U2OS wild - type 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 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . correlations between transcription inhibition 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 ) 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 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 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . correlations between transcription inhibition 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 ) 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 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 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 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 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . correlations between transcription inhibition 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 ) 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 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 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 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 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 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . correlations between transcription inhibition biological ICD score ( F ) previously measured 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 ) 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 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 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 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 50 μM spermidine . For assessing transcription , were pre - treated for 1 . 5 h with library followed by 1 h with same drugs in which EU was added . For assessing translation , were pre - treated with library for 12 h followed by 30 min in methionine free - medium , before addition of azidohomoalanine ( AHA ) . of inhibition was calculated transformed as z - scores . correlations between transcription 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 ) 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 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 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)


inhibition of transcription was assessed for negative positive ICD hits identified with predictive algorithm ( Figure 1 ) . U2OS were treated with 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 2 μM RH - 1 for 1 . 5 h followed by 1 h with EU . of transcription inhibition was calculated coefficient of correlation ( R ) associated p - value ( p ) between of inhibition theoretical ICD score was calculated using Pearson method .

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

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.


Jurkat T were transfected with control , FIP3 . 1 or FIP3 . 2 siRNA oligonucleotides . A : Intracellular distribution of endogenous Rab11 Rac1 was assessed by immunofluorescence . A , E , G : Three - dimensional ( 3D ) confocal were post - treated by deconvolution . A 0 . 4 - µ - thick medial stack is shown . pericentrosomal vesicular compartment is zoomed at bottom right - corner . B , F , H : Population of co - localization volume between Rab11 Rac1 within percentriolar vesicular compartment . Each dot represents one . Horizontal bars represent mean ± , Mann - Whitney was used . are representative of three experiments . Bar , 5

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

Jurkat T cells were transfected with control, FIP3.1 or FIP3.2 siRNA oligonucleotides. A: Intracellular distribution of endogenous Rab11 and Rac1 was assessed by immunofluorescence.A, E, G: Three-dimensional (3D) confocal images were post-treated by deconvolution. A 0.4-µ-thick medial stack is shown. The pericentrosomal vesicular compartment is zoomed at the bottom right-hand corner.B, F, H: Population analysis of the co-localization volume between Rab11 and Rac1 within the percentriolar vesicular compartment. Each dot represents one cell. Horizontal bars represent the mean ± SEM, Mann-Whitney test was used. Images are representative of three experiments. Bar, 5 µm


A - C : JurkatT were transfected with control , FIP3 . 1 or FIP3 . 2 siRNA oligonucleotides . A : Intracellular distribution of endogenous Rab11 Rac1 was assessed by immunofluorescence .

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

A-C: JurkatT cells were transfected with control, FIP3.1 or FIP3.2 siRNA oligonucleotides. A: Intracellular distribution of endogenous Rab11 and Rac1 was assessed by immunofluorescence.


C : FIP3 expression was analyzed by Western blot . of bands was performed with ImageJ normalized to a non - specific band of 35 kDa used as loading control .

O O O O O O O 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: FIP3 expression was analyzed by Western blot. Quantification of bands was performed with ImageJ and normalized to a non-specific band of 35 kDa used as loading control.


E , F : Jurkat were transfected with expression vectors encoding GFP , GFP - FIP3 - WT or GFP - FIP3 - I738E . Endogenous Rac1 was detected by immunofluorescence .

O O O O O O O O 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, F: Jurkat cells were transfected with expression vectors encoding GFP, GFP-FIP3-WT or GFP-FIP3-I738E. Endogenous Rac1 was detected by immunofluorescence.


G : Jurkat were transfected with expression vectors encoding GFP , Rab11WT - GFP , or Rab11Q70L - GFP ( constitutively active ) or Rab11S25N - GFP ( dominant negative ) mutants . Endogenous Rac1 was detected by immunofluorescence . A , E , G : Three - dimensional ( 3D ) confocal were post - treated by deconvolution . A 0 . 4 - µ - thick medial stack is shown . pericentrosomal vesicular compartment is zoomed at bottom right - corner . B , F , H : Population of co - localization volume between Rab11 Rac1 within percentriolar vesicular compartment . Each dot represents one . Horizontal bars represent mean ± , Mann - Whitney was used . are representative of three experiments . Bar , 5

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

G: Jurkat cells were transfected with expression vectors encoding GFP, Rab11WT-GFP, or Rab11Q70L-GFP (constitutively active) or Rab11S25N-GFP (dominant negative) mutants. Endogenous Rac1 was detected by immunofluorescence.A, E, G: Three-dimensional (3D) confocal images were post-treated by deconvolution. A 0.4-µ-thick medial stack is shown. The pericentrosomal vesicular compartment is zoomed at the bottom right-hand corner.B, F, H: Population analysis of the co-localization volume between Rab11 and Rac1 within the percentriolar vesicular compartment. Each dot represents one cell. Horizontal bars represent the mean ± SEM, Mann-Whitney test was used. Images are representative of three experiments. Bar, 5 µm


A , B : Effect of FIP3 silencing on Rac1 expression . Jurkat were transfected with control , FIP3 . 1 or FIP3 . 2 siRNA . Three days later were lysed expression of FIP3 , Rac1 β - tubulin analyzed by western blot . of bands corresponding to FIP3 to Rac1 was quantified by ImageJ normalized to β - tubulin band used as loading control . mean ± SD of of band in silenced with respect to controls of three independent experiments is shown .

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

A, B: Effect of FIP3 silencing on Rac1 expression. Jurkat cells were transfected with control, FIP3.1 or FIP3.2 siRNA. Three days later cells were lysed and the expression of FIP3, Rac1 and β-tubulin analyzed by western blot. The intensity of bands corresponding to FIP3 and to Rac1 was quantified by ImageJ and normalized to the β-tubulin band used as loading control. The mean ± SD of the percentage of band intensity in silenced cells with respect to controls of three independent experiments is shown.


B : Rac1 interaction with FIP3 : HEK293T were transfected with GFP , GFP - FIP3 WT or GFP - FIP3I738E expression vectors . 24 h later , were lysed immunoprecipitated with anti - GFP Ab . Immunoprecipitates were analyzed by electrophoresis western blot using anti - GFP Ab ( top panel ) or anti - Rac1 Ab ( bottom panel ) . Total lysates were analyzed ( 100 , 000 ) . position of Rac1 ( 20 kDa ) Ig chain ( 25 kDa ) polypeptides is depicted on right side . Arrowheads at bottom point bands corresponding to Rac . of bands was performed with ImageJ as of with respect to total lysate bands on left , shown below . A representative experiment out of three carried out is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Rac1 interaction with FIP3: HEK293T cells were transfected with GFP, GFP-FIP3 WT or GFP-FIP3I738E expression vectors. 24 h later, cells were lysed and immunoprecipitated with anti-GFP Ab. Immunoprecipitates were analyzed by electrophoresis and western blot using anti-GFP Ab (top panel) or anti-Rac1 Ab (bottom panel). Total cell lysates were analyzed (100,000 cells). The position of Rac1 (20 kDa) and Ig light chain (25 kDa) polypeptides is depicted on the right side. Arrowheads at the bottom point the bands corresponding to Rac. Quantification of bands was performed with ImageJ as the percentage of intensity with respect to the total lysate bands on the left, shown below. A representative experiment out of three carried out is shown.


JurkatT were transfected with expression vectors encoding : A , C : GFP , GFP - FIP3WT , or GFP - FIP3I738E . B , C : Rab11WT - GFP , Rab11Q70L - GFP ( constitutively active ) , or Rab11S25N - GFP ( dominant negative ) .

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

JurkatT cells were transfected with expression vectors encoding: A, C: GFP, GFP-FIP3WT, or GFP-FIP3I738E. B, C: Rab11WT-GFP, Rab11Q70L-GFP (constitutively active), or Rab11S25N-GFP (dominant negative).


C : Rab11WT - GFP , Rab11Q70L - GFP ( constitutively active ) , or Rab11S25N - GFP ( dominant negative ) .

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

C: Rab11WT-GFP, Rab11Q70L-GFP (constitutively active), or Rab11S25N-GFP (dominant negative).


D , E : or transfected with control , or FIP3 . 1 siRNA . were allowed to form immunological synapses with SEE - pulsed Raji for 30 min . Intracellular distribution of endogenous Rac1 surface distribution of CD3 were detected by immunofluorescence . 3D confocalimages were post - treated by deconvolution . A 0 . 4 - µm - thick medial stack is shown . C , E : Population analyses of amount of Rac1 at immunological synapse relative to total Rac1 . Each dot corresponds to one . Horizontal bars represent mean ± . Mann - Whitney . are representative of three experiments . Bar , 5 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 O O O O O O O O O O O O O O O O O O O O

D, E: or transfected with control, or FIP3.1 siRNA. Cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min. Intracellular distribution of endogenous Rac1 and surface distribution of CD3 were detected by immunofluorescence. 3D confocalimages were post-treated by deconvolution. A 0.4-µm-thick medial stack is shown. C, E: Population analyses of the amount of Rac1 at the immunological synapse relative to the total cellular Rac1. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments. Bar, 5 µm.


