WT Cideb - / - mice under chow diet HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

Serum fed glucose levels (G) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Serum fed insulin ( H ) of WT Cideb - / - mice under chow diet HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

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Serum fed insulin levels (H) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


I Glucose tolerance ( GTT ) of WT Cideb - / - mice under HFLF diet ( n = 5 per group ) . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

I Glucose tolerance test (GTT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


J Insulin tolerance ( ITT ) of WT Cideb - / - mice under HFLF diet ( n = 5 per group ) . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

J Insulin tolerance test (ITT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Decreased SREBP - 1 / 2 SCAP in Golgi apparatus in Cideb - / - mice . IB of organelle fractions of indicated of IB data from 3 independent experiments . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

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Decreased SREBP-1/2 and SCAP levels in Golgi apparatus in Cideb-/- mice. IB of organelle fractions of the indicated proteins and quantification of IB data from 3 independent experiments. Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb deficiency impairs packaging of SCAP / SREBP into COPII vesicles . present in vesicle or microsome fractions obtained from WT or Cideb - / - liver were detected by IB . of IB data from 3 independent experiments . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

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Cideb deficiency impairs the packaging of SCAP/SREBP into COPII vesicles. Protein present in the vesicle or microsome fractions obtained from WT or Cideb-/- liver were detected by IB. Quantification of IB data from 3 independent experiments. Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb depletion decreases recruitment of SCAP to COPII machinery in . hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A - Flag - BirA * . were cultured in sterol - depleted medium treated with 80 µM biotin , prior to pulldown with streptavidin - conjugated beads . biotinylated were detected by IB with indicated antibodies following SDS - PAGE .

Cideb depletion decreases the recruitment of SCAP to the COPII machinery in cells. Primary hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A-Flag-BirA*. Cells were cultured in sterol-depleted medium and treated with 80 µM biotin, prior to pulldown with streptavidin-conjugated beads. The biotinylated proteins were detected by IB with the indicated antibodies following SDS-PAGE.


SREBP bypasses Cideb deficiency . WT Cideb - / - mice were injected with adenovirus expressing GFP , SREBP - 1c full ( FL ) or SREBP - 1c active form ( N ) . Mice were sacrificed 7 days after injection , liver TAG hepatic expression SREBP - 1 target genes in indicated groups were determined ( n = 5 per group ) . Data information : Data represent Mean ± ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

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Cleaved SREBP bypasses Cideb deficiency. WT and Cideb-/- mice were injected with adenovirus expressing GFP, SREBP-1c full length (FL) or SREBP-1c active form (N). Mice were sacrificed 7 days after injection, and liver TAG levels and hepatic expression SREBP-1 target genes in the indicated groups were determined (n=5 per group). Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb co - precipitates with SCAP . Flag - tagged Cideb was co - expressed with different HA - tagged ( PLIN2 , Insig , SCAP , SREBP - 1 or SREBP - 2 ) in 293T immuno - precipitated with an anti - Flag antibody , co - immunoprecipitated were detected by an anti - HA antibody following SDS - PAGE .

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Cideb co-precipitates with SCAP. Flag-tagged Cideb was co-expressed with different HA-tagged proteins (PLIN2, Insig, SCAP, SREBP-1 or SREBP-2) in 293T cells and immuno-precipitated with an anti-Flag antibody, and the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.


Interaction between endogenous SCAP Cideb . Endogenous SCAP was immuno - precipitated from hepatocytes under normal medium , 25 - HC depletion or addition conditions . presence of Cideb in immune - was detected by IB . SREBP - 1 was detected as positive control . Grp94 was detected as negative control .

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Interaction between the endogenous SCAP and Cideb. Endogenous SCAP was immuno-precipitated from primary hepatocytes under normal medium, 25-HC depletion or addition conditions. And the presence of Cideb in the immune-complex was detected by IB. SREBP-1 was detected as the positive control. Grp94 was detected as the negative control.


Cideb interacts with 416 - 800 portion of SCAP . Flag - tagged Cideb was co - expressed with indicated HA - tagged SCAP truncation mutants in 293T immuno - precipitated with an anti - Flag antibody . of co - immunoprecipitated were detected by an anti - HA antibody following SDS - PAGE .

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Cideb interacts with the 416-800 portion of SCAP. Flag-tagged Cideb was co-expressed with the indicated HA-tagged SCAP truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.


Sterol - dependent interaction switch of SCAP . SCAP interacts with Insig in presence of sterol , while switches to Cideb binding upon sterol deprivation . Flag - tagged Insig - 1 or Cideb was co - expressed with HA - tagged SREBP - 2 SCAP or SCAP - Y298C in CHO - K1 in sterol - depleted medium . When indicated , 10 µg / ml 25 - HC were supplemented before were immuno - precipitated with an anti - Flag antibody , of co - immunoprecipitated were detected by IB following SDS - PAGE . dotted blue vertical was drawn to separate result between SCAP - WT SCAP - Y298C .

