A Autoradiography of in - organello mitochondrial translation products derived from HEK293 wild - type cells in the presence of tobramycin , incorporation of 35S - Met and 35S - Cys in MT - CO1 protein . Following in - organello translation proteins were immunoprecipitated and analyzed by autoradiography . Lane 1 , 3 : controls ; lanes 2 , 4 : in - organello translation in the presence of 1000 µM tobramycin . MT - CO1 bands were quantified and ratio of 35S - Cys / 35S - Met calculated , the 35S - Cys / 35S - Met ratio in the absence of tobramycin was set as 1 ( n = 3 ) , error bars indicate SEM .

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

A Autoradiography of in-organello mitochondrial translation products derived from HEK293 wild-type cells in the presence of tobramycin, incorporation of 35S-Met and 35S-Cys in MT-CO1 protein. Following in-organello translation proteins were immunoprecipitated and analyzed by autoradiography. Lane 1, 3: controls; lanes 2, 4: in-organello translation in the presence of 1000 µM tobramycin. MT-CO1 bands were quantified and ratio of 35S-Cys / 35S-Met calculated, the 35S-Cys / 35S-Met ratio in the absence of tobramycin was set as 1 (n=3), error bars indicate SEM.


B Ratios of 35S - Cys / 35S - Met labelled MT - CO1 and MT - CO2 proteins synthesized in in - organello translation and effect of tobramycin . Following in - organello translation in the presence of tobramycin ( 1000 µM , 2000 µM ) proteins were immunoprecipitated and analyzed by autoradiography . MT - CO1 and MT - CO2 bands were quantified and ratio of 35S - Cys / 35S - Met incorporation calculated ( n = 3 ) , error bars indicate SEM ; * p < 0 . 05 , ns , not significant ( Student ' s t - test ) .

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

B Ratios of 35S-Cys / 35S-Met labelled MT-CO1 and MT-CO2 proteins synthesized in in-organello translation and effect of tobramycin. Following in-organello translation in the presence of tobramycin (1000 µM, 2000 µM) proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 and MT-CO2 bands were quantified and ratio of 35S-Cys / 35S-Met incorporation calculated (n=3), error bars indicate SEM; *p<0.05, ns, not significant (Student's t-test).


Ratio of 35S - Cys and 35S - Met labelled MT - CO1 protein synthesized in in - organello translation using mitochondria from HEK293 MRPS5 wild - type cells and HEK MRPS5 mutant V336Y cells . Following in - organello translation , proteins were immunoprecipitated and analyzed by autoradiography . MT - CO1 bands quantified and ratios calculated . The ratio of the MRPS5 wild - type was set as 1 ( n = 8 clones ) , error bars indicate SD ; * * * p < 0 . 0001 ( Student ' s t - test ) .

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

Ratio of 35S-Cys and 35S-Met labelled MT-CO1 protein synthesized in in-organello translation using mitochondria from HEK293 MRPS5 wild-type cells and HEK MRPS5 mutant V336Y cells. Following in-organello translation, proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 bands quantified and ratios calculated. The ratio of the MRPS5 wild-type was set as 1 (n=8 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).


D Autoradiography of in - organello mitochondrial translation products derived from HEK293 MRPS5 wild - type cells in the presence of tobramycin ( 0 - 750 µM ) . Proteins were 35S - Met labeled and loaded either directly on SDS - PAGE ( total , top ) or following poly - lysine immunoprecipitation ( bottom ) . * MT - CO1 and * * MT - CO1 with poly - lysine extension .

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

D Autoradiography of in-organello mitochondrial translation products derived from HEK293 MRPS5 wild-type cells in the presence of tobramycin (0-750 µM). Proteins were 35S-Met labeled and loaded either directly on SDS-PAGE (total, top) or following poly-lysine immunoprecipitation (bottom). * MT-CO1 and ** MT-CO1 with poly-lysine extension.


E Quantification of MT - CO1 proteins in HEK293 MRPS5 wild - type cells and HEK293 MRPS5 mutant V336Y cells . In - organello mitochondrial translation in the presence of 35S - Met and 750 µM tobramycin . Proteins were loaded either directly on SDS - PAGE ( total ) or following poly - lysine immunoprecipitation and analyzed by autoradiography . For total protein samples the MT - CO1 band was quantified , for poly - lysine immunoprecipitated samples the extended MT - CO1 band was quantified and the ratio of extended MT - CO1 / total MT - CO1 calculated . The ratio of MRPS5 wild - type was set as 1 ( MRPS5 wild - type , n = 5 clones , MRPS5 V336Y , n = 7 clones ) , error bars indicate SD ; * * * p < 0 . 0001 ( Student ' s t - test ) .

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

E Quantification of MT-CO1 proteins in HEK293 MRPS5 wild-type cells and HEK293 MRPS5 mutant V336Y cells. In-organello mitochondrial translation in the presence of 35S-Met and 750 µM tobramycin. Proteins were loaded either directly on SDS-PAGE (total) or following poly-lysine immunoprecipitation and analyzed by autoradiography. For total protein samples the MT-CO1 band was quantified, for poly-lysine immunoprecipitated samples the extended MT-CO1 band was quantified and the ratio of extended MT-CO1/total MT-CO1 calculated. The ratio of MRPS5 wild-type was set as 1 (MRPS5 wild-type, n=5 clones, MRPS5 V336Y, n=7 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).


A Generation time ( n = 8 clones , ± SD ) .

A Generation time (n=8 clones, ± SD).


B Whole cell in - vivo mitochondrial translation as determined by 35S - Met incorporation ( n = 4 clones , ± SEM ) .

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

B Whole cell in-vivo mitochondrial translation as determined by 35S-Met incorporation (n=4 clones, ± SEM).


C Autoradiography of in - organello translation using 35S - Met labelling .

C Autoradiography of in-organello translation using 35S-Met labelling.


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . A OCR in brain mitochondria from 19 - months mutant mice ( black circle ) compared with OCR from wild - type mice ( white circle ) . The sequential injection of mitochondrial substrates and inhibitors is indicated by dotted lines Values corresponding to the different respiratory states are shown .

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

Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. A OCR in brain mitochondria from 19-months mutant mice (black circle) compared with OCR from wild-type mice (white circle). The sequential injection of mitochondrial substrates and inhibitors is indicated by dotted lines Values corresponding to the different respiratory states are shown.


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . Baseline OCR , effect of age ( p < 0 . 0001 ) and genotype ( p < 0 . 05 ) .

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

Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. Baseline OCR, effect of age (p<0.0001) and genotype (p<0.05).


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . C State 3 OCR ( after addition of ADP ) , effect of age ( p < 0 . 001 ) and genotype ( p < 0 . 0001 ) .

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

Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. C State 3 OCR (after addition of ADP), effect of age (p<0.001) and genotype (p<0.0001).


Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . D State 3 uncoupled OCR ( state 3u after addition of FCCP ) , effect of genotype ( p < 0 . 0001 ) and age - genotype interaction ( p < 0 . 0001 ) .

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

Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. D State 3 uncoupled OCR (state 3u after addition of FCCP), effect of genotype (p<0.0001) and age-genotype interaction (p<0.0001).


E Ratio of mitochondrial complex IV and complex II activities .

E Ratio of mitochondrial complex IV and complex II activities.


A Average distance to center as measure of anxiety in the open field ( ANOVA : genotype F1 , 24 = 16 . 24 p = 0 . 0005 , age F2 , 24 = 5 . 217 p = 0 . 0131 ) . Mutants show increased anxiety levels at 9 - and 14 - months .

A Average distance to center as measure of anxiety in the open field (ANOVA: genotype F1,24=16.24 p=0.0005, age F2,24=5.217 p=0.0131). Mutants show increased anxiety levels at 9- and 14-months.


B Estimated frequency of rearing activity as measure of exploratory drive in the open field ( ANOVA : genotype F1 , 24 = 39 . 40 p < 0 . 0001 , age x genotype F2 , 26 = 8 . 204 p = 0 . 0017 ) . Mutants showed little exploratory activity at 9 - and 14 - months .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Estimated frequency of rearing activity as measure of exploratory drive in the open field (ANOVA: genotype F1, 24=39.40 p<0.0001, age x genotype F2,26=8.204 p=0.0017). Mutants showed little exploratory activity at 9- and 14-months.


C Light - dark transition test , % time spent in the bright compartment ( ANOVA : genotype F1 , 16 = 7 . 393 p = 0 . 0152 ) . Reduced scores at 9 - months old animals indicate increased anxiety .

C Light-dark transition test, % time spent in the bright compartment (ANOVA: genotype F1,16=7.393 p=0.0152). Reduced scores at 9-months old animals indicate increased anxiety.


D Exploratory corner visits in dark and light phase during adaptation to the IntelliCage ( ANOVA : phase F1 , 18 = 617 . 2 p < 0 . 0001 ) . Corner visits during the dark phase as a correlate of exploratory activity showed premature reduction in 9 - months old animals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Exploratory corner visits in dark and light phase during adaptation to the IntelliCage (ANOVA: phase F1,18=617.2 p<0.0001). Corner visits during the dark phase as a correlate of exploratory activity showed premature reduction in 9-months old animals.


E Learning in the hippocampus - dependent IntelliCage serial reversal task ( ANOVA : day F2 , 56 = 26 . 71 p < 0 . 0001 ) . Scores provided no evidence for a mutation effect on learning rate or performance .

