F No polarization of DHHC2 in dhhc2 : : 6HA gametocytes with only either XA or 22 ° C stimulation . Three replicates performed , values are means ± SD .

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F No polarization of DHHC2 in the dhhc2::6HA gametocytes with only either XA or 22°C stimulation. Three replicates performed, values are means ± SD.


G No change in DHHC2 expression in zygotes of dhhc2 : : 6HA - derived mutants with gene disruption of cdpk4 , hap2 , dozi .

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G No change in DHHC2 expression in zygotes of the dhhc2::6HA parasite-derived mutants with individual gene disruption of cdpk4, hap2, and dozi.


H of with DHHC2 polarization in P28 positive . are indicated in ( G ) or from a genetic cross ( dhhc2 : : 6HA / cdpk4 WT ) . x / y : number of with DHHC2 polarization number of analyzed . Values are means ± SD from three , two - t , * * * , P < 0 . 001 , * * * * , P < 0 . 0001 , ns , not significant .

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H Quantification of parasite with DHHC2 polarization in P28 positive cells. Parasites are indicated in (G) or from a genetic cross (dhhc2::6HA/cdpk4 and WT). x/y: the number of cells with DHHC2 polarization and the number of cells analyzed. Values are means ± SD from three tests, two-tailed t test, ***, P<0.001, ****, P<0.0001, ns, not significant.


I Expression localization of DHHC2 GAP45 during gamete to ookinete development of dhhc2 : : 6HA . GAP45 is an IMC marker is only detected in zygote after fertilization , but not gametes . Scale bar = 5 μm .

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I Expression and localization of DHHC2 and GAP45 during female gamete to ookinete development of dhhc2::6HA parasite. GAP45 is an IMC marker and is only detected in zygote after fertilization, but not gametes. Scale bar = 5 μm.


A Transmission electron microscopy ( ) cross sections of WT , dhhc2kd ∆ isp1 / 3 showing arrangement of SPM underneath IMC . Approximately 60 recognizable hollow microtubules ( green ) are associated with IMC ( red ) , distributing evenly around circumference with an interval of 100 - 120 nm in WT ookinetes . Microtubules lost association with IMC in ookinetes of dhhc2kd ∆ isp1 / 3 . Left : one representative in each is shown ; Right : two areas are magnified to show details . Lower panel is of between adjacent microtubules associated with IMC . Values are mean ± SD for n = 204 ( WT ) , 189 ( dhhc2kd ) , 211 ( ∆ isp1 / 3 ) measurements in 20 each group from two independent experiments ; Kolmogorov - Smirnov , * * * * , P < 0 . 0001 .

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A Transmission electron microscopy (TEM) cross sections of WT, dhhc2kd and ∆isp1/3 parasites showing the arrangement of SPM underneath IMC. Approximately 60 recognizable hollow microtubules (green) are associated with IMC (red), distributing evenly around circumference with an interval of 100-120 nm in WT ookinetes. Microtubules lost association with IMC in the ookinetes of dhhc2kd and ∆isp1/3 parasites. Left: one representative cell in each parasite is shown; Right: two areas are magnified to show the details. Lower panel is the quantification of distance between adjacent microtubules associated with IMC. Values are mean ± SD for n=204 (WT), 189 (dhhc2kd), and 211 (∆isp1/3) measurements in 20 cells each group from two independent experiments; Kolmogorov-Smirnov test, ****, P<0.0001.


B Apical polar ring emanating SPM in WT , dhhc2kd ∆ isp1 / 3 examined by negative . A dome - like SPM structure radiating from apical polar ring was observed in early ( 3 hours ) or mature ( 12 hours ) WT ookinetes , but impaired in mutant . n is number of analyzed from three independent experiments . More in Appendix Figure S5 . Scale bar = 1μm .

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B Apical polar ring and the emanating SPM in WT, dhhc2kd and ∆isp1/3 parasites examined by negative staining and TEM. A dome-like SPM structure radiating from the apical polar ring was observed in early (3 hours) or mature (12 hours) WT ookinetes, but impaired in mutant parasites. n is the number of cells analyzed from three independent experiments. More images in the Appendix Figure S5. Scale bar = 1μm.


C SiR - tubulin show SPM structures of WT , dhhc2kd ∆ isp1 / 3 . Arrowheads point to apical . Scale bar = 5 μm .

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C SiR-tubulin staining show the SPM structures of WT, dhhc2kd and ∆isp1/3 parasites. Arrowheads point to cell apical. Scale bar = 5 μm.


A GST - ISP1 pulls down endogenous β - Tubulin from lysate of WT ookinetes . Lower panel indicates Coomassie blue stain of recombinant GST GST - ISP1 .

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A GST-ISP1 pulls down endogenous β-Tubulin from cell lysate of WT ookinetes. Lower panel indicates the Coomassie blue stain of recombinant GST and GST-ISP1.


B Episomally expressed ISP1 : : HA immunoprecipitated with β - Tubulin in WT ookinetes . Episomal vector as control .

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B Episomally expressed ISP1::HA immunoprecipitated with β-Tubulin in WT ookinetes. Episomal vector as control.


C Endogenous ISP1 : : 3V5 immunoprecipitated with β - Tubulin in TTS ookinetes .

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C Endogenous ISP1::3V5 immunoprecipitated with β-Tubulin in the TTS ookinetes.


D Co - localization of ISP1 : : HA β - Tubulin at ookinete periphery in IFA . Scale bar = 5 μm .

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D Co-localization of ISP1::HA and β-Tubulin at ookinete periphery in IFA. Scale bar = 5 μm.


E Proximity ligation ( PLA ) detecting interaction of ISP1 - HA β - Tubulin at ookinete . Episomal vector as control . Scale bar = 5 μm .

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E Proximity ligation assay (PLA) detecting interaction of ISP1-HA and β-Tubulin at ookinete. Episomal vector as control. Scale bar = 5 μm.


F Solubility detecting association of α - β - Tubulins in WT , dhhc2kd , ∆ isp1 / 3 . GAPDH is a cytosol soluble , P28 is a plasma integral .

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F Solubility assay detecting membrane association of α- and β-Tubulins in WT, dhhc2kd, and ∆isp1/3 parasites. GAPDH is a cytosol soluble protein, P28 is a plasma membrane integral protein.


G of α - β - Tubulins association in F . association index is defined as of Carb fraction to Hypo fraction in F , normalized to that of WT . Values are means ± from three independent .

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G Quantification of α- and β-Tubulins membrane association in F. The membrane association index is defined as the ratio of Carb fraction to Hypo fraction in F, and normalized to that of WT. Values are means ± SEM from three independent tests.


H Linear correlation of association index of α - Tubulin β - Tubulin with conversion rate to mature ookinete among WT , dhhc2kd ∆ isp1 / 3 . Horizontal vertical values are means ± SD .

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H Linear correlation of membrane association index of α-Tubulin and β-Tubulin with the conversion rate to mature ookinete among WT, dhhc2kd and ∆isp1/3 parasites. Horizontal and vertical values are means ± SD.


I association of α - β - Tubulins in WT ookinete culture after DMSO , 2 - BP , or colchicine treatment .

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I Membrane association of α- and β-Tubulins in WT ookinete culture after DMSO, 2-BP, or colchicine treatment.


J of α - β - Tubulins association Values are means ± from three independent repeats .

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J Quantification of α- and β-Tubulins membrane association Values are means ± SEM from three independent repeats.


( D - E ) Measurements of ciliary cAMP using cilia - Pink Flamindo . ( D ) Exemplary are shown for cilia ( cilia - bPAC or cyto - bPAC ) expressing cilia - Pink Flamindo biosensor after stimulation . Scale bars are indicated ; cAMP are color - coded using a high - low look - up table . ( E ) of data exemplified in ( D ) . cAMP level is determined as mean ciliary during first 60 s after stimulation ( measurement interval 10 s ) ; p value for a Kolmogorov - Smirnov is indicated . Each data point represents an cilium . Bars display median interquartile range ; cilia - bPAC : 40 cilia ( biological replicates ) from n = 14 independent experiments ; cyto - bPAC : 31 cilia ( biological replicates ) from n = 31 independent experiments .

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(D-E) Measurements of ciliary cAMP levels using cilia-Pink Flamindo. (D) Exemplary images are shown for cilia (cilia-bPAC or cyto-bPAC cells) expressing the cilia-Pink Flamindo biosensor after light stimulation. Scale bars are indicated; cAMP levels are color-coded using a high-low look-up table. (E) Quantification of data exemplified in (D). The cAMP level is determined as the mean ciliary fluorescence intensity during the first 60 s after light stimulation (measurement interval 10 s); p value for a Kolmogorov-Smirnov test is indicated. Each data point represents an individual cilium. Bars display median and interquartile range; cilia-bPAC: 40 cilia (biological replicates) from n = 14 independent experiments; cyto-bPAC: 31 cilia (biological replicates) from n = 31 independent experiments.


( A ) Wild - type ( WT ) mIMCD - 3 cultured in a 3D matrix during continuous exposure to DMSO ( control ) or 10 µM of Forskolin . Exemplary are shown ( n = 5 biological replicates ) . of cyst number is shown on right Data are shown as mean ± S . D . , each datapoint corresponds to an independent experiment , p values calculated using an unpaired , two - sided Student ' s t - are indicated .

