( H - J ) Blood ketone body levels 6h after normoxia and hypoxia in female C57BL / 6J and ADX mice ( H ) , in female C57BL / 6J mice injected with 5 mg RU486 or vehicle ( DMSO ) ( I ) , and in GRdim / dim mice and their wild - type littermates ( J ) . N = 5 - 9 per group . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

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

(H-J) Blood ketone body levels 6h after normoxia and hypoxia in female C57BL/6J and ADX mice (H), in female C57BL/6J mice injected with 5 mg RU486 or vehicle (DMSO) (I), and in GRdim/dim mice and their wild-type littermates (J). N=5-9 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


( A ) Female C57BL / 6J mice ( n = 3 per group ) were put in normoxia or hypoxia for 24h , injected with PBS or DEX ( 10 mg / kg ) and 2h later , indicated organs were isolated and gene expression was measured via RT - qPCR . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . Survival curves were analysed with Fisher ' s exact test . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Female C57BL/6J mice (n=3 per group) were put in normoxia or hypoxia for 24h, injected with PBS or DEX (10 mg/kg) and 2h later, indicated organs were isolated and gene expression was measured via RT-qPCR. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. Survival curves were analysed with Fisher's exact test. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


( B ) LPS LD100 dose - response in female C57BL / 6J mice injected with indicated LPS doses after 24h hypoxia . During the follow - up of lethality , mice remained under hypoxic conditions . N - values are indicated in the legend .

O O O O O O O O O O O O O O O O O O O O O O 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) LPS LD100 dose-response in female C57BL/6J mice injected with indicated LPS doses after 24h hypoxia. During the follow-up of lethality, mice remained under hypoxic conditions. N-values are indicated in the legend.


( E ) Female C57BL / 6J mice were injected with 14 . 5 mg / kg LPS ( normoxia ) or 5 mg / kg LPS ( hypoxia ) after 24h normoxia or hypoxia . Liver was isolated 6h after injected and typical GRE genes were measured via RT - qPCR . N = 4 - 5 per group . ( F ) Female ADX mice were put in normoxia or hypoxia for 24h , injected i . p . with PBS or DEX ( 10 mg / kg ) and 2h later , liver was isolated for RT - qPCR analyses of typical GRE genes . N = 3 - 4 per group . Data information : All bars represent mean ± SEM . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons , except if otherwise stated . Survival curves were analysed with Fisher ' s exact test . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Female C57BL/6J mice were injected with 14.5 mg/kg LPS (normoxia) or 5 mg/kg LPS (hypoxia) after 24h normoxia or hypoxia. Liver was isolated 6h after injected and typical GRE genes were measured via RT-qPCR. N=4-5 per group. (F) Female ADX mice were put in normoxia or hypoxia for 24h, injected i.p. with PBS or DEX (10 mg/kg) and 2h later, liver was isolated for RT-qPCR analyses of typical GRE genes. N=3-4 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. Survival curves were analysed with Fisher's exact test. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.


( G ) Female C57BL / 6J mice were injected with 14 . 5 mg / kg LPS ( normoxia ) or 5 mg / kg LPS ( hypoxia ) after 24h normoxia or hypoxia , with or without pre - treatment with 10 mg / kg DEX 1h before LPS injection . During the follow - up of lethality , mice remained under normoxic or hypoxic conditions . Mice in normoxia : black circles ( LPS ) , black squares ( DEX - LPS ) ; mice in hypoxia : white circles ( LPS ) , white squares ( DEX - LPS ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Female C57BL/6J mice were injected with 14.5 mg/kg LPS (normoxia) or 5 mg/kg LPS (hypoxia) after 24h normoxia or hypoxia, with or without pre-treatment with 10 mg/kg DEX 1h before LPS injection. During the follow-up of lethality, mice remained under normoxic or hypoxic conditions. Mice in normoxia: black circles (LPS), black squares (DEX-LPS); mice in hypoxia: white circles (LPS), white squares (DEX-LPS).


( H ) Female ADX mice were injected with 0 . 05 mg / kg LPS after 24h normoxia or hypoxia , , with or without 10 mg / kg DEX pre - treatment 1h before LPS injection . During the follow - up of lethality , mice remained under normoxic or hypoxic conditions . ADX mice in normoxia : black circles ( LPS ) , black squares ( DEX - LPS ) ; ADX mice in hypoxia : white circles ( LPS ) , white squares ( DEX - LPS ) .

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

(H) Female ADX mice were injected with 0.05 mg/kg LPS after 24h normoxia or hypoxia, , with or without 10 mg/kg DEX pre-treatment 1h before LPS injection. During the follow-up of lethality, mice remained under normoxic or hypoxic conditions. ADX mice in normoxia: black circles (LPS), black squares (DEX-LPS); ADX mice in hypoxia: white circles (LPS), white squares (DEX-LPS).


( B ) The Western blot analysis of GFP , Spd2 and αTub ( loading control ) in the brain extracts from wild type ( Oregon R , OR ) , Spd2WT - OE and Spd2DK - OE third instar larva . Representative immune blot images are shown on the left . The signal intensities of Spd2 bands were quantified and normalised against αTub values . The values relative to control of each biological replicate ( n = 2 ) and their means are shown as dots and bars , respectively , in a scatted dot plot on the right . The numbers above the bars indicate means ± Range . GFP - Spd2DK proteins in Spd2DK - OE larval brain extracts showed approximately 2 . 04 - fold higher accumulation than GFP - Spd2WT proteins in Spd2WT - OE brain extracts . ( C ) The Western blot analysis of Spd2 , CycB and αTub in OR , fzrRNAi , Spd2WT - RES and Spd2DK - RES brain extracts . The signal intensities of Spd2 bands were quantified and normalised against αTub values . The values relative to control and their means are presented in a scatted dot plot on the right ( biological replicate n = 2 ) . Means ± Range are shown above the bars . RFP - Spd2DK proteins in Spd2DK - RES larval brain extracts showed approximately 2 . 08 - fold higher accumulation than RFP - Spd2WT proteins in Spd2WT - RES brain extracts .

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

(B) The Western blot analysis of GFP, Spd2 and αTub (loading control) in the brain extracts from wild type (Oregon R, OR), Spd2WT-OE and Spd2DK-OE third instar larva. Representative immune blot images are shown on the left. The signal intensities of Spd2 bands were quantified and normalised against αTub values. The values relative to control of each biological replicate (n = 2) and their means are shown as dots and bars, respectively, in a scatted dot plot on the right. The numbers above the bars indicate means ± Range. GFP-Spd2DK proteins in Spd2DK-OE larval brain extracts showed approximately 2.04-fold higher accumulation than GFP-Spd2WT proteins in Spd2WT-OE brain extracts. (C) The Western blot analysis of Spd2, CycB and αTub in OR, fzrRNAi, Spd2WT-RES and Spd2DK-RES brain extracts. The signal intensities of Spd2 bands were quantified and normalised against αTub values. The values relative to control and their means are presented in a scatted dot plot on the right (biological replicate n = 2). Means ± Range are shown above the bars. RFP-Spd2DK proteins in Spd2DK-RES larval brain extracts showed approximately 2.08-fold higher accumulation than RFP-Spd2WT proteins in Spd2WT-RES brain extracts.


( D ) Larval brains of OR , Spd2WT - RES , fzrRNAi and Spd2WT - OE flies were stained by an anti - Spd2 antibody . Representative images of NBs are shown on the top . Scale bar : 10 µm . The local signal intensities of Spd2 immunofluorescence at the centrosomes and in the cytoplasm of individual NBs in each brain were measured , and the normalised Spd2 signal intensities in individual NBs were presented as scattered dot plots ( see Materials and Methods for detail ) . The lines indicate means ± SD . The numbers of NBs ( n ) and the numbers of brains analysed ( N ) are control : n = 29 ( N = 6 ) , Spd2WT - RES : n = 30 ( N = 4 ) , fzrRNA : n = 30 ( N = 6 ) , and Spd2WT - OE : n = 27 ( N = 10 ) . P values were calculated using non - parametric two - tailed Mann - Whitney tests . Based on these results , the relative centrosomal Spd2 protein levels in NBs can be estimated as wildtype ( or control ) : Spd2WT - RES : fzrRNAi : Spd2DK - RES : Spd2WT - OE : Spd2DK - OE = 1 : 1 . 00 ~ 1 . 18 : 1 . 06 ~ 1 . 29 : 2 . 08 ~ 2 . 45 ( = 1 . 00 ~ 1 . 18 x 2 . 08 ) : 1 . 93 ~ 3 . 62 : 3 . 94 ~ 7 . 38 ( = 1 . 93 ~ 3 . 62 x 2 .

