( H ) Co - IP of myc - STIM1 Orai1 - HA , myc - STIM1 ANO8 - YFP , Orai1 - HA ANO8 - YFP was measured in transfected HEK before ( R , resting ) in response to store depletion ( S , stimulated ) . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Co-IP of myc-STIM1 and Orai1-HA, myc-STIM1 and ANO8-YFP, and Orai1-HA and ANO8-YFP was measured in transfected HEK cells before (R, resting) and in response to store depletion (S, stimulated). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( I ) Effect of ANO8 - YFP on myc - STIM1 Orai1 - HA at ER / PM junction . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O 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) Effect of ANO8-YFP on myc-STIM1 and Orai1-HA at the ER/PM junction. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( J - L ) Junctional Orai1 ( J ) , STIM1 ( K ) ANO8 ( L ) in resting ( R ) store - depleted ( S ) . results are mean ± s . e . m of 4 experiments difference were analyzed by unpaired t . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O B-SUBCELLULAR B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L) Junctional Orai1 (J), STIM1 (K) and ANO8 (L) in resting (R) and store-depleted cells (S). The results are mean±s.e.m of 4 experiments and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( M ) Example EM recorded from HEK transfected with empty vector ( left ) or ANO8 ( right ) . Red arrows mark ER / PM junctions . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Example EM images recorded from HEK cells transfected with empty vector (left) or ANO8 (right). Red arrows mark ER/PM junctions. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( N , O ) ( N ) density ( O ) of ER / PM junctions in vector - transfected ( control ) ANO8 - transfected . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N, O) The size (N) and density (O) of ER/PM junctions in vector-transfected (control) and ANO8-transfected cells. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( A , B ) Knockdown of ANO8 ( siA8 ) reduced CRAC current in transfected with Orai1 ( O1 ) STIM1 ( S1 ) buffered with 3 mM EGTA . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed difference were analyzed by unpaired t .

O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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, B) Knockdown of ANO8 (siA8) reduced CRAC current in cells transfected with Orai1 (O1) and STIM1 (S1) and buffered with 3 mM EGTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( C ) Knockdown of ANO8 reduces native store - dependent Ca2 + influx measured in store depleted by Ca2 + add - back . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed difference were analyzed by unpaired t .

O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Knockdown of ANO8 reduces the native store-dependent Ca2+ influx measured in store depleted cells by Ca2+ add-back. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( D , E ) Knockdown of ANO8 reduced number of store - dependent STIM1 at TIRF plane in expressing STIM1 Orai1 . Panel ( D ) shows representative ( E ) is summary of 7 experiments . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed difference were analyzed by unpaired t .

O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D,E) Knockdown of ANO8 reduced the number of store-dependent STIM1 puncta at the TIRF plane in cells expressing STIM1 and Orai1. Panel (D) shows representative images and (E) is the summary of 7 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( F , G ) Current was measured with pipette solution contacting fast strong Ca2 + buffer 10 mM BAPTA in HEK transfected with STIM1 , Orai1 , with ( red ) or without ANO8 ( black ) . Panel ( G ) shows increase in current density at peak current . Note prominent current inactivation in presence of ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed difference were analyzed by unpaired t .

O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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) Current was measured with pipette solution contacting the fast and strong Ca2+ buffer 10 mM BAPTA in HEK cells transfected with STIM1, Orai1, and with (red) or without ANO8 (black). Panel (G) shows the increase in current density at peak current. Note the prominent current inactivation in the presence of ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( H ) Knockdown of SARAF ( red ) in wild - type had no effect on current inactivation in presence of 10 mM BAPTA . ( I ) Knockdown of SARAF did not prevent ANO8 - dependent current inactivation in presence of 10 mM BAPTA . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed difference were analyzed by unpaired t .

O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Knockdown of SARAF (red) in wild-type cells had no effect on current inactivation in the presence of 10 mM BAPTA. (I) Knockdown of SARAF did not prevent the ANO8-dependent current inactivation in the presence of 10 mM BAPTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


( A , B ) Depletion of plasma PI ( 4 , 5 ) P2 with FRB / FKBP system prevented ANO8 - mediated increase in STIM1 - Orai1 current measured in pipette solutions containing 3 mM EGTA ( A ) or 10 mM BAPTA ( B ) . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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, B) Depletion of plasma membrane PI(4,5)P2 with the FRB/FKBP system prevented the ANO8-mediated increase in STIM1-Orai1 current measured in pipette solutions containing 3 mM EGTA (A) or 10 mM BAPTA (B). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( C ) HEK transfected with ANO8 - YFP , STIM1 - CFP Orai1 - HA FRB mCherry - FKBP PI ( 4 , 5 ) P2 depleting constructs were treated with 0 . 2 µM rapamycin ( rapa ) for 5 min then with 25 µM CPA for 10 min before fixation imaging by confocal microscopy . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) HEK cells transfected with ANO8-YFP, STIM1-CFP Orai1-HA and FRB and mCherry-FKBP PI(4,5)P2 depleting constructs were treated with 0.2 µM rapamycin (rapa) for 5 min and then with 25 µM CPA for 10 min before fixation and imaging by confocal microscopy. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( D ) Mutating ANO8 R948 , R950 , R951 in RPRRP , a region that is predicted to include an ANO8 PI ( 4 , 5 ) P2 binding site , prevented increased STIM1 - Orai1 current inactivation . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O 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) Mutating ANO8 R948, R950, R951 in RPRRP, a region that is predicted to include an ANO8 PI(4,5)P2 binding site, prevented increased STIM1-Orai1 current and inactivation. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( E ) Mutating ANO8 R949 , 950 , 951 to Q reduced translocation of ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Mutating ANO8 R949,950,951 to Q reduced translocation of ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( A ) Plot of slope of inactivation as a function of current density in presence ( red ) absence of ANO8 ( black ) . Increasing current density was obtained by varying external Ca2 + between 2 - 50 mM . results are mean ± s . e . m difference were analyzed by unpaired t . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plot of the slope of inactivation as a function of current density in the presence (red) and absence of ANO8 (black). Increasing current density was obtained by varying external Ca2+ between 2-50 mM. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( B , C ) CRAC current was measured in HEK expressing STIM1 Orai1 in pipette solution containing 3 mM EGTA ( b ) or 10 mM BAPTA ( c ) . SERCA pump was inhibited with 25 µM CPA in red traces where indicated 100 µM IP3 was included in pipette solution ( green in c ) . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O B-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) CRAC current was measured in HEK cells expressing STIM1 and Orai1 and in pipette solution containing 3 mM EGTA (b) or 10 mM BAPTA (c). SERCA pump was inhibited with 25 µM CPA in the red traces where indicated and 100 µM IP3 was included in the pipette solution (green in c). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( D , E ) Experimental protocol conditions except that were also transfected with ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Experimental protocol and conditions except that cells were also transfected with ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( F , G ) HEK expressing STIM1 , Orai1 , ANO8 were used to measure current in pipette solution containing 10 mM BAPTA with ( red ) or without ( black ) 100 µM IP3 or 10 mM of ER Ca2 + chelator TPEN ( green ) . current density ( F ) normalized current ( G ) are shown , illustrating delay in current inactivation by IP3 TPEN . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed .

O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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) HEK cells expressing STIM1, Orai1, and ANO8 were used to measure current in pipette solution containing 10 mM BAPTA and with (red) or without (black) 100 µM IP3 or 10 mM of the ER Ca2+ chelator TPEN (green). The current density (F) and the normalized current (G) are shown, illustrating the delay in current inactivation by IP3 and TPEN. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


( A , B ) : ER Ca2 + content was measured with ER - GECO1 in periphery ( A ) center ( B ) or transfected with M3 receptors with ( red , green ) or without ( black , blue ) ANO8 . Receptor mediated store depletion was initiated by stimulating with 0 . 5 mM carbachol in Ca2 + - free solution . stimulation was terminated with 10 µM atropine ER Ca2 + uptake was initiated by perfusing with a solution containing 5 mM Ca2 + . was measured as F / F0 normalized to initial in presence of ANO8 . Panel ( C ) shows traces of ER Ca2 + uptake at expanded time scale in periods marked by rectangular in ( A B ) averaged slopes of ER Ca2 + influx ( D ) shows averaged slope of Ca2 + release at periphery ( Peri ) center ( Center ) . In ( C ) traces were aligned along Y axis to better show difference in uptake rate . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B): ER Ca2+ content was measured with ER-GECO1 in the periphery (A) and the center (B) or cells transfected with M3 receptors and with (red, green) or without (black, blue) ANO8. Receptor mediated store depletion was initiated by stimulating the cells with 0.5 mM carbachol in Ca2+-free solution. Cell stimulation was terminated with 10 µM atropine and ER Ca2+ uptake was initiated by perfusing the cells with a solution containing 5 mM Ca2+. The fluorescence was measured as F/F0 and normalized to the initial fluorescence in the presence of ANO8. Panel (C) shows traces of ER Ca2+ uptake at expanded time scale in the periods marked by rectangular in (A and B) and the averaged slopes of ER Ca2+ influx and (D) shows the averaged slope of Ca2+ release at the cell periphery (Peri) and cell center (Center). In (C) the traces were aligned along the Y axis to better show the difference in uptake rate. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( E ) Enhancement by ANO8 of Co - IP of expressed STIM1 with PMCA4a SERCA2 of Orai1 with SERCA2 in resting store depleted . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Enhancement by ANO8 of the Co-IP of expressed STIM1 with PMCA4a and SERCA2 and of Orai1 with SERCA2 in resting and store depleted cells. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( F ) Effect of ANO8 expression on Co - IP of native SERCA2 STIM1 in resting store depleted . columns are mean ± s . e . m of 3 experiments . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of ANO8 expression on the Co-IP of the native SERCA2 and STIM1 in resting and store depleted cells. The columns are the mean±s.e.m of 3 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( G ) FRET between STIM1 SERCA2 in response to store depletion its enhancement by ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) FRET ratio between STIM1 and SERCA2 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( H , I ) Simultaneous measurements of ER Ca2 + with ER - GECO1 PI ( 4 , 5 ) P2 hydrolysis with PHPLCδ - GFP in response to stimulation of M3 receptors . Traces are shown in ( h ) average slopes are in ( i ) . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H, I) Simultaneous measurements of ER Ca2+ with ER-GECO1 and PI(4,5)P2 hydrolysis with PHPLCδ-GFP in response to cell stimulation of M3 receptors. Traces are shown in (h) and the average slopes are in (i). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( J ) Effect of ANO8 on Co - IP of native STIM1 IP3Rs . columns are mean ± s . e . m of 3 experiments . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of ANO8 on the Co-IP of the native STIM1 and IP3Rs. The columns are the mean±s.e.m of 3 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( K ) FRET between expressed STIM1 IP3R3 in response to store depletion its enhancement by ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) FRET ratio between expressed STIM1 and IP3R3 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( L ) FRET between expressed STIM1 PMCA4 in response to store depletion its enhancement by ANO8 . Data information : first number in parenthesis indicates number of similar experiments performed second number is number of analyzed . results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

(L) FRET ratio between expressed STIM1 and PMCA4 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( A - D ) HeLa transfected with GFP ( black ) or ANO8 ( red ) were stimulated with 0 . 5 then 1 µM ATP to activate native P2Y2 receptors . oscillations were analyzed in terms of % of responding ( B ) , oscillation frequency ( C ) , amplitude of Ca2 + signal ( D ) . Data information : results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

O O O O O B-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O 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) HeLa cells transfected with GFP (black) or ANO8 (red) were stimulated with 0.5 and then 1 µM ATP to activate the native P2Y2 receptors. The oscillations were analyzed in terms of the % of responding cells (B), oscillation frequency (C), and the amplitude of the Ca2+ signal (D). Data information: All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( E - H ) : HeLa were treated with scrambled siRNA ( black ) or siANO8 ( red ) were stimulated with 1 then 5 µM ATP . oscillations were analyzed in terms of % of responding ( F ) , oscillations frequency ( G ) , amplitude of Ca2 + signal ( H ) . Data information : results are given as mean ± s . e . m of indicated number of experiments or analyzed , difference were analyzed by unpaired t .

O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-H): HeLa cells were treated with scrambled siRNA (black) or siANO8 (red) and were stimulated with 1 and then 5 µM ATP. The oscillations were analyzed in terms of the % of responding cells (F), oscillations frequency (G), and the amplitude of the Ca2+ signal (H). Data information: All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


( A ) Total lysates from indicated lines cultured in regular conditions at a steady state were subjected to western blotting . s . e . , short exposure ; l . e . , long exposure . major Merlin band is indicated by an asterisk . of mono - ubiquitinated to native Merlin in each lane of short exposure blot was quantified by Image J is shown under blot . same lysates were also used in Figure EV1A . Asterisks indicate native Merlin , arrows show ubiquitinated Merlin .

