In vitro direct binding assay between FLAG - LUZP1 and GST - MLC . Representative immunoblots are shown . S , supernatant ; P , pellet . A representative plot in which the amount of bound GST - WT - , AA - , or DD - MLC / FLAG - LUZP1 was plotted with the Michaelis - Menten fitted curve . Bar plots with dot density plots showing that the dissociation constant ( Kd ) of LUZP1 from DD - MLC was significantly lower than that from WT - MLC and AA - MLC [ 4 . 04 ± 1 . 05 μM ( WT - MLC ) vs . 4 . 88 ± 1 . 79 μM ( AA - MLC ) vs . 0 . 85 ± 0 . 39 μM ( DD - MLC ) ] . n = 5 ( WT - MLC and AA - MLC ) and n = 4 ( DD - MLC ) . * p < 0 . 05 [ Kruskal - Wallis test followed by Steel - Dwass test ] . Bars and error bars represent the mean ± SD .

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

In vitro direct binding assay between FLAG-LUZP1 and GST-MLC. Representative immunoblots are shown. S, supernatant; P, pellet.   A representative plot in which the amount of bound GST-WT-, AA-, or DD-MLC/FLAG-LUZP1 was plotted with the Michaelis-Menten fitted curve.   Bar plots with dot density plots showing that the dissociation constant (Kd) of LUZP1 from DD-MLC was significantly lower than that from WT-MLC and AA-MLC [4.04 ± 1.05 μM (WT-MLC) vs. 4.88 ± 1.79 μM (AA-MLC) vs. 0.85 ± 0.39 μM (DD-MLC)]. n = 5 (WT-MLC and AA-MLC) and n = 4 (DD-MLC). *p < 0.05 [Kruskal-Wallis test followed by Steel-Dwass test]. Bars and error bars represent the mean ± SD.


Representative immunoblot of WT , LUZP1 KO , and Venus - LUZP1 - expressing LUZP1 knockout ( REV ) Eph4 cells treated with 100 nM calyculin A for 30 min . Quantification of the ppMLC / MLC ratio relative to WT control , confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A [ WT , 1 . 00 ( control ) vs . 1 . 40 ± 0 . 06 ( calyculin A ) vs . 1 . 14 ± 0 . 33 ( washout ) ; KO , 0 . 09 ± 0 . 04 ( control ) vs . 1 . 49 ± 0 . 06 ( calyculin A ) vs . 0 . 81 ± 0 . 99 ( washout ) ; REV , 2 . 06 ± 1 . 78 ( control ) vs . 1 . 82 ± 1 . 50 ( calyculin A ) vs . 1 . 80 ± 1 . 14 ( washout ) ] . n = 3 . Bars and error bars represent the mean ± SD .

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

Representative immunoblot of WT, LUZP1 KO, and Venus-LUZP1-expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A [WT, 1.00 (control) vs. 1.40 ± 0.06 (calyculin A) vs. 1.14 ± 0.33 (washout); KO, 0.09 ± 0.04 (control) vs. 1.49 ± 0.06 (calyculin A) vs. 0.81 ± 0.99 (washout); REV, 2.06 ± 1.78 (control) vs. 1.82 ± 1.50 (calyculin A) vs. 1.80 ± 1.14 (washout)]. n = 3. Bars and error bars represent the mean ± SD.


In vitro MLC phosphorylation assay using 1 μg GST - PP1c β / δ in addition to 25 ng GST - MLC , 4 ng GST - ROCK1 catalytic domain , 1 mM ATP , and 0 - 5 μg GST - LUZP1 . Quantification of the di - phosphorylated MLC ( ppMLC ) / MLC ratio relative to the control showed that LUZP1 up - regulated ppMLC / MLC levels in a dose - dependent manner [ 1 . 00 ( 1st lane , control ) vs . 1 . 27 ± 0 . 33 ( 2nd lane ) vs . 1 . 76 ± 0 . 68 ( 3rd lane ) vs . 2 . 53 ± 1 . 65 ( 4th lane ) vs . 2 . 93 ± 2 . 45 ( 5th lane ) ] . n = 3 or 6 . * * p < 0 . 01 [ Kruskal - Wallis test followed by Steel test ( compared with 1st lane ) ] . Bars and error bars represent the mean ± SD . In vitro Merlin phosphorylation assay using 1 μg GST - PP1c β / δ , 100 ng GST - Merlin , 2 pg p21 - activated kinase 1 ( PAK1 ) , and 5 μg GST - LUZP1 . Quantification of the phosphorylated Merlin ( pMerlin ) / Merlin ratio relative to the control showed that LUZP1 up - regulated pMerlin / Merlin levels [ 0 . 23 ± 0 . 15 ( 1st lane ) vs . 1 . 00 ( 2nd lane , control ) vs . 0 . 32 ± 0 . 17 ( 3rd lane ) vs . 0 . 97 ± 0 . 42 ( 4th lane ) vs . 1 . 25 ± 0 . 39 ( 5th lane ) ] . n = 4 or 9 . * p < 0 . 05 , * * p < 0 . 01 [ Kruskal - Wallis test followed by Steel test ( compared with 3rd lane ) ] . Bars and error bars represent the mean ± SD .

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

In vitro MLC phosphorylation assay using 1 μg GST-PP1c β/δ in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, and 0-5 μg GST-LUZP1. Quantification of the di-phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 up-regulated ppMLC/MLC levels in a dose-dependent manner [1.00 (1st lane, control) vs. 1.27 ± 0.33 (2nd lane) vs. 1.76 ± 0.68 (3rd lane) vs. 2.53 ± 1.65 (4th lane) vs. 2.93 ± 2.45 (5th lane)]. n = 3 or 6. **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.   In vitro Merlin phosphorylation assay using 1 μg GST-PP1c β/δ, 100 ng GST-Merlin, 2 pg p21-activated kinase 1 (PAK1), and 5 μg GST-LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 up-regulated pMerlin/Merlin levels [0.23 ± 0.15 (1st lane) vs. 1.00 (2nd lane, control) vs. 0.32 ± 0.17 (3rd lane) vs. 0.97 ± 0.42 (4th lane) vs. 1.25 ± 0.39 (5th lane)]. n = 4 or 9. *p < 0.05, **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 3rd lane)]. Bars and error bars represent the mean ± SD.


Box plots with dot density plots showing the ratio of the apical area / basal area in co - cultures of Venus - LUZP1 - expressing LUZP1 knockout ( REV ) and LUZP1 knockout ( LUZP1 KO ) Eph4 cells ; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells [ REV , 0 . 65 ± 0 . 16 ( control ) vs . 0 . 90 ± 0 . 18 ( nocodazole ) vs . 0 . 64 ± 0 . 16 ( washout ) ; KO , 1 . 30 ± 0 . 17 ( control ) vs . 1 . 07 ± 0 . 13 ( nocodazole ) vs . 1 . 32 ± 0 . 19 ( washout ) ] . * * p < 0 . 01 ( Kruskal - Wallis test followed by Steel - Dwass test ) . The solid lines represent the medians and the boxes represent the inter - quartile ranges . The error bars extending from the box represents the data within 1 . 5 times of the interquartile range . Representative confocal micrographs of co - cultures of LUZP1 - expressing wild - type ( WT ) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min . Nocodazole treatment partially reversed the difference in di - phosphorylated MLC ( ppMLC ) levels within circumferential rings ( CRs ) between WT and LUZP1 KO cells . Scale bar , 10 μm . Bar plots with dot density plots showing that ppMLC levels within CRs were significantly down - regulated in WT Eph4 cells after nocodazole treatment . Importantly , ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment [ WT , 21 . 43 ± 6 . 96 arbitrary units ( a . u . ) ( control ) vs . 17 . 67 ± 5 . 40 a . u . ( nocodazole ) vs . 20 . 84 ± 7 . 19 a . u . ( washout ) ; KO , 8 . 74 ± 1 . 71 a . u . ( control ) vs . 8 . 67 ± 1 . 89 a . u . ( nocodazole ) vs . 7 . 96 ± 2 . 35 a . u . ( washout ) ] . n = 3 . * * p < 0 . 01 ( Kruskal - Wallis test followed by Steel - Dwass test ) . Bars and error bars represent the mean ± standard deviation ( SD ) .

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

Box plots with dot density plots showing the ratio of the apical area/basal area in co-cultures of Venus-LUZP1-expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells [REV, 0.65 ± 0.16 (control) vs. 0.90 ± 0.18 (nocodazole) vs. 0.64 ± 0.16 (washout); KO, 1.30 ± 0.17 (control) vs. 1.07 ± 0.13 (nocodazole) vs. 1.32 ± 0.19 (washout)]. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). The solid lines represent the medians and the boxes represent the inter-quartile ranges. The error bars extending from the box represents the data within 1.5 times of the interquartile range. Representative confocal micrographs of co-cultures of LUZP1-expressing wild-type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di-phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm.   Bar plots with dot density plots showing that ppMLC levels within CRs were significantly down-regulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment [WT, 21.43 ± 6.96 arbitrary units (a.u.) (control) vs. 17.67 ± 5.40 a.u. (nocodazole) vs. 20.84 ± 7.19 a.u. (washout); KO, 8.74 ± 1.71 a.u. (control) vs. 8.67 ± 1.89 a.u. (nocodazole) vs. 7.96 ± 2.35 a.u. (washout)]. n = 3. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). Bars and error bars represent the mean ± standard deviation (SD).


