( C , D ) Three - day growth curves of WT and Sas - 4 − / − ( C ) or p53 − / − and Sas - 4 − / − p53 − / − ( D ) primary mESCs in the indicated conditions starting with 105 cells on Day 0 ( Four independent experiments ) . * * p < 0 . 01 , * p < 0 . 05 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . For details of the measurements , see Materials and Methods ( Tables 3 and 4 ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) Three-day growth curves of WT and Sas-4−/− (C) or p53−/− and Sas-4−/− p53−/− (D) primary mESCs in the indicated conditions starting with 105 cells on Day 0 (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test). Bars represent mean ± s.d. For details of the measurements, see Materials and Methods (Tables 3 and 4).


( A ) Sagittal planes of whole - mount immunostaining and quantification for TUBG on WT mouse embryos from E3 . 5 to E6 . 5 . The insets are magnifications of selected centrosomes from the dotted areas denoting the inner cell mass ( E3 . 5 ) or epiblast ( E5 . 5 and E6 . 5 ) . Bars represent mean ± s . d ( two - tailed student ' s T - test or one - way ANOVA with Tukey ' s multiple comparisons tests ) . Scale bars : E3 . 5 and E5 . 5 = 15 μm ; E6 . 5 = 30 μm and 3 μm ( insets ) . E3 . 5 : 88 ± 26 % ( n = 73 cells from 5 embryos ) ; E5 . 5 : 92 ± 10 % ( n = 270 from 3 embryos ) ; E6 . 5 : 79 ± 12 % ( n = 12 , 082 from 3 embryos ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O 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) Sagittal planes of whole-mount immunostaining and quantification for TUBG on WT mouse embryos from E3.5 to E6.5. The insets are magnifications of selected centrosomes from the dotted areas denoting the inner cell mass (E3.5) or epiblast (E5.5 and E6.5). Bars represent mean ± s.d (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests). Scale bars: E3.5 and E5.5 = 15 μm; E6.5 = 30 μm and 3 μm (insets). E3.5: 88 ± 26% (n=73 cells from 5 embryos); E5.5: 92 ± 10% (n=270 from 3 embryos); E6.5: 79 ± 12% (n=12,082 from 3 embryos).


( B , C ) Representative gSTED super - resolution images of immunostaining for TUBG and either CEP164 ( B ) or ODF2 ( C ) . Whole - mount staining was used for the CEP164 data as well as for the E3 . 5 blastocysts for ODF2 , whereas staining on sections was used for ODF2 at E5 . 5 and E6 . 5 . Quantification of the percentage of centrosomes ( TUBG ) with CEP164 or ODF2 are shown on the right . * * * * p < 0 . 0001 , * * * p < 0 . 001 , * p < 0 . 05 ( two - tailed student ' s T - test or one - way ANOVA with Tukey ' s multiple comparisons tests ; the ODF2 data between E5 . 5 and E6 . 5 was not significant using the latter test ) . Bars represent mean ± s . d . Scale bar = 0 . 5 μm . CEP164 at E3 . 5 : 2 ± 2 % , ( n = 92 centrosomes from 4 embryos ) ; E5 . 5 : 50 ± 2 % ( n = 249 from 3 embryos ) ; E6 . 5 : 62 ± 0 % ( n = 856 from 3 embryos ) . ODF2 at E3 . 5 : 20 ± 9 % ( n = 108 from 5 embryos ) ; E5 . 5 : 51 ± 4 % ( n = 294 from 5 embryos ) ; E6 . 5 : 60 ± 2 % ( n = 1 , 183 from 4 embryos ) .

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

(B, C) Representative gSTED super-resolution images of immunostaining for TUBG and either CEP164 (B) or ODF2 (C). Whole-mount staining was used for the CEP164 data as well as for the E3.5 blastocysts for ODF2, whereas staining on sections was used for ODF2 at E5.5 and E6.5. Quantification of the percentage of centrosomes (TUBG) with CEP164 or ODF2 are shown on the right. **** p < 0.0001, *** p < 0.001, * p < 0.05 (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests; the ODF2 data between E5.5 and E6.5 was not significant using the latter test). Bars represent mean ± s.d. Scale bar = 0.5 μm. CEP164 at E3.5: 2 ± 2%, (n=92 centrosomes from 4 embryos); E5.5: 50 ± 2% (n=249 from 3 embryos); E6.5: 62 ± 0% (n=856 from 3 embryos). ODF2 at E3.5: 20 ± 9% (n=108 from 5 embryos); E5.5: 51 ± 4% (n=294 from 5 embryos); E6.5: 60 ± 2% (n=1,183 from 4 embryos).


( D ) Immunostaining and quantification of the area of TUBG and PCNT signals from E5 . 5 and E6 . 5 centrosomes in epiblast cells . The separate channels for the marked centriole are shown in the insets . * * * p < 0 . 001 , * * p < 0 . 01 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . Scale bar = 2 μm . Quantification of PCNT at E5 . 5 : 0 . 22 ± 0 . 01 μm ( n = 103 centrosomes from 3 embryos ) ; E6 . 5 : 0 . 31 ± 0 . 02 μm ( n = 342 from 3 embryos ) . Quantification of TUBG at E5 . 5 : 0 . 15 ± 0 . 02 μm ( n = 108 from 4 embryos ) ; E6 . 5 : 0 . 25 ± 0 . 01 μm ( n = 311 from 3 embryos ) .

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

(D) Immunostaining and quantification of the area of TUBG and PCNT signals from E5.5 and E6.5 centrosomes in epiblast cells. The separate channels for the marked centriole are shown in the insets. *** p < 0.001, ** p < 0.01 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bar = 2 μm. Quantification of PCNT at E5.5: 0.22 ± 0.01 μm (n=103 centrosomes from 3 embryos); E6.5: 0.31 ± 0.02 μm (n=342 from 3 embryos). Quantification of TUBG at E5.5: 0.15 ± 0.02 μm (n=108 from 4 embryos); E6.5: 0.25 ± 0.01 μm (n=311 from 3 embryos).


MEFs expressing vector , TRPML - 1 , or GRP1 - DD were treated with BSA or palmitate ( 250 μM ) for 3 h and stained as in ( A ) . aspect ratio ( O ) , branch length ( P ) , and roundness ( Q ) of mitochondria in the cells in ( N ) .

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

MEFs expressing vector, TRPML-1, or GRP1-DD were treated with BSA or palmitate (250 μM) for 3 h and stained as in (A). aspect ratio (O), branch length (P), and roundness (Q) of mitochondria in the cells in (N).


TMRE ( 100 nM ) and MitoTracker green ( 200 nM ) staining in MEFs treated with BSA or palmitate ( 250 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . TMRE intensity in ( A ) on a per cell basis . MitoSOX ( 5 μM ) and MitoTracker Green ( 200 nM ) staining in MEFs treated with vehicle ( DMSO ) or C2 - ceramide ( 50 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . MitoSOX intensity for ( C ) on a per cell basis .

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

TMRE (100 nM) and MitoTracker green (200 nM) staining in MEFs treated with BSA or palmitate (250 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). TMRE intensity in (A) on a per cell basis. MitoSOX (5 μM) and MitoTracker Green (200 nM) staining in MEFs treated with vehicle (DMSO) or C2-ceramide (50 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). MitoSOX intensity for (C) on a per cell basis.


NAD ( P ) H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in ( A ) 4 - 8 h after treatment with vehicle ( n = 5 ) or 120 mg / kg SH - BC - 893 ( n = 4 ) by gavage at ZT8 . 5 . Mice were sacrificed and imaged in pairs ( SD and HFD ) .

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

NAD(P)H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in (A) 4-8 h after treatment with vehicle (n=5) or 120 mg/kg SH-BC-893 (n=4) by gavage at ZT8.5. Mice were sacrificed and imaged in pairs (SD and HFD).


Citrate synthase staining in the arcuate nucleus of mice in ( E ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 3 .

B-MEASURED_VAR I-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

Citrate synthase staining in the arcuate nucleus of mice in (E) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=3.


imaging was performed in the areas of the cortex


NAD ( P ) H autofluorescence was evaluated in epididymal fat ( eWAT ) freshly resected from the mice in ( A ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 5 . NAD ( P ) H intensity from ( B ) calculated on a per cell basis ; 16 - 25 cells from each of the 5 mice per group were quantified ( 87 - 109 total cells per treatment group ) .

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

NAD(P)H autofluorescence was evaluated in epididymal fat (eWAT) freshly resected from the mice in (A) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=5. NAD(P)H intensity from (B) calculated on a per cell basis; 16 - 25 cells from each of the 5 mice per group were quantified (87 - 109 total cells per treatment group).


