E Endogenous LAPTM4B is targeted to both early late endosomes . MDA ‐ MB ‐ 231 were fixed costained for endogenous LAPTM4B ( red ) EEA1 or LAMP1 ( green ) . Boxes are selected regions for magnified view . Note : non ‐ specific by LAPTM4B anti ‐ sera . Scale bar : 10 μm . F of LAPTM4B colocalization with EEA1 LAMP1 ( mean + SD ; n ≥ 4 ) .

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E Endogenous LAPTM4B is targeted to both early and late endosomes. MDA‐MB‐231 cells were fixed and costained for endogenous LAPTM4B (red) and EEA1 or LAMP1 (green). Boxes are selected regions for magnified view. Note: non‐specific nuclear staining by the LAPTM4B anti‐sera. Scale bar: 10 μm.F Quantification of LAPTM4B colocalization with EEA1 and LAMP1 (mean + SD; n ≥ 4).


H LAPTM4B partially colocalizes with PIPKIγi5 . MDA ‐ MB ‐ 231 expressing Myc ‐ tagged PIPKIγi5 were stained with LAPTM4B anti ‐ sera ( red ) , anti ‐ Myc ( green ) , DAPI . Box is selected region for magnified view . Scale bar : 10 μm .

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H LAPTM4B partially colocalizes with PIPKIγi5. MDA‐MB‐231 cells expressing Myc‐tagged PIPKIγi5 were stained with LAPTM4B anti‐sera (red), anti‐Myc (green), and DAPI. Box is selected region for magnified view. Scale bar: 10 μm.


I MDA ‐ MB ‐ 231 stably expressing Flag ‐ LAPTM4B were stained with LAPTM4B anti ‐ sera followed by silver ‐ enhanced immuno ‐ electron microscopy . early late MVEs were defined by number of intraluminal vesicles . N , nucleus ; M , mitochondria ; MVE , multi ‐ vesicular endosome ; PM , plasma . Scale bars : 2 μm ( left ) ; 200 nm ( middle right ) .

I MDA‐MB‐231 cells stably expressing Flag‐LAPTM4B were stained with LAPTM4B anti‐sera followed by silver‐enhanced immuno‐electron microscopy. The early and late MVEs were defined by the number of intraluminal vesicles. N, nucleus; M, mitochondria; MVE, multi‐vesicular endosome; and PM, plasma membrane. Scale bars: 2 μm (left); 200 nm (middle and right).


A MDA ‐ MB ‐ 231 transfected with control or LAPTM4B siRNA were starved stimulated with 100 ng / ml EGF for indicated time periods , followed by whole ‐ lysate harvest for immunoblotting of EGFR . B - D of of EGFR ( B ) , pEGFR ( Y1068 ) ( C ) , pAKT ( S473 ) ( D ) from in ( A ) ( mean ± SD ; n = 3 ) .

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A MDA‐MB‐231 cells transfected with control or LAPTM4B siRNA were starved and stimulated with 100 ng/ml EGF for indicated time periods, followed by whole‐cell lysate harvest for immunoblotting analysis of EGFR levels.B-D Quantification of the levels of EGFR (B), pEGFR (Y1068)(C), and pAKT (S473)(D) from the analysis in (A) (mean ± SD; n = 3).


E Control or LAPTM4B siRNA ‐ transfected MDA ‐ MB ‐ 231 were starved , pulsed with 25 ng / ml Alexa ‐ 555 ‐ EGF for 3 min , washed , chased for indicated time periods followed by fixation , DAPI , microscopy . Scale bar : 10 μm . F of relative amounts of Alexa ‐ 555 ‐ EGF internalized in indicated conditions ( mean + SD , n = 3 ) . G of Alexa ‐ 555 ‐ EGF degradation in control LAPTM4B knockdown in ( E ) ( mean ± SD , n = 3 ) .

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E Control or LAPTM4B siRNA‐transfected MDA‐MB‐231 cells were starved, pulsed with 25 ng/ml Alexa‐555‐EGF for 3 min, washed, and chased for indicated time periods followed by fixation, DAPI staining, and fluorescence microscopy. Scale bar: 10 μm.F Quantification of the relative amounts of Alexa‐555‐EGF internalized in the indicated conditions (mean + SD, n = 3).G Quantification of the Alexa‐555‐EGF degradation in control and LAPTM4B knockdown cells in (E) (mean ± SD, n = 3).


H Control or LAPTM4B ‐ overexpressing MDA ‐ MB ‐ 231 were starved then stimulated with 100 ng / ml EGF for 1 - 4 h . EGFR degradation signaling were analyzed by Western blot . Specific antibodies recognizing pEGFR ( Y1068 ) pAKT ( S473 ) were used . I - K for of EGFR ( I ) pEGFR ( J ) normalized to actin pAKT ( K ) normalized to AKT in control or LAPTM4B ‐ overexpressing ( mean ± SD , n = 3 ) .

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H Control or LAPTM4B‐overexpressing MDA‐MB‐231 cells were starved and then stimulated with 100 ng/ml EGF for 1-4 h. EGFR degradation and signaling were analyzed by Western blot. Specific antibodies recognizing pEGFR (Y1068) and pAKT (S473) were used.I-K Quantification for the levels of EGFR (I) and pEGFR (J) normalized to actin and pAKT (K) normalized to AKT in control or LAPTM4B‐overexpressing cells (mean ± SD, n = 3).


A - D Control or LAPTM4B siRNA ‐ transfected MDA ‐ MB ‐ 231 were starved , pretreated or not with chloroquine for 2 h , stimulated with 100 ng / ml EGF for 15 min , washed , chased for indicated time periods before fixation for costaining of EGFR ( red ) with EEA1 ( A , green ) or LAMP1 ( C , green ) . of average percentages of EGFR signals colocalized with EEA1 ( B ) LAMP1 ( D ) at indicated time points ; mean + SD ; n = 3 ; * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 , one ‐ t ‐ .

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A-D Control or LAPTM4B siRNA‐transfected MDA‐MB‐231 cells were starved, pretreated or not with chloroquine for 2 h, stimulated with 100 ng/ml EGF for 15 min, washed, and chased for indicated time periods before fixation for costaining of EGFR (red) with EEA1 (A, green) or LAMP1 (C, green). Quantification of the average percentages of EGFR signals colocalized with EEA1 (B) and LAMP1 (D) at indicated time points; mean + SD; n = 3; *P 0.05, **P 0.01, ***P 0.001, one‐tailed t‐test.


E MDA ‐ MB ‐ 231 were starved , stimulated with 100 ng / ml EGF for 15 or 120 min , fixed , costained for EGFR LAPTM4B , followed by microscopy . with higher lower LAPTM4B expression were marked with arrows arrowheads , respectively . F amounts of total EGFR in in ( E ) were plotted against LAPTM4B at 15 min 120 min , respectively . Note : for LAPTM4B , non ‐ specific was not included . Trend lines Pearson ' s correlation coefficients are shown . AU , arbitrary unit .

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E MDA‐MB‐231 cells were starved, stimulated with 100 ng/ml EGF for 15 or 120 min, fixed, and costained for EGFR and LAPTM4B, followed by fluorescence microscopy. Cells with higher and lower LAPTM4B expression were marked with arrows and arrowheads, respectively.F The amounts of total EGFR staining in individual cells in (E) were plotted against LAPTM4B levels at 15 min and 120 min, respectively. Note: for LAPTM4B quantification, the non‐specific nuclear staining was not included. Trend lines and Pearson's correlation coefficients are shown. AU, arbitrary unit.


MDA ‐ MB ‐ 231 expressing GFP ‐ Rab5Q79L were starved stimulated with 100 ng / ml Alexa ‐ 555 ‐ EGF for 90 min followed by fixation immunostaining for LAPTM4B ( blue ) . percentages of luminal Alexa ‐ 555 ‐ EGF in endosomes were plotted against endosomal LAPTM4B . Trend lines Pearson ' s correlation coefficients are shown . Data are representative for three independent experiments .

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MDA‐MB‐231 cells expressing GFP‐Rab5Q79L were starved and stimulated with 100 ng/ml Alexa‐555‐EGF for 90 min followed by fixation and immunostaining for LAPTM4B (blue). The percentages of luminal Alexa‐555‐EGF in individual endosomes were plotted against endosomal LAPTM4B levels. Trend lines and Pearson's correlation coefficients are shown. Data are representative for three independent experiments.


C Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 were starved overnight , surface EGFR was labeled with immuno ‐ gold on ice . were then stimulated with EGF for 1 h at 37 ° C fixed for EM study . Scale bar , 200 nm . See Materials Methods for details . D Relative amounts of immuno ‐ gold ‐ labeled EGFR in MVE lumen versus MVE limiting were quantified . Over 80 endosomes for each siRNA treatment from three independent experiments were used for ( mean + SD ; * * * P 0 . 001 , one ‐ t ‐ ) .

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C Control or LAPTM4B knockdown MDA‐MB‐231 cells were starved overnight, cell surface EGFR was labeled with immuno‐gold on ice. Cells were then stimulated with EGF for 1 h at 37°C and fixed for the EM study. Scale bar, 200 nm. See Materials and Methods for details.D Relative amounts of immuno‐gold‐labeled EGFR in the MVE lumen versus MVE limiting membrane were quantified. Over 80 endosomes for each siRNA treatment from three independent experiments were used for quantification (mean + SD; ***P 0.001, one‐tailed t‐test).


MDA ‐ MB ‐ 231 were transfected with GFP ‐ Rab5Q79L Flag ‐ LAPTM4B , starved , stimulated with Alexa ‐ 555 ‐ EGF for 90 min followed by intraluminal sorting . See Materials Methods for details . of EGF localization in LAPTM4B ‐ positive LAPTM4B ‐ negative endosomes in ( E ) ( mean + SD ; n = 3 ; * * * P 0 . 0007 , one ‐ t ‐ ) .

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MDA‐MB‐231 cells were transfected with GFP‐Rab5Q79L and Flag‐LAPTM4B, starved, and stimulated with Alexa‐555‐EGF for 90 min and followed by intraluminal sorting analysis. See Materials and Methods for details. Quantification of EGF localization in LAPTM4B‐positive and LAPTM4B‐negative endosomes in (E) (mean + SD; n = 3; ***P 0.0007, one‐tailed t‐test).


A Co ‐ immunoprecipitation ( co ‐ IP ) of full ‐ or N ‐ terminus ‐ deleted LAPTM4B with PIPKIγi5 in HEK293 .

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A Co‐immunoprecipitation (co‐IP) of full‐length or N‐terminus‐deleted LAPTM4B with PIPKIγi5 in HEK293 cells.


B GST ‐ tagged LAPTM4B N ‐ terminus ( 93 ) T7 ‐ tagged PIPKIγi5 C ‐ terminus ( 223 ) were purified from E . coli for in vitro GSTpull ‐ down .