JurkatT were transfected with either control or FIP3 . 1 siRNA . A : were allowed to form immunological synapses with SEE - pulsed Raji for 30 min endogenous Lck was detected by immunofluorescence . 3D confocalimages were post - treated by deconvolution . A 0 . 4 - µm - thick medial stack is shown .

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

JurkatT cells were transfected with either control or FIP3.1 siRNA. A: Cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min and endogenous Lck was detected by immunofluorescence. 3D confocalimages were post-treated by deconvolution. A 0.4-µm-thick medial stack is shown.


B , C : minimal angle formed by axis connecting synapse edges axis connecting mass centers of T APC nuclei was measured , as schematized in B .

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

B, C: The minimal angle formed by the axis connecting the synapse edges and the axis connecting the mass centers of the T cell and the APC nuclei was measured, as schematized in B.


D : Transfected Jurkat T were allowed to spread on anti - CD3 - coated coverslips for indicated time , F - actin was detected with FITC - Phalloidin . A single optical section at level of contact surface is shown . E , F : Population analyses of spreading of at level of contact surface . Each dot corresponds to one . Horizontal bars represent mean ± . Mann - Whitney . are representative of three experiments . Bar , 5 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

D: Transfected Jurkat T cells were allowed to spread on anti-CD3-coated coverslips for the indicated time, and F-actin was detected with FITC-Phalloidin. A single optical section at the level of the contact surface is shown. E, F: Population analyses of the spreading area of cells at the level of the contact surface. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments. Bar, 5 µm.


G : FIP3 expression was analyzed by Western blot . of bands was performed with ImageJ normalized to a non - specific band of 35 kDa used as loading control .

G: FIP3 expression was analyzed by Western blot. Quantification of bands was performed with ImageJ and normalized to a non-specific band of 35 kDa used as loading control.


A , B , C : JurkatT transfected with siRNA control , FIP3 . 1 or FIP3 . 2 were allowed to spread on poly - lysine - coated coverslips for indicated times . Intracellular actin was detected with FITC - coupled Phalloidin . A : A single optical section at level of contact surface is shown . Bar , 5 µm . B : Population analyses of spreading of at level of contact surface . Each dot represents one . Horizontal bars represent mean ± . C : Time course representation of B , showing mean ± .

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

A, B, C: JurkatT cells transfected with siRNA control, FIP3.1 or FIP3.2 were allowed to spread on poly-lysine-coated coverslips for the indicated times. Intracellular actin was detected with FITC-coupled Phalloidin. A: A single optical section at the level of the contact surface is shown. Bar, 5 µm. B: Population analyses of the spreading area of cells at the level of the contact surface. Each dot represents one cell. Horizontal bars represent the mean ± SEM. C:Time course representation of B, showing mean ± SEM.


D - F : humanCD4T were transfected with control or FIP3 . 1 siRNA . D , E : were allowed to spread on poly - lysine - coated coverslips for 5 min . Intracellular Lck was detected by immunofluoresecence . D : A single optical section at level of contact surface is shown . Bar , 10 µm . E : Population analyses of spreading of at level of contact surface .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F: Primary humanCD4T cells were transfected with control or FIP3.1 siRNA. D, E: Cells were allowed to spread on poly-lysine-coated coverslips for 5 min. Intracellular Lck was detected by immunofluoresecence. D: A single optical section at the level of the contact surface is shown. Bar, 10 µm. E: Population analyses of the spreading area of cells at the level of the contact surface.


F : were stained with CMFDA dye , suspended in a paraformaldehyde solution submitted to centrifugation at 3 , 724 xg onto poly - lysine - coated coverslips . Confocal optical sections separated by 0 . 2 µm were acquired . X - Z were built with ImageJ . maximal sections in ' x ' ( width ) ' z ' ( thickness ) were determined for each ( upper panel ) . Plots represent deformability index ( x / z ) . Each dot corresponds to one . Horizontal bars represent mean ± . Mann - Whitney . are representative of three experiments .

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F: Cells were stained with CMFDA dye, suspended in a paraformaldehyde solution and submitted to centrifugation at 3,724 xg onto poly-lysine-coated coverslips. Confocal optical sections separated by 0.2 µm were acquired. X-Z images were built with ImageJ. The maximal sections in 'x' (width) and 'z' (thickness) were determined for each cell (upper panel). Plots represent the deformability index (x/z). Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments.


Jurkat T were transfected with control or FIP3 . 1 siRNA , then transfected again with empty vector or with expression vectors encoding Myc - tagged - Rac1WT , - Rac1G12V ( constitutively active mutant ) , or - Rac1Rac1T17N ( dominant negative mutant ) . were allowed to spread on poly - lysine - coated coverslips for indicated time spreading of measured . A - C , H : Population analyses of spreading of at level of contact surface . A - C : Time course plots representing mean ± of data shown as in I . D - G : for nucleus ( blue = Dapi ) , F - actin ( green = phalloidin ) Rac1 - Myc ( red = anti - Myc ) is shown . A single optical section at level of contact surface is shown . Bar , 5 µm .

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Jurkat T cells were transfected with control or FIP3.1 siRNA, then transfected again with empty vector or with expression vectors encoding Myc-tagged-Rac1WT, -Rac1G12V (constitutively active mutant), or -Rac1Rac1T17N (dominant negative mutant). Cells were allowed to spread on poly-lysine-coated coverslips for the indicated time and the spreading area of cells measured. A-C, H: Population analyses of the spreading area of cells at the level of the contact surface. A-C: Time course plots representing the mean ± SEM of the data shown as in I. D-G: Staining for nucleus (blue = Dapi), F-actin (green = phalloidin) and Rac1-Myc (red = anti-Myc) is shown. A single optical section at the level of the contact surface is shown. Bar, 5 µm.


H : Dot plots corresponding to measurements at 15 min of spreading : Each dot represents one . Horizontal bars represent mean ± . Mann - Whitney . Data are representative of three experiments .

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H: Dot plots corresponding to measurements at 15 min of spreading: Each dot represents one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Data are representative of three experiments.


A : Jurkat T transfected with control or FIP3 . 1 were treated 1 h with Rac1 inhibitor NSC23766 at 100 µM allowed to spread on poly - lysine - coated coverslips for 15 min . F - actin was detected with FITC - Phalloidin spreading of at contact surface was measured .

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A: Jurkat T cells transfected with control or FIP3.1 were treated 1 h with the Rac1 inhibitor NSC23766 at 100 µM and allowed to spread on poly-lysine-coated coverslips for 15 min. F-actin was detected with FITC-Phalloidin and the spreading area of cells at the contact surface was measured.


B : Transfected were allowed to form immunological synapses with SEE - pulsed Raji for 30 min intracellular distribution of Lck was detected by immunofluorescence . Synapse angle was measured as in figure 4 . Each dot corresponds to one . Horizontal bars represent mean ± . Mann - Whitney .

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B: Transfected cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min and intracellular distribution of Lck was detected by immunofluorescence. Synapse angle was measured as in figure 4. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test.


A : Jurkat T transfected with control or FIP3 . 1 were activated on anti - CD3 - coated plastic plates in presence of soluble anti - CD28 during 16 h . of IL2 released in supernatant was measured by ELISA . Histograms represent mean ± SD of three experiments . Mann - Whitney .

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A: Jurkat T cells transfected with control or FIP3.1 were activated on anti-CD3-coated plastic plates in the presence of soluble anti-CD28 during 16 h. Concentration of IL2 released in the supernatant was measured by ELISA. Histograms represent the mean ± SD of three experiments. Mann-Whitney test.


B : T were activated with soluble anti - CD3 Erk1 / 2 phosphorylation was analyzed by Western blot . Graph represents of each band with respect to that of transfected with control siRNA left non - stimulated ( t = 0 ) . each band from Western blot was normalized with respect to of β - actin for each time point . Representative experiment out of three performed .

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B: T cells were activated with soluble anti-CD3 and Erk1/2 phosphorylation was analyzed by Western blot. Graph represents the percentage of each band intensity with respect to that of cells transfected with control siRNA and left non-stimulated (t=0). The intensity each band from Western blot was normalized with respect to the intensity of β-actin for each time point. Representative experiment out of three performed.


A . Representative of of PCIF1 in three CRC tissues adjacent noncancerous tissues . Scale bar , 50 μm ( left ) . of PCIF1 expression in normal CRC tissues . Each dot represents one sample ( right ) . Data are mean ± . * * P < 0 . 01 , by Student ' s t - .

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A. Representative images of IHC staining of PCIF1 in three CRC tissues and adjacent noncancerous tissues. Scale bar, 50 μm (left). Analysis of the PCIF1 protein expression in normal and CRC tissues. Each dot represents one sample (right). Data are the mean ± SEM. **P < 0.01, by Student's t-test.


B . Kaplan - Meier plots of overall survival of 27 CRC with low PCIF1 expression ( 0 - 4 . 5 score , n = 18 ) or high PCIF1 expression ( 4 . 6 - 9 . 0 score , n = 9 ) . Data are mean ± . * * * P < 0 . 001 by Student ' s t - .