In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST - MLC , 4 ng GST - ROCK1 catalytic domain , 1 mM ATP , 1 μg GST - protein phosphatase 1c β / δ ( PP1c β / δ ) , and 0 - 5 μg GST - LUZP1 . Quantification of the relative ppMLC / MLC ratio to the control showed that MTs promote LUZP1 - mediated inhibition of PP1c β / δ [ 1 . 00 ( 1st - lane , control ) vs . 1 . 42 ± 0 . 59 ( 2nd - lane ) vs . 1 . 72 ± 0 . 76 ( 3rd - lane ) vs . 1 . 99 ± 0 . 56 ( 4th - lane ) vs . 1 . 14 ± 0 . 37 ( 5th - lane ) vs . 2 . 87 ± 1 . 51 ( 6th - lane ) vs . 2 . 74 ± 1 . 19 ( 7th - lane ) vs . 2 . 50 ± 0 . 88 ( 8th - lane ) ] . n = 6 . * p < 0 . 05 [ Kruskal - Wallis test followed by Steel test ( compared with 1st lane ) ] . Bars and error bars represent the mean ± SD .

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

In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, 1 μg GST-protein phosphatase 1c β/δ (PP1c β/δ), and 0-5 μg GST-LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1-mediated inhibition of PP1c β/δ [1.00 (1st-lane, control) vs. 1.42 ± 0.59 (2nd-lane) vs. 1.72 ± 0.76 (3rd-lane) vs. 1.99 ± 0.56 (4th-lane) vs. 1.14 ± 0.37 (5th-lane) vs. 2.87 ± 1.51 (6th-lane) vs. 2.74 ± 1.19 (7th-lane) vs. 2.50 ± 0.88 (8th-lane)]. n = 6. *p < 0.05 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.


( A ) Representative double - plotted actograms showing wheel - running activity of wild type ( WT ) and CRY - deficient ( CRY Knockout ; CKO ) mice during constant light ( yellow shading ) and thereafter in constant darkness . Note the 48 hour x - axis for WT versus 32 hour for CKO .

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

(A) Representative double-plotted actograms showing wheel-running activity of wild type (WT) and CRY-deficient (CRY Knockout; CKO) mice during constant light (yellow shading) and thereafter in constant darkness. Note the 48 hour x-axis for WT versus 32 hour for CKO.


( B ) Mean period and amplitude ( ± SEM ) of mouse behavioural data ( n = 4 ) .

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(B) Mean period and amplitude (±SEM) of mouse behavioural data (n=4).


( C ) Longitudinal bioluminescence recordings of organotypic SCN slices from WT ( black ) and CKO ( red ) PER2 : : LUC mice ( RLU ; relative light units ) .

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(C) Longitudinal bioluminescence recordings of organotypic SCN slices from WT (black) and CKO (red) PER2::LUC mice (RLU; relative light units).


( D ) Mean period and amplitude ( ± SEM ) of rhythmic SCN bioluminescence traces .

(D) Mean period and amplitude (±SEM) of rhythmic SCN bioluminescence traces.


( E ) Circadian PER2 : : LUC expression in immortalised WT and CKO adult lung fibroblasts . Left panel shows two raw traces of a representative longitudinal bioluminescence recording , right panel shows same data detrended with a 24 - hour moving average to remove differences in baseline expression .

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

(E) Circadian PER2::LUC expression in immortalised WT and CKO adult lung fibroblasts. Left panel shows two raw traces of a representative longitudinal bioluminescence recording, right panel shows same data detrended with a 24-hour moving average to remove differences in baseline expression.


( F ) Period of rhythmic fibroblast bioluminescence traces from at least 31 experiments ( n ≥ 3 per experiment , individual values ± SEM shown ) . P - values were calculated by an unpaired t test with Welch correction . Standard deviations differ significantly between WT and CKO ( F test : p < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Period of rhythmic fibroblast bioluminescence traces from at least 31 experiments (n≥3 per experiment, individual values ±SEM shown). P-values were calculated by an unpaired t test with Welch correction. Standard deviations differ significantly between WT and CKO (F test: p <0.0001).


( A ) Detrended traces of bioluminescence recordings of WT and CKO fibroblast at different constant temperature conditions within the physiological range ( n = 4 , solid lines : mean , dashed lines : ± SEM ) . Temperature was changed from 37 ° C to 32 ° C halfway through the experiment , as depicted by red / blue shading . Arrows represent medium changes . Note the lack of rhythmicity in the first three days in CKO and the appearance of rhythmicity after the first medium change . ( B ) Quantification of period from recordings presented in ( A ) . Both WT and CKO oscillations are temperature compensated with respective Q10s of 1 . 05 and 0 . 95 ( n = 3 , mean ± SEM ) . P - values were determined by two - tailed t - test . ( C ) Bioluminescence of WT and CKO PER2 : : LUC cells during temperature entrainment ( 12h 32 ° C ( blue ) - 12h 37 ° C ( red ) ) ( n = 3 , solid lines : mean , dashed lines : ± SEM ) .

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

(A) Detrended traces of bioluminescence recordings of WT and CKO fibroblast at different constant temperature conditions within the physiological range (n=4, solid lines: mean, dashed lines: ±SEM). Temperature was changed from 37°C to 32°C halfway through the experiment, as depicted by red/blue shading. Arrows represent medium changes. Note the lack of rhythmicity in the first three days in CKO and the appearance of rhythmicity after the first medium change. (B) Quantification of period from recordings presented in (A). Both WT and CKO oscillations are temperature compensated with respective Q10s of 1.05 and 0.95 (n=3, mean ±SEM). P-values were determined by two-tailed t-test. (C) Bioluminescence of WT and CKO PER2::LUC cells during temperature entrainment (12h 32°C (blue) - 12h 37°C (red)) (n=3, solid lines: mean, dashed lines: ±SEM).


( A ) Mean number of PER2 : : LUC molecules per cell at the estimated peak of PER2 expression for each cell line ( mean of three experiments , n = 3 each ) .

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 O O O O O O O O O

(A) Mean number of PER2::LUC molecules per cell at the estimated peak of PER2 expression for each cell line (mean of three experiments, n=3 each).


( B ) PER2 : : LUC binding to BMAL1 in WT and CKO cells . Cells were harvested at the peak of PER2 expression , BMAL1 was immunoprecipitated , and PER2 : : LUC binding was measured by bioluminescence measurements ( n = 3 , mean ± SD ) .

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 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 O O O O O O O

(B) PER2::LUC binding to BMAL1 in WT and CKO cells. Cells were harvested at the peak of PER2 expression, BMAL1 was immunoprecipitated, and PER2::LUC binding was measured by bioluminescence measurements (n=3, mean ±SD).


( C ) Per2 mRNA levels in WT ( left ) and CKO ( right ) cells were determined by qPCR over one circadian cycle ( bottom ) , while PER2 : : LUC bioluminescence ( top , min - max normalised ) was recorded from parallel cultures . Per2 mRNA reported relative to Rns18s ( bottom ) , n = 3 , ± SEM ; PER2 : : LUC ( top ) presented as mean ( solid ) ± SEM ( dashed ) , n = 3 . The WT mRNA trace was preferentially fit by a circadian damped sine wave compared with straight line ( p = 0 . 0412 , extra sum - of - square F - test ) whereas CKO data was not ( ns ) .

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 B-MEASURED_VAR 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 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

(C) Per2 mRNA levels in WT (left) and CKO (right) cells were determined by qPCR over one circadian cycle (bottom), while PER2::LUC bioluminescence (top, min-max normalised) was recorded from parallel cultures. Per2 mRNA reported relative to Rns18s (bottom), n=3, ±SEM; PER2::LUC (top) presented as mean (solid) ±SEM (dashed), n=3. The WT mRNA trace was preferentially fit by a circadian damped sine wave compared with straight line (p=0.0412, extra sum-of-square F-test) whereas CKO data was not (ns).


( D ) Detrended Per2 and Nr1d1 promoter activity in WT , CKO and quadruple Cry1 / 2 - Per1 / 2 knockout ( CPKO ) mouse embryonic fibroblasts ( MEFs ) recorded at 37 ° C , n = 3 , mean ( solid ) ± SEM ( dashed ) . Nr1d1 data were preferentially fit with a circadian damped sine wave over straight line ( p < 0 . 0001 , extra sum - of - squares F - test ) ( right hand graphs , solid lines ; error bars , SEM ) .