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(A) Wild-type (WT) mIMCD-3 cells cultured in a 3D matrix during continuous exposure to DMSO (control) or 10 µM of Forskolin. Exemplary images are shown (n = 5 biological replicates). Quantification of cyst number and area is shown on the right Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment, p values calculated using an unpaired, two-sided Student's t-test are indicated.


( D ) mIMCD - 3 stably expressing cilia - bPAC or cyto - bPAC , cultured in a 3D matrix in , during exposure ( 1 h / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² ) or in incubated with 10 µM of Forskolin . Exemplary are shown ( n > 3 ) . ( E ) of data exemplified in ( D ) . Data are shown as mean ± S . D . , each datapoint corresponds to an independent experiment ; p values calculated using an unpaired , two - sided Student ' s t - are indicated .

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(D) mIMCD-3 cells stably expressing cilia-bPAC or cyto-bPAC, cultured in a 3D matrix in the dark, during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) or in the dark and incubated with 10 µM of Forskolin. Exemplary images are shown (n > 3). (E) Quantification of the data exemplified in (D). Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment; p values calculated using an unpaired, two-sided Student's t-test are indicated.


( D ) Immunoblotting of lysates from mIMCD - 3 wild - type ( WT ) mIMCD - 3 stably expressing cilia - bPAC for S6 ribosomal ( S6RP ) phosphorylation at Ser235 / 236 ( pS6RP ) . of total S6RP ( S6RP ) beta - Tubulin have been determined as controls . Cilia - bPAC were treated with DMSO ( control ) or with 10 nM of Rapamycin . were stimulated with ( 1 h / 1 h , 48 h , 465 nm , 38 . 8 µW / cm² ) ( E ) of pS6RP / S6RP . Data are shown as mean ± S . D . , n > 3 ; p values were calculated using a paired , two - sided Student ' s t - . Each data point shows an independent

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(D) Immunoblotting of lysates from mIMCD-3 wild-type (WT) cells and mIMCD-3 cells stably expressing cilia-bPAC for S6 ribosomal protein (S6RP) phosphorylation at Ser235/236 (pS6RP). Levels of total S6RP protein (S6RP) and beta-Tubulin have been determined as controls. Cilia-bPAC cells were treated with DMSO (control) or with 10 nM of Rapamycin. All cells were stimulated with light (1 h light/1 h dark, 48 h, 465 nm, 38.8 µW/cm²) (E) Quantification of the pS6RP/S6RP ratio. Data are shown as mean ± S.D., n > 3; p values were calculated using a paired, two-sided Student's t-test. Each data point shows an independent experiment


( G ) mIMCD - 3 stably expressing cilia - bPAC , cultured in a 3D matrix in , during exposure ( 1 h / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² ) incubation with 10 nM rapamycin . exposure started 2 d later than incubation with 10 nM rapamycin . Exemplary are shown ( n = 3 ) ( H ) of data exemplified in ( G ) . Data are shown as mean ± S . D . , each datapoint corresponds to an independent experiment ; p values calculated using a paired , two - sided Student ' s t - are

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(G) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark, during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubation with 10 nM rapamycin. Light exposure started 2 d later than incubation with 10 nM rapamycin. Exemplary images are shown (n = 3) (H) Quantification of the data exemplified in (G). Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment; p values calculated using a paired, two-sided Student's t-test are indicated


( A ) mIMCD - 3 stably expressing cilia - bPAC , cultured in a 3D matrix in or during exposure ( 1 h / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² ) incubated with DMSO ( control ) , 250 µM IBMX , a ubiquitous PDE inhibitor , or 10 µM Rolipram , a PDE4 - specific inhibitor . Exemplary are shown ( n = 3 ) . Scale bars are indicated . ( B ) of data exemplified in ( A ) . Data are shown as mean ± S . D . , p values calculated using an unpaired , two - sided Student ' s t - are indicated . Data points show experiments .

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(A) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark or during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubated with DMSO (control), 250 µM IBMX, a ubiquitous PDE inhibitor, or 10 µM Rolipram, a PDE4-specific inhibitor. Exemplary images are shown (n = 3). Scale bars are indicated. (B) Quantification of data exemplified in (A). Data are shown as mean ± S.D., p values calculated using an unpaired, two-sided Student's t-test are indicated. Data points show individual experiments.


( C ) mIMCD - 3 stably expressing cyto - bPAC , cultured in a 3D matrix during exposure ( 1 h / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² , 1 day after pharmacological stimulus ) incubation with DMSO or 10 µM rolipram . Exemplary are shown ( n = 4 - 5 ) . Scale bars are indicated . ( D ) of data exemplified in ( C ) Data are shown as mean ± S . D . , p values calculated using an unpaired Student ' s t - are indicated . Data points show experiments .

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(C) mIMCD-3 cells stably expressing cyto-bPAC, cultured in a 3D matrix during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO or 10 µM rolipram. Exemplary images are shown (n = 4-5). Scale bars are indicated. (D) Quantification of data exemplified in (C) Data are shown as mean ± S.D., p values calculated using an unpaired Student's t-test are indicated. Data points show individual experiments.


( F ) of Pde4 isoform expression ( long short isoforms ) in wild - type mIMCD - 3 , revealed by immunoblotting against PDE4A , PDE4B , PDE4D , PDE4D .

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(F) Analysis of Pde4 isoform expression (long and short isoforms) in wild-type mIMCD-3 cells, revealed by immunoblotting against PDE4A, PDE4B, PDE4D, and PDE4D.


( H ) mIMCD - 3 stably expressing cilia - bPAC , cultured in a 3D matrix during exposure ( 1 h / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² , 1 day after pharmacological stimulus ) incubation with DMSO ( 0 µM ) , 3 µM , or 10 µM of Pde4 - long - isoform activator MR - L8 . Exemplary are shown ( n = 3 ) . Scale bars are indicated . ( I ) of data exemplified in ( H ) . Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - are indicated . Data have been normalized to , 0 µM MR - L8 condition ( Set to 100 % ) within each experiment . Data points show experiments .

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(H) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO (0 µM), 3 µM, or 10 µM of the Pde4-long-isoform activator MR-L8. Exemplary images are shown (n = 3). Scale bars are indicated. (I) Quantification of data exemplified in (H). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data have been normalized to the Light, 0 µM MR-L8 condition (Set to 100%) within each experiment. Data points show individual experiments.


( D ) 3D culture of mIMCD - 3 WT continuously exposed to acetone ( control ) or 100 nM PGE2 . Exemplary are shown ( n = 3 biological replicates ) . ( E ) of exemplified in ( D ) . Data are shown as mean ± S . D . , p values calculated using an unpaired , two - sided Student ' s t - are indicated . Data points show biological replicates . ( F ) of cyst in PGE2 - stimulated mIMCD - 3 WT in presence of PDE4 long isoform - specific activator MR - L8 . Data were normalized to PGE2 - control - treated are shown as mean ± S . D . p values calculated using a one - sample t - compared to 100 % are indicated , n = 4 biological replicates .

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(D) 3D culture of mIMCD-3 WT cells continuously exposed to acetone (control) or 100 nM PGE2. Exemplary images are shown (n = 3 biological replicates). (E) Quantification of images exemplified in (D). Data are shown as mean ± S.D., p values calculated using an unpaired, two-sided Student's t-test are indicated. Data points show individual biological replicates. (F) Quantification of the cyst area in PGE2-stimulated mIMCD-3 WT cells in the presence of the PDE4 long isoform-specific activator MR-L8. Data were normalized to PGE2- and control-treated cells and are shown as mean ± S.D. p values calculated using a one-sample t-test compared to 100% are indicated, n = 4 biological replicates.


( G ) 3D culture of mIMCD - 3 WT continuously exposed to acetone ( control ) or 100 nM PGE2 , DMSO ( control ) or 10 µM of EP4 inhibitor L161 , 982 . PGE2 treatment started 2 d after seeding . Exemplary are shown ( n = 3 ) . Scale bars are indicated . ( H ) for exemplified in ( G ) , including addition of 3 µM AH6809 , an EP2 inhibitor Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - are indicated ( p value of n / a indicates that is not applicable because both conditions showed no variance ) . Data points show experiments have been normalized to addition of 100 nM PGE2 ( set to 100 % ) within each experiment .

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(G) 3D culture of mIMCD-3 WT cells continuously exposed to acetone (control) or 100 nM PGE2, and DMSO (control) or 10 µM of the EP4 inhibitor L161,982. PGE2 treatment started 2 d after seeding. Exemplary images are shown (n = 3). Scale bars are indicated. (H) Quantification for images exemplified in (G), including addition of 3 µM AH6809, an EP2 inhibitor Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated (p value of n/a indicates that the test is not applicable because both conditions showed no variance). Data points show individual experiments and have been normalized to addition of 100 nM PGE2 (set to 100%) within each experiment.


( I ) 3D culture of mIMCD - 3 stably expressing cilia - bPAC , incubated with DMSO ( control ) or EP4 - inhibitor L161 , 982 in or during exposure ( 1 / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² , started 2 d after pre - incubation with DMSO or L161 , 982 ) . Exemplary are shown ( n = 3 ) . ( J ) of exemplified in ( I ) . Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - are indicated . Data points show experiments have been normalized to / DMSO condition ( Set to 100 % ) within each experiment .