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B GST‐tagged LAPTM4B N‐terminus (93 amino acids) and T7‐tagged PIPKIγi5 C‐terminus (223 amino acids) were purified from E. coli for in vitro GSTpull‐down assay.


C Co ‐ IP of full ‐ or N ‐ terminal deletion mutants of LAPTM4B with PIPKIγi5 in HEK293 . ∆ 25 : 1 - 25 deleted .

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C Co‐IP of full‐length or N‐terminal deletion mutants of LAPTM4B with PIPKIγi5 in HEK293 cells. ∆25: amino acids 1-25 deleted.


E Coomassie Brilliant Blue of purified wild ‐ type mutated LAPTM4B N ‐ termini .

E Coomassie Brilliant Blue staining of purified wild‐type and mutated LAPTM4B N‐termini.


F Purified LAPTM4B N ‐ termini from ( E ) were used in PIP strips . LPA , lysophosphatidic acid ; LPC , lysophosphocholine ; PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; S1P , sphingosine 1 ‐ phosphate ; PI3 , 4 , 5P3 , PtdIns ( 3 , 4 , 5 ) P3 , phosphatidylinositol ( 3 , 4 , 5 ) ‐ trisphosphate ; PA , phosphatidic acid ; PS , phosphatidylserine .

F Purified LAPTM4B N‐termini from (E) were used in PIP strips assay. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1‐phosphate; PI3,4,5P3, PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)‐trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine.


GST pull ‐ down of wild ‐ type or 6RQ mutant of LAPTM4B N ‐ termini with PIPKIγi5 C ‐ terminus .

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GST pull‐down assay of wild‐type or 6RQ mutant of LAPTM4B N‐termini with PIPKIγi5 C‐terminus.


H Co ‐ IP of wild ‐ type or PMB ‐ mutated LAPTM4B with wild ‐ type or kinase dead PIPKIγi5 in HEK293 .

H Co‐IP of wild‐type or PMB‐mutated LAPTM4B with wild‐type or kinase dead PIPKIγi5 in HEK293 cells.


I Top : GSTpull ‐ down of LAPTM4B N ‐ terminus PIPKIγi5 C ‐ terminus with increased of PtdIns ( 4 , 5 ) P2 addition . Bottom : of relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean ± SD ; n = 4 ) .

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 I-CONTROLLED_VAR I-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

I Top: GSTpull‐down assay of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with increased concentration of PtdIns(4,5)P2 addition. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean ± SD; n = 4).


J LAPTM4B N ‐ terminus binds multiple phosphoinositides including PtdIns ( 4 , 5 ) P2 in liposome ‐ binding .

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

J LAPTM4B N‐terminus binds multiple phosphoinositides including PtdIns(4,5)P2 in liposome‐binding assay.


K Top : GSTpull ‐ down of LAPTM4B N ‐ terminus PIPKIγi5 C ‐ terminus with 0 . 5 μM addition of different phosphoinositides . Bottom : of relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean + SD ; n = 4 ) .

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K Top: GSTpull‐down assay of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with 0.5 μM addition of different phosphoinositides. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean + SD; n = 4).


L Top : GSTpull ‐ down of wild ‐ type or 6RQ mutant of LAPTM4B N ‐ terminus PIPKIγi5 C ‐ terminus with 0 . 5 μM addition of PtdIns ( 4 , 5 ) P2 . Bottom : of relative amounts of PIPKIγi5 C ‐ terminus bound to LAPTM4B N ‐ terminus in top panel ( mean + SD ; n = 4 ) .

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L Top: GSTpull‐down assay of wild‐type or 6RQ mutant of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with 0.5 μM addition of PtdIns(4,5)P2. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean + SD; n = 4).


A Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 were starved stimulated with 100 ng / ml EGF for 30 min , whole ‐ lysates were subject to co ‐ immunoprecipitation ( co ‐ IP ) .

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A Control or LAPTM4B knockdown MDA‐MB‐231 cells were starved and stimulated with 100 ng/ml EGF for 30 min, and whole‐cell lysates were subject to co‐immunoprecipitation (co‐IP) assay.


B Control or LAPTM4B knockdown MDA ‐ MB ‐ 231 were transfected with His ‐ tagged ubiquitin , starved , stimulated with 100 ng / ml EGF for 30 min . Total ubiquitinated were purified from whole ‐ lysates by Ni ‐ NTA agarose analyzed by Western blot .

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B Control or LAPTM4B knockdown MDA‐MB‐231 cells were transfected with His‐tagged ubiquitin, starved, and stimulated with 100 ng/ml EGF for 30 min. Total ubiquitinated proteins were purified from whole‐cell lysates by Ni‐NTA agarose and analyzed by Western blot.


C Control or LAPTM4B ‐ WT / 6RQ ‐ overexpressing MDA ‐ MB ‐ 231 were transfected with His ‐ tagged ubiquitin myc ‐ tagged Hrs , starved , stimulated with 100 ng / ml EGF for 30 min before whole ‐ lysate harvest . Total ubiquitinated were purified by Ni ‐ NTA agarose analyzed by Western blot . D of Hrs ubiquitination from Western blot in ( C ) ( mean + SD ; n = 5 ) .

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C Control or LAPTM4B‐WT/6RQ‐overexpressing MDA‐MB‐231 cells were transfected with His‐tagged ubiquitin and myc‐tagged Hrs, starved, and stimulated with 100 ng/ml EGF for 30 min before whole‐cell lysate harvest. Total ubiquitinated proteins were purified by Ni‐NTA agarose and analyzed by Western blot.D Quantification of Hrs ubiquitination levels from the Western blot in (C) (mean + SD; n = 5).


E co ‐ IP of Myc ‐ Hrs with Flag ‐ LAPTM4B ‐ WT or 6RQ mutant in HEK293 .

E The co‐IP of Myc‐Hrs with Flag‐LAPTM4B‐WT or 6RQ mutant in HEK293 cells.


F co ‐ IP between Hrs Nedd4 with Flag ‐ LAPTM4B ‐ WT or 6RQ mutant overexpression in HEK293 .

F The co‐IP between Hrs and Nedd4 with Flag‐LAPTM4B‐WT or 6RQ mutant overexpression in HEK293 cells.


G effects of LAPTM4B ‐ 6RQ mutant overexpression on EGF ‐ stimulated EGFR degradation signaling . Flag ‐ LAPTM4B ‐ WT or 6RQ mutant overexpression was accomplished by lentivirus ‐ mediated infection approach . with low of expression were selected as polyclonal pools for comparison . Control or overexpressing were starved stimulated with 100 ng / ml EGF for indicated time periods whole ‐ lysates were analyzed by Western blot .

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G The effects of LAPTM4B‐6RQ mutant overexpression on EGF‐stimulated EGFR degradation and signaling. Flag‐LAPTM4B‐WT or 6RQ mutant overexpression was accomplished by lentivirus‐mediated infection approach. Cells with low levels of expression were selected as polyclonal pools for comparison. Control or overexpressing cells were starved and stimulated with 100 ng/ml EGF for indicated time periods and whole‐cell lysates were analyzed by Western blot.


H of EGFR pAKT from Western blot in ( G ) ( mean ± SD ; n = 5 ) .

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H Quantification of EGFR and pAKT levels from the Western blot in (G) (mean ± SD; n = 5).


I PY motif mutant LAPTM4B ‐ 2PA loses interaction with Nedd4 but keeps interaction with Hrs in HEK293 cotransfected with indicated .

I The PY motif mutant LAPTM4B‐2PA loses interaction with Nedd4 but keeps interaction with Hrs in HEK293 cells cotransfected with the indicated proteins.


J effects of LAPTM4B ‐ 2PA mutant overexpression on EGF ‐ stimulated EGFR degradation signaling . Overexpression of Flag ‐ LAPTM4B ‐ WT or 2PA mutant overexpression was accomplished by lentivirus ‐ mediated infection approach . with high expression of Flag ‐ LAPTM4B were selected as polyclonal pools for comparison . were starved stimulated with 100 ng / ml EGF for indicated time periods , whole ‐ lysates were analyzed by Western blot . K of EGFR degradation from Western blot in ( J ) ( mean ± SD ; n = 3 ) .

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J The effects of LAPTM4B‐2PA mutant overexpression on EGF‐stimulated EGFR degradation and signaling. Overexpression of Flag‐LAPTM4B‐WT or 2PA mutant overexpression was accomplished by lentivirus‐mediated infection approach. Cells with high expression of Flag‐LAPTM4B were selected as polyclonal pools for comparison. Cells were starved and stimulated with 100 ng/ml EGF for the indicated time periods, and whole‐cell lysates were analyzed by Western blot.K Quantification of EGFR degradation from Western blot in (J) (mean ± SD; n = 3).


A - C Co ‐ immunoprecipitation ( co ‐ IP ) of wild ‐ type ( WT ) or C ‐ terminal deletion mutants of HA ‐ PIPKIγi5 with SNX5 ( A ) or LAPTM4B ( B C ) in HEK293 cotransfected with indicated . Δ675 indicates a deletion from acid 675 to C ‐ terminus .

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A-C Co‐immunoprecipitation (co‐IP) of wild‐type (WT) or C‐terminal deletion mutants of HA‐PIPKIγi5 with SNX5 (A) or LAPTM4B (B and C) in HEK293 cells cotransfected with indicated proteins. Δ675 indicates a deletion from amino acid 675 to the C‐terminus.


D PIPKIγi5 promotes SNX5 association with LAPTM4B . HEK293 cotransfected with indicated were harvested for co ‐ IP to interaction between Myc ‐ SNX5 Flag ‐ LAPTM4B .

D PIPKIγi5 promotes SNX5 association with LAPTM4B. HEK293 cells cotransfected with indicated proteins were harvested for co‐IP to assay the interaction between Myc‐SNX5 and Flag‐LAPTM4B.


E SNX5 inhibits Hrs association with LAPTM4B . HEK293 cotransfected with indicated were harvested for co ‐ IP to interaction between Myc ‐ Hrs Flag ‐ LAPTM4B .

E SNX5 inhibits Hrs association with LAPTM4B. HEK293 cells cotransfected with indicated proteins were harvested for co‐IP to assay the interaction between Myc‐Hrs and Flag‐LAPTM4B.


( C ) Flag - tagged Raptor was transfected with HA - tagged ArfGAP1 or HA - tagged RFP1 in wildtype ( WT ) or RagA / B knockout ( KO ) HEK293A for 24 hours . lysates were immunoprecipitated ( IP ) with anti - Flag beads . IP or WCL samples were probed for HA - tagged ArfGAP1 or HA - tagged RFP1 , Flag - tagged Raptor , RagA , RagB , Vinculin loading control .