O O O O B-MEASURED_VAR O B-MEASURED_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 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

(D) Detrended Per2 and Nr1d1 promoter activity in WT, CKO and quadruple Cry1/2-Per1/2 knockout (CPKO) mouse embryonic fibroblasts (MEFs) recorded at 37°C, n=3, mean (solid) ±SEM (dashed). Nr1d1 data were preferentially fit with a circadian damped sine wave over straight line (p<0.0001, extra sum-of-squares F-test) (right hand graphs, solid lines; error bars, SEM).


( A ) Actual PER2 : : LUC levels ( dark symbols ( 3 - hour moving average , n = 3 ± SEM , 4 outliers removed ) ) as assayed in acute luciferase assays on cell lysates from cells harvested every hour over 48 hours , compared with parallel longitudinal co - recordings from cells in the presence of 0 . 1 mM luciferin ( light lines ( n = 6 , mean ± SEM ) ) .

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

(A) Actual PER2::LUC levels (dark symbols (3-hour moving average, n=3 ±SEM, 4 outliers removed)) as assayed in acute luciferase assays on cell lysates from cells harvested every hour over 48 hours, compared with parallel longitudinal co-recordings from cells in the presence of 0.1 mM luciferin (light lines (n=6, mean ±SEM)).


( B ) PER2 : : LUC recording of asynchronous WT and CKO cells pulsed with proteasome inhibitor MG132 ( 10 µM , applied at the arrow ) ( n = 3 , mean ± SEM ) . ( C ) Quantification of relative PER2 : : LUC induction upon proteasome inhibition , n = 3 , mean ( solid ) ± SEM ( dashed ) .

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(B) PER2::LUC recording of asynchronous WT and CKO cells pulsed with proteasome inhibitor MG132 (10 µM, applied at the arrow) (n=3, mean ±SEM). (C) Quantification of relative PER2::LUC induction upon proteasome inhibition, n=3, mean (solid) ±SEM (dashed).


( D ) Phase - dependent PER2 : : LUC half - life was determined by inhibiting translation at different circadian phases and fitting the resulting data with a one - phase exponential decay curve ( n = 3 , mean ± SEM ) . Left image depicts the timing of cycloheximide ( CHX , 10 µM ) pulses ( labelled I ( PER2 levels going up ) and II ( PER2 levels going down ) ) , plotted on PER2 : : LUC bioluminescence traces of control cells ( dark colours ) . A representative trace of CHX - treated cells at time point I is shown in light colours . Right image shows quantifications , p - values were calculated by unpaired t test .

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 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 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 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

(D) Phase-dependent PER2::LUC half-life was determined by inhibiting translation at different circadian phases and fitting the resulting data with a one-phase exponential decay curve (n=3, mean ±SEM). Left image depicts the timing of cycloheximide (CHX, 10 µM) pulses (labelled I (PER2 levels going up) and II (PER2 levels going down)), plotted on PER2::LUC bioluminescence traces of control cells (dark colours). A representative trace of CHX-treated cells at time point I is shown in light colours. Right image shows quantifications, p-values were calculated by unpaired t test.


( A ) Period analyses of WT and CKO PER2 : : LUC cells in the presence or absence of CK1δ / ε inhibitor PF670462 ( 0 . 3 µM ; PF ) .

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

(A) Period analyses of WT and CKO PER2::LUC cells in the presence or absence of CK1δ/ε inhibitor PF670462 (0.3 µM; PF).


Period analyses of WT and CKO PER2 : : LUC cells in the presence or absence of GSK3 inhibitor CHIR99021 ( 5 µM ; CHIR ) .

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Period analyses of WT and CKO PER2::LUC cells in the presence or absence of GSK3 inhibitor CHIR99021 (5 µM; CHIR).


Period analyses of WT and CKO PER2 : : LUC cells in the presence or absence of CRY inhibitor KL001 ( 1 µM ) .

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Period analyses of WT and CKO PER2::LUC cells in the presence or absence of CRY inhibitor KL001 (1 µM).


( B ) Western blot revealed with anti - I - Abβ JV2 antibody . WCL : whole cell lysate of MutuDC + pRBC ; S0 : supernatant following incubation with protein - G beads ; S1 : 1st wash of protein - G beads ; IP MHC II : eluates from Y - 3P - immunoprecipitated MHC II , boiled or not . Representative of 2 independent experiments .

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(B) Western blot revealed with anti-I-Abβ JV2 antibody. WCL: whole cell lysate of MutuDC + pRBC ; S0: supernatant following incubation with protein-G beads ; S1: 1st wash of protein-G beads ; IP MHC II: eluates from Y-3P-immunoprecipitated MHC II, boiled or not. Representative of 2 independent experiments.


( C ) Venn diagram of unique peptides from P . berghei ( left ) and Mus musculus ( right ) identified in the experiments performed with PbK pRBC ( blue ) and PbA pRBC ( orange ) .

O O O O O O O O O O O O O O O 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) Venn diagram of unique peptides from P. berghei (left) and Mus musculus (right) identified in the experiments performed with PbK pRBC (blue) and PbA pRBC (orange).


( B ) Gating strategy to calculate the proportion of CD11a + CD49d + activated CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC , PbA pRBC , OVA peptide ( IR17 ) or ETRAMP10 . 2 peptide ( NL17 ) .

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

(B) Gating strategy to calculate the proportion of CD11a+ CD49d+ activated CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC, PbA pRBC, OVA peptide (IR17) or ETRAMP10.2 peptide (NL17).


( C ) Percentage of IFN / TNF - double producing cells among activated CD4 T cells for each peptide . Basal level with MutuDC alone was subtracted . C , D : Data show the mean + / - SEM . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon tests in comparison with OVA peptide . AMA1 , P = 0 . 0098 ; TIM14 , P = 0 . 078 ; ENO , P = 0 . 016 ; GAPDH . 1 , P = 0 . 002 ; MAHRP , P = 0 . 0039 ; EF1α , P = 0 . 0039 ; M1 , P = 0 . 016 ; MSP1 , P = 0 . 031 ; LDH , P = 0 . 062 ; ETRAMP , P = 0 . 001 ; ATPSYN , P = 0 . 25 ; END70 , P = 0 . 25 ; PDI , P = 0 . 16 ; GAPDH . 2 , P = 0 . 99 ; pRBC , P = 0 . 001 . N = 6 mice for ATPSYN , END70 , PDI , GAPDH . 2 , N = 11 mice for all other peptides , pooled from 3 replicates .

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

(C) Percentage of IFN/TNF-double producing cells among activated CD4 T cells for each peptide. Basal level with MutuDC alone was subtracted. C,D: Data show the mean +/- SEM. Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.0098 ; TIM14, P = 0.078 ; ENO, P = 0.016 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.0039 ; EF1α, P = 0.0039 ; M1, P = 0.016 ; MSP1, P = 0.031 ; LDH, P = 0.062 ; ETRAMP, P = 0.001 ; ATPSYN, P = 0.25 ; END70, P = 0.25 ; PDI, P = 0.16 ; GAPDH.2, P = 0.99 ; pRBC, P = 0.001. N = 6 mice for ATPSYN, END70, PDI, GAPDH.2, N = 11 mice for all other peptides, pooled from 3 replicates.


( D ) Hierarchy of immunodominance , depicted as the percentage of each peptide - specific response with respect to the total parasite - specific response obtained with PbA pRBC - loaded MutuDC . C , D : Data show the mean + / - SEM . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon tests in comparison with OVA peptide . AMA1 , P = 0 . 0098 ; TIM14 , P = 0 . 078 ; ENO , P = 0 . 016 ; GAPDH . 1 , P = 0 . 002 ; MAHRP , P = 0 . 0039 ; EF1α , P = 0 . 0039 ; M1 , P = 0 . 016 ; MSP1 , P = 0 . 031 ; LDH , P = 0 . 062 ; ETRAMP , P = 0 . 001 ; ATPSYN , P = 0 . 25 ; END70 , P = 0 . 25 ; PDI , P = 0 . 16 ; GAPDH . 2 , P = 0 . 99 ; pRBC , P = 0 . 001 . N = 6 mice for ATPSYN , END70 , PDI , GAPDH . 2 , N = 11 mice for all other peptides , pooled from 3 replicates .

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

(D) Hierarchy of immunodominance, depicted as the percentage of each peptide-specific response with respect to the total parasite-specific response obtained with PbA pRBC-loaded MutuDC. C,D: Data show the mean +/- SEM. Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.0098 ; TIM14, P = 0.078 ; ENO, P = 0.016 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.0039 ; EF1α, P = 0.0039 ; M1, P = 0.016 ; MSP1, P = 0.031 ; LDH, P = 0.062 ; ETRAMP, P = 0.001 ; ATPSYN, P = 0.25 ; END70, P = 0.25 ; PDI, P = 0.16 ; GAPDH.2, P = 0.99 ; pRBC, P = 0.001. N = 6 mice for ATPSYN, END70, PDI, GAPDH.2, N = 11 mice for all other peptides, pooled from 3 replicates.