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

(D) Larval brains of OR, Spd2WT-RES, fzrRNAi and Spd2WT-OE flies were stained by an anti-Spd2 antibody. Representative images of NBs are shown on the top. Scale bar: 10 µm. The local signal intensities of Spd2 immunofluorescence at the centrosomes and in the cytoplasm of individual NBs in each brain were measured, and the normalised Spd2 signal intensities in individual NBs were presented as scattered dot plots (see Materials and Methods for detail). The lines indicate means ± SD. The numbers of NBs (n) and the numbers of brains analysed (N) are control: n= 29 (N = 6), Spd2WT-RES: n = 30 (N = 4), fzrRNA: n = 30 (N = 6), and Spd2WT-OE: n = 27 (N = 10). P values were calculated using non-parametric two-tailed Mann-Whitney tests. Based on these results, the relative centrosomal Spd2 protein levels in NBs can be estimated as wildtype (or control) : Spd2WT-RES: fzrRNAi: Spd2DK-RES: Spd2WT-OE: Spd2DK-OE = 1: 1.00~1.18: 1.06~1.29: 2.08~2.45 (= 1.00~1.18 x 2.08): 1.93~3.62: 3.94 ~ 7.38 (= 1.93~3.62 x 2.04


( C ) In time lapse movies , NBs that showed an active centrosome that was attached to the cortex ( " Attached " ) and NBs with both two centrosomes being inactivated and detached from the cortex ( " Detached " ) in interphase were counted in third instar larval brains of the indicated fly lines and their proportions are presented in bar graphs . The numbers of NBs analysed from at least four brains ( n ) are Control : 29 , fzrRNAi : 80 , Spd2WT - RES : 45 , Spd2DK - RES : 52 , Spd2WT - OE : 43 , Spd2DK - OE : 40 , Spd2WT - OE + fzrRNAi : 72 . p - values are fzrRNAi : 0 . 0069 , Spd2WT - RES : 0 . 2479 , Spd2DK - RES : 0 . 0116 , Spd2WT - OE : < 0 . 0001 , Spd2DK - OE : < 0 . 0001 , Spd2WT - OE + fzrRNAi : 0 . 0008 ( Pearson ' s chi - squared tests were performed between control and each condition ) .

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

(C) In time lapse movies, NBs that showed an active centrosome that was attached to the cortex ("Attached") and NBs with both two centrosomes being inactivated and detached from the cortex ("Detached") in interphase were counted in third instar larval brains of the indicated fly lines and their proportions are presented in bar graphs. The numbers of NBs analysed from at least four brains (n) are Control: 29, fzrRNAi: 80, Spd2WT-RES: 45, Spd2DK-RES: 52, Spd2WT-OE: 43, Spd2DK-OE: 40, Spd2WT-OE + fzrRNAi: 72. p-values are fzrRNAi: 0.0069, Spd2WT-RES: 0.2479, Spd2DK-RES: 0.0116, Spd2WT-OE: <0.0001, Spd2DK-OE: <0.0001, Spd2WT-OE + fzrRNAi: 0.0008 (Pearson's chi-squared tests were performed between control and each condition).


( E ) The signal intensities of the indicated centriolar and PCM proteins at two centrosomes were quantified in individual interphase NBs in Spd2WT - RES and Spd2DK - RES larval brains , and their asymmetric distributions of their centrosomal signals between the two centrosomes are presented as Asymmetric Indexes ( see Materials and Method ) in a scatted dot plot . Red bars represent means ± SD . The numbers of NBs analysed from at least 3 different brains of Spd2WT - RES and Spd2DK - RES ( n ) were 40 and 40 for Asl , 40 and 41 for RFP - Spd2 , 42 and 37 for Cnn , 40 and 41 for γTub , and 40 and 40 for AurA .

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

(E) The signal intensities of the indicated centriolar and PCM proteins at two centrosomes were quantified in individual interphase NBs in Spd2WT-RES and Spd2DK-RES larval brains, and their asymmetric distributions of their centrosomal signals between the two centrosomes are presented as Asymmetric Indexes (see Materials and Method) in a scatted dot plot. Red bars represent means ± SD. The numbers of NBs analysed from at least 3 different brains of Spd2WT-RES and Spd2DK-RES (n) were 40 and 40 for Asl, 40 and 41 for RFP-Spd2, 42 and 37 for Cnn, 40 and 41 for γTub, and 40 and 40 for AurA.


( D ) Division axis deviations were separately analysed in NBs with apically attached interphase centrosomes ( " attached " ) and those with centrosomes being detached from the cortex ( " detached " ) for each line , using the same dataset as in Fig . 3C . The same control data as in Fig . 3C is shown for reference . fzrRNA detached ( mean ± SD : 34 . 62 ° ± 23 . 39 ) , Spd2DK - OE detached ( 64 . 01 ° ± 33 . 93 ) , and Spd2WT - OE + fzrRNAi attached ( 39 . 55 ° ± 27 . 08 ) and detached ( 59 . 08 ° ± 34 . 88 ) brains showed statistically significant differences from control , while NBs with cortically attached centrosomes in any of the lines did not show significant differences . n : the number of NB divisions analysed from at least four brains for each line . p - values were calculated using Mann - Whitney U tests comparing with control and are shown above the dots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Division axis deviations were separately analysed in NBs with apically attached interphase centrosomes ("attached") and those with centrosomes being detached from the cortex ("detached") for each line, using the same dataset as in Fig. 3C. The same control data as in Fig. 3C is shown for reference. fzrRNA detached (mean ± SD: 34.62° ± 23.39), Spd2DK-OE detached (64.01° ± 33.93), and Spd2WT-OE+fzrRNAi attached (39.55° ± 27.08) and detached (59.08° ± 34.88) brains showed statistically significant differences from control, while NBs with cortically attached centrosomes in any of the lines did not show significant differences. n: the number of NB divisions analysed from at least four brains for each line. p-values were calculated using Mann-Whitney U tests comparing with control and are shown above the dots.


( B ) Selected images from time - lapse microscopy of NBs in control ( wor > mCh - Tub , pUbq - GFP - Fzr , top panels ) and Spd2DK - OE ( lower panels ) larval brains ( Movies EV1 , EV10 ) . In the control NB , the daughter centrosomes ( blue arrowheads ) first matured ( - 5 . 0 min ) and was segregated into the daughter NB ( 12 . 0 min ) , while the mother centrosome ( yellow arrowheads ) matured a little later ( 0 . 0 min ) and segregated into the GMC . In contrast , in the Spd2DK - OE NB , both centrosomes lost microtubule nucleation activity in interphase ( red arrowheads ) . The centrosome that matured earlier ( blue arrowheads , - 6 . 0 min ) was incorrectly segregated into the GMC ( 12 . 0 min ) while the centrosome that matured later ( yellow arrowheads , 0 . 0 min ) was segregated into the NB ( 12 . 0 min ) . Dotted white lines outline the dividing NBs and the newly formed GMC , which are also marked by asterisks .

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

(B) Selected images from time-lapse microscopy of NBs in control (wor>mCh-Tub, pUbq-GFP-Fzr, top panels) and Spd2DK-OE (lower panels) larval brains (Movies EV1, EV10). In the control NB, the daughter centrosomes (blue arrowheads) first matured (-5.0 min) and was segregated into the daughter NB (12.0 min), while the mother centrosome (yellow arrowheads) matured a little later (0.0 min) and segregated into the GMC. In contrast, in the Spd2DK-OE NB, both centrosomes lost microtubule nucleation activity in interphase (red arrowheads). The centrosome that matured earlier (blue arrowheads, -6.0 min) was incorrectly segregated into the GMC (12.0 min) while the centrosome that matured later (yellow arrowheads, 0.0 min) was segregated into the NB (12.0 min). Dotted white lines outline the dividing NBs and the newly formed GMC, which are also marked by asterisks.


( C , D ) Representative images of interphase NBs in fixed wor > lacZ ( control , top panels ) and HA - Spd2WT - OE larval brains expressing Polo - GFP from the native promoter and stained for DAPI and Spd2 ( C ) . Asymmetric indexes of Polo - GFP in interphase NBs wor > lacZ ( control , top panels ) and HA - Spd2WT - OE larval brains ( D ) . 38 and 39 NBs ( n ) from at least three brains were analysed in each line . The asymmetric distribution of Polo - GFP at interphase centrosomes was unaffected by HA - Spd2WT overexpression .

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

(C, D) Representative images of interphase NBs in fixed wor>lacZ (control, top panels) and HA-Spd2WT-OE larval brains expressing Polo-GFP from the native promoter and stained for DAPI and Spd2 (C). Asymmetric indexes of Polo-GFP in interphase NBs wor>lacZ (control, top panels) and HA-Spd2WT-OE larval brains (D). 38 and 39 NBs (n) from at least three brains were analysed in each line. The asymmetric distribution of Polo-GFP at interphase centrosomes was unaffected by HA-Spd2WT overexpression.


