( E ) Quantification of the number of newly renewed ECs in the indicated genotypes after 7 days Increase in EB numbers and EC turnover in the EGFRact midgut reflects both suppression of EB deaths and the non - autonomous stimulation of ISC in this background . Data information : ( n = 17 , 13 , 12 , 10 , 21 , 24 ) . P values were calculated using one - way ANOVA Bonferroni corrected ( * * * * , P < 0 , 0001 ) .

O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the number of newly renewed ECs in the indicated genotypes after 7 days Increase in EB numbers and EC turnover in the EGFRact midgut reflects both suppression of EB deaths and the non-autonomous stimulation of ISC in this background. Data information: (n= 17, 13, 12, 10, 21, 24). P values were calculated using one-way ANOVA Bonferroni corrected (****, P < 0,0001).


( F ) Representative images acquired using the same laser intensity are shown for Diap1 - GFP in the indicated illustrative examples . Scale bar , 100µm .

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

(F) Representative images acquired using the same laser intensity are shown for Diap1-GFP in the indicated illustrative examples. Scale bar, 100µm.


Full - length TLN ( but not APP ) accumulates in a distinct compartment in PS1 − / − hippocampal neurons . Double - immunofluorescence staining for endogenous TLN ( green , B36 . 1 ) and APP ( red , mAb C1 6 . 1 ) in wild - type ( top ) and PS1 − / − ( bottom ) hippocampal neurons ( 15 - d culture ) . The inset shows an overview of the neuron . In wild - type neurons TLN labeling is essentially localized to the somatodendritic plasmalemma in contrast to APP , which is found in intracellular compartments . In addition , PS1 deficiency causes TLN , but not APP , to cluster in large somatic accumulations . Bars , 10 μm .

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

Full-length TLN (but not APP) accumulates in a distinct compartment in PS1−/−hippocampal neurons. Double-immunofluorescence staining for endogenous TLN (green, B36.1) and APP (red, mAb C1 6.1) in wild-type (top) and PS1−/− (bottom) hippocampal neurons (15-d culture). The inset shows an overview of the neuron. In wild-type neurons TLN labeling is essentially localized to the somatodendritic plasmalemma in contrast to APP, which is found in intracellular compartments. In addition, PS1 deficiency causes TLN, but not APP, to cluster in large somatic accumulations. Bars, 10 μm.


( A ) Western blot analysis of endogenous TLN and APP in wild - type and PS1 − / − hippocampal neurons . PS1 deficiency results in the accumulation of APP - CTF . No TLN - CTF ( estimated ± 7 kD ) was detected , neither was it detected after SFV - induced exogenous overexpression . Also , overexpression of a TLNΔE fragment did not generate a PS1 - dependent TLN intracellular domain .

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

(A) Western blot analysis of endogenous TLN and APP in wild-type and PS1−/− hippocampal neurons. PS1 deficiency results in the accumulation of APP-CTF. No TLN-CTF (estimated ± 7 kD) was detected, neither was it detected after SFV-induced exogenous overexpression. Also, overexpression of a TLNΔE fragment did not generate a PS1-dependent TLN intracellular domain.


( B ) Different constructs fused to the Gal4VP16 domain were used in a γ - secretase reporter assay . Control transfections ( pIPspAdApt empty vector and pIPspAdApt - APP without Gal4VP16 ) show no luciferase activity . APP - Gal4VP16 and NotchΔE - Gal4VP16 activate the luciferase gene upon cleavage and are inhibited by DAPT , L658 , 458 , or compound 32 ( 125 nM ) . No luciferase activity was detected using TLNΔE or APP in which the transmembrane domain was replaced by that of TLN ( APP / TLNTMR chimaera ) , indicating that this region is not cleaved by γ - secretase .

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

(B) Different constructs fused to the Gal4VP16 domain were used in a γ-secretase reporter assay. Control transfections (pIPspAdApt empty vector and pIPspAdApt-APP without Gal4VP16) show no luciferase activity. APP-Gal4VP16 and NotchΔE-Gal4VP16 activate the luciferase gene upon cleavage and are inhibited by DAPT, L658,458, or compound 32 (125 nM). No luciferase activity was detected using TLNΔE or APP in which the transmembrane domain was replaced by that of TLN (APP/TLNTMR chimaera), indicating that this region is not cleaved by γ-secretase.


( C ) Left : γ - secretase inhibitors do not induce the formation of TLN accumulations . Wild - type hippocampal neurons were treated with γ - secretase inhibitors ( 125 nM , daily from d 7 - 14 post - plating ) , fixed , and immunostained for TLN and the percentage of neurons with TLN accumulations was scored . Culturing neurons for 25 d increases the size and number ( insets ) , but not the frequency of TLN accumulations ( mean ± SEM , n = 3 , 200 - 700 neurons per time point ) . Right : Western blot showing that only APP - CTF accumulates after chronic treatment with γ - secretase inhibitors .

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

(C) Left: γ-secretase inhibitors do not induce the formation of TLN accumulations. Wild-type hippocampal neurons were treated with γ-secretase inhibitors (125 nM, daily from d 7-14 post-plating), fixed, and immunostained for TLN and the percentage of neurons with TLN accumulations was scored. Culturing neurons for 25 d increases the size and number (insets), but not the frequency of TLN accumulations (mean ± SEM, n = 3, 200-700 neurons per time point). Right: Western blot showing that only APP-CTF accumulates after chronic treatment with γ-secretase inhibitors.


( D ) Adenoviral reintroduction of human PS1 suppresses TLN accumulations . 30 - 35 % of PS1 − / − neurons display TLN accumulations , in contrast to < 5 % in PS1 + / + and + / − cultures as well as in cultures expressing human PS1 in a PS1 − / − background . However , suppression of TLN accumulations was obtained by adenoviral introduction of human PS1 . A clear dose - dependent inhibition was seen with increasing MOI of adenoviral ( AV ) human PS1 , but also the familial Alzheimer ' s disease - linked PS1G384A and dominant - negative PS1D257A mutants . Control adenoviral infections with eGFP did not have a significant effect . After 9 - 11 d post - infection ( 15 d post - plating ) , neurons displayed significant PS1 protein expression as demonstrated by double staining for TLN and human PS1 ( mAb 5 . 2 ) . Bars , 20 μm .