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(C) Flag-tagged Raptor was transfected with HA-tagged ArfGAP1 or HA-tagged RFP1 in wildtype (WT) or RagA/B knockout (KO) HEK293A cells for 24 hours. Cell lysates were immunoprecipitated (IP) with anti-Flag beads. IP or WCL samples were probed for HA-tagged ArfGAP1 or HA-tagged RFP1, Flag-tagged Raptor, RagA, RagB, and Vinculin the loading control.


( G ) RagA / B KO HEK293A were transfected with HA - tagged ArfGAP1 , HA - tagged ArfGAP2 , or HA - tagged RFP1 ( control ) for 72 hours . lysates were immunoprecipitated ( IP ) with anti - HA beads . IP or WCL samples were probed for HA mTORC1 components mTOR , Raptor mLST8 . Vinculin was probed as a control .

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(G) RagA/B KO HEK293A cells were transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) for 72 hours. Cell lysates were immunoprecipitated (IP) with anti-HA beads. IP or WCL samples were probed for HA and mTORC1 components mTOR, Raptor and mLST8. Vinculin was probed as a control.


( D ) ArfGAP1 was stably knocked down in HEK293A using shRNAs against ArfGAP1 ( shArfGAP1 ) or GFP ( shGFP ) control . were cultured under normal condition ( NC ; lanes 1 7 ) , or starved of for 1 hour ( - AA 1h ; lanes 2 8 ) , followed by either maintaining in acid starvation media ( - AA ; lanes 3 9 ) , or addition of 1 mM of Leu ( + Leu ; lanes 4 10 ) , Gln ( + Gln ; lanes 5 11 ) , or Asn ( + Asn ; lanes 6 12 ) for 2 hours . mTORC1 activity was analyzed similar to ( B ) . of ArfGAP1 were confirmed by immunoblotting . S6K1 Actin were probed as controls .

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(D) ArfGAP1 was stably knocked down in HEK293A cells using shRNAs against ArfGAP1 (shArfGAP1) or GFP (shGFP) control. Cells were cultured under normal condition (NC; lanes 1 and 7), or starved of amino acids for 1 hour (-AA 1h; lanes 2 and 8), followed by either maintaining in amino acid starvation media (-AA; lanes 3 and 9), or addition of 1 mM of Leu (+Leu; lanes 4 and 10), Gln (+Gln; lanes 5 and 11), or Asn (+Asn; lanes 6 and 12) for 2 hours. mTORC1 activity was analyzed similar to (B). Protein levels of ArfGAP1 were confirmed by immunoblotting. S6K1 and Actin were probed as controls.


( E ) RagA / B KO HEK293A were co - transfected with HA - tagged ArfGAP1 , HA - tagged ArfGAP2 , or HA - tagged RFP1 ( control ) with Myc - tagged S6K1 . were starved of ( - AA ) for 2 hours then 4mM of Gln or Asn were added for 2 hours . mTORC1 was analyzed similar to ( B ) . IP or WCL samples were probed for HA , Myc , phosphorylated S6K1 at Thr 389 . Actin serve as a loading control .

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

(E) RagA/B KO HEK293A cells were co-transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) with Myc-tagged S6K1. Cells were starved of amino acids (-AA) for 2 hours and then 4mM of Gln or Asn were added for 2 hours. mTORC1 was analyzed similar to (B). IP or WCL samples were probed for HA, Myc, and phosphorylated S6K1 at Thr 389. Actin serve as a loading control.


( B ) Lifetime Imaging Microscopy ( FLIM ) Förster resonance energy transfer ( FRET ) were performed in wildtype ( WT , left ) or RagA / B KO ( middle ) HAP1 expressing mCherry - tagged ArfGAP1 endogenously GFP - tagged Raptor . either had ( + AA ) or were starved of ( - AA ) . At least 20 were analyzed in each group . Box plots shows of pixels with ≥ 30 % FRET efficiency of co - transfected . RFP1 was expressed analyzed as a negative control . whiskers of boxplot represent minimum maximum values , while box covers data between 25th 75th percentiles with a in middle plotted at median . Right - Expression level of RagA / B was confirmed Western blot . Actin was used as a control .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence Lifetime Imaging Microscopy (FLIM) and Förster resonance energy transfer (FRET) analysis were performed in wildtype (WT, left) or RagA/B KO (middle) HAP1 cells expressing mCherry-tagged ArfGAP1 and endogenously GFP-tagged Raptor. Cells either had amino acids (+AA) or were starved of amino acids (-AA). At least 20 cells were analyzed in each group. Box plots shows the percentage of pixels with ≥ 30% FRET efficiency of co-transfected cells. RFP1 was expressed and analyzed as a negative control. The whiskers of the boxplot represent the minimum and maximum values, while the box covers the data between 25th and 75th percentiles with a line in the middle plotted at the median. Right- Expression level of RagA/B was confirmed Western blot analysis. Actin was used as a control.


( A ) Mouse embryonic fibroblast ( MEF ) were transfected with HA - tagged ArfGAP1 . Immunofluorescence was assessed for HA - tagged ArfGAP1 ( blue ) , mTOR ( green ) , LAMP2 ( red , lysosomal marker ) . Left - A representative example of immunofluorescence ( scale bar , 10 μm ) . top left image shows of HA - tagged ArfGAP1 , with a outline highlighting region that is positive for HA - tagged ArfGAP1 . This outline was overlaid with mTOR channel ( top right ) LAMP2 channel ( bottom left ) . mTOR LAMP2 channels were then merged processed using Squassh segmentation algorithm ( Rizk et al . , 2014 ; bottom right ) . co - localization of mTOR LAMP2 was analyzed for region within outline ( HA - ArfGAP1 positive ) region outside of outline ( HA - ArfGAP1 - negative ) , respectively . Right - In each microscopy field , image was processed as described above , co - localization between mTOR LAMP2 was quantified normalized by LAMP2 positive using Squassh algorithm ( Rizk et al . , 2014 ) . was performed for subgroups of that are positively or negatively stained for HA - tagged ArfGAP1 , respectively , compared using Student ' s t - . Columns represent Mean ± ( N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mouse embryonic fibroblast (MEF) cells were transfected with HA-tagged ArfGAP1. Immunofluorescence staining was assessed for HA-tagged ArfGAP1 (blue), mTOR (green), and LAMP2 (red, lysosomal marker). Left- A representative example of the immunofluorescence staining (scale bar, 10 μm). The top left image shows the staining of HA-tagged ArfGAP1, with a white outline highlighting the region that is positive for HA-tagged ArfGAP1. This outline was overlaid with the mTOR channel (top right) and the LAMP2 channel (bottom left). The mTOR and LAMP2 channels were then merged and processed using the Squassh segmentation algorithm (Rizk et al., 2014; bottom right). The co-localization of mTOR and LAMP2 was analyzed for the region within the outline (HA-ArfGAP1 positive) and the region outside of the outline (HA-ArfGAP1-negative), respectively. Right- In each microscopy field, the image was processed as described above, and the co-localization between mTOR and LAMP2 was quantified and normalized by LAMP2 positive puncta using the Squassh algorithm (Rizk et al., 2014). The quantification was performed for the subgroups of cells that are positively or negatively stained for HA-tagged ArfGAP1, respectively, and compared using Student's t-test. Columns represent Mean ± SEM (N = 3).


( D ) MIA Paca - 2 stably expressing FLAG - tagged or HA - tagged TMEM192 were cultured under normal condition ( NC ) or starved of ( - AA ) , then stimulated with 2 mM glutamine asparagine ( + Gln / Asn ) for 2 hours . HA - tagged TMEM192 was immunoprecipitated ( IP ) with HA beads IP whole lysates ( WCL ) were probed for ArfGAP1 , HA , RagA , or p18 ( LAMTOR1 ) . Flag , pS6K1 ( mTORC1 activity ) , S6K1 , Vinculin were probed for as controls .

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

(D) MIA Paca-2 cells stably expressing FLAG-tagged or HA-tagged TMEM192 cells were cultured under normal condition (NC) or starved of amino acids (-AA), and then stimulated with 2 mM glutamine and asparagine (+Gln/Asn) for 2 hours. HA-tagged TMEM192 was immunoprecipitated (IP) with HA beads and IP and whole cell lysates (WCL) were probed for ArfGAP1, HA, RagA, or p18 (LAMTOR1). Flag, pS6K1 (mTORC1 activity), S6K1, and Vinculin were probed for as controls.


( D ) Top - Input of GST , GST - tagged ArfGAP1 , GST - tagged ArfGAP1 L207D / V279D that was us in in vitro binding assays . Bottom - Flag - tagged mTOR , Flag - tagged Raptor , Flag - tagged mLST8 were overexpressed in HEK293A . lysates were immunoprecipitated ( IP ) with anti - Flag beads incubated with either GST ( control ) , GST - tagged ArfGAP1 , or GST - ArfGAP1 L207D / V279D . IP or WCL samples were probed for Flag or GST .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top - Input of GST, GST-tagged ArfGAP1, and GST-tagged ArfGAP1 L207D/V279D that was us in the in vitro binding assays. Bottom - Flag-tagged mTOR, Flag-tagged Raptor, and Flag-tagged mLST8 were overexpressed in HEK293A cells. Cell lysates were immunoprecipitated (IP) with anti-Flag beads and incubated with either GST (control), GST-tagged ArfGAP1, or GST-ArfGAP1 L207D/V279D. IP or WCL samples were probed for Flag or GST.


( A ) TCGA cohort of pancreatic cancer were divided into two groups according to median level of ARFGAP1 mRNA expression . Overall survival disease - free survival were compared between these two groups , as shown in Kaplan - Meier curves . Median survivals ( in months ) , log - rank P values , hazard ratios ( HR ) with 95 % confidence intervals are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TCGA cohort of pancreatic cancer patients were divided into two groups according to the median level of ARFGAP1 mRNA expression. Overall survival and disease-free survival were compared between these two groups, as shown in Kaplan-Meier curves. Median survivals (in months), log-rank P values, and hazard ratios (HR) with 95% confidence intervals are indicated.


( D ) MIA Paca - 2 stably expressing short hairpin RNAs ( shRNAs ) against ArfGAP1 or GFP ( control ) were plated treated with mTOR inhibitor Torin 1 ( 250 nM ) or vehicle control for 48 hours prior to counting . Columns represent Mean ± ( N = 6 ) . Student ' s t - shGFP Vehicle vs . shGFP Torin 1 ( P = 2 . 83 E - 07 ) , shGFP Vehicle vs . shArfGAP1 Vehicle ( * P = 0 . 025 ) , shGFP Torin 1 vs , shArfGAP1 Torin 1 ( P = 0 . 139 ) , shArfGAP1 Vehicle vs . shArfGAP1 Torin 1 ( P = 1 . 22 E - 07 ) . " ns " stands for " not significant " .