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Sterol-dependent interaction switch of SCAP. SCAP interacts with Insig in the presence of sterol, while switches to Cideb binding upon sterol deprivation. Flag-tagged Insig-1 or Cideb was co-expressed with HA-tagged SREBP-2 and SCAP or SCAP-Y298C in CHO-K1 cells in sterol-depleted medium. When indicated, 10 µg/ml 25-HC were supplemented before cells were immuno-precipitated with an anti-Flag antibody, and the levels of co-immunoprecipitated proteins were detected by IB following SDS-PAGE. The dotted blue vertical line was drawn to separate the result between SCAP-WT and SCAP-Y298C.


Cideb relieves sterol - dependent suppression of SREBP processing . Hepatocytes infected with adenovirus expressing GFP or Flag - Cideb were cultured in sterol deprived condition . When indicated , were treated with different doses of 25 - HC before subjected to IB with indicated antibodies following SDS - PAGE . of IB data from 3 independent experiments . Data represents Mean ± .

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Cideb relieves sterol-dependent suppression of SREBP processing. Hepatocytes infected with adenovirus expressing GFP or Flag-Cideb were cultured in sterol deprived condition. When indicated, cells were treated with different doses of 25-HC before subjected to IB with the indicated antibodies following SDS-PAGE. Quantification of IB data from 3 independent experiments. Data represents Mean ± SEM.


Cideb promotes Golgi localization of SCAP . HepG2 transfected with GFP - SCAP alone or with HA - Cideb were cultured in sterol deprived conditions or treated with 1 µg / ml 25 - HC . were fixed subjected to confocal microscopy following with antibodies against GM130 or HA . Scale bars represent 10 µm ( Merge ) , 2 µm ( Inlay ) . Bottom : of of SCAP localized in Golgi - positive region per ( left ) of of containing Golgi - localized GFP - SCAP ( right ) under indicated conditions , from 3 independent experiments ; Data represent Mean ± ; * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - Student ' s t .

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Cideb promotes Golgi localization of SCAP. HepG2 cells transfected with GFP-SCAP alone or with HA-Cideb were cultured in sterol deprived conditions or treated with 1 µg/ml 25-HC. Cells were fixed and subjected to confocal microscopy following staining with antibodies against GM130 or HA. Scale bars represent 10 µm (Merge), and 2 µm (Inlay). Bottom: quantification of the percentage of SCAP localized in Golgi-positive region per cell (left) and quantification of the percentage of cells containing Golgi-localized GFP-SCAP (right) under indicated conditions, from 3 independent experiments; Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb interacts with Sec12 . 293T transfected with Cideb GFP - tagged Sec12 , Sec23 , Sec16S , or Sec16L were subjected to immuno - precipitation with an anti - Cideb antibody , of co - immunoprecipitated were detected by an anti - GFP antibody .

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Cideb interacts with Sec12. 293T cells transfected with Cideb and GFP-tagged Sec12, Sec23, Sec16S, or Sec16L were subjected to immuno-precipitation with an anti-Cideb antibody, and levels of the co-immunoprecipitated protein were detected by an anti-GFP antibody.


Co - localization of SCAP Cideb at ER exit sites . HepG2 transfected with GFP - SCAP HA - Cideb were arrested with 10 µM BFA 1 µM Nocodazole for 2 hours , then fixed with methanol stained with antibodies against Sec12 or HA . 3D reconstruction was obtained by processing 3D - original image with Imaris . Scale bars represent 10 µm ( Merge ) , 1 µm ( Inlay 3D reconstruction ) .

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Co-localization of SCAP and Cideb at the ER exit sites. HepG2 cells transfected with GFP-SCAP and HA-Cideb were arrested with 10 µM BFA and 1 µM Nocodazole for 2 hours, then fixed with methanol and stained with antibodies against Sec12 or HA. 3D reconstruction was obtained by processing 3D-original image with Imaris. Scale bars represent 10 µm (Merge), and 1 µm (Inlay and 3D reconstruction).


Localization of Cideb at ER exit sites . AML12 knocked - in with a GFP tag at C - terminus of Cideb genome was treated with 10 µM BFA for 1 hour , then fixed with methanol stained with antibodies against Sec12 . Scale bars represent 10 µm ( Merge ) , 1 µm ( Inlay ) .

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Localization of Cideb at the ER exit sites. AML12 cells knocked-in with a GFP tag at the C-terminus of Cideb genome was treated with 10 µM BFA for 1 hour, then fixed with methanol and stained with antibodies against Sec12. Scale bars represent 10 µm (Merge), and 1 µm (Inlay).