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(I) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO (control) or the EP4-inhibitor L161,982 in the dark or during light exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO or L161,982). Exemplary images are shown (n = 3). (J) Quantification of images exemplified in (I). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments and have been normalized to the light/DMSO condition (Set to 100%) within each experiment.


( F ) 3D culture of mIMCD - 3 stably expressing cilia - bPAC , incubated with DMSO ( control ) or 20 µM Celecoxib , a COX - 2 - inhibitor , in or during exposure ( 1 / 1 h , 9 days , 465 nm , 38 . 8 µW / cm² , started 2 d after pre - incubation with DMSO or Celecoxib ) . Exemplary are shown ( n = 3 ) . Scale bars are indicated . ( G ) of exemplified in ( F ) from 3D cultures incubated with 1 μM celecoxib . Data are shown as mean ± S . D . , p values calculated using a paired , two - sided Student ' s t - are indicated . Data points show experiments .

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(F) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO (control) or 20 µM Celecoxib, a COX-2-inhibitor, in the dark or during light exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO or Celecoxib). Exemplary images are shown (n = 3). Scale bars are indicated. (G) Quantification of images exemplified in (F) and from 3D cultures incubated with 1 μM celecoxib. Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments.


( A ) Wild - type ( WT ) , cilia - bPAC , or cyto - bPAC ( magenta ) mIMCD - 3 were stimulated by ( for 1 h prior to fixation , 465 nm , 38 . 8 µW / cm² ) , treated with DMSO ( control ) or 50 µM Ciliobrevin - D , a dynein - inhibitor . were labeled with DAPI ( blue ) to label , an ARL13B antibody ( ) to label cilia , a phospho - specific ( Ser133 ) CREB antibody ( pCREB , green ) . Arrows indicate direction of shift of respective channel . box indicates position of magnified view shown on right of each panel . B ) of ciliary pCREB signal in cilia - bPAC cyto - bPAC . Data are shown as mean ± S . D . , n > 3 . Each data point represents an independent experiment corresponds to median of > 73 cilia ( biological replicates ) ; p values for an unpaired , two - sided Student ' s t - with Welch ' s correction are indicated .

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(A) Wild-type (WT), cilia-bPAC, or cyto-bPAC (magenta) mIMCD-3 cells were stimulated by light (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²), and treated with DMSO (control) or 50 µM Ciliobrevin-D, a dynein-inhibitor. Cells were labeled with DAPI (blue) to label the DNA, an ARL13B antibody (white) to label cilia, and a phospho-specific (Ser133) CREB antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel. B) Quantification of the ciliary pCREB signal in cilia-bPAC and cyto-bPAC cells. Data are shown as mean ± S.D., n > 3. Each data point represents an independent experiment and corresponds to the median of > 73 cilia (biological replicates); p values for an unpaired, two-sided Student's t-test with Welch's correction are indicated.


( C ) mIMCD - 3 Ift27 - / - with or without stable cilia - bPAC expression ( magenta ) were stimulated with ( for 1 h prior to fixation , 465 nm , 38 . 8 µW / cm² ) . were labeled with DAPI ( blue ) to label , an ARL13B antibody ( ) to label cilia , a phospho - specific ( Ser133 ) CREB antibody ( pCREB , green ) . Arrows indicate direction of shift of respective channel . box indicates position of magnified view shown on right of each panel . ( D ) of ciliary pCREB signal . Data are shown as mean ± S . D . , n > 3 . Each data point represents median of > 90 cilia from same experiment ; p values are indicated for an unpaired , two - sided Student ' s t - with Welch ' s correction .

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(C) mIMCD-3 Ift27-/- cells with or without stable cilia-bPAC expression (magenta) were stimulated with light (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²). Cells were labeled with DAPI (blue) to label the DNA, an ARL13B antibody (white) to label cilia, and a phospho-specific (Ser133) CREB antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel. (D) Quantification of the ciliary pCREB signal. Data are shown as mean ± S.D., n > 3. Each data point represents the median of > 90 cilia from the same experiment; p values are indicated for an unpaired, two-sided Student's t-test with Welch's correction.


B . Validation of TNF - RSC interacting . A549 were stimulated with FLAG - TNF - α for indicated time TNF - RSCs were isolated as described in Figure 3A . TNF - α - stimulation - dependent interaction of indicated was confirmed by isolating TNF - RSC blotting with indicated antibodies . amount of analyzed in input material used for TNF - RSC isolation is also shown .

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B. Validation of the TNF-RSC interacting proteins. A549 cells were stimulated with FLAG-TNF-α for the indicated time and the TNF-RSCs were isolated as described in Figure 3A. TNF-α-stimulation-dependent interaction of the indicated proteins was confirmed by isolating the TNF-RSC and blotting with the indicated antibodies. The amount of the analyzed proteins in the input material used for the TNF-RSC isolation is also shown.


C . of ubiquitin linkage peptides in TNF - RSC . box plot shows relative abundance of K48 , K63 linear ubiquitin linkage - specific peptides in TNF - RSCs isolated from TNF - α - stimulated ( 15 min ) control quantified by SILAC - MS .

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C. Quantification of ubiquitin linkage peptides in the TNF-RSC. The box plot shows the relative abundance of K48, K63 and linear ubiquitin linkage-specific peptides in the TNF-RSCs isolated from TNF-α-stimulated (15 min) and control cells quantified by SILAC-based MS.


E . list of enriched in SPATA2pull - down . table shows that were enriched in GFP - SPATA2pull - down . columns under " Unstimulated " shows SILAC ratios of indicated in SPATA2 pull - downs from unstimulated , whereas columns under " Stimulated ( TNF - α 15 min ) " shows SILAC ratios of indicated in SPATA2 pull - downs from stimulated with TNF - α for 15 minutes .

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E. The list of proteins enriched in SPATA2pull-down. The table shows proteins that were enriched in GFP-SPATA2pull-down. The columns under "Unstimulated" shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from unstimulated cells, whereas the columns under "Stimulated (TNF-α 15 min)" shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from cells stimulated with TNF-α for 15 minutes.


F . Validation of TNF - α - stimulation - independent association of SPATA2 with CYLD . GFP - SPATA2 was immunoprecipitated from unstimulated TNF - α - stimulated , probed with CYLD antibody .

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F. Validation of TNF-α-stimulation-independent association of SPATA2 with CYLD. GFP-SPATA2 was immunoprecipitated from unstimulated and TNF-α-stimulated cells, and probed with CYLD antibody.


G . HOIP interacts with SPATA2 . FLAG - HOIP expressing A20 . 2J were SILAC labeled FLAG - HOIP was immunoprecipitated from unstimulated associated were quantified using LC - MS / MS . table contains that were enriched in HOIPpull - down compared to control pull - down .

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G. HOIP interacts with SPATA2. FLAG-HOIP expressing A20.2J cells were SILAC labeled and FLAG-HOIP was immunoprecipitated from unstimulated cells and associated proteins were quantified using LC-MS/MS. The table contains proteins that were enriched in HOIPpull-down compared to control pull-down.


H . Validation of SPATA2 interaction with HOIP . GFP - tagged RNF31 was purified from unstimulated or TNF - a - stimulated ( 15 min ) HCT116 immunoprecipitates were blotted with SPATA2 , CYLD GFP ( HOIP ) antibodies . lower panels show expression of indicated in input . asterisk indicates unspecific band .

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H. Validation of SPATA2 interaction with HOIP. GFP-tagged RNF31 was purified from unstimulated or TNF-a-stimulated (15 min) HCT116 cells and the immunoprecipitates were blotted with SPATA2, CYLD and GFP (HOIP) antibodies. The lower panels show expression of the indicated proteins in the input. The asterisk indicates unspecific band.


B . predicted PUB domain in N - terminus of SPATA2 interacts with CYLD , but not with LUBAC or other TNF - RSC components . GFP - SPATA2 indicated deletion mutants were purified from A549 that were stimulated with TNF - α for 15 minutes , enriched were quantified using SILAC - MS .

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B. The predicted PUB domain in the N-terminus of SPATA2 interacts with CYLD, but not with LUBAC or other TNF-RSC components. GFP-SPATA2 and the indicated deletion mutants were purified from A549 cells that were stimulated with TNF-α for 15 minutes, and the enriched proteins were quantified using SILAC-based MS.


C . Validation of SPATA2 N - terminus interaction with CYLD . GFP - SPATA2 indicated deletion mutants were purified from A549 as described in Figure 5A , association of CYLD was analyzed by immunoblotting . lower panels show controls .

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C. Validation of SPATA2 N-terminus interaction with CYLD. GFP-SPATA2 and the indicated deletion mutants were purified from A549 cells as described in Figure 5A, and association of CYLD was analyzed by immunoblotting. The lower panels show controls.


D . USP domain of CYLD interacts with SPATA2 . STREP - FLAG - CYLD indicated deletion mutants were purified from A549 using Step - Tactin sepharose blotted with SPATA2 antibody . lower panels show controls .

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D. The USP domain of CYLD interacts with SPATA2. STREP-FLAG-CYLD and the indicated deletion mutants were purified from A549 cells using Step-Tactin sepharose and blotted with SPATA2 antibody. The lower panels show controls.


E . C - terminus of SPATA2 interacts with HOIP . A549 were transiently transfected with GFP - tagged SPATA2 , SPATA2 D200 SPATA2 200 * , SPATA2 mutants were immunoprecipitated using GFP - trap beads . immunoprecipitates were immunoblotted with HOIP , CYLD GFP ( HOIP ) antibodies . lower panels show input control , * shows unspecific band .