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

(D) MIA Paca-2 cells stably expressing short hairpin RNAs (shRNAs) against ArfGAP1 or GFP (control) were plated and treated with mTOR inhibitor Torin 1 (250 nM) or vehicle control for 48 hours prior to cell counting. Columns represent Mean ± SEM (N = 6). Student's t-test shGFP Vehicle vs. shGFP Torin 1 (P = 2.83 E-07), shGFP Vehicle vs. shArfGAP1 Vehicle (*P = 0.025), shGFP Torin 1 vs, shArfGAP1 Torin 1 (P = 0.139), and shArfGAP1 Vehicle vs. shArfGAP1 Torin 1 (P = 1.22 E-07). "ns" stands for "not significant".


Tau in cortex ( A , C - E ) hippocampus ( B , F - H ) of 4 - 10 - month - old mice were determined by western blot . ( A , B ) Representative western blots of cortical ( A ) hippocampal ( B ) homogenates from mice of indicated genotypes show of hTau ( HT7 ) total tau ( Tau5 ) . Actin served as a loading control . ( C - H ) Quantitation of western blot signals for full - tau detected with mTau / hTau cross - reactive antibodies Tau5 ( C , F ) or EP2456Y ( D , G ) or hTau - specific antibody Tau12 ( E , H ) . n = 3 - 8 mice per group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - unpaired t ( E , H ) or by Welch ' s one - way ANOVA with post - hoc two - Welch ' s t ( C , D , F , G ) . P values were Holm - adjusted for multiple comparisons . Values are means ± .

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

Tau levels in the cortex (A, C-E) and hippocampus (B, F-H) of 4-10-month-old mice were determined by western blot analysis. (A, B) Representative western blots of cortical (A) and hippocampal (B) homogenates from mice of the indicated genotypes show levels of hTau (HT7) and total tau (Tau5). Actin served as a loading control. (C-H) Quantitation of western blot signals for full-length tau detected with the mTau/hTau cross-reactive antibodies Tau5 (C, F) or EP2456Y (D, G) or the hTau-specific antibody Tau12 (E, H). n = 3-8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed unpaired t test (E, H) or by Welch's one-way ANOVA with post-hoc two-tailed Welch's t test (C, D, F, G). P values were Holm-adjusted for multiple comparisons. Values are means ± SEM.


Tau expression in cortex ( A , C , E ) hippocampus ( B , D , F ) of 4 - 10 - month - old mice was determined by quantitative RT - PCR ( A , B ) . ( A , B ) hTau mRNA in tissue homogenates were determined by quantitative RT - PCR .

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

Tau expression in the cortex (A, C, E) and hippocampus (B, D, F) of 4-10-month-old mice was determined by quantitative RT-PCR (A, B). (A, B) hTau mRNA levels in tissue homogenates were determined by quantitative RT-PCR.


Tau expression in cortex ( A , C , E ) hippocampus ( B , D , F ) of 4 - 10 - month - old mice was determined by western blot ( C - F ) . ( C , D ) Western blots from Figure 1A B were enhanced to better reveal tau fragments of ~ 40 kDa . ( E , F ) Quantitation of western blot signals for tau fragments of ~ 40 kDa detected with Tau5 in tissue homogenates . n = 3 - 4 mice per group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - unpaired t ( A , B ) or by Welch ' s one - way ANOVA with post - hoc two - Welch ' s t ( E , F ) . P values were Holm - adjusted . Values are means ± .

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

Tau expression in the cortex (A, C, E) and hippocampus (B, D, F) of 4-10-month-old mice was determined by western blot analysis (C-F).(C, D) Western blots from Figure 1A and B were enhanced to better reveal tau fragments of ~40 kDa. (E, F) Quantitation of western blot signals for tau fragments of ~40 kDa detected with Tau5 in tissue homogenates. n = 3-4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed unpaired t test (A, B) or by Welch's one-way ANOVA with post-hoc two-tailed Welch's t test (E, F). P values were Holm-adjusted. Values are means ± SEM.


Coronal brain sections from 2 - 4 - month - old mice of indicated genotypes were immunostained with different tau antibodies . ( A - I ) Sections of hemibrain ( A - C ) , whole hippocampus ( D - F ) , CA1 region ( G - I ) immunostained for hTau ( HT7 ) . ( J - O ) Hippocampal sections immunostained with phosphorylation - dependent tau antibodies : PHF1 ( p396 , 404 ) ( J - L ) , AT8 ( pSer 199 , 202 , pThr 205 ) ( M - O ) .

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

Coronal brain sections from 2-4-month-old mice of the indicated genotypes were immunostained with different tau antibodies. (A-I) Sections of hemibrain (A-C), whole hippocampus (D-F), and CA1 region (G-I) immunostained for hTau (HT7). (J-O) Hippocampal sections immunostained with phosphorylation-dependent tau antibodies: PHF1 (p396, 404) (J-L), AT8 (pSer 199, 202, and pThr 205) (M-O).


( P - R ) Representative photomicrographs of GFAP immunostaining of CA1 region ( P , Q ) quantitation of GFAP immunoreactivity ( R ) .

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

(P-R) Representative photomicrographs of GFAP immunostaining of the CA1 region (P, Q) and quantitation of GFAP immunoreactivity (R).


Six - month - old hTau - A152T ( L1 ) mice NTG controls were or were not treated with DOX for 2 months analyzed at 8 months of by immunohistochemistry . ( A - I ) Untreated hTau - A152T ( L1 ) mice had increased neuronal labeling with hTau antibody HT7 ( A ) , phosphorylation - dependent tau antibody PHF1 ( D ) , conformation - dependent tau antibody MC1 ( G ) . HT7 ( A ) PHF1 ( D ) patterns were similar . MC1 ( G ) specifically stained hippocampal mossy . In hTau - A152T ( L1 ) mice , DOX reduced hippocampal with HT7 , PHF1 , MC1 ( B , E , H ) to in DOX - treated NTG controls ( C , F , I ) .

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

Six-month-old hTau-A152T(L1) mice and NTG controls were or were not treated with DOX for 2 months and analyzed at 8 months of age by immunohistochemistry. (A-I) Untreated hTau-A152T(L1) mice had increased neuronal labeling with the hTau antibody HT7 (A), the phosphorylation-dependent tau antibody PHF1 (D), and the conformation-dependent tau antibody MC1 (G). HT7 (A) and PHF1 (D) staining patterns were similar. MC1 (G) specifically stained hippocampal mossy fibers. In hTau-A152T(L1) mice, DOX reduced hippocampal staining with HT7, PHF1, and MC1 (B, E, H) to the levels in DOX-treated NTG controls (C, F, I).


( J - M ) DOX also normalized CA1 of GFAP immunoreactivity ( IR ) in hTau - A152T ( L1 ) mice , as shown by representative photomicrographs ( J - L ) quantitation of GFAP immunoreactivity ( M ) . Scale bars : 300 µm ( A - I ) , 25 µm ( J - L ) . n = 3 - 4 mice per genotype . * P < 0 . 05 by two - Welch ' s t without Holm adjustment . Values are means ± . a . u . , arbitrary units .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-M) DOX also normalized CA1 levels of GFAP immunoreactivity (IR) in hTau-A152T(L1) mice, as shown by representative photomicrographs (J-L) and quantitation of GFAP immunoreactivity (M). Scale bars: 300 µm (A-I), 25 µm (J-L). n = 3-4 mice per genotype. *P < 0.05 by two-tailed Welch's t test without Holm adjustment. Values are means ± SEM. a.u., arbitrary units.


Nesting behavior of mice at 10 - 14 months of was scored 0 - 7 by an investigator blinded to genotype . n = 17 NTG , 12 CaMKII - tTA , 13 hTau - WT ( L32 ) , 10 hTau - A152T ( L1 ) . Nest building was significantly impaired only in hTau - A152T ( L1 ) mice ( P < 0 . 01 at 2 6 h ) CaMKII - tTA mice ( P < 0 . 05 at 2 h , P < 0 . 01 at 6 hours ) by nonparametric Wilcoxon rank - sum with gatekeeping approach Holm adjustment . Values are means ± .

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

Nesting behavior of mice at 10-14 months of age was scored 0-7 by an investigator blinded to genotype. n = 17 NTG, 12 CaMKII-tTA, 13 hTau-WT(L32), and 10 hTau-A152T(L1). Nest building was significantly impaired only in hTau-A152T(L1) mice (P < 0.01 at 2 and 6 h) and CaMKII-tTA mice (P < 0.05 at 2 h, P < 0.01 at 6 hours) by nonparametric Wilcoxon rank-sum test with gatekeeping approach and Holm adjustment. Values are means ± SEM.


Mice from cohorts 1 - 3 were tested in MWM at indicated ages . ( A - D , M , N ) Learning curves of mice in cohorts 1 ( A - D ) 3 ( M , N ) . Data at day 0 are from first trial on day 1 . Data from cohort 2 are shown in EV5B - G . Compared with NTG controls , only hTau - A152T ( L1 ) mice showed significant learning impairments at 4 - 6 , 17 - 19 ( cohort 1 , P < 0 . 001 ) 17 - 21 ( cohort 3 , P < 0 . 01 ) months of by Cox proportional hazards model with mixed effects Holm adjustment for following comparisons for each range : NTG versus CaMKII - tTA , NTG versus TRE - hTau - A152T ( L1 ) , NTG versus hTau - A152T ( L1 ) for cohort 1 , NTG versus CaMKII - tTA , NTG versus hTau - WT ( L32 ) , NTG versus hTau - A152T ( L1 ) , hTau - WT ( L32 ) versus hTau - A152T ( L1 ) for cohort 3 . ( E - L , O - V ) Performance of mice from cohorts 1 ( E - L ) , 3 ( O - R ) 1 - 3 combined ( S - V ) in a probe trial ( platform removed ) 24 h after last training trial . ( E - H , O , P , S , T ) Latency to reach original platform location . ( I - L , Q , R , U , V ) Number of times mice crossed original platform location ( black bars ) or average of their crossings of corresponding locations in three other quadrants ( bars ) . Cohort 1 : n = 12 ( A - L ) NTG ; 13 ( A , B , E , F , I , J ) , 11 ( C , G , K ) , or 9 ( D , H , L ) TRE - hTau - A152T ( L1 ) ; 11 ( A , B , E , F , I , J ) , 10 ( C , G , K ) , or 9 ( D , H , L ) CaMKII - tTA ; 14 ( A , B , E , F , I , J ) , 13 ( C , G , K ) , or 12 ( D , H , L ) hTau - A152T ( L1 ) mice . Cohort 3 : n = 17 ( M - R ) NTG ; 12 ( M , O , Q ) or 11 ( N , P , R ) CaMKII - tTA ; 13 ( M , O , Q ) or 12 ( N , P , R ) hTau - WT ( L32 ) ; 10 ( M - R ) hTau - A152T ( L1 ) mice . Cohorts 1 + 2 + 3 : n = 47 ( S , U ) , or 39 ( T , V ) NTG ; 11 ( S , U ) or 9 ( T , V ) TRE - hTau - A152T ( L1 ) ; 34 ( S , U ) or 31 ( T , V ) CaMKII - tTA ; 13 ( S , U ) or 12 ( T , V ) hTau - WT ( L32 ) ; 43 ( S , U ) or 36 ( T , V ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Cox proportional hazards / mixed effects model ( A - D , M , N ) , one - way ANOVA with post - hoc Tukey ( E - H , O , P ) or Dunnett ' s ( S , T ) , or one - paired t with Holm adjustment ( I - L , Q , R , U , V ) . Values are means ± .