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

(D) Adenoviral reintroduction of human PS1 suppresses TLN accumulations. 30-35% of PS1−/− neurons display TLN accumulations, in contrast to <5% in PS1+/+ and +/− cultures as well as in cultures expressing human PS1 in a PS1−/− background. However, suppression of TLN accumulations was obtained by adenoviral introduction of human PS1. A clear dose-dependent inhibition was seen with increasing MOI of adenoviral (AV) human PS1, but also the familial Alzheimer's disease-linked PS1G384A and dominant-negative PS1D257A mutants. Control adenoviral infections with eGFP did not have a significant effect. After 9-11 d post-infection (15 d post-plating), neurons displayed significant PS1 protein expression as demonstrated by double staining for TLN and human PS1(mAb 5.2). Bars, 20 μm.


Wild - type and PS1 − / − cortical neurons were transduced with SFV - TLN ( A ) or - APP ( B ) , pulse labeled for 15 min with [ 35S ] methionine , and chased for different time periods . ImmunoprecipitatedTLN and APP were treated with EndoH and analyzed by SDS - PAGE and phosphorimaging . The accumulation of an EndoH - resistant band indicates progressive maturation during Golgi passage ; however , the ratio of EndoH - resistant to - sensitive TLN ( A , top ) and APP ( B , top ) revealed no difference ( mean ± SEM , n = 3 ) . Instead , the half - life of overexpressed TLN ( A , bottom ) , but not full - length APP ( B , bottom ) , is significantly prolonged in neurons − / − neurons ( mean ± SEM , n = 3 ) .

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

Wild-type and PS1−/− cortical neurons were transduced with SFV-TLN (A) or -APP (B), pulse labeled for 15 min with [35S]methionine, and chased for different time periods. ImmunoprecipitatedTLN and APP were treated with EndoH and analyzed by SDS-PAGE and phosphorimaging. The accumulation of an EndoH-resistant band indicates progressive maturation during Golgi passage; however, the ratio of EndoH-resistant to -sensitive TLN (A, top) and APP (B, top) revealed no difference (mean ± SEM, n = 3). Instead, the half-life of overexpressed TLN (A, bottom), but not full-length APP (B, bottom), is significantly prolonged in neurons−/−neurons (mean ± SEM, n = 3).


( C ) Wild - type and PS1 − / − hippocampal neurons ( 12 d ) were scraped , pelleted , and treated with 10 mU EndoH , followed by Western blot detection of APP , TLN , PS1 , nicastrin ( NCT ) , synaptophysin , and ductin . TLN levels are increased almost fourfold in PS1 − / − neurons . No EndoH - sensitive TLN was detected in PS1 − / − neurons , indicating a complete maturation . APP - CTF shows a dramatic accumulation ( 44 - fold ; mean ± SEM , n = 3 ) .

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

(C) Wild-type and PS1−/− hippocampal neurons (12 d) were scraped, pelleted, and treated with 10 mU EndoH, followed by Western blot detection of APP, TLN, PS1, nicastrin (NCT), synaptophysin, and ductin. TLN levels are increased almost fourfold in PS1−/− neurons. No EndoH-sensitive TLN was detected in PS1−/− neurons, indicating a complete maturation. APP-CTF shows a dramatic accumulation (44-fold; mean ± SEM, n = 3).


TLN accumulation occurs in a compartment distinct from classic biosynthetic and endosomal pathways . ( A - F ) Early compartments . No colocalization was observed between TLN ( green in all panels ) and nuclei ( A , TOPRO - 3 ) , ER - markers BIP ( B ) and calnexin ( C ) , ERGIC - 53 ( D ) , β - COP ( E ) or GM130 ( F ) , and labeling cis - Golgi . ( G - L ) Late compartments . TLN accumulations are not accessible for exogenous biotin ( G ) . Early and recycling endosomes ( H , EEA1 ) and transferrin receptor ( I , TFR ) , late endosomes ( J , LBPA ) , and lysosomes ( K , Lamp - 2 ; L , catD ) also stained negative for TLN . Vertical sections ( x - z , arrowheads in I and J ) clearly distinguish TLN accumulations from recycling ( I , TFR ) and late endosomes ( J , LBPA ) . Bars , 10 μm .

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

TLN accumulation occurs in a compartment distinct from classic biosynthetic and endosomal pathways. (A-F) Early compartments. No colocalization was observed between TLN (green in all panels) and nuclei (A, TOPRO-3), ER-markers BIP (B) and calnexin (C), ERGIC-53 (D), β-COP (E) or GM130 (F), and labeling cis-Golgi. (G-L) Late compartments. TLN accumulations are not accessible for exogenous biotin (G). Early and recycling endosomes (H, EEA1) and transferrin receptor (I, TFR), late endosomes (J, LBPA), and lysosomes (K, Lamp-2; L, catD) also stained negative for TLN. Vertical sections (x-z, arrowheads in I and J) clearly distinguish TLN accumulations from recycling (I, TFR) and late endosomes (J, LBPA). Bars, 10 μm.


Correlative light immuno - EM analysis of TLN accumulations . ( A ) Insert : overview of the selected neuron as seen by immunocytochemistry . The bright signal for TLN reflects the numerous membranous accumulations seen at the ultrastructural immuno - EM level ( star ) . ( B ) Higher magnification of the boxed area in A . ( C ) High magnification of the boxed structure seen in B . 10 - nm gold particles label TLN . Note the complex composition of the internal membranes of the TLN - positive compartments . ( D ) Example of a TLN ( 15 - nm gold ) - containing structure obtained by the conventional cryosectioning technique . Note some heterogeneity in the internal membranes between C and D . Lamp - 1 ( 10 - nm gold ) is absent from the TLN - containing compartment . Arrows point to invaginations of the cytoplasm . N , nucleus ; P , plasma membrane . Bars : A , 5 μm ; B , 2 μm ; C , 500 nm ; D , 200 nm .