( E ) Representative images of interphase NBs in fixed mCh - Spd2WT - RES ( top panels ) and mCH - Spd2DK - RES larval brains and stained for DAPI , γTub and Cnn . Both multichannel and single - channel images of mCh - Spd2 , γTub and Cnn are shown . Dotted yellow squares highlight centrosomes and their magnified images are shown in insets . Similar to Spd2DK - RES NBs , in mCh - Spd2CONS - RES NBs , Spd2 and γTub were more symmetrically accumulated at the two centrosomes than in mCh - Spd2WT - RES NBs , but Cnn was still asymmetrically distributed .

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

(E) Representative images of interphase NBs in fixed mCh-Spd2WT-RES (top panels) and mCH-Spd2DK-RES larval brains and stained for DAPI, γTub and Cnn. Both multichannel and single-channel images of mCh-Spd2, γTub and Cnn are shown. Dotted yellow squares highlight centrosomes and their magnified images are shown in insets. Similar to Spd2DK-RES NBs, in mCh-Spd2CONS-RES NBs, Spd2 and γTub were more symmetrically accumulated at the two centrosomes than in mCh-Spd2WT-RES NBs, but Cnn was still asymmetrically distributed.


( G , H ) FRAP analyses of the centrosomal Spd2 fluorescent signals in Spd2WT - RES and Spd2DK - OE NBs . ( G ) Representative images from time - lapse movies ( Movies EV13 , EV14 ) of Spd2 fluorescent signals in Spd2WT - RES NBs ( top panels ) and Spd2DK - OE NBs ( lower panels ) upon photobleaching . ( H ) The recoveries of the fluorescent intensities of centrosomal Spd2 signals after photobleaching were measured in Spd2WT - RES ( n = 7 ) and Spd2DK - OE NBs ( n = 5 ) . Means ± SEM of the normalised fluorescent intensities are shown in a line graph . Centrosomal Spd2 signals recovered more fully after photobleaching in Spd2DK - OE NBs than in Spd2WT - RES NBs .

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

(G, H) FRAP analyses of the centrosomal Spd2 fluorescent signals in Spd2WT-RES and Spd2DK-OE NBs. (G) Representative images from time-lapse movies (Movies EV13, EV14) of Spd2 fluorescent signals in Spd2WT-RES NBs (top panels) and Spd2DK-OE NBs (lower panels) upon photobleaching. (H) The recoveries of the fluorescent intensities of centrosomal Spd2 signals after photobleaching were measured in Spd2WT-RES (n = 7) and Spd2DK-OE NBs (n = 5). Means ± SEM of the normalised fluorescent intensities are shown in a line graph. Centrosomal Spd2 signals recovered more fully after photobleaching in Spd2DK-OE NBs than in Spd2WT-RES NBs.


H . Endogenous levels of Stim1 in testes from C57BL / 6 wt and splice deficient 10A mice , using an antibody detecting the N - term of STIM1 , detecting STIM1A ( A , red arrow ) , STIM1 and its glycosylated ( wt ) and unglycosylated ( wt gl . ) form .

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 B-MEASURED_VAR 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

H. Endogenous levels of Stim1 in testes from C57BL/6 wt and splice deficient 10A mice, using an antibody detecting the N-term of STIM1, detecting STIM1A (A, red arrow), STIM1 and its glycosylated (wt) and unglycosylated (wt gl.) form.


A . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura2 ratio ) over time in response to perfusion of different [ Ca2 + ] o as indicated in the upper bar in MEF Stim1 / Stim2 - / - cells transfected with Stim1 - ( black trace , n = 111 ) , Stim1A ( red trace , n = 116 ) , the combination Stim1 with Stim1A ( blue trace , n = 86 ) or with vector only ( grey trace , n = 79 ) . B . Quantification of changes in ratio of resting , influx rate ( Δratio / time ) , Δpeak and Δplateau measured in A . * * * p < 0 . 001 , Kruskal - Wallis Anova . C . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura 2 ratio ) in HEK293 cells co - transfected with Orai1 and either Stim1 - ( black trace , n = 119 ) or Stim1A IRES - mCherry ( red trace , n = 118 ) . D . Quantification of changes in ratio of resting , influx rate ( Δratio / time ) , Δpeak and Δplateau measured in C . * * p < 0 . 01 * * * p < 0 . 001 Mann - Whitney test . E . Data information : Data were obtained from three biological replicates ( e . g . transfections ) with each three measured dishes ( technical replicates ) with multiple cells each ( yielding a total number n ) and is shown as mean ± s . e . m .

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

A. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar in MEF Stim1/Stim2-/- cells transfected with Stim1- (black trace, n=111), Stim1A (red trace, n=116), the combination Stim1 with Stim1A (blue trace, n=86) or with vector only (grey trace, n=79). B. Quantification of changes in ratio of resting, influx rate (Δratio/time), Δpeak and Δplateau measured in A. *** p<0.001, Kruskal-Wallis Anova. C. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in HEK293 cells co-transfected with Orai1 and either Stim1- (black trace, n=119) or Stim1A IRES-mCherry (red trace, n=118). D. Quantification of changes in ratio of resting, influx rate (Δratio/time), Δpeak and Δplateau measured in C. ** p<0.01 *** p<0.001 Mann-Whitney test. E. Data information: Data were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m.


I . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura 2 ratio ) in primary astrocytes from C57BL / 6 wt ( CTRL , black trace , n = 49 ) and splice deficient C57BL / 6 10ACon mice ( 10ACon , red trace , n = 82 ) . J . Quantification of parameters determined in [ I ] . * * * p < 0 . 001 Mann - Whitney test . Data information : D were obtained from three biological replicates ( e . g . transfections ) with each three measured dishes ( technical replicates ) with multiple cells each ( yielding a total number n ) and is shown as mean ± s . e . m .

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

I. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in primary astrocytes from C57BL/6 wt (CTRL,black trace, n=49) and splice deficient C57BL/6 10ACon mice (10ACon, red trace, n=82). J. Quantification of parameters determined in [I]. *** p<0.001 Mann-Whitney test. Data information: D were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m.


C . Images showing representative HEK293 cells expressing Orai1 - GFP ( green = donor molecule ) or either STIM1 - mCherry ( upper panel = acceptor molecule ) or STIM1A - mCherry ( lower panel = acceptor molecule ) after stimulation with TG . Also shown is the overlay of the two channels , as well as the FRET signal .

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

C. Images showing representative HEK293 cells expressing Orai1-GFP (green = donor molecule) or either STIM1-mCherry (upper panel = acceptor molecule) or STIM1A-mCherry (lower panel = acceptor molecule) after stimulation with TG. Also shown is the overlay of the two channels, as well as the FRET signal.


B . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura2 ratio ) over time in response to perfusion of different [ Ca2 + ] o as indicated in the upper bar with constructs as indicated expressed in HEKO1 . STIM1 ( black , n = 145 ) , STIM1A ( red , n = 157 ) and STIM1A _ D503A ( n = 185 ) . C . Relative fluorescence intensities of mCherry - tagged constructs measured in ( B ) . D . Quantification of changes in resting ratio , TG peak ( ∆ ratio ) and SOCE parameters measured in B . E . Data information : * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 Kruskal - Wallis Anova with Dunn ' s multiple comparisons test . Data points ( total n ) were obtained from three biological replicates with each three technical replicates ( each multiple cells ) and shown as mean ± s . e . m for traces and as scatter plots with the underlying boxes showing the means for individual parameters .

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

B. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar with constructs as indicated expressed in HEKO1. STIM1 (black, n=145), STIM1A (red, n=157) and STIM1A_D503A (n=185). C. Relative fluorescence intensities of mCherry-tagged constructs measured in (B). D. Quantification of changes in resting ratio, TG peak (∆ ratio) and SOCE parameters measured in B. E. Data information: *P<0.05, **P<0.01 ***P<0.001 Kruskal-Wallis Anova with Dunn's multiple comparisons test. Data points (total n) were obtained from three biological replicates with each three technical replicates (each multiple cells) and shown as mean±s.e.m for traces and as scatter plots with the underlying boxes showing the means for individual parameters.


E . Average traces showing whole - cell current density ( CD ) over time extracted at - 80 mV in HEKO1 cells transfected with STIM1 ( black ) , STIM1A ( red ) or STIM1A _ D503A ( blue ) . F . Average maximum CDs recorded from cells measured in E ( n within bars ) . G Data information : * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 Kruskal - Wallis Anova with Dunn ' s multiple comparisons test . Data points ( total n ) were obtained from three biological replicates with each three technical replicates ( each multiple cells ) and shown as mean ± s . e . m for traces and as scatter plots with the underlying boxes showing the means for individual parameters .