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

Mice from cohorts 1-3 were tested in the MWM at the indicated ages. (A-D, M, N) Learning curves of mice in cohorts 1 (A-D) and 3 (M, N). Data at day 0 are from the first trial on day 1. Data from cohort 2 are shown in EV5B-G. Compared with NTG controls, only hTau-A152T(L1) mice showed significant learning impairments at 4-6, 17-19 (cohort 1, P < 0.001) and 17-21 (cohort 3, P < 0.01) months of age by Cox proportional hazards model analysis with mixed effects and Holm adjustment for the following comparisons for each age range: NTG versus CaMKII-tTA, NTG versus TRE-hTau-A152T(L1), and NTG versus hTau-A152T(L1) for cohort 1, and NTG versus CaMKII-tTA, NTG versus hTau-WT(L32), NTG versus hTau-A152T(L1), and hTau-WT(L32) versus hTau-A152T(L1) for cohort 3. (E-L, O-V) Performance of mice from cohorts 1 (E-L), 3 (O-R) and 1-3 combined (S-V) in a probe trial (platform removed) 24 h after the last training trial. (E-H, O, P, S, T) Latency to reach original platform location. (I-L, Q, R, U, V) Number of times mice crossed the original platform location (black bars) or the average of their crossings of corresponding locations in the three other quadrants (white bars). Cohort 1: n = 12 (A-L) NTG; 13 (A, B, E, F, I, J), 11 (C, G, K), or 9 (D, H, L) TRE-hTau-A152T(L1); 11 (A, B, E, F, I, J), 10 (C, G, K), or 9 (D, H, L) CaMKII-tTA; and 14 (A, B, E, F, I, J), 13 (C, G, K), or 12 (D, H, L) hTau-A152T(L1) mice. Cohort 3: n = 17 (M-R) NTG; 12 (M, O, Q) or 11 (N, P, R) CaMKII-tTA; 13 (M, O, Q) or 12 (N, P, R) hTau-WT(L32); and 10 (M-R) hTau-A152T(L1) mice. Cohorts 1+2+3: n = 47 (S, U), or 39 (T, V) NTG; 11 (S, U) or 9 (T, V) TRE-hTau-A152T(L1); 34 (S, U) or 31 (T, V) CaMKII-tTA; 13 (S, U) or 12 (T, V) hTau-WT(L32); and 43 (S, U) or 36 (T, V) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by Cox proportional hazards/mixed effects model analysis (A-D, M, N), one-way ANOVA with post-hoc Tukey (E-H, O, P) or Dunnett's (S, T) test, or one-tailed paired t test with Holm adjustment (I-L, Q, R, U, V). Values are means ± SEM.


( A - C ) One cohort of mice was assessed with social approach at young ( A ) old ( B ) another cohort of mice at middle ( C ) . Mice were placed individually into empty center chamber of a three - chamber apparatus . One side - chamber contained an empty cup , other an identical cup with an unfamiliar live mouse in it . Close contact with each cup was monitored for 10 min . n = 12 ( A , B ) or 17 ( C ) NTG ; 13 ( A ) or 10 ( B ) TRE - hTau - A152T ( L1 ) ; 12 ( A ) , 9 ( B ) , or 12 ( C ) CamKII - tTA ; 13 ( C ) hTau - WT ( L32 ) ; 14 ( A ) , 12 ( B ) , or 10 ( C ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by one - paired t with Holm adjustment . Values are means ± .

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

(A-C) One cohort of mice was assessed with the social approach test at young (A) and old (B) age and another cohort of mice at middle age (C). Mice were placed individually into the empty center chamber of a three-chamber apparatus. One side-chamber contained an empty cup, and the other an identical cup with an unfamiliar live mouse in it. Close contact with each cup was monitored for 10 min. n = 12 (A, B) or 17 (C) NTG; 13 (A) or 10 (B) TRE-hTau-A152T(L1); 12 (A), 9 (B), or 12 (C) CamKII-tTA; 13 (C) hTau-WT(L32); and 14 (A), 12 (B), or 10 (C) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by one-tailed paired t test with Holm adjustment. Values are means ± SEM.


( A - F ) Coronal brain sections obtained from mice at 4 - 6 ( A - C ) or 20 - 23 ( D - F ) months of were immunostained for NeuN . number of NeuN - positive with neuronal was determined in dentate gyrus ( A , D ) , CA3 ( B , E ) , CA1 ( C , F ) . n = 11 ( A - C ) or 18 ( D - F ) NTG ; 11 ( A - C ) or 5 ( D - F ) CamKII - tTA ; 7 ( A - C ) or 9 ( D - F ) hTau - WT ( L32 ) ; 14 ( A - C ) or 10 ( D - F ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by one - way ANOVA Tukey . Values are means ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Coronal brain sections obtained from mice at 4-6 (A-C) or 20-23 (D-F) months of age were immunostained for NeuN. The number of NeuN-positive cells with neuronal morphology was determined in dentate gyrus (A, D), CA3 (B, E), and CA1 (C, F). n = 11 (A-C) or 18 (D-F) NTG; 11 (A-C) or 5 (D-F) CamKII-tTA; 7 (A-C) or 9 (D-F) hTau-WT(L32); and 14 (A-C) or 10 (D-F) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA and Tukey test. Values are means ± SEM.


fEPSPs in CA3 stratum lucidum were induced by stimulation of mossy in acute hippocampal slices . ( A ) Representative fEPSP traces from slices of 4 - 8 - month - old mice . ( B , C ) Input / output curves reflecting change in fEPSP slope as a function of fiber volley amplitude from slices obtained at 4 - 8 ( B ) or 20 ( C ) months of . ( D , E ) Short - term plasticity assessed by measuring PPR ( second / first ) with 50 - msec interpulse interval at 4 - 8 ( D ) or 20 ( E ) months of .

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

fEPSPs in CA3 stratum lucidum were induced by stimulation of mossy fibers in acute hippocampal slices. (A) Representative fEPSP traces from slices of 4-8-month-old mice. (B, C) Input/output curves reflecting the change in the fEPSP slope as a function of the fiber volley amplitude from slices obtained at 4-8 (B) or 20 (C) months of age. (D, E) Short-term plasticity assessed by measuring the PPR (second pulse/first pulse) with 50-msec interpulse interval at 4-8 (D) or 20 (E) months of age.


( F ) Mossy fiber LTP at 20 months of . n ( mice / slices ) = 5 - 9 / 8 - 21 ( NTG ) , 2 - 5 / 5 - 12 ( CaMKII - tTA ) , 3 - 6 / 8 - 14 ( TRE - hTau ) , 3 - 6 / 8 - 14 ( hTau - WT ) , 5 - 6 / 8 - 14 ( hTau - A152T ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus NTG by linear regression with Holm adjustment ( B , C ) or by one - way ANOVA Dunnett ' s ( D , E ) . No significant difference was detected by two - way ANOVA in ( F ) . Values are means ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mossy fiber LTP at 20 months of age. n (mice/slices) = 5-9/8-21 (NTG), 2-5/5-12 (CaMKII-tTA), 3-6/8-14 (TRE-hTau), 3-6/8-14 (hTau-WT), and 5-6/8-14 (hTau-A152T). *P < 0.05, **P < 0.01, ***P < 0.001 versus NTG by linear regression analysis with Holm adjustment (B, C) or by one-way ANOVA and Dunnett's test (D, E). No significant difference was detected by two-way ANOVA in (F). Values are means ± SEM.


( A - C ) Subdural EEG recordings from freely behaving mice at 4 - 9 months of before ( A top trace , B ) after ( A bottom trace , C ) injection of PTZ at a dose ( 30 mg / kg ) that did not produce convulsions . ( A ) Representative EEG traces from an hTau - A152T ( L1 ) mouse . Arrows indicate epileptic discharges . ( B , C ) Quantitation of spikes per hour at baseline ( B ) of spikes per 20 - min intervals after PTZ injection ( C ) . n = 31 ( B ) or 23 ( C ) NTG ; 13 ( B ) or 11 ( C ) CaMKII - tTA ; 12 ( B ) or 10 ( C ) hTau - WT ( L32 ) ; 22 ( B ) or 21 ( C ) hTau - A152T ( L1 ) mice . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Welch ' s one - way ANOVA two - Welch ' s t with Holm adjustment ( B ) or by two - way repeated - measures ANOVA Tukey ( C ) . Values are means ± .

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

(A-C) Subdural EEG recordings from freely behaving mice at 4-9 months of age before (A top trace, B) and after (A bottom trace, C) injection of PTZ at a dose (30 mg/kg) that did not produce convulsions. (A) Representative EEG traces from an hTau-A152T(L1) mouse. Arrows indicate epileptic discharges. (B, C) Quantitation of spikes per hour at baseline (B) and of spikes per 20-min intervals after PTZ injection (C). n = 31 (B) or 23 (C) NTG; 13 (B) or 11 (C) CaMKII-tTA; 12 (B) or 10 (C) hTau-WT(L32); and 22 (B) or 21 (C) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by Welch's one-way ANOVA and two-tailed Welch's t test with Holm adjustment (B) or by two-way repeated-measures ANOVA and Tukey test (C). Values are means ± SEM.


( A - D ) Numbers of offspring from crosses of hAPP - J20 mice with hTau - A152T ( L1 ) ( A , B ) or hTau - WT ( L32 ) ( C , D ) mice that were ( A , C ) or were not ( B , D ) treated with DOX . ( E ) Numbers of offspring from crosses of lower expresser hAPP - J9 mice with hTau - A152T ( L1 ) mice that were not treated with DOX . n = 141 ( A ) , 112 ( B ) , 94 ( C ) , 83 ( D ) , 282 ( E ) mice per cohort . Yields from two crosses indicated significant deviations from Mendelian inheritance : hAPP - J20 x TRE - hTau - A152T ( L1 ) x CaMKII - tTA without DOX ( Holm - adjusted P = 0 . 003 , unadjusted P = 0 . 0008 ) hAPP - J9 x TRE - hTau - WT ( L32 ) x CaMKII - tTA without DOX ( adjusted unadjusted P < 0 . 0001 ) by chi - square goodness - of - fit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Numbers of offspring from crosses of hAPP-J20 mice with hTau-A152T(L1) (A, B) or hTau-WT(L32) (C, D) mice that were (A, C) or were not (B, D) treated with DOX. (E) Numbers of offspring from crosses of lower expresser hAPP-J9 mice with hTau-A152T(L1) mice that were not treated with DOX. n = 141 (A), 112 (B), 94 (C), 83 (D), and 282 (E) mice per cohort. Yields from two crosses indicated significant deviations from Mendelian inheritance: hAPP-J20 x TRE-hTau-A152T(L1) x CaMKII-tTA without DOX (Holm-adjusted P = 0.003, unadjusted P = 0.0008) and hAPP-J9 x TRE-hTau-WT(L32) x CaMKII-tTA without DOX (adjusted and unadjusted P < 0.0001) by chi-square goodness-of-fit test.