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

Correlative light immuno-EM analysis of TLN accumulations. (A) Insert: overview of the selected neuron as seen by immunocytochemistry. The bright signal for TLN reflects the numerous membranous accumulations seen at the ultrastructural immuno-EM level (star). (B) Higher magnification of the boxed area in A. (C) High magnification of the boxed structure seen in B. 10-nm gold particles label TLN. Note the complex composition of the internal membranes of the TLN-positive compartments. (D) Example of a TLN (15-nm gold)-containing structure obtained by the conventional cryosectioning technique. Note some heterogeneity in the internal membranes between C and D. Lamp-1 (10-nm gold) is absent from the TLN-containing compartment. Arrows point to invaginations of the cytoplasm. N, nucleus; P, plasma membrane. Bars: A, 5 μm; B, 2 μm; C, 500 nm; D, 200 nm.


TLN accumulations are distinct from lysosomes . A TLN ( 15 - nm gold ) - containing structure as obtained by the conventional cryosectioning technique double labeled with Lamp - 1 ( 10 - nm gold ) . The TLN - positive compartment is clearly distinct in size and from lysosomes ( L ) and lacks Lamp - 1 . P , plasma membrane . Bar , 200 nm .

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

TLN accumulations are distinct from lysosomes. A TLN (15-nm gold)-containing structure as obtained by the conventional cryosectioning technique double labeled with Lamp-1 (10-nm gold). The TLN-positive compartment is clearly distinct in size and from lysosomes (L) and lacks Lamp-1. P, plasma membrane. Bar, 200 nm.


( B and C ) Double immunostaining of wild - type and PS1 − / − hippocampal neurons for TLN ( green ) and autophagic vacuole - associated Apg12p ( B ) and LC3 ( C ) . To detect both sets of pAbs , an alternative method was developed using biotinylated anti - TLN ( see Materials and methods ) . Each experiment included controls for specificity ( not depicted ) . In wild - type neurons , both Apg12p and LC3 ( B and C , top row ) were broadly distributed in cell bodies and neurites , but did not colocalize with TLN . Colocalization was only observed in multiple TLN accumulations in PS1 − / − hippocampal neurons ( second row in B and C , respectively ) . For Apg12p , coimmunostaining was also noticed in small discrete structures in the proximal dendrites ( B , bottom row ) . Bars , 10 μm .

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

(B and C) Double immunostaining of wild-type and PS1−/− hippocampal neurons for TLN (green) and autophagic vacuole-associated Apg12p (B) and LC3 (C). To detect both sets of pAbs, an alternative method was developed using biotinylated anti-TLN (see Materials and methods). Each experiment included controls for specificity (not depicted). In wild-type neurons, both Apg12p and LC3 (B and C, top row) were broadly distributed in cell bodies and neurites, but did not colocalize with TLN. Colocalization was only observed in multiple TLN accumulations in PS1−/− hippocampal neurons (second row in B and C, respectively). For Apg12p, coimmunostaining was also noticed in small discrete structures in the proximal dendrites (B, bottom row). Bars, 10 μm.


( A ) Intracellular sorting and maturation of catD . Wild - type and PS1 − / − neurons were metabolically labeled and chased for the indicated time periods . Phosphorimaging of immunoprecipitated radiolabeled catD did not reveal a difference in the ratio of proteolytically processed 46 - kD intermediate vs . 52 - kD precursor ( mean ± SEM , n = 3 ) .

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

(A) Intracellular sorting and maturation of catD. Wild-type and PS1−/− neurons were metabolically labeled and chased for the indicated time periods. Phosphorimaging of immunoprecipitated radiolabeled catD did not reveal a difference in the ratio of proteolytically processed 46-kD intermediate vs. 52-kD precursor (mean ± SEM, n = 3).


( B - D ) TLN localizes to autophagic vacuoles of catD − / − hippocampal neurons . Lysotracker staining revealed sparsely distributed small - sized organelles in wild - type and PS1 − / − neurons in contrast to catD − / − neurons where high numbers of large Lysotracker - positive organelles were found ( B , arrowheads ) . Although TLN accumulations in PS1 − / − were fully negative for Lysotracker , some acidic organelles tended to closely associate ( C , arrowheads , middle ) . In catD − / − neurons , TLN was clearly detected in the large acidic organelles ( C , bottom ) . Bars , 20 μm . ( D ) At the immuno - EM level , the large acidic organelles seen in catD − / − neurons represent dense autophagic vacuole - like structures ( star ) that label positive for TLN ( 10 - nm gold ) . M , mitochondrion . Bars , 200 nm .

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

(B-D) TLN localizes to autophagic vacuoles of catD−/−hippocampal neurons. Lysotracker staining revealed sparsely distributed small-sized organelles in wild-type and PS1−/− neurons in contrast to catD−/−neurons where high numbers of large Lysotracker-positive organelles were found (B, arrowheads). Although TLN accumulations in PS1−/− were fully negative for Lysotracker, some acidic organelles tended to closely associate (C, arrowheads, middle). In catD−/− neurons, TLN was clearly detected in the large acidic organelles (C, bottom). Bars, 20 μm. (D) At the immuno-EM level, the large acidic organelles seen in catD−/−neurons represent dense autophagic vacuole-like structures (star) that label positive for TLN (10-nm gold). M, mitochondrion. Bars, 200 nm.


TLN mediates phagocytic uptake of microbeads in hippocampal neurons . ( A ) Microbeads align along neurites of hippocampal neurons as demonstrated by phase contrast ( DIC ) . Already after 4 h , many beads stain positive for TLN ( arrowheads , top ) . At 48 h ( bottom ) , most TLN immunoreactivity was associated with microbeads at the expense of its typical plasma membrane staining . * , cell body . Note that microbeads were not found associated with preexisting TLN accumulations ( asterisk in top panels , 4 h ) . ( B ) Microbeads ( 24 h ) accumulate TLN and actin , as shown by phalloidin - Alexa 568 . An overview of the neuron is given in each top left inset , next to two detailed areas ( white boxes ) . The differential interference contrast ( DIC ) clearly demonstrates a complete colocalization of microbeads with TLN and phalloidin ( arrowheads ) . TOPRO - 3 ( blue ) marks the nucleus ( see also Fig . S1 , available at http : / / www . jcb . org / cgi / content / full / jcb . 200406060 / DC1 ) . ( C ) Similar as in B , but immunostained for TLN and endogenous PIP2 . The overview demonstrates the overall recruitment of PIP2 to microbeads ( arrows ) . The parallel insets show a detailed area ( white box ) with PIP2 being colocalized with TLN on individual microbeads ( arrowheads ) . Note that PIP2 occasionally colocalizes with TLN at the plasma membrane ( asterisk ) . Bars ( A - C ) , 10 μm .

