Figure 5 ( A ) Immunoblot of cytosol - enriched cerebral tissue homogenate ( Input ) and eluate of Abeta42 or DnaJA1 using Abeta - specific antibody ( 6E10 ) and DnaJA1 - specific antibody ( DnaJA1 ) , respectively , showing optimal and long exposure ( Long exp . ) . Immunoprecipitation using magnetic beads without antibody , DnaJA1 or 6E10 antibody of female ( 15 months old ) wild type ( WT ) or 3xTg ( PS1M146V / APPSwe / tauP301L ) mice . Sections showing monomer and low - n oligomers , dodecamer and full - length amyloid precursor protein ( APP ) are from 1 immunoblot . ( B ) Representative immunoblot of DnaJA1 levels from the hippocampi of AD patients ( AD ) and aged non - demented controls ( ctrl ) . PonceauS serves as a loading control . ( C ) Quantification by densitometry of DnaJA1 normalized to PonceauS from the hippocampi of AD patients ( AD ) and aged non - demented controls ( ctrl ) . Representative immunoblot is shown in ( B ) . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 4 - 5 independent patients . * , p < 0 . 05 . Unpaired , two - tailed t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Immunoblot of cytosol-enriched cerebral tissue homogenate (Input) and eluate of Abeta42 or DnaJA1 using Abeta-specific antibody (6E10) and DnaJA1-specific antibody (DnaJA1), respectively, showing optimal and long exposure (Long exp.). Immunoprecipitation using magnetic beads without antibody, DnaJA1 or 6E10 antibody of female (15 months old) wild type (WT) or 3xTg (PS1M146V/APPSwe/tauP301L) mice. Sections showing monomer and low-n oligomers, dodecamer and full-length amyloid precursor protein (APP) are from 1 immunoblot.(B) Representative immunoblot of DnaJA1 levels from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). PonceauS serves as a loading control. (C) Quantification by densitometry of DnaJA1 normalized to PonceauS from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). Representative immunoblot is shown in (B). Dot plots show all data points along with the mean (bar) ± s.d.. n = 4-5 independent patients. *, p < 0.05. Unpaired, two-tailed t-test.


Figure 6 ( A ) Representative confocal microscopy of 10 - day - old male fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) and reference nuclei staining with DAPI ( blue ) of Droj2 knockdown flies ( Droj2 + / - ) flies and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) . ( B ) Representative confocal and gSTED deconvolved ( decon ) images of Kenyon cells in 18 - day - old male fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) and reference nuclei staining with DAPI ( blue ) of Droj2 knockdown flies ( Droj2 + / - ) flies and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) . ( C - D ) Average intensity ( C ) and total area ( D ) of Abeta ( 6E10 ) signal from confocal images representatively shown in ( A ) from 12 brains of w1118 wild type ( Droj2 + / + ) and knockdown ( Droj2 + / - ) 10 - day - old male flies . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 12 . * , p < 0 . 05 . Unpaired , two - tailed t - test . ( E - F ) qPCR analysis of Droj2 - mRNA levels of 3 - 6 days old female ( E ) and male ( F ) flies expressing human Abeta42 ( UAS - A42 ) of w1118 wild type flies ( Droj2 + / + ) normalized to corresponding isogenic w1118 wild type flies without Abeta42 expression ( Droj2 + / + ctrl ) . Reference gene is Rpl32 . Dot plots show all data points along with the mean ( line ) ± s . d . . n = 3 . * * , p < 0 . 01 . One sample t - test against 1 . ( G ) Immunoprecipitation ( IP : 6E10 ) of w1118 wild type flies ( Droj2 + / + ) using synthetic Abeta42 added to the fly head extract ( Input ) . Abeta - specific antibody 6E10 was used for Abeta42 and DnaJA1 - specific antibody for Droj2 immunoblot detection . Showing input , supernatant , supernatant after washing ( WS ) and eluate .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Representative confocal microscopy of 10-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).(B) Representative confocal and gSTED deconvolved (decon) images of Kenyon cells in 18-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).(C-D) Average intensity (C) and total area (D) of Abeta (6E10) signal from confocal images representatively shown in (A) from 12 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 10-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 12. *, p < 0.05. Unpaired, two-tailed t-test.(E-F) qPCR analysis of Droj2-mRNA levels of 3-6 days old female (E) and male (F) flies expressing human Abeta42 (UAS-A42) of w1118 wild type flies (Droj2+/+) normalized to corresponding isogenic w1118 wild type flies without Abeta42 expression (Droj2+/+ ctrl). Reference gene is Rpl32. Dot plots show all data points along with the mean (line) ± s.d.. n = 3. **, p < 0.01. One sample t-test against 1.(G) Immunoprecipitation (IP: 6E10) of w1118 wild type flies (Droj2+/+) using synthetic Abeta42 added to the fly head extract (Input). Abeta-specific antibody 6E10 was used for Abeta42 and DnaJA1-specific antibody for Droj2 immunoblot detection. Showing input, supernatant, supernatant after washing (WS) and eluate.