( F ) Subdural EEG recordings from untreated 3 - 10 - month - old mice were analyzed to compare their number of spikes at baseline . n = 8 NTG , 6 hAPP - J9 , 6 hTau - A152T ( L1 ) , 8 hAPP - J9 / hTau - A152T ( L1 ) . * P < 0 . 05 , * * P < 0 . 01 by logistic regression for three - way interaction of three transgenes with Holm adjustment ( A - E ) or by two - Welch ' s t with Holm adjustment ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Subdural EEG recordings from untreated 3-10-month-old mice were analyzed to compare their number of spikes at baseline. n = 8 NTG, 6 hAPP-J9, 6 hTau-A152T(L1), and 8 hAPP-J9/hTau-A152T(L1). *P < 0.05, **P < 0.01 by logistic regression analysis for three-way interaction of the three transgenes with Holm adjustment (A-E) or by two-tailed Welch's t test with Holm adjustment (F).


Cumulative distribution function of differential ribosome occupancy as detected by ribosome profiling in miRNA _ KO mutants versus WT . Of 759 predicted miRNA target genes , 728 ( 96 % ) were detectable in ribosome profiling data appear in plot . Data information : In A , student ' s t - was applied to assess significance for increase in ribosome occupancy of 728 / 759 miRNA targets ( p - value < 1e51 for every mutant ) .

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

Cumulative distribution function of differential ribosome occupancy as detected by ribosome profiling in miRNA_KO mutants versus WT. Of 759 predicted miRNA target genes, 728 (96%) were detectable in the ribosome profiling data and appear in the plot. Data information: In A, student's t-test was applied to assess the significance for the increase in ribosome occupancy of the 728/759 miRNA targets (p-value<1e51 for every mutant).


Top : Immunoblot of CIC in WT , Drosha _ KO , Dicer _ KO Ago2 & 1 _ KO mESCs . TUBULIN was used as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing of CIC , normalized to TUBULIN relative to WT sample in three biological replicates . Data information : Bar graph in Fig 2F shows mean of CIC signal ± SD normalized to TUBULIN . Values are relative to WT , which was set to 1 . P - values were calculated using a student ' s t - comparing each value to WT . * p - value < 0 . 05 .

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

Top: Immunoblot analysis of CIC in WT, Drosha_KO, Dicer_KO and Ago2&1_KO mESCs. TUBULIN was used as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of CIC intensity, normalized to TUBULIN and relative to the WT sample in three biological replicates. Data information: Bar graph in Fig 2F shows mean intensity of CIC signal ± SD normalized to TUBULIN. Values are relative to WT, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05.


MA plot of DGE in miR - 290 - 295 _ KO mESCs . Data information : In A , significant genes in MA plot were determined using an adjusted p - value of 0 . 1 .

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

MA plot of the DGE analysis in miR-290-295_KO mESCs. Data information: In A, significant genes in MA plot were determined using an adjusted p-value of 0.1.


F , G . Integrated data for Tfap4 gene shows multiple lines of evidence for its regulation by miR - 290 - 295 . In addition to data shown described in Fig 1D , E , a second AGO2 - binding dataset , on cross - linking immunoprecipitation sequencing ( CLIP - seq ) , has been integrated . Response elements for miR - 290 - 295 cluster members are denoted in red labeled .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G. Integrated data for Tfap4 gene shows multiple lines of evidence for its regulation by miR-290-295. In addition to the data shown and described in Fig 1D, E, a second AGO2-binding dataset, based on cross-linking immunoprecipitation and sequencing (CLIP-seq), has been integrated. Response elements for miR-290-295 cluster members are denoted in red and labeled.


Top : Immunoblot of TFAP4 in WT , Drosha _ KO , Dicer _ KO , Ago2 & 1 _ KO miR - 290 - 295 _ KO mESCs . LAMINB1 was used as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing of CIC , normalized to LAMINB1 relative to WT sample in three biological replicates . Data information : In H bar graphs show mean of TFAP4 signal ± SD normalized to LAMINB1 Values are relative to WT or negative control mimic , which was set to 1 . P - values were calculated using a student ' s t - comparing each value to WT . * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * * p - value < 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Top: Immunoblot analysis of TFAP4 in WT, Drosha_KO, Dicer_KO, Ago2&1_KO and miR-290-295_KO mESCs. LAMINB1 was used as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of CIC intensity, normalized to LAMINB1 and relative to the WT sample in three biological replicates. Data information: In H bar graphs show mean intensity of TFAP4 signal ± SD normalized to LAMINB1 Values are relative to WT or the negative control mimic, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05, **p-value<0.01, ****p-value<0.0001.


Top : Immunoblot of TFAP4 after transfection of miRNA mimics ( miR - 291a - 5p , miR - 291a - 3p miR - 291a - 5p + miR - 291a - 3p combined ) in miR - 290 - 295 _ KO mESCs . Immunoblots were stained with Coomassie blue dye as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing of TFAP4 , normalized to Coomassie relative to WT sample in three biological replicates . Data information : In I , bar graphs show mean of TFAP4 signal ± SD normalized to Coomassie . Values are relative to WT or negative control mimic , which was set to 1 . P - values were calculated using a student ' s t - comparing each value to WT . * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 .

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Top: Immunoblot analysis of TFAP4 after transfection of miRNA mimics (miR-291a-5p, miR-291a-3p and miR-291a-5p+miR-291a-3p combined) in miR-290-295_KO mESCs. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of TFAP4 intensity, normalized to Coomassie and relative to the WT sample in three biological replicates. Data information: In I, bar graphs show mean intensity of TFAP4 signal ± SD normalized to Coomassie. Values are relative to WT or the negative control mimic, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05, **p-value<0.01, ****p-value<0.0001.


Top : Mature sequences of 5p 3p miRNAs of miR - 290 - 295 family mature of human miR - 302c - 3p . Colors indicate identical seed sequences . Bottom : AGO2 CLIP - seq shows a peak at miR - 302c - 3p binding site on human TFAP4 .

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Top: Mature sequences of 5p and 3p miRNAs of the miR-290-295 family and the mature sequence of the human miR-302c-3p. Colors indicate identical seed sequences. Bottom: AGO2 CLIP-seq shows a peak at the miR-302c-3p binding site on human TFAP4.


Immunoblot validation of siPOOL - mediated knock down of Tfap4 . TFAP4 were compared between untreated WT versus miR - 290 - 295 _ KO treated with a negative control a Tfap4 - targeted siPOOL . Immunoblots were stained with Coomassie blue dye as a loading control . Blot is a representative image of two biological replicates .

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Immunoblot validation of siPOOL-mediated knock down of Tfap4. TFAP4 levels were compared between untreated WT versus miR-290-295_KO cells treated with a negative control and a Tfap4-targeted siPOOL. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of two biological replicates.


Scatterplot of DEG in miR - 290 - 295 _ KO control versus siPOOL - Tfap4 . SiPOOL experiments were performed in miR - 290 - 295 _ KO DE was assessed relative to a negative control siPOOL transfection . Only genes that are statistically significantly differentially expressed in miR - 290 - 295 _ KO are shown . Genes predicted to be targeted by miR - 290 - 295 are marked in blue . Genes are defined as rescued ( orange ) if log2FoldChange - between miR - 290 - 295 _ KO control siPOOL - Tfap4 is in range [ - 2 , - 1 / 2 ] . Tfap4 is marked by a red dot .

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Scatterplot of DEG in miR-290-295_KO control versus siPOOL-Tfap4. SiPOOL experiments were performed in miR-290-295_KO cells and DE was assessed relative to a negative control siPOOL transfection. Only genes that are statistically significantly differentially expressed in miR-290-295_KO are shown. Genes predicted to be targeted by miR-290-295 are marked in blue. Genes are defined as rescued (orange) if the log2FoldChange-ratio between miR-290-295_KO control and siPOOL-Tfap4 is in the range [-2, -1/2]. Tfap4 is marked by a red dot.


Representative of NANOG ( red ) OCT4 ( green ) immunofluorescence in WT , Drosha _ KO Drosha & Tfap4 _ KO mESCs . Scale bar = 20 μm . of NANOG signal in WT , Drosha _ KO Drosha & Tfap4 _ KO mESCs show no significant rescue of NANOG in Drosha & Tfap4 _ KO Data information : In E , box plots show of NANOG in ~ 300 per genotype . P - values were generated using ordinary one - way ANOVA . * * * * p - value < 0 . 0001 . In panel E , box plot ' s central band represents median relative for each genotype . boxes represent interquartile range whiskers represent maximum minimum relative values .

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Representative images of NANOG (red) and OCT4 (green) immunofluorescence staining in WT, Drosha_KO and Drosha & Tfap4_KO mESCs. Scale bar = 20 μm. Quantification of NANOG fluorescence signal intensity in WT, Drosha_KO and Drosha & Tfap4_KO mESCs show no significant rescue of NANOG intensity in Drosha & Tfap4_KO Data information: In E, box plots show nuclear intensity of NANOG in ~300 cells per genotype. P-values were generated using ordinary one-way ANOVA. ****p-value<0.0001. In panel E, the box plot's central band represents the median relative intensity for each genotype. The boxes represent the interquartile range and the whiskers represent the maximum and minimum relative intensity values.


Confocal of mPS - PI stained 6 - day - old seedlings of Col - 0 ( A ) , bravo - 2 ( B ) , wox5 - 1 ( C ) bravo - 2 wox5 - 1 ( D ) mutants . Left black arrows indicate QC right arrows indicate CSC . Scale bar : 50 µm .

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Confocal images of mPS-PI stained 6-day-old seedlings of Col-0 (A), bravo-2 (B), wox5-1 (C) and bravo-2 wox5-1 (D) mutants. Left black arrows indicate QC cells and right white arrows indicate CSC. Scale bar: 50 µm.


of QC divisions in 6 - day - old roots expressed in ( n > 50 , 3 replicates ) . D : QC divided ; ND : QC non divided .