O O O O O O O O O O O O O O O O O O O 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 Learning in the hippocampus-dependent IntelliCage serial reversal task (ANOVA: day F2,56=26.71 p<0.0001). Scores provided no evidence for a mutation effect on learning rate or performance.


F Learning in the hippocampus - dependent IntelliCage chaining task ( ANOVA : day F2 , 54 = 450 . 1 p < 0 . 0001 ) . Scores provided no evidence for a mutation effect on learning rate or performance .

O O O O O O O O O O O O O O O O O O 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 Learning in the hippocampus-dependent IntelliCage chaining task (ANOVA: day F2,54=450.1 p<0.0001). Scores provided no evidence for a mutation effect on learning rate or performance.


G Water - maze place navigation , swim path during acquisition and reversal ( ANOVA : day F4 , 104 = 21 . 14 p < 0 . 0001 , genotype F1 , 26 = 13 . 11 p = 0 . 0013 , age x genotype F2 , 26 = 8 . 204 p = 0 . 0017 ) . All groups learned : controls showed reduced performance with increasing age , mutants showed poor performance already at 9 - and 14 - months .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Water-maze place navigation, swim path during acquisition and reversal (ANOVA: day F4,104=21.14 p<0.0001, genotype F1,26=13.11 p=0.0013, age x genotype F2,26=8.204 p=0.0017). All groups learned: controls showed reduced performance with increasing age, mutants showed poor performance already at 9- and 14-months.


H Number of wall approaches as indicator of poor coping with the test and non - spatial strategy choice ( ANOVA : genotype F1 , 26 = 18 . 31 p = 0 . 0002 , age F2 , 26 = 4 . 486 p = 0 . 0212 , age x genotype F2 , 26 = 7 . 068 p = 0 . 0035 ) . Number of wall approaches increased with age in controls and was prematurely increased in mutants at 9 - and 14 - months .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Number of wall approaches as indicator of poor coping with the test and non-spatial strategy choice (ANOVA: genotype F1,26=18.31 p=0.0002, age F2,26=4.486 p=0.0212, age x genotype F2,26=7.068 p=0.0035). Number of wall approaches increased with age in controls and was prematurely increased in mutants at 9- and 14-months.


Water - maze probe trial , spatial retention score for target zones 1 - 3 comprising 5 , 10 and 10 % of the pool surface versus remaining 75 % of surface ( ANOVA : zone F3 , 78 = 36 . 04 p < 0 . 0001 , genotype x zone F3 , 78 = 7 . 807 p = 0 . 0001 ) . Spatial selectivity declined with age in controls and was prematurely reduced in mutants at 9 - and 14 - months .

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

Water-maze probe trial, spatial retention score for target zones 1-3 comprising 5, 10 and 10% of the pool surface versus remaining 75% of surface (ANOVA: zone F3,78=36.04 p<0.0001, genotype x zone F3,78=7.807 p=0.0001). Spatial selectivity declined with age in controls and was prematurely reduced in mutants at 9- and 14-months.


Noise exposure and ABR measurements . Exposure of mice to broadband noise ( 2 - 10 kHz ) of either 108 or 110 dB SPL resulted in elevations of auditory thresholds . Thresholds in Mrps5V338Y / V338Y mutant mice ( black bars ) were significantly higher after exposure than thresholds in littermates ( open bars ) , ( p < 0 . 0001 , two - way ANOVA for combined 108 and 110 dB data ) . Frequencies of 12 , 24 and 32 kHz were selected for measurement to cover the mid - frequency to high - frequency regions of the cochlea . n = 6 for baseline littermates ; n = 6 for baseline mutants ; n = 3 for all noise exposures .

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

Noise exposure and ABR measurements. Exposure of mice to broadband noise (2-10 kHz) of either 108 or 110 dB SPL resulted in elevations of auditory thresholds. Thresholds in Mrps5V338Y/V338Y mutant mice (black bars) were significantly higher after exposure than thresholds in littermates (open bars), (p<0.0001, two-way ANOVA for combined 108 and 110 dB data). Frequencies of 12, 24 and 32 kHz were selected for measurement to cover the mid-frequency to high-frequency regions of the cochlea. n=6 for baseline littermates; n=6 for baseline mutants; n=3 for all noise exposures.


( A ) Fed rat liver lysosomes were extracted with 1 % of the indicated detergents and then subjected to sucrose gradient centrifugation . Aliquots collected from top to bottom were subjected to SDS - PAGE and immunoblotted for LAMP ‐ 2A and flotillin ‐ 1 . Stars indicate the aliquots of the gradient of translucent appearance ( highly enriched in lipids ) .

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

(A) Fed rat liver lysosomes were extracted with 1% of the indicated detergents and then subjected to sucrose gradient centrifugation. Aliquots collected from top to bottom were subjected to SDS-PAGE and immunoblotted for LAMP‐2A and flotillin‐1. Stars indicate the aliquots of the gradient of translucent appearance (highly enriched in lipids).


( B ) The distribution of LAMP ‐ 2A through the sucrose gradient after extraction with 1 % Triton X ‐ 114 was calculated from the densitometric quantification of immunoblots as the one shown here . Values are expressed as percentage of the total lysosomal levels and are mean + s . e . of four experiments .

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

(B) The distribution of LAMP‐2A through the sucrose gradient after extraction with 1% Triton X‐114 was calculated from the densitometric quantification of immunoblots as the one shown here. Values are expressed as percentage of the total lysosomal levels and are mean+s.e. of four experiments.


( A ) Lysosomes from livers of fed ( Fed ) or 24 h starved rats ( Stv ) were extracted with 1 % Triton X ‐ 114 and processed as in Figure 1 or the aliquots in the translucent region were pooled together as the detergent ‐ resistant ( DR ) fraction , and anything below this fraction was pooled , as the solubilized fraction ( Sol ) ( B ) . Lanes 5 - 6 are one ‐ tenth of the amount of lysosomes loaded in the gradient .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysosomes from livers of fed (Fed) or 24 h starved rats (Stv) were extracted with 1% Triton X‐114 and processed as in Figure 1 or the aliquots in the translucent region were pooled together as the detergent‐resistant (DR) fraction, and anything below this fraction was pooled, as the solubilized fraction (Sol) (B). Lanes 5-6 are one‐tenth of the amount of lysosomes loaded in the gradient.


( C ) Distribution of LAMP ‐ 2A ( L ‐ 2A ) and flotillin ‐ 1 ( Flot ‐ 1 ) in fed rats liver lysosomes extracted with 1 % Triton X ‐ 114 at 4 or 37 ° C . DR fractions were pooled as in ( B ) , and the aliquots below this region were divided in three fractions ( a soluble ( Sol ; aliquot 15 ) and two intermediate fractions ( light : aliquots 11 - 12 ; heavy : aliquots 13 - 14 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Distribution of LAMP‐2A (L‐2A) and flotillin‐1 (Flot‐1) in fed rats liver lysosomes extracted with 1% Triton X‐114 at 4 or 37°C. DR fractions were pooled as in (B), and the aliquots below this region were divided in three fractions (a soluble (Sol; aliquot 15) and two intermediate fractions (light: aliquots 11-12; heavy: aliquots 13-14).


( D ) Fed ratliverlysosomes were incubated with MBCD or filipin , as described under Material and methods , and then processed as in ( C ) . The mean value of the densitometric quantification of the LAMP ‐ from two experiments is shown at the bottom in ( C ) and ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fed ratliverlysosomes were incubated with MBCD or filipin, as described under Material and methods, and then processed as in (C). The mean value of the densitometric quantification of the LAMP‐ from two experiments is shown at the bottom in (C) and (D).


( A ) Lysosomalmembranes from fed ratlivers and their corresponding 1 % Triton X ‐ 114 ‐ resistant regions , isolated as described under Material and methods , were subjected to one ( left ) ‐ or two ‐ dimensional ( right ) electrophoresis and stained with Sypro Ruby .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysosomalmembranes from fed ratlivers and their corresponding 1% Triton X‐114‐resistant regions, isolated as described under Material and methods, were subjected to one (left)‐ or two‐dimensional (right) electrophoresis and stained with Sypro Ruby.


( B ) Immunoblot for the indicated proteins of the detergent resistant ( DR ) , intermediate ( Int ) and solubilized ( Sol ) fractions of lysosomal membranes obtained as in Figure 2 .

O O O O O O O O O O O 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) Immunoblot for the indicated proteins of the detergent resistant (DR), intermediate (Int) and solubilized (Sol) fractions of lysosomal membranes obtained as in Figure 2.


LAMP ‐ 2A localizes in cholesterol ‐ and GM ‐ 1 ‐ enriched intracellular membrane domains . Mouse fibroblasts grown on coverslips in the presence ( + ) or absence ( − ) of serum were fixed and labeled with filipin ( red ) ( A ) or incubated with Texas ‐ Red ‐ cholera toxin B subunit to label GM ‐ 1 ‐ enriched membrane regions ( red ) and then fixed .

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

LAMP‐2A localizes in cholesterol‐ and GM‐1‐enriched intracellular membrane domains. Mouse fibroblasts grown on coverslips in the presence (+) or absence (−) of serum were fixed and labeled with filipin (red) (A) or incubated with Texas‐Red‐cholera toxin B subunit to label GM‐1‐enriched membrane regions (red) and then fixed.


