Human osteosarcoma U2OS cells were pre - treated with dactinomycin ( DACT ) , bortezomib ( BTZ ) , daunorubicin ( DAUN ) , docetaxel ( DOC ) , doxorubicin ( DOXO ) , epirubicin ( EPI ) , mitoxantrone ( MTX ) , paclitaxel ( PACL ) , vinblastine ( VB ) and vincristine ( VC ) at 0 . 5 , 1 and 5 µM ; with cisplatin ( CDDP ) at 75 , 150 and 300 µM , with oxaliplatin ( OXA ) at 250 , 500 and 1000 µM and with crizotinib ( CRIZ ) at 10 , 20 and 40 µM for 1 . 5 to 2 . 5 h and followed by an additional hour of treatment in the presence of 100 mM 5 - ethynyl uridine ( EU ) . After fixation cells were permeabilized and EU was stained with an AlexaFluor - 488 - coupled azide . Representative images are shown for each treatment ( A ) . The EU intensity in the nucleus of each condition was ranked between the untreated control ( Ctr , 0 % transcription inhibition ) and the control that was not incubated with EU ( corresponding to 100 % transcription inhibition ) ( B ) . Representative images of DACT 1 µM , BTZ 1 µM , CDDP 150 µM , CRIZ 20 µM , DAUN 0 . 5 µM , DOC 1 µM , DOXO 1 µM , EPI 1 µM , MTX 1 µM , OXA 500 µM , PACL 1 µM , VB 1 µM , VC 1 µM are

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

Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h and followed by an additional hour of treatment in the presence of 100 mM 5-ethynyl uridine (EU). After fixation cells were permeabilized and EU was stained with an AlexaFluor-488-coupled azide. Representative images are shown for each treatment (A). The EU intensity in the nucleus of each condition was ranked between the untreated control (Ctr, 0 % transcription inhibition) and the control that was not incubated with EU (corresponding to 100 % transcription inhibition) (B). Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown


Human osteosarcoma U2OS cells were pre - treated with dactinomycin ( DACT ) , bortezomib ( BTZ ) , daunorubicin ( DAUN ) , docetaxel ( DOC ) , doxorubicin ( DOXO ) , epirubicin ( EPI ) , mitoxantrone ( MTX ) , paclitaxel ( PACL ) , vinblastine ( VB ) and vincristine ( VC ) at 0 . 5 , 1 and 5 µM ; with cisplatin ( CDDP ) at 75 , 150 and 300 µM , with oxaliplatin ( OXA ) at 250 , 500 and 1000 µM and with crizotinib ( CRIZ ) at 10 , 20 and 40 µM for 1 . 5 to 2 . 5 h Cells were treated for 2 . 5 h and before fixation and permeabilization . Then , cells were stained with a rabbit anti - fibrillarin antibody followed by a staining with an anti - rabbit AlexaFluor - 647 - or AlexaFluor - 546 - coupled secondary antibody as well as with a mouse anti - nucleolin antibody followed by a staining with an anti - mouse AlexaFluor - 488 - coupled secondary antibody . Then images were acquired and colocalization between both signals was assessed ( C ) . The surface overlap coefficient ( SOC ) was calculated and ranked between the untreated control ( Ctr ) and the positive control ( DACT ) Representative images of DACT 1 µM , BTZ 1 µM , CDDP 150 µM , CRIZ 20 µM , DAUN 0 . 5 µM , DOC 1 µM , DOXO 1 µM , EPI 1 µM , MTX 1 µM , OXA 500 µM , PACL 1 µM , VB 1 µM , VC 1 µM are

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

Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h Cells were treated for 2.5 h and before fixation and permeabilization. Then, cells were stained with a rabbit anti-fibrillarin antibody followed by a staining with an anti-rabbit AlexaFluor-647- or AlexaFluor-546-coupled secondary antibody as well as with a mouse anti-nucleolin antibody followed by a staining with an anti-mouse AlexaFluor-488-coupled secondary antibody. Then images were acquired and colocalization between both signals was assessed (C). The surface overlap coefficient (SOC) was calculated and ranked between the untreated control (Ctr) and the positive control (DACT) Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown


Human osteosarcoma U2OS cells were pre - treated with dactinomycin ( DACT ) , bortezomib ( BTZ ) , daunorubicin ( DAUN ) , docetaxel ( DOC ) , doxorubicin ( DOXO ) , epirubicin ( EPI ) , mitoxantrone ( MTX ) , paclitaxel ( PACL ) , vinblastine ( VB ) and vincristine ( VC ) at 0 . 5 , 1 and 5 µM ; with cisplatin ( CDDP ) at 75 , 150 and 300 µM , with oxaliplatin ( OXA ) at 250 , 500 and 1000 µM and with crizotinib ( CRIZ ) at 10 , 20 and 40 µM for 1 . 5 to 2 . 5 h Cells were pre - treated overnight with the aforementioned compounds in complete medium followed by washout and treatment pursued in methionine - free medium for 30 min . Afterwards , the treatments were continued in methionine - free medium supplemented with 25 µM L - azidohomoalanine ( AHA ) for additional 1 . 5 h . AHA incorporation was detected after fixation , permeabilization and blocking by the addition of an AlexaFluor - 488 - coupled azide . Then images were acquired ( E ) and AHA intensity in the cells was ranked between the untreated control ( Ctr , 0 % translation inhibition ) and the untreated control without AHA ( corresponding to 100 % translation inhibition ) ( F ) . Representative images of DACT 1 µM , BTZ 1 µM , CDDP 150 µM , CRIZ 20 µM , DAUN 0 . 5 µM , DOC 1 µM , DOXO 1 µM , EPI 1 µM , MTX 1 µM , OXA 500 µM , PACL 1 µM , VB 1 µM , VC 1 µM are

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

Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h Cells were pre-treated overnight with the aforementioned compounds in complete medium followed by washout and treatment pursued in methionine-free medium for 30 min. Afterwards, the treatments were continued in methionine-free medium supplemented with 25 µM L-azidohomoalanine (AHA) for additional 1.5 h. AHA incorporation was detected after fixation, permeabilization and blocking by the addition of an AlexaFluor-488-coupled azide. Then images were acquired (E) and AHA intensity in the cells was ranked between the untreated control (Ctr, 0 % translation inhibition) and the untreated control without AHA (corresponding to 100 % translation inhibition) (F). Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown


Correlation between the transcription measured by EU incorporation and measured by fibrillarin and nucleolin colocalization is depicted with Pearson correlation coefficient ( R ) and p - value ( p ) ( G ) . The same parameters are shown for the correlation between transcription measured by EU incorporation and translation measured with AHA

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

Correlation between the transcription measured by EU incorporation and measured by fibrillarin and nucleolin colocalization is depicted with Pearson correlation coefficient (R) and p-value (p) (G). The same parameters are shown for the correlation between transcription measured by EU incorporation and translation measured with AHA incorporation


U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and ICD prediction score Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR )

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

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and ICD prediction score Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and peIF2α expression Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR )

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

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and peIF2α expression Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and CALR exposure Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR )

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and CALR exposure Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and ATP decrease Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR )

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

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and ATP decrease Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and HMBG1 exodus Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR )

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and HMBG1 exodus Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . The correlations between transcription inhibition and biological ICD score ( F ) previously measured and expressed as z - scores ( except for ICD prediction score ) Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR )

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

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and biological ICD score (F) previously measured and expressed as z-scores (except for ICD prediction score)  Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


U2OS wild - type cells were treated with a custom made anti - cancer library previously described Bezu et al , 2018 ( ) at 3 μM , supplemented with 500 μM oxaliplatin ( OXA ) , 150 μM cisplatin , 50 μM resveratrol and 50 μM spermidine . For assessing transcription , cells were pre - treated for 1 . 5 h with the library followed by 1 h with the same drugs in which EU was added . For assessing translation , cells were pre - treated with the library for 12 h followed by 30 min in methionine free - medium , before addition of azidohomoalanine ( AHA ) . Percentage of inhibition was calculated and transformed as z - scores . The correlations between transcription and translation inhibitions Known immunogenic drugs are indicated with colors : dactinomycin ( DACT ) , mitoxantrone ( MTX ) , doxorubicin ( DOXO ) , daunorubicin ( DAUN ) , OXA , docetaxel ( DOC ) , paclitaxel ( PACL ) , vinblastine ( VB ) , vincristine ( VC ) and vinorelbine ( VR )

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

U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. For assessing translation, cells were pre-treated with the library for 12 h followed by 30 min in methionine free-medium, before addition of azidohomoalanine (AHA). Percentage of inhibition was calculated and transformed as z-scores. The correlations between transcription and translation inhibitions Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)


The inhibition of transcription was assessed for the negative and positive ICD hits identified with the predictive algorithm ( Figure 1 ) . U2OS cells were treated with the agents at concentrations corresponding to their IC60 : 1 μM dactinomycin ( DACT ) , 50 μM topotecan , 1 μM becatecarin , 0 . 5 μM trabectedin , 5 μM UCN - 01 , 30 μM mycophenolate mofetil , 30 μM nonoxynol - 9 , 25 μM dactolisib , 2 . 5 μM β - lapachone , 5 μM 5 - fluorodeoxycytidine and 2 μM RH - 1 for 1 . 5 h followed by 1 h with EU . The percentage of transcription inhibition was calculated and the coefficient of correlation ( R ) and associated p - value ( p ) between the percentage of inhibition and the theoretical ICD score was calculated using the Pearson method .