TMRE ( 100 nM ) and MitoTracker Green staining ( 200 nM ) in eWAT resected from mice in ( D ) treated as in ( B , C ) ; n = 5 . TMRE ( F ) or MitoTracker ( G ) intensity for ( E ) calculated on a per cell basis ; 15 - 24 cells from each of the 5 mice in each group were quantified ( 99 - 104 total cells per treatment group ) .

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

TMRE (100 nM) and MitoTracker Green staining (200 nM) in eWAT resected from mice in (D) treated as in (B,C); n=5. TMRE (F) or MitoTracker (G) intensity for (E) calculated on a per cell basis; 15 - 24 cells from each of the 5 mice in each group were quantified (99 - 104 total cells per treatment group).


MitoSOX ( 5 μM ) and MitoTracker Green staining in eWAT resected from the mice in ( D ) treated as in ( B and E ) ; n = 5 . MitoSOX ( I ) or MitoTracker ( J ) intensity calculated as in ( F and G ) ; 10 - 13 cells from each of the 5 mice in each group were quantified ( 57 - 58 total cells per treatment group ) .

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

MitoSOX (5 μM) and MitoTracker Green staining in eWAT resected from the mice in (D) treated as in (B and E); n=5. MitoSOX (I) or MitoTracker (J) intensity calculated as in (F and G); 10 - 13 cells from each of the 5 mice in each group were quantified (57 - 58 total cells per treatment group).


Chop , Xbp1s , Atf6 , and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg / kg SH - BC - 893 on Monday , Wednesday , and Friday for the final 3 weeks ; n = 7 - 9 .

B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR 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

Chop, Xbp1s, Atf6, and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg/kg SH-BC-893 on Monday, Wednesday, and Friday for the final 3 weeks; n=7-9.


Plasma adiponectin ( A ) or leptin ( B ) in mice maintained on a SD or HFD for 8 - 9 weeks then gavaged with vehicle or 120 mg / kg SH - BC - 893 4 h prior to blood collection . Pair - fed mice were provided with the amount of food consumed by SH - BC - 893 - treated mice in 4 h but left untreated ; n = 8 - 10 .

O B-MEASURED_VAR 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

Plasma adiponectin (A) or leptin (B) in mice maintained on a SD or HFD for 8-9 weeks then gavaged with vehicle or 120 mg/kg SH-BC-893 4 h prior to blood collection. Pair-fed mice were provided with the amount of food consumed by SH-BC-893-treated mice in 4 h but left untreated; n=8-10.


Average daily distance run by HFD - fed mice treated with vehicle ( n = 8 ) or SH - BC - 893 ( 120 mg / kg , n = 6 ) on Mondays , Wednesdays , and Fridays over 4 weeks when housed with a running wheel .

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

Average daily distance run by HFD-fed mice treated with vehicle (n=8) or SH-BC-893 (120 mg/kg, n=6) on Mondays, Wednesdays, and Fridays over 4 weeks when housed with a running wheel.


Body weight change in the mice in ( O , P ) over the 24 h after the first treatment ( Q ) or after the 3 week course of treatment ( R ) ; n = 9 .

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

Body weight change in the mice in (O,P) over the 24 h after the first treatment (Q) or after the 3 week course of treatment (R); n=9.


Representative H & E stained liver sections from the mice shown in Fig . 6F after 4 weeks of treatment with vehicle or 120 mg / kg SH - BC - 893 . Scale bar , 10 μm .

Representative H&E stained liver sections from the mice shown in Fig. 6F after 4 weeks of treatment with vehicle or 120 mg/kg SH-BC-893. Scale bar, 10 μm.


Blood glucose levels ( F ) or area under the curve ( AUC , ( G ) ) during an oral glucose tolerance test performed in the same mice as in ( E ) .

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

Blood glucose levels (F) or area under the curve (AUC, (G)) during an oral glucose tolerance test performed in the same mice as in (E).


Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH - BC - 893 from the mice in Fig . 5D , E or in 9 week old SD - fed mice treated with vehicle ( same mice as in Fig . 5J , K , 0 weeks on HFD ) ; n = 8 . Insulin levels in the plasma collected 4 h after treatment with vehicle or SH - BC - 893 from the same mice as in Fig . 5A , B ; n = 8 .

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

Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH-BC-893 from the mice in Fig. 5D,E or in 9 week old SD-fed mice treated with vehicle (same mice as in Fig. 5J,K, 0 weeks on HFD); n=8. Insulin levels in the plasma collected 4 h after treatment with vehicle or SH-BC-893 from the same mice as in Fig. 5A,B; n=8.


( B ) Flow cytometry analysis of WT to Il21r - / - splenic SWHEL B cells over time . ( C ) WT to Il21r - / - SWHEL B cell ratio within CTV division peaks . Data information : Data in ( B ) is representative of 8 - 9 biological replicates ( n = 8 - 9 ) pooled from two independent experiments with statistical analysis by one - way ANOVA with Tukey ' s post - test . * * p ≤ 0 . 01 ; * * * * p ≤ 0 . 0001 . Data show concatenated data from 4 - 5 biological replicates ( n = 4 - 5 ) and are representative for two independent experiments .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometry analysis of WT to Il21r-/- splenic SWHEL B cells over time. (C) WT to Il21r-/- SWHEL B cell ratio within CTV division peaks. Data information: Data in (B) is representative of 8-9 biological replicates (n = 8-9) pooled from two independent experiments with statistical analysis by one-way ANOVA with Tukey's post-test. ** p ≤ 0.01; **** p ≤ 0.0001. Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.


( E ) Rate of apoptosis measured by detecting active caspase 3 by flow cytometry across CTV division peaks . Data information : Data show concatenated data from 4 - 5 biological replicates ( n = 4 - 5 ) and are representative for two independent experiments .

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

(E) Rate of apoptosis measured by detecting active caspase 3 by flow cytometry across CTV division peaks. Data information: Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.


( B ) Flow cytometry analysis showing WT to Il21r - / - SWHEL B cell ratio of cells that have been in S phase and thus incorporated BrdU . Data information : Data are representative of 5 - 7 biological replicates ( n = 5 - 7 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 .

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

(B) Flow cytometry analysis showing WT to Il21r-/- SWHEL B cell ratio of cells that have been in S phase and thus incorporated BrdU. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.


( D ) Ratio of the proportion of BrdU positive WT and Il21r - / - SWHEL B cells with 2N DNA content 4 , 10 and 12 hours post BrdU pulse on day 3 . 5 post immunization . Data information : Data are representative of 5 - 7 biological replicates ( n = 5 - 7 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 .

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

(D) Ratio of the proportion of BrdU positive WT and Il21r-/- SWHEL B cells with 2N DNA content 4, 10 and 12 hours post BrdU pulse on day 3.5 post immunization. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.


( C ) Rate of de novo cell cycle entry 10 hours post in vivo IL - 21 ( 2 µg ) or saline pulse . The ratio of WT to Il21r - / - cells not in S / G2 at the time of pulse ( BrdU - cells ) and containing > 2N DNA content 10 hours after pulse is shown . Data information : Data are representative of 10 - 12 biological replicates ( n = 10 - 12 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 .

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

(C) Rate of de novo cell cycle entry 10 hours post in vivo IL-21 (2 µg) or saline pulse. The ratio of WT to Il21r-/- cells not in S/G2 at the time of pulse (BrdU- cells) and containing >2N DNA content 10 hours after pulse is shown. Data information: Data are representative of 10-12 biological replicates (n = 10-12) from two independent experiments. Statistical analysis by one-sample t test. * p ≤ 0.05.


Phosphoflow analysis of naïve WT or Il21r - / - B cells following in vitro culture for 3 hours with or without IL - 21 ( 20 ng / mL ) and / or in the presence of BCR cross - linking ( biotinylated anti - Igκ + anti - Igλ and avidin - mediated cross - linking ) or agonistic anti - CD40 . ( A ) Exemplary p - AKT ( S473 ) staining and ( B ) quantification of p - AKT median fluorescence intensity ( MedFI ) . ( C ) Exemplary p - S6 ( Ser235 / 236 ) staining and ( D ) quantification of frequency of p - S6 positive cells Data information : Data are representative of 4 biological replicates ( n = 4 ) . Statistical analysis by one - way ANOVA with Tukey ' s post - test ( D ) , t test ( B , E ) * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

Phosphoflow analysis of naïve WT or Il21r-/- B cells following in vitro culture for 3 hours with or without IL-21 (20 ng/mL) and/or in the presence of BCR cross-linking (biotinylated anti-Igκ + anti-Igλ and avidin-mediated cross-linking) or agonistic anti-CD40. (A) Exemplary p-AKT (S473) staining and (B) quantification of p-AKT median fluorescence intensity (MedFI). (C) Exemplary p-S6 (Ser235/236) staining and (D) quantification of frequency of p-S6 positive cells Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by one-way ANOVA with Tukey's post-test (D), t test (B, E) * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


( F , In vitro cell culture of CTV labeled SWHEL B cells with or without IL - 21 ( 20 ng / mL ) and anti - CD40 for three days and analysis by flow cytometry . ( F ) Exemplary CTV division peaks .