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

E. Average traces showing whole-cell current density (CD) over time extracted at -80 mV in HEKO1 cells transfected with STIM1 (black), STIM1A (red) or STIM1A_D503A (blue). F. Average maximum CDs recorded from cells measured in E (n within bars). G Data information: *P<0.05, **P<0.01 ***P<0.001 Kruskal-Wallis Anova with Dunn's multiple comparisons test. Data points (total n) were obtained from three biological replicates with each three technical replicates (each multiple cells) and shown as mean±s.e.m for traces and as scatter plots with the underlying boxes showing the means for individual parameters.


G . Average current - voltage ( I - V ) relationship of all cells recorded in E .

G. Average current-voltage (I- V) relationship of all cells recorded in E.


F . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura2 ratio ) over time in response to perfusion of different [ Ca2 + ] o as indicated in the upper bar with constructs as indicated expressed in HEKS1 / S2 - / - . G . Quantification of the relative reduction of ratio / time or ratio for cells ( 135 H Data information : , * * * P < 0 . 001 , Kruskal - Wallis Anova with Dunn ' s multiple comparisons test Data points ( total n ) were obtained from three biological replicates with each three technical replicates ( with multiple cells ) and shown as mean ± s . e . m for traces ( F ) and as scatter plots with the underlying boxes showing the means for individual parameters ( G ) .

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

F. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar with constructs as indicated expressed in HEKS1/S2-/-. G. Quantification of the relative reduction of ratio/time or ratio for cells (135 H Data information: , ***P<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test Data points (total n) were obtained from three biological replicates with each three technical replicates (with multiple cells) and shown as mean±s.e.m for traces (F) and as scatter plots with the underlying boxes showing the means for individual parameters (G).


H , I . Average traces ( mean ± s . e . m . ) showing whole - cell current density ( CD ) over time extracted at - 80 mV in in HEKS1 / S2 - / - cells co - transfected either with STIM1 ( black ) or STIM1A ( red ) and ORAI1 ( H ) or with ORAI1 R77E ( I ) and recorded using extracellular solution containing 2 mM Ca2 + . J . Average maximum CDs recorded from cells measured in [ H , I ] ( n within bars ) . Data information : I , * P < 0 . 05 , * * P < 0 . 01 , unpaired T test with Welch ' s correction . For patch - clamp experiments , number in bars indicate measured cells , which were from three independent transfections .

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

H,I. Average traces (mean±s.e.m.) showing whole- cell current density (CD) over time extracted at -80 mV in in HEKS1/S2-/- cells co-transfected either with STIM1 (black) or STIM1A (red) and ORAI1 (H) or with ORAI1 R77E (I) and recorded using extracellular solution containing 2 mM Ca2+. J. Average maximum CDs recorded from cells measured in [H,I] (n within bars). Data information: I , *P<0.05, **P<0.01, unpaired T test with Welch's correction. For patch-clamp experiments, number in bars indicate measured cells, which were from three independent transfections.


B . Hits from ( A ) tested for interaction with commercially available constructs quantified with bimolecular fluorescence complementation ( BiFC ) via flow cytometry . HEKS1 / S2 - / - were transfected with STIM1 - YFPC ( black ) or STIM1A - YFPC ( red ) in combination with POI - YFPN and screened for YFP + cells via FACS , results ( mean ± SD ) from three transfections with each 10 . 000 sorted cells .

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

B. Hits from (A) tested for interaction with commercially available constructs quantified with bimolecular fluorescence complementation (BiFC) via flow cytometry. HEKS1/S2-/- were transfected with STIM1-YFPC (black) or STIM1A-YFPC (red) in combination with POI-YFPN and screened for YFP+ cells via FACS, results (mean ± SD) from three transfections with each 10.000 sorted cells.


D . Ratio nuclear NFAT - GFP vs . cytosolic GFP intensity normalized to t = 0 ( mean ± s . e . m . ) after transfection of SH - SY5Y S1 - / - cells with STIM ( n = 42 ) , STIM1A ( n = 82 ) , STIM1A _ D503A ( n = 49 ) or vector only ( ØSTIM , n = 15 ) IRES mCherry and induction of SOCE after stimulation with 1 µM TG . Data ( total n as indicated ) from at least three independent transfections .

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

D. Ratio nuclear NFAT-GFP vs. cytosolic GFP intensity normalized to t=0 (mean±s.e.m.) after transfection of SH-SY5Y S1-/- cells with STIM (n=42), STIM1A (n=82), STIM1A_D503A (n=49) or vector only (ØSTIM, n=15) IRES mCherry and induction of SOCE after stimulation with 1 µM TG. Data (total n as indicated) from at least three independent transfections.


A . Time course of translocation ( Nuclear NFAT - GFP signal / cytosolic signal normalized to TG addition at t = 0 , mean ± s . e . m , for each time point ) after transfection of indicated constructs and preincubation with 1 µM PF - 04957325 ( analyzed cells : STIM1 + PF n = 75 , STIM1A + PF n = 50 , ØSTIM + PF n = 9 ) Data ( total n as indicated ) from at least three independent transfections . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Kruskal - Wallis Anova with Dunn ' s multiple comparisons test . B . PF - 04957325 induced difference of the nuclear NFAT - GFP signal vs . cytosolic signal from ( A ) to mean of control without the blocker ( see Fig . 6D ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney test of differences . . C Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , 2way Anova Data ( total number of cells , n ) were obtained from at least three independent transfections with several technical replicates each .

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

A. Time course of translocation (Nuclear NFAT-GFP signal/cytosolic signal normalized to TG addition at t=0, mean±s.e.m, for each time point) after transfection of indicated constructs and preincubation with 1 µM PF-04957325 (analyzed cells: STIM1 + PF n = 75, STIM1A + PF n = 50, ØSTIM + PF n = 9) Data (total n as indicated) from at least three independent transfections. * p< 0.05, **p<0.01, ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test. B. PF-04957325 induced difference of the nuclear NFAT-GFP signal vs. cytosolic signal from (A) to mean of control without the blocker (see Fig. 6D). * p< 0.05, **p<0.01, ***p<0.001,Mann-Whitney test of differences.. C Data information: *p<0.05, **p<0.01, ***p<0.001, 2way Anova Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.


C . Endpoint value with addition of PF or indicated cAMP analoga loaded for 30 min as AM esters before the experiment , shown as mean ± s . e . m , ØSTIM + 6Bnz - cAMP / 8 - pCPT - cAMP n = 15 / 14 cells ; STIM1 + 6Bnz - cAMP / 8 - pCPT - cAMP : n = 87 / 66 , STIM1A + 6Bnz - cAMP : n = 78 / 67 ; from three independent transfections . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , , Kruskal - Wallis Anova with Dunn ' s multiple comparison test Data ( total number of cells , n ) were obtained from at least three independent transfections with several technical replicates each .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O 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. Endpoint value with addition of PF or indicated cAMP analoga loaded for 30 min as AM esters before the experiment, shown as mean±s.e.m , ØSTIM + 6Bnz-cAMP/8-pCPT-cAMP n = 15/14 cells; STIM1 + 6Bnz-cAMP/8-pCPT-cAMP: n = 87/66, STIM1A + 6Bnz-cAMP : n = 78/67; from three independent transfections. Data information: *p<0.05, **p<0.01, ***p<0.001, , Kruskal-Wallis Anova with Dunn's multiple comparison test Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.


G . Normalized fluorescence signal change ( mean ± s . e . m ) of R - FlincA , SH - SY5Y S1 - / - cells expressing STIM1 ( n = 20 ) or STIM1A ( n = 22 ) IRES GFP variants and the indicator were stimulated by TG ( 1 μM ) . To avoid photobleaching , values were only recorded after 5 and 30 min , respectively The mutant R - Flinc ( n = 15 ) was measured in SH - SY5Y wt cells after TG stimulation . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Kruskal - Wallis Anova with Dunn ' s multiple comparison test Data ( total number of cells , n ) were obtained from at least three independent transfections with several technical replicates each .

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

G. Normalized fluorescence signal change (mean±s.e.m) of R-FlincA, SH-SY5Y S1-/- cells expressing STIM1 (n=20) or STIM1A (n=22) IRES GFP variants and the indicator were stimulated by TG (1 μM). To avoid photobleaching, values were only recorded after 5 and 30 min, respectively The mutant R-Flinc (n=15) was measured in SH-SY5Y wt cells after TG stimulation. Data information: *p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparison test Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.


