( B - G ) Munc18 - 1 null hippocampal neurons were rescued with wildtype Munc18 - 1a or a Munc18 - 1 mutant , Y473A , in which Y473 was replaced with alanine . ( E ) Release by hyperosmotic sucrose application ( 500mM , 3 . 5sec . ) was used to assess the RRP . Left : RRP charge ( M18WT : 1 . 68 ± 0 . 64 nC , n = 13 ; M18Y473A : 0 . 27 ± 0 . 08 nC , n = 13 , t - test with Welch correction , p = 0 . 0487 ) . Typical responses are depicted in the middle . Right : vesicular release probability per neuron ( EPSC charge / RRP charge ) . Mean Pves ( M18WT : 6 . 75 ± 0 . 77 % , n = 13 ; M18Y473A : 1 . 98 ± 0 . 72 % , n = 13 , t - test , p = 0 . 0001 ) .

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

(B-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(E) Release by hyperosmotic sucrose application (500mM, 3.5sec.) was used to assess the RRP. Left: RRP charge (M18WT: 1.68 ± 0.64 nC, n=13; M18Y473A: 0.27 ± 0.08 nC, n=13, t-test with Welch correction, p = 0.0487). Typical responses are depicted in the middle. Right: vesicular release probability per neuron (EPSC charge / RRP charge). Mean Pves (M18WT: 6.75 ± 0.77%, n=13; M18Y473A: 1.98 ± 0.72%, n=13, t-test, p = 0.0001).


( B - G ) Munc18 - 1 null hippocampal neurons were rescued with wildtype Munc18 - 1a or a Munc18 - 1 mutant , Y473A , in which Y473 was replaced with alanine . ( F ) Top : example traces of spontaneous release of single vesicles ( mEPSCs ) . Average mEPSC frequency ( M18WT : 20 . 18 ± 5 . 18 Hz , n = 15 ; M18Y473A : 0 . 48 ± 0 . 15 Hz , n = 20 , t - test with Welch correction , p = 0 . 0019 ) , amplitude ( M18WT : 26 . 0 ± 1 . 7 pA , n = 15 ; M18Y473A : 24 . 5 ± 1 . 6 pA , n = 20 , t - test , p = 0 . 5334 ) and decay time ( M18WT : 1 . 98 ± 0 . 10 ms , n = 15 ; M18Y473A : 2 . 13 ± 0 . 17 ms , n = 20 , t - test with Welch correction , p = 0 . 4499 ) .

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

(B-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(F) Top: example traces of spontaneous release of single vesicles (mEPSCs). Average mEPSC frequency (M18WT: 20.18 ± 5.18 Hz, n=15; M18Y473A: 0.48 ± 0.15 Hz, n=20, t-test with Welch correction, p = 0.0019), amplitude (M18WT: 26.0 ± 1.7 pA, n=15; M18Y473A: 24.5 ± 1.6 pA, n=20, t-test, p = 0.5334) and decay time (M18WT: 1.98 ± 0.10 ms, n=15; M18Y473A: 2.13 ± 0.17 ms, n=20, t-test with Welch correction, p = 0.4499).


( B - G ) Munc18 - 1 null hippocampal neurons were rescued with wildtype Munc18 - 1a or a Munc18 - 1 mutant , Y473A , in which Y473 was replaced with alanine . ( G ) Typical electron microscopy images from rescued autaptic hippocampal Munc18 - 1 null neurons ( scale bar = 100 nm ) . Neurons expressing M18Y473A have less synaptic vesicles ( SV ) docked at the number of docked SV . Average

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

(B-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(G) Typical electron microscopy images from rescued autaptic hippocampal Munc18-1 null neurons (scale bar = 100 nm). Neurons expressing M18Y473A have less synaptic vesicles (SV) docked at the number of docked SV. Average


Autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies . ( A ) Absolute EPSC amplitudes during a 10 Hz train .

O O B-CONTROLLED_VAR I-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

Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies. (A) Absolute EPSC amplitudes during a 10 Hz train.


Autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies . ( B ) EPSC amplitudes during a 10 Hz train normalized to the first EPSC .

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Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(B) EPSC amplitudes during a 10 Hz train normalized to the first EPSC.


Autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies . ( C ) Top : example traces of the currents evoked by 40 Hz stimulation . Bottom right : first 5 pulses of the 40 Hz train . Far right : transferred charge during 40 Hz train . Insert shows total charge transferred during entire 40 Hz train ( M18WT : 2 . 63 ± 0 . 38 nC ; M18Y473A : 2 . 93 ± 0 . 54 nC , t test , p = 0 . 6630 ) .

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

Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(C) Top: example traces of the currents evoked by 40 Hz stimulation. Bottom right: first 5 pulses of the 40 Hz train. Far right: transferred charge during 40 Hz train. Insert shows total charge transferred during entire 40 Hz train (M18WT: 2.63 ± 0.38 nC; M18Y473A: 2.93 ± 0.54 nC, t test, p = 0.6630).


Autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies . ( D ) EPSC augmentation is calculated by dividing the EPSC amplitude of a single pulse after a stimulation train by the amplitude of the first EPSC within this train . Mean augmentation after 5 Hz train ( M18WT : 0 . 54 ± 0 . 05 , n = 8 ; M18Y473A : 2 . 88 ± 0 . 47 , n = 10 ; Unpaired t - test with Welch correction , p = 0 . 0008 ) , 10 Hz train ( M18WT : 0 . 70 ± 0 . 10 , n = 8 ; M18Y473A : 5 . 39 ± 0 . 77 , n = 10 ; Unpaired t - test with Welch correction , p = 0 . 0002 ) and 40 Hz train ( M18WT : 0 . 93 ± 0 . 11 , n = 8 ; M18Y473A : 8 . 77 ± 1 . 52 , n = 10 ; Unpaired t - test with Welch correction , p = 0 . 0006 ) .

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

Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(D) EPSC augmentation is calculated by dividing the EPSC amplitude of a single pulse after a stimulation train by the amplitude of the first EPSC within this train. Mean augmentation after 5 Hz train (M18WT: 0.54 ± 0.05, n=8; M18Y473A: 2.88 ± 0.47, n=10; Unpaired t-test with Welch correction, p = 0.0008), 10 Hz train (M18WT: 0.70 ± 0.10, n=8; M18Y473A: 5.39 ± 0.77, n=10; Unpaired t-test with Welch correction, p = 0.0002) and 40 Hz train (M18WT: 0.93 ± 0.11, n=8; M18Y473A: 8.77 ± 1.52, n=10; Unpaired t-test with Welch correction, p = 0.0006).


Autaptic excitatory munc18 - 1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies . ( E ) A single action potential or sucrose application ( 500 mM , 3 . 5 sec . ) was given before ( naive ) or two seconds after HFS ( 100 pulses at 40 Hz ) . Average EPSC charge M18WT ( Naive : 70 . 9 ± 19 . 5 pC , after HFS : 75 . 4 ± 17 . 4 pC , n = 12 , Paired t - test , p = 0 . 4049 ) . Average EPSC charge M18Y473A ( naive : 10 . 3 ± 4 . 5 pC , after HFS : 46 . 2 ± 14 . 5 pC , n = 13 , Wilcoxon matched - pairs signed - ranks test , p = 0 . 0005 ) . Average RRP size M18WT ( naive : 1 . 73 ± 0 . 69 nC , after HFS : 1 . 34 ± 0 . 54 nC , n = 12 , paired t - test , p = 0 . 0557 ) Average HFS size M18Y473A ( naive : 0 . 268 ± 0 . 075 nC , after HFS : 0 . 439 ± 0 . 107 nC , n = 13 , paired t - test , p = 0 . 0100 ) . Average Pves M18WT ( EPSC charge / RRP charge * 100 ) ( Naive : 7 . 07 ± 0 . 76 % , after HFS : 10 . 6 ± 1 . 6 % , n = 12 , paired t - test , p = 0 . 0201 ) . Average Pves M18Y473A ( naive : 1 . 98 ± 0 . 72 % , 12 . 2 ± 2 . 8 % , n = 13 , paired t - test , p = 0 . 0021 ) .

O O B-CONTROLLED_VAR I-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 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 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 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 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 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 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

Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(E) A single action potential or sucrose application (500 mM, 3.5 sec.) was given before (naive) or two seconds after HFS (100 pulses at 40 Hz). Average EPSC charge M18WT (Naive: 70.9 ± 19.5 pC, after HFS: 75.4 ± 17.4 pC, n=12, Paired t-test, p = 0.4049). Average EPSC charge M18Y473A (naive: 10.3 ± 4.5 pC, after HFS: 46.2 ± 14.5 pC, n=13, Wilcoxon matched-pairs signed-ranks test, p = 0.0005). Average RRP size M18WT (naive: 1.73 ± 0.69 nC, after HFS: 1.34 ± 0.54 nC, n=12, paired t-test, p = 0.0557) Average HFS size M18Y473A (naive: 0.268 ± 0.075 nC, after HFS: 0.439 ± 0.107 nC, n=13, paired t-test, p = 0.0100). Average Pves M18WT (EPSC charge / RRP charge * 100) (Naive: 7.07 ± 0.76 %, after HFS: 10.6 ± 1.6 %, n=12, paired t-test, p = 0.0201). Average Pves M18Y473A (naive: 1.98 ± 0.72 %, 12.2 ± 2.8 %, n=13, paired t-test, p = 0.0021).