O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY B-GENEPROD O

(A) Total lysates from the indicated cell lines cultured in regular conditions at a steady state were subjected to western blotting. s.e., short exposure; l.e., long exposure. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot. The same lysates were also used in Figure EV1A. Asterisks indicate native Merlin, arrows show ubiquitinated Merlin.


( B ) Total lysates from LN229 in which Merlin was or was not knocked out ( KO ) were subjected to western blotting .

O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(B) Total lysates from LN229 cells in which Merlin was or was not knocked out (KO) were subjected to western blotting.


LN229 ( C ) were detached ( denoted by sus . , referring to suspension ) by trypsinization ( see Methods ) reseeded ( attach ) for two ( LN229 ) hours . Total lysates from these were subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot . Ubiquitinated Merlin is marked with arrows .

B-CELL_LINE O O O O 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-CELL_LINE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O

LN229 (C) cells were detached (denoted by sus., referring to suspension) by trypsinization (see Methods) and reseeded (attach) for two (LN229) hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with arrows.


Met5 - A ( D ) were detached ( denoted by sus . , referring to suspension ) by trypsinization ( see Methods ) reseeded ( attach ) for four ( Met5 - A ) hours . Total lysates from these were subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot . Ubiquitinated Merlin is marked with arrows .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O 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-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O

Met5-A (D) cells were detached (denoted by sus., referring to suspension) by trypsinization (see Methods) and reseeded (attach) for four (Met5-A) hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with arrows.


( E ) LN229 transduced with a Merlin or scrambled shRNA were treated with DMSO , ionomycin ( Iono ) or thapsigargin ( TG ) . Total lysates of these were subjected to western blotting . major Merlin band is indicated by an asterisk . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot . Ubiquitinated Merlin is marked with an arrow .

O O O B-CELL_LINE O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O O

(E) LN229 cells transduced with a Merlin or scrambled shRNA were treated with DMSO, ionomycin (Iono) or thapsigargin (TG). Total lysates of these cells were subjected to western blotting. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with an arrow.


( F ) LN229 treated with DMSO or thapsigargin ( TG ) were lysed subjected to immunoprecipitation with a Merlin antibody or IgG . immunoprecipitated products were subjected to western blotting . number of conjugated ubiquitin ( Ub ) moieties was marked . Tetra - ubiquitinated Merlin is indicated by an arrow .

O O O B-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O

(F) LN229 cells treated with DMSO or thapsigargin (TG) were lysed and subjected to immunoprecipitation with a Merlin antibody or IgG. The immunoprecipitated products were subjected to western blotting. The number of conjugated ubiquitin (Ub) moieties was marked. Tetra-ubiquitinated Merlin is indicated by an arrow.


( G ) LN229 stably transduced with 6x - Histidine - tagged ubiquitin ( His - Ubi ) were treated with DMSO or thapsigargin ( TG ) . These were lysed subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . Both arrowhead arrow point to mono - ubiquitinated Merlin . of mono - ubiquitinated to native Merlin in each lane of lysate blot was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

(G) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were treated with DMSO or thapsigargin (TG). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Both arrowhead and arrow point to the mono-ubiquitinated Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( H ) LN229 stably transduced with 6x - Histidine - tagged ubiquitin ( His - Ubi ) were detached or reseeded as in ( C ) . These were lysed subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . Arrowheads arrows point to mono - or di - ubiquitinated Merlin ( see a longer exposed blotting results in Figure EV1D ) . of mono - ubiquitinated to native Merlin in each lane of lysate blot was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

(H) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were detached or reseeded as in (C). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Arrowheads and arrows point to the mono- or di-ubiquitinated Merlin (see a longer exposed blotting results in Figure EV1D). The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( A ) LN229 were detached ( denoted by sus . , referring to suspension ) by trypsinization reseeded ( denoted by attach ) for two hours . Total lysates from these were subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(A) LN229 cells were detached (denoted by sus., referring to suspension) by trypsinization and reseeded (denoted by attach) for two hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( B ) LN229 treated with DMSO , ionomycin ( Iono ) or thapsigargin ( TG ) were lysed subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(B) LN229 cells treated with DMSO, ionomycin (Iono) or thapsigargin (TG) were lysed and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( C ) LN229 treated with DMSO or thapsigargin ( TG ) were subjected to active RAC1 pulldown followed by western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(C) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to active RAC1 pulldown assay followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


LN229 transduced with vector or EGFP - RAC1 ( Q61L ) ( D ) were treated with DMSO or thapsigargin ( TG ) subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

LN229 cells transduced with vector or EGFP-RAC1(Q61L) (D) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( E ) LN229 transduced with vector or Myc - PAK1 ( T423E ) ( E ) were treated with DMSO or thapsigargin ( TG ) subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(E) LN229 cells transduced with vector or Myc-PAK1(T423E) (E) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( F ) LN229 stably transduced with wild - type Merlin or indicated mutants were treated with DMSO or thapsigargin ( TG ) analyzed by western blot . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot . Native Merlin ( asterisk ) ; ubiquitinated Merlin ( arrow ) .

O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-EXP_ASSAY B-GENEPROD O O O O

(F) LN229 cells stably transduced with wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG) and analyzed by western blot. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Native Merlin (asterisk); ubiquitinated Merlin (arrow).


( G ) Merlin - depleted LN229 stably transduced with Flag - tagged wild - type Merlin or indicated mutants were treated with DMSO or thapsigargin ( TG ) . were lysed subjected to immunoprecipitation with a Flag antibody . lysate immunoprecipitated products were subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane of lysate blot was quantified by Image J is shown under blot .

O O O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

(G) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( B ) LN229 transduced with a pool of four siRNAs against NEDD4L ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane of short exposure blot was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

(B) LN229 cells transduced with a pool of four siRNAs against NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot.