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E. The C-terminus of SPATA2 interacts with HOIP. A549 cells were transiently transfected with GFP-tagged SPATA2, SPATA2 D200 and SPATA2 200*, and SPATA2 and the mutants were immunoprecipitated using GFP-trap beads. The immunoprecipitates were immunoblotted with HOIP, CYLD and GFP (HOIP) antibodies. The lower panels show input control, and the * shows unspecific band.


B . Effect of SPATA2 knockdown on association of with TNF - RSC . list shows SILAC ratios of TNF - RSC - associated in control SPATA2 knockdown . that showed reduced association with TNF - RSC after SPATA2 knockdown are highlighted red .

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B. Effect of SPATA2 knockdown on association of proteins with TNF-RSC. The list shows SILAC ratios of TNF-RSC-associated proteins in control cells and SPATA2 knockdown cells. The proteins that showed reduced association with the TNF-RSC after SPATA2 knockdown are highlighted red.


C . Validation of SPATA2 - dependent association of CYLD with TNF - RSC . TNF - RSC was purified under indicated conditions association of SPATA2 , CYLD , RIPK1 HOIP with TNF - RSC was analyzed by immunoblotting . input controls show expression of analyzed in lysates . asterisk denotes unspecific bands .

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C. Validation of SPATA2-dependent association of CYLD with the TNF-RSC. The TNF-RSC was purified under the indicated conditions and association of SPATA2, CYLD, RIPK1 and HOIP with the TNF-RSC was analyzed by immunoblotting. The input controls show expression of the analyzed proteins in cell lysates. The asterisk denotes unspecific bands.


A . SPATA2 regulates TNF - a - induced NF - κB activation . HEK293T were transfected with SPATA2 siRNA ( SMARTpool or with to siRNA ) or a non - targeting control siRNA , NF - kB activation was monitored with luciferase - NF - kB reporter assays . Error bars specify standard error of mean ( ) of three independent experiments . p * * < 0 . 05 ( two - , unpaired Student ´ s t - ) .

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A. SPATA2 regulates TNF-a-induced NF-κB activation. HEK293T cells were transfected with the SPATA2 siRNA (SMARTpool or with to individual siRNA) or a non-targeting control siRNA, and NF-kB activation was monitored with luciferase-based NF-kB reporter assays. Error bars specify the standard error of the mean (SEM) of three independent experiments. p** < 0.05 (two-tailed, unpaired Student´s t-test).


B . Knockdown of SPATA2 enhances expression of NF - κB target genes . HEK293T were transfected with SPATA2 siRNA or a non - targeting control siRNA expression of indicated mRNAs was measured in stimulated with TNF - a for 0 , 3 6 hours with qPCR .

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B. Knockdown of SPATA2 enhances expression of the NF-κB target genes. HEK293T cells were transfected with SPATA2 siRNA or a non-targeting control siRNA and the expression of the indicated mRNAs was measured in cells stimulated with TNF-a for 0, 3 and 6 hours with qPCR.


C . SPATA2 is involved in TNF - α - induced necroptosis . SPATA2 was knocked down in L929 using RNAi , were pretreated with DMSO , Z - VAD , or Z - VAD + NEC1 for 1 hour followed by treatment with TNF - α for 2 . 5 hours . viability was assessed with propidium iodide flow cytometry . Knockdown of SPATA2 mRNA was verified using real time PCR . p * * < 0 . 05 ( two - , unpaired Student ´ s t - ) .

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C. SPATA2 is involved in TNF-α-induced necroptosis. SPATA2 was knocked down in L929 cells using RNAi, and cells were pretreated with DMSO, Z-VAD, or Z-VAD+NEC1 for 1 hour followed by treatment with TNF-α for 2.5 hours. Cell viability was assessed with propidium iodide staining and flow cytometry. Knockdown of SPATA2 mRNA was verified using real time PCR. p** < 0.05 (two-tailed, unpaired Student´s t-test).


D . Knockdown of SPATA2 reduces phosphorylation of MLKL in TNF - α - induced necroptosis . TNF - α - dependent necroptosis was induced in L929 as described in Figure 6C . Phosphorylation of MLKL was analyzed using S358 phospho - specific antibody .

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D. Knockdown of SPATA2 reduces phosphorylation of MLKL in TNF-α-induced necroptosis. TNF-α-dependent necroptosis was induced in L929 as described in Figure 6C. Phosphorylation of MLKL was analyzed using S358 phospho-specific antibody.


( a ) Representative 2D μ - CT scans of RasV12 ; ctrl at day 6 , RasV12 ; scrib - / - larvae at day 6 day 10 . Muscle ( green ) , fat ( blue ) eye - antennal discs / ( red ) are outlined . Scale bar : 1mm . Anterior ( A ) , Right ( R ) .

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(a) Representative 2D μ-CT scans of RasV12; ctrl at day 6, RasV12; scrib-/- larvae at day 6 and day 10. Muscle (green), fat body (blue) and eye-antennal discs/tumor (red) are outlined.Scale bar: 1mm. Anterior (A), Right (R).


( a ) of growth of RasV12 ; ctrl day 6 ( n = 15 ) RasV12 ; scrib - / - tumors , day 6 ( n = 15 ) , day 7 ( n = 5 ) , day 8 ( n = 4 ) , day 9 ( n = 5 ) day 10 ( n = 5 ) .

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(a) Quantification of tumor growth of RasV12; ctrl day 6 (n=15) RasV12; scrib-/- tumors, day 6 (n=15), day 7 (n=5), day 8 (n=4), day 9 (n=5) and day 10 (n=5).


( b ) Quantifications of muscle volume , of larvae carrying tumors of RasV12 ; ctrl ( n = 15 ) , RasV12 ; scrib - / - at day 6 ( n = 15 ) , day 7 ( n = 5 ) , day 8 ( n = 5 ) , day 9 ( n = 5 ) dat 10 ( n = 5 ) .

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(b) Quantifications of muscle volume, of larvae carrying tumors of RasV12; ctrl (n=15), and RasV12; scrib-/- at day 6(n=15), day 7 (n=5), day 8 (n=5), day 9 (n=5) and dat 10 (n=5).


( c ) of width of Dorsal Oblique 3 ( DO3 ) muscle in larvae carrying tumors of RasV12 ; ctrl at day 6 ( n = 29 ) RasV12 ; scrib - / - at days 6 ( n = 29 ) day 10 ( n = 31 ) .

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(c) Quantification of width of Dorsal Oblique 3 (DO3) muscle in larvae carrying tumors of RasV12; ctrl at day 6 (n=29) and RasV12; scrib-/-at days 6 (n=29) and day 10 (n=31).


( d ) of adipose tissue volume in larvae carrying tumors of RasV12 ; ctrl at day 6 RasV12 ; scrib - / - at days 6 day 10 .

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(d) Quantification of adipose tissue volume in larvae carrying tumors of RasV12; ctrl at day 6 and RasV12; scrib-/-at days 6 and day 10.


( e ) Representative confocal of adipose tissue in RasV12 ; ctrl RasV12 ; scrib - / - tumors bearing at indicated ages . Lipid droplets are highlighted with Lipid Tox . Scale bar :

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(e) Representative confocal images of adipose tissue in RasV12; ctrl and RasV12; scrib-/- tumors bearing animals at indicated ages. Lipid droplets are highlighted with Lipid Tox staining. Scale bar: 50μm


( f ) of dry of RasV12 ; ctrl ( n = 9 ) , RasV12 ; scrib - / - day 6 ( n = 15 ) day 10 ( n = 18 ) bearing larvae excluding .

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(f) Quantification of dry weight of RasV12; ctrl (n=9), RasV12; scrib-/- day 6 (n=15) and day 10 (n=18) tumor bearing larvae excluding the tumor weight.


( g ) of larval motility measure by crawling crawling pattern in RasV12 ; ctrl ( n = 14 ) , RasV12 ; scrib - / - day 6 ( n = 15 ) , day 8 ( n = 16 ) day 10 ( n = 14 ) , each colored represents a single larva .

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(g) Quantification of larval motility measure by crawling distance and crawling pattern in RasV12; ctrl (n=14), RasV12; scrib-/- day 6 (n=15), day 8 (n=16) and day 10 (n=14), each colored line represents a single larva.


( h ) Coomassie feeding to asses food intake of RasV12 ; scrib - / - over time , three repeated measurements of an average food intake of 20 larvae .

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(h) Coomassie feeding assay to asses food intake of RasV12; scrib-/- over time, three repeated measurements of an average food intake of 20 larvae.


( a - e ) Cartoon ( top ) illustrates genotypes of larvae , eye - antennal disc ( EAD , circle : in green microenvironment in black ) , systemic are illustrated as a square ( wild type in brown atg13 mutant in orange ) at indicated ages . Cartoon ( left ) illustrates structure of cephalic attached to mouth hook . Representative of Larva ( image of larva using backlight of microscope ) , cephalic ( green highlights GFP - labelled clones ) , muscle ( Phalloidin in green stains actin Hoechst in blue stains nucleus ) adipose tissue ( Lipid Tox in red stains for lipid droplets , Hoechst stains nucleus ) from top to bottom . ( a ) RasV12 ; ctrl bearing at day 6 . ( b ) RasV12 ; scrib - / - bearing at day 6 . ( c ) RasV12 ; scrib - / - bearing at day 8 . ( d ) RasV12 , scrib - / - , atg13 - / - / / atg13 - / - at day 8 . ( e ) RasV12 , scrib - / - , atg13 - / - / / atg13 - / - complemented with transgenic atg13 , rescuing growth , at day 8 .