( B ) Both groups of cells were then subjected to indirect immunofluorescence for LAMP ‐ 2A ( green ) . Where indicated , cells were treated with MBCD ( 25 mM ) for 30 min before fixation . The merged images of both fluorophores are shown on the right . Insets show vesicles at higher magnification . Bar : 10 μm .

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

(B) Both groups of cells were then subjected to indirect immunofluorescence for LAMP‐2A (green). Where indicated, cells were treated with MBCD (25 mM) for 30 min before fixation. The merged images of both fluorophores are shown on the right. Insets show vesicles at higher magnification. Bar: 10 μm.


The quantification of the colocalization of the two fluorophores is shown in ( C ) . Values are expressed as the mean + s . e . of percentage of colocalization in 20 - 40 cells .

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

The quantification of the colocalization of the two fluorophores is shown in (C). Values are expressed as the mean+s.e. of percentage of colocalization in 20-40 cells.


( A , B ) Regions of the lysosomalmembrane resistant to 1 % Triton X ‐ 114 recovered from the sucrose gradients were fixed and processed for electron microscopy ( A ) or visualized without fixation by electron microscopy ( B ) . Arrows point to the membrane bilayer .

(A, B) Regions of the lysosomalmembrane resistant to 1% Triton X‐114 recovered from the sucrose gradients were fixed and processed for electron microscopy (A) or visualized without fixation by electron microscopy (B). Arrows point to the membrane bilayer.


( C ) Intact ratliverlysosomes ( top ) and DR regions of the lysosomal membranes ( bottom ) were fixed and subjected to immunogold labeling for LAMP ‐ 2A and LAMP ‐ 2B , and a gold ‐ conjugated secondary antibody as described under Material and methods . Top right panels show ' clusters ' of gold particles ( black arrows ) in individual lysosomes at higher magnification . Graph shows the labeling density ( gold particles per μm ) in the indicated fractions for both proteins . Values are mean + s . e . from the quantification of 15 different micrographs .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Intact ratliverlysosomes (top) and DR regions of the lysosomal membranes (bottom) were fixed and subjected to immunogold labeling for LAMP‐2A and LAMP‐2B, and a gold‐conjugated secondary antibody as described under Material and methods. Top right panels show 'clusters' of gold particles (black arrows) in individual lysosomes at higher magnification. Graph shows the labeling density (gold particles per μm) in the indicated fractions for both proteins. Values are mean+s.e. from the quantification of 15 different micrographs.


( D ) Intact rat liver lysosomes were fixed and subjected to double immunogold labeling for LAMP ‐ 2A and flotillin ‐ 1 . Representative lysosomes are shown . Far right panel : zoomed ‐ in image of the distribution of LAMP ‐ 2A in regions containing ( black arrows ) and not containing ( white arrows ) flotillin ‐ 1 . Bars : 0 . 2 μm .

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

(D) Intact rat liver lysosomes were fixed and subjected to double immunogold labeling for LAMP‐2A and flotillin‐1. Representative lysosomes are shown. Far right panel: zoomed‐in image of the distribution of LAMP‐2A in regions containing (black arrows) and not containing (white arrows) flotillin‐1. Bars: 0.2 μm.


( A ) Fed ratliverlysosomes were incubated in an isotonic buffer without additions , or in the presence of ratlivercytosol ( 25 μg ) , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ( GAPDH ) ( 25 μg ) , GST ‐ hsc70 ( 10 μg / ml ) , ATP ( 5 mM ) or the indicated combinations for 15 min at 37 ° C . Lysosomes incubated directly with apyrase or with cytosol treated with apyrase are shown in the right bottom panel . At the end of the incubation , lysosomes were subjected to Triton X ‐ 114 extraction , apyrase density gradient centrifugation and immunoblot as in Figure 2C . Graph shows changes in LAMP ‐ 2A distribution expressed as times the value in untreated lysosomes and are the mean of the densitometric quantification of 3 - 6 experiments as the one shown here . Because all the incubations were performed in the presence of protease inhibitors , total levels of LAMP ‐ 2A in lysosomes remained constant throughout the incubation .

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

(A) Fed ratliverlysosomes were incubated in an isotonic buffer without additions, or in the presence of ratlivercytosol (25 μg), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (25 μg), GST‐hsc70 (10 μg/ml), ATP (5 mM) or the indicated combinations for 15 min at 37°C. Lysosomes incubated directly with apyrase or with cytosol treated with apyrase are shown in the right bottom panel. At the end of the incubation, lysosomes were subjected to Triton X‐114 extraction, apyrase density gradient centrifugation and immunoblot as in Figure 2C. Graph shows changes in LAMP‐2A distribution expressed as times the value in untreated lysosomes and are the mean of the densitometric quantification of 3-6 experiments as the one shown here. Because all the incubations were performed in the presence of protease inhibitors, total levels of LAMP‐2A in lysosomes remained constant throughout the incubation.


( B ) Lysosomes from livers of fed rats , untreated or treated with paraquat ( + PQ ) , were processed as in ( A ) . Graph shows changes in LAMP ‐ 2A distribution induced by the PQ treatment , expressed as times the value in untreated animals and are the mean of three experiments .

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

(B) Lysosomes from livers of fed rats, untreated or treated with paraquat (+PQ), were processed as in (A). Graph shows changes in LAMP‐2A distribution induced by the PQ treatment, expressed as times the value in untreated animals and are the mean of three experiments.


( C ) Lysosomes with high ( CMA + ) and low ( CMA − ) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in ( A ) . Top left : Levels of two CMA components ( LAMP ‐ 2A and hsc70 ) and of an unrelated lysosomalmembrane protein ( LAMP ‐ 1 ) in each of the four groups of lysosomes . Bottom left : Percentage of total lysosomal LAMP ‐ 2A present in each fraction , determined by densitometric quantification of the immunoblots .

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

(C) Lysosomes with high (CMA+) and low (CMA−) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in (A). Top left: Levels of two CMA components (LAMP‐2A and hsc70) and of an unrelated lysosomalmembrane protein (LAMP‐1) in each of the four groups of lysosomes. Bottom left: Percentage of total lysosomal LAMP‐2A present in each fraction, determined by densitometric quantification of the immunoblots.


( A ) Degradation of long ‐ lived proteins in mouse fibroblasts ( NIH3T3 cells ) treated or not with 25 mM MBCD . Where indicated , 15 mM NH4Cl / 100 μM leupeptin ( NH4Cl + Leup ) were added into the medium . Values are the mean + s . e . of three experiments with triplicate samples .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Degradation of long‐lived proteins in mouse fibroblasts (NIH3T3 cells) treated or not with 25 mM MBCD. Where indicated, 15 mM NH4Cl/100 μM leupeptin (NH4Cl+Leup) were added into the medium. Values are the mean+s.e. of three experiments with triplicate samples.


( B ) Effect of MBCD on total rates of protein degradation , measured as in ( A ) , in wild ‐ type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP ‐ 2A ( LAMP ‐ 2A ( − ) ) . The ratio of degradation in MBCD ‐ treated and untreated cells is shown for cells maintained with or without 10 mM 3 ‐ methyladenine ( 3MA ) .

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

(B) Effect of MBCD on total rates of protein degradation, measured as in (A), in wild‐type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP‐2A (LAMP‐2A(−)). The ratio of degradation in MBCD‐treated and untreated cells is shown for cells maintained with or without 10 mM 3‐methyladenine (3MA).


( C ) Proteolysis of [ 14C ] GAPDH or a pool of [ 3H ] labeled cytosolic proteins ( pool proteins ) by isolated rat liver lysosomes , treated or not with MBCD . Broken : lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins . Values are expressed as percentage of the degradation by untreated lysosomes and are the mean + s . e . of three experiments with triplicate samples .

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

(C) Proteolysis of [14C]GAPDH or a pool of [3H]labeled cytosolic proteins (pool proteins) by isolated rat liver lysosomes, treated or not with MBCD. Broken: lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins. Values are expressed as percentage of the degradation by untreated lysosomes and are the mean+s.e. of three experiments with triplicate samples.


( D ) Lysosomal - mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol ( 50 μM ) . Top : Distribution of LAMP ‐ 2A after Triton X ‐ 114 extraction and sucrose density gradient centrifugation . Bottom : Ability of intact or disrupted lysosomal - mitochondrial fractions to proteolize [ 14C ] GAPDH . Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean ± s . e . of triplicate samples .

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

(D) Lysosomal-mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol (50 μM). Top: Distribution of LAMP‐2A after Triton X‐114 extraction and sucrose density gradient centrifugation. Bottom: Ability of intact or disrupted lysosomal-mitochondrial fractions to proteolize [14C]GAPDH. Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean±s.e. of triplicate samples.


( A ) Lysosomes from mouse fibroblasts transfected with wild ‐ type hLAMP ‐ 2A and maintained in the presence ( Serum + ) or absence ( serum − ) of serum were collected by centrifugation and subjected to Triton X ‐ 114 extraction , sucrose density gradient centrifugation and immunobloting for human ( hL2A ) or mouse ( mL2A ) LAMP ‐ 2A . Lane 1 : homogenate .