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Quantification of the QC divisions in 6-day-old roots expressed in percentage (n>50, 3 replicates). D: QC divided; ND: QC non divided.


of CSC layers in 6 - day - old roots expressed in ( n > 50 , 3 replicates ) .

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Quantification of CSC layers in 6-day-old roots expressed in percentage (n>50, 3 replicates).


Lateral root density ( number of lateral roots per mm of root ) of 7 - day - olf WT , bravo - 2 , wox5 - 1 bravo - 2 wox5 - 1 mutants ( n > 40 , 2 replicates ) . Different letters indicate statistically significant differences ( p - value < 0 . 05 Student ´ s t - ) . In boxplot , box width represents interquartile range ( IQR = Q3 - Q1 ) , with horizontal denoting median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . dots are outliers .

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Lateral root density (number of lateral roots per mm of root length) of 7-day-olf WT, bravo-2, wox5-1 and bravo-2 wox5-1 mutants (n>40, 2 replicates). Different letters indicate statistically significant differences (p- value < 0.05 Student´s t-test). In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers.


Confocal of PI - stained 6 - day - old roots . GFP - tagged expression is shown in yellow . A - C ) pBRAVO : GFP in WT ( A ) , bravo - 2 ( B ) wox5 - 1 ( C ) knockout backgrounds . D - G ) pWOX5 : GFP in WT ( D ) , bravo - 2 ( E ) , wox5 - 1 ( F ) bravo - 2 wox5 - 1 ( G ) knockout backgrounds . Scale bar : 50 µm . of GFP signal of roots in A - C ( H ) D - G ( I ) . Boxplot indicating average pixel of GFP in niche ( n > 25 , 3 biological replicates , * p - value < 0 . 05 Student ´ s t - for each genotype versus WT in same condition ) . was done by integrating GFP signal in each root across defined areas that included whole SCN ( Appendix Figure S7 ) . In boxplot , box width represents interquartile range ( IQR = Q3 - Q1 ) , with horizontal denoting median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . dots are outliers black dots are experimental observations .

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Confocal images of PI-stained 6-day-old roots. GFP-tagged expression is shown in yellow. A-C) pBRAVO:GFP in WT (A), bravo-2 (B) and wox5-1 (C) knockout backgrounds. D-G) pWOX5:GFP in the WT (D), bravo-2 (E), wox5-1 (F) and bravo-2 wox5-1 (G) knockout backgrounds. Scale bar: 50 µm. Quantification of the GFP fluorescent signal of the roots in A-C (H) and D-G (I). Boxplot indicating the average pixel intensity of the GFP in the stem cell niche (n>25, 3 biological replicates, *p-value < 0.05 Student´s t- test for each genotype versus the WT in the same condition). Quantification was done by integrating the GFP signal in each root across defined areas that included the whole SCN (Appendix Figure S7). In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers and black dots are the experimental observations.


Parameter space exploration of ( C ) alleviation ( D ) activation models . Boxplots showing distribution of stationary promoter pBRAVO ( pB ) pWOX5 ( pW ) fold - changes in each mutant overexpression lines over WT obtained for different parameter values . Superindexes denote genotype , standing bravo for bravo mutant , wox5 for wox5 mutant , dm for bravo wox5 double mutant , Woe for WOX5 overexpression Boe for BRAVO overexpression . Box width represents interquartile range ( IQR = Q3 - Q1 ) , with horizontal denoting median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . results are obtained by solving N = 1000 different runs of each model at stationary state ( red blue stripplots ) for WT , mutants overexpressor scenarios . These N runs differ in parameter values , which are chosen at random from a uniform distribution

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Parameter space exploration of (C) the alleviation and the (D) activation models. Boxplots showing the distribution of stationary promoter pBRAVO (pB) and pWOX5 (pW) fold-changes in each mutant and overexpression lines over the WT obtained for different parameter values. Superindexes denote the genotype, standing bravo for bravo mutant, wox5 for wox5 mutant, dm for the bravo wox5 double mutant, Woe for WOX5 overexpression and Boe for BRAVO overexpression. Box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. The results are obtained by solving N=1000 different runs of each model at the stationary state (red and blue stripplots) for the WT, mutants and overexpressor scenarios. These N runs differ in the parameter values, which are chosen at random from a uniform distribution between


Interaction of BRAVO with WOX5 ( B ) , BES1 ( C ) , BES1D ( D ) TPL ( E ) ; interaction of WOX5 with BRAVO ( G ) , BES1 ( H ) , BES1D ( I ) TPL ( J ) measured by FRET - FLIM . GFP lifetime τ [ ns ] was measured in transiently expressing Nicotiana benthamiana leaf epidermal . GFP lifetime fitted pixel - wise with a mono - exponential model of BRAVO WOX5 interactions . mV , mVenus ; mCh , mCherry . Scale bar : 5 µm .

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Interaction of BRAVO with WOX5 (B), BES1 (C), BES1D (D) and TPL (E); and interaction of WOX5 with BRAVO (G), BES1 (H), BES1D (I) and TPL (J) measured by FRET-FLIM. GFP fluorescence lifetime τ [ns] was measured in transiently expressing Nicotiana benthamiana leaf epidermal cells. GFP fluorescence lifetime fitted pixel-wise with a mono-exponential model of BRAVO and WOX5 interactions. mV, mVenus; mCh, mCherry. Scale bar: 5 µm.


- weighted average lifetimes of BRAVO WOX5 interactions fitted with a double - exponential model of indicated samples are summarized in box plots . Statistical significance was tested by one - way ANOVA with a Sidakholm post - hoc . Different letters indicate statistically significant differences ( p - value < 0 . 01 ) . For each combination two to three independent experiments were carried out in total more than 20 biological replicates were measured . In boxplot , box width represents interquartile range ( IQR = Q3 - Q1 ) , with horizontal denoting median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . dots are outliers .

Fluorescence-weighted average lifetimes of BRAVO and WOX5 interactions fitted with a double-exponential model of the indicated samples are summarized in box plots. Statistical significance was tested by one-way ANOVA with a Sidakholm post-hoc test. Different letters indicate statistically significant differences (p-value < 0.01). For each combination two to three independent experiments were carried out and in total more than 20 biological replicates were measured. In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers.


Yeast two - hybrid showing BRAVO interacting with WOX5 , BES1 - D TPL ; BES1 - D interacting with TPL . In left column yeast were grown on control media , in right column yeast were grown on control media lacking Leu , Trp His , indicating an interaction between .

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Yeast two-hybrid assay showing BRAVO interacting with WOX5, BES1-D and TPL; and BES1-D interacting with TPL. In the left column yeast cells were grown on control media, and in the right column yeast cells were grown on control media lacking Leu, Trp and His, indicating an interaction between the proteins.


Expression of 380 common BRAVO WOX5 regulated genes from ( B ) . Expression of 53 genes from ( C ) showing similar expression in bravo wox5 mutants . Expression of 41 genes from ( C ) showing opposite expression in bravo wox5 mutants .

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Expression of the 380 common BRAVO and WOX5 regulated genes from (B). Expression of the 53 genes from (C) showing similar expression in bravo and wox5 mutants. Expression of the 41 genes from (C) showing opposite expression levels in bravo and wox5 mutants.


LPS - primed WT Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . Culture supernatants ( SN ) lysates ( Lysate ) were collected immunoblotted with indicated antibodies （ A ） .

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LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) and cell lysates(Lysate) were collected and immunoblotted with the indicated antibodies（A）.


LPS - primed WT Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . Culture supernatants ( SN ) were collected ( B - E ) IL - 1β secretion ( B ) , IL - 18 secretion ( C ) TNF - ɑ secretion ( D ) LDH release ( E ) in supernatants . Data are shown as means ± ; determined by Student ' s t - ; * , P < 0 . 05 ; * * , P < 0 . 01 In each panel , data are representative of at least three independent experiments .

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LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) were collected and (B-E) IL-1β secretion(B), IL-18 secretion (C) and TNF-ɑ secretion (D) and LDH release(E) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.


LPS - primed WT Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . ( F ) HBVLV - Vdr expression in Vdr - / - BMDMs by lentivirus - mediated transduction primed with LPS infected with 50 MOI S . Typhimurium for 2 h , lysates , culture supernatants ( SN ) were collected immunoblotted with indicated antibodies .

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LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. (F) HBVLV-Vdr expression in Vdr-/- BMDMs by lentivirus-mediated transduction primed with LPS and infected with 50 MOI S. Typhimurium for 2 h, cell lysates, and culture supernatants (SN) were collected and immunoblotted with the indicated antibodies.


( G ) LPS - primed WT Vdr - / - BMDMs were treated with 1 µg / ml FLIC ( LFn - Flagellin PA ) or PrgJ （ 1 µg / ml LFn - PrgJ PA ） for 1 h , total mixtures ( culture supernatants lysates ) were collected immunoblotted with indicated antibodies .

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(G) LPS-primed WT and Vdr-/- BMDMs were treated with 1 µg/ml FLIC (LFn-Flagellin and PA) or PrgJ （1 µg/ml LFn- PrgJ and PA）for 1 h, total mixtures( culture supernatants and cell lysates) were collected and immunoblotted with the indicated antibodies.


( H ) LPS - primed WT , Vdr - / - , NLRC4 - / - Vdr - / - NLRC4 - / - BMDMs were treated with FLIC for 1 h , Culture supernatants ( SN ) lysates were collected immunoblotted with indicated antibodies .

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(H) LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs were treated with FLIC for 1 h, Culture supernatants (SN) and cell lysates were collected and immunoblotted with the indicated antibodies.


LPS - primed WT , Vdr - / - , NLRC4 - / - Vdr - / - NLRC4 - / - BMDMs were treated with FLIC for 1 h , Culture supernatants ( SN ) were collected ( I - L ) IL - 1β secretion ( I ) , IL - 18 secretion ( J ) TNF - ɑ secretion ( K ) LDH release ( L ) in supernatants . Data are shown as means ± ; determined by Student ' s t - ; * , P < 0 . 05 ; * * , P < 0 . 01 In each panel , data are representative of at least three independent experiments .

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LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs were treated with FLIC for 1 h, Culture supernatants (SN) were collected and (I-L) IL-1β secretion(I), IL-18 secretion (J) and TNF-ɑ secretion (K) and LDH release(L) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.


LPS - primed WT , Vdr - / - , NLRP3 - / - NLRP3 - / - Vdr - / - BMDMs were infected with different MOI ( 0 , 10 , 50 ) S . Typhimurium for 2 h . Culture supernatants ( SN ) lysates were collected immunoblotted with indicated antibodies ( A ) .