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(a-e) Cartoon (top) illustrates the genotypes of the larvae, eye-antennal disc (EAD, circle: tumor cells in green and microenvironment cell in black), systemic cells are illustrated as a square (wild type cells in light brown and atg13 mutant cells in light orange) at indicated ages. Cartoon (left) illustrates the structure of cephalic complex attached to mouth hook. Representative images of Larva (image of larva using the backlight of microscope), cephalic complex (green highlights the GFP-labelled tumor clones), muscle (Phalloidin in green stains actin and Hoechst in blue stains nucleus) and adipose tissue (Lipid Tox in red stains for lipid droplets, Hoechst stains nucleus) from top to bottom. (a) RasV12; ctrl tumor bearing animal at day 6. (b) RasV12; scrib-/- tumor bearing animal at day 6. (c) RasV12; scrib-/- tumor bearing animal at day 8. (d) RasV12, scrib-/-, atg13-/-//atg13-/- at day 8. (e) RasV12, scrib-/-, atg13-/-//atg13-/- animal complemented with transgenic atg13, rescuing the tumor growth, at day 8.


( f ) of of space occupied with lobes of fat within larval segments 4 to 8 ( shown in yellow dashed in 2a ) , of bearing tumors at day 6 RasV12 ; ctrl ( n = 45 ) , RasV12 ; scrib - / - ( n = 30 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 30 ) ey3 . 5 - atg13 ; RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 29 ) at day 10 RasV12 ; scrib - / - ( n = 40 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 25 ) ey3 . 5 - atg13 ; RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 28 ) .

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(f) Quantification of area of space occupied with lobes of fat body within larval segments 4 to 8 (shown in yellow dashed line in 2a), of animals bearing tumors at day 6 RasV12; ctrl (n=45), RasV12; scrib-/- (n=30), RasV12, scrib-/-, atg13-/-//atg13-/- (n=30) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=29) and at day 10 RasV12; scrib-/- (n=40), RasV12, scrib-/-, atg13-/-//atg13-/- (n=25) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=28).


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(g) Quantification of tumor volumes day 6 RasV12; ctrl (n=7), RasV12; scrib-/- (n=11) and day 8 RasV12; scrib-/- (n=13), RasV12, scrib-/-, atg13-/-//atg13-/- (n=9) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=9).


( h ) of Ventral Longitudinal muscle 4 ( VL4 ) ( shown in yellow dashed in 2a ) of third segment of larvae carrying at day 6 RasV12 ; ctrl ( n = 15 ) , RasV12 ; scrib - / - ( n = 14 ) , at day8 RasV12 ; scrib - / - ( n = 11 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 12 ) ey3 . 5 - atg13 ; RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - ( n = 14 ) .

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(h) Quantification of Ventral Longitudinal muscle 4 (VL4) (shown in yellow dashed line in 2a) of third segment of larvae carrying at day 6 RasV12; ctrl (n=15), RasV12; scrib-/- (n=14), and at day8 RasV12; scrib-/- (n=11), RasV12, scrib-/-, atg13-/-//atg13-/- (n=12) and ey3.5-atg13; RasV12; scrib-/-, atg13-/-//atg13-/- (n=14).


( i ) of dry excluding of RasV12 ; scrib - - , atg13 - / - / / atg13 - / - at day 6 ( n = 8 ) day 10 ( n = 7 ) .

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(i) Quantification of dry weight excluding tumor of RasV12; scrib- -, atg13-/-//atg13-/-at day 6 (n=8) and day 10 (n=7).


( j ) of larval motility measured by crawling crawling pattern for RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - at day 6 ( n = 15 ) at day 8 ( n = 15 ) , each colored represents a single larva .

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(j) Quantification of larval motility measured by crawling distance and crawling pattern for RasV12; scrib-/-, atg13-/-//atg13-/- at day 6 (n=15) and at day 8 (n=15), each colored line represents a single larva.


( a ) Changes in groups ( carbohydrates , fatty ) of storage metabolites measured in circulating haemolymph with progressing wasting , shown as log2 changes measured by LC - MS calculated per larvae relative to RasV12 ; ctrl at day 6 .

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(a) Changes in groups (carbohydrates, amino acids and fatty acids) of storage metabolites measured in circulating haemolymph with progressing wasting, shown as log2 changes measured by LC-MS and calculated per larvae relative to RasV12; ctrl at day 6.


( b ) Volcano plot showing autophagy - dependent changes to amounts of circulating metabolites at day 6 . X - axis shows log2 of fold change of RasV12 ; atg13 - / - scrib - / - / / atg13 - / - vs . RasV12 ; scrib - / - , y - axis shows - log10 p - value , calculated by t - . Metabolite names are shown for metabolites with log2 ( FC ) > ± 1 / or - log10 ( P ) < 2 . Green points indicate log2 ( FC ) > ± 1 , blue indicate - log10 ( P ) < 2 red indicates for above both thresholds .

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(b) Volcano plot showing autophagy-dependent changes to amounts of circulating metabolites at day 6. X-axis shows the log2 of fold change of RasV12;atg13 -/-scrib-/- // atg13 -/- vs. RasV12; scrib-/- , y-axis shows -log10 p-value, calculated by t-test. Metabolite names are shown for metabolites with log2(FC) >± 1 and/or -log10(P) < 2. Green points indicate log2(FC) >± 1, blue indicate -log10(P) < 2 and red indicates for above both thresholds.


( c ) Autophagy - driven wasting releases metabolites into circulation of 113 reliably detected metabolites , those showing significant differences in any of three comparisons are shown . Color indicates log2 ( fold change ) difference numbers show p - value of comparison . statistical to define significance was FDR - adjusted t - p - value < 0 .

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(c) Autophagy-driven wasting releases metabolites into circulation of 113 reliably detected metabolites, those showing significant differences in any of the three comparisons are shown. Color indicates the log2(fold change) difference and the numbers show the p-value of the comparison. The statistical test to define significance was FDR-adjusted t-test p-value < 0.05


( a ) amount of glycogen in whole larvae measured by biochemical ( n = 4 ) , normalized to RasV12 ; ctrl at day 6 , per number of larvae .

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(a) The amount of glycogen in the whole larvae measured by biochemical assay (n=4), normalized to RasV12; ctrl at day 6, and per number of larvae.


( b - e ) Representative confocal of muscle adipose tissue of larvae carrying RasV12 ; ctrl , RasV12 ; scrib - / - RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - showing glycogen ( ) at day 6 day 8 .

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(b-e) Representative confocal images of muscle and adipose tissue of larvae carrying RasV12; ctrl, RasV12; scrib-/- and RasV12; scrib-/-, atg13-/-//atg13-/- showing glycogen levels (white) at day 6 and day 8.


( g ) Sources of carbon incorporated into biomass were differentiated by changing isotopic carbon content of food source 25 hours before measuring total carbon content isotopic of . A similar , but independent experiment is reported in Holland P . , et al . , 2021 BMC Biol .

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(g) Sources of carbon incorporated into tumor biomass were differentiated by changing the isotopic carbon content of the food source 25 hours before measuring the total carbon content and isotopic ratio of the tumor. A similar, but independent experiment is reported in Holland P., et al., 2021 BMC Biol.


HSATIII lncRNA - dependent localization of m6A - related factors . Control HSATIII knockdown HeLa were exposed to thermal stress ( 42 ° C for 2 h recovery for 1 h at 37 ° C ) stained by HSATIII - FISH immunofluorescence using an anti - WTAP antibody ( B ) or anti - YTHDC1 antibody ( C ) . nuclei were stained with DAPI . Scale bar : 10 µm .

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HSATIII lncRNA-dependent localization of m6A-related factors. Control and HSATIII knockdown HeLa cells were exposed to thermal stress (42°C for 2 h and recovery for 1 h at 37°C) and stained by HSATIII-FISH and immunofluorescence using an anti-WTAP antibody (B) or anti-YTHDC1 antibody (C). The nuclei were stained with DAPI. Scale bar: 10 µm.


YTHDC1 accumulation within nSBs was arrested by METTL3 knockdown . HeLa were stained as described in Figure 1C . Scale bar : 10 µm . Box plot of relative intensities of YTHDC1 to HSATIII within nSBs in nuclei ( n = 30 ) . mean is indicated with X . first third quartiles are ends of box , median is indicated with a vertical in box , minimum maximum except for outliers are ends of whiskers . outliers are indicated with open circles . P - values ( Kruskal - Wallis , followed by Dunn ' s multiple comparison ) are shown above .

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YTHDC1 accumulation within nSBs was arrested by METTL3 knockdown. HeLa cells were stained as described in Figure 1C. Scale bar: 10 µm. Box plot of relative intensities of YTHDC1 to HSATIII within nSBs in individual nuclei (n=30). The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above.