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

TLN mediates phagocytic uptake of microbeads in hippocampal neurons. (A) Microbeads align along neurites of hippocampal neurons as demonstrated by phase contrast (DIC). Already after 4 h, many beads stain positive for TLN (arrowheads, top). At 48 h (bottom), most TLN immunoreactivity was associated with microbeads at the expense of its typical plasma membrane staining. *, cell body. Note that microbeads were not found associated with preexisting TLN accumulations (asterisk in top panels, 4 h). (B) Microbeads (24 h) accumulate TLN and actin, as shown by phalloidin-Alexa 568. An overview of the neuron is given in each top left inset, next to two detailed areas (white boxes). The differential interference contrast (DIC) clearly demonstrates a complete colocalization of microbeads with TLN and phalloidin (arrowheads). TOPRO-3 (blue) marks the nucleus (see also Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406060/DC1). (C) Similar as in B, but immunostained for TLN and endogenous PIP2. The overview demonstrates the overall recruitment of PIP2 to microbeads (arrows). The parallel insets show a detailed area (white box) with PIP2 being colocalized with TLN on individual microbeads (arrowheads). Note that PIP2 occasionally colocalizes with TLN at the plasma membrane (asterisk). Bars (A-C), 10 μm.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( A ) PS1 , PS2 and NCT were analyzed in cell lysates by immunoblotting using antibodies PS1N ( PS1 ) , BI - HF5C ( PS2 ) and N1660 ( NCT ) , respectively . ( B ) Full length APP and APP CTFs were analyzed by immunoblotting using antibody 6687 . ( C ) Conditioned media were analyzed for secreted APPs by immunoblotting using antibody 22C11 and for total Aβ by combined immunoprecipitation / immunoblotting using antibodies 3552 / 2D8 .

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Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(A) PS1, PS2 and NCT were analyzed in cell lysates by immunoblotting using antibodies PS1N (PS1), BI-HF5C (PS2) and N1660 (NCT), respectively.(B) Full length APP and APP CTFs were analyzed by immunoblotting using antibody 6687. (C) Conditioned media were analyzed for secreted APPs by immunoblotting using antibody 22C11 and for total Aβ by combined immunoprecipitation/immunoblotting using antibodies 3552/2D8.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( D ) Conditioned media were analyzed for individual Aβ species on Tris - Bicine urea SDS - PAGE by combined immunoprecipitation / immunoblotting of Aβ using antibodies 3552 / 2D8 . Pooled clones of HEK293 / sw cells stably transfected with PS1 L166P were used as reference . Note that more sample was loaded for the PS1 L435F mutant to facilitate analysis .

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

Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (D) Conditioned media were analyzed for individual Aβ species on Tris-Bicine urea SDS-PAGE by combined immunoprecipitation/immunoblotting of Aβ using antibodies 3552/2D8. Pooled clones of HEK293/sw cells stably transfected with PS1 L166P were used as reference. Note that more sample was loaded for the PS1 L435F mutant to facilitate analysis.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( E ) PS1 expression levels were analyzed in cell lysates of HEK293 / sw cells stably expressing PS1 WT or PS1 L435F by immunoblotting using antibodies PS1NT and 5E12 , respectively .

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

Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (E) PS1 expression levels were analyzed in cell lysates of HEK293/sw cells stably expressing PS1 WT or PS1 L435F by immunoblotting using antibodies PS1NT and 5E12, respectively.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( F ) Conditioned media were analyzed by ELISA specific for Aβ40 , Aβ42 and Aβ43 . Data represent mean ± s . e . m . ( n = 6 ) . Absolute levels and Aβ ratios are shown .

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

Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (F) Conditioned media were analyzed by ELISA specific for Aβ40, Aβ42 and Aβ43. Data represent mean ± s.e.m. (n=6). Absolute levels and Aβ ratios are shown.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( G ) Secreted Aβ was analyzed as in ( D ) . To verify individual Aβ species , Aβ standards were co - migrated . Conditioned media were analyzed for individual Aβ species on Tris - Bicine urea SDS - PAGE by combined immunoprecipitation / immunoblotting of Aβ using antibodies 3552 / 2D8 .

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

Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(G) Secreted Aβ was analyzed as in (D). To verify individual Aβ species, Aβ standards were co-migrated. Conditioned media were analyzed for individual Aβ species on Tris-Bicine urea SDS-PAGE by combined immunoprecipitation/immunoblotting of Aβ using antibodies 3552/2D8.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( H ) Total Aβ in conditioned media was analyzed by MALDI - TOF MS following immunoprecipitation with antibody 4G8 . Observed ( Aβ42 , 4513 . 6 ; Aβ43 , 4615 . 3 ) and predicted molecular masses ( Aβ42 , 4514 . 1 ; Aβ43 , 4615 . 2 ) were in good agreement .

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

Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(H) Total Aβ in conditioned media was analyzed by MALDI-TOF MS following immunoprecipitation with antibody 4G8. Observed (Aβ42, 4513.6; Aβ43, 4615.3) and predicted molecular masses (Aβ42, 4514.1; Aβ43, 4615.2) were in good agreement.


( A ) PS1 / 2 - / - MEF cells stably transduced with PS1 WT or PS1 L435F were analyzed for PS1 expression and APP processing by immunoblotting as in Fig 1A . Antibody 5E12 was used for the detection of the PS1 CTF .

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

(A) PS1/2-/- MEF cells stably transduced with PS1 WT or PS1 L435F were analyzed for PS1 expression and APP processing by immunoblotting as in Fig 1A. Antibody 5E12 was used for the detection of the PS1 CTF.


( B ) Stably transduced PS1 / 2 - / - MEF cells of A were transiently transfected with APPsw - 6myc and conditioned media were analyzed for Aβ species by Tris - Bicine urea SDS - PAGE . Individual Aβ species were verified by co - migration with Aβ standards ( left panel ) and quantified ( right panel ) . Data represent mean ± s . e . m . of n = 3 independently performed transfections . Note that more sample was loaded for the PS1 L435F mutant to facilitate immunoblot analysis .