( B ) IFN / TNF - double producing cells among activated CD4 T cells , after subtracting the basal level with MutuDC alone ( mean + / - SEM ) . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon tests in comparison with OVA peptide . AMA1 , P = 0 . 027 ; TIM14 , P = 0 . 84 ; ENO , P = 0 . 84 ; GAPDH . 1 , P = 0 . 002 ; MAHRP , P = 0 . 71 ; EF1α , P = 0 . 001 ; M1 , P = 0 . 032 ; MSP1 , P = 0 . 23 ; LDH , P = 0 . 016 ; ETRAMP , P = 0 . 064 ; ATPSYN , P = 0 . 77 ; END70 , P = 0 . 097 ; PDI , P = 0 . 0052 ; GAPDH . 2 , P = 0 . 19 ; pRBC , P = 0 . 001 . N = 15 mice pooled from 3 replicates .

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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 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 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 B-CONTROLLED_VAR 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

(B) IFN/TNF-double producing cells among activated CD4 T cells, after subtracting the basal level with MutuDC alone (mean +/- SEM). Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.027 ; TIM14, P = 0.84 ; ENO, P = 0.84 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.71; EF1α, P = 0.001 ; M1, P = 0.032 ; MSP1, P = 0.23 ; LDH, P = 0.016 ; ETRAMP, P = 0.064 ; ATPSYN, P = 0.77 ; END70, P = 0.097 ; PDI, P = 0.0052 ; GAPDH.2, P = 0.19 ; pRBC, P = 0.001. N = 15 mice pooled from 3 replicates.


( C ) Blood parasitemia ( mean + / - SEM ) monitored by flow cytometry after inoculation of 5x104 PbA . GFP spz in B6 mice , treated ( open squares ) or not ( black squares ) with CQ at day 6 and 7 pi , in order to avoid ECM without blunting infection . Day 6 , P = 0 . 29 ; Day 7 , P = 0 . 0013 by multiple unpaired t - tests .

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(C) Blood parasitemia (mean +/- SEM) monitored by flow cytometry after inoculation of 5x104 PbA.GFP spz in B6 mice, treated (open squares) or not (black squares) with CQ at day 6 and 7 pi, in order to avoid ECM without blunting infection. Day 6, P = 0.29 ; Day 7, P = 0.0013 by multiple unpaired t-tests.


( D ) IFN / TNF - double producing cells among activated CD4 T cells , at day 14 pi following CQ treatment ( mean + / - SEM ) . Basal level with MutuDC alone was subtracted . GAPDH . 1 , P = 0 . 99 ; EF1α , P = 0 . 062 ; ETRAMP , P = 0 . 031 ; pRBC , P = 0 . 031 by paired non - parametric Wilcoxon tests in comparison with OVA peptide .

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

(D) IFN/TNF-double producing cells among activated CD4 T cells, at day 14 pi following CQ treatment (mean +/- SEM). Basal level with MutuDC alone was subtracted. GAPDH.1, P = 0.99 ; EF1α, P = 0.062 ; ETRAMP, P = 0.031 ; pRBC, P = 0.031 by paired non-parametric Wilcoxon tests in comparison with OVA peptide.


Figure 4 . cDC1 are superior to other spleen antigen - presenting cells for MHC II presentation of PbA and non - PbA antigens ( A ) Gating strategy for FACS - sorting pDC ( Lin - CD11clo B220 + Ly6C + ) , cDC1 ( Lin - CD11c + CD8α + ) , cDC2 ( Lin - CD11c + CD8α - ) and a mix of macrophages moDC ( Lin - CD11c - CD11b + Ly6C + ) from spleens of mice infected for 6 days with PbA pRBC .

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Figure 4. cDC1 are superior to other spleen antigen-presenting cells for MHC II presentation of PbA and non-PbA antigens (A) Gating strategy for FACS-sorting pDC (Lin- CD11clo B220+ Ly6C+), cDC1 (Lin- CD11c+ CD8α+), cDC2 (Lin- CD11c+ CD8α-) and a mix of macrophages moDC (Lin- CD11c- CD11b+ Ly6C+) from spleens of mice infected for 6 days with PbA pRBC.


( B ) I - Ab presentation of ETRAMP10 . 2 peptide by the indicated APC , measured with BEZ hybridomas . PbA pRBC - loaded MutuDC were used as controls . Representative of 2 experiments with 4 infected mice pooled per replicate .

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(B) I-Ab presentation of ETRAMP10.2 peptide by the indicated APC, measured with BEZ hybridomas. PbA pRBC-loaded MutuDC were used as controls. Representative of 2 experiments with 4 infected mice pooled per replicate.


( C ) Fold - change surface expression of I - Ab in cDC2 over cDC1 in day 6 - infected mice ( mean + / - SEM of 5 experiments ) . Difference is not significant based on unpaired t - test with Welch ' s correction .

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

(C) Fold-change surface expression of I-Ab in cDC2 over cDC1 in day 6-infected mice (mean +/- SEM of 5 experiments). Difference is not significant based on unpaired t-test with Welch's correction.


( D , E , F ) I - Ab presentation of non - PbA antigens by cDC isolated from day 6 PbA - infected mice . ( D ) I - Ab - AS15 presentation after exogenous addition of AS15 peptide at the indicated concentrations , assessed with the BTg01Z hybridomas . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen .

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

(D,E,F) I-Ab presentation of non-PbA antigens by cDC isolated from day 6 PbA-infected mice. (D) I-Ab-AS15 presentation after exogenous addition of AS15 peptide at the indicated concentrations, assessed with the BTg01Z hybridomas.Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( D , E , F ) I - Ab presentation of non - PbA antigens by cDC isolated from day 6 PbA - infected mice . ( E ) I - Ab - AS15 presentation by DC incubated with heat - killed T . gondii at the indicated ratio , assessed with the BTg01Z hybridomas . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen .

O O O O O O 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 I-MEASURED_VAR I-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

(D,E,F) I-Ab presentation of non-PbA antigens by cDC isolated from day 6 PbA-infected mice. (E) I-Ab-AS15 presentation by DC incubated with heat-killed T. gondii at the indicated ratio, assessed with the BTg01Z hybridomas. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( D , E , F ) I - Ab presentation of non - PbA antigens by cDC isolated from day 6 PbA - infected mice . ( F ) I - Ab - IR17 presentation by DC incubated with OVA - expressing E . coli at the indicated ratio , assessed with the OTIIZ hybridomas . PbA pRBC - loaded MutuDC were used as controls . D , E , F : representative of 2 experiments with 4 infected mice pooled per replicate . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen .

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

(D,E,F) I-Ab presentation of non-PbA antigens by cDC isolated from day 6 PbA-infected mice. (F) I-Ab-IR17 presentation by DC incubated with OVA-expressing E. coli at the indicated ratio, assessed with the OTIIZ hybridomas. PbA pRBC-loaded MutuDC were used as controls. D,E,F: representative of 2 experiments with 4 infected mice pooled per replicate. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( G ) In vivo pRBC uptake by cDC1 ( XCR1 + CD172a - ) and cDC2 ( XCR1 - CD172a + ) analyzed at 6 days post - infection with PbA . GFP . Gates were positioned based on the auto - fluorescence of the cDC subset in day 6 PbA - infected mice . As GFP + events are rare , 107 events were acquired for each mouse . Symbols show the mean percentage + / - SEM ( N = 4 mice per group ) of GFP + cells in each DC subset . * , P = 0 . 0005 by unpaired t - test without assuming consistent SD .

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

(G) In vivo pRBC uptake by cDC1 (XCR1+ CD172a-) and cDC2 (XCR1- CD172a+) analyzed at 6 days post-infection with PbA.GFP. Gates were positioned based on the auto-fluorescence of the cDC subset in day 6 PbA-infected mice. As GFP+ events are rare, 107 events were acquired for each mouse. Symbols show the mean percentage +/- SEM (N = 4 mice per group) of GFP+ cells in each DC subset. *, P = 0.0005 by unpaired t-test without assuming consistent SD.


( H , I ) I - Ab presentation by cDC isolated from naïve mice . ( H ) I - Ab - NL17 presentation by DC incubated with PbA pRBC at a ratio of 5 : 1 , assessed with the BEZ hybridomas . H , I : representative of 2 independent experiments with 20 naïve mice pooled per experiment . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen .

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

(H,I) I-Ab presentation by cDC isolated from naïve mice. (H) I-Ab-NL17 presentation by DC incubated with PbA pRBC at a ratio of 5:1, assessed with the BEZ hybridomas. H,I: representative of 2 independent experiments with 20 naïve mice pooled per experiment. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( H , I ) I - Ab presentation by cDC isolated from naïve mice . ( I ) I - Ab - IR17 presentation by DC incubated with OVA - expressing E . coli at a ratio of 20 : 1 , assessed with the OTIIZ hybridomas . H , I : representative of 2 independent experiments with 20 naïve mice pooled per experiment . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen .

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

(H,I) I-Ab presentation by cDC isolated from naïve mice. (I) I-Ab-IR17 presentation by DC incubated with OVA-expressing E. coli at a ratio of 20:1, assessed with the OTIIZ hybridomas. H,I: representative of 2 independent experiments with 20 naïve mice pooled per experiment. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( J ) Fold - change surface expression of I - Ab in cDC2 over cDC1 in naïve mice ( mean + / - SEM of 4 experiments ) . Difference is not significant based on unpaired t - test with Welch ' s correction .