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

(F, In vitro cell culture of CTV labeled SWHEL B cells with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by flow cytometry. (F) Exemplary CTV division peaks.


G ) In vitro cell culture of CTV labeled SWHEL B cells with or without IL - 21 ( 20 ng / mL ) and anti - CD40 for three days and analysis by flow cytometry . ( G ) Quantification of the proportion SWHEL B that had divided and thus diluted CTV . Data information : Data are representative of 4 biological replicates ( n = 4 ) . Statistical analysis by two - way ANOVA with correction for multiple comparison using Šidák method ( G ) . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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G) In vitro cell culture of CTV labeled SWHEL B cells with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by flow cytometry. (G) Quantification of the proportion SWHEL B that had divided and thus diluted CTV. Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by two-way ANOVA with correction for multiple comparison using Šidák method (G). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


( B , C ) Analysis of splenic OTII Tfh cells by flow cytometry . ( B ) Tfh cell count and ( C ) Proportion of Il21Gfp / + OTII Tfh cells transcribing the Il21 locus and thus expressing GFP . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . Statistical analysis by one - way ANOVA with Tukey ' s post - test ( B , C ) . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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(B, C) Analysis of splenic OTII Tfh cells by flow cytometry. (B) Tfh cell count and (C) Proportion of Il21Gfp/+ OTII Tfh cells transcribing the Il21 locus and thus expressing GFP. Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. Statistical analysis by one-way ANOVA with Tukey's post-test (B, C). * p ≤ 0.05; ** p ≤ 0.01.


Analysis of splenic WT and Il21r - / - SWHEL B cells by flow cytometry . ( D ) CTV profile of and ( E ) absolute cell count in pooled CTV division peaks ( 0 - 4 , 5 - 8 , 9 - 11 + ) . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . D , show concatenated data representative of 3 - 5 biological replicates . Data in E were analyzed after log transformation using Multiple paired t tests and p values corrected for multiple comparisons using Holm - Šídák method . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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Analysis of splenic WT and Il21r-/- SWHEL B cells by flow cytometry. (D) CTV profile of and (E) absolute cell count in pooled CTV division peaks (0-4, 5-8, 9-11+). Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. D, show concatenated data representative of 3-5 biological replicates. Data in E were analyzed after log transformation using Multiple paired t tests and p values corrected for multiple comparisons using Holm-Šídák method. * p ≤ 0.05; ** p ≤ 0.01.


( F ) Cell surface expression ( median fluorescence intensity , MedFI ) of CD38 , IgD and FAS across individual CTV division peaks . ( G ) Representative flow cytometry plot of plasma cell differentiation indicated by CD138 expression . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . F and G show concatenated data representative of 3 - 5 biological replicates .

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(F) Cell surface expression (median fluorescence intensity, MedFI) of CD38, IgD and FAS across individual CTV division peaks. (G) Representative flow cytometry plot of plasma cell differentiation indicated by CD138 expression. Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. F and G show concatenated data representative of 3-5 biological replicates.


( A - E ) Analysis of splenic SWHEL B cells and PC by flow cytometry . ( A ) Proportion of SWHEL B cells with a FAS + GL7 + GC phenotype , ( B ) SWHEL GC B cell count and ( C ) representation of IL - 21R deficient cells among total SWHEL GC B cells . ( D ) CD98 + CD138 + _ SWHEL PC count and ( E ) representation of IL - 21R deficient cells among total SWHEL PC . Data information : Data are representative of 6 - 10 biological replicates ( n = 6 - 10 ) from two independent experiments . Data in B , D were analyzed after log transformation with with statistical analysis by Multiple paired t tests ( A , B , D ) and p - values corrected for multiple comparisons using Holm - Šídák method or One - way ANOVA with Tukey ' s post - test ( C ) or t test ( E ) .

O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Analysis of splenic SWHEL B cells and PC by flow cytometry. (A) Proportion of SWHEL B cells with a FAS+GL7+ GC phenotype, (B) SWHEL GC B cell count and (C) representation of IL-21R deficient cells among total SWHEL GC B cells. (D) CD98+ CD138+_SWHEL PC count and (E) representation of IL-21R deficient cells among total SWHEL PC. Data information: Data are representative of 6-10 biological replicates (n = 6-10) from two independent experiments. Data in B, D were analyzed after log transformation with with statistical analysis by Multiple paired t tests (A, B, D) and p-values corrected for multiple comparisons using Holm-Šídák method or One-way ANOVA with Tukey's post-test (C) or t test (E).


Western blotting in the WT , Trim71 knockout ( Trim71 _ KO ) , and the Trim71 mutant ( R783H ) mESCs . The signals from the pluripotency factors Nanog and Oct4 were obtained through short ( ~ 10 seconds ) exposure .

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Western blotting in the WT, Trim71 knockout (Trim71_KO), and the Trim71 mutant (R783H) mESCs. The signals from the pluripotency factors Nanog and Oct4 were obtained through short (~10 seconds) exposure.


Colony formation assay for mESCs . The mESCs were cultured in 15 % FBS + Lif for 7 days , and the resultant colonies were fixed and stained for AP .

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Colony formation assay for mESCs. The mESCs were cultured in 15%FBS + Lif for 7 days, and the resultant colonies were fixed and stained for AP.


Exit pluripotency assay for mESCs . The mESCs were induced to exit pluripotency in medium without Lif for 2 days and then switched to 2i + Lif medium for 5 days . The resultant colonies were fixed and stained for AP .

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Exit pluripotency assay for mESCs. The mESCs were induced to exit pluripotency in medium without Lif for 2 days and then switched to 2i+Lif medium for 5 days. The resultant colonies were fixed and stained for AP.


Western blotting of pluripotency factors during EB formation . The signals from the pluripotency factors Nanog and Oct4 were obtained through long ( 5 - 10min ) exposure .

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Western blotting of pluripotency factors during EB formation. The signals from the pluripotency factors Nanog and Oct4 were obtained through long (5-10min) exposure.


Immunofluorescence ( IF ) staining showing the expression level of Rex1 in mESCs cultured in the stemness ( 2i + Lif ) and differentiating ( 15 % FBS - Lif ) conditions .

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Immunofluorescence (IF) staining showing the expression level of Rex1 in mESCs cultured in the stemness (2i + Lif) and differentiating (15% FBS - Lif) conditions.


Relative intensity of IF signals from individual cells in experiment associated with E . The results represent the means ( ± SD ) of three independent IF stainings .

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Relative intensity of IF signals from individual cells in experiment associated with E. The results represent the means (± SD) of three independent IF stainings.


Expression of neural lineage markers during mESCs neural differentiation .

Expression of neural lineage markers during mESCs neural differentiation.


IF staining of Nestin . The quantifications represent the means ( ± SD ) of three independent experiments .

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IF staining of Nestin. The quantifications represent the means (± SD) of three independent experiments.


Distribution of the WT Trim71 and the R783H Trim71 mutant binding regions in the mouse genome . In each CLIP - seq , there are two biological replicates . The binding regions present in both of the biological replicates are used for the analysis .

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Distribution of the WT Trim71 and the R783H Trim71 mutant binding regions in the mouse genome. In each CLIP-seq, there are two biological replicates. The binding regions present in both of the biological replicates are used for the analysis.


CLIP - qRT - PCR for the identified target mRNAs of the Trim71 mutant R783H . The results represent the means ( ± SD ) of three independent experiments .

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CLIP-qRT-PCR for the identified target mRNAs of the Trim71 mutant R783H. The results represent the means (±SD) of three independent experiments.


UCSC genome browser snapshot for the CLIP - seq data from Trim71 and the Trim71 mutant R783H in the Lsd1 locus . The red box indicates the binding region of the Trim71 mutant R783H . The inputs are from the size - matched input samples in the CLIP - seq analysis .

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UCSC genome browser snapshot for the CLIP-seq data from Trim71 and the Trim71 mutant R783H in the Lsd1 locus. The red box indicates the binding region of the Trim71 mutant R783H. The inputs are from the size-matched input samples in the CLIP-seq analysis.