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

(B) Stably transduced PS1/2-/- MEF cells of A were transiently transfected with APPsw-6myc and conditioned media were analyzed for Aβ species by Tris-Bicine urea SDS-PAGE. Individual Aβ species were verified by co-migration with Aβ standards (left panel) and quantified (right panel). Data represent mean ± s.e.m. of n=3 independently performed transfections. Note that more sample was loaded for the PS1 L435F mutant to facilitate immunoblot analysis.


( C ) Secreted Aβ from cells in B were quantified by ELISA . Data represent mean ± s . e . m . of n = 5 ( PS1 WT ) or 6 ( PS1 L435F ) independently performed transfections .

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(C) Secreted Aβ from cells in B were quantified by ELISA. Data represent mean ± s.e.m. of n=5 (PS1 WT) or 6 (PS1 L435F) independently performed transfections.


Immunohistochemical detection of Aβ40 ( left column ) , Aβ42 ( medium column ) and Aβ43 ( right column ) in consecutive frontal cortex paraffin sections of two FAD cases with PS1 L435F mutation ( cases # 1 and # 2 , 1st and 2nd rows ) , another FAD case with different PS1 mutation ( case # 3 , 3rd row ) and one SAD case ( case # 4 , forth row ) . In both PS1 L435F mutation cases the numerous Aβ plaques mainly contain Aβ42 and Aβ43 but less Aβ40 . This is in contrast to Aβ plaques of the control FAD and SAD cases , in which solely Aβ42 predominates and Aβ43 is sparse ; even levels of Aβ40 seem to be lower than in the PS1 L435F cases . Note that in both PS1 L435F cases several plaques are larger than those seen in cases # 3 and # 4 , representing cotton wool plaques . Scale bar = 500 µm . Magnification is identical in all pictures .

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

Immunohistochemical detection of Aβ40 (left column), Aβ42 (medium column) and Aβ43 (right column) in consecutive frontal cortex paraffin sections of two FAD cases with PS1 L435F mutation (cases #1 and #2, 1st and 2nd rows), another FAD case with different PS1 mutation (case #3, 3rd row) and one SAD case (case #4, forth row). In both PS1 L435F mutation cases the numerous Aβ plaques mainly contain Aβ42 and Aβ43 but less Aβ40. This is in contrast to Aβ plaques of the control FAD and SAD cases, in which solely Aβ42 predominates and Aβ43 is sparse; even levels of Aβ40 seem to be lower than in the PS1 L435F cases. Note that in both PS1 L435F cases several plaques are larger than those seen in cases #3 and #4, representing cotton wool plaques. Scale bar = 500 µm. Magnification is identical in all pictures.


B , phase contrast images of the morphological changes in MDR cells , residual MDR cells and regrown cells for Du145TXR or MCF - 7ADR . Red circles mark magnified areas ( bottom ) . Scale bars , 50 μm ( low magnification images ) .

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

B, phase contrast images of the morphological changes in MDR cells, residual MDR cells and regrown cells for Du145TXR or MCF-7ADR. Red circles mark magnified areas (bottom). Scale bars, 50 μm (low magnification images).


C - D , cell viability of residual MDR cells ( RMCs ) and MDR cells ( MCs ) of ( C ) Du145TXR or ( D ) MCF - 7ADR cells using the same combination treatment for 24 hours on the indicated days . Student ' s t test was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

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

C-D, cell viability of residual MDR cells (RMCs) and MDR cells (MCs) of (C) Du145TXR or (D) MCF-7ADR cells using the same combination treatment for 24 hours on the indicated days. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.


F - G , drug tolerant rate of ( F ) Du145 / Du145TXR ( 20 nM taxol / 20 nM taxol plus 50 μM verapamil ) or ( G ) MCF - 7 / MCF - 7ADR ( 200 nM adriamycin / 200 nM adriamycin plus 50 μM verapamil ) cells treated with drugs on the indicated days . Drug tolerant rate defined by comparing cell viability followed treatment , RMCs VS . MCs or CCs VS . PeCs , > 1 representing drug tolerant . Induction Time indicates the time of entering DTP state ; Sustaining Time indicates the time of maintaining DTP state . Blue colors represent control groups ; Red colors represent MDR cancer cells . Mean with ± SD .

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

F-G, drug tolerant rate of (F) Du145/Du145TXR (20 nM taxol/20 nM taxol plus 50 μM verapamil) or (G) MCF-7/MCF-7ADR (200 nM adriamycin/200 nM adriamycin plus 50 μM verapamil) cells treated with drugs on the indicated days. Drug tolerant rate defined by comparing cell viability followed treatment, RMCs VS. MCs or CCs VS. PeCs, > 1 representing drug tolerant. Induction Time indicates the time of entering DTP state; Sustaining Time indicates the time of maintaining DTP state. Blue colors represent control groups; Red colors represent MDR cancer cells. Mean with ± SD.


D , heat map based on scoring rank indicated consistently expressed genes identified by RNA - seq of MCs VS . MPCs in Du145TXR and MCF - 7ADR .

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D, heat map based on scoring rank indicated consistently expressed genes identified by RNA-seq of MCs VS. MPCs in Du145TXR and MCF-7ADR.


F , immunoblotting of NPC1L1 in MCs , MPCs and RCs of Du145TXR or MCF - 7ADR . G , immunoblotting of NPC1L1 in Du145TXR or MCF - 7ADR cells transfected with siNPC1L1 or siScramble .

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

F, immunoblotting of NPC1L1 in MCs, MPCs and RCs of Du145TXR or MCF-7ADR. G, immunoblotting of NPC1L1 in Du145TXR or MCF-7ADR cells transfected with siNPC1L1 or siScramble.


H - I , colony formation assay and quantification of ( H ) Du145TXR or ( I ) MCF - 7ADR cells transfected with siNPC1L1 or siScramble followed by treatment with indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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

H-I, colony formation assay and quantification of (H) Du145TXR or (I) MCF-7ADR cells transfected with siNPC1L1 or siScramble followed by treatment with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


A - C , flow cytometric analysis of ROS levels in Du145TXR or MCF - 7ADR cells treated with indicated agents for 12 and 36 hours . ( A ) Representative images and quantification of ROS in ( B ) Du145TXR or ( C ) MCF - 7ADR cells are shown . One - way ANOVA was used to analyze statistical differences . Mean with ± SD Data information : Results are representative of three independent experiments .