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B. Kaplan-Meier plots of overall survival of 27 CRC patients with low tumor PCIF1 expression (0-4.5 staining score, n = 18) or high tumor PCIF1 expression (4.6-9.0 staining score, n = 9). Data are the mean ± SEM. ***P < 0.001 by Student's t-test.


C . Western blot of PCIF1 in normal colon ( CCD841CON ) various CRC lines . GAPDH served as a loading control .

C. Western blot analysis of PCIF1 in normal colon cells (CCD841CON) and various CRC cell lines. GAPDH served as a loading control.


D . qRT - PCR of PCIF1 mRNA in indicated lines . were normalized to GAPDH expression . Data are mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

D. qRT-PCR analysis of PCIF1 mRNA in the indicated cell lines. Levels were normalized to GAPDH expression. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


E . Representative immunofluorescence of HT29 HCT116 stained for endogenous PCIF1 ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm .

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E. Representative immunofluorescence images of HT29 and HCT116 cells stained for endogenous PCIF1 protein (green). Nuclei were stained with DAPI (blue). Scale bar, 30 μm.


F . Western blot of PCIF1 in whole extract ( W ) , ( C ) , ( N ) fractions of HCT116 HT29 . Tubulin lamin A / C served as markers , respectively .

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F. Western blot analysis of PCIF1 in whole extract (W), cytosolic (C), and nuclear (N) fractions of HCT116 and HT29 cells. Tubulin and lamin A/C served as cytosolic and nuclear markers, respectively.


A . Western blot of PCIF1 in HT29 HCT116 depleted of PCIF1 using four different sgRNAs . GAPDH served as a loading control .

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A. Western blot analysis of PCIF1 in HT29 and HCT116 cells depleted of PCIF1 using four different sgRNAs. GAPDH served as a loading control.


Matrigel invasion ( B ) , of indicated control PCIF1 - depleted CRC lines . Representative of are shown at left right , respectively , of each panel . Data are mean ± SD of n = 3 replicates / condition . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

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Matrigel invasion assay (B), of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


fibronectin adhesion ( C ) , of indicated control PCIF1 - depleted CRC lines . Representative of are shown at left right , respectively , of each panel . Data are mean ± SD of n = 3 replicates / condition . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

fibronectin adhesion assay (C), of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


colony formation ( D ) of indicated control PCIF1 - depleted CRC lines . Representative of are shown at left right , respectively , of each panel . Data are mean ± SD of n = 3 replicates / condition . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

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colony formation assay (D) of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


wound healing migration ( E ) of indicated control PCIF1 - depleted CRC lines . Representative of are shown at left right , respectively , of each panel . Data are mean ± SD of n = 3 replicates / condition . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

wound healing migration assay (E) of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


F . Growth of indicated HCT116 tumors after subcutaneous injection into athymic nude mice . volume was recorded on indicated days . Data are mean ± of number of mice / group indicated on each panel . n , numbers of mice . * P < 0 . 05 , * * P < 0 . 01 by Student ' s t - .

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F. Growth of the indicated HCT116 tumors after subcutaneous injection into athymic nude mice. Tumor volume was recorded on the indicated days. Data are the mean ± SEM of the number of mice/group indicated on each panel. n, the numbers of mice. *P < 0.05, **P < 0.01 by Student's t-test.


G . Survival of mice injected bearing indicated HCT116 tumors . Data are mean ± of number of mice / group indicated on each panel . n , numbers of mice . * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

G. Survival analysis of mice injected bearing the indicated HCT116 tumors. Data are the mean ± SEM of the number of mice/group indicated on each panel. n, the numbers of mice. **P < 0.01, ***P < 0.001 by Student's t-test.


A . LC - MS / MS identification of m6Am or m6A in mRNA from control PCIF1 - depleted HCT116 . Data are presented as enrichment of methylated vs unmethylated mRNA . Data are mean ± SD of n = 3 biological replicates . * * P < 0 . 01 by Student ' s t - .

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A. LC-MS/MS identification and quantification of m6Am or m6A levels in mRNA from control and PCIF1-depleted HCT116 cells. Data are presented as the enrichment of methylated vs unmethylated mRNA. Data are the mean ± SD of n=3 biological replicates. **P < 0.01 by Student's t-test.


B . Metagene plots of m6Am enrichment around transcription start site ( TSS ) of expressed genes in control PCIF1 - depleted HCT116 .

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B. Metagene plots analysis of m6Am enrichment around the transcription start site (TSS) of all expressed genes in control and PCIF1-depleted HCT116 cells.


E . Western blot of FOS PCIF1 in control PCIF1 - depleted HCT116 HT29 . GAPDH served as a loading control .

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E. Western blot analysis of FOS and PCIF1 in control and PCIF1-depleted HCT116 and HT29 cells. GAPDH served as a loading control.


F . Genome browser views of FOS with m6Am sites . Read coverage of input sample IP sample are shown in blue red , respectively , green rectangle indicates m6Am peaks located near TSS .

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F. Genome browser views of FOS with m6Am sites. Read coverage of input sample and IP sample are shown in blue and red, respectively, and green rectangle indicates the m6Am peaks located near the TSS.


G . m6Am - exo - qPCR of m6Am enrichment in FOS mRNAs from indicated HCT116 . GAPDH served as a negative control . Data are mean ± SD of n = 3 biological replicates . * * P < 0 . 01 by Student ' s t - .

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G. m6Am-exo-qPCR analysis of m6Am enrichment in FOS mRNAs from the indicated HCT116 cells. GAPDH served as a negative control. Data are the mean ± SD of n=3 biological replicates. **P < 0.01 by Student's t-test.


H . qRT - PCR of FOS mRNA stability in control PCIF1 - depleted HCT116 HT29 treated with actinomycin D for indicated times . Data are mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 by Student ' s t - .

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H. qRT-PCR analysis of FOS mRNA stability in control and PCIF1-depleted HCT116 and HT29 cells treated with actinomycin D for the indicated times. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01 by Student's t-test.


I . Growth of tumors after subcutaneous injection of indicated HCT116 into athymic nude mice . volumes were recorded on indicated days . Data are mean ± of n = 5 . * P < 0 . 05 by Student ' s t - .

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I. Growth of tumors after subcutaneous injection of the indicated HCT116 cells into athymic nude mice. Tumor volumes were recorded on the indicated days. Data are the mean ± SEM of n=5. *P < 0.05 by Student's t-test.


A . Western blot of FOS in control FOS - depleted HCT116 HT29 . GAPDH served as a loading control .

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A. Western blot analysis of FOS in control and FOS-depleted HCT116 and HT29 cells. GAPDH served as a loading control.


MTS proliferation ( B ) , of control FOS - depleted HCT116 . Data are mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

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MTS cell proliferation assay (B), of control and FOS-depleted HCT116 cells. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


wound healing migration ( C ) , of control FOS - depleted HCT116 . Representative of colonies are shown to left right , respectively . Data are mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

wound healing migration assay (C), of control and FOS-depleted HCT116 cells. Representative images and quantification of colonies are shown to the left and right, respectively. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


colony formation ( D ) of control FOS - depleted HCT116 . Representative of colonies are shown to left right , respectively . Data are mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

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colony formation assay (D) of control and FOS-depleted HCT116 cells. Representative images and quantification of colonies are shown to the left and right, respectively. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


E . Growth of control FOS - depleted HCT116 tumors after subcutaneous injection into athymic nude mice . volume was recorded on indicated days . Data are mean ± of n = 10 mice / group . * * P < 0 . 01 by Student ' s t - .

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E. Growth of control and FOS-depleted HCT116 tumors after subcutaneous injection into athymic nude mice. Tumor volume was recorded on the indicated days. Data are the mean ± SEM of n=10 mice/group. **P < 0.01 by Student's t-test.


F . Survival of mice bearing control FOS - depleted HCT116 tumors . Data are mean ± of n = 5 mice / group . * P < 0 . 05 by Student ' s t - .

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F. Survival analysis of mice bearing control and FOS-depleted HCT116 tumors. Data are the mean ± SEM of n= 5 mice/group. *P < 0.05 by Student's t-test.


G . Positive correlation between FOS PCIF1 in tumors from 29 with CRC ( R2 = 0 . 6902 , P < 0 . 001 ) .

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G. Positive correlation between FOS and PCIF1 protein levels in tumors from 29 patients with CRC (R2 =0.6902, P < 0.001).


B . mRNA expression of PCIF1 was quantified by qRT - PCR in HCT116 transfected with gradient amount of control PCIF1 siRNAs . Data are mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - .

B. mRNA expression of PCIF1 was quantified by qRT-PCR in the HCT116 cells transfected with gradient amount of control and PCIF1 siRNAs. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.


C . Western blot of PCIF1 in HCT116 with control PCIF1 siRNAs . GAPDH served as a loading control .