Relative expression and co - localization of IL6 mRNA and IL - 6 Signaling Scoring in integrated skin cells by blended UMAPs .

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

Relative expression and co-localization of IL6 mRNA and IL-6 Signaling Scoring in integrated skin cells by blended UMAPs.


Quantification by real - time qPCR analysis of Il6 mRNA in the skin of naïve and irradiated ( 15Gy ) WT mice at indicated times , ( n = 3 - 6 ) . Data information : Data represent mean ± SD ( B , * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by one - way ANOVA with Dunnet ' s multiple comparisons test ( B ) , five independent experiments .

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

Quantification by real-time qPCR analysis of Il6 mRNA in the skin of naïve and irradiated (15Gy) WT mice at indicated times, (n=3-6). Data information: Data represent mean ± SD (B, *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by one-way ANOVA with Dunnet's multiple comparisons test (B), five independent experiments.


Quantification of ventral hair loss area 21 days post - IR ( 15Gy ) in WT and IL - 6 - / - mice , ( n = 11 ) . Photographic images showing hair loss ( area demarked by yellow dashed line ) 21 days post - IR ( 15 Gy ) in wild type ( WT ) and IL - 6 - / - mice . Data information : Data represent mean ± SD C , * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by , two - tailed Mann - Whitney test ( C , ; five independent experiments .

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Quantification of ventral hair loss area 21 days post-IR (15Gy) in WT and IL-6-/- mice, (n=11). Photographic images showing hair loss (area demarked by yellow dashed line) 21 days post-IR (15 Gy) in wild type (WT) and IL-6-/- mice. Data information: Data represent mean ± SD C, *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by , two-tailed Mann-Whitney test (C, ; five independent experiments.


Histochemical ( H & E ) and immunostaining ( red ) showing skin morphology , and T cell ( CD3 + ) and neutrophil ( Ly6b + ) infiltration in WT or IL - 6 - / - mice before ( naïve ) , and 14 days ( H & E , Ly6b + ) , or 21 days ( CD3 + ) post - IR . Infiltrating CD3 + T cells ( yellow arrows ) within degenerating hair follicles ( HF ; dashed lines ) are indicated . Scale bars , 100 μm . Quantification of epidermal thickness at 14 days post - IR ( n = 5 - 8 ) . Kinetics of neutrophil ( Ly6b + ) ( n = 3 - 9 ) and T cell ( CD3 + ) ( n = 3 ) infiltration . Data information : Data represent mean ± SD F , mean ± SD ( G ) . * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by two - tailed Mann - Whitney test F , or two - way ANOVA with Bonferroni ' s posthoc multiple comparisons test ( G ) ; five independent experiments .

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Histochemical (H&E) and immunostaining (red) showing skin morphology, and T cell (CD3+) and neutrophil (Ly6b+) infiltration in WT or IL-6-/- mice before (naïve), and 14 days (H&E, Ly6b+), or 21 days (CD3+) post-IR. Infiltrating CD3+ T cells (yellow arrows) within degenerating hair follicles (HF; dashed lines) are indicated. Scale bars, 100 μm. Quantification of epidermal thickness at 14 days post-IR (n = 5-8). Kinetics of neutrophil (Ly6b+) (n = 3-9) and T cell (CD3+) (n = 3) infiltration. Data information: Data represent mean ± SD F, mean ± SD (G). *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by two-tailed Mann-Whitney test F, or two-way ANOVA with Bonferroni's posthoc multiple comparisons test (G); five independent experiments.


Quantification by real - time qPCR analysis of Tnfa , Ccl3 , and Ifng mRNAs in whole skin of WT and IL - 6 - / - mice at indicated times post - IR ( 15Gy ) , ( n = 5 - 7 ) . Data information : Data represent mean ± SD H ) * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by , two - tailed Mann - Whitney test H ) , five independent experiments .

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Quantification by real-time qPCR analysis of Tnfa, Ccl3, and Ifng mRNAs in whole skin of WT and IL-6-/- mice at indicated times post-IR (15Gy), (n = 5-7). Data information: Data represent mean ± SD H) *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by , two-tailed Mann-Whitney test H), five independent experiments.


Western blot analysis shows phosphorylated STAT3 ( p - Stat3 ) , Akt ( p - Akt ) ) , Erk1 and Erk2 ( p - Erk1 / 2 ) ) , and β - Actin in the skin of WT , IL - 6 - / - , and IL - 1R - / - mice at indicated times ( days ) post - IR ( 15Gy ) .

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Western blot analysis shows phosphorylated STAT3 (p-Stat3), Akt (p-Akt)), Erk1 and Erk2 (p-Erk1/2)), and β-Actin in the skin of WT, IL-6-/-, and IL-1R-/- mice at indicated times (days) post-IR (15Gy).


Histochemical ( H & E ) and immunostaining ( red ) staining of T cells ( CD3 + ) , neutrophils ( Ly6b + ) and phosphorylated Stat3 ( p - Stat3 ) ( red nuclear staining , black arrows ) in skin thin sections prepared at 14 days post - IR from mice treated by daily topical application of acquosum ( control ) , ruxolitinib or tofacitinib following irradiation ( 15 Gy ) . Scale bar , 100 μm . ( CD3 and Ly6b immunostaining images from Tofacitinib treated mice represent seperate analyses performed on serial thin sections . ) Quantification of epidermal thickness , T cells ( CD3 + ) and neutrophils ( Ly6b + ) , and p - STAT3 + cells in ( B ) , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , by two - tailed Student ' s t test ; two independent experiments .

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Histochemical (H&E) and immunostaining (red) staining of T cells (CD3+), neutrophils (Ly6b+) and phosphorylated Stat3 (p-Stat3) (red nuclear staining, black arrows) in skin thin sections prepared at 14 days post-IR from mice treated by daily topical application of acquosum (control), ruxolitinib or tofacitinib following irradiation (15 Gy). Scale bar, 100 μm. (CD3 and Ly6b immunostaining images from Tofacitinib treated mice represent seperate analyses performed on serial thin sections.) Quantification of epidermal thickness, T cells (CD3+) and neutrophils (Ly6b+), and p-STAT3+ cells in (B), (n = 4). Data information: Data represent mean ± SD. *P<0.05, **P<0.01, by two-tailed Student's t test; two independent experiments.


Photographic images showing hair loss in mice at 14 days post - IR ( 15 Gy ) following treatment by daily topical application of acquosum ( carrier control ) or JAK inhibitors , ruxolitinib or tofacitinib . Chest and neck hairs of mice in all groups were clipped prior to irradiation to enhance drug absorption . Quantification of the area of ventral hair loss ( in pixels ) in mice in ( D ) , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , by two - tailed Student ' s t test ; two independent experiments .

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Photographic images showing hair loss in mice at 14 days post-IR (15 Gy) following treatment by daily topical application of acquosum (carrier control) or JAK inhibitors, ruxolitinib or tofacitinib. Chest and neck hairs of mice in all groups were clipped prior to irradiation to enhance drug absorption. Quantification of the area of ventral hair loss (in pixels) in mice in (D), (n = 4). Data information: Data represent mean ± SD. *P<0.05, **P<0.01, by two-tailed Student's t test; two independent experiments.


UMAP plots depicting relative expression and localization of Il1a and Il1b mRNAs in irradiated and naïve skin - derived cell clusters .

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UMAP plots depicting relative expression and localization of Il1a and Il1b mRNAs in irradiated and naïve skin-derived cell clusters.


Photographic images showing hair loss 14 days post - IR ( 15 Gy ) in WT and IL - 1R - / - mice . Quantification of ventral hair loss area 14 days post - IR ( 15Gy ) in WT and IL - 1R - / - mice , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , by two - tailed Mann - Whitney test ( C ) five independent experiments .

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Photographic images showing hair loss 14 days post-IR (15 Gy) in WT and IL-1R-/- mice. Quantification of ventral hair loss area 14 days post-IR (15Gy) in WT and IL-1R-/- mice, (n = 4). Data information: Data represent mean ± SD. *P<0.05, by two-tailed Mann-Whitney test (C) five independent experiments.


Histochemical ( H & E ) and immunostaining ( red ) showing morphology and neutrophil ( Ly6b ) infiltration in the skin of mice in ( B ) . Scale bar , 50 μm . Quantification of epidermal thickness , neutrophils ( Ly6b ) and T cells ( CD3 + ) in specimens in ( D ) , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , by Student ' s t test ( E ) ; five independent experiments .

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Histochemical (H&E) and immunostaining (red) showing morphology and neutrophil (Ly6b) infiltration in the skin of mice in (B). Scale bar, 50 μm. Quantification of epidermal thickness, neutrophils (Ly6b) and T cells (CD3+) in specimens in (D), (n = 4). Data information: Data represent mean ± SD. *P<0.05, by Student's t test (E); five independent experiments.