( C ) LN229 stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were treated with DMSO or thapsigargin ( TG ) subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(C) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( D ) LN229 stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were detached by trypsinization ( see Methods ) subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(D) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were detached by trypsinization (see Methods) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( E ) LN229 stably transduced with 6x - Histidine - tagged Ubiquitin ( His - Ubi ) were then stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA . These were detached as in ( D ) , lysed subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . of mono - ubiquitinated to native Merlin in each lane of lysate blot was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

(E) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA. These cells were detached as in (D), lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( F ) HEK293T were transfected with indicated genes subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(F) HEK293T cells were transfected with indicated genes and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( G ) HEK293T were transfected with indicated genes . were lysed subjected to immunoprecipitation with a Flag antibody . immunoprecipitated products were subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane of lysate blot was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

(G) HEK293T cells were transfected with indicated genes. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( H ) LN229 were lysed subjected to immunoprecipitation with a Merlin antibody , a NEDD4L antibody , or IgG . immunoprecipitated products were subjected to western blotting .

O O O B-CELL_LINE O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O

(H) LN229 cells were lysed and subjected to immunoprecipitation with a Merlin antibody, a NEDD4L antibody, or IgG. The immunoprecipitated products were subjected to western blotting.


( A ) LN229 transduced with a pool of four siRNAs against AMOTL1 ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(A) LN229 cells transduced with a pool of four siRNAs against AMOTL1 (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( B ) LN229 stably transduced with 6x - Histidine - tagged Ubiquitin ( His - Ubi ) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control ( sh - Co . ) . These were detached , lysed subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . of mono - ubiquitinated to native Merlin in each lane of lysate blot was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

(B) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control (sh-Co.). These cells were detached, lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( D ) Merlin - depleted LN229 stably expressing Flag - HA - tagged Merlin were transduced with scrambled shRNA ( sh - Co . ) or two distinct shRNAs targeting AMOTL1 , treated with DMSO or thapsigargin ( TG ) subjected to Flag immunoprecipitation followed by western blotting .

O O O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O

(D) Merlin-depleted LN229 cells stably expressing Flag-HA-tagged Merlin were transduced with scrambled shRNA (sh-Co.) or two distinct shRNAs targeting AMOTL1, treated with DMSO or thapsigargin (TG) and subjected to Flag immunoprecipitation followed by western blotting.


( E ) HEK293T were transfected with indicated constructs . were lysed subjected to immunoprecipitation with a Flag antibody . lysate immunoprecipitated products were subjected to western blotting . PY : PPEY motif ; LY : LPTY motif . of mono - ubiquitinated to native Merlin in each lane of lysate blot was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

(E) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. PY: PPEY motif; LY: LPTY motif. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( F ) Merlin - depleted LN229 stably transduced with Flag - tagged wild - type Merlin or indicated mutants were treated with DMSO or thapsigargin ( TG ) . were lysed subjected to immunoprecipitation with a Flag antibody . lysate immunoprecipitated products were subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane of lysate blot was quantified by Image J is shown under blot .

(F) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( G ) HEK293T were transfected with indicated constructs . were lysed subjected to immunoprecipitation with a Flag antibody . lysate immunoprecipitated products were subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane of lysate blot was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

(G) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( A ) LN229 stably transduced with Flag - tagged wild - type Merlin or S518D mutant were treated with thapsigargin subjected to immunoprecipitation with a Flag antibody followed by SDS - PAGE . Outlined gel pieces were analyzed by mass spectrometry . ( B ) Numbers of unique peptides of Merlin or Ubiquitin from each outlined gel pieces in ( A ) are shown .

O O O B-CELL_LINE O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O B-GENEPROD O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O

(A) LN229 cells stably transduced with Flag-tagged wild-type Merlin or the S518D mutant were treated with thapsigargin and subjected to immunoprecipitation with a Flag antibody and followed by SDS-PAGE. Outlined gel pieces were analyzed by mass spectrometry. (B) Numbers of unique peptides of Merlin or Ubiquitin from each outlined gel pieces in (A) are shown.


( D ) ( E ) Mass spectrometry spectrums of indicated peptides from each immunoprecipitated product are shown .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O

(D) and (E) Mass spectrometry spectrums of the indicated peptides from each immunoprecipitated product are shown.


( F ) Merlin - depleted LN229 expressing His - Ubiquitin were stably transduced with indicated HA - tagged Merlin forms or an empty vector . These treated with DMSO or thapsigargin were subjected to nickel affinity purification in a denaturing step followed by western blotting for HA . Arrows indicate mono - ubiquitinated Merlin . red asterisk indicates native Merlin black asterisks indicate non - specific signals . of mono - ubiquitinated to native Merlin in each lane of input blot was quantified by Image J is shown under blot .

O O O B-GENEPROD O O B-CELL_LINE O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

(F) Merlin-depleted LN229 cells expressing His-Ubiquitin were stably transduced with the indicated HA-tagged Merlin forms or an empty vector. These cells treated with DMSO or thapsigargin were subjected to nickel affinity purification in a denaturing step followed by western blotting for HA. Arrows indicate mono-ubiquitinated Merlin. The red asterisk indicates native Merlin and black asterisks indicate non-specific signals. The ratio of mono-ubiquitinated to native Merlin in each lane of the input blot was quantified by Image J and is shown under the blot.


( G ) Merlin - depleted LN229 were stably transduced with an empty vector , Flag - tagged wild - type ( WT ) Merlin or its K396R mutant . These were detached ( S , suspension ) by trypsinization using procedure described in Methods section reseeded ( A , attached ) for two hours . lysates were subjected to immunoprecipitation with a Flag antibody followed by western blotting . of mono - ubiquitinated to native Merlin in each lane of lysate blot was quantified by Image J is shown under blot .

O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O

(G) Merlin-depleted LN229 cells were stably transduced with an empty vector, Flag-tagged wild-type (WT) Merlin or its K396R mutant. These cells were detached (S, suspension) by trypsinization using the procedure described in the Methods section and reseeded (A, attached) for two hours. Cell lysates were subjected to immunoprecipitation with a Flag antibody followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( H ) Merlin - depleted LN229 stably transduced with wild - type ( WT ) Merlin or its K396R mutant were treated with DMSO or thapsigargin ( TG ) subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(H) Merlin-depleted LN229 cells stably transduced with wild-type (WT) Merlin or its K396R mutant were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( I ) Merlin - depleted LN229 stably transduced with HA - tagged wild - type ( WT ) Merlin or its K396R mutant were treated as described in ( G ) subjected to western blotting . of mono - ubiquitinated to native null in each lane was quantified by Image J is shown under blot .