B . A representative set of Western blots in the WT and the R783H Trim71 mESCs . C . Quantification of the target proteins in the WT and the R783H Trim71 mESCs . Data information : The results represent the means ( ± SD ) of three independent experiments . One - Way ANOVA was used to determine the significance of the difference , * p < 0 . 05 . The Western blots listed in B is the Biological Replicate

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B. A representative set of Western blots in the WT and the R783H Trim71 mESCs. C. Quantification of the target proteins in the WT and the R783H Trim71 mESCs. Data information: The results represent the means (± SD) of three independent experiments. One-Way ANOVA was used to determine the significance of the difference, *p<0.05. The Western blots listed in B is the Biological Replicate 1


Western blotting in the WT and the Trim71 ( R783H ) mESCs .

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Western blotting in the WT and the Trim71 (R783H) mESCs.


Quantification of Lsd1 protein and mRNA levels . Gapdh and 18S rRNA were used for normalization in protein and mRNA quantifications , respectively .

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Quantification of Lsd1 protein and mRNA levels. Gapdh and 18S rRNA were used for normalization in protein and mRNA quantifications, respectively.


Polysome analysis in the WT and the Trim71 ( R783H ) mESCs .

Polysome analysis in the WT and the Trim71 (R783H) mESCs.


Quantification of the indicated mRNA distribution in the RNP , 80S , and polysome fractions from the WT and the Trim71 ( R783H ) mESCs .

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Quantification of the indicated mRNA distribution in the RNP, 80S, and polysome fractions from the WT and the Trim71 (R783H) mESCs.


Western blotting in the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) .

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Western blotting in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).


Quantification of Lsd1 protein and mRNA in the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) .

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Quantification of Lsd1 protein and mRNA in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).


Polysome analysis in the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) . Quantification of the indicated mRNA distribution in the RNP , 80S , and polysome fractions from the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) .

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Polysome analysis in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H). Quantification of the indicated mRNA distribution in the RNP, 80S, and polysome fractions from the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).


Deletion of the Trim71 mutant R783H binding site in Lsd1 mRNA ' s 3 ' UTR .

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Deletion of the Trim71 mutant R783H binding site in Lsd1 mRNA's 3'UTR.


CLIP - RIP followed by qRT - PCR to examine mRNAs associated with the Trim71 and the Trim71 mutant R783H in the WT , Trim71 ( R783H ) , CLIP ∆ , and Trim71 ( R783H ) / CLIP ∆ mESCs . The mRNA signals from the E14 mESCs were set as 1 for relative comparison .

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CLIP-RIP followed by qRT-PCR to examine mRNAs associated with the Trim71 and the Trim71 mutant R783H in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs. The mRNA signals from the E14 mESCs were set as 1 for relative comparison.


Western blotting in the WT , Trim71 ( R783H ) , CLIP ∆ , and Trim71 ( R783H ) / CLIP ∆ mESCs .

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Western blotting in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs.


Quantification of Lsd1 protein and mRNA in the WT , Trim71 ( R783H ) , CLIP ∆ , and Trim71 ( R783H ) / CLIP ∆ mESCs . Beta - Tubulin and 18S rRNA were used for normalization in the protein and mRNA quantification , respectively .

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Quantification of Lsd1 protein and mRNA in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs. Beta-Tubulin and 18S rRNA were used for normalization in the protein and mRNA quantification, respectively.


Exit pluripotency assay for mESCs.


Representative Western blotting and quantification of pluripotency factors during EB formation .

Representative Western blotting and quantification of pluripotency factors during EB formation.


Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation .

Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation.


Western blotting in the WT and the Trim71 ( R783H ) mESCs with dox - inducible expression of Lsd1 - GFP .

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Western blotting in the WT and the Trim71(R783H) mESCs with dox-inducible expression of Lsd1-GFP.


Representative Western blotting and quantification of pluripotency factors during the monolayer differentiation of mESCs .

Representative Western blotting and quantification of pluripotency factors during the monolayer differentiation of mESCs.


Representative Western blotting and quantification in the WT and the R783H / + mESCs .

Representative Western blotting and quantification in the WT and the R783H/+ mESCs.


Exit pluripotency assay for the WT and the R783H / + mESCs .

Exit pluripotency assay for the WT and the R783H/+ mESCs.


Representative Western blotting and quantification of pluripotency factors in differentiating mESCs .

Representative Western blotting and quantification of pluripotency factors in differentiating mESCs.


Western blotting in the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP .

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Western blotting in the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.


Exit pluripotency assay for the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP .

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Exit pluripotency assay for the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.


Representative Western blotting and quantification of pluripotency factors during the differentiation of the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP .

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Representative Western blotting and quantification of pluripotency factors during the differentiation of the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.


Representative Western blotting and quantification of neural lineage markers during the neural differentiation of the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP .

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Representative Western blotting and quantification of neural lineage markers during the neural differentiation of the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.


( C ) Electrophoretic mobility - shift assay ( EMSA ) with biotin - labeled RORA consensus , rs2978980T or rs2978980G probes and HGC - 27 or MGC80 - 3 nuclear extracts . HGC - 27 or MGC80 - 3 left panel : EMSA with RORA consensus or rs2978980T probes . Lane 1 - 8 : from the first lane at the left side to the right side . Lanes 4 and 8 , probe only ; lanes 2 and 6 , probe and nuclear extracts ; lanes 1 , 3 , 5 and 7 , probe and nuclear extracts plus 100 × unlabeled rs2978980T ( lanes 1 and 5 ) or RORA consensus probes ( lanes 3 and 7 ) . HGC - 27 or MGC80 - 3 right panel : EMSA with rs2978980T or rs2978980G probes . Lanes 1 and 6 , probe only ; lanes 2 and 7 , probe and nuclear extracts ; lanes 3 - 5 and 8 - 10 , probe and nuclear extracts plus 100 × unlabeled rs2978980T ( lanes 5 and 8 ) , rs2978980G ( lanes 3 and 10 ) or RORA consensus probes ( lanes 4 and 9 ) .

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(C) Electrophoretic mobility-shift assay (EMSA) with biotin-labeled RORA consensus, rs2978980T or rs2978980G probes and HGC-27 or MGC80-3 nuclear extracts. HGC-27 or MGC80-3 left panel: EMSA with RORA consensus or rs2978980T probes. Lane 1-8: from the first lane at the left side to the right side. Lanes 4 and 8, probe only; lanes 2 and 6, probe and nuclear extracts; lanes 1, 3, 5 and 7, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 1 and 5) or RORA consensus probes (lanes 3 and 7). HGC-27 or MGC80-3 right panel: EMSA with rs2978980T or rs2978980G probes. Lanes 1 and 6, probe only; lanes 2 and 7, probe and nuclear extracts; lanes 3-5 and 8-10, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 5 and 8), rs2978980G (lanes 3 and 10) or RORA consensus probes (lanes 4 and 9).


( F ) Suppression of RORA using siRNAs ( siRORA - 1 and siRORA - 2 ) could markedly down - regulate lncPSCA expression in MGC80 - 3 cells but not in HGC - 27 cells . In detail , MGC80 - 3 and HGC - 27 cells were transfected with siRORA - 1 and siRORA - 2 , lncPSCA and RORA expression levels were measured by RT - qPCR and Western Blot .

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(F) Suppression of RORA using siRNAs (siRORA-1 and siRORA-2) could markedly down-regulate lncPSCA expression in MGC80-3 cells but not in HGC-27 cells. In detail, MGC80-3 and HGC-27 cells were transfected with siRORA-1 and siRORA-2, lncPSCA and RORA expression levels were measured by RT-qPCR and Western Blot.


( E ) lncPSCA - knockout significantly promoted growth of MGC80 - 3 ( n = 8 ) or HGC - 27 ( n = 7 ) xenografts compared with control xenografts after 27 or 18 days .

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(E) lncPSCA-knockout significantly promoted growth of MGC80-3 (n = 8) or HGC-27 (n = 7) xenografts compared with control xenografts after 27 or 18 days.


( F ) HE and Ki67 staining of MGC80 - 3 or HGC - 27 xenografts . Scale bar = 100 μm .

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(F) HE and Ki67 staining of MGC80-3 or HGC-27 xenografts. Scale bar = 100 μm.


( B ) lncPSCA inhibits invasion abilities of MGC80 - 3 and HGC - 27 cells . Cells on the lower surface of the chamber were stained by crystal violet . Scale bar = 100 μm .

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(B) lncPSCA inhibits invasion abilities of MGC80-3 and HGC-27 cells. Cells on the lower surface of the chamber were stained by crystal violet. Scale bar = 100 μm.