C. Western blot analysis of PCIF1 in HCT116 cells with control and PCIF1 siRNAs. GAPDH served as a loading control.


D . viability assays were performed in in HCT116 transfected with 100pmol control PCIF1 siRNAs . Data are mean ± SD of n = 3 biological replicates . * * * P < 0 . 001 by Student ' s t - .

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D. Cell viability assays were performed in in the HCT116 cells transfected with 100pmol control and PCIF1 siRNAs. Data are the mean ± SD of n=3 biological replicates. ***P < 0.001 by Student's t-test.


F . growth of HCT116 treated with 1mg / kg control PCIF1 LNP - siRNAs starting on day 9 mice were intratumorally treated twice a week . Data are mean ± of n = 5 mice / group . * P < 0 . 05 , * * P < 0 . 01 by Student ' s t - .

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F. Tumor growth of HCT116 cells treated with 1mg/kg control and PCIF1 LNP-siRNAs starting on day 9 and mice were intratumorally treated twice a week. Data are the mean ± SEM of n=5 mice/group. *P < 0.05, **P < 0.01 by Student's t-test.


G . Immunoblots of PCIF1 FOS were carried out from indicated tumors in four replicates with GAPDH as a loading control .

G. Immunoblots of PCIF1 and FOS were carried out from the indicated tumors in four replicates with GAPDH as a loading control.


( b - c ) Western blot for p53 select p53 targets post - radiation in ( b ) oscillatory ( c ) rising conditions . Actin is shown as a loading control .

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(b-c) Western blot for p53 and select p53 targets post-radiation in (b) oscillatory and (c) rising conditions. Actin is shown as a loading control.


( d - e ) Average of ( d ) mRNA quantified by RNAseq ( red ) or qRT - PCR ( grey ) or ( e ) quantified by mass spectrometry ( green ) or western blot ( grey ) for select p53 target genes under oscillatory p53 . Data information : , n = 2 biological replicates , error bars represent std dev .

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(d-e) Average levels of (d) mRNA quantified by RNAseq (red) or qRT-PCR (grey) or (e) protein quantified by mass spectrometry (light green) or western blot (grey) for select p53 target genes under oscillatory p53. Data information: , n=2 biological replicates, error bars represent std dev.


( f - g ) Average of ( f ) mRNA quantified by RNAseq ( blue ) or qRT - PCR ( grey ) or ( g ) quantified by mass spectrometry ( green ) or western blot ( grey ) for select p53 target genes under rising p53 . Data information : n = 2 biological replicates , error bars represent std dev .

(f-g) Average levels of (f) mRNA quantified by RNAseq (blue) or qRT-PCR (grey) or (g) protein quantified by mass spectrometry (dark green) or western blot (grey) for select p53 target genes under rising p53. Data information: n=2 biological replicates, error bars represent std dev.


a , Pancreatic sections stained with Atg7 , LC3 p62 showing Atg7 − / − next to Atg7 + / + regions after Pdx - Cre - mediated recombination . Right - side are cropped magnified regions ( boxed ) from left - side . magnification is × 2 . 5 .

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a, Pancreatic sections stained with Atg7, LC3 and p62 showing Atg7−/− next to Atg7+/+ regions after Pdx-Cre-mediated recombination. Right-side images are cropped and magnified regions (boxed) from left-side images. The magnification is × 2.5.


b , Haematoxylin eosin ( HE ) , p53 caspase - 3 of exocrine tissue in Atg7 + / + Atg7 − / − pancreases .

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b, Haematoxylin and eosin (HE), p53 and caspase-3 staining of exocrine tissue in Atg7+/+ and Atg7−/− pancreases.


c , ( median , s . e . m . ) of p53 caspase - 3 activation in Atg7 - deficient acinar tissue .

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c, Quantification (median, s.e.m.) of p53 and caspase-3 activation in Atg7-deficient acinar tissue.


d , Kaplan - Meier comparing overall survival of mice either wild type ( blue ) or Atg7 − / − ( red ) in pancreas . Mantel - Cox was used for statistics . ' n / a ' , 14 / 16 mice were killed > 500 days when completely healthy .

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d, Kaplan-Meier analysis comparing overall survival of mice either wild type (blue) or Atg7−/− (red) in the pancreas. Mantel-Cox test was used for statistics. 'n/a', 14/16 mice were killed >500 days when completely healthy.


e , Biochemical of endocrine function in moribund Atg7 − / − or - matched healthy control mice . Detailed information about mice is tabulated . A Mann - Whitney U - was used for statistics . Scale bars : 50 µm . * * P 0 . 01 * * * P 0 . 001 .

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e, Biochemical analysis of endocrine function in moribund Atg7−/− or age-matched healthy control mice. Detailed information about the mice is tabulated. A Mann-Whitney U-test was used for statistics. Scale bars: 50 µm. **P 0.01 and ***P 0.001.


a , Alcian blue / PAS ( AB / PAS ) in pancreases from 150 - day - old Atg7 - proficient or Atg7 - deficient KrasG12D / + mice ( wild - type mice with expression of KrasG12D in their pancreases ) .

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a, Alcian blue/PAS (AB/PAS) staining in pancreases from 150-day-old Atg7-proficient or Atg7-deficient KrasG12D/+ mice (wild-type mice with expression of KrasG12D in their pancreases).


b , of PanIN from mice of indicated genotype . Fold increase / ratios are shown . Box plots express first ( Q1 ) third ( Q3 ) quartiles set by upper lower limits of box , whereas median is shown by horizontal inside box . whiskers extend upward downward to highest or lowest observation within upper ( Q3 + 1 . 5 × interquartile range ) lower ( Q1 − 1 . 5 × interquartile range ) limits . Values outside upper lower limits are ' outliers ' are shown by symbols .

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b, Quantification of PanIN from mice of the indicated genotype and age. Fold increase and male/female ratios are shown. Box plots express the first (Q1) and third (Q3) quartiles set by the upper and lower limits of the box, whereas the median is shown by the horizontal line inside the box. The whiskers extend upward and downward to the highest or lowest observation within the upper (Q3 + 1.5 × the interquartile range) and lower (Q1 − 1.5 × interquartile range) limits. Values outside the upper and lower limits are 'outliers' and are shown by individual symbols.


c , d , Kaplan - Meier comparing overall survival ( c ) or PDAC - free survival ( d ) of Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / + mice .

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c, d, Kaplan-Meier analysis comparing overall survival (c) or PDAC-free survival (d) of Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ mice.


c , d , Kaplan - Meier comparing overall survival ( c ) or PDAC - free survival ( d ) of Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / +

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c, d, Kaplan-Meier analysis comparing overall survival (c) or PDAC-free survival (d) of Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ mice


e , PanIN grading of pancreases from 150 - day - old Atg7 + / + or Atg7 − / − Pdx - Cre KrasG12D / + mice . Values are mean error bars are s . d . b , e , A Mann - Whitney U - was used for statistics .

e, PanIN grading of pancreases from 150-day-old Atg7+/+ or Atg7−/− Pdx-Cre KrasG12D/+ mice. Values are mean and error bars are s.d. b, e, A Mann-Whitney U-test was used for statistics.


f , Representative p53 from 150 - day - old mice of both genotypes .

f, Representative p53 staining from 150-day-old mice of both genotypes.


g , PDAC - free survival of autophagy - proficient ( blue ) Atg5 - deficient ( red ) KrasG12D / + mice . d , g , Median survival ± s . d . / ( m / f ) are provided . ' n / a ' , no succumbed to PDAC . A log - rank ( Mantel - Cox ) was used for statistics . a , f , Scale bars : 500 µm ( a ) , 50 µm ( f ) . * P 0 . 05 , * * P 0 . 01 ; NS , not significant .

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g, PDAC-free survival of autophagy-proficient (blue) and Atg5-deficient (red) KrasG12D/+ mice. d, g, Median survival ± s.d. and male/female (m/f) ratio are provided. 'n/a', no animals succumbed to PDAC. A log-rank test (Mantel-Cox) was used for statistics. a, f, Scale bars: 500 µm (a), 50 µm (f). *P  0.05, **P  0.01; NS, not significant.


a , Kaplan - Meier comparing PDAC - free survival in Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / + p53 − / − mice .

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a, Kaplan-Meier analysis comparing PDAC-free survival in Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ p53−/− mice.


b , Pancreatic tumours of indicated genotypes stained for Atg7 , LC3 p62 . Insets are magnified crops from same to show specifics of LC3 p62 . magnification is × 4 .

b, Pancreatic tumours of indicated genotypes stained for Atg7, LC3 and p62. Insets are magnified crops from the same images to show specifics of LC3 and p62 staining. The magnification is × 4.


c , Tumour incidence tumour ( median , s . e . m . ) in mice from indicated genotypes , killed at 21 , 29 38 days of . Numbers in incidence diagram represent tumour - bearing mice versus mice . Significance was assessed with Fisher ' s exact ( incidence ) Mann - Whitney U - ( ) .