O O O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O

(I) Merlin-depleted LN229 cells stably transduced with HA-tagged wild-type (WT) Merlin or its K396R mutant were treated as described in (G) and subjected to western blotting. The ratio of mono-ubiquitinated to native null in each lane was quantified by Image J and is shown under the blot.


( A ) LN229 were treated with DMSO or thapsigargin ( TG ) for indicated time subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(A) LN229 cells were treated with DMSO or thapsigargin (TG) for the indicated time and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( B ) Merlin - depleted LN229 were reconstituted with wild type ( WT ) Merlin or its mutants , treated with DMSO or thapsigargin ( TG ) , subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(B) Merlin-depleted LN229 cells were reconstituted with wild type (WT) Merlin or its mutants, treated with DMSO or thapsigargin (TG), and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( C ) Merlin - depleted LN229 were reconstituted with wild type ( WT ) Merlin or its mutants , treated with DMSO or thapsigargin ( TG ) , subjected to western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

(C) Merlin-depleted LN229 cells were reconstituted with wild type (WT) Merlin or its mutants, treated with DMSO or thapsigargin (TG), and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( D ) LN229 transfected with a pool of four siRNAs targeting NEDD4L ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) subjected to western blotting .

O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O

(D) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting.


( E ) Merlin - depleted LN229 were stably transduced with empty vector , Flag - tagged wild - type ( WT ) Merlin , or its K396R mutant . These were detached ( S , suspension ) by trypsinization using procedure described in Methods reseeded ( A , attached ) for two hours . lysates were subjected to western blotting .

O O O B-GENEPROD O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O

(E) Merlin-depleted LN229 cells were stably transduced with empty vector, Flag-tagged wild-type (WT) Merlin, or its K396R mutant. These cells were detached (S, suspension) by trypsinization using the procedure described in Methods and reseeded (A, attached) for two hours. Cell lysates were subjected to western blotting.


( F ) LN229 transfected with a pool of four siRNAs targeting NEDD4L ( + ) or a scrambled siRNA ( - ) were detached ( Susp . , suspension ) by trypsinization using procedure described in Methods reseeded ( Attach , attached ) for two hours . Total lysates from these were subjected to western blotting .

O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O

(F) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L (+) or a scrambled siRNA (-) were detached (Susp., suspension) by trypsinization using the procedure described in Methods and reseeded (Attach, attached) for two hours. Total lysates from these cells were subjected to western blotting.


( G ) LN229 transfected with indicated siRNAs were seeded for 3 hours . CTGF or CYR61 mRNA in these was quantified by q - RT - PCR . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 . Mean ± s . e . m , N = 4 biological repeats , unpaired t - .

O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) LN229 cells transfected with indicated siRNAs were seeded for 3 hours. CTGF or CYR61 mRNA in these cells was quantified by q-RT-PCR. *P<0.05, **P<0.01, ****P<0.0001. Mean±s.e.m, N=4 biological repeats, unpaired t-test.


( A ) LN229 stably transduced withHA - Lats1 ( two leftmost panels ) or vector only ( right panel ) were treated with DMSO or thapsigargin ( TG ) subjected to PLA using HA Merlin antibodies . Scale bar = 10 μm . ( B ) PLA signals ( dots ) in each from results in ( A ) were quantified . Mean ± s . e . m , two - way ANOVA . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each data point represents an image field containing an average of 10 . N = 4 - 9 for each condition as indicated . were collected from one experiment . Two independent experiments were performed gave similar results .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O 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) LN229 cells stably transduced withHA-Lats1 (two leftmost panels) or vector only (right panel) were treated with DMSO or thapsigargin (TG) and subjected to PLA using HA and Merlin antibodies. Scale bar = 10 μm. (B) PLA signals (dots) in each cell from the results in (A) were quantified. Mean±s.e.m, two-way ANOVA. *P<0.05, ***P<0.001, ****P<0.0001. Each data point represents an image field containing an average of 10 cells. N=4-9 images for each condition as indicated. All images were collected from one experiment. Two independent experiments were performed and gave similar results.


( C ) Merlin - depleted LN229 were stably transduced with HA - tagged wild - type ( WT ) Merlin or its mutants . These were treated with thapsigargin ( TG ) for 15 minutes subjected to PLA using HA Lats1 antibodies . Scale bar = 20 μm . ( D ) PLA signals ( dots ) in each from results in ( C ) were quantified . Mean ± s . e . m , Ordinary one - way ANOVA . * * * * P < 0 . 0001 . Each data point represents an image field containing an average of 10 . NVector = 8 , NWT = 16 , NK396R = 18 , NK159R = 17 . were collected from one experiment . Two independent experiments were performed gave similar results .

O O O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Merlin-depleted LN229 cells were stably transduced with HA-tagged wild-type (WT) Merlin or its mutants. These cells were treated with thapsigargin (TG) for 15 minutes and subjected to PLA using HA and Lats1 antibodies. Scale bar = 20 μm. (D) PLA signals (dots) in each cell from the results in (C) were quantified. Mean±s.e.m, Ordinary one-way ANOVA. ****P<0.0001. Each data point represents an image field containing an average of 10 cells. NVector=8, NWT=16, NK396R=18, NK159R=17 images. All images were collected from one experiment. Two independent experiments were performed and gave similar results.


( E ) Merlin - depleted LN229 stably transduced with empty vector , Flag - tagged wild - type ( WT ) Merlin , or its K396R mutant were subjected to immunoprecipitation with a Flag antibody followed by western blotting .

O O O B-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(E) Merlin-depleted LN229 cells stably transduced with empty vector, Flag-tagged wild-type (WT) Merlin, or its K396R mutant were subjected to immunoprecipitation with a Flag antibody followed by western blotting.


( F ) HEK293T were transfected with indicated subjected to immunoprecipitation with a Flag antibody . lysates immunoprecipitated products were subjected to western blotting .

O O O B-CELL_LINE O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O

(F) HEK293T cells were transfected with indicated proteins and subjected to immunoprecipitation with a Flag antibody. The lysates and immunoprecipitated products were subjected to western blotting.