( C ) In MGC80 - 3 and HGC - 27 cells , expression changes of different markers ( β - catenin , ZO - 1 , SNAIL and VIMENTIN ) of epithelial - to - mesenchymal transition were examined after overexpression or knockout of lncPSCA .

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(C) In MGC80-3 and HGC-27 cells, expression changes of different markers (β-catenin, ZO-1, SNAIL and VIMENTIN) of epithelial-to-mesenchymal transition were examined after overexpression or knockout of lncPSCA.


( F ) Gastric cancer cells after lncPSCA - knockout or stably overexpressing lncPSCA and control cells were treated with cycloheximide ( CHX ) or vehicle for the indicated periods of time . DDX5 levels were analyzed by Western blot .

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(F) Gastric cancer cells after lncPSCA-knockout or stably overexpressing lncPSCA and control cells were treated with cycloheximide (CHX) or vehicle for the indicated periods of time. DDX5 levels were analyzed by Western blot.


( I ) Knockdown of DDX5 with siRNA ( siD - 1 and siD - 2 ) substantially reduced the proliferation of gastric cancer cells . Data show one representative example of three biological replicates . ( J ) Silencing of DDX5 with shRNA ( shD ) significantly reduced the proliferation of gastric cancer cells . Data show one representative example of three biological replicates . (

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(I) Knockdown of DDX5 with siRNA (siD-1 and siD-2) substantially reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates. (J) Silencing of DDX5 with shRNA (shD) significantly reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates. (


( I ) Knockdown of DDX5 with siRNA ( siD - 1 and siD - 2 ) substantially reduced the proliferation of gastric cancer cells . Data show one representative example of three biological replicates .

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(I) Knockdown of DDX5 with siRNA (siD-1 and siD-2) substantially reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates.


( E ) RNA Pol II ChIPseq and RNAseq signals of PMAIP1 , CDKN1A , SESN2 and SESN3 in HGC - 27 cells . Red bars represent the RNA Pol II ChIPseq signal . Orange or blue bars represent the RNAseq signals of HGC - 27 cells overexpressing lncPSCA ( lncP ) or with silenced DDX5 ( shD ) , respectively .

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(E) RNA Pol II ChIPseq and RNAseq signals of PMAIP1, CDKN1A, SESN2 and SESN3 in HGC-27 cells. Red bars represent the RNA Pol II ChIPseq signal. Orange or blue bars represent the RNAseq signals of HGC-27 cells overexpressing lncPSCA (lncP) or with silenced DDX5 (shD), respectively.


( F ) Validation of candidate downstream genes of the lncPSCA - DDX5 axis identified through the integrated analyses . qRT - PCR was performed for PMAIP1 , CDKN1A and SESN2 genes upon overexpression of lncPSCA ( lncP ) , silencing of DDX5 ( shD ) or lncPSCA konckout . Error bars indicate the SEM . Data show one representative example of three biological replicates .

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(F) Validation of candidate downstream genes of the lncPSCA-DDX5 axis identified through the integrated analyses. qRT-PCR was performed for PMAIP1, CDKN1A and SESN2 genes upon overexpression of lncPSCA (lncP), silencing of DDX5 (shD) or lncPSCA konckout. Error bars indicate the SEM. Data show one representative example of three biological replicates.


( A ) Swelling of neurons in the vestibular nucleus due to fine vesiculation ( arrows ) and clear vacuolization ( arrowhead ) of the cytoplasm . HE , scale bar 100 μm .

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(A) Swelling of neurons in the vestibular nucleus due to fine vesiculation (arrows) and clear vacuolization (arrowhead) of the cytoplasm. HE, scale bar 100 μm.


( B ) Clear cytoplasmic vacuolization ( arrows ) in cerebellar corticalPurkinje cells . HE , scale bar 100 μm .

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(B) Clear cytoplasmic vacuolization (arrows) in cerebellar corticalPurkinje cells. HE, scale bar 100 μm.


( C ) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells ( arrows ) and a dense granular cell layer . HE , scale bar 100 μm . ( D ) Marked neuronal loss is present the cerebellar cortex of an affected dog . The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain ( arrow ) . HE , scale bar 100 μm .

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(C) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells (arrows) and a dense granular cell layer. HE, scale bar 100 μm. (D) Marked neuronal loss is present the cerebellar cortex of an affected dog. The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain (arrow). HE, scale bar 100 μm.


( E ) Axonal spheroids of varying quality were seen in the white matter ( arrows ) of cerebellum and brainstem . HE , scale bar 100 μm .

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(E) Axonal spheroids of varying quality were seen in the white matter (arrows) of cerebellum and brainstem. HE, scale bar 100 μm.


( F ) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells . HE , scale bar 100 μm .

(F) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells. HE, scale bar 100 μm.


( G ) Purkinje cell with numerous single - membrane bound , cytoplasmic vacuoles tethering to each other ( arrows , inset ) . Electronmicrograph , scale bar 2 μm . Inset : scale bar 1 μm .

(G) Purkinje cell with numerous single-membrane bound, cytoplasmic vacuoles tethering to each other (arrows, inset). Electronmicrograph, scale bar 2 μm. Inset: scale bar 1 μm.


( H ) Axonal spheroid containing aggregated degenerated mitochondria , occasional double - membrane - bound autophagosomes ( arrows ) and free electron dense material , compressed by a peripheral clear vacuolar space . Electronmicrograph , scale bar 0 . 5 μm . Abbreviations : n , nucleus ; ml , molecular layer ; pl , Purkinje cell layer ; gl , granular cell layer .

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(H) Axonal spheroid containing aggregated degenerated mitochondria, occasional double-membrane-bound autophagosomes (arrows) and free electron dense material, compressed by a peripheral clear vacuolar space. Electronmicrograph, scale bar 0.5 μm. Abbreviations: n, nucleus; ml, molecular layer; pl, Purkinje cell layer; gl, granular cell layer.


( A ) Axonal spheroids stain diffusely positive for LC3B . IHCLC3B , scale bar 100 μm . ( B , C ) The granular cores of the spheroids are positive for ( B ) ubiquitin ( arrow ) and ( C ) p62 . IHCubiquitin and p62 , scale bars 20 and 100 μm , respectively . ( D ) Smooth axonal swellings in the cerebellar cortex contain ATG4D . Inset : control . IHCATG4D , scale bar 100 μm . ( E ) Affected neurons show increased perinuclear granular LC3B positivity . Inset : control . IHCLC3B , scale bar 100 μm . ( F ) Neuronalvacuoles are partially LAMP2 positive ( arrow ) and partially negative ( arrow head ) . Inset : control . IHCLAMP2 , scale bar 20 μm .

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(A) Axonal spheroids stain diffusely positive for LC3B. IHCLC3B, scale bar 100 μm. (B,C) The granular cores of the spheroids are positive for (B) ubiquitin (arrow) and (C) p62. IHCubiquitin and p62, scale bars 20 and 100 μm, respectively. (D) Smooth axonal swellings in the cerebellar cortex contain ATG4D. Inset: control. IHCATG4D, scale bar 100 μm. (E) Affected neurons show increased perinuclear granular LC3B positivity. Inset: control. IHCLC3B, scale bar 100 μm. (F) Neuronalvacuoles are partially LAMP2 positive (arrow) and partially negative (arrow head). Inset: control. IHCLAMP2, scale bar 20 μm.


( A - F ) Lateral views of control and morphant zebrafishembryos . ( A , B ) Control embryos appear normal at 1 dpf . ( C , D ) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf . Black arrowheads denote the developing cerebellum . Purple arrows indicate the hindbrain irregularities . ( E ) Control embryo at 2 dpf . ( F ) A morphant embryo at 2 dpf displaying hydrocephalus ( yellow arrow ) and small head and eye .

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(A-F) Lateral views of control and morphant zebrafishembryos. (A,B) Control embryos appear normal at 1 dpf. (C,D) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf. Black arrowheads denote the developing cerebellum. Purple arrows indicate the hindbrain irregularities. (E) Control embryo at 2 dpf. (F) A morphant embryo at 2 dpf displaying hydrocephalus (yellow arrow) and small head and eye.


( G ) RT - PCR assay showing the efficiency of the atg4daSMO .

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(G) RT-PCR assay showing the efficiency of the atg4daSMO.


Immunostaining with ( A - C ) anti - Pvalb7 and ( D - F ) anti - zebrinII antibody show loss of cerebellarPurkinje cells in atg4da morphants . ( A , D ) Control embryos at 4 . 5 dpf . ( B , E ) Morphants with mild phenotype show partial loss of cerebellarPurkinje cells . ( C , F ) Morphants with strong phenotype show either total loss of Purkinje cells or presence of few differentiated neurons , which are laterally located in the cerebellum . ( G - I ) Labeling of cerebellar granule cells with anti - Vglut1 antibody in control and morphant embryos . ( H ) Mildly affected morphants show reduced expression of Vglut1 in the cerebellum . ( I ) Vglut1 expression is strongly reduced in embryos showing severe phenotype . White arrowheads indicate the region of cerebellum . Abbreviations : hb , hindbrain .