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

A-C, flow cytometric analysis of ROS levels in Du145TXR or MCF-7ADR cells treated with indicated agents for 12 and 36 hours. (A) Representative images and quantification of ROS in (B) Du145TXR or (C) MCF-7ADR cells are shown. One-way ANOVA was used to analyze statistical differences. Mean with ± SD Data information: Results are representative of three independent experiments.


D - E , cell viability of ( D ) Du145TXR or ( E ) MCF - 7ADR treated with indicated agents for 72 hours . One - way ANOVA was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

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

D-E, cell viability of (D) Du145TXR or (E) MCF-7ADR treated with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.


J - K , cell viability of ( J ) Du145TXR or ( K ) MCF - 7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 72 hours . One - way ANOVA was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J-K, cell viability of (J) Du145TXR or (K) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD . Data information: Results are representative of three independent experiments.


C - D , endocytosis analysis of ( C ) Du145TXR or ( D ) MCF - 7ADR cells treated with indicated agents for 8 hours , followed by staining with 250 μg / mL FITC - DEX for 4 hours . Scale bars , 10 μm . Data information : Results are representative of three independent experiments .

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

C-D, endocytosis analysis of (C) Du145TXR or (D) MCF-7ADR cells treated with indicated agents for 8 hours, followed by staining with 250 μg/mL FITC-DEX for 4 hours. Scale bars, 10 μm. Data information: Results are representative of three independent experiments.


E - F , phase contrast images of the morphological changes in ( E ) Du145TXR and ( F ) MCF - 7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 12 hours . Scale bars , 10 or 25 μm . The red arrows indicate vacuoles . Data information : Results are representative of three independent experiments .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

E-F, phase contrast images of the morphological changes in (E) Du145TXR and (F) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 12 hours. Scale bars, 10 or 25 μm. The red arrows indicate vacuoles. Data information: Results are representative of three independent experiments.


I - J , colony formation assay and quantification of ( I ) Du145TXR or ( J ) MCF - 7ADR cells treated with indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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

I-J, colony formation assay and quantification of (I) Du145TXR or (J) MCF-7ADR cells treated with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


C , immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF - 7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours . Data information : Results are representative of three independent experiments .

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

C, immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours. Data information: Results are representative of three independent experiments.


F - G , relative luciferase activities of wild - type ( WT ) or mutant ( MUT ) NPC1L1 promoter ( - 205 ~ - 215 , # 1 ) reporter constructs in ( F ) Du145TXR or ( G ) MCF - 7ADR cells treated with indicated agents . Student ' s t test was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

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

F-G, relative luciferase activities of wild-type (WT) or mutant (MUT) NPC1L1 promoter (-205~-215, #1) reporter constructs in (F) Du145TXR or (G) MCF-7ADR cells treated with indicated agents. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.


J - K , colony formation assay and quantification of ( J ) Du145TXR or ( K ) MCF - 7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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

J-K, colony formation assay and quantification of (J) Du145TXR or (K) MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


immunohistochemical staining of Ki67 in indicated groups . ( D ) Representative images are shown . Scale bar , 50 μm . , immunohistochemical staining of cleaved - caspase 3 ( CC3 ) in indicated groups . ( F ) Representative images are shown . Scale bar , 50 μm .

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

immunohistochemical staining of Ki67 in indicated groups. (D) Representative images are shown. Scale bar, 50 μm. , immunohistochemical staining of cleaved-caspase 3 (CC3) in indicated groups. (F) Representative images are shown. Scale bar, 50 μm.


( I ) Corresponding images and isolated tumors are shown . The red dotted line represents cured tumors .

(I) Corresponding images and isolated tumors are shown. The red dotted line represents cured tumors.


A ) Deletion of the CID domain dramatically reduces the interaction of Nrd1 with Sen1 . Coimmunoprecipitation ( CoIP ) experiments using TAP - tagged Nrd1 ( either wt or ∆ CID ) as the bait . Representative gel of one out of two independent experiments .

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A) Deletion of the CID domain dramatically reduces the interaction of Nrd1 with Sen1. Coimmunoprecipitation (CoIP) experiments using TAP-tagged Nrd1 (either wt or ∆CID) as the bait. Representative gel of one out of two independent experiments.


C ) Deletion of the NIM decreases substantially the association of Nrd1 with Sen1 . CoIP experiments using Nrd1 - TAP as the bait in a SEN1 or sen1 ∆ NIM background . Representative gel of one out of two independent experiments .

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C) Deletion of the NIM decreases substantially the association of Nrd1 with Sen1. CoIP experiments using Nrd1-TAP as the bait in a SEN1 or sen1∆NIM background. Representative gel of one out of two independent experiments.


D ) CoIP experiments using HA - tagged Sen1 , either wt or ∆ NIM , as the bait . Representative gel of one out of two independent experiments . Protein extracts were treated with RNaseA prior to immunoprecipitation . In these experiment , Sen1 could not be detected in the input extracts .

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

D) CoIP experiments using HA-tagged Sen1, either wt or ∆NIM, as the bait. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA prior to immunoprecipitation. In these experiment, Sen1 could not be detected in the input extracts.


A ) Deletion of the N - terminal of Sen1 is lethal . A ∆ sen1 strain ( YDL2767 ) covered by an URA3 - containing plasmid ( pFL38 ) expressing wt Sen1 was transformed with a TRP1 - plasmid ( pFL39 ) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left . After over - night growth in non - selective medium , cells were plated on minimal medium ( CSM ) containing 5 - fluorootic acid ( 5 - FOA ) to select those that have lost the URA3 plasmid . The absence of growth in 5 - FOA implies that the SEN1 version expressed in the TRP1 - plasmid does not support viability .

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

A) Deletion of the N-terminal of Sen1 is lethal. A ∆sen1 strain (YDL2767) covered by an URA3-containing plasmid (pFL38) expressing wt Sen1 was transformed with a TRP1-plasmid (pFL39) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left. After over-night growth in non-selective medium, cells were plated on minimal medium (CSM) containing 5-fluorootic acid (5-FOA) to select those that have lost the URA3 plasmid. The absence of growth in 5-FOA implies that the SEN1 version expressed in the TRP1-plasmid does not support viability.