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The inhibition of transcription was assessed for the negative and positive ICD hits identified with the predictive algorithm (Figure 1). U2OS cells were treated with the agents at concentrations corresponding to their IC60: 1 μM dactinomycin (DACT), 50 μM topotecan, 1 μM becatecarin, 0.5 μM trabectedin, 5 μM UCN-01, 30 μM mycophenolate mofetil, 30 μM nonoxynol-9, 25 μM dactolisib, 2.5 μM β-lapachone, 5 μM 5-fluorodeoxycytidine and 2 μM RH-1 for 1.5 h followed by 1 h with EU. The percentage of transcription inhibition was calculated and the coefficient of correlation (R) and associated p-value (p) between the percentage of inhibition and the theoretical ICD score was calculated using the Pearson method.


G Verify the mobility of GFP ( control ) mRNA and GFP - MdCAX3 mRNA by RT - PCR in Mx leaf , stem , and root . The recombinant vectors ( F ) were transformed into A . tumefaciens GV3101 and introduced into Mx leaves by vacuum infiltration .

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G Verify the mobility of GFP (control) mRNA and GFP-MdCAX3 mRNA by RT-PCR in Mx leaf, stem, and root. The recombinant vectors (F) were transformed into A. tumefaciens GV3101 and introduced into Mx leaves by vacuum infiltration.


H GFP fluorescence image of the Mx roots under different treatment . Nuclei were labeled using DAPI . Scale Bar : 100 μm .

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H GFP fluorescence image of the Mx roots under different treatment. Nuclei were labeled using DAPI. Scale Bar: 100 μm.


H Ratio ( 488 / 440 ) image of BCECF fluorescence in the roots of MdCAX3 - suppressed in leaves Md / Mx . Scale bars : 100 μm .

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H Ratio (488/440) image of BCECF fluorescence in the roots of MdCAX3-suppressed in leaves Md/Mx. Scale bars: 100 μm.


I Rhizosphere acidification of MdCAX3 - suppressed in leaves Md / Mx . Bromocresol purple was used as a pH indicator for visualization . Scale bars : 1 cm .

I Rhizosphere acidification of MdCAX3-suppressed in leaves Md/Mx. Bromocresol purple was used as a pH indicator for visualization. Scale bars: 1 cm.


F GUS staining in N . benthamiana leaves after transient co - expression of pro35S : : GFP with proMxCXIP1 : GUS or proMbCXIP1 : GUS , and co - expression with pBI101 : GUS as a control .

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F GUS staining in N. benthamiana leaves after transient co-expression of pro35S::GFP with proMxCXIP1:GUS or proMbCXIP1:GUS, and co-expression with pBI101:GUS as a control.


G Interaction between MdCXIP1 and MdCAX3 tested by yeast two - hybrid ( Y2H ) assays . A dilution series of NMY51 yeast cells co - expressing MdCXIP1 and MdCAX3 cultured on SD / II and SDIV screening media . pPBT3 - N ( empty vector ) co - expressed with MdCXIP1 was used as a negative control . X - gal was used in SD / - Trp / - Leu / - His / - Ade screening medium to further test for possible interactions .

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G Interaction between MdCXIP1 and MdCAX3 tested by yeast two-hybrid (Y2H) assays. A dilution series of NMY51 yeast cells co-expressing MdCXIP1 and MdCAX3 cultured on SD/II and SDIV screening media. pPBT3-N (empty vector) co-expressed with MdCXIP1 was used as a negative control. X-gal was used in SD/-Trp/-Leu/-His/-Ade screening medium to further test for possible interactions.


I Interaction between MdCXIP1 and the MdCAX3 N - terminal autoinhibitory domain in an in vitro pull - down assay . The His - MdCXIP1 , GST and GST - N - MdCAX3 fusion proteins were expressed in the Escherichia coli BL21 strain , and the GST protein alone was used as a negative control . ' + ' indicates presence and " − " indicates absence . IB , immunoblot .

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I Interaction between MdCXIP1 and the MdCAX3 N-terminal autoinhibitory domain in an in vitro pull-down assay. The His-MdCXIP1, GST and GST-N-MdCAX3 fusion proteins were expressed in the Escherichia coli BL21 strain, and the GST protein alone was used as a negative control. '+' indicates presence and "−" indicates absence. IB, immunoblot.


C Fluorescence ratio ( 488 / 440 nm excitations ) of BCECF fluorescence in the roots of MdCAX3 and MdCXIP1 co - overexpressing Mb . The central band represents the median and the box ranges showing the interquartile range and covers the central 50 % of the data . The whiskers showing the minimum and maximum of the data . Three biological replicates ; 3 roots were quantified for each replicate . Asterisks indicate statistically significant differences ( * * * , P < 0 . 001 ; ANOVA , Tukey correction ) .

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C Fluorescence ratio (488/440 nm excitations) of BCECF fluorescence in the roots of MdCAX3 and MdCXIP1 co-overexpressing Mb. The central band represents the median and the box ranges showing the interquartile range and covers the central 50% of the data. The whiskers showing the minimum and maximum of the data. Three biological replicates; 3 roots were quantified for each replicate. Asterisks indicate statistically significant differences (***, P < 0.001; ANOVA, Tukey correction).


D Ratio ( 488 / 440 ) image of BCECF fluorescence in MdCAX3 and MdCXIP1 co - overexpressing Mb . Scale Bar : 100 μm . E Rhizosphere acidification in MdCAX3 and MdCXIP1 co - overexpressing Mb . Bromocresol purple was used as a pH indicator for visualization . Scale Bar : 1 cm .

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D Ratio (488/440) image of BCECF fluorescence in MdCAX3 and MdCXIP1 co-overexpressing Mb. Scale Bar: 100 μm. E Rhizosphere acidification in MdCAX3 and MdCXIP1 co-overexpressing Mb. Bromocresol purple was used as a pH indicator for visualization. Scale Bar: 1 cm.


D Cellular localization of Zn2 + ( blue ) in Mx and MdCAX3 - suppressed Mx roots under Fe deficient conditions . Arrowhead indicates the cytoplasm . Scale Bars : 20 μm . E Fluorescence intensity detection at the position indicated by the long and narrow arrow in the Fig 6D .

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D Cellular localization of Zn2+ (blue) in Mx and MdCAX3-suppressed Mx roots under Fe deficient conditions. Arrowhead indicates the cytoplasm. Scale Bars: 20 μm. E Fluorescence intensity detection at the position indicated by the long and narrow arrow in the Fig 6D.


HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( A ) Concentrations of IL - 6 were determined ( CTRL , n = 9 ; Bax / Bak , n = 6 ; Bcl - XL , n = 3 ; STING , n = 3 ) .

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HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (A) Concentrations of IL-6 were determined (CTRL, n=9; Bax/Bak, n=6; Bcl-XL, n=3; STING, n=3).


HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( B ) Concentrations of IL - 8 were determined ( CTRL , n = 7 ; Bax / Bak , n = 4 ) .

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HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (B) Concentrations of IL-8 were determined (CTRL, n=7; Bax/Bak, n=4).


HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( C ) Concentrations of CXCL1 were determined ( CTRL , n = 8 ; Bax / Bak , n = 5 ) .

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HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (C) Concentrations of CXCL1 were determined (CTRL, n=8; Bax/Bak, n=5).