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LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants (SN) and Cell lysates were collected and immunoblotted with the indicated antibodies(A).ELISA


LPS - primed WT , Vdr - / - , NLRP3 - / - NLRP3 - / - Vdr - / - BMDMs were infected with different MOI ( 0 , 10 , 50 ) S . Typhimurium for 2 h . Culture supernatants ( SN ) were collected antibodies ( A ) . ELISA detected TNF - ɑ ( B ) andIL - 1β ( C ) in supernatants . Data information : n ≥ 3 biological replicates . Data are presented as mean ± ; determined by Student ' s t - ; * * , P < 0 . 01 .

LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants (SN) were collected antibodies(A).ELISA detected TNF-ɑ(B)andIL-1β (C) in supernatants. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.


LPS - primed WT , Vdr - / - , NLRP3 - / - NLRP3 - / - Vdr - / - BMDMs were infected with S . Typhimurium burden was detected in spleens ( D ) , PCF ( E ) , blood ( F ) of WT , Vdr - / - , NLRP3 - / - , NLRP3 - / - Vdr - / - mice 24 h after S . Typhimurium infection . Data information : n ≥ 3 biological replicates . Data are presented as mean ± ; determined by Student ' s t - ; * * , P < 0 . 01 .

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LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with S. Typhimurium Bacterial burden was detected in spleens(D), PCF(E), and blood (F)of WT, Vdr-/-, NLRP3-/-, and NLRP3-/- Vdr-/- mice 24 h after S. Typhimurium infection. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.


A LPS - primed WT Vdr - / - BMDMs were infected with S . Typhimurium at an MOI of 50 for 2 h . Endogenous ASC specks ( arrows ) were represented quantified by immunofluorescence . data show representative results from three independent experiments : scale bar , 20 μm . Data are presented as mean ± ; determined by Student ' s t - ; * * * , p < 0 . 001 .

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A LPS-primed WT and Vdr-/- BMDMs were infected with S. Typhimurium at an MOI of 50 for 2 h. Endogenous ASC specks (arrows) were represented and quantified by immunofluorescence images. The data show representative results from three independent experiments: scale bar, 20 μm. Data are presented as the mean ± SEM; determined by Student's t-test ; ***,p < 0.001.


B ASC oligomerization induced by indicated stimuli in WT Vdr − / − BMDMs primed with LPS .

B ASC oligomerization induced by the indicated stimuli in WT and Vdr −/− BMDMs primed with LPS.


LPS - primed BMDMs were infected with S . Typhimurium at an MOI of 50 for 2h . Lysates were immunoprecipitated ( IP ) using an anti - VDR antibody were immunoblotted with indicated antibodies ( A ) . Whole - lysates were shown as input . Data are representative of three independent experiments .

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LPS-primed BMDMs were infected with S. Typhimurium at an MOI of 50 for 2h. Lysates were immunoprecipitated (IP) using an anti-VDR antibody and were immunoblotted with the indicated antibodies(A). Whole-cell lysates were shown as the input. Data are representative of three independent experiments.


( B - C ) Flag - VDR was co - expressed with Myc - NLRC4 in HEK293T ; were immunoprecipitated analyzed by immunoblotting with indicated antibodies . Whole - lysates were shown as input . Data are representative of three independent experiments .

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(B-C) Flag-VDR was co-expressed with Myc-NLRC4 in HEK293T cells; proteins were immunoprecipitated and analyzed by immunoblotting with indicated antibodies. Whole-cell lysates were shown as the input. Data are representative of three independent experiments.


( D ) Purified GST - VDR was incubated with purified His - NLRC4 for 2 h . His - NLRC4 - bound to GST - VDR was pulled down by glutathione beads subjected to immunoblot . Data are representative of three independent experiments .

(D) Purified GST-VDR was incubated with purified His-NLRC4 for 2 h. His-NLRC4- bound to GST-VDR was pulled down by glutathione beads and subjected to immunoblot analysis. Data are representative of three independent experiments.


( E ) WT or mutant NLRC4 ( CARD , NACHT , or LRR ( leucine - rich repeat ) ) HA - VDR were expressed in HEK293T , immunoprecipitated , analyzed by immunoblotting with indicated antibodies . * no specific band . Data are representative of three independent experiments .

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(E) WT or mutant NLRC4 (CARD, NACHT, or LRR (leucine-rich repeat)) and HA-VDR were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting with indicated antibodies.*no specific band. Data are representative of three independent experiments.


( F ) WT or mutant VDR ( DBD or LBD ) Myc - NLRC4 were expressed in HEK293T , immunoprecipitated , analyzed by immunoblotting with indicated antibodies . Data are representative of three independent experiments .

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(F) WT or mutant VDR (DBD or LBD) and Myc-NLRC4 were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting with indicated antibodies. Data are representative of three independent experiments.


A - B HEK293T were transfected with Flag - NLRC4 , VDR , Myc - FLagellin , HA - NAIP5 ( A ) or Flag - NLRC4 , VDR , Myc - PrgJ HA - NAIP2 ( B ) Samples were immunoprecipitated with anti - Flag analyzed by immunoblotting with indicated antibodies .

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A-B HEK293T cells were transfected with Flag-NLRC4, VDR, Myc-FLagellin, and HA-NAIP5 (A) or Flag-NLRC4, VDR, Myc- PrgJ and HA-NAIP2(B) Samples were immunoprecipitated with the anti-Flag and analyzed by immunoblotting with indicated antibodies.


HEK293T were transfected with Flag - NLRC4 , LBD - VDR , Myc - FLagellin , HA - NAIP5 ( C ) or Flag - NLRC4 , LBD - VDR , Myc - PrgJ , HA - NAIP2 ( D ) . Samples were immunoprecipitated with anti - Flag analyzed by immunoblotting with indicated antibodies .

HEK293T cells were transfected with Flag-NLRC4, LBD-VDR, Myc-FLagellin, and HA-NAIP5 (C) or Flag-NLRC4, LBD-VDR, Myc-PrgJ, and HA-NAIP2 (D). Samples were immunoprecipitated with the anti-Flag and analyzed by immunoblotting with indicated antibodies.


B VDR or mutant VDR ( K123A ) Myc - NLRC4 were expressed in HEK293T , immunoprecipitated with anti - Flag antibody , analyzed by immunoblotting with indicated antibodies .

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B VDR or mutant VDR(K123A)and Myc-NLRC4 were expressed in HEK293T cells, immunoprecipitated with the anti-Flag antibody, and analyzed by immunoblotting with indicated antibodies.


C Transfected VDR or mutant VDR ( K123A ) Flag - NLRC4 , Myc - PrgJ , HA - NAIP2 in HEK293T , immunoprecipitated with anti - HA antibody , analyzed by immunoblotting with indicated antibodies .

C Transfected VDR or mutant VDR(K123A) and Flag-NLRC4, Myc- PrgJ, and HA-NAIP2 in HEK293T cells, immunoprecipitated with the anti-HA antibody, and analyzed by immunoblotting with indicated antibodies.


D Vdr - / - BMDMs were infected with lentivirus , which expressed Vector ( NC ) , VDR , VDR - K123A , infected with S . Typhimurium at an MOI of 50 for 2 h , lysates ( Lysate ) culture supernatants ( SN ) were collected immunoblotted with indicated antibodies .

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D Vdr-/- BMDMs were infected with lentivirus, which expressed Vector(NC), VDR, and VDR-K123A, infected with S. Typhimurium at an MOI of 50 for 2 h, cell lysates(Lysate) and culture supernatants(SN) were collected and immunoblotted with the indicated antibodies.


E - H LDH release ( E ) , IL - 1β secretion ( F ) , IL - 18 secretion ( G ) TNF - ɑ secretion ( H ) in supernatants . Data are shown as means ± ; determined by Student ' s t - ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; In each panel , data are representative of at least three independent experiments .

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E-H LDH release(E), IL-1β secretion(F), IL-18 secretion (G) and TNF-ɑ secretion (H) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; **, P < 0.01; ***, P <0.001; In each panel, data are representative of at least three independent experiments.


B . Relative of four histone in each tissue replicate ( order of appearance per replicate : H2A , H2B , H3 . 3 H4 ) .

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B. Relative quantification of all four histone proteins in each tissue replicate (order of appearance per replicate: H2A, H2B, H3.3 and H4).


C . Immunohistochemistry from Human Atlas ( www . proteinatlas . org ) for ANXA1 with ( blue ) for three selected tissues .

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C. Immunohistochemistry images from the Human Protein Atlas (www.proteinatlas.org) for protein ANXA1 with nuclear staining (blue) for three selected tissues.


D . Calibration curves for two of four histone peptides , with decreasing amount of QPrEST standard spiked into a U2OS lysate .

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D. Calibration curves for two of the four histone peptides, with decreasing amount of QPrEST standard spiked into a U2OS cell lysate.


A . Absolute copy number of in kidney tissue human embryonal kidney ( HEK293 ) , liver tissue liver cancer ( HepG2 ) , lung tissue lung cancer ( A549 ) , breast tissue breast cancer ( MCF7 ) . order of are same in tissue corresponding have been order according to abundance in respective tissue .

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A. Absolute copy number of protein in kidney tissue and human embryonal kidney cells (HEK293), liver tissue and liver cancer cell line (HepG2), lung tissue and lung cancer cell line (A549), and breast tissue and breast cancer cell line (MCF7). The order of proteins are the same in the tissue and corresponding cell line and the proteins have been order according to the abundance in the respective tissue.


B . direct correlation between ( TPM ) abundances ( copy number ) for quantified genes in same tissues lines . Spearman ( ρ ) Pearson ( r ) correlation between two values across quantified genes are shown . other seven tissues five lines are shown in Figure EV4 .

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B. The direct correlation between RNA (TPM) and protein abundances (copy number) for all quantified genes in the same tissues and cell lines. Spearman (ρ) and Pearson (r) correlation between the two values across the quantified genes are shown. The other seven tissues and five cell lines are shown in Figure EV4.


Subcellular localization by immunofluorescence immunohistochemistry in tissue sections by three different antibodies ( SELENBP1 , HPA011731 ; STOM , HPA010961 ; ASS1 , HPA020896 ) . Microtubule - probes are visualized in red blue respectively . Antibody is shown in green . to across nine lines 11 tissues with Spearman rho , Pearson r R2 for each gene . other genes can be found in Figure EV1 .

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Subcellular localization by immunofluorescence staining and immunohistochemistry staining in tissue sections by three different antibodies (SELENBP1, HPA011731; STOM, HPA010961; ASS1, HPA020896). Microtubule- and nuclear probes are visualized in red and blue respectively. Antibody staining is shown in green. RNA to protein ratio across nine cell lines and 11 tissues with Spearman rho, Pearson r and R2 for each gene. All other genes can be found in Figure EV1.


A : Volcano plot showing significantly regulated genes between control ATP - treated msEGCs .

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A: Volcano plot showing significantly regulated genes between control and ATP-treated msEGCs.


G - I : qPCR of indicated gliosis genes in ATP - treated EGCs .

G-I: qPCR analysis of indicated gliosis genes in ATP-treated EGCs.