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c, Tumour incidence and tumour weight (median, s.e.m.) in mice from indicated genotypes, killed at 21, 29 and 38 days of age. Numbers in the incidence diagram represent tumour-bearing mice versus all mice. Significance was assessed with Fisher's exact test (incidence) and Mann-Whitney U-test (weight).


d , Representative haematoxylin eosin stainings of pancreases from indicated time points genotypes .

d, Representative haematoxylin and eosin stainings of pancreases from indicated time points and genotypes.


e , Kaplan - Meier comparing PDAC - free survival of Atg5 + / + ( blue ) or Atg5 − / − ( green ) KrasG12D / + p53 − / − mice .

e, Kaplan-Meier analysis comparing PDAC-free survival of Atg5+/+ (blue) or Atg5−/− (green) KrasG12D/+ p53−/− mice.


f , Kaplan - Meier comparing PDAC - free survival of KrasG12D / + p53 − / − mice that were chloroquine - treated ( CQ , red ) , vehicle - treated ( PBS , black ) or untreated ( blue ) . a , e , f , blue curve in Kaplan - Meier plots represents same colony of mice ( KrasG12D / + , p53 − / − , Atg7 + / + , Atg5 + / + ) . A Mantel - Cox was used for statistics . b , d , Scale bars : 100 µm ( b ) , 200 µm ( d ) . * P 0 . 05 , * * P 0 . 01 .

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f, Kaplan-Meier analysis comparing PDAC-free survival of KrasG12D/+ p53−/− mice that were chloroquine-treated (CQ, red), vehicle-treated (PBS, black) or untreated (blue). a, e, f, The blue curve in all Kaplan-Meier plots represents the same colony of mice (KrasG12D/+, p53−/−, Atg7+/+, Atg5+/+). A Mantel-Cox test was used for statistics. b, d, Scale bars: 100 µm (b), 200 µm (d). *P 0.05, **P 0.01.


a , LC3 of representative pancreatic tumours from mice of indicated genotypes . Scale bars : 50 µm .

a, LC3 staining of representative pancreatic tumours from mice of indicated genotypes. Scale bars: 50 µm.


b , Western blotting for LC3 , p53 Atg7 in lines derived from tumours which developed in absence or presence of p53 .

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b, Western blotting for LC3, p53 and Atg7 in cell lines derived from individual tumours which developed in the absence or presence of p53.


c , d , Average oxygen consumption rate ( OCR , c ) extracellular acidification rate ( ECAR ,

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c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR,


c , d , Average oxygen consumption rate ( OCR , c ) extracellular acidification rate ( ECAR , d ) of eight Atg7 + / + seven Atg7 − / − lines revealed increased ECAR in autophagy - deficient . Error bars are s . e . m .

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c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR, d) of eight Atg7+/+ and seven Atg7−/− cell lines revealed increased ECAR in autophagy-deficient cells. Error bars are s.e.m.


e , Treatment with 2 - deocyglucose ( 2 - DG ) reduces ECAR abrogates difference between lines from Atg7 + / + Atg7 − / − tumours . Values are average of three biological replicates . Error bars are s . e . m .

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e, Treatment with 2-deocyglucose (2-DG) reduces ECAR and abrogates the difference between cell lines from Atg7+/+ and Atg7−/− tumours. Values are the average of three biological replicates. Error bars are s.e.m.


, g , LC - MS revealed increased glucose consumption from medium ( f ) extracellular lactate accumulation ( g ) in Atg7 - null lines . Values are average of eight ( Atg7 + / + ) seven ( Atg7 − / − ) biological replicates .

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, g, LC-MS analysis revealed increased glucose consumption from medium (f) and extracellular lactate accumulation (g) in Atg7-null cell lines. Values are the average of eight (Atg7+/+) and seven (Atg7−/−) biological replicates.


h , LC - MS shows increased accumulation of 2 - DG its metabolite 2 - DG - 6 - phosphate in Atg7 - deficient lines . Values are averages of five biological replicates for each genotype .

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h, LC-MS analysis shows increased accumulation of 2-DG and its metabolite 2-DG-6-phosphate in Atg7-deficient cell lines. Values are averages of five biological replicates for each genotype.


i , Background of 18F - FDG uptake in brain heart can be seen in sagittal coronal views ( middle right panels , respectively ) of wild - type Atg7 mice ( top row ) . In contrast , intense 18F - FDG uptake is seen in pancreatic tumours of Atg7 deficient mice ( bottom row ) .

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i, Background levels of 18F-FDG uptake in brain and heart can be seen in sagittal and coronal views (middle and right panels, respectively) of wild-type Atg7 mice (top row). In contrast, intense 18F-FDG uptake is seen in pancreatic tumours of Atg7 deficient mice (bottom row).


j , LC - MS of intracellular glycolytic pentose phosphate pathway intermediates shows enhanced accumulation in Atg7 − / − tumour lines . Values are average of eight ( Atg7 + / + ) six ( Atg7 − / − ) biological replicates . f - h , j , Error bars are s . e . m . a , f - h , j , A Mann - Whitney U - was used to ascertain significance . * P 0 . 05 , * * P 0 . 01 .

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j, LC-MS of intracellular glycolytic and pentose phosphate pathway intermediates shows enhanced accumulation in Atg7−/− tumour cell lines. Values are average of eight (Atg7+/+) and six (Atg7−/−) biological replicates. f-h, j, Error bars are s.e.m. a, f-h, j, A Mann-Whitney U-test was used to ascertain significance. *P  0.05, **P  0.01.


( f ) A549 - ACE2 or control A549 - Venus were pretreated with Tubercidin or vehicle ( DMSO ) 3h prior to infection with SARS - CoV - 2 at MOI 0 . 1 . After 24 hours , abundances of SARS - CoV - 2 nucleoprotein ( N ) , ACE2 , Venus β - actin ( ACTB , loading control ) were visualized using Western blotting . Presented data is representative of 3 independent repeats .

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(f) A549-ACE2 or control A549-Venus cells were pretreated with Tubercidin or vehicle (DMSO) 3h prior to infection with SARS-CoV-2 at MOI 0.1. After 24 hours, abundances of SARS-CoV-2 nucleoprotein (N), ACE2, Venus and β-actin (ACTB, loading control) were visualized using Western blotting. Presented data is representative of 3 independent repeats.


( i ) NHBEs derived from three independent donors were pretreated for 6 h with indicated concentrations of DZNep or vehicle infected with SARS - CoV - 2 for 24 h . were fixed abundance of SARS - CoV - 2 N was quantified by immunofluorescent . Shown are vehicle - normalized integrated anti - N confluence , error bars represent mean + / - sd of n = 3 donors .

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(i) NHBEs derived from three independent donors were pretreated for 6 h with indicated concentrations of DZNep or vehicle and infected with SARS-CoV-2 for 24 h. Cells were fixed and the abundance of SARS-CoV-2 N was quantified by immunofluorescent staining. Shown are vehicle-normalized integrated anti-N fluorescent intensity and cell confluence, error bars represent mean +/- sd of n=3 donors.


Mass spectrometry of treated with DZNep infected with SARS - CoV - 2 SARS - CoV . ( b ) Number of significantly up - or downregulated in indicated comparisons according to ( a ) .

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Mass spectrometry based analysis of cells treated with DZNep and infected with SARS-CoV-2 and SARS-CoV. (b) Number of significantly up- or downregulated proteins in indicated comparisons according to (a).


Mass spectrometry of treated with DZNep infected with SARS - CoV - 2 SARS - CoV . ( c ) Donor - normalized LFQ abundances of viral nucleoprotein ( N ) spike ( S ) in indicated conditions . Error bars represent mean + / - sd of n = 4 donors ( NHBE ) or n = 4 independently infected A549 - ACE2 cultures . Statistics were calculated using Student ' s two - sided t - as indicated .

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Mass spectrometry based analysis of cells treated with DZNep and infected with SARS-CoV-2 and SARS-CoV. (c) Donor-normalized LFQ abundances of viral nucleoprotein (N) and spike (S) in the indicated conditions. Error bars represent mean +/- sd of n=4 donors (NHBE) or n=4 independently infected A549-ACE2 cultures. Statistics were calculated using Student's two-sided t-test as indicated.


( d ) Expression patterns according to ( a ) of a selection of genes related to disease - relevant pathways as annotated .

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(d) Expression patterns according to (a) of a selection of genes related to the disease-relevant pathways as annotated.


( e ) NHBEs ( 6 independent donors ) were pretreated for 6 h with 0 . 75 μM DZNep or vehicle infected with SARS - CoV or SARS - CoV - 2 at MOI 3 . 24 h later , accumulation of IL - 6 IP - 10 was measured in supernatant by ELISA . Donor - wise IL - 6 IP - 10 secretion , normalized to vehicle treated uninfected controls ( as further described in materials methods ) is shown .

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(e) NHBEs (6 independent donors) were pretreated for 6 h with 0.75 μM DZNep or vehicle and infected with SARS-CoV or SARS-CoV-2 at MOI 3. 24 h later, accumulation of IL-6 and IP-10 was measured in the supernatant by ELISA. Donor-wise IL-6 and IP-10 secretion, normalized to vehicle treated uninfected controls (as further described in materials and methods) is shown.