HeLa cell lines were treated with the small - molecule Bcl - 2 / Bcl - XL - inhibitor ABT - 737 for 72 h . Cytokines were measured by ELISA . ( D ) Concentrations of FGF - 2 were determined ( CTRL , n = 7 ; Bax / Bak , n = 4 ) .

HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (D) Concentrations of FGF-2 were determined (CTRL, n=7; Bax/Bak, n=4).


HeLa cell lines were treated with the Mcl - 1 - inhibitor S63845 for 72 h . Cytokines were measured by ELISA . ( E ) HeLa CTRL cells and Bax / Bak - deficient cells were treated with the Mcl - 1 - inhibitor S63845 . Concentrations of IL - 6 in supernatants are shown ( CTRL , n = 8 ; Bax / Bak , n = 7 ) .

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HeLa cell lines were treated with the Mcl-1-inhibitor S63845 for 72 h. Cytokines were measured by ELISA. (E) HeLa CTRL cells and Bax/Bak-deficient cells were treated with the Mcl-1-inhibitor S63845. Concentrations of IL-6 in supernatants are shown (CTRL, n=8; Bax/Bak, n=7).


F 1205Lu human melanoma cells or 1205Lu melanoma cells over - expressing Bcl - XL were treated with ABT - 737 . Concentrations of IL - 6 in the supernatants were measured after 48 h ( n = 5 ) .

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F 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing Bcl-XL were treated with ABT-737. Concentrations of IL-6 in the supernatants were measured after 48 h (n=5).


G 1205Lu human melanoma cells or 1205Lu melanoma cells over - expressing mouse Bcl - XL were treated with various concentrations of ABT - 737 . To some aliquots the caspase - inhibitor zVAD - fmk ( 50 μM ) was added . Relative numbers of apoptotic cells were determined as in EV 1B after 48 h . Data are means / SEM of at least three independent experiments . ( 1205Lu , n = 4 ; 1205Lu 5 µM ABT - 737 , n = 3 ; 1205Lu / Bcl - XL and 1205Lu + zVAD , n = 3 ) .

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G 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing mouse Bcl-XL were treated with various concentrations of ABT-737. To some aliquots the caspase-inhibitor zVAD-fmk (50 μM) was added. Relative numbers of apoptotic cells were determined as in EV 1B after 48 h. Data are means/SEM of at least three independent experiments. (1205Lu, n=4; 1205Lu 5 µM ABT-737, n=3; 1205Lu/Bcl-XL and 1205Lu + zVAD, n=3).


H HeLa - UL12 . 5 cells untreated or treated with tamoxifen ( 100 nM , 24 h ) were stained with Picogreen ( 3 µl / ml ) to detect mitochondrial DNA . Scale bare , 10

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H HeLa-UL12.5 cells untreated or treated with tamoxifen (100 nM, 24 h) were stained with Picogreen (3 µl/ml) to detect mitochondrial DNA. Scale bare, 10 µm


I HeLa CTRL cells and HeLa - UL12 . 5 cells were treated with ABT - 737 ( 10µM ) . Concentration of IL - 6 in the supernatant were measured after 72h ( n = 3 ) .

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I HeLa CTRL cells and HeLa-UL12.5 cells were treated with ABT-737 (10µM). Concentration of IL-6 in the supernatant were measured after 72h (n=3).


A , The caspase - 3 - reporter cell line ( labelled DEVD ) and the control cell line ( DEVG ; see methods ) were infected and analysed at various time points post - infection by flow cytometry ( Chlamydia trachomatis ( Ctr ; MOI = 1 , 30 h ) ; Salmonella Typhimurium ( STy ; MOI = 50 , 24h ) ; Toxoplasma gondii ( MOI = 5 , 16 h ) ; MVA ( MOI = 10 , 16h ) ; Influenza A virus ( IAV ; MOI = 1 , 16h ) ; Herpes simplex virus 1 ( HSV - 1 ; MOI = 1 , 16h ) . Staurosporine treated cells ( 1 μM , 3h ) were used as a positive control . Percentages of cells positive for caspase - 3 - activation ( FRET - loss ) were determined . Data are means / SEM of three independent experiments . ( n = 3 )

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A, The caspase-3-reporter cell line (labelled DEVD) and the control cell line (DEVG; see methods) were infected and analysed at various time points post-infection by flow cytometry (Chlamydia trachomatis (Ctr; MOI=1, 30 h); Salmonella Typhimurium (STy; MOI=50, 24h); Toxoplasma gondii (MOI=5, 16 h); MVA (MOI=10, 16h); Influenza A virus (IAV; MOI=1, 16h); Herpes simplex virus 1 (HSV-1; MOI=1, 16h). Staurosporine treated cells (1 μM, 3h) were used as a positive control. Percentages of cells positive for caspase-3-activation (FRET-loss) were determined. Data are means/SEM of three independent experiments. (n=3)


B , C Cells were infected with modified vaccinia virus Ankara ( MVA ) ( MOI = 10 ) . The caspase - inhibitor zVAD - fmk was used at 100µM ; cells were analysed 16 h post - infection .

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B, C Cells were infected with modified vaccinia virus Ankara (MVA) (MOI=10). The caspase-inhibitor zVAD-fmk was used at 100µM; cells were analysed 16 h post-infection.


D , E Cells were infected with influenza A virus ( IAV ) ( MOI = 1 ; analysis was 16 h post - infection ) . D , detection of viral M2 protein was used as infection control .

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D, E Cells were infected with influenza A virus (IAV) (MOI=1; analysis was 16 h post-infection). D, detection of viral M2 protein was used as infection control.


F , G Cells were infected with Herpes simplex virus 1 ( HSV - 1 ) ( MOI = 1 ; analysis was 16 h post - infection ) .

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F, G Cells were infected with Herpes simplex virus 1 (HSV-1) (MOI=1; analysis was 16 h post-infection).


H Cells were infected with Chlamydia trachomatis ( Ctr ) ( MOI = 1 ; analysis was 30 h post - infection ) .

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H Cells were infected with Chlamydia trachomatis (Ctr) (MOI=1; analysis was 30 h post-infection).


I Cells were infected with Salmonella Typhimurium ( Sty ) ( MOI = 50 ; analysis was 24 h post - infection ) .

I Cells were infected with Salmonella Typhimurium (Sty) (MOI=50; analysis was 24 h post-infection).


J Cells were infected with the protozoan parasite Toxoplasma gondii ( MOI = 5 ; analysis was 16 h post - infection ) .

J Cells were infected with the protozoan parasite Toxoplasma gondii (MOI=5; analysis was 16 h post-infection).


A Infection with MVA ( MOI = 10 for 16 h ; IL - 6 ) .

A Infection with MVA (MOI=10 for 16 h; IL-6).


B infection with HSV - 1 ( MOI = 1 for 16 h ; IL - 6 ) .

B infection with HSV-1 (MOI=1 for 16 h; IL-6).


Infection with Chlamydia trachomatis ( Ctr ; MOI = 1 for 30 h ; IL -

Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; IL-6


Infection with Chlamydia trachomatis ( Ctr ; MOI = 1 for 30 h ; , IL - 8 ) .

Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; , IL-8).


Infection with Salmonella Typhimurium ( STy ; MOI = 50 for 24 h ; IL -

Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; IL-6


Infection with Salmonella Typhimurium ( STy ; MOI = 50 for 24 h ; CXCL - 1 ) .

Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; CXCL-1).


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; IL -

Infection with Toxoplasma gondii (MOI=5 for 16 h; IL-6


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; , IL -

Infection with Toxoplasma gondii (MOI=5 for 16 h; , IL-8


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; CXCL -

Infection with Toxoplasma gondii (MOI=5 for 16 h; CXCL-1


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; FGF - 2 ) .

Infection with Toxoplasma gondii (MOI=5 for 16 h; FGF-2).


( A ) HeLa cell lines with an intact ( CTRL , carrying a non - coding gRNA ) or inactive ( Bax / Bak - double deficient ) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP ( MOI = 0 . 2 ) . Cells were analyzed by flow cytometry at the indicated time points . Cells were gated on the GFP - positive ( infected ) host cell population , and mean fluorescence intensity was recorded . Data are means / SEM of six experiments . ( A ) Growth of C . trachomatis was measured in HeLa cells as increase in GFP - fluorescence between 18 and 38 h . The data show means / SEM of six independent experiments ( genotype effect ( significance of the difference of GFP - expression between the cell lines overall , p = 0 . 08 ) . Red line indicates the fold change calculation of the individual different time points and cell lines ( significance tested ( one sample t - test ) for each time point for a difference between Bax / Bak vs CTRL ) .