Figure 7 ( A - B ) Survival of female ( A ) and male ( B ) w1118 wild type flies ( Droj2 + / + ) and Droj2 knockdown flies ( Droj2 + / - ) with expression of human Abeta42 ( UAS - A42 ) or control flies without expression ( ctrl ) , upon supplementation of sugar ( 10 % sucrose ) with 20 mM MnCl2 . Survival was determined at indicated time points . n = 6 with 100 - 120 flies per experiment . The indicated p - value refers to the interaction ( int . ) term of a Cox Proportional Hazards model comparing Abeta42 toxicity ( UAS - A42 vs . ctrl ) and Droj2 expression ( Droj2 + / + vs . Droj2 + / - ) as main factors . The following pairwise comparisons of the indicated groups survival were done by Log Rank test ( * * * * , p < 0 . 0001 ; ns , p > 0 . 05 ) . ( C ) Aversive associative memory performance 2 min after training of aged ( 18 days old ) male Droj2 knockdown flies ( Droj2 + / - ) and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) both expressing human Abeta42 ( UAS - A42 ) of six independent biological replicates . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 6 . * , p < 0 . 05 . Unpaired , two - tailed t - test . ( D ) Representative confocal and gSTED deconvolved images of Kenyon cells in 15 - day - old male fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) and mitochondrial marker ATP5A - specific antibody ( ATP5A , green ) of Droj2 knockdown flies ( Droj2 + / - ) flies and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) . ( E - F ) Solidity ( E ) and circularity ( F ) of ATP5A - stained mitochondria from fly brain gSTED deconvolved images representatively shown in ( D ) from 8 - 10 brains of w1118 wild type ( Droj2 + / + ) and knockdown ( Droj2 + / - ) 15 - day - old male flies . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 8 - 10 . * , p < 0 . 05 . Unpaired , two - tailed t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A-B) Survival of female (A) and male (B) w1118 wild type flies (Droj2+/+) and Droj2 knockdown flies (Droj2+/-) with expression of human Abeta42 (UAS-A42) or control flies without expression (ctrl), upon supplementation of sugar (10% sucrose) with 20 mM MnCl2. Survival was determined at indicated time points. n = 6 with 100-120 flies per experiment. The indicated p-value refers to the interaction (int.) term of a Cox Proportional Hazards model comparing Abeta42 toxicity (UAS-A42 vs. ctrl) and Droj2 expression (Droj2+/+ vs. Droj2+/-) as main factors. The following pairwise comparisons of the indicated groups survival were done by Log Rank test (****, p < 0.0001; ns, p > 0.05).(C) Aversive associative memory performance 2 min after training of aged (18 days old) male Droj2 knockdown flies (Droj2+/-) and corresponding isogenic w1118 wild type flies (Droj2+/+) both expressing human Abeta42 (UAS-A42) of six independent biological replicates. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6. *, p < 0.05. Unpaired, two-tailed t-test.(D) Representative confocal and gSTED deconvolved images of Kenyon cells in 15-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and mitochondrial marker ATP5A-specific antibody (ATP5A, green) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).(E-F) Solidity (E) and circularity (F) of ATP5A-stained mitochondria from fly brain gSTED deconvolved images representatively shown in (D) from 8-10 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 15-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8-10. *, p < 0.05. Unpaired, two-tailed t-test.