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

(A) Lysosomes from mouse fibroblasts transfected with wild‐type hLAMP‐2A and maintained in the presence (Serum+) or absence (serum−) of serum were collected by centrifugation and subjected to Triton X‐114 extraction, sucrose density gradient centrifugation and immunobloting for human (hL2A) or mouse (mL2A) LAMP‐2A. Lane 1: homogenate.


( B ) Amino - acid sequence of the transmembrane and cytosolic region of hLAMP - 2A and the mutations performed . Replaced amino acids are shadowed .

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

(B) Amino-acid sequence of the transmembrane and cytosolic region of hLAMP-2A and the mutations performed. Replaced amino acids are shadowed.


( C ) Lysosomes from mouse fibroblasts transfected with the indicated forms of hLAMP ‐ 2A and maintained in the presence of serum were processed as in ( A ) .

O O O O O O O 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) Lysosomes from mouse fibroblasts transfected with the indicated forms of hLAMP‐2A and maintained in the presence of serum were processed as in (A).


( D ) The percentage of total lysosomal hLAMP ‐ 2A present in each fraction was determined by densitometric quantification of immunoblots as the ones shown here . Values , corrected for the amount of mLAMP ‐ 2A in each fraction , are mean + s . e . of 3 - 4 experiments .

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

(D) The percentage of total lysosomal hLAMP‐2A present in each fraction was determined by densitometric quantification of immunoblots as the ones shown here. Values, corrected for the amount of mLAMP‐2A in each fraction, are mean+s.e. of 3-4 experiments.


( E ) Changes in hLAMP ‐ 2A distribution induced by serum removal . Values are expressed as times the values in cells maintained in the presence of serum and are the mean of two experiments . Changes in the three nonDR regions are shown together ( no DR ) .

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

(E) Changes in hLAMP‐2A distribution induced by serum removal. Values are expressed as times the values in cells maintained in the presence of serum and are the mean of two experiments. Changes in the three nonDR regions are shown together (no DR).


( F ) Proteolysis of a pool of 3 [ H ] labeled cytosolic proteins by intact ( left ) or disrupted ( right ) lysosomes isolated from untransfected mouse fibroblasts ( none ) or fibroblasts transfected with wild ‐ type ( WT ) or the indicated mutant forms of hLAMP ‐ 2A . Values are the mean + s . e . of two experiments with triplicate samples .

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

(F) Proteolysis of a pool of 3[H]labeled cytosolic proteins by intact (left) or disrupted (right) lysosomes isolated from untransfected mouse fibroblasts (none) or fibroblasts transfected with wild‐type (WT) or the indicated mutant forms of hLAMP‐2A. Values are the mean+s.e. of two experiments with triplicate samples.


( G ) Part of the lysosomes from ( F ) was treated with MBCD before the incubation with the radiolabeled proteins . Values are expressed as percentage of degradation by untreated lysosomes and are the mean + s . e . of two experiments with triplicate samples .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Part of the lysosomes from (F) was treated with MBCD before the incubation with the radiolabeled proteins. Values are expressed as percentage of degradation by untreated lysosomes and are the mean+s.e. of two experiments with triplicate samples.


( A ) The Triton X ‐ 114 ‐ resistant and soluble regions of the lysosomalmembrane were subjected to centrifugation through a continuous ( 10 - 80 % ) sucrose density gradient , SDS - PAGE and immunoblot for LAMP ‐ 2A . The molecular weight range of each fraction is as follows : fraction 1 ( 100 kDa ) ; fraction 2 ( 100 - 200 kDa ) ; fraction 3 ( 200 - 400 kDa ) ; fraction 4 ( 400 - 600 kDa ) ; fraction 5 ( > 600 kDa ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The Triton X‐114‐resistant and soluble regions of the lysosomalmembrane were subjected to centrifugation through a continuous (10-80%) sucrose density gradient, SDS-PAGE and immunoblot for LAMP‐2A. The molecular weight range of each fraction is as follows: fraction 1 (100 kDa); fraction 2 (100-200 kDa); fraction 3 (200-400 kDa); fraction 4 (400-600 kDa); fraction 5 (>600 kDa).


( B ) Fed rat liver lysosomes were incubated with 1 mM CaCl2 ( + Ca2 + ) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors ( PI ) . Samples were subjected to Triton X ‐ 114 extraction , sucrose density gradients centrifugation and LAMP ‐ 2A immunoblot .

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

(B) Fed rat liver lysosomes were incubated with 1 mM CaCl2 (+Ca2+) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors (PI). Samples were subjected to Triton X‐114 extraction, sucrose density gradients centrifugation and LAMP‐2A immunoblot.


( C ) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS - PAGE and immunobloted with antibodies against the cytosolic ( Top ; L ‐ 2A ) and luminal ( Bottom ; L ‐ 2 ) regions of LAMP ‐ 2A . Arrow : LAMP ‐ 2A proteolytic fragment ( Cuervo and Dice , 2000a ) .

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

(C) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS-PAGE and immunobloted with antibodies against the cytosolic (Top; L‐2A) and luminal (Bottom; L‐2) regions of LAMP‐2A. Arrow: LAMP‐2A proteolytic fragment (Cuervo and Dice, 2000a).


( D , E ) Rat liver lysosomes incubated or not with 1 mM CaCl2 ( + Ca2 + ) were subjected to Triton X ‐ 114 extraction and sucrose density gradients centrifugation . Levels of cathepsin A ( D ) and carboxypeptidase A activity ( E ) were measured in each fraction by immunoblot or using a standardized colorimetric assay , respectively . Values in ( E ) are expressed as times the activity in untreated membranes and are mean + s . e . of two experiments .

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

(D, E) Rat liver lysosomes incubated or not with 1 mM CaCl2 (+Ca2+) were subjected to Triton X‐114 extraction and sucrose density gradients centrifugation. Levels of cathepsin A (D) and carboxypeptidase A activity (E) were measured in each fraction by immunoblot or using a standardized colorimetric assay, respectively. Values in (E) are expressed as times the activity in untreated membranes and are mean+s.e. of two experiments.


C . Cathepsin activity assay in WT and Tpl2 [ D270A ] BMDMs 0 . 5 h after uptake of latex beads . BMDMs were stained with the Magic Red cathepsin L substrate ( red ) . Average fluorescence intensity of the cathepsin probe per cell was quantified ( n = 40 - 51 cells ) . E . pH assay in WT and Tpl2 [ D270A ] BMDMs upon 0 . 5 h after incubation with latex beads . BMDMs were stained with the LysoTracker Red DND - 99 dye ( red ) . Average fluorescence intensity of the Lysotracker Red probe per cell was quantified ( n = 95 - 126 cells ) . Data information : One representative experiment out of three shown . Error bars and shaded areas represent SEM . * * * * P < 0 . 0001 . Student ' s unpaired t - test .

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

C. Cathepsin activity assay in WT and Tpl2[D270A] BMDMs 0.5 h after uptake of latex beads. BMDMs were stained with the Magic Red cathepsin L substrate (red). Average fluorescence intensity of the cathepsin probe per cell was quantified (n = 40-51 cells). E. pH assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the LysoTracker Red DND-99 dye (red). Average fluorescence intensity of the Lysotracker Red probe per cell was quantified (n = 95-126 cells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Student's unpaired t-test.


G . Reactive oxygen species assay in WT and Tpl2 [ D270A ] BMDMs upon 0 . 5 h after incubation with latex beads . BMDMs were stained with the ROS Deep Red dye . Average fluorescence intensity of the ROS Deep Red probe per cell was quantified ( n = 80 - 110 cells ) Data information : One representative experiment out of three shown . Error bars and shaded areas represent SEM . * * * * P < 0 . 0001 . Student ' s unpaired t - test .

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

G. Reactive oxygen species assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the ROS Deep Red dye. Average fluorescence intensity of the ROS Deep Red probe per cell was quantified (n = 80-110 cells) Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Student's unpaired t-test.


Experiments were performed using murine BMDMs . ( C ) Cell extracts from LPS - stimulated Nfkb1 [ SSAA ] and Nfkb1 [ SSAA ] / Tpl2 [ D270A ] BMDMs were immunoblotted for the indicated antigens .

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

Experiments were performed using murine BMDMs. (C) Cell extracts from LPS-stimulated Nfkb1[SSAA] and Nfkb1[SSAA]/Tpl2[D270A] BMDMs were immunoblotted for the indicated antigens.


Experiments were performed using human primary monocyte - derived macrophages . ( K ) Cell extracts from LPS - stimulated , inhibitor - treated primary human macrophages were immunoblotted for p - ERK1 / 2 , ERK1 / 2 , p - p38 , p38 and HSP90 .

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

Experiments were performed using human primary monocyte-derived macrophages. (K) Cell extracts from LPS-stimulated, inhibitor-treated primary human macrophages were immunoblotted for p-ERK1/2, ERK1/2, p-p38, p38 and HSP90.


C . Protein intensities of V - ATPase subunits from phagosomes purified from WT and Tpl2 [ D270A ] BMDMs , ( n = 3 biological replicates ) . Data information : Data were analysed by Student ' s t - test . Error bars represent SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

C. Protein intensities of V-ATPase subunits from phagosomes purified from WT and Tpl2[D270A] BMDMs, (n = 3 biological replicates). Data information: Data were analysed by Student's t-test. Error bars represent SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.