UMAP plots depicting relative expression and localization of Ccr6 and Ccl20 mRNAs in irradiated and naïve skin - derived cell clusters . Quantification by real - time qPCR analysis of Ccr6 and Ccl20 mRNAs in whole skin of naïve and irradiated ( 15Gy ) WT and IL - 6 - / - mice at indicated times post - IR , ( n = 5 - 7 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two independent experiments .

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UMAP plots depicting relative expression and localization of Ccr6 and Ccl20 mRNAs in irradiated and naïve skin-derived cell clusters. Quantification by real-time qPCR analysis of Ccr6 and Ccl20 mRNAs in whole skin of naïve and irradiated (15Gy) WT and IL-6-/- mice at indicated times post-IR, (n=5-7). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.


Photographic images showing the development of radiodermatitis and alopecia in irradiated ( 15 Gy ) WT and CCR6 - / - mice at indicated times post - IR . Escalating radiodermatitis , which manifests on day 7 - 10 as serous exudates on the chin and neck , is visible in both strains and progresses in WT mice to alopecia by day 14 post - IR but resolves spontaneously in the CCR6 - / - mice . Quantification ( lower right ) of the area of hair loss at 14 days post - IR , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two independent experiments .

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

Photographic images showing the development of radiodermatitis and alopecia in irradiated (15 Gy) WT and CCR6-/- mice at indicated times post-IR. Escalating radiodermatitis, which manifests on day 7-10 as serous exudates on the chin and neck, is visible in both strains and progresses in WT mice to alopecia by day 14 post-IR but resolves spontaneously in the CCR6-/- mice. Quantification (lower right) of the area of hair loss at 14 days post-IR, (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.


Histochemical staining ( H & E ) and immunostaining ( red ) show morphology and infiltration of neutrophils ( Ly6b + ) and T cells ( CD3 + ) ( red arrows ) to the skin and to the hair follicle ( HF ) ( dashed lines ) in WT or CCR6 - / - mice 14 days post - IR . Scale bars , 50 μm ( black ) and 25 μm ( red ) . Quantification of epidermal thickness , neutrophils ( Ly6b + ) , total T cell ( CD3 + ) infiltration to the skin , and ( F ) CD3 + cells infiltration to the hair follicle in ( D ) , ( n = 5 - 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t test ( E - Ly6b , by Mann - Whitney test Two independent experiments .

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Histochemical staining (H&E) and immunostaining (red) show morphology and infiltration of neutrophils (Ly6b+) and T cells (CD3+) (red arrows) to the skin and to the hair follicle (HF) (dashed lines) in WT or CCR6-/- mice 14 days post-IR. Scale bars, 50 μm (black) and 25 μm (red). Quantification of epidermal thickness, neutrophils (Ly6b+), total T cell (CD3+) infiltration to the skin, and (F) CD3+ cells infiltration to the hair follicle in (D), (n = 5-6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t test (E-Ly6b, by Mann-Whitney test Two independent experiments.


MHC class I immunostaining ( red ) of skin thin sections prepared at 14 days post - IR from mice in ( C ) , and quantification ( right ) of MHC I positive hair follicles , ( n = 4 - 5 ) . Scale bar , 50 μm . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two independent experiments .

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MHC class I immunostaining (red) of skin thin sections prepared at 14 days post-IR from mice in (C), and quantification (right) of MHC I positive hair follicles, (n = 4-5). Scale bar, 50 μm. Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.


Quantification by real - time qPCR analysis of Il6 , Il17a , Ccl20 , and Ccl3 mRNAs in the skin of naïve WT and irradiated ( 15Gy ) WT and CCR6 - / - mice at 14 days post - IR , ( n = 3 - 5 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two independent experiments .

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Quantification by real-time qPCR analysis of Il6, Il17a, Ccl20, and Ccl3 mRNAs in the skin of naïve WT and irradiated (15Gy) WT and CCR6-/- mice at 14 days post-IR, (n = 3-5). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.


Photographic images showing hair loss in control ( PBS ) and cyclosporine ( CsA ) treated WT mice at 14 days post - IR ( 15 Gy ) . Quantification of the area of ventral hair loss in mice in ( A ) , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Mann - Whitney test ; two independent experiments .

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Photographic images showing hair loss in control (PBS) and cyclosporine (CsA) treated WT mice at 14 days post-IR (15 Gy). Quantification of the area of ventral hair loss in mice in (A), (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.


Histochemical ( H & E ) and immunostaining ( red ) show T cell ( CD3 + ) and neutrophil ( Ly6b + ) infiltration in the skin of mice in ( A ) . Scale bar , 100 μm . ( Images of CD3 and Ly6b immunostaining from CsA treated mice represent seperate analyses performed on serial thin sections . ) Quantification of epidermal thickness , and T cell ( CD3 + ) and neutrophil ( Ly6b + ) infiltration ( red staining ) in skin thin sections from ( C ) , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Mann - Whitney test ; two independent experiments .

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Histochemical (H&E) and immunostaining (red) show T cell (CD3+) and neutrophil (Ly6b+) infiltration in the skin of mice in (A). Scale bar, 100 μm. (Images of CD3 and Ly6b immunostaining from CsA treated mice represent seperate analyses performed on serial thin sections.) Quantification of epidermal thickness, and T cell (CD3+) and neutrophil (Ly6b+) infiltration (red staining) in skin thin sections from (C), (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.


Quantification by real - time qPCR analysis of Il6 , Il17a , II22 , Ccl3 , Ccr6 , and Ccl20 mRNAs in whole skin of naïve and irradiated ( 15Gy ) control ( PBS ) and CSA treated WT mice at 14 days post - IR , ( n = 5 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Mann - Whitney test ; two independent experiments .

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Quantification by real-time qPCR analysis of Il6, Il17a, II22, Ccl3, Ccr6, and Ccl20 mRNAs in whole skin of naïve and irradiated (15Gy) control (PBS) and CSA treated WT mice at 14 days post-IR, (n = 5). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.


( A - B ) Immunofluorescence ( IF ) analysis of IκBα in sections from murine small intestine of 2 month - old WT ( A ) and Lgr5 - GFP reporter mice ( B ) . Scale bars , 50μm .

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(A-B) Immunofluorescence (IF) analysis of IκBα in sections from murine small intestine of 2 month-old WT (A) and Lgr5-GFP reporter mice (B). Scale bars, 50μm.


IF analysis of P - IκBα in the intestine of 2 month - old WT ( C ) and quantification of P - IκBα positivity Scale bars in C , 25

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IF analysis of P-IκBα in the intestine of 2 month-old WT (C) and quantification of P-IκBα positivity Scale bars in C, 25 μm


IF analysis of P - IκBα in the intestine of 2 month - old Lgr5 - GFP reporter mice ( D ) , and quantification of P - IκBα positivity inside the Lgr5 - GFP population . A minimum of 30 crypts was counted in 3 Lgr5 - GFP mice . Scale bars in D , 50 μm .

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IF analysis of P-IκBα in the intestine of 2 month-old Lgr5-GFP reporter mice (D), and quantification of P-IκBα positivity inside the Lgr5-GFP population. A minimum of 30 crypts was counted in 3 Lgr5-GFP mice. Scale bars in D, 50 μm.


( E ) Distribution of P - IκBα positive cells according to their position in the intestinal crypt from 200 crypts counted .

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(E) Distribution of P-IκBα positive cells according to their position in the intestinal crypt from 200 crypts counted.


( F ) Double IF analysis of 2 month - old murine small intestine sections with the indicated antibodies and quantification of P - IκBα positivity inside the EphB2 + population . 70 crypts were counted . Bars represent mean values ± standard error of the mean ( s . e . m ) . Scale bars , 50 μm .

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(F) Double IF analysis of 2 month-old murine small intestine sections with the indicated antibodies and quantification of P-IκBα positivity inside the EphB2+ population. 70 crypts were counted. Bars represent mean values ± standard error of the mean (s.e.m). Scale bars, 50 μm.


( G ) Double IF analysis of murine small intestine sections with the indicated antibodies . Scale bars , 50 μm .

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(G) Double IF analysis of murine small intestine sections with the indicated antibodies. Scale bars, 50 μm.


( H ) Western blot analysis of cytoplasmic ( CYT ) and nuclear ( NUC ) extracts from isolated intestinal crypt cells .

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(H) Western blot analysis of cytoplasmic (CYT) and nuclear (NUC) extracts from isolated intestinal crypt cells.


( I ) Western blot analysis of crypt nuclear extracts immunoprecipitated with anti - SUMO2 / 3 antibody .