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

(J) Fold-change surface expression of I-Ab in cDC2 over cDC1 in naïve mice (mean +/- SEM of 4 experiments). Difference is not significant based on unpaired t-test with Welch's correction.


( B ) Gating strategy to assess efficiency of cDC1 depletion .

(B) Gating strategy to assess efficiency of cDC1 depletion.


( C ) Percentage ( mean + / - SEM ) of CD8α + out of Lin - B220 - CD11c + MHC IIhi cells . * , P = 0 . 0043 by Mann - Whitney test .

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(C) Percentage (mean +/- SEM) of CD8α+ out of Lin- B220- CD11c+ MHC IIhi cells. *, P = 0.0043 by Mann-Whitney test.


( D ) FACS plots showing the proportion of CD11a + CD49d + CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC , PbA pRBC or ETRAMP10 . 2 peptide .

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(D) FACS plots showing the proportion of CD11a+ CD49d+ CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC, PbA pRBC or ETRAMP10.2 peptide.


( E ) Proportion of CD11a + CD49d + CD4 T cells that co - produce IFN and TNF ( mean + / - SEM ) in response to MutuDC loaded with the indicated antigen , isolated from Karma mice treated ( open circles ) or not ( black circles ) with DT . Asterisks show significant differences between no DT and + DT group by multiple unpaired t - tests without assuming consistent SD . RBC , P = 0 . 18 ; GAPDH . 1 , P = 0 . 019 ; EF1a , 0 . 039 ; ETRAMP , P = 0 . 00054 ; OVA , P = 0 . 25 ; pRBC , P = 0 . 015 .

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(E) Proportion of CD11a+ CD49d+ CD4 T cells that co-produce IFN and TNF (mean +/- SEM) in response to MutuDC loaded with the indicated antigen, isolated from Karma mice treated (open circles) or not (black circles) with DT. Asterisks show significant differences between no DT and + DT group by multiple unpaired t-tests without assuming consistent SD. RBC, P = 0.18 ; GAPDH.1, P = 0.019 ; EF1a, 0.039 ; ETRAMP, P = 0.00054 ; OVA, P = 0.25 ; pRBC, P = 0.015.


( F ) Absolute numbers of IFN + TNF + CD11a + CD49d + CD4 + T cells responding to the indicated stimuli ( mean + / - SEM ) . GAPDH . 1 , P = 0 . 0047 ; EF1a , 0 . 00039 ; ETRAMP , P = 0 . 00022 ; pRBC , P = 0 . 00019 by multiple unpaired t - tests without assuming consistent SD .

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(F) Absolute numbers of IFN+ TNF+ CD11a+ CD49d+CD4+ T cells responding to the indicated stimuli (mean +/- SEM). GAPDH.1, P = 0.0047 ; EF1a, 0.00039 ; ETRAMP, P = 0.00022 ; pRBC, P = 0.00019 by multiple unpaired t-tests without assuming consistent SD.


( G ) Blood parasitemia ( mean + / - SEM ) at day 4 and 6 pi in Karma mice treated ( open circles ) or not ( black circles ) with DT . Day 4 , P = 0 . 022 ; Day 6 , P = 0 . 77 by multiple unpaired t - tests without assuming consistent SD . C , E , F , G : No DT group , N = 5 mice ; + DT group , N = 6 mice . Representative of 3 independent experiments .

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(G) Blood parasitemia (mean +/- SEM) at day 4 and 6 pi in Karma mice treated (open circles) or not (black circles) with DT. Day 4, P = 0.022 ; Day 6, P = 0.77 by multiple unpaired t-tests without assuming consistent SD. C,E,F,G: No DT group, N = 5 mice ; + DT group, N = 6 mice. Representative of 3 independent experiments.


( H ) Absolute numbers of CD4 + T cells producing IFN , TNF or IL - 10 , either individually or in combination , following restimulation with MutuDC + PbA pRBC ( mean + / - SEM ) . These cells are called cytokine + in the next panels . * , P = 0 . 0023 by unpaired t - test without assuming consistent SD .

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(H) Absolute numbers of CD4+ T cells producing IFN, TNF or IL-10, either individually or in combination, following restimulation with MutuDC+PbA pRBC (mean +/- SEM). These cells are called cytokine+ in the next panels. *, P = 0.0023 by unpaired t-test without assuming consistent SD.


( I ) Multifunctionality analysis ( IFN , TNF , IL - 10 ) of cytokine + T cells responding to MutuDC loaded with PbA pRBC .

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(I) Multifunctionality analysis (IFN, TNF, IL-10) of cytokine+ T cells responding to MutuDC loaded with PbA pRBC.


( J ) Proportion of IL - 10 + IFN - TNF - among cytokine + T cells , in response to MutuDC loaded with the indicated antigen ( mean + / - SEM ) . GAPDH . 1 , P = 0 . 32 ; EF1a , 0 . 16 ; ETRAMP , P = 8 . 5x10 - 5 ; OVA , P = 0 . 11 ; pRBC , P = 0 . 0015 by multiple unpaired t - tests without assuming consistent SD . H , I , J : N = 3 mice per group .

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(J) Proportion of IL-10+ IFN- TNF- among cytokine+ T cells, in response to MutuDC loaded with the indicated antigen (mean +/- SEM). GAPDH.1, P = 0.32 ; EF1a, 0.16 ; ETRAMP, P = 8.5x10-5 ; OVA, P = 0.11 ; pRBC, P = 0.0015 by multiple unpaired t-tests without assuming consistent SD. H,I,J: N = 3 mice per group.


D . The morphology of oocytes and embryos from female patients with MOS variants . The day of oocyte retrieval was defined as day 0 . Polar bodies were indicated by black arrows . Scale bar = 20 µm .

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D. The morphology of oocytes and embryos from female patients with MOS variants. The day of oocyte retrieval was defined as day 0. Polar bodies were indicated by black arrows. Scale bar=20 µm.


B . The immunofluorescence images show the pERK1 / 2 ( red ) dynamics in immature ( GV , n = 5 ) , mature ( MII , n = 10 ) , and fertilized oocytes from unidentified control patients ( n = 6 ) , as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants . pERK1 / 2 activation was inhibited after U0126 treatment ( n = 6 ) . FITC - α - tubulin ( Green ) and DAPI ( blue ) were used for co - staining . Scale bar = 10 µm .

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B. The immunofluorescence images show the pERK1/2 (red) dynamics in immature (GV, n=5), mature (MII, n=10), and fertilized oocytes from unidentified control patients (n=6), as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants. pERK1/2 activation was inhibited after U0126 treatment (n=6). FITC-α-tubulin (Green) and DAPI (blue) were used for co-staining. Scale bar=10 µm.


C . Western blot analysis of the MEK1 / 2 and ERK1 / 2 activation after transfection of FLAG - tagged wild - type MOS and identified MOS variants in HEK 293 cells .

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C. Western blot analysis of the MEK1/2 and ERK1/2 activation after transfection of FLAG-tagged wild-type MOS and identified MOS variants in HEK 293 cells.


F . Immunofluorescence of FLAG ( green ) and pERK1 / 2 ( grey ) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs . The signal of mCherry was directly captured after fixation , with DAPI staining for DNA visualization ( blue ) . ( n = over 30 oocytes in each group ) . Scale bar = 10 µm .

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F. Immunofluorescence of FLAG (green) and pERK1/2 (grey) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs. The signal of mCherry was directly captured after fixation, with DAPI staining for DNA visualization (blue). (n=over 30 oocytes in each group). Scale bar=10 µm.


A . Immunofluorescence of FLAG ( red ) and F - actin ( gray ) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild - type human MOS or MOS variants mRNAs and culture in medium with 2 . 5 µM milrinone for 24 h , followed by release to maturation ( n = 15 - 20 oocytes each group ) . Scale bar = 10 µm .

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A. Immunofluorescence of FLAG (red) and F-actin (gray) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild-type human MOS or MOS variants mRNAs and culture in medium with 2.5 µM milrinone for 24 h, followed by release to maturation (n=15-20 oocytes each group). Scale bar= 10 µm.


C . The immunofluorescence of F - actin ( red ) and TPX2 ( green ) in WT and Erk1 / 2oo - / - oocytes , using DAPI ( blue ) co - staining ( n = 15 - 20 oocytes for each group ) . The zoomed images of F - actin are displayed in gray . Scale bar = 10 µm . D . The box plot summarizing the relative intensities of F - actin between control and ERK1 / 2oo - / - oocytes ( n = 7 - 10 oocytes for each group ) .

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C. The immunofluorescence of F-actin (red) and TPX2 (green) in WT and Erk1/2oo-/- oocytes, using DAPI (blue) co-staining (n=15-20 oocytes for each group). The zoomed images of F-actin are displayed in gray. Scale bar=10 µm. D. The box plot summarizing the relative intensities of F-actin between control and ERK1/2oo-/- oocytes (n=7-10 oocytes for each group).


E . Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1 / 2oo - / - mice . The MII oocyte , zygote , 2 - cell and blastocyst embryos were harvested in vivo at 14 , 24 , 48 , and 96 h post - hCG , respectively ( n = 3 mice for each timepoints in two groups ) . Scale bar = 100 µm . F , G . Bar graphs shows the blastocyst percentage ( F ) and fragment percentage ( G ) in wild - type ( n = 51 ) and maternal ERK1 / 2 deletion embryos ( n = 63 ) .

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E. Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1/2oo-/- mice. The MII oocyte, zygote, 2-cell and blastocyst embryos were harvested in vivo at 14, 24, 48, and 96 h post-hCG, respectively (n=3 mice for each timepoints in two groups). Scale bar=100 µm. F, G. Bar graphs shows the blastocyst percentage (F) and fragment percentage (G) in wild-type (n=51) and maternal ERK1/2 deletion embryos (n=63).


H . The bar graphs show the expression levels of selected MOS - regulated decay genes ( n = 2 or 3 biological replicates ) .

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H. The bar graphs show the expression levels of selected MOS-regulated decay genes (n=2 or 3 biological replicates).


H . Heatmap showing the expression level of selected maternal genes , mitochondrial translation genes and mitochondrial respiratory chain genes regulated by MOS mutation or ERK1 / 2 inactivation .

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H. Heatmap showing the expression level of selected maternal genes, mitochondrial translation genes and mitochondrial respiratory chain genes regulated by MOS mutation or ERK1/2 inactivation.


A . The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO ( control , Ctrl , n = 11 ) or 20 uM U0126 treatment ( n = 12 ) . B . The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO ( control , Ctrl , n = 19 ) or 20 uM U0126 ( n = 17 ) .

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A. The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO (control, Ctrl, n=11) or 20 uM U0126 treatment (n=12). B. The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO (control, Ctrl, n=19) or 20 uM U0126 (n=17). C


C . The representative images showing the JC - 1 monomers ( green ) and aggregates ( red ) of mouse oocytes after treatment with DMSO ( control , Ctrl , n = 34 ) or U0126 ( n = 40 ) in in vitro culture medium for 16 h . Scale bar = 100 µm . D . The bar graph of ratio of intensities of JC - 1 aggregates to those of monomers in ( C ) . The intensities of JC - 1 were quantified by Image J software . The oocytes number used was labeled .

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C. The representative images showing the JC-1 monomers (green) and aggregates (red) of mouse oocytes after treatment with DMSO (control, Ctrl, n=34) or U0126 (n=40) in in vitro culture medium for 16 h. Scale bar=100 µm. D. The bar graph of ratio of intensities of JC-1 aggregates to those of monomers in (C). The intensities of JC-1 were quantified by Image J software. The oocytes number used was labeled. E


E . Representative images displaying JC - 1 monomers ( green ) , aggregates ( red ) and bright filed images of GV and MII oocytes from wild - type ( WT ) and Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big and multiple polar bodies . Scale bar = 100 µm .

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E. Representative images displaying JC-1 monomers (green), aggregates (red) and bright filed images of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies. Scale bar=100 µm.


F . Confocal images of JC - 1 monomers ( green ) and aggregates ( red ) of GV and MII oocytes from wild - type ( WT ) and Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big and multiple polar bodies , indicated by red arrows . Hoechst 33342 ( blue ) was co - stained to represent nucleus or chromosome . Scale bar = 10 µm .

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F. Confocal images of JC-1 monomers (green) and aggregates (red) of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies, indicated by red arrows. Hoechst 33342 (blue) was co-stained to represent nucleus or chromosome. Scale bar=10 µm.


( A Representative confocal images of HeLa and TRIM16KO cells treated with ( A ) MG132 ( 20 µM , 2h ) , scale bar : 7 . 5 µm . and the samples were processed for IF analysis with Ub and p62 antibody . ( B Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with ( B ) MG132 ( 20 µM , 2h ) , where IF analysis was conducted with Ub and p62 antibodies . ( C The graph shows the the percentage of cells with Ub - p62 co - localized dots . Data from ≥ 10 fields ( 40X ) , n = 3 , Mean ± SD , * * p < 0 . 0003 , * p < 0 . 002 ( Student ' s unpaired t - test ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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(A Representative confocal images of HeLa and TRIM16KO cells treated with (A) MG132 (20 µM, 2h), scale bar: 7.5 µm. and the samples were processed for IF analysis with Ub and p62 antibody. (B Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (B) MG132 (20 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. (C The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.


D ) Representative confocal images of HeLa and TRIM16KO cells treated with ( D ) H2O2 ( 200 µM , 2h ) and the samples were processed for IF analysis with Ub and p62 antibody . E ) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with ( E ) H2O2 , ( 200 µM , 2h ) , where IF analysis was conducted with Ub and p62 antibodies . F ) The graph shows the the percentage of cells with Ub - p62 co - localized dots . Data from ≥ 10 fields ( 40X ) , n = 3 , Mean ± SD , * * p < 0 . 0003 , * p < 0 . 002 ( Student ' s unpaired t - test ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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D) Representative confocal images of HeLa and TRIM16KO cells treated with (D) H2O2 (200 µM, 2h) and the samples were processed for IF analysis with Ub and p62 antibody. E) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (E) H2O2, (200 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. F) The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.


( G ) Western blot ( WB ) analysis of detergent - soluble and - insoluble fractions of HeLa and TRIM16KO cells treated with MG132 ( 50 µM , 1h ) and probed with indicated antibodies .

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(G) Western blot (WB) analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells treated with MG132 (50 µM, 1h) and probed with indicated antibodies.


( H , I ) Densitometric analysis ( using ImageJ ) of WB normalized with α - Tubulin or Lamin - B1 . Data information : Unless otherwise stated , scale bar : 10 µm .

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(H, I) Densitometric analysis (using ImageJ) of WB normalized with α-Tubulin or Lamin-B1. Data information: Unless otherwise stated, scale bar: 10 µm.


( J ) Representative confocal images of HeLa and TRIM16KO cells treated with MG132 ( 20 µM , 2h ) and processed for IF analysis with Ub and LC3B antibody .

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(J) Representative confocal images of HeLa and TRIM16KO cells treated with MG132 (20 µM, 2h) and processed for IF analysis with Ub and LC3B antibody.


( K , L ) WB analysis of LC3B levels in lysates of HeLa and TRIM16KO cells treated with ( K ) Bafilomycin A1 ( 300 nM , 3h ) alone or ( L ) with MG132 ( 10 µM , 4h ) as indicated . L . E , Low exposure ; H . E , high exposure .

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(K, L) WB analysis of LC3B levels in lysates of HeLa and TRIM16KO cells treated with (K) Bafilomycin A1 (300 nM, 3h) alone or (L) with MG132 (10 µM, 4h) as indicated. L.E, Low exposure; H.E, high exposure.


( M ) Representative confocal images HeLa and TRIM16KO cells treated with H2O2 ( 200 µM , 2h ) and processed for IF analysis with Proteostat dye , Ub , and p62 antibody . To show the cellular details , the brightness of TRIM16KO cells images was increased more than control

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(M) Representative confocal images HeLa and TRIM16KO cells treated with H2O2 (200 µM, 2h) and processed for IF analysis with Proteostat dye, Ub, and p62 antibody. To show the cellular details, the brightness of TRIM16KO cells images was increased more than control cells


( A ) Western blot analysis of HeLa and TRIM16KO cell lysates probed with indicated antibodies .

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(A) Western blot analysis of HeLa and TRIM16KO cell lysates probed with indicated antibodies.


( B ) Western blot analysis of lysates from HeLa and TRIM16KO cells treated with or without MG132 and probed with indicated antibodies .

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(B) Western blot analysis of lysates from HeLa and TRIM16KO cells treated with or without MG132 and probed with indicated antibodies.


( C ) Left panel , western blotting with HeLa and TRIM16KO cell lysates probed with indicated antibodies . Right panel , densitometric analysis ( mean ± SD ) of protein band intensity relative to actin , n = 3 , * * * p < 0 . 0005 ( Student ' s unpaired t - test ) .

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(C) Left panel, western blotting with HeLa and TRIM16KO cell lysates probed with indicated antibodies. Right panel, densitometric analysis (mean ±SD) of protein band intensity relative to actin, n=3, ***p < 0.0005 (Student's unpaired t-test).


D - G ( D , F ) HEK293T cells were either transiently transfected with an empty vector or a Flag - tagged - TRIM16 expressing vector and immunoblotted with Flag or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . ( E , G ) Densitometric analysis ( mean ± SD ) of protein band intensity relative to actin , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) .

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D-G (D, F) HEK293T cells were either transiently transfected with an empty vector or a Flag-tagged-TRIM16 expressing vector and immunoblotted with Flag or indicated antibodies. L.E, Low exposure; H.E, High Exposure. (E, G) Densitometric analysis (mean ±SD) of protein band intensity relative to actin, n=3, *p < 0.05 (Student's unpaired t-test).