D . Protein intensities of RAB5 and LAMP - 1 from phagosome proteome analysis of WT and Tpl2 [ D270A ] BMDMs ( n = 3 biological replicates ) ( left ) . Immunoblot of isolated phagosomes from WT and Tpl2 [ D270A ] BMDMs probed for RAB5 , LAMP - 1 and vimentin . Phagosomal fractions of two biological replicates were pooled . One representative experiment out of two shown ( right ) ( n = 2 ) . Data information : Data were analysed by Student ' s t - test . Error bars represent SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Protein intensities of RAB5 and LAMP-1 from phagosome proteome analysis of WT and Tpl2[D270A] BMDMs (n = 3 biological replicates) (left). Immunoblot of isolated phagosomes from WT and Tpl2[D270A] BMDMs probed for RAB5, LAMP-1 and vimentin. Phagosomal fractions of two biological replicates were pooled. One representative experiment out of two shown (right) (n = 2). Data information: Data were analysed by Student's t-test. Error bars represent SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.


A . TPL - 2 - dependent phosphoproteome following phagocytosis of latex beads ( 0 . 5 h ) was determined by TMT mass spectrometry . Volcano plot representing the significance ( - log10 P - values after Welch ' s t - test ) vs . phosphorylation fold change ( Welch difference ratios ) between WT and Tpl2 [ D270A ] BMDMs . Five biological replicates were analysed per genotype ( n = 5 ) . Three of the most highly and significantly downregulated phospho - sites in Tpl2 [ D270A ] BMDMs relative to WT , as well as unaltered DMXL1 phospho - sites , are shown .

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

A. TPL-2-dependent phosphoproteome following phagocytosis of latex beads (0.5 h) was determined by TMT mass spectrometry. Volcano plot representing the significance (-log10 P-values after Welch's t-test) vs. phosphorylation fold change (Welch difference ratios) between WT and Tpl2[D270A] BMDMs. Five biological replicates were analysed per genotype (n = 5). Three of the most highly and significantly downregulated phospho-sites in Tpl2[D270A] BMDMs relative to WT, as well as unaltered DMXL1 phospho-sites, are shown.


BMDMs of the indicated genotypes were infected with YFP - S . aureus ( MOI 10 ) for 1 h . ( B ) Representative images of YFP + BMDMs labelled with Magic Red cathepsin L substrate ( red ) and DAPI ( nuclear stain ) 1h post - infection . ( C ) Quantification of Magic Red cathepsin L staining from panel ( A ) . Average fluorescence intensity of Magic Red cathepsin L substrate per cell relative to fluorescence intensity of YFP - S . aureus ( n = 26 - 29 cells ) . ( D ) Quantification of Magic Red cathepsin L staining co - localised with YFP - S . aureus from panel ( A ) ( n = 26 - 29 cells ) . Data information : One representative experiment out of three shown . Error bars represent SEM . P < 0 . 01 , * * * * P < 0 . 0001 . Student ' s unpaired t - test .

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

BMDMs of the indicated genotypes were infected with YFP-S. aureus (MOI 10) for 1 h. (B) Representative images of YFP+ BMDMs labelled with Magic Red cathepsin L substrate (red) and DAPI (nuclear stain) 1h post-infection. (C) Quantification of Magic Red cathepsin L staining from panel (A). Average fluorescence intensity of Magic Red cathepsin L substrate per cell relative to fluorescence intensity of YFP-S.aureus (n = 26-29 cells). (D) Quantification of Magic Red cathepsin L staining co-localised with YFP-S. aureus from panel (A) (n = 26-29 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, **** P < 0.0001. Student's unpaired t-test.


BMDMs of the indicated genotypes were infected with YFP - S . aureus ( MOI 10 ) for 1 h . ( E ) Representative images of YFP + BMDMs labelled with LysoTracker Red DND - 99 dye ( red ) and DAPI , 1h post - infection with YFP - S . aureus ( green ) . ( F ) Quantification of LysoTracker Red signal from panel ( D ) . Average fluorescence intensity of LysoTracker Red per cell relative to fluorescence intensity of YFP - S . aureus ( n = 66 - 98 cells ) . ( G ) Quantification of LysoTracker Red co - localisation with YFP - S . aureus from panel ( D ) ( n = 66 - 98 cells ) . Data information : One representative experiment out of three shown . Error bars represent SEM . P < 0 . 01 , * * * * P < 0 . 0001 . Student ' s unpaired t - test .

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

BMDMs of the indicated genotypes were infected with YFP-S. aureus (MOI 10) for 1 h. (E) Representative images of YFP+ BMDMs labelled with LysoTracker Red DND-99 dye (red) and DAPI, 1h post-infection with YFP-S. aureus (green).(F) Quantification of LysoTracker Red signal from panel (D). Average fluorescence intensity of LysoTracker Red per cell relative to fluorescence intensity of YFP-S. aureus (n = 66-98 cells). (G) Quantification of LysoTracker Red co-localisation with YFP-S. aureus from panel (D) (n = 66-98 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, **** P < 0.0001. Student's unpaired t-test.


BMDMs of the indicated genotypes were infected with YFP - S . aureus ( MOI 10 ) for 1 h . ( H ) Representative images of BMDMs stained with an anti - EEA1 antibody ( red ) and DAPI , 1h post - infection with YFP - S . aureus ( green ) . ( I ) Quantification of EEA1 + S . aureus - containing phagosomes from panel ( G ) ( n = 27 cells ) . ( J ) Representative images of BMDMs stained with an anti - LAMP - 1 antibody ( red ) and DAPI , 1h post - infection with YFP - S . aureus ( green ) . ( K ) Quantification of LAMP - 1 + S . aureus phagosomes from panel ( I ) ( n = 25 cells ) . Data information : One representative experiment out of three shown . Error bars represent SEM . P < 0 . 01 , * * * * P < 0 . 0001 . Student ' s unpaired t - test .

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

BMDMs of the indicated genotypes were infected with YFP-S. aureus (MOI 10) for 1 h. (H) Representative images of BMDMs stained with an anti-EEA1 antibody (red) and DAPI, 1h post-infection with YFP-S. aureus (green). (I) Quantification of EEA1+ S. aureus-containing phagosomes from panel (G) (n = 27 cells). (J) Representative images of BMDMs stained with an anti-LAMP-1 antibody (red) and DAPI, 1h post-infection with YFP-S. aureus (green). (K) Quantification of LAMP-1+ S. aureus phagosomes from panel (I) (n = 25 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, **** P < 0.0001. Student's unpaired t-test.


E . BMDMs of the indicated genotypes were infected with GFP - C . rodentium ( MOI 2 ) for 0 . 5h . C . rodentium CFU were assessed at 2 h after infection and normalised to CFU at 0 . 5 h ( n = 12 ; 2 biological and 6 technical replicates per condition per experiment ) . As a control , WT cells were treated with bafilomycin A . Data information : One representative experiment out of three shown . Error bars represent SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , not significant ( ns ) . one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. BMDMs of the indicated genotypes were infected with GFP-C. rodentium (MOI 2) for 0.5h. C. rodentium CFU were assessed at 2 h after infection and normalised to CFU at 0.5 h (n = 12; 2 biological and 6 technical replicates per condition per experiment). As a control, WT cells were treated with bafilomycin A. Data information: One representative experiment out of three shown. Error bars represent SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001, not significant (ns). one-way ANOVA.


F . Representative images of GFP + BMDMs labelled with LysoTracker Red DND - 99 dye ( red ) and DAPI , 1h post - infection with GFP - C . rodentium ( green ) . G . Quantification of LysoTracker Red co - localisation with GFP - C . rodentium from panel ( F ) ( n = 54 - 63 cells ) . Data information : One representative experiment out of three shown . Error bars represent SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , not significant ( ns ) . P one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of GFP+ BMDMs labelled with LysoTracker Red DND-99 dye (red) and DAPI, 1h post-infection with GFP-C. rodentium (green). G. Quantification of LysoTracker Red co-localisation with GFP- C. rodentium from panel (F) (n = 54-63 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001, not significant (ns). P one-way ANOVA.


Kaplan Meier plots representing the survival difference between SKCM patients stratified according to the 40 % highest or lowest BNIP3 expression .

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

Kaplan Meier plots representing the survival difference between SKCM patients stratified according to the 40% highest or lowest BNIP3 expression.


Representative images of BNIP3 immunostaining in a benign nevus ( n = 5 ) , primary melanoma and their paired metastasis ( n = 7 ) . In the representative pictures , nevi or tumor tissue is circled in yellow and highlighted with a yellow asterisk ( * ) . Histopathological scoring of BNIP3 protein levels in nevi and melanoma patients with paired primary and metastases harboring weak and in gradient ( + / - ) , weak and homogeneous ( + ) or strong and homogeneous ( + + ) BNIP3 protein levels based on histopathological staining intensity and distribution scoring . Scale bars represent 150µm .

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

Representative images of BNIP3 immunostaining in a benign nevus (n=5), primary melanoma and their paired metastasis (n=7). In the representative pictures, nevi or tumor tissue is circled in yellow and highlighted with a yellow asterisk (*). Histopathological scoring of BNIP3 protein levels in nevi and melanoma patients with paired primary and metastases harboring weak and in gradient (+/-), weak and homogeneous (+) or strong and homogeneous (++) BNIP3 protein levels based on histopathological staining intensity and distribution scoring. Scale bars represent 150µm.