B ) Deletion of the N - terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo . Northern blot analyses of three well - characterized NNS - targets , the CUT NEL025c and the snoRNAs SNR13 and SNR33 , in a Sen1 - AID ( auxin - induced degron ) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1 . Sen1 - AID was depleted for 2h by the addition of 500 μM indole - 3 - acetic acid ( IAA ) to monitor the capacity of the plasmid - borne versions of SEN1 to induce transcription termination . The expected RNA species resulting from inefficient termination ( RT for readthrough species ) are indicated . assays were performed in a ∆ rrp6 background to detect the primary products of NNS - dependent termination and the U4 RNA is used as a loading control .

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

B) Deletion of the N-terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo. Northern blot analyses of three well-characterized NNS-targets, the CUT NEL025c and the snoRNAs SNR13 and SNR33, in a Sen1-AID (auxin-induced degron) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1. Sen1-AID was depleted for 2h by the addition of 500 μM indole-3-acetic acid (IAA) to monitor the capacity of the plasmid-borne versions of SEN1 to induce transcription termination. The expected RNA species resulting from inefficient termination (RT for readthrough species) are indicated. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.


C ) Overexpression of sen1 ∆ Nter restores viability . Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter ( pGAL ) in the presence of galactose .

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

C) Overexpression of sen1∆Nter restores viability. Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter (pGAL) in the presence of galactose.


D ) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N - terminal domain . Northern blot analysis of a typical NNS - target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose . assays were performed in a ∆ rrp6 background to detect the primary products of NNS - dependent termination and the U4 RNA is used as a loading control .

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

D) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N-terminal domain. Northern blot analysis of a typical NNS-target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . A ) Metagene analysis of the RNAPII distribution relative to the annotated polyadenylation site ( PAS ) of mRNAs . Values on the y axis correspond to the median coverage . B ) Box - plot representation of the readthrough ( RT ) index as a readout of the termination efficiency at each mRNA . The RT index was calculated as the average signal over a window of 200 nt downstream of the PAS , divided by the average signal from the TSS to the PAS of each mRNA . Boxes include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median . The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1 . 5 * IQR ( where IQR is the interquartile distance or distance between the first and the third quartile ) . The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1 . 5 * IQR . For each mRNA ( n = 5752 ) , the values correspond to the average of two independent biological replicates . Data populations were compared using a t - test ( bilateral distributions , unpaired data ) and p - values ≥ 0 . 05 are indicated as ns ( not significant ) .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. A) Metagene analysis of the RNAPII distribution relative to the annotated polyadenylation site (PAS) of mRNAs. Values on the y axis correspond to the median coverage. B) Box-plot representation of the readthrough (RT) index as a readout of the termination efficiency at each mRNA. The RT index was calculated as the average signal over a window of 200 nt downstream of the PAS, divided by the average signal from the TSS to the PAS of each mRNA. Boxes include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR (where IQR is the interquartile distance or distance between the first and the third quartile). The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. For each mRNA (n=5752), the values correspond to the average of two independent biological replicates. Data populations were compared using a t-test (bilateral distributions, unpaired data) and p-values≥ 0.05 are indicated as ns (not significant).


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . C - D ) IGV screenshots of two well - characterized protein - coding genes displaying substantial defects in premature termination in some mutants . The values correspond to the counts per million of counts ( CPM ) multiplied by 10 .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. C-D) IGV screenshots of two well-characterized protein-coding genes displaying substantial defects in premature termination in some mutants. The values correspond to the counts per million of counts (CPM) multiplied by 10.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . E ) Metagene analyses of transcribing RNAPII at CUTs performed as for mRNAs but using the annotated transcription termination site ( TTS ) as the reference point . F ) Analysis of termination defects in the different mutants at CUTs . Box - plots performed as in ( B ) . The RT index is calculated as the average signal over a window of 400 nt downstream of the TTS , divided by the average signal over a window of 100 nt downstream from the TSS ( CUT100 ) . For each CUT ( n = 329 ) , the values correspond to the average of two independent biological replicates . P - values < 0 . 001 are indicated by three asterisks .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. E) Metagene analyses of transcribing RNAPII at CUTs performed as for mRNAs but using the annotated transcription termination site (TTS) as the reference point. F) Analysis of termination defects in the different mutants at CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 400 nt downstream of the TTS, divided by the average signal over a window of 100 nt downstream from the TSS (CUT100). For each CUT (n=329), the values correspond to the average of two independent biological replicates. P-values < 0.001 are indicated by three asterisks.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . G - H ) IGV screenshots of examples of CUTs displaying substantial termination defects in the mutants .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. G-H) IGV screenshots of examples of CUTs displaying substantial termination defects in the mutants.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . Analysis of RNAPII occupancy around snoRNAs performed as for CUTs . Box - plots performed as in ( B ) . The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS , divided by the average signal over the mature snoRNA .

B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS, divided by the average signal over the mature snoRNA.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . Analysis of RNAPII occupancy around snoRNAs performed as for CUTs .

B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs.


A ) Deletion of Sen1 N - terminal domain does not prevent the interaction of Sen1 with RNAPII . CoIP experiments using Rbp3 - FLAG as the bait . Assays were performed in a Sen1 - AID strain harboring a plasmid expressing either SEN1 or sen1 ∆ Nter upon depletion of Sen1 - AID in the presence of IAA for 2h . An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1 ∆ Nter . Nrd1 is detected as a positive control . Representative gel of one out of two independent experiments . Protein extracts were treated with RNaseA before immunoprecipitation .

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

A) Deletion of Sen1 N-terminal domain does not prevent the interaction of Sen1 with RNAPII. CoIP experiments using Rbp3-FLAG as the bait. Assays were performed in a Sen1-AID strain harboring a plasmid expressing either SEN1 or sen1∆Nter upon depletion of Sen1-AID in the presence of IAA for 2h. An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1∆Nter. Nrd1 is detected as a positive control. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.


B ) Deletion of the Sen1 N - terminal domain reduces the interaction of Sen1 with the S5P - CTD . CoIP experiments using TAP - Sen1 as the bait . Sen1 proteins were expressed from pGAL in the presence of galactose . Nab3 is detected as a positive control . Representative gel of one out of three independent experiments . Protein extracts were treated with RNaseA before immunoprecipitation .