( H ) WB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide ( 100 µM ) for the indicated period and probed with different antibodies as indicated . The blots of control and TRIM16KO cells are exposed for the equal duration and developed together on the same X - ray film . ( I ) Quantification of NRF2 , p62 , and KEAP1 band intensities relative to actin . n = 3 , * * p < 0 . 005 , * p < 0 . 05 ( Student ' s unpaired t - test ) .

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(H) WB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide (100 µM) for the indicated period and probed with different antibodies as indicated. The blots of control and TRIM16KO cells are exposed for the equal duration and developed together on the same X-ray film. (I) Quantification of NRF2, p62, and KEAP1 band intensities relative to actin. n=3, **p < 0.005, *p < 0.05 (Student's unpaired t-test).


( J ) Immunoprecipitation ( IP ) analysis of interaction between endogenous TRIM16 and endogenous p62 in HeLa cell lysates in absence and presence of MG132 ( 10 µM , 4h ) .

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(J) Immunoprecipitation (IP) analysis of interaction between endogenous TRIM16 and endogenous p62 in HeLa cell lysates in absence and presence of MG132 (10 µM, 4h).


IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in ( L ) absence of MG132 ( 10 µM , 4h ) .

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IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (L) absence of MG132 (10 µM, 4h).


IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in ( M ) presence of MG132 ( 10 µM , 4h ) .

IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (M) presence of MG132 (10 µM, 4h).


( N ) IP analysis of the interaction between endogenous TRIM16 and endogenous NRF2 in HeLa cell lysates . Mr , Molecular Weight Marker .

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(N) IP analysis of the interaction between endogenous TRIM16 and endogenous NRF2 in HeLa cell lysates. Mr, Molecular Weight Marker.


( O ) Co - IP analysis to map the interaction between NRF2 and TRIM16 domains in HEK293T cell lysates .

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(O) Co-IP analysis to map the interaction between NRF2 and TRIM16 domains in HEK293T cell lysates.


( P ) IP analysis of the interaction between endogenous TRIM16 and endogenous KEAP1 in HeLa cell lysates .

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(P) IP analysis of the interaction between endogenous TRIM16 and endogenous KEAP1 in HeLa cell lysates.


( A , B ) Co - IP analysis of the interaction between NRF2 and KEAP1 in absence and presence of TRIM16 .

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(A, B) Co-IP analysis of the interaction between NRF2 and KEAP1 in absence and presence of TRIM16.


( C ) Co - IP analysis of the interaction between NRF2 and p62 in absence and presence of TRIM16 .

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(C) Co-IP analysis of the interaction between NRF2 and p62 in absence and presence of TRIM16.


( D ) Co - IP analysis of the interaction between KEAP1 and p62 in absence and presence of TRIM16 .

(D) Co-IP analysis of the interaction between KEAP1 and p62 in absence and presence of TRIM16.


( A - C ) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Co - IP assays using transiently transfected plasmid constructs as indicated . Two different variants of ubiquitin protein are used , one that can only be ubiquitinated at Lysine 48 residue ( HA - K48 - UB ) and other that can be only ubiquitinated at Lysine 63 residue ( HA - K63 - UB ) . All other lysine residues are mutated . L . E , Low exposure ; H . E , High Exposure .

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(A-C) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Co-IP assays using transiently transfected plasmid constructs as indicated. Two different variants of ubiquitin protein are used, one that can only be ubiquitinated at Lysine 48 residue (HA-K48-UB) and other that can be only ubiquitinated at Lysine 63 residue (HA-K63-UB). All other lysine residues are mutated. L.E, Low exposure; H.E, High Exposure.


Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni - NTA pull - down assays using transiently transfected plasmid constructs as indicated . His - tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays .

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Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni-NTA pull-down assays using transiently transfected plasmid constructs as indicated. His-tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays.


( F ) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 deletion variants by Co - IP assays using transiently transfected plasmid constructs as indicated .

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(F) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 deletion variants by Co-IP assays using transiently transfected plasmid constructs as indicated.


( G ) Western blot analysis of lysates from HeLa , TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 ( 20 µM , 2h ) and the blot is probed with indicated antibodies .

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(G) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.


( H ) Western blot analysis of lysates from HeLa , TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 ( 20 µM , 2h ) and the blot is probed with indicated antibodies .

(H) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.


( A ) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with increasing concentrations of H2O2 as specified and probed with antibodies as indicated .

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(A) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with increasing concentrations of H2O2 as specified and probed with antibodies as indicated.


( B ) WB analysis of HeLa and TRIM16KO lysates of cells treated with 400 µM of H2O2 for different durations as indicated and probed with antibodies as shown . The red arrows indicate the time point where the cells were retreated with 400 µM of H2O2 . L . E , Low exposure ; H . E , High Exposure .

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(B) WB analysis of HeLa and TRIM16KO lysates of cells treated with 400 µM of H2O2 for different durations as indicated and probed with antibodies as shown. The red arrows indicate the time point where the cells were retreated with 400 µM of H2O2. L.E, Low exposure; H.E, High Exposure.


( C ) Densitometric analysis of NRF2 band intensity normalized with actin .

(C) Densitometric analysis of NRF2 band intensity normalized with actin.


( D ) WB analysis of HeLa and TRIM16KO lysates of cells treated with 2 . 5 µM ( 2 h ) of As2O3 .

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(D) WB analysis of HeLa and TRIM16KO lysates of cells treated with 2.5 µM (2 h) of As2O3.


( E - G ) RNA isolated from HeLa and TRIM16KO cells , untreated or treated with MG132 ( 20 µM , 2h ) were subjected to qRT - PCR with primers of genes as indicated . The fold induction in MG132 treated samples is calculated relative to untreated samples . Mean ± SD , n = 3 , * p < 0 . 05 , * * p < 0 . 005 ( Student ' s unpaired t - test ) .

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(E-G) RNA isolated from HeLa and TRIM16KO cells, untreated or treated with MG132 (20 µM, 2h) were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05, **p < 0.005 (Student's unpaired t-test).


RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc - TRIM16 subjected to qRT - PCR with primers of genes as indicated . Mean ± SD , n = 3 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Student ' s unpaired t - test ) .

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RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc-TRIM16 subjected to qRT-PCR with primers of genes as indicated. Mean ±SD, n=3, **p < 0.005, ***p < 0.0005 (Student's unpaired t-test).


( K ) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with h increasing concentrations of H2O2 as specified and probed with antibodies as indicated .

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(K) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with h increasing concentrations of H2O2 as specified and probed with antibodies as indicated.


( L ) Densitometric analysis of Ub smear intensity normalized with actin . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) .

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(L) Densitometric analysis of Ub smear intensity normalized with actin. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).


( M ) Representative confocal images of control , Ubb and Ube2n siRNA transfected cells treated with H2O2 ( 200 µM , 2h ) and IF was performed with Ub and p62 antibodies . Scale bar : 10 µm .

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(M) Representative confocal images of control, Ubb and Ube2n siRNA transfected cells treated with H2O2 (200 µM, 2h) and IF was performed with Ub and p62 antibodies. Scale bar: 10 µm.


( N ) The graph shows the percentage of cells with protein aggregates . Data from ≥ 10 microscopic fields for each condition ( 40X ) , n = 2 , Mean ± SD .

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(N) The graph shows the percentage of cells with protein aggregates. Data from ≥ 10 microscopic fields for each condition (40X), n=2, Mean ±SD.


( O ) Western blot analysis of Ubb and Ube2n siRNA transfected cell lysates with antibodies as indicated .

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(O) Western blot analysis of Ubb and Ube2n siRNA transfected cell lysates with antibodies as indicated.


( A ) Control or NRF2 siRNA transfected HeLa cells lysates were subjected to western blotting with indicated antibodies .

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(A) Control or NRF2 siRNA transfected HeLa cells lysates were subjected to western blotting with indicated antibodies.


( B , C ) RNA isolated from untreated or MG132 - treated ( 20 µM , 2h ) control or NRF2 siRNA transfected cells were subjected to qRT - PCR with primers of genes as indicated . The fold induction in MG132 treated samples is calculated relative to untreated samples . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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(B, C) RNA isolated from untreated or MG132-treated (20 µM, 2h) control or NRF2 siRNA transfected cells were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.


WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells , untreated or treated with MG132 ( 20 µM , 2h ) as indicated and probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure .

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WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with MG132 (20 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.


WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells , untreated or treated with H2O2 ( 200 µM , 2h ) as indicated and probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure .

WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with H2O2 (200 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.


Left panels : Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with ( F ) MG132 ( 20 µM , 2h ) , where IF was conducted with Ub and p62 antibodies . Right panels : fluorescence intensity line tracing corresponding to a white line in zoom panel .

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Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (F) MG132 (20 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.


Left panels : Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with ( G ) H2O2 ( 200 µM , 2h ) , where IF was conducted with Ub and p62 antibodies . Right panels : fluorescence intensity line tracing corresponding to a white line in zoom panel .

Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (G) H2O2 (200 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.