Figure 1A WT and Ccnc - / - MEF cultures were treated with 0 . 4 mM H2O2 as indicated , extracts prepared and immunoprecipitated with antibody recognizing the active conformation of Bax . The immunoprecipitates were subjected to Western blot analysis and probed for total Bax or cyclin C . Whole cell extracts ( WCE ) were subjected to Western blot analysis as indicated to control for protein concentrations in the extracts . Molecular weight markers ( kDa ) are indicated on the left . B Cyclin C and Bax directly interact . Recombinant Bax and SUMO - His6 - cyclin C were either incubated together or separately at room temperature then passed over Co + + resin . The elutions were subjected to Western blot analysis probing for Bax and cyclin C ( lanes 4 - 6 ) . Input controls for each sample ( 1 / 10 ) are included in lanes 1 - 3 . Molecular weight markers ( kDa ) are indicated on the left . C Mitochondrial relocalization of cyclin C is required for Bax activation . The experiment described in ( A ) was repeated except that H2O2 treated cells were treated with 1 µM pifithrin - µ ( PFT ) for 24 h as indicated . Molecular weight markers ( kDa ) are indicated on the left . D Cyclin C is required for efficient Bax - Bcl - XL disassociation . Extracts prepared from MEF cells with the indicated genotypes following treatment with H2O2 ( 0 . 4 mM , 4 h ) were immunoprecipitated with active conformation Bax antibodies . The immunoprecipitates were subjected to Western blot analysis probing with Bcl - XL antibodies . Western blot analysis of WCE was performed as input concentration controls . Molecular weight markers ( kDa ) are indicated on the left . E The mitochondrial fission complex is required for cyclin C - dependent Bax activation . Extracts were prepared MEF cultures with the indicated genotypes treated with H2O2 ( 0 . 4 mM , 4 h ) . The extracts were treated as in A . Molecular weight markers ( kDa ) are indicated on the left . F Drp1 is required for cyclin C - Bax interaction . Extracts were prepared from wild type or Drp1 - / - MEF cultures treated or not with H2O2 and analyzed for the presence of Bax and cyclin C as described in A . Molecular weight markers ( kDa ) are indicated on the left .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A WT and Ccnc-/- MEF cultures were treated with 0.4 mM H2O2 as indicated, extracts prepared and immunoprecipitated with antibody recognizing the active conformation of Bax. The immunoprecipitates were subjected to Western blot analysis and probed for total Bax or cyclin C. Whole cell extracts (WCE) were subjected to Western blot analysis as indicated to control for protein concentrations in the extracts. Molecular weight markers (kDa) are indicated on the left.B Cyclin C and Bax directly interact. Recombinant Bax and SUMO-His6-cyclin C were either incubated together or separately at room temperature then passed over Co++ resin. The elutions were subjected to Western blot analysis probing for Bax and cyclin C (lanes 4-6). Input controls for each sample (1/10) are included in lanes 1-3. Molecular weight markers (kDa) are indicated on the left.C Mitochondrial relocalization of cyclin C is required for Bax activation. The experiment described in (A) was repeated except that H2O2 treated cells were treated with 1 µM pifithrin-µ (PFT) for 24 h as indicated. Molecular weight markers (kDa) are indicated on the left.D Cyclin C is required for efficient Bax-Bcl-XL disassociation. Extracts prepared from MEF cells with the indicated genotypes following treatment with H2O2 (0.4 mM, 4 h) were immunoprecipitated with active conformation Bax antibodies. The immunoprecipitates were subjected to Western blot analysis probing with Bcl-XL antibodies. Western blot analysis of WCE was performed as input concentration controls. Molecular weight markers (kDa) are indicated on the left.E The mitochondrial fission complex is required for cyclin C-dependent Bax activation. Extracts were prepared MEF cultures with the indicated genotypes treated with H2O2 (0.4 mM, 4 h). The extracts were treated as in A. Molecular weight markers (kDa) are indicated on the left.F Drp1 is required for cyclin C-Bax interaction. Extracts were prepared from wild type or Drp1-/- MEF cultures treated or not with H2O2 and analyzed for the presence of Bax and cyclin C as described in A. Molecular weight markers (kDa) are indicated on the left.