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Immunostaining with (A-C) anti-Pvalb7 and (D-F) anti-zebrinII antibody show loss of cerebellarPurkinje cells in atg4da morphants. (A,D) Control embryos at 4.5 dpf. (B,E) Morphants with mild phenotype show partial loss of cerebellarPurkinje cells. (C,F) Morphants with strong phenotype show either total loss of Purkinje cells or presence of few differentiated neurons, which are laterally located in the cerebellum. (G-I) Labeling of cerebellar granule cells with anti-Vglut1 antibody in control and morphant embryos. (H) Mildly affected morphants show reduced expression of Vglut1 in the cerebellum. (I) Vglut1 expression is strongly reduced in embryos showing severe phenotype. White arrowheads indicate the region of cerebellum. Abbreviations: hb, hindbrain.


A ) Transmission electron microscopy ( TEM ) of MoDCs infected with P . gingivalis for 2 , 12 and 24 hours ( left , middle and right panels ) . The sections show the intra - and extra - cellular contents of Cont . ( un - infected ) , Pg381 , Mfa1 + Pg and FimA + Pg infected MoDCs for the different time

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A) Transmission electron microscopy (TEM) of MoDCs infected with P. gingivalis for 2, 12 and 24 hours (left, middle and right panels). The sections show the intra-and extra-cellular contents of Cont. (un-infected), Pg381, Mfa1+Pg and FimA+Pg infected MoDCs for the different time points


B ) The figure shows the intracellular content of P . gingivalis strains after 24 hours of infection . MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re - suspended and maintained in anaerobic broth for 5 days . The data represents CFU within MoDCs harvested from three healthy individuals . The means ± standard deviation ( in triplicates ) were analyzed by One - way ANOVA of different groups and Tukey ' s test for multiple group comparisons within 3 different experiments ( * statistical significance at the p < 0 . 001 ) .

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B) The figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey's test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001).


A ) DC - SIGN mRNA expression in P . gingivalis - infected MoDCs at 0 . 1 , 1 and 10 MOIs . The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections . The target gene ( DC - SIGN ) was normalized using the endogenous control GAPDH ( ΔCt ) and fold regulations were calculated using 2 - ( ΔΔCt ) method . The statistical analysis was performed using the t - test , which accounts for the clustering of infected and un - infected controls within 3 different experiments ( * p < 0 . 001 ) .

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A) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001).


B ) Immuno - electron microscopy of un - infected MoDCs ( Cont . ) ( upper panel ) , MoDCs infected with Pg381 ( middle panel ) and Mfa1 + Pg mutants ( lower panel ) . Gold particles ( marked with red rings ) for positive DC - SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1 + Pg strains . Minimal positive staining for DC - SIGN was detected in the membranes of MoDCs infected with Pg381 , while no cytoplasmic gold labeling was detected in these cells .

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B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells.


C ) Flow cytometry analysis of surface DC - SIGN in human MoDCs after infection with Pg381 , Mfa1 + Pg and FimA + Pg . The analysis of the intensity used Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons 3 different experiments ( * p < 0 . 01 ) .

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C) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons 3 different experiments (* p<0.01).


A ) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with / without Rapamycin treatment . The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days . The plot represents the means ± standard deviation of CFU within MoDCs harvested from three healthy individuals ( * P < 0 . 001 ) . The analysis of readings used One - way AVOVA analysis of different groups and Tukey ' s test for multiple comparisons .

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A) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey's test for multiple comparisons.


B ) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P . gingivalis infections . LC3 - II ( red - fluorescent dye ) and the bacterial strains ( green CFSE ) were studied in MoDCs 11 hours after Rapamycin treatment ( 12 hours after infections ) .

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B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections).


C ) Quantifications of the fluorescent intensity of CFSE - labeled P . gingivalis and LC3 - II signals within infected MoDCs using NIS - Elements BR software . One - way ANOVA analysis was used to compare the means of intensity of different groups and Tukey ' s test for multiple comparisons ( * P < 0 . 001 ) .

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C) Quantifications of the fluorescent intensity of CFSE-labeled P. gingivalis and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons (* P<0.001).


A ) Survival of P . gingivalis strains within MoDCs after 6 , 12 , 24 and 48 hours . Blue lines show P . gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines .

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A) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines.


The effect of rapamycin on Mfa1 + Pg , Pg381 and FimA + Pg survival within MoDCs are shown in figures B , C and D respectively . A three - factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading . The survival curves for the strains are showed in blue , while the effect of rapamycin treatments are in red . Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black , respectively . Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain ( Mfa1 + Pg , Pg381 and FimA + Pg ) between treated ( rapamycin ) and untreated were significantly different overtime ( p - value < 0 . = 001 ) .

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The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).


A ) Epifluorescence microscopy images of MoDCs infected with Pg381 , Mfa1 + Pg , FimA + Pg and MFB strains after 12 hours . LC3 - II was detected in red - fluorescent ( Texas red ) dye and the bacterial strains were pre - labeled with CFSE ( green ) . Co - localization of P . gingivalis and LC3 - II showed in the right panels .

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A) Epifluorescence microscopy images of MoDCs infected with Pg381, Mfa1+Pg, FimA+Pg and MFB strains after 12 hours. LC3-II was detected in red-fluorescent (Texas red) dye and the bacterial strains were pre-labeled with CFSE (green). Co-localization of P. gingivalis and LC3-II showed in the right panels.


B ) Quantifications of the fluorescent intensity of LC3 - II within infected MoDCs using NIS - Elements BR software . One - way ANOVA analysis was used to compare the means of intensity of different groups and Tukey ' s test for multiple comparisons of three different experiments ( * p < 0 . 001 , # p < 0 . 01 ) .

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B) Quantifications of the fluorescent intensity of LC3-II within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons of three different experiments (* p<0.001, # p<0.01).


C ) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity ( * p < 0 . 001 , # p < 0 . 01 ) .

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C) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity (* p<0.001, # p<0.01).


A ) Immuno - fluorescence images of LC3 - II and P . gingivalis within MoDCs pre - treated with GP120 ( DC - SIGN blocker ) after 12 hours of infection . LC3 - II was detected in red - fluorescent dye and the bacterial strains were pre - labeled with green CFSE .

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A) Immuno-fluorescence images of LC3-II and P. gingivalis within MoDCs pre-treated with GP120 (DC-SIGN blocker) after 12 hours of infection. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE.


B ) Fluorescent intensity of LC3 - II and P . gingivalis strains signal ( in triplicates ) were statistically analyzed ( * and # p < 0 . 001 ) .

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B) Fluorescent intensity of LC3-II and P. gingivalis strains signal (in triplicates) were statistically analyzed (* and # p<0.001).


C ) Flow cytometry of MoDCs treated with siRNA for DC - SIGN ( si - DC - SIGN ) after 12 hours . Left panels show the decrease of DC - SIGN in Cont . ( un - infected ) , Pg381 and Mfa1 + Pg - infected MoDCs .

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C) Flow cytometry of MoDCs treated with siRNA for DC-SIGN (si-DC-SIGN) after 12 hours. Left panels show the decrease of DC-SIGN in Cont. (un-infected), Pg381 and Mfa1+Pg-infected MoDCs.


D ) Epifluorescence microscopy images of MoDCs ( si - DC - SIGN ) infected with Pg381 and Mfa1 + Pg . LC3 - II was detected in red - fluorescent dye and the bacterial strains were pre - labeled with green CFSE .

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D) Epifluorescence microscopy images of MoDCs (si-DC-SIGN) infected with Pg381 and Mfa1+Pg. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE.


E ) The figure shows the CFU counts of the P . gingivalis strains with MoDCs that lack DC - SIGN ( si - DC - SIGN ) . The analysis of readings used One - way ANOVA analysis of different groups and Tukey ' s test for multiple comparisons ( * p < 0 . 001 ) .

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E) The figure shows the CFU counts of the P. gingivalis strains with MoDCs that lack DC-SIGN (si-DC-SIGN). The analysis of readings used One-way ANOVA analysis of different groups and Tukey's test for multiple comparisons (* p<0.001).


A ) Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours . B ) The mean ± standard deviation of LC3 - II intensity represents three different experiments and the values were analyzed using Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons .

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A) Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours. B) The mean ±standard deviation of LC3-II intensity represents three different experiments and the values were analyzed using Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.


C ) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs . Cells were treated with Bafilomycin for 4 hours at 4nM . D ) LC3 - II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin . The analysis of the intensity of three different experiments used Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons .

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C) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs. Cells were treated with Bafilomycin for 4 hours at 4nM. D) LC3-II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin. The analysis of the intensity of three different experiments used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.


A ) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand ( E . coli LPS ) and TLR1 and 2 ligand ( Pam3csk4 ) for 4 hour .

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A) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand (E. coli LPS) and TLR1 and 2 ligand (Pam3csk4) for 4 hour.


B ) Immuno - fluorescence images of LC3 - II ( red ) within MoDCs after incubation with TLR4 and TLR1 & amp ; amp ; 2 ligands ( E . coli LPS and Pam3csk4 )

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B) Immuno-fluorescence images of LC3-II (red) within MoDCs after incubation with TLR4 and TLR1&amp;2 ligands (E. coli LPS and Pam3csk4)


C ) The plot represents the means ± standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours ( * * p < 0 . 001 ) .

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C) The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours (** p<0.001).


A ) Scanning electron microscopy ( SEM ) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1 + Pg ( upper panel 10000x and lower panel 25000x ) . B ) SEM for MoDCs interacting with Pg381 ( green stains for bacteria are computer generated ) .

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A) Scanning electron microscopy (SEM) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1+Pg (upper panel 10000x and lower panel 25000x). B) SEM for MoDCs interacting with Pg381 (green stains for bacteria are computer generated).


C ) Transmission electron microscopy ( TEM ) of autophagosome like structures within MoDCs infected with Pg381 , Mfa1 + Pg and FimA + Pg for 12 hours ( upper , middle and lower panels , respectively ) . The right and left sections show the different magnifications of randomly selected sections . Pg381 and FimA + Pg strains are mostly enclosed in the characteristics double - membrane intracellular vesicles ( Orange arrows ) . Contrary , Mfa1 + Pg escaped these autophagic ( double - membrane ) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm ( Green arrows ) .

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C) Transmission electron microscopy (TEM) of autophagosome like structures within MoDCs infected with Pg381, Mfa1+Pg and FimA+Pg for 12 hours (upper, middle and lower panels, respectively). The right and left sections show the different magnifications of randomly selected sections. Pg381 and FimA+Pg strains are mostly enclosed in the characteristics double-membrane intracellular vesicles (Orange arrows). Contrary, Mfa1+Pg escaped these autophagic (double-membrane) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm (Green arrows).


D ) The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage . Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection . Each strain was counted in three randomly selected grids for each sample . The analysis of the bacterial counts used Kruskal - Wallis test of different groups and Dunn ' s test for multiple comparisons ( * p < 0 . 01 ) .

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D) The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage. Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection. Each strain was counted in three randomly selected grids for each sample. The analysis of the bacterial counts used Kruskal-Wallis test of different groups and Dunn's test for multiple comparisons (*p<0.01).


A ) Epifluorescence microscopy images of MoDCs infected with pre - labeled bacteria ( green - CFSE ) . LAMP1 detected by transduction of red fluorescent protein ( RFP ) chimera using baculovirus transgenes to MoDCs .

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A) Epifluorescence microscopy images of MoDCs infected with pre-labeled bacteria (green-CFSE). LAMP1 detected by transduction of red fluorescent protein (RFP) chimera using baculovirus transgenes to MoDCs.


B ) The mean of fluorescent intensities ± standard deviation of LAMP1 were quantified in three different experiments ( * p < 0 . 001 ) .

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B) The mean of fluorescent intensities ±standard deviation of LAMP1 were quantified in three different experiments (* p<0.001).


C ) The upper scattered plot shows the co - localization of red and green signals in sections of MoDCs infected with Pg381 and mutants strains . The lower plot shows the average of Pearson ' s correlation measures of three randomly selected region of interest ( ROI ) within each field within 3 different experiments ( * p < 0 . 0001 ) ( Table 4 and 5 ) . All analysis of fluorescence intensity used One - way ANOVA analysis of different groups and Tukey ' s test for multiple comparisons .

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C) The upper scattered plot shows the co-localization of red and green signals in sections of MoDCs infected with Pg381 and mutants strains. The lower plot shows the average of Pearson's correlation measures of three randomly selected region of interest (ROI) within each field within 3 different experiments (* p<0.0001) (Table 4 and 5). All analysis of fluorescence intensity used One-way ANOVA analysis of different groups and Tukey's test for multiple comparisons.


C , D . Immunolabeling of HH25 chick embryo sections 50 hours post - engraftment of A375P : : GFP cells , using an anti - GFP antibody ( in green in C , in brown in D , A375P : GFP cells ) , an anti - HNK1 antibody ( in white , migrating and early post - migrating NCCs ) , an anti - Ki67 antibody ( in red in C , cycling cells ) , an anti - SOX10 antibody ( in red in D , also stains chick endogenous NCCs ) and an anti - MITF antibody ( in yellow in D ) . Nuclei were stained with Hoechst ( in blue ) . In C , the upper left photo shows a grafted embryo prior to cryosection . In D , right panels are enlargements of the lower left panel . Scale bar : 200 µm .

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C,D. Immunolabeling of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody (in green in C, in brown in D, A375P:GFP cells), an anti-HNK1 antibody (in white, migrating and early post-migrating NCCs), an anti-Ki67 antibody (in red in C, cycling cells), an anti-SOX10 antibody (in red in D, also stains chick endogenous NCCs) and an anti-MITF antibody (in yellow in D). Nuclei were stained with Hoechst (in blue). In C, the upper left photo shows a grafted embryo prior to cryosection. In D, right panels are enlargements of the lower left panel. Scale bar: 200 µm.


F . 3D views ( light - sheet imaging ) of HH25 chick embryos engrafted with different melanoma patient samples , showing different patterns of tumor cell localization 50 hours post - engraftment .

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F. 3D views (light-sheet imaging) of HH25 chick embryos engrafted with different melanoma patient samples, showing different patterns of tumor cell localization 50 hours post-engraftment.


G . Immunofluorescent labeling of HH25 chick embryo sections 50 hours post - engraftment of OF - MEL - 020 patient sample , labeled with CFSE ( in green ) prior to the graft . An anti - SOX10 antibody ( in red ) and an anti - MITF antibody ( in yellow ) were used . Nuclei were stained with Hoechst ( in blue ) . Right panels are enlargements of the dotted area in the left panel .

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G. Immunofluorescent labeling of HH25 chick embryo sections 50 hours post-engraftment of OF-MEL-020 patient sample, labeled with CFSE (in green) prior to the graft. An anti-SOX10 antibody (in red) and an anti-MITF antibody (in yellow) were used. Nuclei were stained with Hoechst (in blue). Right panels are enlargements of the dotted area in the left panel.


A , B . Immunofluorescent labeling ( A ) of HH25 chick embryo sections 50 hours post - engraftment of A375P : : GFP cells , using an anti - GFP antibody , an anti - MITF antibody ( pink ) and an anti - SOX10 ( white ) antibody . Nuclei were stained with Hoechst . Right panels are enlargements of the dotted area in left panel . In B , the mean fraction of MITF - negative ( MITFneg ) , - low ( MITFlow ) and - positive ( MITFpos ) A375P : : GFP cells in the tumor mass and in migrating cells was quantified ( n = 16 slices from 3 embryos ) . Error bars show SEM . Mann - Whitney test comparing tumors vs migrating cells for each class of MITF expression was performed , p - values are indicated on the graph .

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A,B. Immunofluorescent labeling (A) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody, an anti-MITF antibody (pink) and an anti-SOX10 (white) antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in left panel. In B, the mean fraction of MITF-negative (MITFneg), -low (MITFlow) and -positive (MITFpos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 16 slices from 3 embryos). Error bars show SEM. Mann-Whitney test comparing tumors vs migrating cells for each class of MITF expression was performed, p-values are indicated on the graph.


C , D . Immunofluorescent labeling ( C ) of HH25 chick embryo sections 50 hours post - engraftment of A375P : : GFP cells , using an anti - GFP antibody and an anti - Ki67 antibody . Nuclei were stained with Hoechst . Right panels are enlargements of the dotted area in the left panel . In D , the mean fraction of Ki67 - positive ( Ki67pos ) A375P : : GFP cells in the tumor mass and in migrating cells was quantified ( n = 25 slices from 3 embryos ) . Arrows point at cells having a mesenchymal morphology . Error bars show SEM . Student t - test , * * p = 0 . 0070 .