( H ) Hela cells , TRIM16KO cells , and TRIM16KO cells complemented with Myc - NRF2 or GFP - NRF2 were treated with MG132 ( 20 µM , 2h ) and subjected to WB analysis with Myc , GFP and indicated antibodies .

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(H) Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 or GFP-NRF2 were treated with MG132 (20 µM, 2h) and subjected to WB analysis with Myc, GFP and indicated antibodies.


( I ) Representative confocal images of Hela cells , TRIM16KO cells , and TRIM16KO cells complemented with Myc - NRF2 which were treated with MG132 ( 20 µM , 2h ) and subjected to IF with Ub and p62 antibodies .

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(I) Representative confocal images of Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 which were treated with MG132 (20 µM, 2h) and subjected to IF with Ub and p62 antibodies.


( J ) Graph represent % of cells with Ub - p62 dots , data from ≥ 10 microscopic fields ( 40X ) , n = 3 , mean ± SD , * p < 0 . 005 ( one - way ANOVA test ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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(J) Graph represent % of cells with Ub-p62 dots, data from ≥ 10 microscopic fields (40X), n=3, mean ±SD, *p < 0.005 (one-way ANOVA test). Data information: Unless otherwise stated, scale bar: 10 µm.


WB analysis of detergent - soluble and - insoluble fractions of HeLa and TRIM16KO cells expressing either ( A ) Flag - p62 probed with Flag or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric analysis of WB ' s normalized with α - Tubulin or Lamin - B1 .

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WB analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells expressing either (A) Flag-p62 probed with Flag or indicated antibodies. L.E, Low exposure; H.E, High Exposure. Densitometric analysis of WB's normalized with α-Tubulin or Lamin-B1.


WB analysis of detergent - soluble and - insoluble fractions of HeLa and TRIM16KO cells expressing either ( C ) GFP - polyQ74 probed with or GFP or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric analysis of WB ' s normalized with α - Tubulin or Lamin - B1 .

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WB analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells expressing either (C) GFP-polyQ74 probed with or GFP or indicated antibodies. L.E, Low exposure; H.E, High Exposure. Densitometric analysis of WB's normalized with α-Tubulin or Lamin-B1.


( E ) Confocal images of GFP - TRIM16 expressing HeLa cells , untreated or treated with MG132 ( 10 µM , 4h ) and / or Bafilomycin A1 ( 300 nM , 3h ) and IF analysis is performed with antibodies as indicated . Right panel graphs : fluorescence intensity line tracing corresponding to a white line in zoom panel . Scale bar : 10 µm . ( F ) The graph shows the percentage of cells with TRIM16 - Ub or TRIM16 - Ub - p62 co - localized dots / aggregates . Data from ≥ 10 microscopic fields , n = 2 , mean ± SD .

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(E) Confocal images of GFP-TRIM16 expressing HeLa cells, untreated or treated with MG132 (10 µM, 4h) and/or Bafilomycin A1 (300 nM, 3h) and IF analysis is performed with antibodies as indicated. Right panel graphs: fluorescence intensity line tracing corresponding to a white line in zoom panel. Scale bar: 10 µm. (F) The graph shows the percentage of cells with TRIM16-Ub or TRIM16-Ub-p62 co-localized dots/aggregates. Data from ≥ 10 microscopic fields, n=2, mean ±SD.


( G ) Representative confocal images of GFP - TRIM16 expressing HeLa cells , untreated or treated with MG132 ( 10 µM , 4h ) and / or Bafilomycin A1 ( 300 nM , 3h ) and IF analysis is performed with antibodies as indicated . Right panel graphs : fluorescence intensity line tracing corresponding to a white line in zoom panel . Scale bar : 10 µm .

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(G) Representative confocal images of GFP-TRIM16 expressing HeLa cells, untreated or treated with MG132 (10 µM, 4h) and/or Bafilomycin A1 (300 nM, 3h) and IF analysis is performed with antibodies as indicated. Right panel graphs: fluorescence intensity line tracing corresponding to a white line in zoom panel. Scale bar: 10 µm.


( H ) Co - IP analysis of the interaction between two differently tagged TRIM16 variants .

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(H) Co-IP analysis of the interaction between two differently tagged TRIM16 variants.


( I ) WB analysis of detergent - soluble and - insoluble DSS - cross - linked fractions of HeLa and TRIM16KO cells , untreated or treated with MG132 ( 10 µM , 4h ) and probed with indicated antibodies .

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(I) WB analysis of detergent-soluble and -insoluble DSS-cross-linked fractions of HeLa and TRIM16KO cells, untreated or treated with MG132 (10 µM, 4h) and probed with indicated antibodies.


( J ) Co - IP analysis of the interaction between Flag - tagged TRIM16 and Myc - tagged TRIM16 full length and its domain deletion proteins .

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(J) Co-IP analysis of the interaction between Flag-tagged TRIM16 and Myc-tagged TRIM16 full length and its domain deletion proteins.


( A ) MTT assays performed at different time points with HeLa and TRIM16KO cells , untreated or treated with H2O2 ( 400 µM , 2h ) . Data , mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) .

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(A) MTT assays performed at different time points with HeLa and TRIM16KO cells, untreated or treated with H2O2 (400 µM, 2h). Data, mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).


( B ) Images of HeLa and TRIM16KO cells , untreated or treated with As2O3 ( 2 . 5 µM , 4h ) . Scale bar : 400 µm .

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(B) Images of HeLa and TRIM16KO cells, untreated or treated with As2O3 (2.5 µM, 4h). Scale bar: 400 µm.


Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( C ) As2O3 ( 2 . 5 µM , 4h )

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Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (C) As2O3 (2.5 µM, 4h)


Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( D ) H2O2 ( 400 µM , 2h )

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Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (D) H2O2 (400 µM, 2h)


Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( E ) puromycin ( 5 µg / ml , 6h )

Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (E) puromycin (5 µg/ml, 6h)


Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( F ) MG132 ( 20 µM , 8h ) .

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Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (F) MG132 (20 µM, 8h).


( G ) Immunoblot blot analysis of HeLa and TRIM16KO lysates of cells treated with 5 µM of As2O3 for different durations as indicated and probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure . Arrowheads indicate the cleaved form of PARP - 1 and Casapse - 3 .

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(G) Immunoblot blot analysis of HeLa and TRIM16KO lysates of cells treated with 5 µM of As2O3 for different durations as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure. Arrowheads indicate the cleaved form of PARP-1 and Casapse-3.


( A , B ) Clonogenic assays were performed with HeLa and TRIM16KO cells , untreated or treated with As2O3 or MG132 .

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(A, B) Clonogenic assays were performed with HeLa and TRIM16KO cells, untreated or treated with As2O3 or MG132.


( C ) Tumor volumes of HeLa and TRIM16KO tumors at indicated time points . As2O3 is injected when tumor volume of all groups was > 100mm3 ( day 0 in the graph ) . Tumor volumes were measured on an interval as indicated . Mean ± SE , n = 6 ( each group ) , * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( ANOVA ) . ( D ) Graph shows the % tumor regression ( Tumor volume on day 1 of As2O3 treatment / Tumor volume on day of sacrifice X 100 ) . Mean ± SE , n = 6 ( each group ) , * * p < 0 . 005 ( Student ' s unpaired t - test ) .

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(C) Tumor volumes of HeLa and TRIM16KO tumors at indicated time points. As2O3 is injected when tumor volume of all groups was >100mm3 (day 0 in the graph). Tumor volumes were measured on an interval as indicated. Mean ±SE, n=6 (each group), *p < 0.05, **p < 0.005, ***p < 0.0005 (ANOVA). (D) Graph shows the % tumor regression (Tumor volume on day 1 of As2O3 treatment / Tumor volume on day of sacrifice X 100). Mean ±SE, n=6 (each group), **p < 0.005 (Student's unpaired t-test).


( E ) Representative images of HeLa and TRIM16KO tumors formed in the nude mice in the absence and presence of As2O3 . ( F ) Representative pictures of dissected tumors .

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(E) Representative images of HeLa and TRIM16KO tumors formed in the nude mice in the absence and presence of As2O3. (F) Representative pictures of dissected tumors.


( G ) Immunohistochemistry analysis performed with KI - 67 cell proliferation marker and hematoxylin nuclear stain . The graph represents the % of cells with KI - 67 staining . > 400 cells were counted for this analysis from three different sections from different animals , mean ± SD , * p < 0 . 05 ( Student ' s unpaired t - test ) . Scale bar : 50 µm .

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(G) Immunohistochemistry analysis performed with KI-67 cell proliferation marker and hematoxylin nuclear stain. The graph represents the % of cells with KI-67 staining. >400 cells were counted for this analysis from three different sections from different animals, mean ±SD, *p < 0.05 (Student's unpaired t-test). Scale bar: 50 µm.


( H ) WB analysis of tumor tissue lysates from two different mice with antibodies as indicated . L . E , Low exposure ; H . E , High Exposure .

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(H) WB analysis of tumor tissue lysates from two different mice with antibodies as indicated. L.E, Low exposure; H.E, High Exposure.