- MS of HSATIII lncRNAs digested by RNase T1 . Base peak chromatogram ( BPC , top panel ) extracted chromatograms ( XIC , lower panels ) for RNase T1 - digested fragments of isolated HSATIII lncRNAs . XICs detecting for negatively - charged ions of AAUGp tetramer without modification ( second panel ) , with one methylation ( third panel ) , two methylations ( bottom panel ) . , mass to charge ( m / z ) charge state are indicated . NL ; normalization level .

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RNA-MS analysis of HSATIII lncRNAs digested by RNase T1. Base peak chromatogram (BPC, top panel) and extracted chromatograms (XIC, lower panels) for the RNase T1-digested fragments of the isolated HSATIII lncRNAs. XICs detecting for the negatively-charged ions of AAUGp tetramer without modification (second panel), with one methylation (third panel), and two methylations (bottom panel) . The sequence, mass to charge ratio (m/z) and charge state are indicated. NL; normalization level.


Changes of nSB localization of m6A - related factors at various time points during after thermal stress exposure . HeLa were exposed to thermal stress ( 42 ° C for 2 h ) cultured at 37 ° C for indicated periods . m6A - related factors were visualized by HSATIII - FISH IF using anti - WTAP antibody ( D ) or anti - YTHDC1 antibody ( F ) . Nuclei were stained with DAPI . Scale bar : 10 µm . ( E , G ) Box plot of relative intensities of WTAP ( D ) YTHDC1 ( F ) to HSATIII within nSB areas in nuclei . nSB areas were defined by binarized of HSATIII - FISH . mean is indicated with X . first third quartiles are ends of box , median is indicated with a vertical in box , minimum maximum except for outliers are ends of whiskers . outliers are indicated with open circles . P - values ( Kruskal - Wallis , followed by Dunn ' s multiple comparison ) are shown above ( n = 30 ) .

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Changes of nSB localization of m6A-related factors at various time points during and after thermal stress exposure. HeLa cells were exposed to thermal stress (42°C for 2 h) and cultured at 37°C for the indicated periods. m6A-related factors were visualized by HSATIII-FISH and IF using anti-WTAP antibody (D) or anti-YTHDC1 antibody (F). Nuclei were stained with DAPI. Scale bar: 10 µm. (E, G) Box plot of relative intensities of WTAP (D) and YTHDC1 (F) to HSATIII within nSB areas in individual nuclei. nSB areas were defined by binarized images of HSATIII-FISH. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above (n=30).


HSATIII dependent sequestration of nucleoplasmic YTHDC1 ( G ) WTAP ( H ) at four time points during thermal stress recovery . experiments same as A C were performed at indicated timepoints . mean is indicated with X . first third quartiles are ends of box , median is indicated with a vertical in box , minimum maximum except for outliers are ends of whiskers . outliers are indicated with open circles . P - values ( Kruskal - Wallis , followed by Dunn ' s multiple comparison ) are shown above ( n = 40 ) .

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HSATIII dependent sequestration of nucleoplasmic YTHDC1 (G) and WTAP (H) at four time points during thermal stress and recovery. The experiments same as A and C were performed at the indicated timepoints. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above (n=40).


Scatter plot of LogFC of whole introns ( grey ) HSATIII - target introns ( blue ) upon SRSF9 KD ( X - axis ) HSATIII KD ( Y - axis ) . 51 , 469 548 introns are plotted as whole introns previously - reported HSATIII - target introns , respectively . Pearson ' s correlation ( r ) is indicated above .

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Scatter plot of LogFC of whole introns (grey) and HSATIII-target introns (blue) upon SRSF9 KD (X-axis) and HSATIII KD (Y-axis). 51,469 and 548 introns are plotted as whole introns and previously-reported HSATIII-target introns, respectively. Pearson's correlation (r) is indicated above.


RT - qPCR validation of splicing of nascent RNAs . Splicing efficiency of nascent RNAs was quantified as of spliced form to intron retaining isoform ( IR ) form normalized to control . Control siRNA HSATIII control oligonucleotide are indicated with ( - ) . Data are shown as mean ± SD ( n = 3 ) ; p - values ( Tukey ' s multiple comparison ) are shown above .

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RT-qPCR validation of splicing of nascent RNAs. Splicing efficiency of nascent RNAs was quantified as the ratio of the spliced form to the intron retaining isoform (IR) form and normalized to the control. Control siRNA and HSATIII control oligonucleotide are indicated with (-). Data are shown as the mean ± SD (n=3); p-values (Tukey's multiple comparison test) are shown above.


Read maps represent - seq data of poly ( A ) + RNAs ( upper panel , biological triplicate ) m6A - RIP RNAs ( lower panel , biological duplicate ) in control HSATIII KD . exon numbers of FAM214A gene are indicated below . retaining intron m6A peak are marked by a red arrow , respectively . For entire FAM214A region , see also Figure EV4B .

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Read maps represent RNA-seq data of nuclear poly (A)+ RNAs (upper panel, biological triplicate) and m6A-RIP RNAs (lower panel, biological duplicate) in control and HSATIII KD cells. The exon numbers of the FAM214A gene are indicated below. The retaining intron and the m6A peak are marked by a red line and arrow, respectively. For the entire FAM214A region, see also Figure EV4B.


In vitro pulldown of RNP complexes with various repeat RNAs . In vitro pulldown was performed using biotinylated ( GGAAU ) × 20 other three repeat RNAs incubated in HeLa extract . co - precipitated were detected by western blotting using specific antibodies . ALYREF , a non - specific - binding , was used as a control . of data in A . Relative co - precipitation efficiency of each was normalized by maximum value . Data are shown as mean ± SD ( n = 3 ) .

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In vitro pulldown assay of RNP complexes with various repeat RNAs. In vitro RNA pulldown was performed using biotinylated (GGAAU)×20 and other three repeat RNAs incubated in HeLa nuclear extract. The co-precipitated proteins were detected by western blotting using specific antibodies. ALYREF, a non-specific RNA-binding protein, was used as a control. Quantification of data in A. Relative co-precipitation efficiency of each protein was normalized by the maximum value. Data are shown as the mean ± SD (n=3).


In vitro pulldown with methylated GGAAU repeat . In vitro pulldown was performed using biotinylated GGAAU GGm6Am6AU repeat RNAs as in A . of data in C . Relative co - precipitation efficiency of each was normalized by maximum value . Data are shown as mean ± SD ( n = 3 ) .

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In vitro pulldown assay with methylated GGAAU repeat RNA. In vitro RNA pulldown assay was performed using biotinylated GGAAU and GGm6Am6AU repeat RNAs as in A. Quantification of data in C. Relative co-precipitation efficiency of each protein was normalized by the maximum value. Data are shown as the mean ± SD (n=3).


Serial diluted recombinant S1 of SARS - CoV - 2 , SARS - CoV MERS - CoV were coated on 96 well plates , incubated with recombinant Fc - tagged ACE2 ( ACE2 - Fc ) for binding evaluation .

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Serial diluted recombinant S1 proteins of SARS-CoV-2, SARS-CoV and MERS-CoV were coated on 96 well plates, incubated with the recombinant Fc-tagged ACE2 (ACE2-Fc) for binding evaluation.


Recombinant S1 of SARS - CoV - 2 SARS - CoV were incubated with 293T - ACE2 subjected to FACS evaluation for binding .

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Recombinant S1 proteins of SARS-CoV-2 and SARS-CoV were incubated with 293T-ACE2 cells and subjected to FACS evaluation for binding.


Binding of SARS nAbs to SARS - CoV S1 were tested by ELISA . Recombinant S1 of SARS - CoV were coated on plates , serial diluted nAbs were added for binding to recombinant S1 . B . Binding of SARS nAbs to SARS - CoV - 2 S1 were tested by ELSIA . Recombinant S1 of SARS - CoV - 2 were coated on plates , serial diluted nAbs were added for binding to recombinant S1 .

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Binding of SARS nAbs to SARS-CoV S1 protein were tested by ELISA. Recombinant S1 protein of SARS-CoV were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein.B. Binding of SARS nAbs to SARS-CoV-2 S1 protein were tested by ELSIA. Recombinant S1 protein of SARS-CoV-2 were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein.


Neutralization of SARS - CoV nAbs against SARS - CoV - 2 PSV .

Neutralization of SARS-CoV nAbs against SARS-CoV-2 PSV.


Antibody competition with SARS - CoV RBD binding to ACE2 . Recombinant SARS - CoV RBD was coated on plates , nAbs recombinant ACE2 were then added for RBD binding competition measurements .

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Antibody competition with SARS-CoV RBD binding to ACE2. Recombinant SARS-CoV RBD protein was coated on plates, nAbs and recombinant ACE2 were then added for RBD binding competition measurements.


PCP - MS strategy to identify interacting with specific proteasome subtypes . U937 were cross - linked with formaldehyde lysed , were concentrated ultrafiltrated on a 100 kDa cutoff device . complexes were then separated on a 15 - 40 % glycerol gradient . Each fraction of gradient was analyzed by nano - LC - MS / MS . was performed using mean XIC of three most intense validated peptides for each , after internal standard calibration using a mix of 8 isotopically labeled peptides . PCP was performed as described in Materials Methods section .

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PCP-MS strategy to identify proteins interacting with specific proteasome subtypes. U937 cells were cross-linked with formaldehyde and lysed, and proteins were concentrated and ultrafiltrated on a 100 kDa cutoff device. Protein complexes were then separated on a 15-40% glycerol gradient. Each fraction of the gradient was analyzed by nano-LC-MS/MS. Protein quantification was performed using the mean XIC of the three most intense validated peptides for each protein, after internal standard calibration using a mix of 8 isotopically labeled peptides. The PCP analysis was performed as described in the Materials and Methods section.