Representative images of BNIP3 immunohistochemistry in TMAs containing ( n = 158 ) melanoma metastases and related Kaplan Meier plot of survival difference comparing melanoma patients with different BNIP3 histopathological scores . Scale bars represent 50µm .

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

Representative images of BNIP3 immunohistochemistry in TMAs containing (n=158) melanoma metastases and related Kaplan Meier plot of survival difference comparing melanoma patients with different BNIP3 histopathological scores. Scale bars represent 50µm.


Growth curves of B16 - F10 tumors generated with cells transduced with either empty vector ( CntlKD ) , shRNA against BNIP3 ( BNIP3KD ) or ATG5 ( ATG5KD ) ( n = 7 per condition ) and analyzed with a two - way ANOVA with Tukey ' s multiple comparisons test .

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

Growth curves of B16-F10 tumors generated with cells transduced with either empty vector (CntlKD), shRNA against BNIP3 (BNIP3KD) or ATG5 (ATG5KD) (n=7 per condition) and analyzed with a two-way ANOVA with Tukey's multiple comparisons test.


Haematoxylin and eosin staining of sections representative of CntlKD , BNIP3KD or ATG5KD B16 - F10 tumors . Necrotic areas are highlighted with an asterisk ( * ) . Quantification of TUNEL + tumor area ( representing the fraction of dying / dead cell population ) of CntlKD , BNIP3KD or ATG5KD B16 - F10 tumors ( n = 5 per condition ) . Quantification of the Ki67 + cells within the tumor area ( representing the fraction of proliferative cell populations in non - necrotic areas ) of CntlKD ( n = 7 ) , BNIP3KD ( n = 6 ) and ATG5KD ( n = 8 ) B16 - F10 tumors .

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

Haematoxylin and eosin staining of sections representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors. Necrotic areas are highlighted with an asterisk (*). Quantification of TUNEL+ tumor area (representing the fraction of dying/dead cell population) of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors (n= 5 per condition). Quantification of the Ki67+ cells within the tumor area (representing the fraction of proliferative cell populations in non-necrotic areas) of CntlKD (n=7), BNIP3KD (n=6) and ATG5KD (n=8) B16-F10 tumors.


Immunofluorescent staining for Ki67 ( red ) representative of CntlKD , BNIP3KD or ATG5KD B16 - F10 tumor sections . Immunofluorescent staining for LC3B ( orange granularity ) representative of CntlKD , BNIP3KD or ATG5KD B16 - F10 tumor sections . A zoomed - in area is shown in the lower - left corner .

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

Immunofluorescent staining for Ki67 (red) representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumor sections. Immunofluorescent staining for LC3B (orange granularity) representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumor sections. A zoomed-in area is shown in the lower-left corner.


Confocal microscopy images of co - localization analysis of the BNIP3 - proficient ( shCntl , n = 27 cells ) or deficient ( shBNIP3 , n = 31 cells ) B16 - F10 cells transiently transfected with GFP - LC3 and immunostained for TOMM20 ( mitochondrial marker ) . Colocalization events of GFP - LC3 puncta ( green ) with TOMM20 ( red ) are highlighted with white arrows . The percentage of GFP - LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann - Whitney ' s non - parametric test . Scale bars represent 10µm .

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

Confocal microscopy images of co-localization analysis of the BNIP3-proficient (shCntl, n=27 cells) or deficient (shBNIP3, n=31 cells) B16-F10 cells transiently transfected with GFP-LC3 and immunostained for TOMM20 (mitochondrial marker). Colocalization events of GFP-LC3 puncta (green) with TOMM20 (red) are highlighted with white arrows. The percentage of GFP-LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann-Whitney's non-parametric test. Scale bars represent 10µm.


Glut1 , Hk2 , Pkm1 , Pkm2 , Pdk1 , Mct1 , Ldha , Ldhb and Mct4 transcript levels measured by qPCR from the corresponding B16 - F10 cell lysates . Transcript expression is represented as the fold - change relative to their corresponding shCntl value . Heatmap shows the average values in each condition ( n = 4 ) and they were analyzed with a one - sample T - test only against shCntl , except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test .

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

Glut1, Hk2, Pkm1, Pkm2, Pdk1, Mct1, Ldha, Ldhb and Mct4 transcript levels measured by qPCR from the corresponding B16-F10 cell lysates. Transcript expression is represented as the fold-change relative to their corresponding shCntl value. Heatmap shows the average values in each condition (n=4) and they were analyzed with a one-sample T-test only against shCntl, except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA and ACTIN protein levels from lysates of B16 - F10 cells ( n = 4 ) or B16 - F10 tumors [ CntlKD ( n = 5 ) , BNIP3KD ( n = 5 ) and ATG5KD ( n = 7 ) ] ) . The HIF - 1α - OH / HIF - 1α ratio is shown in a bar graph below . Densitometric quantifications relative to ACTIN levels are shown below each corresponding band . Analysis was performed as indicated in the Data Information section , unless for HIF - 1α / Actin ratio ( in vivo ) , which was analyzed with Kruskal - Wallis non - parametric test with Dunn ' s multiple comparisons test , and LDHA / Actin ratio for shATG5 condition ( in vitro ) , which was analyzed with Wilcoxon ' s rank test .

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

Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from lysates of B16-F10 cells (n=4) or B16-F10 tumors [CntlKD (n=5), BNIP3KD (n=5) and ATG5KD (n=7)]). The HIF-1α-OH /HIF-1α ratio is shown in a bar graph below. Densitometric quantifications relative to ACTIN levels are shown below each corresponding band. Analysis was performed as indicated in the Data Information section, unless for HIF-1α/Actin ratio (in vivo), which was analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test, and LDHA/Actin ratio for shATG5 condition (in vitro), which was analyzed with Wilcoxon's rank test.


Immunohistochemical staining for HIF - 1α ( brown ) representative from CntlKD , BNIP3KD and ATG5KD , B16 - F10 tumor tissues . Higher magnification of the highlighted ( with a white square ) stained sections is shown below .

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

Immunohistochemical staining for HIF-1α (brown) representative from CntlKD, BNIP3KD and ATG5KD, B16-F10 tumor tissues. Higher magnification of the highlighted (with a white square) stained sections is shown below.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA , PHD2 and ACTIN protein levels from normoxic lysates of B16 - F10 cells transfected in presence of non - targeting siRNA sequences ( Scr ) or siRNA against PHD2 .

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

Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, PHD2 and ACTIN protein levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against PHD2.


Relative intracellular Fe2 + levels in normoxic B16 - F10 cells ( n = 4 ) . Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one - way ANOVA ( Geisser - Greenhouse correction ) with Holm - Sidak ' s multiple comparisons test . Scale bars represent 50µm .

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

Relative intracellular Fe2+ levels in normoxic B16-F10 cells (n=4). Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test. Scale bars represent 50µm.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA , NCOA4 and ACTIN levels from normoxic lysates of B16 - F10 cells transfected in presence of non - targeting siRNA sequences ( Scr ) or siRNA against NCOA4 ( n = 4 ) .

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

Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, NCOA4 and ACTIN levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against NCOA4 (n=4).


Growth curves of B16 - F10 tumors generated by shCntl or shBNIP3 cells expressing Luciferase ( Luc ) or an undegradable HIF - 1α ( HIF - 1α - AA ) construct ( n = 7 for CntlKD Luc / BNIP3KD HIF - 1α - AA , n = 8 CntlKD HIF - 1α - AA and n = 5 for BNIP3KD Luc ) and analyzed with a two - way ANOVA with Tukey ' s multiple comparisons test . Representative double immunostaining for CD31 and αSMA in B16 - F10 tumors ( n = 5 per condition ) . Scale bars represent 50µm .

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

Growth curves of B16-F10 tumors generated by shCntl or shBNIP3 cells expressing Luciferase (Luc) or an undegradable HIF-1α (HIF-1α-AA) construct (n=7 for CntlKD Luc/BNIP3KD HIF-1α-AA, n=8 CntlKD HIF-1α-AA and n=5 for BNIP3KD Luc) and analyzed with a two-way ANOVA with Tukey's multiple comparisons test. Representative double immunostaining for CD31 and αSMA in B16-F10 tumors (n=5 per condition). Scale bars represent 50µm.


HEK293T cells were transfected with 200 nM siRNAs per well . After 24 h , the cells were infected with HIV - luc / 2019 - nCoV pseudoviruses . After washing and incubation with fresh medium for 48 h , the intracellular luciferase activity was measured . Ordinary one - way ANOVA test was used for this analysis . The results are shown as the mean and SEM . * P < 0 . 05 .

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

HEK293T cells were transfected with 200 nM siRNAs per well. After 24 h, the cells were infected with HIV-luc/2019-nCoV pseudoviruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. Ordinary one-way ANOVA test was used for this analysis. The results are shown as the mean and SEM. *P< 0.05.


assay . B , Teicoplanin inhibit the entry of 2019 - nCoV and SARS - nCoV . Chemical structure of teicoplanin ( Left ) . A549 cells were seeded in a 96 - well plate , and 24 h later , the cells were infected with HIV - luc / 2019 - nCoV , HIV - luc / SARS - nCoV or HIV - luc / VSVG pseudoviruses . After washing and incubation with fresh medium for 48 h , the intracellular luciferase activity was measured . Ordinary one - way ANOVA test was used for this analysis . The results are shown as the mean and SEM . * P < 0 . 05 .