Figure 2A Wild type MEF cells were fixed before ( control ) and following S - HAD treatment ( 10 µM , 4 h ) then nuclei ( DAPI ) , cyclin C ( indirect immunofluorescence ) and mitochondria ( MitoTracker Red ) were visualized by fluorescence microscopy . Merge panels present DAPI staining ( blue ) only . Zoom panels ( 4X ) are indicated by the boxes in the merge panels . Green arrows indicate cyclin C and mitochondrial co - localization . Bar indicates 10 µM . B Timecourse experiment with wild type and Ccnc - / - MEF cells treated with S - HAD ( 10 µM ) for the times indicated ( n = 3 ) . Error bars indicate SD . Asterisks indicate p values < 0 . 05 from pretreatment control ( Student ' s T - Test ) . MEF cells were fixed before ( control ) and following S - HAD treatment ( 10 µM , 4 h ) then nuclei ( DAPI ) , cyclin C ( indirect immunofluorescence ) and mitochondria ( MitoTracker Red ) were visualized by fluorescence microscopy . Merge panels present DAPI staining ( blue ) only . S - HAD peptide activity requires cyclin C . The experiment described in A was repeated with Ccnc - / - MEF cultures . D S - HAD treatment increases Bax mitochondrial localization . Western blot analysis of the indicated proteins in nuclear and mitochondrial fractions before and following S - HAD treatment ( 10 µM , 4 h ) . Atp5A and fibrillarin levels controlled for mitochondrial and nuclear loadings , respectively . Molecular weight markers ( kDa ) are indicated on the left . E Bax activation was monitored in wild - type MEF cultures treated with S - HAD ( 10 µM ) for the times indicated . The anti - oxidant N - acetyl cysteine ( NAC ) was added ( 1 mM ) 2 h prior to S - HAD treatment . Active Bax immunoprecipitates were subjected to Western blot analysis for Bax and cyclin C as indicated . Bax , cyclin C and ß - actin levels were monitored in extract preparations as input controls . Molecular weight markers ( kDa ) are indicated on the left .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Wild type MEF cells were fixed before (control) and following S-HAD treatment (10 µM, 4 h) then nuclei (DAPI), cyclin C (indirect immunofluorescence) and mitochondria (MitoTracker Red) were visualized by fluorescence microscopy. Merge panels present DAPI staining (blue) only. Zoom panels (4X) are indicated by the boxes in the merge panels. Green arrows indicate cyclin C and mitochondrial co-localization. Bar indicates 10 µM.B Timecourse experiment with wild type and Ccnc-/- MEF cells treated with S-HAD (10 µM) for the times indicated (n=3). Error bars indicate SD. Asterisks indicate p values <0.05 from pretreatment control (Student's T-Test).MEF cells were fixed before (control) and following S-HAD treatment (10 µM, 4 h) then nuclei (DAPI), cyclin C (indirect immunofluorescence) and mitochondria (MitoTracker Red) were visualized by fluorescence microscopy. Merge panels present DAPI staining (blue) only. S-HAD peptide activity requires cyclin C. The experiment described in A was repeated with Ccnc-/- MEF cultures.D S-HAD treatment increases Bax mitochondrial localization. Western blot analysis of the indicated proteins in nuclear and mitochondrial fractions before and following S-HAD treatment (10 µM, 4 h). Atp5A and fibrillarin levels controlled for mitochondrial and nuclear loadings, respectively. Molecular weight markers (kDa) are indicated on the left.E Bax activation was monitored in wild-type MEF cultures treated with S-HAD (10 µM) for the times indicated. The anti-oxidant N-acetyl cysteine (NAC) was added (1 mM) 2 h prior to S-HAD treatment. Active Bax immunoprecipitates were subjected to Western blot analysis for Bax and cyclin C as indicated. Bax, cyclin C and ß-actin levels were monitored in extract preparations as input controls. Molecular weight markers (kDa) are indicated on the left.


Figure 3A Caspase activation was measured by fluorescent cell analysis in wild - type MEF cells treated with S - HAD ( 10 µM , 24 h ) or cisplatin ( CP , 30 µM , 16 h ) . n = 3 . Error bars indicate SD with * and * * indicate p < 0 . 05 and p < 0 . 01 , respectively using Student ' s T test . B PARP cleavage was not induced by S - HAD treatment . PARP cleavage was monitored by Western blot analysis in Hela cells treated with S - HAD or cisplatin ( CP ) as described in ( A ) . The blot was stripped and reprobed with ß - actin antibodies as a loading control . Molecular weight markers ( kDa ) are indicated on the left . C Wild type and Ccnc - / - MEF cultures were treated with S - HAD and cisplatin ( CP ) as just described . Cells were imaged and examined for a " rounding up " phenotype diagnostic for death . D Cyclin C is required for normal Bax oligomerization in response to cisplatin . Whole cell extracts prepared from DSP crosslinked wild type and CCNC - / - MEF cells exposed to cisplatin ( 30 µM , 16 h ) were probed for total Bax . Bax multimers are indicated . Molecular weight markers ( kDa ) are indicated on the left . E S - HAD does not induce Bax oligomerization . Experiments described in D were repeated with cultures treated with S - HAD ( 10 µM , 24 h ) . Bax multimers are indicated . Molecular weight markers ( kDa ) are indicated on the left . F S - HAD treatment sensitizes Hela cells to cisplatin . Hela cultures were treated with cisplatin ( 30 µM ) , S - HAD ( 10 µM ) and N - acetyl cysteine ( NAC , 1 mM ) as indicated . Fluorescent cell analysis was used to quantitate the apoptotic ( Annexin V positive ) and necrotic ( PI positive ) population percentages , respectively . n = 3 independent cultures . Error bars indicate SD . Asterisk indicates p < 0 . 