PCP of 19S regulatory . abundance profiles of 16 of 19S RP ( Rpt1 - 6 , Rpn1 - 3 , Rpn5 , 7 - 9 , 11 - 13 , gray lanes ) of their median abundance ( black lane ) ( left panel ) . PCP is performed by plotting χ2 values ( representing Euclidian between abundance profile of each reference profile ) of experimental replicate 2 as a function of χ2 values of experimental replicate 1 ( middle left panel ) . median profile of 19S subunits was used as reference profile for calculation of χ2 values . Different zooms of graph are represented ( middle right right panels ) . gray dots represent quantified in fractions of density gradient blue dots represent 19S subunits ( right panel ) .

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PCP analysis of the 19S regulatory complex. Protein abundance profiles of 16 proteins of the 19S RP (Rpt1-6, Rpn1-3, Rpn5, 7-9, 11-13, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values (representing the Euclidian distance between the abundance profile of each protein and the reference profile) of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 19S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and blue dots represent 19S subunits (right panel).


PCP of proteasome 20S . abundance profiles of 17 of 20S CP ( α1 - α7 , β1 - β7 , β1i , β2i , β5i , gray lanes ) of their median abundance ( black lane ) ( left panel ) . PCP is performed by plotting χ2 values of experimental replicate 2 as a function of χ2 values of experimental replicate 1 ( middle left panel ) . median profile of 20S subunits was used as reference profile for calculation of χ2 values . Different zooms of graph are represented ( middle right right panels ) . gray dots represent quantified in fractions of density gradient red dots represent 20S subunits .

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PCP analysis of proteasome 20S complex. Protein abundance profiles of 17 proteins of the 20S CP (α1-α7, β1-β7, β1i, β2i, β5i, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 20S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and red dots represent 20S subunits.


correlation profiling ( PCP ) using median profile of PA28αβ regulator as reference profileProfiles of PA28α , PA28β , β2i ( blue , red green lines , respectively ) .

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Protein correlation profiling (PCP) analysis using the median profile of the PA28αβ regulator as the reference profileProfiles of the PA28α, PA28β, and the β2i proteins (blue, red and green lines, respectively).


Plot of χ2 values of experimental replicate 2 as a function of χ2 values of experimental replicate 1 . A zoom of graph in ( B ) is represented χ2 coordinates for PA28α , PA28β , β2i are highlighted as blue , red , green dots , respectively . gray dots represent χ2 coordinates of quantified in fractions of gradient . median profile of PA28α PA28β subunits was used as reference profile for calculation of χ2 values .

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Plot of the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1.A zoom of the graph in (B) is represented and χ2 coordinates for PA28α, PA28β, and β2i proteins are highlighted as blue, red, and green dots, respectively. Light gray dots represent the χ2 coordinates of the proteins quantified in all the fractions of the gradient. The median profile of the PA28α and PA28β subunits was used as the reference profile for the calculation of the χ2 values.


A Proteasome complexes were immunopurified from nine formaldehyde - crosslinked human lines analyzed by nano - LC - MS / MS . abundance indexes ( PAIs ) were used to represent abundance of in purified proteasome samples . correlation between two different was quantified using coefficients of determination ( R2 ) .

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A Proteasome complexes were immunopurified from nine formaldehyde-crosslinked human cell lines and analyzed by nano-LC-MS/MS. Protein abundance indexes (PAIs) were used to represent the abundance of proteins in purified proteasome samples. The correlation between two different proteins was quantified using coefficients of determination (R2).


B - E Correlations of abundances of α7 α6 ( B ) , Rpn3 Rpn1 ( C ) , PA28β PA28α ( D ) , Rpn3 PA28β ( E ) .

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B-E Correlations of abundances of α7 and α6 (B), Rpn3 and Rpn1 (C), PA28β and PA28α (D), and Rpn3 and PA28β (E).


Heat - map representing correlations ( expressed as R2 ) between abundances of 73 known proteasome - interacting ( PIPs ) abundances of 8 reference or complexes , PA28γ , β2i ( representing iP20S ) , PA28αβ , ncP20S ( median of α1 - α7 , β3 , β4 , β6 , β7 profiles ) , 19S ( median of Rpt1 - 6 , Rpn1 - 3 , 5 - 14 profiles ) , PI31 , β5 ( representing sP20S ) , PA200 . For complexes , median PAI of their subunits in each of 24 AP - MS experiments was used : α1 - α7 , β3 , β4 , β6 , β7 subunits for ncP20S , Rpt1 - 6 , Rpn1 - 3 , 5 - 14 for 19S RP , PA28α PA28β subunits for PA28αβ RP . R2 values were hierarchically clustered . Three distinct clusters of composition detailed hereafter could be obtained . Cluster 1 ( from top to bottom ) : Rpt3 , Rpn13 , α2 , Rpn7 , USP14 , hHR23B , α1 , β6 , β3 , α4 , α7 , Rpn6 , Rpn3 , Rpt4 , Rpn10 , Rpn5 , Rpt5 , Rpn1 , Rpn11 , Rpt1 , Rpn9 , Rpn2 , Rpn8 , α3 , α6 , β4 , Rpt6 , PDC6 , Rpn12 , APEH , Ubiquilin - 1 , α5 , β7 , CCT7 , CCT4 , CCT2 , CCT3 , CCT5 , CCT6A , DNAJA1 , HSP90AB1 , HSP90AA1 , PNP , Rpt2 . Cluster 2 ( from top to bottom ) : 14 - 3 - 3ζ / δ , CAND1 , GSR , UBE3C , β2 , ATP5A1 , ATP5B , β1 , PA200 , β5 , FBXO7 , UCHL5 , TXNL1 , ECM29 , PI31 . Cluster 3 ( from top to bottom ) : PA28β , PITH1 , β2i , PA28α , PA28γ , β5i , β1i .

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

Heat-map representing the correlations (expressed as the R2) between the abundances of 73 known proteasome-interacting proteins (PIPs) and the abundances of 8 reference proteins or protein complexes, PA28γ, β2i (representing the iP20S), PA28αβ, ncP20S (median of α1-α7, β3, β4, β6, and β7 profiles), 19S (median of Rpt1-6, Rpn1-3, 5-14 profiles), PI31, β5 (representing the sP20S), and PA200. For protein complexes, the median PAI of their subunits in each of the 24 AP-MS experiments was used: α1-α7, β3, β4, β6, and β7 subunits for the ncP20S, Rpt1-6, Rpn1-3, 5-14 for the 19S RP, and PA28α and PA28β subunits for the PA28αβ RP. The R2 values were hierarchically clustered. Three distinct clusters of composition detailed hereafter could be obtained. Cluster 1 (from top to bottom): Rpt3, Rpn13, α2, Rpn7, USP14, hHR23B, α1, β6, β3, α4, α7, Rpn6, Rpn3, Rpt4, Rpn10, Rpn5, Rpt5, Rpn1, Rpn11, Rpt1, Rpn9, Rpn2, Rpn8, α3, α6, β4, Rpt6, PDC6, Rpn12, APEH, Ubiquilin-1, α5, β7, CCT7, CCT4, CCT2, CCT3, CCT5, CCT6A, DNAJA1, HSP90AB1, HSP90AA1, PNP, Rpt2. Cluster 2 (from top to bottom): 14-3-3ζ/δ, CAND1, GSR, UBE3C, β2, ATP5A1, ATP5B, β1, PA200, β5, FBXO7, UCHL5, TXNL1, ECM29, PI31. Cluster 3 (from top to bottom): PA28β, PITH1, β2i, PA28α, PA28γ, β5i, β1i.


two HEK EBNA lines express only standard proteasome or immunoproteasome subunits . Western blots against immuno - ( β1i , β2i , β5i ) standard ( β1 , β2 , β5 ) catalytic subunits of 20S proteasome . Calnexin is used as a loading control . Black lines delineate boundary between vertically sliced that juxtapose lanes that were non - adjacent in gel . Importantly , bands were assembled from same blot .

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The two HEK EBNA cell lines express only standard proteasome or immunoproteasome subunits. Western blots against the immuno- (β1i, β2i, β5i) and standard (β1, β2, β5) catalytic subunits of the 20S proteasome. Calnexin is used as a loading control. Black lines delineate the boundary between vertically sliced images that juxtapose lanes that were non-adjacent in the gel. Importantly, the bands were assembled from the same blot.


Relative normalized abundance indexes of proteasome regulators in HEK EBNA containing only immunoproteasome compared to HEK EBNA containing only standard proteasome . normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions ( n = 4 ) .

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Relative normalized abundance indexes of proteasome regulators in HEK EBNA cells containing only immunoproteasome compared to HEK EBNA cells containing only standard proteasome. The normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions (n = 4).


Kinetics of IFN - γ treatment on HeLa . HeLa were stimulated for 0 , 24 , 48 , or 72 h with IFN - γ . Western blots were performed on total lysates with antibodies against β2i , α2 , α5 subunits . IRF - 1 was used to control IFN - γ treatment efficiency , GAPDH was used as a loading control .