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

assay.B, Teicoplanin inhibit the entry of 2019-nCoV and SARS-nCoV. Chemical structure of teicoplanin (Left). A549 cells were seeded in a 96-well plate, and 24 h later, the cells were infected with HIV-luc/2019-nCoV, HIV-luc/SARS-nCoV or HIV-luc/VSVG pseudoviruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. Ordinary one-way ANOVA test was used for this analysis. The results are shown as the mean and SEM. *P< 0.05.C


, A549 cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software .

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

, A549 cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.


types . A , HEK293T cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software .

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

types.A, HEK293T cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.B


software . B , Huh7 cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software .

software.B, Huh7 cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.C


A549 cells were incubated with dalbavancin ( D ) at various concentrations and infected with HIV - luc / 2019 - nCoV pseudoviruses . The intracellular luciferase activity was measured at 48 h post - infection . Ordinary one - way ANOVA test was used for this analysis .

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

A549 cells were incubated with dalbavancin (D) at various concentrations and infected with HIV-luc/2019-nCoV pseudoviruses. The intracellular luciferase activity was measured at 48 h post-infection. Ordinary one-way ANOVA test was used for this analysis. dalbavancin


D , A549 cells were incubated with vancomycin at various concentrations and infected with HIV - luc / 2019 - nCoV pseudoviruses . The intracellular luciferase activity was measured at 48 h post - infection . Ordinary one - way ANOVA test was used for this analysis . Left : Chemical structure of

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

D, A549 cells were incubated with vancomycin at various concentrations and infected with HIV-luc/2019-nCoV pseudoviruses. The intracellular luciferase activity was measured at 48 h post-infection. Ordinary one-way ANOVA test was used for this analysis. Left: Chemical structure of vancomycin


( A ) NIH3T3 fibroblasts co - expressing DsRed - CA7 and EGFP - CA2 ( n = 4 independent replicates ) .

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

(A) NIH3T3 fibroblasts co-expressing DsRed-CA7 and EGFP-CA2 (n = 4 independent replicates).


( B - E ) Co - localization of EGFP and the two CA isoforms with filamentous actin studied in fibroblasts expressing ( B ) EGFP , ( C ) EGFP - CA2 , or ( D , E ) EGFP - CA7 and stained with phalloidin - 594 to visualize F - actin ( n = 4 , 10 and 8 independent replicates , respectively ) . A magnification of the area marked with the yellow rectangle in ( C ) and ( D ) shows the localization of EGFP - CA2 and EGFP - CA7 compared to phalloidin - 594 . ( E ) EGFP - CA7 caused a prominent overexpression phenotype with thick and curvy cytosolic actin bundles ( arrow ) and plasmalemmal protrusions ( arrowhead ) .

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

(B-E) Co-localization of EGFP and the two CA isoforms with filamentous actin studied in fibroblasts expressing (B) EGFP, (C) EGFP-CA2, or (D, E) EGFP-CA7 and stained with phalloidin-594 to visualize F-actin (n = 4, 10 and 8 independent replicates, respectively). A magnification of the area marked with the yellow rectangle in (C) and (D) shows the localization of EGFP-CA2 and EGFP-CA7 compared to phalloidin-594. (E) EGFP-CA7 caused a prominent overexpression phenotype with thick and curvy cytosolic actin bundles (arrow) and plasmalemmal protrusions (arrowhead).


( F - H ) The normalized fluorescence emission intensity profiles for F - actin ( red line ) and ( F ) EGFP , ( G ) EGFP - CA2 , or ( H ) EGFP - CA7 ( black 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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O

(F-H) The normalized fluorescence emission intensity profiles for F-actin (red line) and (F) EGFP, (G) EGFP-CA2, or (H) EGFP-CA7 (black line).


( A ) Actin co - sedimentation assay shows that CA7 binds to F - actin . The binding is enhanced at more acidic pH ( 6 . 5 vs . 7 . 4 ) n = 3 independent replicates at each actin concentration , two - way ANOVA , P < 0 . 001 .

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

(A) Actin co-sedimentation assay shows that CA7 binds to F-actin. The binding is enhanced at more acidic pH (6.5 vs. 7.4) n = 3 independent replicates at each actin concentration, two-way ANOVA, P < 0.001.


( B ) Fluorescence time ‐ lapse images of F - actin bundling in an in vitro bundling assay . A mixture of unlabeled and Rhodamine labelled non - muscle actin was polymerized in the absence ( PBS control , upper panel ) or presence of mCA7 ( lower panel ) . Numbers in images indicate the time after the onset of the experiment ( 0 , 5 and 23 min ) . Intensity based Fire - coloring ( Fiji ) was used to visualize intensity changes . Scale bar 10 µm . ( C ) Quantification of the mean increase in filament length ( n = 10 filaments at each time point ) and the mean relative fluorescence intensity values of cross - sections for individual filaments / bundles ( n = 30 - 31 ) in the absence and presence of mCA7 ( 1 . 12 µM ) . The data were analyzed using a general mixed model with time as a within - unit factor and the presence of CA7 as a between - unit factor . n = 3 independent repetitions , experiment repeats were included as a covariate and were non - significant .

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

(B) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the absence (PBS control, upper panel) or presence of mCA7 (lower panel). Numbers in images indicate the time after the onset of the experiment (0, 5 and 23 min). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. Scale bar 10 µm. (C) Quantification of the mean increase in filament length (n = 10 filaments at each time point) and the mean relative fluorescence intensity values of cross-sections for individual filaments /bundles (n = 30 - 31) in the absence and presence of mCA7 (1.12 µM). The data were analyzed using a general mixed model with time as a within-unit factor and the presence of CA7 as a between-unit factor. n = 3 independent repetitions, experiment repeats were included as a covariate and were non-significant.


( D ) Kymographs showing different time points in the line of interest ( line width 1 µm ) from the experiments analyzed in ( C ) . Kymographs were generated with Fiji Multi Kymograph function . Total time is 159 frames = 26 . 5 minutes . Scale bar 5 μm ( full height is 20 μm ) . F - actin bundles can be detected as clear lines in kymographs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kymographs showing different time points in the line of interest (line width 1 µm) from the experiments analyzed in (C). Kymographs were generated with Fiji Multi Kymograph function. Total time is 159 frames = 26.5 minutes. Scale bar 5 μm (full height is 20 μm). F-actin bundles can be detected as clear lines in kymographs.


( E ) Fluorescence time ‐ lapse images of F - actin bundling in an in vitro bundling assay . A mixture of unlabeled and Rhodamine labelled non - muscle actin was polymerized in the presence of mCA7 ( 0 . 11 µM ) . Numbers above images indicate the time after the onset of the experiment ( 22 min - 23 min 20 sec ) . Intensity based Fire - coloring ( Fiji ) was used to visualize intensity changes . White arrows highlight the bundling filaments . Scale bar 5 μm . This is a representative video from 8 similar experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the presence of mCA7 (0.11 µM). Numbers above images indicate the time after the onset of the experiment (22 min -23 min 20 sec). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. White arrows highlight the bundling filaments. Scale bar 5 μm. This is a representative video from 8 similar experiments.


( A , B ) NIH3T3 cells transfected with DsRed ( A ) or DsRed - CA7 ( B ) were incubated in growth medium with 5 µM Latrunculin B for 0 , 2 , 5 , 10 , or 30 minutes , or in an equal amount of DMSO for 60 minutes . Analyses of experiments show that in cells transfected with DsRed - CA7 F - actin structures collapse more slowly ( 0 min : 83 % " normal " ; 2 min : 80 % , p = 0 . 44 ; 5 min : 74 % , p = 0 . 39 ; 10 min : 27 % , p = 0 . 08 ; 30 min : 16 % , p = 0 . 02 ; DMSO : 82 % , p = 0 . 99 ; tested against 0 min with two - way ANOVA , Dunnett ' s multiple comparison test ) than in the DsRed - transfected ones ( 0 min : 89 % " normal " ; 2 min : 48 % , p = 0 . 14 ; 5 min : 31 % , p = 0 . 04 ; 10 min : 2 % , p = 0 . 001 ; 30 min : 0 . 7 % , p = 0 . 001 ; DMSO : 88 % , p = 0 . 8 ; tested against 0 min with two - way ANOVA , Dunnett ' s multiple comparison test ) . For the analysis , cells were categorized to three groups as " normal " , " some shape / F - actin left " and " round " . The upper panel shows example images of the cells in all three categories for ( A ) DsRed - or ( B ) DsRed - CA7 transfected cells ( actin visualized with Phalloidin - 488 ) .

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

(A,B) NIH3T3 cells transfected with DsRed (A) or DsRed-CA7 (B) were incubated in growth medium with 5 µM Latrunculin B for 0, 2, 5, 10, or 30 minutes, or in an equal amount of DMSO for 60 minutes. Analyses of experiments show that in cells transfected with DsRed-CA7 F-actin structures collapse more slowly (0 min: 83% "normal"; 2 min: 80%, p = 0.44; 5 min: 74%, p = 0.39; 10 min: 27%, p = 0.08; 30 min: 16%, p = 0.02; DMSO: 82%, p = 0.99; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) than in the DsRed-transfected ones (0 min: 89% "normal"; 2 min: 48%, p = 0.14; 5 min: 31%, p = 0.04; 10 min: 2%, p = 0.001; 30 min: 0.7%, p = 0.001; DMSO: 88%, p = 0.8; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) . For the analysis, cells were categorized to three groups as "normal", "some shape/F-actin left" and "round". The upper panel shows example images of the cells in all three categories for (A) DsRed- or (B) DsRed-CA7 transfected cells (actin visualized with Phalloidin-488).