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(A) HeLa cell lines with an intact (CTRL, carrying a non-coding gRNA) or inactive (Bax/Bak-double deficient) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP (MOI=0.2). Cells were analyzed by flow cytometry at the indicated time points. Cells were gated on the GFP-positive (infected) host cell population, and mean fluorescence intensity was recorded. Data are means/SEM of six experiments. (A) Growth of C. trachomatis was measured in HeLa cells as increase in GFP-fluorescence between 18 and 38 h. The data show means/SEM of six independent experiments (genotype effect (significance of the difference of GFP-expression between the cell lines overall, p = 0.08). Red line indicates the fold change calculation of the individual different time points and cell lines (significance tested (one sample t-test) for each time point for a difference between Bax/Bak vs CTRL).


AGS cell lines with an intact ( CTRL , carrying a non - coding gRNA ) or inactive ( Bax / Bak - double deficient ) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP ( MOI = 0 . 2 ) . Cells were analyzed by flow cytometry at the indicated time points . Cells were gated on the GFP - positive ( infected ) host cell population , and mean fluorescence intensity was recorded . Data are means / SEM of 4 - 5 experiments . ( B ) Growth of C . trachomatis was measured in AGS cells as increase in GFP - fluorescence . The data show means / SEM of 4 to 5 independent experiments ( genotype effect : p < 0 . 0001 ; time - genotype interaction : p < 0 . 0001 ) ( 12 h and 38 h , n = 4 ; 18 h , n = 5 ) .

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AGS cell lines with an intact (CTRL, carrying a non-coding gRNA) or inactive (Bax/Bak-double deficient) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP (MOI=0.2). Cells were analyzed by flow cytometry at the indicated time points. Cells were gated on the GFP-positive (infected) host cell population, and mean fluorescence intensity was recorded. Data are means/SEM of 4-5 experiments. (B) Growth of C. trachomatis was measured in AGS cells as increase in GFP-fluorescence. The data show means/SEM of 4 to 5 independent experiments (genotype effect: p<0.0001; time-genotype interaction: p<0.0001) (12 h and 38 h, n=4; 18 h, n=5).


C Chlamydial genomes per cell culture ( expressed as chlamydial DNA in 50 ng of total DNA , determined by quantitative PCR ) . * , p < 0 . 05 between control and mutant cells . ( n = 3 )

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C Chlamydial genomes per cell culture (expressed as chlamydial DNA in 50 ng of total DNA, determined by quantitative PCR). *, p<0.05 between control and mutant cells. (n=3)


D CTRL , Bax / Bak - deficient or Bcl - XL - over - expressing HeLa cells were infected with Salmonella Typhimurium ( MOI = 50 ) . Extracellular bacteria were killed using non cell - permeable antibiotic . At the indicated time points cells were lysed , and aliquots were plated on agar plates . Recovered colony forming units were calculated . Data are means / SEM of four independent experiments . Significant genotype effects were found between mutant and control cells ( Bax / Bak p < 0 . 0001 ; Bcl - XL p 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 CTRL, Bax/Bak-deficient or Bcl-XL-over-expressing HeLa cells were infected with Salmonella Typhimurium (MOI=50). Extracellular bacteria were killed using non cell-permeable antibiotic. At the indicated time points cells were lysed, and aliquots were plated on agar plates. Recovered colony forming units were calculated. Data are means/SEM of four independent experiments. Significant genotype effects were found between mutant and control cells (Bax/Bak p<0.0001; Bcl-XL p 0.0001).


E CTRL , Bax / Bak - deficient or Apaf - 1 - deficient HeLa cells were infected with Salmonella Typhimurium ( MOI = 50 ) . CFU / well were calculated as in ( D ) . n = 3 .

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

E CTRL, Bax/Bak-deficient or Apaf-1-deficient HeLa cells were infected with Salmonella Typhimurium (MOI=50). CFU/well were calculated as in (D). n=3.


B Yeast two - hybrid analysis for interaction between the above - shown Rabaptin5 segments ( Rbpt5 # , fused to LexA on the bait plasmid ) and residues 257 - 444 of FIP200 ( FIP , fused to the Gal4 activation domain on the prey plasmid ) to drive HIS3 expression . Three different clones each were replica - plated on medium with His or without His , but containing 3 - amino - 1 , 2 , 4 - triazole ( 3AT ; an inhibitor of His synthesis to increase stringency ) and grown in the absence of Trp and leucine as a control . As negative controls , empty bait or prey plasmids were used . The asterisk indicates a clone invalidated by recombination .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Yeast two-hybrid analysis for interaction between the above-shown Rabaptin5 segments (Rbpt5#, fused to LexA on the bait plasmid) and residues 257-444 of FIP200 (FIP, fused to the Gal4 activation domain on the prey plasmid) to drive HIS3 expression. Three different clones each were replica-plated on medium with His or without His, but containing 3-amino-1,2,4-triazole (3AT; an inhibitor of His synthesis to increase stringency) and grown in the absence of Trp and leucine as a control. As negative controls, empty bait or prey plasmids were used. The asterisk indicates a clone invalidated by recombination.


D FIP200 was immunoprecipitated ( IP ) from lysates of HeLa or HEK293A cells and probed for FIP200 , Rabaptin5 ( Rbpt5 ) , and EEA1 ( early endosome antigen 1 ) by immunoblotting . Input lysate ( 10 % ) was immunoblotted blotted parallel . As a negative control , the immunoprecipitation was performed using an anti - GAPDH antibody . E - H Lysates of HeLa cells transiently transfected with full - length FIP200 - mCherry ( FIP200 - mCh ) or a deletion mutant lacking the segment interacting with Rabaptin5 ( ∆ 280 - 440 ) were immuprecipitated with anti - mCherry ( IP FIP200 - mCh ) or , as a control , with anti - FLAG antibodies ( IP FLAG ) . Immunoprecipitates and input lysates ( 10 % ) were immunoblotted for mCherry and Rabaptin5 ( E ) , ATG13 ( F ) , or ULK1 ( G ) . Co - immunoprecipitation of Rabaptin5 , ATG13 , and ULK1 with FIP200 ∆ 280 - 440 ( FIP ∆ ) was quantified in comparison to that with wild - type FIP200 ( H ; signals normalized to that of the immunoprecipitated protein ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FIP200 was immunoprecipitated (IP) from lysates of HeLa or HEK293A cells and probed for FIP200, Rabaptin5 (Rbpt5), and EEA1 (early endosome antigen 1) by immunoblotting. Input lysate (10%) was immunoblotted blotted parallel. As a negative control, the immunoprecipitation was performed using an anti-GAPDH antibody. E-H Lysates of HeLa cells transiently transfected with full-length FIP200-mCherry (FIP200-mCh) or a deletion mutant lacking the segment interacting with Rabaptin5 (∆280-440) were immuprecipitated with anti-mCherry (IP FIP200-mCh) or, as a control, with anti-FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for mCherry and Rabaptin5 (E), ATG13 (F), or ULK1 (G). Co-immunoprecipitation of Rabaptin5, ATG13, and ULK1 with FIP200∆280-440 (FIP∆) was quantified in comparison to that with wild-type FIP200 (H; signals normalized to that of the immunoprecipitated protein;


To more easily visualize Rabaptin5 , a stable HEK293A cell line overexpressing Rabaptin5 ( HEK + Rbpt5 ) was generated . Immunofluorescence microscopy of Rabaptin5 and transferrin receptor ( TfR ) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min ( + CQ , panel A ) compared to untreated cells ( - CQ , A '

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

To more easily visualize Rabaptin5, a stable HEK293A cell line overexpressing Rabaptin5 (HEK+Rbpt5) was generated. Immunofluorescence microscopy of Rabaptin5 and transferrin receptor (TfR) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min (+CQ, panel A) compared to untreated cells (-CQ, A'


HEK + Rbpt5 cells , untransfected or 24 h after transfection with mCherry - galectin3 ( mCh - Gal3 ) , mRuby3 - galectin8 ( mRuby - Gal8 ) , mCherry - FIP200 , or - ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry - galectin3 or mRuby3 - galectin8 ( C ) , ubiquitin ( Ub ; D ) , p62 ( E ) , mCherry - FIP200 ( F ) , WIPI2 ( G ) , mCherry - ATG16L1 ( H ) , or LC3B ( I ) . Scale bar , 10 µm . In the enlarged insets , arrowheads point out chloroquine - induced enlarged , ring - like early endosomes . Rabaptin5 - positive enlarged endosomes positive for mCherry - galectin3 ( Gal3 ) or mRuby3 - galectin8 ( Gal8 ) were quantified ( C ' ; mean ± S . D .