B An Integrative Genomics Viewer ( IGV ) screenshot of Notch de novo TE insertion site from sample P47 ( clonal neoplasia ) its head control , sample P48 . Bars represent sequencing reads . Reads supporting TE insertion are colored according to homology to a specific TE insertion . Multiple colors at a putative insert site frequently indicate homology to different reference copies of same TE family . Two types of supporting reads can be seen : soft - clipped reads - spanning insertion site mapping partially to reference genome partially to TE , mate pair support reads - flanking insertion site mapping to reference genome but with mates ( not seen ) mapping to TE .

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B An Integrative Genomics Viewer (IGV) screenshot of the Notch de novo TE insertion site from sample P47 (clonal neoplasia) and its head control, sample P48. Bars represent sequencing reads. Reads supporting the TE insertion are colored according to homology to a specific TE insertion sequence. Multiple colors at a putative insert site frequently indicate homology to different reference copies of the same TE family. Two types of supporting reads can be seen: soft-clipped reads - spanning the insertion site and mapping partially to the reference genome and partially to the TE, and mate pair support reads - flanking the insertion site and mapping to the reference genome but with mates (not seen) mapping to the TE.


D PCR validation of 4 somatic , neoplasia - specific TE insertions . Primers were designed to target regions flanking insertion sites . Yellow arrowheads indicate PCR products containing an insertion amplified in clonal but not in neighboring gut tissue ( non - clonal ) , head or thorax for same fly . Short wild - type amplicon was detectable in samples . Thorax sample was not available for sample P15 .

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D PCR validation of 4 somatic, neoplasia-specific TE insertions. Primers were designed to target regions flanking the insertion sites. Yellow arrowheads indicate PCR products containing an insertion amplified in the clonal DNA but not in the neighboring gut tissue (non-clonal), head or thorax for the same fly. Short wild-type amplicon was detectable in all samples. Thorax DNA sample was not available for sample P15.


D TSD distribution for somatic insertions of most frequent TE families . Insertions from both genotypes were pooled .

D TSD length distribution for somatic insertions of most frequent TE families. Insertions from both genotypes were pooled.


C distribution of somatic insertions with estimated fraction higher or lower than Notch - inactivating mutations for most represented LTR - elements ( rover copia ) - like I - elements . ( p - values were calculated with Fisher ' s exact , two - , n = number of insertions )

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C The distribution of somatic insertions with estimated cell fraction higher or lower than Notch-inactivating mutations for the most represented LTR-elements (rover and copia) and LINE-like I-elements. (p-values were calculated with Fisher's exact test, two-tailed, n= number of insertions)


A Heatmaps representing normalized TE expression ( in log2 ( TPM ) , transcripts per million ) mobility ( log2 ( insertion ) ) in Pros > 2xGFP midguts . TEs with TPM values below 0 . 05 ( log2 ( TPM ) < - 4 . 3 ) are not depicted . Crossed out represent no somatic insertions of that family identified .

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A Heatmaps representing normalized TE expression levels (in log2(TPM), transcripts per million) and mobility (log2(insertion counts)) in Pros>2xGFP midguts. TEs with TPM values below 0.05 (log2(TPM)< -4.3) are not depicted. Crossed out cells represent no somatic insertions of that family identified.


B Normalized read coverage over full - canonical of selected TE families .

B Normalized read coverage over the full-length canonical sequence of selected TE families.


C Normalized expression of siRNA pathway genes .

C Normalized expression levels of siRNA pathway genes.


D distribution of sense antisense reads from gut small fractions mapping to TEs ( upper panel ) or selected TE families mobilizing in gut . R1 R2 are two biological replicates .

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D The size distribution of sense and antisense reads from gut small RNA fractions mapping to all TEs (upper panel) or selected TE families mobilizing in the gut. R1 and R2 are two biological replicates.


E Scatter plot of normalized transcript ( mRNA ) antisense , 21nt siRNA for TE families . Transposons generating somatic insertions are highlighted in red ( LTR - elements ) or blue ( - like ) , with a symbol reflecting somatic insertion .

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E Scatter plot of normalized transcript (mRNA) levels and antisense, 21nt siRNA levels for all TE families. Transposons generating somatic insertions are highlighted in red (LTR-elements) or blue (LINE-like), with a symbol size reflecting the somatic insertion counts.


B TE families with tissue - specific , singleton TSD - bearing insertions detected in pooled gut or head .

B TE families with tissue-specific, singleton TSD-bearing insertions detected in pooled gut or head DNA.


B Somatic TE insertion sites of rover LTR elements were depleted from intergenic exonic sequences enriched in introns 3 ' UTR regions of fly genome . Insertions of copia LTR elements were also enriched in introns .

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B Somatic TE insertion sites of rover LTR elements were depleted from intergenic and exonic sequences and enriched in introns and 3'UTR regions of the fly genome. Insertions of copia LTR elements were also enriched in introns.


D Correlations of somatic insertion sites of three most represented TE families ( rover , copia I - element ) with modENCODE tracks for adult fly tissues .

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D Correlations of somatic insertion sites of the three most represented TE families (rover, copia and I-element) with modENCODE tracks for adult fly tissues.


E Correlations of somatic insertion sites of three most represented TE families ( rover , copia I - element ) with DamID tracks for adult fly intestinal ( ISC ) .

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E Correlations of somatic insertion sites of the three most represented TE families (rover, copia and I-element) with DamID tracks for adult fly intestinal stem cells (ISC).


[ Ca2 + ] i changes in BMDMs were measured ( A ) using fura - 2 during frustrated phagocytosis ( n = 46 - 113 ) ( B ) by dropping beads onto adherent loaded with Calbryte 520 ( n = 64 - 159 ) , in 1 . 8 mM extracellular Ca2 + ( + Ca2 + o ) or Ca2 + - free medium containing 100 μM EGTA ( - Ca2 + o ) with or without prior loading with 25 μM EGTA / AM or 25 μM BAPTA / AM . maximum fura - 2 changes ( ∆ 350 / 380 ) or changes normalized to initial ( ∆ F / F0 ) are plotted .

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[Ca2+]i changes in individual BMDMs were measured (A) using fura-2 during frustrated phagocytosis (n = 46-113) and (B) by dropping beads onto adherent cells loaded with Calbryte 520 (n = 64-159), in 1.8 mM extracellular Ca2+ (+Ca2+o) or Ca2+-free medium containing 100 μM EGTA (-Ca2+o) with or without prior loading with 25 μM EGTA/AM or 25 μM BAPTA/AM. The maximum fura-2 ratio changes (∆ 350/380) or fluorescence changes normalized to initial fluorescence (∆ F/F0) are plotted.


FcγR - mediated phagocytosis was monitored using beads conjugated to Alexa Fluor 488 ( in green ) . External beads were differentially labelled with anti - IgG ( in red ) distinguished by their dual labelling ( in yellow ) . Cytochalasin D ( 10 μM , Cyt D ) prevented bead internalization .

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FcγR-mediated phagocytosis was monitored using beads conjugated to Alexa Fluor 488 (in green). External beads were differentially labelled with anti-IgG (in red) and distinguished by their dual labelling (in yellow). Cytochalasin D (10 μM, Cyt D) prevented bead internalization.


Bead uptake in 15 min , under conditions as in ( A , B ) , n = 80 - 91 with representative of delineated by a dotted .

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Bead uptake in 15 min, under conditions as in (A, B), n = 80-91 cells with representative images of individual cells delineated by a dotted line.


Experiments conducted in - Ca2 + o : ( F ) 1 μM thapsigarin ( Tg ) or 10 μM CPA pre - treatment , but eliminated 1 μM ionomycin ( iono ) response ; single - Ca2 + traces mean ∆ F / F0 , n = 450 - 603 . ( G ) FcγR activation ( as in B ) evoked Ca2 + oscillations that were blocked with Tg or CPA treatment , whilst bead phagocytosis was not affected ( H ) , n = 312 - 428 . ( I ) 1 μM bafilomycin A1 ( Baf ) for 3h , or Ca2 + - binding dextran ( Cal520 - dx ) non - Ca2 + - binding dextrans ( FITC - dx Texas red - dx ) trafficked to lysosomes , minimally affected global Ca2 + signals evoked by FcγR activation ( J ) ; in parallel , bead uptake was only reduced by bafilomycin A1 or by chelating Ca2 + within lysosome with Cal520 - dx ( K ) , n = 100 - 162 . are WT BMDM beads are 3 - μm IgG - opsonized .

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

Experiments conducted in -Ca2+o: (F) 1 μM thapsigarin (Tg) or 10 μM CPA pre-treatment, all but eliminated the 1 μM ionomycin (iono) response; single-cell Ca2+ traces and mean ∆ F/F0, n = 450-603 cells. (G) FcγR activation (as in B) evoked Ca2+ oscillations that were blocked with Tg or CPA treatment, whilst bead phagocytosis was not affected (H), n = 312-428 cells. (I) 1 μM bafilomycin A1 (Baf) for 3h, or Ca2+- binding dextran (Cal520-dx) and non-Ca2+-binding dextrans (FITC-dx and Texas red-dx) trafficked to lysosomes, minimally affected global Ca2+ signals evoked by FcγR activation (J); in parallel, bead uptake was only reduced by bafilomycin A1 or by chelating Ca2+ within the lysosome with Cal520-dx (K), n = 100-162 cells. All cells are WT BMDM and all beads are 3-μm IgG-opsonized.