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Kinetics of IFN-γ treatment on HeLa cells. HeLa cells were stimulated for 0, 24, 48, or 72 h with IFN-γ. Western blots were performed on total cell lysates with antibodies against the β2i, α2, and α5 subunits. IRF-1 was used to control IFN-γ treatment efficiency, and GAPDH was used as a loading control.


For each time point of IFN - γ treatment , proteasome complexes were purified analyzed by LC - MS / MS . Proteasome complexes dynamics was measured by label - free quantitative proteomics . normalized abundance index of each or obtained at each time point was compared to one obtained at 0 h time point to obtain a regulator relative normalized PAI ( n = 3 ) .

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For each time point of the IFN-γ treatment, proteasome complexes were purified and analyzed by LC-MS/MS. Proteasome complexes dynamics was measured by label-free quantitative proteomics. The normalized abundance index of each protein or protein complex obtained at each time point was compared to the one obtained at the 0 h time point to obtain a regulator relative normalized PAI (n = 3).


Dual - color SMLM shows co - localization of BAX DRP1 at nanoscale . B ) Magnified SMLM of GFP - BAX DRP1 assemblies co - localizing up to 30 nm during apoptosis . Scale bar 100 nm . C ) Co - localization control of DRP1 immunostaining with same different secondary antibodies ( labelled with AF647 CF680 ) in HeLa . Scale bar 100 nm . D ) of from GFP - BAX to DRP1 structures ( measured at center of each structure ) from dual - color SMLM . Data are quantified from n = 4 independent experiments with a total of 720 BAX structure .

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Dual-color SMLM shows co-localization of BAX and DRP1 at the nanoscale. B) Magnified SMLM images of GFP-BAX and DRP1 assemblies co-localizing up to 30 nm during apoptosis. Scale bar 100 nm. C) Co-localization control of DRP1 immunostaining with the same primary and different secondary antibodies (labelled with AF647 and CF680) in HeLa cells. Scale bar 100 nm. D) Quantification of the distance from GFP-BAX to DRP1 structures (distance measured at the center of each structure) from dual-color SMLM images. Data are quantified from n=4 independent experiments with a total of 720 BAX structure.


E , F ) Representative confocal microscopy of HeLa transfected with ddFP RA - BAX GB - DRP1 ( E ) in untreated or apoptotic ( STS ) conditions , or with RA - BAX GB - BAX during apoptosis ( F ) . RA - BAX / GB - DRP1 RA - BAX / GB - BAX complexes shown in green , mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm . Right panels are zoomed areas representing merged channels . Scale bar 5 μm .

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E,F) Representative confocal microscopy images of HeLa cells transfected with ddFP RA-BAX and GB-DRP1 (E) in untreated or apoptotic (STS) conditions, or with RA-BAX and GB-BAX during apoptosis (F). RA-BAX/GB-DRP1 and RA-BAX/GB-BAX complexes shown in green, mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm. Right panels are zoomed areas representing individual and merged channels. Scale bar 5 μm.


G ) BAX / DRP1 interaction compared to BAX / BAX BCL ‑ xL / DRP1 by ddFP . % mito - BFP transfected that show RA / GB signal was quantified in HeLa untreated ( untr . ) 3 h after apoptosis induction ( STS ) . Box plots represent interquartile ( box ) , median ( ) SD ( whiskers ) of n = 3 independent experiments ( with n = 100 each ) . of significance were determined by paired two - Student ´ s t - ( * p < 0 . 05 , * * * p < 0 . 001 ) compared to BAX / DRP1 after apoptotic induction ( STS ) .

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G) BAX/DRP1 interaction compared to BAX/BAX and BCL‑xL/DRP1 by ddFP. % mito-BFP transfected cells that show RA/GB signal was quantified in HeLa cells untreated (untr.) and 3 h after apoptosis induction (STS). Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, *** p < 0.001) compared to BAX/DRP1 after apoptotic induction (STS).


A ) Confocal microscopy time - series of increase in Smac - GFP ( magenta ) in relation to detection of RA - BAX / GB - DRP1 complexes ( green ) during apoptosis induction in MEF DRP1 KO . Scale bar 10 μm . are representative for n = 3 independent experiments .

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A) Confocal microscopy time-series images of the increase in cytosolic Smac-GFP (magenta) in relation to the detection of RA-BAX/GB-DRP1 complexes (green) during apoptosis induction in MEF DRP1 KO cells. Scale bar 10 μm. Images are representative for n=3 independent experiments.


C ) of BAX / DRP1 interaction ( % with RA - BAX / GB - DRP1 foci normalized to mito - BFP positive , n = 100 per experiment ) during inhibition of effector caspases ( Q - VD - OPh ) , mitochondrial permeability transition pore ( CsA ) versus control ( DMSO ) , with or without apoptosis induction ( + / - STS ) . Box plots represent interquartile ( box ) , median ( ) SD ( whiskers ) . Significance was determined from n = 3 independent experiments ( with n = 100 each ) by paired two - Student ´ s t - ( n . s . indicating p > 0 . 05 ) compared to DMSO control after apoptotic induction ( STS ) .

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C) Quantification of BAX/DRP1 interaction (% cells with RA-BAX/GB-DRP1 foci normalized to the mito-BFP positive cells, n=100 cells per experiment) during inhibition of effector caspases (Q-VD-OPh), the mitochondrial permeability transition pore (CsA) versus control (DMSO), with or without apoptosis induction (+/- STS). Box plots represent the interquartile (box), median (line) and SD (whiskers). Significance was determined from n=3 independent experiments (with n=100 cells each) by paired two-tailed Student´s t-test (n.s. indicating p > 0.05) compared to DMSO control after apoptotic induction (STS).


C ) of % CC between DRP1 - AF488 BAX - AF633 in solution ( grey ) , in ( violet ) , in ( beige ) in presence of excess unlabeled cBID . Box plots represent interquartile ( outer box ) , mean ( inner box ) , median ( ) range ( whiskers ) . of significance were determined by paired two - Student ´ s t - ( * p < 0 . 05 , * * p < 0 . 01 ) from n = 9 measurements in solution , n = 46 individually measured GUVs in or n = 17 GUVs in presence of cBID .

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C) Quantification of %CC between DRP1-AF488 and BAX-AF633 in solution (grey), in the membrane (violet), and in the membrane (beige) in presence of excess unlabeled cBID. Box plots represent the interquartile (outer box), mean (inner box), median (line) and range (whiskers). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01) from n=9 measurements in solution, n=46 individually measured GUVs in the membrane or n=17 GUVs in presence of cBID.


E ) Effect of DRP1 on BAX - induced GUV permeabilization . Left : % GUVs permeabilized to Cytochrome c488 ( Cyt c , 12 kDa , beige ) allophycocianin ( APC , 104 kDa , blue ) in absence or presence of cBID , BAX DRP1 combined as indicated . Data are presented as mean ± SD of n = 4 independent experiments . * p < 0 . 05 ( paired two - Student ´ s t - ) . Right : representative confocal microscopy showing GUVs ( grey ) in a solution of Cyt c ( green ) APC ( magenta ) . Scale bar 10 µm .

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E) Effect of DRP1 on BAX-induced GUV permeabilization. Left: % GUVs permeabilized to Cytochrome c488 (Cyt c, 12 kDa, beige) and allophycocianin (APC, 104 kDa, blue) in the absence or presence of cBID, BAX and DRP1 combined as indicated. Data are presented as mean ± SD of n=4 independent experiments. * p < 0.05 (paired two-tailed Student´s t-test). Right: representative confocal microscopy images showing GUVs (grey) in a solution of Cyt c (green) and APC (magenta). Scale bar 10 µm.


Effect of BAX on DRP1 density DRP1 - induced shape alterations of liposomes measured by flow cytometry . G ) density of DRP1 ( corrected units , cFU , left graph ) DRP1 - induced tethering ( indicated by a shape index > 1 , right graph ) in LUVs in absence or presence of different concentrations of BAX . Data are presented as mean ± SD of n = 3 independent experiments . * p < 0 . 05 ( paired two - Student ´ s t - ) vs . DRP1 without BAX .

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Effect of BAX on DRP1 membrane density and DRP1-induced shape alterations of liposomes measured by flow cytometry. G) Membrane density of DRP1 (corrected fluorescence units, cFU, left graph) and DRP1-induced membrane tethering (indicated by a shape index > 1, right graph) in LUVs in the absence or presence of different concentrations of BAX. Data are presented as mean ± SD of n=3 independent experiments. * p < 0.05 (paired two-tailed Student´s t-test) vs. DRP1 without BAX.


BAX peptide array to define interaction - site with DRP1 . D ) of relative of DRP1 - AF488 at rim of GUVs for each BAX peptide of n = 3 experiments ( with n = 100 vesicles each ) . Black lines indicate mean value . Interacting BAX peptides are highlighted in beige .

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BAX peptide array to define interaction-site with DRP1. D) Quantification of the relative fluorescence intensity of DRP1-AF488 at the rim of the GUVs for each BAX peptide of n=3 individual experiments (with n=100 vesicles each). Black lines indicate the mean value. Interacting BAX peptides are highlighted in beige.


B ) Representative confocal microscopy of U2OS BAX / BAK DKO of interactions between RA - BAX variants GB - DRP1 , shown as green signal , 3 h after apoptosis induction . Mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm .

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

B) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX variants and GB-DRP1, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.