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

HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-galectin3 (mCh-Gal3), mRuby3-galectin8 (mRuby-Gal8), mCherry-FIP200, or -ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry-galectin3 or mRuby3-galectin8 (C), ubiquitin (Ub; D), p62 (E), mCherry-FIP200 (F), WIPI2 (G), mCherry-ATG16L1 (H), or LC3B (I). Scale bar, 10 µm. In the enlarged insets, arrowheads point out chloroquine-induced enlarged, ring-like early endosomes. Rabaptin5-positive enlarged endosomes positive for mCherry-galectin3 (Gal3) or mRuby3-galectin8 (Gal8) were quantified (C'; mean ± S.D.


J , K HEK + Rbpt5 cells , untransfected or 24 h after transfection with mCherry - ATG16L1 , were treated with 60 µM chloroquine for 0 , 15 and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry - ATG16L1 . Manders ' colocalization coefficients were determined , M1 showing the fraction of Rabaptin5 - positive structures also positive for WIPI2 ( J ) or mCherry - ATG16L1 ( K ) , and M2 showing the respective inverse ( mean ± S . D . of three independent experiments ;

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

J, K HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-ATG16L1, were treated with 60 µM chloroquine for 0, 15 and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry-ATG16L1. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for WIPI2 (J) or mCherry-ATG16L1 (K), and M2 showing the respective inverse (mean ± S.D. of three independent experiments;


A HEK293A cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing Rabaptin5 ( siRbpt5 ) or FIP200 ( siFIP200 ) for 72 h and treated without ( - ) or with 60 µM chloroquine ( + CQ ) or 250 nM Torin1 for 150 min . Scale bar , 10 µm . Below , the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin ( Tub ) as a loading control . B WIPI2 or LC3B puncta per cell were quantified for each condition ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 , * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h and treated without (-) or with 60 µM chloroquine (+CQ) or 250 nM Torin1 for 150 min. Scale bar, 10 µm. Below, the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin (Tub) as a loading control. B WIPI2 or LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, **P<0.01). C


C HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy . Cells were fixed and immunostained for endogenous WIPI2 and LC3B . Scale bar , 10 µm . D WIPI2 or LC3B puncta per cell were quantified ( mean ± S . D . of three independent experiments ) .

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

C HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy. Cells were fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. D WIPI2 or LC3B puncta per cell were quantified (mean ± S.D. of three independent experiments).


A HEK293A cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing ATG13 ( siATG13 ) for 72 h , treated without ( - ) or with 60 µM chloroquine ( + CQ ) for 150 minutes , and fixed and immunostained for endogenous WIPI2 and LC3B . Scale bar , 10 µm . Efficiency of ATG13 knockdown was assayed by immunoblotting below on the right using GAPDH as loading controls . B WIPI2 of LC3B puncta per cell were quantified for each condition ( mean ± S . D . of three independent experiments ; ANOVA : * * P < 0 . 01 ) .

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

A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ATG13 (siATG13) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ATG13 knockdown was assayed by immunoblotting below on the right using GAPDH as loading controls. B WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: **P<0.01). C


HEK293A cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing ULK1 ( siULK1 ) , ULK2 ( siULK2 ) , or both ( siULK1 + 2 ) for 72 h , treated without ( - ) or with 60 µM chloroquine ( + CQ ) for 150 minutes , and fixed and immunostained for endogenous WIPI2 and LC3B . Scale bar , 10 µm . Efficiency of ULK1 knockdown was assayed by immunoblotting The efficiency of ULK2 knockdown was assayed by quantitative RT - PCR and the result is shown in panel C D WIPI2 of LC3B puncta per cell were quantified for each condition ( mean ± S . D . of three independent

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

HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ULK1 (siULK1), ULK2 (siULK2), or both (siULK1+2) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ULK1 knockdown was assayed by immunoblotting The efficiency of ULK2 knockdown was assayed by quantitative RT-PCR and the result is shown in panel C D WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments


E HEK293A cells were incubated without ( - ) , with 60 µM chloroquine ( + CQ ) , or with chloroquine and 5 µM MRT68921 ( + CQ + MRT ) for 150 min and fixed and stained for WIPI2 and LC3B . F WIPI2 of LC3B puncta per cell were quantified for each condition as in panel B ( mean ± S . D . of three independent experiments ) .

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E HEK293A cells were incubated without (-), with 60 µM chloroquine (+CQ), or with chloroquine and 5 µM MRT68921 (+CQ +MRT) for 150 min and fixed and stained for WIPI2 and LC3B. F WIPI2 of LC3B puncta per cell were quantified for each condition as in panel B (mean ± S.D. of three independent experiments).


B Co - immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR / Cas9 knockout cells lacking FIP200 ( FIP - KO ) . Anti - HA antibodies were used as a control ( IP HA ) . On the right , HEK293A and FIP200 knockout cells were immunoblotted for FIP200 and as a loading control of tubulin ( Tub ) . Signals were quantified and the ratios of mCherry - ATG16L1 / Rabaptin5 normalized to that of HEK293A cells without chloroquine

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

B Co-immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR/Cas9 knockout cells lacking FIP200 (FIP-KO). Anti-HA antibodies were used as a control (IP HA). On the right, HEK293A and FIP200 knockout cells were immunoblotted for FIP200 and as a loading control of tubulin (Tub). Signals were quantified and the ratios of mCherry-ATG16L1/Rabaptin5 normalized to that of HEK293A cells without chloroquine treatment


Lysates of HEK293A or HeLa cells transiently transfected with full - length mCherry - ATG16L1 ( wt ) or a mutant lacking the WD domain ( ∆ WD ; residues 1 - 319 of ATG16L1 , precisely deleting only the WD40 repeats residues 320 - 607 ) were immunoprecipitated with anti - Rabaptin5 or anti - FLAG antibodies , and immunoblotted for Rabaptin5 and mCherry - ATG16L1 , or immunoprecipitated with anti - FIP200 or anti - HA antibodies Co - immunoprecipitation of ATG16L1 ∆ WD with Rabaptin5 ( C ) was reduced to 6 . 6 ± 2 . 1 % and 3 . 4 ± 2 . 1 % in HEK293A and HeLa cells , respectively , relative to that of full - length ATG16L1 ( signals normalized to the immunoprecipitated protein ; mean ± S . D . deviation of three independent experiments each ) .

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

Lysates of HEK293A or HeLa cells transiently transfected with full-length mCherry-ATG16L1 (wt) or a mutant lacking the WD domain (∆WD; residues 1-319 of ATG16L1, precisely deleting only the WD40 repeats residues 320-607) were immunoprecipitated with anti-Rabaptin5 or anti-FLAG antibodies, and immunoblotted for Rabaptin5 and mCherry-ATG16L1 , or immunoprecipitated with anti-FIP200 or anti-HA antibodies Co-immunoprecipitation of ATG16L1∆WD with Rabaptin5 (C) was reduced to 6.6±2.1% and 3.4±2.1% in HEK293A and HeLa cells, respectively, relative to that of full-length ATG16L1 (signals normalized to the immunoprecipitated protein; mean ± S.D. deviation of three independent experiments each).


F Lysates of HeLa cells transiently transfected with myc - tagged wild - type Rabaptin5 ( wt ) or triple - alanine mutant ( AAA ) were immunoprecipitated with anti - myc ( IP myc ) or anti - FLAG antibodies ( IP FLAG ) , and immunoblotted for myc and ATG16L1 . Co - immunoprecipitation of ATG16L1 with Rabaptin5 - AAA triple mutant was reduced to 1 . 5 ± 1 . 2 % relative to that with wild - type Rabaptin5 ( signals normalized to that of the immunoprecipitated protein ; mean ± S . D . of three independent experiments ) .

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

F Lysates of HeLa cells transiently transfected with myc-tagged wild-type Rabaptin5 (wt) or triple-alanine mutant (AAA) were immunoprecipitated with anti-myc (IP myc) or anti-FLAG antibodies (IP FLAG), and immunoblotted for myc and ATG16L1. Co-immunoprecipitation of ATG16L1 with Rabaptin5-AAA triple mutant was reduced to 1.5±1.2% relative to that with wild-type Rabaptin5 (signals normalized to that of the immunoprecipitated protein; mean ± S.D. of three independent experiments).