( G ) Merlin - depleted LN229 were stably transduced by HA - tagged Merlin . These were then transduced with a pool of four siRNAs against NEDD4L or a scrambled control siRNA treated with DMSO or thapsigargin ( TG ) . PLA was performed in these using HA Lats1 antibodies , PLA signals ( dots , showed in Figure EV5E ) in each were quantified . Mean ± s . e . m , two - way ANOVA . * * * * P < 0 . 0001 . Each data point represents an image field containing averagely 10 . N = 12 in conditions , except for Nsi - Control : TG = 13 . were collected from one experiment . Two independent experiments were performed showed similar results .

O O O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Merlin-depleted LN229 cells were stably transduced by HA-tagged Merlin. These cells were then transduced with a pool of four siRNAs against NEDD4L or a scrambled control siRNA and treated with DMSO or thapsigargin (TG). PLA was performed in these cells using HA and Lats1 antibodies, and the PLA signals (dots, showed in Figure EV5E) in each cell were quantified. Mean±s.e.m, two-way ANOVA. ****P<0.0001. Each data point represents an image field containing averagely 10 cells. N=12 images in all conditions, except for Nsi-Control:TG=13 images. All images were collected from one experiment. Two independent experiments were performed and showed similar results.


( H ) LN229 treated with DMSO or thapsigargin ( TG ) were subjected to ( C ) / ( M ) fractionation followed by western blotting . of mono - ubiquitinated to native Merlin in each lane was quantified by Image J is shown under blot .

O O O B-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

(H) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to cytosolic (C)/membrane (M) fractionation followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( A ) Met5 - A stably transduced with indicated shRNAs targeting Merlin or a scrambled shRNA were subjected to BrdU incorporation . Scale bar = 40 μm . ( B ) of BrdU positive from ( A ) was quantified . Mean ± s . e . m , N = 3 biological repeats , paired t - . * * P < 0 . 01 , * * * P < 0 . 001 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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) Met5-A cells stably transduced with the indicated shRNAs targeting Merlin or a scrambled shRNA were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (B) The percentage of BrdU positive cells from (A) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.01, ***P<0.001.


( C ) Met5 - A stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were subjected to BrdU incorporation . Scale bar = 40 μm . ( D ) of BrdU positive from ( C ) was quantified . Mean ± s . e . m , N = 3 biological repeats , paired t - . * * P < 0 . 01 , * * * P < 0 . 001 .

(C) Met5-A cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (D) The percentage of BrdU positive cells from (C) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.01, ***P<0.001.


( E ) Meso - 33 transduced with vector or indicated Merlin forms were subjected to BrdU incorporation . Scale bar = 40 μm . ( F ) of BrdU positive from ( E ) was quantified . Mean ± s . e . m , N = 3 biological repeats , ordinary one - way ANOVA . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Meso-33 cells transduced with vector or the indicated Merlin forms were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (F) The percentage of BrdU positive cells from (E) was quantified. Mean±s.e.m, N=3 biological repeats, ordinary one-way ANOVA. *P<0.05, **P<0.01.


( G ) Meso - 33 stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were then transduced with vector or Merlin prior to BrdU incorporation . of BrdU positive was quantified . Mean ± s . e . m , N = 3 biological repeats , two - way ANOVA .

O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

(G) Meso-33 cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were then transduced with vector or Merlin prior to the BrdU incorporation assay. The percentage of BrdU positive cells was quantified. Mean±s.e.m, N=3 biological repeats, two-way ANOVA.


( H ) FC - 1801 stably transduced with vector or indicated Merlin forms were subjected to sphere formation . Scale bar = 500 μm . ( I ) number of spheres in each well from ( H ) was quantified . Mean ± s . e . m , N = 3 biological repeats , paired t - . * * P < 0 . 05 .

O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) FC-1801 cells stably transduced with vector or the indicated Merlin forms were subjected to the sphere formation assay. Scale bar = 500 μm. (I) The number of spheres in each well from (H) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.05.


( J ) FC - 1801 stably transduced with vector or indicated Merlin forms were subcutaneously injected into nude mice . sizes in each group were quantified plotted . Mean ± s . e . m , NVector = 8 mice , NWT = 10 mice , NK396R = 10 mice , NS518D = 8 mice , two - way ANOVA . P values of last measurements ( day 37 after injection ) are shown . * P < 0 . 05 , * * P < 0 . 01 . ( K ) Tumors were collected when vector group in ( J ) reached end point ( day 37 after injection ) .

O O O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FC-1801 cells stably transduced with vector or the indicated Merlin forms were subcutaneously injected into nude mice. Tumor sizes in each group were quantified and plotted. Mean±s.e.m, NVector=8 mice, NWT=10 mice, NK396R=10 mice, NS518D=8 mice, two-way ANOVA. P values of the last measurements (day 37 after injection) are shown. *P<0.05, **P<0.01. (K) Tumors were collected when the vector group in (J) reached the end point (day 37 after injection).


Confocal microscopy of IBA - 1 ( green ) immunohistochemistry of RPE flatmounts ( RPE autofluorescence visible as orange due to its autofluorescence in red green channel ) from a healthy donor ( A ) , a geographic atrophy lesion ( B ) , large drusen ( C D ) . ( A , B , D ) : orthogonal Z - stack projection ; ( C ) : oblique Z - stack projection dissecting microscope appearance of postmortem large drusen after removal of overlaying retina ( inset ) .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O B-TISSUE O O O O

Confocal microscopy of IBA-1 (green staining) immunohistochemistry of RPE flatmounts (RPE autofluorescence visible as orange due to its autofluorescence in the red and green channel) from a healthy donor (A), a geographic atrophy lesion (B), and large drusen (C and D). (A, B, D): orthogonal Z-stack projection; (C): oblique Z-stack projection and dissecting microscope appearance of postmortem large drusen after removal of the overlaying retina (inset).


Double - labeling on subretinal side of retina ( to avoid masking by RPE autofluorescence ) of IBA - 1 + ( E , green ) CD18 ( F , red ; G , merge ) .

O O O O O O O B-TISSUE O O O O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O

Double-labeling on the subretinal side of the retina (to avoid masking by RPE autofluorescence) of IBA-1+ (E, green fluorescence) and CD18 (F, red fluorescence; G, merge).


Orthogonal lateral Z - stack of a subretinal IBA - 1 + ( green ) MPs adjacent to RPE ( orange autofluorescence ) in vicinity of a large drusen .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O

Orthogonal and lateral Z-stack of a subretinal IBA-1+ (green fluorescence) MPs adjacent to the RPE (orange autofluorescence) in the vicinity of a large drusen.