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(I) Western blot analysis of crypt nuclear extracts immunoprecipitated with anti-SUMO2/3 antibody.


( J ) IF analysis of developing intestines from E12 . 5 to E17 . 5 with the indicated antibodies . Scale bars , 25 μm .

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(J) IF analysis of developing intestines from E12.5 to E17.5 with the indicated antibodies. Scale bars, 25 μm.


( K ) Representation of IκBα distribution in the indicated genomic regions obtained from ChIP - sequencing analysis ( n = 4 ) .

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(K) Representation of IκBα distribution in the indicated genomic regions obtained from ChIP-sequencing analysis (n=4).


( A ) Representative images of P4 and P6 mice of the indicated IκBα genotypes .

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(A) Representative images of P4 and P6 mice of the indicated IκBα genotypes.


Quantification of the animal weight ( B ) from 50 WT / HET and 15 KO mice analyzed . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant .

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Quantification of the animal weight (B) from 50 WT/HET and 15 KO mice analyzed. Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant.


Quantification of the intestinal length ( C ) from 50 WT / HET and 15 KO mice analyzed . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant .

Quantification of the intestinal length (C) from 50 WT/HET and 15 KO mice analyzed. Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant.


D - G ( D ) Immunohistochemical ( IHC ) , IF and Alcian Blue staining with quantification of P6 IκBα WT and KO intestines ( D ) , 2 month - old IκBβ ; IκBε KO ( E ) and 2 month - old IκBαNES mice ( F and G ) . A minimum of 50 crypts / villus was counted per genotype ( 3 mice ) . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant . Scale bars in D , E , F and G , 50 μm .

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D-G (D) Immunohistochemical (IHC), IF and Alcian Blue staining with quantification of P6 IκBα WT and KO intestines (D), 2 month-old IκBβ;IκBε KO (E) and 2 month-old IκBαNES mice (F and G). A minimum of 50 crypts/villus was counted per genotype (3 mice). Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant. Scale bars in D, E, F and G, 50 μm.


( A ) Representative image of the intestinal populations purified in the cell sorting experiments .

(A) Representative image of the intestinal populations purified in the cell sorting experiments.


( B ) IF analysis of EphB2 in P6 WT and IκBα KO intestine . Data information : Scale bars , 25 μm .

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(B) IF analysis of EphB2 in P6 WT and IκBα KO intestine. Data information: Scale bars , 25 μm.


( C ) P - IκBα IF of EphB2 - high or negative sorted cells . Data information : Scale bars , 25 μm .

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(C) P-IκBα IF of EphB2-high or negative sorted cells. Data information: Scale bars , 25 μm.


( D ) Clustering analysis of the indicated cell populations based on their transcriptional profiles obtained in RNA - seq . 3 mice per genotype were initially processed although 1 EphB2neg KO replicate was excluded from the analysis due to insufficient number of reads in the RNA seq .

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(D) Clustering analysis of the indicated cell populations based on their transcriptional profiles obtained in RNA-seq. 3 mice per genotype were initially processed although 1 EphB2neg KO replicate was excluded from the analysis due to insufficient number of reads in the RNA seq.


( E ) Correlation plot of the indicated differentially expressed gene ( DEG ) sets . Values for DEG in fetal and adult ISC was obtained from Nusse et al , 2012 .

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(E) Correlation plot of the indicated differentially expressed gene (DEG) sets. Values for DEG in fetal and adult ISC was obtained from Nusse et al, 2012.


( F ) Table indicating the top 15 up - regulated genes in the IκBα KO EphB2 - high signature including 11 genes in the fetal ISC signature ( in orange ) .

(F) Table indicating the top 15 up-regulated genes in the IκBα KO EphB2-high signature including 11 genes in the fetal ISC signature (in orange).


( G ) GSEA of EphB2 - high cells indicating the enrichment of relevant ISC - related pathways .

(G) GSEA of EphB2-high cells indicating the enrichment of relevant ISC-related pathways.


( H ) Representative image of the Lgr5 + population purified in the cell sorting experiments .

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(H) Representative image of the Lgr5+ population purified in the cell sorting experiments.


( I ) qPCR analysis of the indicated genes in purified Lgr5 + cells from the different IκBα backgrounds . 4 WT and 1 KO mice were analyzed with at least three technical replicates . Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

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(I) qPCR analysis of the indicated genes in purified Lgr5+ cells from the different IκBα backgrounds. 4 WT and 1 KO mice were analyzed with at least three technical replicates. Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, *p-value<0.05, n.s. no significant.


( A ) IF analysis of c - Rel and RelA / p65 in P6 WT and IκBα KO intestines . Scale bars in 25 μm .

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(A) IF analysis of c-Rel and RelA/p65 in P6 WT and IκBα KO intestines. Scale bars in 25 μm.


( B ) Number of peaks from p65 ChIP - sequencing analysis associated with the different genomic localizations was obtained merging two biological replicates per condition ( n = 2 P6 WT and n = 2 P6 IκBα KO intestinal crypt cells ) . Data from individual experiments have been deposited at GSE131187 .

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(B) Number of peaks from p65 ChIP-sequencing analysis associated with the different genomic localizations was obtained merging two biological replicates per condition (n=2 P6 WT and n=2 P6 IκBα KO intestinal crypt cells). Data from individual experiments have been deposited at GSE131187.


IF analysis of active Notch1 ( ICN1 ) ( C ) in the indicated IκBα backgrounds at P6 intestines . Scale bars in 25 μm .

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IF analysis of active Notch1 (ICN1) (C) in the indicated IκBα backgrounds at P6 intestines. Scale bars in 25 μm.


IF analysis of β - catenin ( D ) in the indicated IκBα backgrounds at P6 intestines . Scale bars 25 μm .

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IF analysis of β-catenin (D) in the indicated IκBα backgrounds at P6 intestines. Scale bars 25 μm.


( E ) IF analysis of the PRC2 protein EZH2 and quantification of the EZH2 positive cells per crypt in n = 3 P6 animals per genotype were analyzed ( a minimum of 15 crypts was counted per genotype ) . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 . Scale bars 25 μm .

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(E) IF analysis of the PRC2 protein EZH2 and quantification of the EZH2 positive cells per crypt in n=3 P6 animals per genotype were analyzed (a minimum of 15 crypts was counted per genotype). Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001. Scale bars 25 μm.


( F ) Table indicating the methylation - related genes ( PRC2 members and KDM6 demethylase ) differentially express between IκBα WT and KO EphB2 - high sorted cells ( RNA - seq ) .

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(F) Table indicating the methylation-related genes (PRC2 members and KDM6 demethylase) differentially express between IκBα WT and KO EphB2-high sorted cells (RNA-seq).


( G ) Western blot analysis of IκBα IP from crypt nuclear extracts .

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(G) Western blot analysis of IκBα IP from crypt nuclear extracts.


( I ) Representation of H3K27me3 distribution at embryonic E14 . 5 ( n = 2 WT and n = 2 KO mice ) and postnatal P6 ( n = 2 WT and n = 2 KO mice ) ( from ChIP - sequencing ) and expression levels ( from RNA - sequencing ) in the genomic region of the indicated fetal ISC genes .

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(I) Representation of H3K27me3 distribution at embryonic E14.5 (n=2 WT and n=2 KO mice) and postnatal P6 (n=2 WT and n=2 KO mice) (from ChIP-sequencing) and expression levels (from RNA-sequencing) in the genomic region of the indicated fetal ISC genes.


( J ) Representation of IκBα ( n = 4 ) and H3K27me3 ( n = 2 ) distribution ( from ChIP - sequencing ) and expression levels ( from RNA - sequencing ) in the genomic region of Litaf .

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(J) Representation of IκBα (n=4) and H3K27me3 (n=2) distribution (from ChIP-sequencing) and expression levels (from RNA-sequencing) in the genomic region of Litaf.


( A ) Representative stereoscopic image of P6 WT and IκBα KO - derived intestinal organoids . Scale bars in A , 100

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(A) Representative stereoscopic image of P6 WT and IκBα KO-derived intestinal organoids. Scale bars in A, 100 μm


( B ) IHC analysis with the indicated antibodies and Alcian Blue staining of WT and IκBα KO - derived organoids . Scale bars in B 50 μm .

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(B) IHC analysis with the indicated antibodies and Alcian Blue staining of WT and IκBα KO-derived organoids. Scale bars in B 50 μm.


( C ) qPCR analysis of the indicated ISC genes in WT and IκBα KO - derived organoids . Data information : 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 .

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(C) qPCR analysis of the indicated ISC genes in WT and IκBα KO-derived organoids. Data information: 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, * p-value<0.05.