A By immunoblot analysis , the levels of Rabaptin5 and as a loading control of tubulin ( Tub ) were assessed in wild - type HEK293A cells , HEK + Rbpt5 cells stably overexpressing Rabaptin5 , and Rabaptin5 - knockout cells without ( Rbpt5 - KO ) or with stable re - expression of wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) . B The same stable HEK293A - derived cell lines were treated without ( - CQ ) or with 60 µM chloroquine for 150 min ( + CQ ) and analyzed by immunofluorescence microscopy for WIPI2 or LC3B . Scale bar , 10 µm .

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

A By immunoblot analysis, the levels of Rabaptin5 and as a loading control of tubulin (Tub) were assessed in wild-type HEK293A cells, HEK+Rbpt5 cells stably overexpressing Rabaptin5, and Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA). B The same stable HEK293A-derived cell lines were treated without (-CQ) or with 60 µM chloroquine for 150 min (+CQ) and analyzed by immunofluorescence microscopy for WIPI2 or LC3B. Scale bar, 10 µm. C


D , E The HEK293A - derived cell lines were treated with 60 µM chloroquine for 30min and non - lipidated and lipidated LC3B ( I and II , resp . ) were assayed by immunoblot analysis ( D ) . The increase of the LC3B - II fraction of total LC3B upon chloroquine treatment was quantified ( mean ± S . D . of three independent experiments ;

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

D, E The HEK293A-derived cell lines were treated with 60 µM chloroquine for 30min and non-lipidated and lipidated LC3B (I and II, resp.) were assayed by immunoblot analysis (D). The increase of the LC3B-II fraction of total LC3B upon chloroquine treatment was quantified (mean ± S.D. of three independent experiments;


F , G HEK + Rbpt5 cells and Rabaptin5 - knockout cells stably re - expressing wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) were transfected with mCherry - ATG16L1 , treated with 60 µM chloroquine ( CQ ) for 0 , 15 and 30 min , and analyzed by immunofluorescence microscopy for Rabaptin5 , mCherry - ATG16L1 , and WIPI2 . Manders ' colocalization coefficients were determined , M1 showing the fraction of Rabaptin5 - positive structures also positive for mCherry - ATG16L1 ( F ) or WIPI2 ( G ) and M2 showing the inverse ( mean ± S . D . of three independent

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

F, G HEK+Rbpt5 cells and Rabaptin5-knockout cells stably re-expressing wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were transfected with mCherry-ATG16L1, treated with 60 µM chloroquine (CQ) for 0, 15 and 30 min, and analyzed by immunofluorescence microscopy for Rabaptin5, mCherry-ATG16L1, and WIPI2. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for mCherry-ATG16L1 (F) or WIPI2 (G) and M2 showing the inverse (mean ± S.D. of three independent experiments


H Wild - type HEK293A cells , HEK + Rbpt5 cells , Rabaptin5 - knockout cells without ( Rbpt5 - KO ) or with stable re - expression of wild - type ( Rbpt5 - KO + wt ) or AAA - mutant Rabaptin5 ( Rbpt5 - KO + AAA ) were stained with lysotracker and with DAPI for nuclei . Scale bar , 10 µm . I The number of lysosomes ( lysotracker - positive structures ) per cell was quantified from cells as in panel C ( mean ± S . D . of three independent experiments ) . Images for at least 15 cells per sample were quantified . The value for HEK + Rbpt5 cells is an underestimate , because the density of puncta makes them difficult to distinguish as separate structures .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Wild-type HEK293A cells, HEK+Rbpt5 cells, Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were stained with lysotracker and with DAPI for nuclei. Scale bar, 10 µm. I The number of lysosomes (lysotracker-positive structures) per cell was quantified from cells as in panel C (mean ± S.D. of three independent experiments). Images for at least 15 cells per sample were quantified. The value for HEK+Rbpt5 cells is an underestimate, because the density of puncta makes them difficult to distinguish as separate structures.


A HeLa cells were transfected with nontargeting siRNA ( siCtr ) or siRNAs silencing Rabaptin5 ( siRbpt5 ) or FIP200 ( siFIP200 ) for 72 h . The cells were infected with Salmonella by centrifugation at 500 × g for 5min at 37 ° C and incubation for 10 min at 37 ° C , washed three times , and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0 , 1 , 3 , or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points , shown as a percentage of internalized cells after infection ( mean ± S . D . of three independent experiments ) . The fractions of internalized bacteria alive 1 h after infection are shown separately in the middle ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 ) . On the right , the fraction of infected cells was determined for HeLa cells transfected with siRNAs and infected as above with Salmonella expressing GFP , washed , and immediately fixed for fluorescence microscopy and stained with anti - transferrin receptor and anti - LC3B as cellular markers . Z - stacks for > 5 ' 000cells / sample were acquired and analyzed in Fiji to determine the fraction of infected cells ( mean ± S . D . of three independent experiments ) . The average number of bacteria per infected cell was identical ( 2 . 16 , 2 . 11 , and 2 . 12 bacteria per cell transfected with siCtr , siRbpt5 , and siFIP200 , respectively ) .

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A HeLa cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h. The cells were infected with Salmonella by centrifugation at 500×g for 5min at 37°C and incubation for 10 min at 37°C, washed three times, and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0, 1, 3, or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points, shown as a percentage of internalized cells after infection (mean ± S.D. of three independent experiments). The fractions of internalized bacteria alive 1 h after infection are shown separately in the middle (mean ± S.D. of three independent experiments; ANOVA: *P<0.05). On the right, the fraction of infected cells was determined for HeLa cells transfected with siRNAs and infected as above with Salmonella expressing GFP, washed, and immediately fixed for fluorescence microscopy and stained with anti-transferrin receptor and anti-LC3B as cellular markers. Z-stacks for >5'000cells/sample were acquired and analyzed in Fiji to determine the fraction of infected cells (mean ± S.D. of three independent experiments). The average number of bacteria per infected cell was identical (2.16, 2.11, and 2.12 bacteria per cell transfected with siCtr, siRbpt5, and siFIP200, respectively).


B Wild - type HEK293A , HEK + Rbpt5 , Rbpt5 - KO , Rbpt5 - KO + wt , Rbpt5 - KO + AAA , and FIP200 - KO cells were infected with Salmonella and treated and analyzed as in panels A ( mean ± S . D . of three independent experiments ; ANOVA : * P < 0 . 05 , * * * * P < 0 . 0001 ) .

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B Wild-type HEK293A, HEK+Rbpt5, Rbpt5-KO, Rbpt5-KO+wt, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella and treated and analyzed as in panels A (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, ****P<0.0001).


C Wild - type HEK293A , HEK + Rbpt5 , Rbpt5 - KO , Rbpt5 - KO + AAA , and FIP200 - KO cells were infected with Salmonella expressing GFP as in panel B , incubated in fresh culture medium containing gentamicin for 0 , 5 , 15 , 30 , and 60 min , fixed with methanol and immunostained for transferrin receptor ( TfR ) as a marker of early endosomes and for LC3B as a marker of autophagy . Salmonella were classified according to their association with a TfR - and / or LC3B - positive compartment - as illustrated on the top left ( scale bar , 2 µm ) - during the first hour after infection . In the absence of Rabaptin5 , LC3 - positive SCVs with early endosomal characteristics ( containing TfR ) were strongly reduced . ( Mean ± S . D . of three independent experiments , analyzing > 50 bacteria for each time point . )

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

C Wild-type HEK293A, HEK+Rbpt5, Rbpt5-KO, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella expressing GFP as in panel B, incubated in fresh culture medium containing gentamicin for 0, 5, 15, 30, and 60 min, fixed with methanol and immunostained for transferrin receptor (TfR) as a marker of early endosomes and for LC3B as a marker of autophagy. Salmonella were classified according to their association with a TfR- and/or LC3B-positive compartment - as illustrated on the top left (scale bar, 2 µm) - during the first hour after infection. In the absence of Rabaptin5, LC3-positive SCVs with early endosomal characteristics (containing TfR) were strongly reduced. (Mean ± S.D. of three independent experiments, analyzing >50 bacteria for each time point.)


B . Volcano plot showing the magnitude of differential gene up - regulation , as measured by the respective log 2 - fold change in expression level , at day ( positive ) or night ( negative ) versus statistical significance , as quantified by a negative binomial exact test ( red dots , dashed line p < 0 . 05 adjusted for multiple testing using Benjamin - Hochberg ) . Known circadian - regulated genes are labelled .