APOE ( A ) IBA - 1 ( B , merge in C ) double - labeling on paraffin sections of surgical specimens of human tonsils .

B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O

APOE (A) and IBA-1 (B, merge in C) double-labeling on paraffin sections of surgical specimens of human tonsils.


APOE ( D ) IBA - 1 ( E , merge in F ) double - labeling on subretinal aspect of a large drusen overlaying retina ( to avoid specific signal masking by RPE orange autofluorescence ) .

B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE O O O O O O O B-TISSUE O O O O

APOE (D) and IBA-1 (E, merge in F) double-labeling on the subretinal aspect of a large drusen overlaying retina (to avoid specific signal masking by the RPE orange autofluorescence).


APOE ( red ) immunohistochemistry of control donor tissue ( G ) adjacent to GA lesion ( H ) .

B-GENEPROD O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O

APOE (red staining) immunohistochemistry of control donor tissue (G) and adjacent to GA lesion (H).


IBA - 1 ( green , RPE far - red autofluorescence ) double - labeling of GA lesion shown in ( H ) . Representative from five donor eyes with large drusen , five donor eyes with GA , three healthy donors between 70 89 years old .

B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O B-SUBCELLULAR I-SUBCELLULAR O O B-ORGANISM B-TISSUE O O O O O O O O O O O O

IBA-1 (green staining, RPE far-red autofluorescence) double-labeling of the GA lesion shown in (H). Representative images from five donor eyes with large drusen, five donor eyes with GA, and three healthy donors between 70 and 89 years old.


Immunohistochemistry of APOE ( red ) IBA - 1 ( green ) on a section ( A , blue Hoechst ) subretinal side of a retinal flatmount ( B C ) from a 12 - month - old Cx3cr1GFP / GFP mouse ( representative of 3 independent experiments , experiments omitting antibody immunostaining served as negative controls ) . ONL : outer layer ; RPE : retinal pigment epithelium .

B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O

Immunohistochemistry of APOE (red) and IBA-1 (green) on a section (A, blue Hoechst) and the subretinal side of a retinal flatmount (B and C) from a 12-month-old Cx3cr1GFP/GFP mouse (representative of 3 independent experiments, experiments omitting the primary antibody immunostaining served as negative controls). ONL: outer nuclear layer; RPE: retinal pigment epithelium.


Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J Cx3cr1GFP / GFP monocytes cultured for 24 h in contact with POS of an overlaying retinal explant ( n = 4 / group per experiment ; Mann - Whitney U - , * P = 0 . 0286 , experiment was repeated twice with similar results ) . Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J Cx3cr1GFP / GFP FACS - sorted microglial , freshly extracted from adult brain ( n = 4 - 5 / group ; Mann - Whitney U - , C57BL / 6J versus Cx3cr1GFP / GFP * P = 0 . 0159 ) . Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J Cx3cr1GFP / GFP peritoneal macrophages cultured for 24 h with CX3CL1 . ( n = 4 / group per experiment ; Mann - Whitney U - , * P = 0 . 0286 . experiment was repeated twice with similar results ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O O B-SUBCELLULAR O O O B-TISSUE O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O O O O O B-TISSUE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP monocytes cultured for 24 h in contact with POS of an overlaying retinal explant (n = 4/group per experiment; Mann-Whitney U-test, *P = 0.0286, the experiment was repeated twice with similar results).Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP FACS-sorted microglial cells, freshly extracted from adult brain (n = 4-5/group; Mann-Whitney U-test, C57BL/6J versus Cx3cr1GFP/GFP *P = 0.0159).Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP peritoneal macrophages cultured for 24 h with CX3CL1. (n = 4/group per experiment; Mann-Whitney U-test, *P = 0.0286. The experiment was repeated twice with similar results).


APOE Western blot of equivalent amounts of supernatant from CX3CL1 - exposed C57BL / 6J Cx3cr1GFP / GFP peritoneal macrophages at 24 h . Soluble Mer receptor tyrosine kinase that is released constitutively from cultured macrophages served as a loading control . experiment was repeated twice with similar results .

B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O

APOE Western blot analysis of equivalent amounts of supernatant protein from CX3CL1-exposed C57BL/6J and Cx3cr1GFP/GFP peritoneal macrophages at 24 h. Soluble Mer receptor tyrosine kinase that is released constitutively from cultured macrophages served as a loading control. The experiment was repeated twice with similar results.


12 - month - old IBA - 1 stained RPE flatmounts of Cx3cr1GFP / GFP ( H ) Cx3cr1GFP / GFPApoE − / − ( I ) mice . of subretinal IBA - 1 + mononuclear phagocytes in 2 - month - old ( left ) 12 - month - old ( right ) mice of indicated strains ( n = 9 - 20 / group ANOVA / Dunnett at 12 months : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - at 12 months of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P < 0 . 0001 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

12-month-old IBA-1 stained RPE flatmounts of Cx3cr1GFP/GFP (H) and Cx3cr1GFP/GFPApoE−/− (I) mice.Quantification of subretinal IBA-1+ mononuclear phagocytes in 2-month-old (left) and 12-month-old (right) mice of the indicated strains (n = 9-20/group ANOVA/Dunnett test at 12 months: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test at 12 months of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).


of subretinal IBA - 1 + mononuclear phagocytes after 4 days of challenge of 2 - month - old mice of indicated strains ( n = 10 - 25 / group ANOVA / Dunnett : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P < 0 . 0001 ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

Quantification of subretinal IBA-1+ mononuclear phagocytes after 4 days of light challenge of 2-month-old mice of the indicated strains (n = 10-25/group ANOVA/Dunnett test: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).


IBA - 1 - ( green ) CD102 - ( red ) stained RPE flatmounts 7 days after injury of 2 - month - old Cx3cr1GFP / GFP ( E ) Cx3cr1GFP / GFPApoE − / − ( F ) mice . of subretinal IBA - 1 + mononuclear phagocytes on RPE counted on RPE at a of 0 - 500 μm to CD102 + CNV 7 days after injury of 2 - month - old mice of indicated strains ( n = 8 - 10 eyes / group ANOVA / Dunnett : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P < 0 . 0001 ) .