Ca2 + signal elicited by frustrated phagocytosis was significantly reduced in Ned - 19 ( 10 μM ) - treated WT in TPC - KO BMDMs ; maximum Ca2 + amplitude , expressed as a of WT control ( n = 314 - 641 ) . Phagocytosis of opsonized 3 - µm ( B C ) 6 - μm ( D ) beads was inhibited by Ned - 19 ( B ) in BMDMs from Tpcn - / - mice compared to WT ( n = 134 - 280 ) .

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The Ca2+ signal elicited by frustrated phagocytosis was significantly reduced in Ned-19 (10 μM)-treated WT and in all TPC-KO BMDMs; maximum Ca2+ amplitude, expressed as a percentage of WT control (n = 314-641 cells). Phagocytosis of opsonized 3-µm (B and C) and 6-μm (D) beads was inhibited by Ned-19 (B) and in BMDMs from Tpcn-/- mice compared to WT (n = 134-280 cells).


( E ) Phagocytosis of 3 - µm beads was unchanged in MCOLN1 - / - BMDMs , but was impaired using 6 - µm beads ( F ) , n = 89 - 157 .

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(E) Phagocytosis of 3-µm beads was unchanged in MCOLN1-/- BMDMs, but was impaired using 6-µm beads (F), n = 89-157.


Heterologous expression of mouse TPCs tagged with TagRFP - T ( in red ) rescues phagocytic defect of Tpcn1 - / - Tpcn2 - / - BMDM , but a pore - dead TPC2 - mutant ( N257A ) does not , n = 39 - 94 . taken 40 min post - 3 - μm bead addition .

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Heterologous expression of mouse TPCs tagged with TagRFP-T (in red) rescues the phagocytic defect of Tpcn1-/- and Tpcn2-/- BMDM, but a pore-dead TPC2-mutant (N257A) does not, n = 39-94. Images taken 40 min post- 3-μm bead addition.


Raising Ca2 + with ionomycin ( 1 μM ) rescues Tpcn1 - / - Tpcn2 - / - phagocytic defect , whilst UTP ( 100 μM ) ML - SA1 ( 50 μM ) only weakly rescue ( n = 84 - 192 ) .

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Raising cytosolic Ca2+ with ionomycin (1 μM) rescues the Tpcn1-/- and Tpcn2-/- phagocytic defect, whilst UTP (100 μM) and ML-SA1 (50 μM) only weakly rescue (n = 84-192 cells).


Internalization of is reduced in Tpcn - / - BMDM , as assessed by single - imaging of BMDM allowed to internalise M . smegmatis tagged with mCherry ( in red ) for 1 h . Internalization of assessed discontinuously in BMDM populations : ( B ) resultant growth of colonies on agar plates following lysis of BMDM infected with M . smegmatis ( C ) of colony forming units ( CFU ) expressed as a of WT control ( n = 12 ) ;

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Internalization of bacteria is reduced in Tpcn-/- BMDM, as assessed by single-cell imaging of BMDM allowed to internalise M. smegmatis tagged with mCherry (in red) for 1 h. Internalization of bacteria assessed discontinuously in BMDM populations: (B) The resultant growth of bacteria colonies on agar plates following lysis of BMDM infected with M. smegmatis and (C) quantification of colony forming units (CFU) expressed as a percentage of WT control (n = 12);


Real - time internalization of into BMDM populations : ( D ) Relative Units ( RFU ) of internalised mCherry - tagged M . smegmatis ( E ) mCherry - tagged BCG over time , read using a plate reader ( n = 8 ) .

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Real-time internalization of fluorescent bacteria into BMDM populations: (D) Relative Fluorescence Units (RFU) of internalised mCherry-tagged M. smegmatis (E) and mCherry-tagged BCG over time, read using a plate reader (n = 8).


BMDMs were co - loaded with 2 µM Calbryte590 / AM ( 50 min ) 200 nM Lysotracker Green ( LTG , 5 min ) , at room , presented with 3 - µm opsonized beads labelled with Alexa Fluor 647 ( blue ) . A three - channel image was recorded every 10 s . LTG brightness has been enhanced post hoc to visualize dimmer , peripheral lysosomes . Time Zero is defined as frame when first Ca2 + signal was observed . ( A ) represent Ca2 + responses or an overlay of LTG bead . Times of each image are relative to first Ca2 + spike . ( B ) Enlargement of first contact point between lysosomes bead ( red arrow ) as depicted by dashed box , 80 s post - Ca2 + . ( C ) Whole - Ca2 + responses from in A . ( D ) Collation of post - Ca2 + contact time , displayed as a scatter plot of mean ± S . E . M . ( n = 27 ) .

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BMDMs were co-loaded with 2 µM Calbryte590/AM (50 min) and 200 nM Lysotracker Green (LTG, 5 min) and, at room temperature, presented with 3-µm opsonized beads labelled with Alexa Fluor 647 (blue). A three-channel image was recorded every 10 s. The LTG brightness has been enhanced post hoc to visualize the dimmer, peripheral lysosomes. Time Zero is defined as the frame when the first Ca2+ signal was observed. (A) Images represent Ca2+ responses or an overlay of LTG and bead fluorescence. Times of each image are relative to the first Ca2+ spike. (B) Enlargement of the first contact point between lysosomes and bead (red arrow) as depicted by the dashed box, 80 s post-Ca2+. (C) Whole-cell Ca2+ responses from the cell in A. (D) Collation of the post-Ca2+ contact time, displayed as a scatter plot of individual cells and the mean ± S.E.M. (n = 27 cells).


RAW246 . 7 were transfected with either TPC1 - TagRFP - T or TPC2 - TagRFP - T ( red ) presented with 3 - µm opsonized beads labelled with Alexa Fluor 647 ( blue ) . Acute time - series of bead engagement were collected . Contact between TPCs beads are depicted in time - stamped , where time is arbitrary after start of image stack ( E , F ) i . e . 110 s is still prior to engagement . spatial relationship between TPCs beads was quantified using a target graticule of 0 . 5 - µm bands centred on engulfed bead ; mean within each band was normalized to of maximum band plotted against this radial ( G , H ) ; n = 35 - 36 .

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RAW246.7 cells were transfected with either TPC1-TagRFP-T or TPC2-TagRFP-T (red) and presented with 3-µm opsonized beads labelled with Alexa Fluor 647 (blue). Acute time-series of bead engagement were collected. Contact between TPCs and beads are depicted in the time-stamped micrographs, where time is arbitrary after the start of the image stack (E,F) i.e. 110 s is still prior to engagement. The spatial relationship between TPCs and beads was quantified using a target graticule of 0.5-µm bands centred on the engulfed bead; the mean fluorescence within each band was normalized to the fluorescence of the maximum band and plotted against this radial distance (G,H); n = 35-36 cells.


RAW 247 . 6 macrophages loaded with a chemical dye ( Cyto dye ) to detect global Ca2 + , heterologously expressing ( B ) TPC1 - G - GECO1 . 2 ( TPC1 - GG ) or ( C - E ) TPC2 - G - GECO1 . 2 ( TPC2 - GG ) to detect peri - endo - lysosomal Ca2 + domains , which were not detected by ( F ) a TPC2 mutant ( D276K ) - G - GECO1 . 2 that blocks ion permeation or ( A ) unfused G - GECO1 . 2 ( Cyto GG ) . ( A - F ) Single - Ca2 + traces upon FcγR activation by opsonized 3 - μm beads in - Ca2 + o expressed as a of maximum of indicator , determined by adding 2 μM ionomycin 10 mM CaCl2 at end of each experiment ( % Fmax ) ( D ) TPC2 - G - GECO1 . 2 activity was inhibited by 10 µM Ned - 19 , but was unaffected by ( E ) chelating global Ca2 + with EGTA / AM ( 100 μM for 2 min prior to bead addition ) . Summary data of first spike from A - F ( subsequent spikes show same pattern ; Appendix Fig . S4B ) , n = 87 - 274 ; * * * P < 0 . 001 vs Cyto GG , # # # P < 0 . 001 vs TPC2 - GG ctrl , † † † P < 0 . 001 vs Cyto GG . Fusion of G - GECO1 . 2 to TPC2 did not alter affinity of G - GECO1 . 2 for Ca2 + determined in permeabilized ( n = 34 - 71 ) .