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B. Volcano plot showing the magnitude of differential gene up-regulation, as measured by the respective log 2-fold change in expression level, at day (positive) or night (negative) versus statistical significance, as quantified by a negative binomial exact test (red dots, dashed line p<0.05 adjusted for multiple testing using Benjamin-Hochberg). Known circadian-regulated genes are labelled.


B . Violin plots depicting the distribution of the quantified average cellular expression of Prok2 , ProkR2 , Vip , Vipr2 , Avp , Avpr1a , Grp and Grpr in each sub - cluster in CT7 . 5 ( left ) and CT15 . 5 ( right ) datasets .

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B. Violin plots depicting the distribution of the quantified average cellular expression of Prok2, ProkR2, Vip, Vipr2, Avp, Avpr1a, Grp and Grpr in each sub-cluster in CT7.5 (left) and CT15.5 (right) datasets.


C . The period of Cry1 - luc bioluminescence rhythms in SCN slices transfected with either pProk2 . Cre . T2A . mCherry or pProkR2 . Cre . T2A . Venus does not differ between treatments ( p = 0 . 71 , n = 10 , 9 SCN slices per group , respectively ) . D . Similarly , the amplitude of Cry1 - luc bioluminescence rhythms from Prok2 + SCN cells and ProkR2 + SCN cells , does not differ between treatments ( p = 0 . 67 ) .

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C. The period of Cry1-luc bioluminescence rhythms in SCN slices transfected with either pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus does not differ between treatments (p=0.71, n= 10, 9 SCN slices per group, respectively). D. Similarly, the amplitude of Cry1-luc bioluminescence rhythms from Prok2+ SCN cells and ProkR2+ SCN cells, does not differ between treatments (p=0.67).


C . The period of Cry1 - luc bioluminescence rhythms in SCN slices transfected with either pProk2 . Cre . T2A . mCherry or pProkR2 . Cre . T2A . Venus does not differ between treatments ( p = 0 . 71 , n = 10 , 9 SCN slices per group , respectively ) .

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C. The period of Cry1-luc bioluminescence rhythms in SCN slices transfected with either pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus does not differ between treatments (p=0.71, n= 10, 9 SCN slices per group, respectively).


H . Left : confocal images of representative SCN slices dual - transfected with pProk2 . Cre . T2A . mCherry and Syn . GCaMP6f ( top ) , or pProkR2 . Cre . T2A . Venus and Syn . jRCaMP1a ( below ) ( scale bar = 500µm ) . Right , above : representative detrended plots of signal from GCaMP6f ( green ) traces and pCry1 - DIO - Luc , emitted from Prok2 + SCN cells ( red ) . Right , below : representative detrended plots of jRCaMP1a ( purple ) and pCry1 - DIO - Luc emitted from ProkR2 + cells ( light green ) . Orientation bars depict the horizontal axis of the optic chiasm and the vertical axis of the third ventricle for each SCN .

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H. Left: confocal images of representative SCN slices dual-transfected with pProk2.Cre.T2A.mCherry and Syn.GCaMP6f (top), or pProkR2.Cre.T2A.Venus and Syn.jRCaMP1a (below) (scale bar=500µm). Right, above: representative detrended plots of signal from GCaMP6f (green) traces and pCry1-DIO-Luc, emitted from Prok2+ SCN cells (red). Right, below: representative detrended plots of jRCaMP1a (purple) and pCry1-DIO-Luc emitted from ProkR2+ cells (light green). Orientation bars depict the horizontal axis of the optic chiasm and the vertical axis of the third ventricle for each SCN.


B . Comparison of the Per2 : : Luciferase rhythm amplitude of the cycle preceding and the first and second cycles succeeding vehicle ( applied CT23 - 20 dark grey n = 12 SCN slices , CT - 20 - 23 light grey n = 6 SCN slices ) or Prok2RA ( applied CT23 - 20 purple n = 13 SCN slices , CT20 - 23 pink n = 11 SCN slices ) treatment ( interaction , p < 0 . 0001 ) . A significant decline in amplitude occurred when Prok2RA signalling was blocked between CT20 - 23 ( * * * * p < 0 . 0001 ) .

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B. Comparison of the Per2::Luciferase rhythm amplitude of the cycle preceding and the first and second cycles succeeding vehicle (applied CT23-20 dark grey n=12 SCN slices, CT-20-23 light grey n=6 SCN slices) or Prok2RA (applied CT23-20 purple n=13 SCN slices, CT20-23 pink n=11 SCN slices) treatment (interaction, p<0.0001). A significant decline in amplitude occurred when Prok2RA signalling was blocked between CT20-23 (****p<0.0001).


H . Period lengthening occurred at the same rate when Prok2 . Cre or ProkR2 . Cre was the first AAV to be applied ( p = 0 . 1 ; Prok2 . Cre - baseline vs . 1 AAV , * p = 0 . 01 ; baseline vs . both AAVs , * p = 0 . 04 ; ProkR2 . Cre - baseline vs . 1 AAV , * p = 0 . 03 ; baseline vs . both AAVs , * * p = 0 . 01 .

H. Period lengthening occurred at the same rate when Prok2.Cre or ProkR2.Cre was the first AAV to be applied (p=0.1; Prok2.Cre - baseline vs. 1 AAV, *p=0.01; baseline vs. both AAVs, *p=0.04; ProkR2.Cre - baseline vs. 1 AAV, *p=0.03; baseline vs. both AAVs, **p=0.01.


A . Representative Per2 : : Luciferase traces of Cry1 , 2 - / - SCN slices at baseline ( black , arrhythmic ) , after Cry1 complementation in Prok2 + cells ( red ) and further complementation in ProkR2 + cells ( yellow , rhythmic ) ( cohort n = 8 ) . B . as in A but order of complementation reversed ( green = Cry1 complementation in ProkR2 + cells ) ( cohort n = 8 ) .

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A. Representative Per2::Luciferase traces of Cry1,2-/- SCN slices at baseline (black, arrhythmic), after Cry1 complementation in Prok2+ cells (red) and further complementation in ProkR2+ cells (yellow, rhythmic) (cohort n=8). B. as in A but order of complementation reversed (green =Cry1 complementation in ProkR2+ cells) (cohort n=8).


A . Representative Per2 : : Luciferase traces of Cry1 , 2 - / - SCN slices at baseline ( black , arrhythmic ) , after Cry1 complementation in Prok2 + cells ( red ) and further complementation in ProkR2 + cells ( yellow , rhythmic ) ( cohort n = 8 ) .

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A. Representative Per2::Luciferase traces of Cry1,2-/- SCN slices at baseline (black, arrhythmic), after Cry1 complementation in Prok2+ cells (red) and further complementation in ProkR2+ cells (yellow, rhythmic) (cohort n=8).


Schematic representation of the upstream region of myogenin and amplified regions by RT - PCR ( top ) . RT - PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes ( bottom ) . The presence or absence of reverse transcriptase ( RT ) is shown by ( + ) or ( - ) , respectively . The templates ( cDNA or genomic DNA ) are indicated by C or G , respectively .

Schematic representation of the upstream region of myogenin and amplified regions by RT-PCR (top). RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes (bottom). The presence or absence of reverse transcriptase (RT) is shown by (+) or (-), respectively. The templates (cDNA or genomic DNA) are indicated by C or G, respectively.


The primers used for RT - PCR ( top ) . Strand - specific RT - PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA ( middle and bottom ) and poly ( A ) + RNA ( bottom ) .

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The primers used for RT-PCR (top). Strand-specific RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA (middle and bottom) and poly(A)+ RNA (bottom).


Relative expression of indicated RNAs in differentiating C2C12 cells . The presence or absence of RT is shown by ( + ) or ( - ) , respectively . PCR products were verified by sequencing . n = 3 , mean ± SD . The nuclear / cytoplasmic fractionation is confirmed by the expression of H2B and tubulin , respectively .

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Relative expression of indicated RNAs in differentiating C2C12 cells. The presence or absence of RT is shown by (+) or (-), respectively. PCR products were verified by sequencing. n = 3, mean ± SD. The nuclear/cytoplasmic fractionation is confirmed by the expression of H2B and tubulin, respectively.


Chromatin pellet extract ( CPE ) / soluble nuclear extract ( SNE ) ratio of SRA1 , Malat1 , and Myoparr in C2C12 myotubes on a log scale . n = 3 , mean ± SD .