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

B) Deletion of the Sen1 N-terminal domain reduces the interaction of Sen1 with the S5P-CTD. CoIP experiments using TAP-Sen1 as the bait. Sen1 proteins were expressed from pGAL in the presence of galactose. Nab3 is detected as a positive control. Representative gel of one out of three independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.


C ) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability . Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1 - plasmid carrying the SEN1 versions indicated in the scheme on the left . The growth of the strain expressing the Nrd1 CID - sen1 ∆ Nter chimera in 5 - FOA implies that this gene can support viability .

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C) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability. Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1-plasmid carrying the SEN1 versions indicated in the scheme on the left. The growth of the strain expressing the Nrd1 CID-sen1∆Nter chimera in 5-FOA implies that this gene can support viability.


D ) Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1 ∆ Nter mutant . Northern blot assays performed in a Sen1 - AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in figure 3B . Experiments performed in a ∆ rrp6 background . Representative gel of one out of two independent experiments . The U4 RNA is used as a loading control .

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

D) Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1∆Nter mutant. Northern blot assays performed in a Sen1-AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in figure 3B. Experiments performed in a ∆rrp6 background. Representative gel of one out of two independent experiments.The U4 RNA is used as a loading control.


E ) Sen1 Nter interacts with the C - terminal domain ( Cter ) of Sen1 both in the presence and in the absence of the NIM in vitro . Pull - down experiments using either a wt or a ∆ NIM version of recombinant Sen1 Cter immobilized on glutathione - sepharose beads and a TAP - tagged version of Sen1 Nter expressed in yeast . Representative gel of one out of three independent experiments .

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

E) Sen1 Nter interacts with the C-terminal domain (Cter) of Sen1 both in the presence and in the absence of the NIM in vitro. Pull-down experiments using either a wt or a ∆NIM version of recombinant Sen1 Cter immobilized on glutathione-sepharose beads and a TAP-tagged version of Sen1 Nter expressed in yeast. Representative gel of one out of three independent experiments.


A - C ) Comparison of the distribution of Sen1 and RNAPII ( Rpb1 ) at mRNAs and CUTs . Values on the y axis correspond to the median coverage . Note that the scale of Sen1 and Rpb1 datasets have been modified to be able to visualize both in the same plot .

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

A-C) Comparison of the distribution of Sen1 and RNAPII (Rpb1) at mRNAs and CUTs. Values on the y axis correspond to the median coverage. Note that the scale of Sen1 and Rpb1 datasets have been modified to be able to visualize both in the same plot.


D - F ) IGV screenshots of two mRNAs and one CUT exemplifying the trend observed in the metagene plots .

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D-F) IGV screenshots of two mRNAs and one CUT exemplifying the trend observed in the metagene plots.


G ) Metagene analysis of the distribution of the different Sen1 variants at mRNAs using either the TSS or the PAS as reference point . Values correspond to the median of Sen1 data normalized by the RNAPII signal obtained in the corresponding background .

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

G) Metagene analysis of the distribution of the different Sen1 variants at mRNAs using either the TSS or the PAS as reference point. Values correspond to the median of Sen1 data normalized by the RNAPII signal obtained in the corresponding background.


H - I ) Screenshots of two well - characterized protein - coding genes that are regulated by NNS - dependent premature termination . Values correspond to unnormalized Sen1 occupancy .

O O O O 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-GENEPROD O O

H-I) Screenshots of two well-characterized protein-coding genes that are regulated by NNS-dependent premature termination. Values correspond to unnormalized Sen1 occupancy.


J ) Profile of Sen1 binding at CUTs calculated as for mRNAs .

J) Profile of Sen1 binding at CUTs calculated as for mRNAs.


K - L ) Examples of CUTs exhibiting differences in Sen1 occupancy upon deletion of the NIM or the Nter . Plots correspond to unnormalized data .

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

K-L) Examples of CUTs exhibiting differences in Sen1 occupancy upon deletion of the NIM or the Nter. Plots correspond to unnormalized data.


M - O ) Analysis of the distribution of the different Sen1 versions around snoRNAs performed as for CUTs .

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M-O) Analysis of the distribution of the different Sen1 versions around snoRNAs performed as for CUTs.


P ) Comparison of the total levels of the different Sen1 variants at mRNAs , CUTs and the termination region of snoRNAs ( i . e . a window of 200 nt downstream of the 3 ' end of the mature snoRNA , snoRNA - ter ) . Boxes in box - plots include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median . The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1 . 5 * IQR . The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1 . 5 * IQR . Datasets for the different Sen1 versions were compared using a t - test ( bilateral distributions , unpaired data ) . p - values ≥ 0 . 05 are indicated as ns ( not significant ) , one asterisk is used when 0 . 05 > p - value ≥ 0 . 01 , two asterisks when 0 . 01 > p - value ≥ 0 . 001 and three asterisks for p - value < 0 . 001 .

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

P) Comparison of the total levels of the different Sen1 variants at mRNAs, CUTs and the termination region of snoRNAs (i.e. a window of 200 nt downstream of the 3' end of the mature snoRNA, snoRNA-ter). Boxes in box-plots include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR. The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. Datasets for the different Sen1 versions were compared using a t-test (bilateral distributions, unpaired data). p-values≥ 0.05 are indicated as ns (not significant), one asterisk is used when 0.05>p-value≥0.01, two asterisks when 0.01>p-value≥0.001 and three asterisks for p-value <0.001.


A ) Decreasing the elongation rate alleviates the termination defects associated with deletion of Sen1 Nter . Northern blot assays performed in a Sen1 - AID , ∆ rrp6 strain carrying a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in former experiments . Where indicated , cells were treated with 50 mg / L of 6 - azauracile ( 6AU ) for 2h . Representative gel of one out of two independent biological replicates . The U4 RNA is used as a loading control .

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

A) Decreasing the elongation rate alleviates the termination defects associated with deletion of Sen1 Nter. Northern blot assays performed in a Sen1-AID, ∆rrp6 strain carrying a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in former experiments. Where indicated, cells were treated with 50 mg/L of 6-azauracile (6AU) for 2h. Representative gel of one out of two independent biological replicates.The U4 RNA is used as a loading control.


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

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

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 B-ORGANISM B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY 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 B-ORGANISM O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM B-CELL_TYPE O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 .

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

B-CELL_TYPE O O O O O B-EXP_ASSAY O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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

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

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.