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C,D. Immunofluorescent labeling (C) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody and an anti-Ki67 antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in the left panel. In D, the mean fraction of Ki67-positive (Ki67pos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 25 slices from 3 embryos). Arrows point at cells having a mesenchymal morphology. Error bars show SEM. Student t-test, **p = 0.0070.


H - K . 3D views ( H , J ) and quantification of tumor volumes ( I , K ) of HH25 chick embryos engrafted with GLO ( H , I ) or GLO - R ( J , K ) cells and treated with a combination of Vemurafenib and Cobimetinib , or Vorinostat alone , or a combination of the three molecules . The number of embryos analyzed for each experimental condition are indicated on the graphs . Bars and error bars indicate mean + SEM . Student t - tests , * * : p < 0 . 01 , ns : not significant . Exact p - values are indicated on the graphs .

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H-K. 3D views (H, J) and quantification of tumor volumes (I, K) of HH25 chick embryos engrafted with GLO (H, I) or GLO-R (J, K) cells and treated with a combination of Vemurafenib and Cobimetinib, or Vorinostat alone, or a combination of the three molecules. The number of embryos analyzed for each experimental condition are indicated on the graphs. Bars and error bars indicate mean + SEM. Student t-tests, **: p < 0.01, ns: not significant. Exact p-values are indicated on the graphs.


A , B . 3D views ( A ) and quantification of variations in the mean tumor volume ( B ) of HH25 chick embryos engrafted with BRAFwt ( OF - MEL - 028 ) or BRAFV600E ( OF - MEL - 020 , OF - MEL - 027 ) patient samples and treated with excipient or a combination of Vemurafenib and Cobimetinib . The number of embryos analyzed for each patient sample is indicated on the graphs . Student t - test for OF - MEL - 020 ; Mann - Whintey test for OF - MEL - 028 , * : p < 0 . 05 , ns : not significant . Exact p - values are indicated on the graphs .

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A,B. 3D views (A) and quantification of variations in the mean tumor volume (B) of HH25 chick embryos engrafted with BRAFwt (OF-MEL-028) or BRAFV600E (OF-MEL-020, OF-MEL-027) patient samples and treated with excipient or a combination of Vemurafenib and Cobimetinib. The number of embryos analyzed for each patient sample is indicated on the graphs. Student t-test for OF-MEL-020; Mann-Whintey test for OF-MEL-028, *: p < 0.05, ns: not significant. Exact p-values are indicated on the graphs.


C , D . Survival rate ( left axis ) and mean body surface area ( BSA , right axis ) of chick embryos injected with increasing doses of Dabrafenib ( C ) and Trametinib ( D ) . The maximum tolerated dose ( MTD , in red on the X - axis ) was defined as the higher dose of drug associated with a survival rate higher than 75 % and a mean BSA similar ( i . e . , non - statistically different ) from embryos treated with excipient ( 17 . 1 and 0 . 0228 mg / kg respectively ) . N = 10 replicates per experimental group . Error bars indicate SEM . ns , non - significant using Student ' s t - test compared with excipient .

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C,D. Survival rate (left axis) and mean body surface area (BSA, right axis) of chick embryos injected with increasing doses of Dabrafenib (C) and Trametinib (D). The maximum tolerated dose (MTD, in red on the X-axis) was defined as the higher dose of drug associated with a survival rate higher than 75% and a mean BSA similar (i.e., non-statistically different) from embryos treated with excipient (17.1 and 0.0228 mg/kg respectively). N=10 replicates per experimental group. Error bars indicate SEM. ns, non-significant using Student's t-test compared with excipient.


( F - H ) of HH25 chick embryos engrafted with 3 different patient samples ( OF - MEL - 033 ( F ) , OF - MEL - 034 ( G ) , OF - MEL - 035 ( H ) ) and treated with excipient or a combination of Dabrafenib and Trametinib . The clinical response of each patient after a 3 months treatment with Dabrafenib / Trametinib is indicated above the graphs . Error bars indicate SEM . Mann - Whitney tests , exact p - values are indicated on the graphs .

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(F-H) of HH25 chick embryos engrafted with 3 different patient samples (OF-MEL-033 (F), OF-MEL-034 (G), OF-MEL-035 (H)) and treated with excipient or a combination of Dabrafenib and Trametinib. The clinical response of each patient after a 3 months treatment with Dabrafenib/Trametinib is indicated above the graphs. Error bars indicate SEM. Mann-Whitney tests, exact p-values are indicated on the graphs.


a , Fluorescence microscopyHeLa cells infected with Shigella and Listeria visualized with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) ( pseudo - coloured red ) , and Salmonella carrying mCherry - expressing vector ( red ) . The cis - Golgi ( green ) was detected by α - GM130 antibodies . Scale bar , 10 µm .

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a, Fluorescence microscopyHeLa cells infected with Shigella and Listeria visualized with 4′,6-diamidino-2-phenylindole (DAPI) (pseudo-coloured red), and Salmonella carrying mCherry-expressing vector (red). The cis-Golgi (green) was detected by α-GM130 antibodies. Scale bar, 10 µm.


b , Fluorescence microscopy of HeLa cells transfected with either IpaJ or VirA . The cis - Golgi ( GM130 , green ) and F - actin ( red ) are shown . Scale bar , 10 µm .

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b, Fluorescence microscopy of HeLa cells transfected with either IpaJ or VirA. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm.


c , Fluorescence microscopy of HeLa cells infected with indicated Shigella mutant strains . Scale bar , 10 µm .

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c, Fluorescence microscopy of HeLa cells infected with indicated Shigella mutant strains. Scale bar, 10 µm.


d , Percentage of HeLa cells with disrupted Golgi morphology in 100 cells infected with either wild - type Shigella ( M90T ) or the indicated mutants . Error bars , means ± s . e . m . calculated from three independent experiments .

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d, Percentage of HeLa cells with disrupted Golgi morphology in 100 cells infected with either wild-type Shigella (M90T) or the indicated mutants. Error bars, means ± s.e.m. calculated from three independent experiments.


e , Number of recoverable c . f . u . per mouselung24 h after intranasal inoculation with 1 × 106 of wild - type M90T or each mutant Shigella strain as indicated . Limit of detection ( LOD ) for this assay is indicated by the dotted line . Error bars , geometrical means ± s . e . m . * * P < 0 . 001 .

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e, Number of recoverable c.f.u. per mouselung24 h after intranasal inoculation with 1 × 106 of wild-type M90T or each mutant Shigella strain as indicated. Limit of detection (LOD) for this assay is indicated by the dotted line. Error bars, geometrical means ± s.e.m. **P<0.001.


b , Fluorescence microscopy of HeLa cells transfected with the indicated IpaJ mutants . The cis - Golgi ( GM130 , green ) and F - actin ( red ) are shown . Scale bar , 10 µm .

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b, Fluorescence microscopy of HeLa cells transfected with the indicated IpaJ mutants. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm.


c , IpaJ or its catalytic mutants were expressed from a galactose - inducible promoter ( pGal413 vector ) and assayed for a growth arrest phenotype on galactose or glucose ( control ) carbon source .

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c, IpaJ or its catalytic mutants were expressed from a galactose-inducible promoter (pGal413 vector) and assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.


e , Yeast strain harbouring a galactose - inducible IpaJ gene were transformed with a multi - copy vector containing the indicated genes . Yeast were assayed for a growth arrest phenotype on galactose or glucose ( control ) carbon source .

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e, Yeast strain harbouring a galactose-inducible IpaJ gene were transformed with a multi-copy vector containing the indicated genes. Yeast were assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.


a - c , Mass spectra of purified ARF1 - strep in untreated ( a ) , IpaJ - treated ( b ) or IpaJ C64A - treated ( c ) cells , with the observed monoisotopic mass reported in daltons . * Observed 1 Da shift in molecular mass is accounted for in MS / MS data ( Supplementary Fig . 7 ) .

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a-c, Mass spectra of purified ARF1-strep in untreated (a), IpaJ-treated (b) or IpaJ C64A-treated (c) cells, with the observed monoisotopic mass reported in daltons. *Observed 1 Da shift in molecular mass is accounted for in MS/MS data (Supplementary Fig. 7).


f , In - gel fluorescence assay ( top panel ) visualizing Alexa Fluor 647 - labelled myristoylated ARF1 - His isolated from bacteria either not expressing ( lane 1 ) or expressing ( lane 2 ) NMTp . Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated . The expression levels of ARF1 - His were determined by Coomassie blue stain .

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f, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-His isolated from bacteria either not expressing (lane 1) or expressing (lane 2) NMTp. Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated. The expression levels of ARF1-His were determined by Coomassie blue stain.