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Chromatin pellet extract (CPE)/soluble nuclear extract (SNE) ratio of SRA1, Malat1, and Myoparr in C2C12 myotubes on a log scale. n = 3, mean ± SD.


Quantitative RT - PCR for myogenin ( A ) and Myoparr ( B ) during myogenesis of C2C12 cells , primary mouse myoblasts ( growth medium , GM ) , and mouse embryonic skeletal muscle ( S . K . ) . The x - axis shows days after differentiation induction or embryonic days . n = 3 , mean ± SD .

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Quantitative RT-PCR for myogenin (A) and Myoparr (B) during myogenesis of C2C12 cells, primary mouse myoblasts (growth medium, GM), and mouse embryonic skeletal muscle (S.K.). The x-axis shows days after differentiation induction or embryonic days. n = 3, mean ± SD.


qRT - PCR showing increased Myoparr expression by MyoD in C3H10T1 / 2 fibroblasts . n = 4 , mean ± SD . * * p < 0 . 01 ( unpaired two - tailed Welch ' s t - test ) .

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qRT-PCR showing increased Myoparr expression by MyoD in C3H10T1/2 fibroblasts. n = 4, mean ± SD. **p < 0.01 (unpaired two-tailed Welch's t-test).


Treatment of recombinant TGF - β for 24 h decreased Myoparr expression in differentiating C2C12 cells . n = 3 , mean ± SD . * p < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

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Treatment of recombinant TGF-β for 24 h decreased Myoparr expression in differentiating C2C12 cells. n = 3, mean ± SD. *p < 0.05 (unpaired two-tailed Student's t-test).


Relative luciferase activities of the indicated promoter in differentiating C2C12 cells by exogenous MyoD . n = 3 , mean ± SD . * * p < 0 . 01 . ( unpaired two - tailed Student ' s t - test ) .

Relative luciferase activities of the indicated promoter in differentiating C2C12 cells by exogenous MyoD. n = 3, mean ± SD. **p < 0.01. (unpaired two-tailed Student's t-test).


Relative luciferase activities of the indicated promoter with / without E - box mutations in differentiating C2C12 cells . n = 3 , mean ± SD . * * p < 0 . 01 . ( unpaired two - tailed Student ' s t - test ) .

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Relative luciferase activities of the indicated promoter with/without E-box mutations in differentiating C2C12 cells. n = 3, mean ± SD. **p < 0.01. (unpaired two-tailed Student's t-test).


Relative luciferase activities of the indicated promoter with / without E - box mutations in differentiating C2C12 cells by TGF - β treatment . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 . n . s . , not significant ( unpaired two - tailed Student ' s t - test ) .

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Relative luciferase activities of the indicated promoter with/without E-box mutations in differentiating C2C12 cells by TGF-β treatment. n = 3, mean ± SD. *p < 0.05. **p < 0.01. n.s., not significant (unpaired two-tailed Student's t-test).


C2C12 cells were transfected with 50 nM of indicated siRNAs in growth medium . After additional 24 - h incubation in growth medium , cells were transferred to differentiation medium . The levels of Myoparr and myogenin expression were quantified by qRT - PCR 24 h after differentiation induction . n = 3 , mean ± SD . * p < 0 . 05 . n . s . , not significant . Statistical analyses were performed using unpaired two - tailed Student ' s t - test . In cases of unequal variances , unpaired two - tailed Welch ' s t - test was used .

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C2C12 cells were transfected with 50 nM of indicated siRNAs in growth medium. After additional 24-h incubation in growth medium, cells were transferred to differentiation medium. The levels of Myoparr and myogenin expression were quantified by qRT-PCR 24 h after differentiation induction. n = 3, mean ± SD. *p < 0.05. n.s., not significant. Statistical analyses were performed using unpaired two-tailed Student's t-test. In cases of unequal variances, unpaired two-tailed Welch's t-test was used.


Western blot showing decreased expression of myogenin in differentiating C2C12 cells 48 h after Myoparr KD . Tubulin expression served as an internal control .

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Western blot showing decreased expression of myogenin in differentiating C2C12 cells 48 h after Myoparr KD. Tubulin expression served as an internal control.


The expression levels of myogenin and Myoparr evaluated by qRT - PCR in myogenin - depleted C2C12 cells . n = 3 , mean ± SD . * * p < 0 . 01 . * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

The expression levels of myogenin and Myoparr evaluated by qRT-PCR in myogenin-depleted C2C12 cells. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test).


ChIP - qPCR detection of Pol II occupancy and histone modification status at the myogenin locus in Myoparr - depleted differentiating C2C12 cells . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 ( unpaired two - tailed Student ' s t - test or unpaired two - tailed Welch ' s t - test ) .

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ChIP-qPCR detection of Pol II occupancy and histone modification status at the myogenin locus in Myoparr-depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. **p < 0.01 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test).


Retrieval rate of Myoparr using Myoparr - ChIRP probes from C2C12 cells quantified by qPCR is shown as percent of input values . GAPDH was amplified as a negative control . n = 3 , mean ± SD . ND , not detected .

Retrieval rate of Myoparr using Myoparr-ChIRP probes from C2C12 cells quantified by qPCR is shown as percent of input values. GAPDH was amplified as a negative control. n = 3, mean ± SD. ND, not detected.


ChIRP - qPCR detection of the interaction between endogenous Myoparr and the myogenin promoter . The myogenin 3 ´ UTR and GAPDH promoter were amplified as negative controls . The data were normalized to input values . n = 3 , mean ± SD . * p < 0 . 05 . * * p < 0 . 01 ( unpaired two - tailed Welch ' s t - test ) . ND , not detected .

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ChIRP-qPCR detection of the interaction between endogenous Myoparr and the myogenin promoter. The myogenin 3´UTR and GAPDH promoter were amplified as negative controls. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. ** p < 0.01 (unpaired two-tailed Welch's t-test). ND, not detected.


Immunocytochemistry for the detection of myogenin at 24 h ( G ) or MHC at 72 h ( H ) in Myoparr - depleted C2C12 cells after differentiation induction . Nuclei were counterstained with DAPI . Bar , 100 μm . The percentage of myogenin - positive cells or fusion index is shown as percent of the control . n = 3 , mean ± SD . * * p < 0 . 01 . * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test or unpaired two - tailed Welch ' s t - test ) .

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Immunocytochemistry for the detection of myogenin at 24 h (G) or MHC at 72 h (H) in Myoparr-depleted C2C12 cells after differentiation induction. Nuclei were counterstained with DAPI. Bar, 100 μm. The percentage of myogenin-positive cells or fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test).


Genes significantly differentially expressed in both Myoparr - and myogenin - depleted cells show correlated expression ( R = 0 . 63 , log 2 ratio scale ) .

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Genes significantly differentially expressed in both Myoparr- and myogenin-depleted cells show correlated expression (R = 0.63, log 2 ratio scale).


Following immunoprecipitation , the interaction between Myoparr and endogenous Ddx17 was confirmed by immunoblotting using a Ddx17 - specific antibody . Two different Ddx17 isoforms ( p72 and p82 ) were observed .

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Following immunoprecipitation, the interaction between Myoparr and endogenous Ddx17 was confirmed by immunoblotting using a Ddx17-specific antibody. Two different Ddx17 isoforms (p72 and p82) were observed.


Determination of the Ddx17 - binding region of Myoparr by RNA pull - down analyses . Schematic diagram of full - length or truncated Myoparr used for RNA pull - down ( top ) . In vitro transcribed / translated Ddx17 protein was pulled down by indicated Myoparr and then detected by western blot using a Ddx17 antibody ( bottom ) .

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Determination of the Ddx17-binding region of Myoparr by RNA pull-down analyses. Schematic diagram of full-length or truncated Myoparr used for RNA pull-down (top). In vitro transcribed/translated Ddx17 protein was pulled down by indicated Myoparr and then detected by western blot using a Ddx17 antibody (bottom).


Relative luciferase activities of indicated constructs in differentiating C2C12 cells .

Relative luciferase activities of indicated constructs in differentiating C2C12 cells.


Relative luciferase activities of indicated constructs in differentiating C2C12 cells transfected with control or Myoparr shRNA ( Myoparr KD ) .

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

Relative luciferase activities of indicated constructs in differentiating C2C12 cells transfected with control or Myoparr shRNA (Myoparr KD).