( A - C ) NIH3T3 fibroblasts expressing EGFP - CA7 - mutant1 ( A ) , EGFP - CA7 - mutant2 ( B ) , and EGFP - CA7 - mutant3 ( C ) . F - actin is visualized with Phalloidin - 594 . In the right - most panel of ( A - C ) are the normalized fluorescence intensity profiles of the mutated CA7 EGFP signal ( black ) and actin ( red ) and the yellow line in left - most panels indicates the cross - section from which the pixel intensities were measured . Scale bars 20 µm .

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

(A-C) NIH3T3 fibroblasts expressing EGFP-CA7-mutant1 (A), EGFP-CA7-mutant2 (B), and EGFP-CA7-mutant3 (C). F-actin is visualized with Phalloidin-594. In the right-most panel of (A-C) are the normalized fluorescence intensity profiles of the mutated CA7 EGFP signal (black) and actin (red) and the yellow line in left-most panels indicates the cross-section from which the pixel intensities were measured. Scale bars 20 µm.


( D ) Analysis of the mutated CA7 and F - actin co - localization in cultured fibroblasts . Scatterplots of fluorescent intensities per pixel ( EGFP vs . Phalloidin - 594 ) along a cross section through a representative cell . Pearson ' s correlation coefficient ( r ) for the analyzed cell is given in each panel .

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

(D) Analysis of the mutated CA7 and F-actin co-localization in cultured fibroblasts. Scatterplots of fluorescent intensities per pixel (EGFP vs. Phalloidin-594) along a cross section through a representative cell. Pearson's correlation coefficient (r) for the analyzed cell is given in each panel.


( E ) Pearson ' s correlation coefficient values calculated for the depicted constructs and compared to CA7 . F - actin had a strong positive correlation coefficient with EGFP - CA7 ( r = 0 . 91 ± 0 . 02 , n = 26 cells ) . Neither EGFP alone ( r = 0 . 01 ± 0 . 03 , n = 56 ) nor EGFP - CA2 ( r = 0 . 09 ± 0 . 03 , n = 53 ) co - localized with F - actin ( P < 0 . 0001 for both constructs , when compared to CA7 ) . From the five mutated CA7 constructs , EGFP - CA7 - mutant1 ( r = 0 . 51 ± 0 . 04 , P < 0 . 0001 , n = 25 ) and EGFP - CA7 - mutant3 ( r = 0 . 72 ± 0 . 03 , P = 0 . 05 , n = 21 ) co - localized less with F - actin actin when compared to CA7 . The co - localization of the other four mutated CA7 constructs , EGFP - CA7 - mutant2 ( r = 0 . 92 ± 0 . 02 , P = 0 . 99 , n = 18 ) , EGFP - CA7 - R223E ( r = 0 . 86 ± 0 . 02 , P = 0 . 99 , n = 24 ) and EGFP - CA7 - H96 / 98C ( r = 0 . 79 ± 0 . 04 , P = 0 . 14 , n = 18 ) did not differ significantly from that of CA7 . Data are shown as mean ± SEM . Data did not pass Shapiro - Wilk test for normality , statistical comparison against CA7 was done with Kruskall - Wallis test corrected for multiple comparisons .

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

(E) Pearson's correlation coefficient values calculated for the depicted constructs and compared to CA7. F-actin had a strong positive correlation coefficient with EGFP-CA7 (r = 0.91 ± 0.02, n = 26 cells). Neither EGFP alone (r = 0.01 ± 0.03, n = 56) nor EGFP-CA2 (r = 0.09 ± 0.03, n = 53) co-localized with F-actin (P < 0.0001 for both constructs, when compared to CA7). From the five mutated CA7 constructs, EGFP-CA7-mutant1 (r= 0.51 ± 0.04, P < 0.0001, n = 25) and EGFP-CA7-mutant3 (r = 0.72 ± 0.03, P = 0.05, n = 21) co-localized less with F-actin actin when compared to CA7. The co-localization of the other four mutated CA7 constructs, EGFP-CA7-mutant2 (r = 0.92 ± 0.02, P = 0.99, n = 18), EGFP-CA7-R223E (r = 0.86 ± 0.02, P = 0.99, n = 24) and EGFP-CA7-H96/98C (r = 0.79 ± 0.04, P = 0.14, n = 18) did not differ significantly from that of CA7. Data are shown as mean ± SEM. Data did not pass Shapiro-Wilk test for normality, statistical comparison against CA7 was done with Kruskall-Wallis test corrected for multiple comparisons.


( A ) Isoform - specific subcellular localization shown in cultured hippocampal neurons ( DIV14 ) co - expressing DsRed - CA7 ( left ) and EGFP - CA2 ( middle ) .

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

(A) Isoform-specific subcellular localization shown in cultured hippocampal neurons (DIV14) co-expressing DsRed-CA7 (left) and EGFP-CA2 (middle).


( B , C ) Representative confocal images of precocious in vivo expression of ( B ) EGFP - CA2 and ( C ) EGFP - CA7 in P40 mouse cortical layer 2 / 3 pyramidal neurons . Neurons were transfected at E14 . 5 with EGFP - CA2 or EGFP - CA7 using in utero electroporation and images were taken from fixed slices . Right panels in ( B ) and ( C ) show higher magnification of the primary apical dendrite marked with a box . ( D ) The expression of EGFP - CA7 disrupted the normal spine morphology and induced the formation of thick , filopodia - like protrusions .

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

(B,C) Representative confocal images of precocious in vivo expression of (B) EGFP-CA2 and (C) EGFP-CA7 in P40 mouse cortical layer 2/3 pyramidal neurons. Neurons were transfected at E14.5 with EGFP-CA2 or EGFP-CA7 using in utero electroporation and images were taken from fixed slices. Right panels in (B) and (C) show higher magnification of the primary apical dendrite marked with a box. (D) The expression of EGFP-CA7 disrupted the normal spine morphology and induced the formation of thick, filopodia-like protrusions.


( A ) Comparison of mEPSCs in cortical layer 2 / 3 pyramidal neurons from P30 - P40 WT and CA7 KO mice . Sample traces of mEPSC recordings from WT and CA7 KO neurons , low - pas filtered at 1 kHz ( left ) . The data are summarized in the bar diagrams ( right ) . mEPSC frequency ( P = 0 . 63 ) and amplitude ( P = 0 . 90 ) were not significantly different between WT and CA7 KO and neurons ( n = 7 and 5 neurons , respectively , Student ' s independent samples t - test ) .

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

(A) Comparison of mEPSCs in cortical layer 2/3 pyramidal neurons from P30 - P40 WT and CA7 KO mice. Sample traces of mEPSC recordings from WT and CA7 KO neurons, low-pas filtered at 1 kHz (left). The data are summarized in the bar diagrams (right). mEPSC frequency (P = 0.63) and amplitude (P = 0.90) were not significantly different between WT and CA7 KO and neurons (n = 7 and 5 neurons, respectively, Student's independent samples t-test).


( B ) Representative confocal images of apical dendrites from Lucifer Yellow injected cortical layer 2 / 3 pyramidal neurons from WT and CA7 KO mice . The dendritic spine density and spine head size were examined in fixed slice preparations from P34 - P37 mice . Scale bar 2 µm .

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

(B) Representative confocal images of apical dendrites from Lucifer Yellow injected cortical layer 2/3 pyramidal neurons from WT and CA7 KO mice. The dendritic spine density and spine head size were examined in fixed slice preparations from P34 - P37 mice. Scale bar 2 µm.


( C ) Summary of the spine density analysis done from the Lucifer Yellow injected neurons . Spine density was a higher in CA7 KO neurons both in apical and basal dendrites ( n = 28 neurons for both ) compared to WT ( n = 29 neurons for apical and n = 30 for basal dendrite analysis ) ( P = 0 . 000002 for apical dendrites , analyzed with Mann - Whitney test , and P = 6 , 8 x 10 - 8 for basal dendrites , Student ' s t - test with Welch - correction . ) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice .

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

(C) Summary of the spine density analysis done from the Lucifer Yellow injected neurons. Spine density was a higher in CA7 KO neurons both in apical and basal dendrites (n = 28 neurons for both) compared to WT (n= 29 neurons for apical and n=30 for basal dendrite analysis) (P = 0.000002 for apical dendrites, analyzed with Mann-Whitney test, and P = 6,8 x 10-8 for basal dendrites, Student's t-test with Welch-correction.) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice.


( D ) The spine head width distribution7differed significantly between the genotypes ( n = 467 spines from WT and n = 421 spines from CA7 KO animals , 15 neurons analyzed from both genotypes , Wilcoxon rank sum test with continuity correction , W = 134540 , P < 0 . 001 ) .

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

(D) The spine head width distribution7differed significantly between the genotypes (n= 467 spines from WT and n = 421 spines from CA7 KO animals, 15 neurons analyzed from both genotypes, Wilcoxon rank sum test with continuity correction, W = 134540, P < 0.001).