B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O B-TISSUE O O O O O O O O B-GENEPROD O B-TISSUE O O O O O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

IBA-1- (green) and CD102- (red) stained RPE flatmounts 7 days after laser injury of 2-month-old Cx3cr1GFP/GFP (E) and Cx3cr1GFP/GFPApoE−/− (F) mice.Quantification of subretinal IBA-1+mononuclear phagocytes on the RPE counted on the RPE at a distance of 0-500 μm to CD102+CNV 7 days after the laser injury of 2-month-old mice of the indicated strains (n = 8-10 eyes/group ANOVA/Dunnett test: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).


Representative image of a RPE flatmount 12 h after subretinal injection ( red marking ) of 4 μl PBS with 12 , 000 CFSE - stained thioglycollate - elicited peritoneal that contain 70 % macrophages ( inset close - up view ) . Quantifications of CFSE + F4 / 80 + macrophages at different time points after subretinal injections of C57BL / 6J Cx3cr1GFP / GFP CFSE + macrophages ( n = 5 / per group ( 12 h ) n = 6 / per group thereafter ; Mann - Whitney U - , C57BL / 6J versus Cx3cr1GFP / GFP : 1 day n = 20 / group * P < 0 . 0001 ; 2 day n = 6 / group * P = 0 . 0317 ) .

O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O 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-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O 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 image of a RPE flatmount 12 h after the subretinal injection (red marking) of 4 μl PBS with 12,000 CFSE-stained thioglycollate-elicited peritoneal cells that contain 70% macrophages (inset close-up view).Quantifications of CFSE+F4/80+ macrophages at different time points after subretinal injections of C57BL/6J and Cx3cr1GFP/GFP CFSE+ macrophages (n = 5/per group (12 h) and n = 6/per group thereafter; Mann-Whitney U-test, C57BL/6J versus Cx3cr1GFP/GFP: 1 day n = 20/group *P < 0.0001; 2 day n = 6/group *P = 0.0317).


Representative image of TUNEL / Hoechst double - 12 h after subretinal injection of C57BL / 6J CFSE + macrophages ( experiment repeated three times , inset close - up view ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O

Representative image of TUNEL/Hoechst double-staining 12 h after subretinal injection of C57BL/6J CFSE+ macrophages (experiment repeated three times, inset close-up view).


Representative cytometry of SSC - A / CFSE CD11b / F4 / 80 gated of eye suspensions prepared 24 h after injection of Cx3cr1GFP / GFP CFSE + macrophages cytometric of eye suspensions at 24 h after injection of C57BL / 6J Cx3cr1GFP / GFP macrophages into C57BL / 6J ( n = 16 - 20 / group ; Mann - Whitney U - , * P = 0 . 0024 ) .

O B-EXP_ASSAY O O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O B-TISSUE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD B-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

Representative cytometry images of SSC-A/CFSE and CD11b/F4/80 gated analysis of eye cell suspensions prepared 24 h after the injection of Cx3cr1GFP/GFP CFSE+ macrophages and cytometric quantification of eye cell suspensions at 24 h after the injection of C57BL/6J and Cx3cr1GFP/GFP macrophages into C57BL/6J (n = 16-20/group; Mann-Whitney U-test, *P = 0.0024).


of subretinal CFSE + on RPE retinal flatmounts 24 h after subretinal injections of CFSE + magnetic - bead - sorted bone marrow - derived monocytes ( Mo ) from C57BL / 6J Cx3cr1GFP / GFPmice into C57BL / 6Jmice ( n = 8 - 12 / group ; Mann - Whitney U - , * P = 0 . 0006 ) . of subretinal CFSE + on RPE retinal flatmounts 24 h after subretinal injections of CFSE + CD11bFACS - sorted brainmicroglial from C57BL / 6J Cx3cr1GFP / GFPmice into C57BL / 6Jmice ( n = 9 - 12 / group ; Mann - Whitney U - , * P = 0 . 0087 ) .

O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O 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-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

Quantification of subretinal CFSE+ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+ magnetic-bead-sorted bone marrow-derived monocytes (Mo) from C57BL/6J and Cx3cr1GFP/GFPmice into C57BL/6Jmice (n = 8-12/group; Mann-Whitney U-test, *P = 0.0006).Quantification of subretinal CFSE+ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+CD11bFACS-sorted brainmicroglial cells from C57BL/6J and Cx3cr1GFP/GFPmice into C57BL/6Jmice (n = 9-12/group; Mann-Whitney U-test, *P = 0.0087).


of subretinal CFSE + F4 / 80 + macrophages on RPE retinal flatmounts 24 h after subretinal injections of CFSE + macrophages from C57BL / 6J , Cx3cr1GFP / GFP , Cx3cr1GFP / GFPApoE − / − , ApoE − / − mice into C57BL / 6Jmice ( n = 8 - 12 / group ; one - way ANOVA / Dunnett of Cx3cr1GFP / GFP versus any other group * P ≤ 0 . 0001 ; Mann - Whitney U - , Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P = 0 . 0006 ) .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+macrophages from C57BL/6J, Cx3cr1GFP/GFP, Cx3cr1GFP/GFPApoE−/−, and ApoE−/−mice into C57BL/6Jmice (n = 8-12/group; one-way ANOVA/Dunnett test of Cx3cr1GFP/GFP versus any other group *P ≤ 0.0001; Mann-Whitney U-test, Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P = 0.0006).


of subretinal CFSE + F4 / 80 + macrophages on RPE retinal flatmounts 24 h after subretinal injections of C57BL / 6J CFSE + macrophages into C57BL / 6J with exogenously added APOE3 at 1 , 10 , or 100 μg / ml calculated intraocular concentrations ( n = 6 - 7 / group ; one - way ANOVA / Dunnett : C57BL / 6J versus 10 μg * P = 0 . 0488 ; C57BL / 6J versus 100 μg ‡ P = 0 . 006 . Mann - Whitney U - : C57BL / 6J versus 10 μg * P = 0 . 0012 ; C57BL / 6J versus 100 μg ‡ P = 0 . 0013 ) .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O B-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O O 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-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O

Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injections of C57BL/6J CFSE+ macrophages into C57BL/6J and with exogenously added APOE3 at 1, 10, or 100 μg/ml calculated intraocular concentrations (n = 6-7/group; one-way ANOVA/Dunnett test: C57BL/6J versus 10 μg *P = 0.0488; C57BL/6J versus 100 μg ‡P = 0.006. Mann-Whitney U-test: C57BL/6J versus 10 μg *P = 0.0012; C57BL/6J versus 100 μg ‡P = 0.0013).