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

RAW 247.6 macrophages loaded with a cytosolic chemical dye (Cyto dye) to detect global cytosolic Ca2+, and heterologously expressing (B) TPC1-G-GECO1.2 (TPC1-GG) or (C-E) TPC2-G-GECO1.2 (TPC2-GG) to detect peri-endo-lysosomal Ca2+ domains, which were not detected by (F) a TPC2 mutant (D276K)-G-GECO1.2 that blocks ion permeation or (A) unfused cytosolic G-GECO1.2 (Cyto GG). (A-F) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of the maximum fluorescence of the indicator, determined by adding 2 μM ionomycin and 10 mM CaCl2 at the end of each experiment (% Fmax) (D) TPC2-G-GECO1.2 activity was inhibited by 10 µM Ned-19, but was unaffected by (E) chelating global Ca2+ with EGTA/AM (100 μM for 2 min prior to bead addition). Summary data of the first spike from cells A-F (subsequent spikes show the same pattern; Appendix Fig. S4B), n = 87-274 cells; ***P<0.001 vs Cyto GG, ### P<0.001 vs TPC2-GG ctrl, ††† P<0.001 vs Cyto GG. Fusion of G-GECO1.2 to TPC2 did not alter the affinity of G-GECO1.2 for Ca2+ determined in permeabilized cells (n = 34-71 cells).


( I ) Single - Ca2 + traces upon FcγR activation by opsonized 3 - μm beads in - Ca2 + o expressed as a of Fmax . ( I , J ) Pre - emptying ER stores with CPA or thapsigargin ( Tg ) abolished global Ca2 + response evoked by beads , but a TPC2 - G ‑ GECO1 . 2 component remained , n = 17 - 121 .

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(I) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of Fmax. (I, J) Pre-emptying the ER stores with CPA or thapsigargin (Tg) abolished the global cytosolic Ca2+ response evoked by beads, but a TPC2-G‑GECO1.2 component remained, n = 17 - 121 cells.


No local Ca2 + signal was detected with TPC2 - G - GECO1 . 2 when Ca2 + spiking was evoked by ER - Ca2 + release with 100 μM UTP ( K , L ) or 10 μM CPA ( M ) ; only a bystander response equivalent to unfused G - GECO1 . 2 was detected ( n = 121 - 182 ; * P < 0 . 05 ) .

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No local Ca2+ signal was detected with TPC2-G-GECO1.2 when Ca2+ spiking was evoked by ER-Ca2+ release with 100 μM UTP (K, L) or 10 μM CPA (M); only a bystander response equivalent to unfused cytosolic G-GECO1.2 was detected (n = 121-182 cells; *P<0.05).


Translocation of CB , a non - Ca2 + binding CB mutant ( CB mut ) or a triple mTagBFP2 ( BFP ) to lysosome ( LAMP1 ) , as indicated by increase in Pearson ' s correlation coefficient was induced by 250 nM rapalog ( 30 mins ) compared with ethanol ( EtOH ) control - treated RAW 247 . 6 . n = 11 - 25 ; * * * P < 0 . 001 .

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Translocation of CB, a non-Ca2+ binding CB mutant (CB mut) or a triple mTagBFP2 (BFP) to the lysosome (LAMP1), as indicated by the increase in the Pearson's correlation coefficient was induced by 250 nM rapalog (30 mins) compared with ethanol (EtOH) control-treated RAW 247.6 cells. n = 11-25 cells; ***P<0.001.


of a single RAW 247 . 6 displaying yellow indicative of successful re - positioning of CB ( in green ) to lysosome ( in red ) in presence of rapalog but not EtOH .

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Images of a single RAW 247.6 cell displaying yellow puncta indicative of successful re-positioning of CB (in green) to the lysosome (in red) in the presence of rapalog but not EtOH.


When CB was brought to lysosome ( + rapalog ) uptake of beads was inhibited , whilst non - Ca2 + binding CB ( CB mut ; n = 46 - 187 ) or triple mTagBFP2 ( BFP ; n = 38 - 52 ) did not block phagocytosis ; * * * P < 0 . 001 .

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When CB was brought to the lysosome (+rapalog) uptake of beads was inhibited, whilst the non-Ca2+ binding CB (CB mut; n = 46-187 cells) or triple mTagBFP2 (BFP; n = 38-52 cells) did not block phagocytosis;***P<0.001.


Bead uptake in BMDMs with without loading of 25 µM EGTA / AM 10 μM Ned - 19 . EGTA - insensitive phagocytosis of beads was inhibited by Ned - 19 ( n = 75 - 102 ) in WT BMDM ; * * * P < 0 . 001 .

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Bead uptake in BMDMs with and without loading of 25 µM EGTA/AM and 10 μM Ned-19. EGTA-insensitive phagocytosis of beads was inhibited by Ned-19 (n= 75-102 cells) in WT BMDM; ***P<0.001.


RAW 247 . 6 macrophages expressing G - GECO1 . 2 ( Cyto GG ) , TPC2 - G - GECO1 . 2 ( TPC2 - GG ) or TRPML1 - G - GECO1 . 2 ( TRPML1 - GG ) . ( F ) Single - Ca2 + traces upon FcγR activation by opsonized 3 - μm or 6 - μm beads in - Ca2 + o expressed as a of maximum indicator ( % Fmax = 2 μM ionomycin 10 mM CaCl2 ) . ( G ) Summary of first Ca2 + spike ; n = 69 - 121 ( 3 - μm bead ) , 22 - 46 ( 6 - μm beads ) ; * * * P < 0 . 001 , * * P < 0 . 01 vs Cyto GG , # # # P < 0 . 001 TPC2 - GG vs TRPML1 - GG .

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RAW 247.6 macrophages expressing cytosolic G-GECO1.2 (Cyto GG), TPC2-G-GECO1.2 (TPC2-GG) or TRPML1-G-GECO1.2 (TRPML1-GG). (F) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm or 6-μm beads in -Ca2+o expressed as a percentage of the maximum indicator fluorescence (% Fmax = 2 μM ionomycin and 10 mM CaCl2). (G) Summary of first Ca2+ spike; n = 69-121 (3-μm bead), 22-46 (6-μm beads) cells; ***P<0.001, **P<0.01 vs Cyto GG, ### P<0.001 TPC2-GG vs TRPML1-GG.


Dynamin inhibitors ( 80 μM Dynasore or 20 μM Dyngo - 4a ) inhibit phagocytosis of opsonized 3 - μm beads in WT BMDM ; n = 101 - 149 , * * * P < 0 . 001 .

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Dynamin inhibitors (80 μM Dynasore or 20 μM Dyngo-4a) inhibit phagocytosis of opsonized 3-μm beads in WT BMDM; n = 101-149 cells, ***P<0.001.


Rescue of TPC - KO dynamin defect by dynamin activator , Ryngo - 1 - 23 ( 80 μM ) : mean number of internalised beads per as a of WT control ; n = 128 - 241 , * * P < 0 . 01 , * * * P < 0 . 001 .

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Rescue of the TPC-KO dynamin defect by the dynamin activator, Ryngo-1-23 (80 μM): mean number of internalised beads per cell as a percentage of the WT control; n = 128-241 cells, **P<0.01, ***P<0.001.


In WT BMDM , expression of a dominant - negative dynamin - 2 mutant ( Dyn2 K44A ) reduced phagocytosis ( n = 68 - 184 ) . Expression of wildtype dynamin - 2 ( Dyn2 ) or constitutively active dynamin - 2 ( S764A mutant , mimicking dephosphorylated state ) in Tpcn1 - / - macrophages restored phagocytosis of 3 - μm beads after 10 min , n = 10 - 76 ; * * * P < 0 . 001 vs WT control , # # # P < 0 . 001 vs Tpcn1 - / - control .

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In WT BMDM, expression of a dominant-negative dynamin-2 mutant (Dyn2 K44A) reduced phagocytosis (n = 68-184). Expression of wildtype dynamin-2 (Dyn2) or constitutively active dynamin-2 (S764A mutant, mimicking the dephosphorylated state) in Tpcn1-/- macrophages restored phagocytosis of 3-μm beads after 10 min, n = 10-76 cells; ***P<0.001 vs WT control, ### P<0.001 vs Tpcn1-/- control.


Single WT BMDM expressing HA - tagged dynamin - 2 ( immunolabelled with anti - HA , in red ) undergoing phagocytosis of IgG - 3 - μm beads ( blue ) . were taken 10 or 30 min post - addition of beads ; actin ( green ) labelled with Phalloidin Alexa 488 .

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Single WT BMDM expressing HA-tagged dynamin-2 (immunolabelled with anti-HA, in red) undergoing phagocytosis of IgG-3-μm beads (blue). Images were taken 10 or 30 min post-addition of beads; actin (green) labelled with Phalloidin Alexa 488.


Calcineurin inhibitors , FK506 ( 10 μM ) or cyclosporin A ( CsA , 10 μM ) inhibited phagocytosis in WT , n = 65 - 159 . Ryngo ( 80 μM ) rescued phagocytosis defects in Tpcn1 - / - BMDM even in presence of FK506 or CsA , n = 103 - 208 .

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Calcineurin inhibitors, FK506 (10 μM) or cyclosporin A (CsA, 10 μM) inhibited phagocytosis in WT cells, n = 65-159 cells. Ryngo (80 μM) rescued phagocytosis defects in Tpcn1-/- BMDM even in the presence of FK506 or CsA, n = 103-208 cells.