( D ) Correlation plots of the indicated differentially expressed gene ( DEG ) sets obtained in expression microarray ( 5 technical replicates of a minimum of two organoids per genotype were analyzed ) . Values for DEG in fetal and adult organoids were obtained from Mustata et al , 2013 .

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(D) Correlation plots of the indicated differentially expressed gene (DEG) sets obtained in expression microarray (5 technical replicates of a minimum of two organoids per genotype were analyzed). Values for DEG in fetal and adult organoids were obtained from Mustata et al, 2013.


( E ) qPCR analysis of the indicated fetal genes in WT and IκBα KO - derived organoids . Data information : 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 .

(E) qPCR analysis of the indicated fetal genes in WT and IκBα KO-derived organoids. Data information: 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, * p-value<0.05.


( F ) Western blot analysis of control and IκBα - deleted organoids using CRISPR - Cas9 technology .

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(F) Western blot analysis of control and IκBα-deleted organoids using CRISPR-Cas9 technology.


( G ) Representative stereoscopic image of WT and IκBα - deleted organoids . Scale bars in G , 50 μm .

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(G) Representative stereoscopic image of WT and IκBα-deleted organoids. Scale bars in G, 50 μm.


A ) Representative stereoscopic images and Western blot analysis of IκBα KO organoids infected with the indicated sh - RNA for NF - κB elements . Data information Scale bars in A , 100 μm ,

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A) Representative stereoscopic images and Western blot analysis of IκBα KO organoids infected with the indicated sh-RNA for NF-κB elements. Data information Scale bars in A, 100 μm,


( B ) qPCR analysis of the indicated ISC and differentiation genes in the different sh - RNA treated organoids . The canonical NF - κB target gene Cxcl10 is shown as positive control of the experiment . Data information 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

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(B) qPCR analysis of the indicated ISC and differentiation genes in the different sh-RNA treated organoids. The canonical NF-κB target gene Cxcl10 is shown as positive control of the experiment. Data information 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.


( C ) Representative stereoscopic images of IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ - 6438 . Data information Scale bars in 50 μm .

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(C) Representative stereoscopic images of IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information Scale bars in 50 μm.


( D - E ) qPCR analysis of the indicated adult and fetal ISC genes ( D ) and elements of the Notch and IFN pathway ( E ) ( differentially expressed in IκBα KO organoids ) in IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ - 6438 . Data information : 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

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(D-E) qPCR analysis of the indicated adult and fetal ISC genes (D) and elements of the Notch and IFN pathway (E) (differentially expressed in IκBα KO organoids) in IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.


( F ) Representative stereoscopic images of IκBα KO organoids treated with dexamethasone as IFN inhibitor and DAPT as Notch inhibitor . Data information Scale bars in F , 50 μm .

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(F) Representative stereoscopic images of IκBα KO organoids treated with dexamethasone as IFN inhibitor and DAPT as Notch inhibitor. Data information Scale bars in F, 50 μm.


( G ) qPCR analysis of IκBα KO organoids untreated or treated with dexamethasone and DAPT . Data information : 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

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(G) qPCR analysis of IκBα KO organoids untreated or treated with dexamethasone and DAPT. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.


( A ) IF analysis of colonic tissue from control and DSS - treated animals including acute damaged ( AD ) areas and recovery ( R ) areas . Data information : Scale bars 100

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(A) IF analysis of colonic tissue from control and DSS-treated animals including acute damaged (AD) areas and recovery (R) areas. Data information: Scale bars 100 μm


IF analysis of P - IκBα and the indicated markers in the areas of acute damage and recovery ( B ) Asterisks indicate the presence of rare cells with nuclear P - IκBα . Data information : Scale bars 50 μm .

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IF analysis of P-IκBα and the indicated markers in the areas of acute damage and recovery (B) Asterisks indicate the presence of rare cells with nuclear P-IκBα. Data information: Scale bars 50 μm.


IF analysis of P - IκBα and colocalization of P - IκBα with the ISC marker Olfm4 in acute damaged areas ( C ) . Asterisks indicate the presence of rare cells with nuclear P - IκBα . Data information : Scale bars 50 μm .

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IF analysis of P-IκBα and colocalization of P-IκBα with the ISC marker Olfm4 in acute damaged areas (C). Asterisks indicate the presence of rare cells with nuclear P-IκBα. Data information: Scale bars 50 μm.


( D ) qPCR analysis of the indicated genes in WT and IκBα KO organoids treated with TNFα and collected at the indicated time - points . 3 technical replicates of organoids from each genotype were analyzed .

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(D) qPCR analysis of the indicated genes in WT and IκBα KO organoids treated with TNFα and collected at the indicated time-points. 3 technical replicates of organoids from each genotype were analyzed.


( E - F ) Representative images of the colonic tissue from 2 months - old DSS - treated WT and IκBα KO mice are shown . Alcian Blue staining was used to identify the mucus - secreting goblet cells in the colonic glands and Ki67 as proliferation marker . Nuclear counterstain is Fast Red . The table shows number of ulcerations present in the intestines of mice analysed ( 3 WT , 2 IκBα KO ) . Data information : Scale bars in E and F , 100

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(E-F) Representative images of the colonic tissue from 2 months-old DSS-treated WT and IκBα KO mice are shown. Alcian Blue staining was used to identify the mucus-secreting goblet cells in the colonic glands and Ki67 as proliferation marker. Nuclear counterstain is Fast Red. The table shows number of ulcerations present in the intestines of mice analysed (3 WT, 2 IκBα KO). Data information: Scale bars in E and F, 100 μm


A and B ) Confocal microscopy images showing mitochondrial fragmentation in HBV - and HBx - expressing cells , respectively . Huh7 cells were transiently transfected with HBV ( A ) or the HBx - flag construct ( B ) . At 2 days post - transfection , cells prestained with MitoTracker ( Mito , white ) were immunostained with anti - HBsAg ( A , green ) and anti - flag ( B , green ) antibodies , respectively . In the zoomed images , typical tubular mitochondria in untransfected cells and fragmented mitochondria in transfected cells are shown .

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A and B) Confocal microscopy images showing mitochondrial fragmentation in HBV- and HBx-expressing cells, respectively. Huh7 cells were transiently transfected with HBV (A) or the HBx-flag construct (B). At 2 days post-transfection, cells prestained with MitoTracker (Mito, white) were immunostained with anti-HBsAg (A, green) and anti-flag (B, green) antibodies, respectively. In the zoomed images, typical tubular mitochondria in untransfected cells and fragmented mitochondria in transfected cells are shown.


C ) Whole cell lysates extracted from Huh7 cells with empty vector ( Mock ) and those with HBV construct were analyzed by Western blotting with antibodies against the indicated proteins . β - actin was used as an internal loading control . Mitochondrial fraction ( bottom panel ) isolated from HepAD38 cells ( see the purity in Figure 2B ) was analyzed by Western blotting with phospho - Drp1 ( Ser616 ) antibody .

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C) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct were analyzed by Western blotting with antibodies against the indicated proteins. β-actin was used as an internal loading control. Mitochondrial fraction (bottom panel) isolated from HepAD38 cells (see the purity in Figure 2B) was analyzed by Western blotting with phospho-Drp1 (Ser616) antibody.


( D - F ) Confocal immunofluorescence showing mitochondrial translocation of Drp1 in HBV - and HBx - expressing cells , respectively . Huh7 cells transfected with HBV ( D and E ) or HBx - flag construct ( F ) were immunostained with antibodies specific to TOM20 ( red ) , Drp1 ( D , green ) , and phospho - Drp1 ( Ser616 ) ( E and F , green ) , respectively . HBV and HBx gene expression ( white ) was verified using anti - HBsAg ( D and E ) and anti - flag ( F ) antibodies , respectively . In the zoomed images , the yellow color indicates the merge of Drp1 or phospho - Drp1 ( Ser616 ) with mitochondria . ( A , B , D , E , and F ) Transfected ( + ) and untransfected ( − ) cells are marked . Nuclei were stained with DAPI ( blue ) .

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(D-F) Confocal immunofluorescence showing mitochondrial translocation of Drp1 in HBV- and HBx-expressing cells, respectively. Huh7 cells transfected with HBV (D and E) or HBx-flag construct (F) were immunostained with antibodies specific to TOM20 (red), Drp1 (D, green), and phospho-Drp1 (Ser616) (E and F, green), respectively. HBV and HBx gene expression (white) was verified using anti-HBsAg (D and E) and anti-flag (F) antibodies, respectively. In the zoomed images, the yellow color indicates the merge of Drp1 or phospho-Drp1 (Ser616) with mitochondria. (A, B, D, E, and F) Transfected (+) and untransfected (−) cells are marked. Nuclei were stained with DAPI (blue).