A Endogenous PIPKIγi5 and LAPTM4B were immunoprecipitated from the whole ‐ cell lysates of MDA ‐ MB ‐ 231 cells followed by immunoblotting to examine the co ‐ immunoprecipitatedPIPKIγi5 and LAPTM4B .

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

A Endogenous PIPKIγi5 and LAPTM4B were immunoprecipitated from the whole‐cell lysates of MDA‐MB‐231 cells followed by immunoblotting to examine the co‐immunoprecipitatedPIPKIγi5 and LAPTM4B.


B PIPKIγi5 specifically interacts with LAPTM4B , but not LAPTM4A or LAPTM5 . Top : schematic diagram of all three LAPTM members . Bottom : each Flag ‐ tagged LAPTM protein was immunoprecipitated from HEK293 cells cotransfected with Myc ‐ tagged PIPKIγi5 and empty vector or Flag ‐ tagged LAPTM , and the co ‐ immunoprecipitatedPIPKIγi5 was examined by immunoblotting .

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

B PIPKIγi5 specifically interacts with LAPTM4B, but not LAPTM4A or LAPTM5. Top: schematic diagram of all three LAPTM members. Bottom: each Flag‐tagged LAPTM protein was immunoprecipitated from HEK293 cells cotransfected with Myc‐tagged PIPKIγi5 and empty vector or Flag‐tagged LAPTM, and the co‐immunoprecipitatedPIPKIγi5 was examined by immunoblotting.


C LAPTM4B selectively associates with PIPKIγi5 . Top : schematic diagram of four PIPKI isoforms . Bottom : Myc ‐ tagged PIPKIγi5 was immunoprecipitated from HEK293 cells expressing Flag ‐ tagged LAPTM4B and each isoform of PIPKI , followed by immunoblotting to examine the co ‐ immunoprecipitatedLAPTM4B .

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C LAPTM4B selectively associates with PIPKIγi5. Top: schematic diagram of four PIPKI isoforms. Bottom: Myc‐tagged PIPKIγi5 was immunoprecipitated from HEK293 cells expressing Flag‐tagged LAPTM4B and each isoform of PIPKI, followed by immunoblotting to examine the co‐immunoprecipitatedLAPTM4B.


D Kinase activity of PIPKIγi5 is required for LAPTM4B association . HEK293 cells expressing LAPTM4B and wild ‐ type ( WT ) or kinase ‐ dead ( KD ) PIPKIγi5 were starved overnight , stimulated or not with 100 ng / ml EGF for 15 min , and harvested for immunoprecipitation with anti ‐ myc . The co ‐ immunoprecipitatedLAPTM4B was detected by immunoblotting .

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

D Kinase activity of PIPKIγi5 is required for LAPTM4B association. HEK293 cells expressing LAPTM4B and wild‐type (WT) or kinase‐dead (KD) PIPKIγi5 were starved overnight, stimulated or not with 100 ng/ml EGF for 15 min, and harvested for immunoprecipitation with anti‐myc. The co‐immunoprecipitatedLAPTM4B was detected by immunoblotting.


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 ) .

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

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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 ‐ length or N ‐ terminus ‐ deleted LAPTM4B with PIPKIγi5 in HEK293 cells .

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

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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 ‐ length or N ‐ terminal deletion mutants of LAPTM4B with PIPKIγi5 in HEK293 cells . ∆ 25 : amino acids 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 staining of purified wild ‐ type and mutated LAPTM4B N ‐ termini .

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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 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 .

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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 assay 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 cells .

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


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 ) .

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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 assay .

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


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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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

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


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

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


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 .

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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 Quantification of EGFR and pAKT levels from the 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 The PY motif mutant LAPTM4B ‐ 2PA loses interaction with Nedd4 but keeps interaction with Hrs in HEK293 cells cotransfected with the indicated proteins .

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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 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 ) .

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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 and C ) in HEK293 cells cotransfected with indicated proteins . Δ675 indicates a deletion from amino acid 675 to the 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 cells cotransfected with indicated proteins were harvested for co ‐ IP to assay the interaction between Myc ‐ SNX5 and Flag ‐ LAPTM4B .

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

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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(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 ) 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 .

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(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 ) 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 ) .

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

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(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 , 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 .

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(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 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 .

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(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 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 " .

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(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 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 .

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

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 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 .

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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 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 .

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

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 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 ) .

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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 the CA1 region ( P , Q ) and quantitation of GFAP immunoreactivity ( R ) .

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(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 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 ) .

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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 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 .

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(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 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 .

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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 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 .

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

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 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 .

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

(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 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(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 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 .

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

(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 ) , 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 .

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

(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 ) , 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O 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 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 .

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

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

Representative 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 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 .

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

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.


Quantification of the QC divisions in 6 - day - old roots expressed in percentage ( 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.


Quantification of CSC layers in 6 - day - old roots expressed in percentage ( 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 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 .

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

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.