05 ( Student ' s T - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Caspase activation was measured by fluorescent cell analysis in wild-type MEF cells treated with S-HAD (10 µM, 24 h) or cisplatin (CP, 30 µM, 16 h). n = 3. Error bars indicate SD with * and ** indicate p <0.05 and p<0.01, respectively using Student's T test.B PARP cleavage was not induced by S-HAD treatment. PARP cleavage was monitored by Western blot analysis in Hela cells treated with S-HAD or cisplatin (CP) as described in (A). The blot was stripped and reprobed with ß-actin antibodies as a loading control. Molecular weight markers (kDa) are indicated on the left.C Wild type and Ccnc-/- MEF cultures were treated with S-HAD and cisplatin (CP) as just described. Cells were imaged and examined for a "rounding up" phenotype diagnostic for death.D Cyclin C is required for normal Bax oligomerization in response to cisplatin. Whole cell extracts prepared from DSP crosslinked wild type and CCNC-/- MEF cells exposed to cisplatin (30 µM, 16 h) were probed for total Bax. Bax multimers are indicated. Molecular weight markers (kDa) are indicated on the left.E S-HAD does not induce Bax oligomerization. Experiments described in D were repeated with cultures treated with S-HAD (10 µM, 24 h). Bax multimers are indicated. Molecular weight markers (kDa) are indicated on the left.F S-HAD treatment sensitizes Hela cells to cisplatin. Hela cultures were treated with cisplatin (30 µM), S-HAD (10 µM) and N-acetyl cysteine (NAC, 1 mM) as indicated. Fluorescent cell analysis was used to quantitate the apoptotic (Annexin V positive) and necrotic (PI positive) population percentages, respectively. n = 3 independent cultures. Error bars indicate SD. Asterisk indicates p <0.05 (Student's T-test).


Figure 4A S - HAD treatment induces mitochondrial ROS . Wild type and Ccnc - / - MEF cultures were treated with S - HAD ( 10 µM , 24 h ) , rotenone or vehicle control as indicated . Mitochondrial ROS was measured by MitoSox staining ( 4 µM , 15 min ) and quantitative confocal imaging . Error bars indicate SD . n = 3 , asterisk indicates p < 0 . 05 ( Student ' s T - test ) , n . s . , not significantly different . B Quenching mitochondrial ROS reduces cyclin C - Bax interaction . Wild type and Ccnc - / - MEF cultures were treated with S - HAD ( 10 µM , 16 h ) and 100 nM mitochondrial - specific reactive oxygen scavenger MitoTempol . Western blot analysis was conducted as described in Fig . 1 . Molecular weight markers ( kDa ) are indicated on the left . C Insulin treatment stimulates S - HAD induced Bax activation . Wild type and Ccnc - / - MEF cultures were grown with or without insulin ( 10 µg / ml ) then treated with S - HAD ( 10 µM , 2 h ) . Mitochondrial and cytosolic fractions were prepared and probed for the indicated proteins . Atp5A and ß - actin levels control for mitochondrial and cytosolic loading , respectively . The two blots were prepared , processed and developed identically . Molecular weight markers ( kDa ) are indicated on the left .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A S-HAD treatment induces mitochondrial ROS. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 24 h), rotenone or vehicle control as indicated. Mitochondrial ROS was measured by MitoSox staining (4 µM, 15 min) and quantitative confocal imaging. Error bars indicate SD. n=3, asterisk indicates p<0.05 (Student's T-test), n.s., not significantly different.B Quenching mitochondrial ROS reduces cyclin C-Bax interaction. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 16 h) and 100 nM mitochondrial-specific reactive oxygen scavenger MitoTempol. Western blot analysis was conducted as described in Fig. 1. Molecular weight markers (kDa) are indicated on the left.C Insulin treatment stimulates S-HAD induced Bax activation. Wild type and Ccnc-/- MEF cultures were grown with or without insulin (10 µg/ml) then treated with S-HAD (10 µM, 2 h). Mitochondrial and cytosolic fractions were prepared and probed for the indicated proteins. Atp5A and ß-actin levels control for mitochondrial and cytosolic loading, respectively. The two blots were prepared, processed and developed identically. Molecular weight markers (kDa) are indicated on the left.


Figure 1A , Primary HBMEC were challenged with type I ( RH ) or type II ( ME49 ) strains of T . gondii for the indicated time points . Cell lysates were used to probe for total Akt and phospho - Ser473 Akt by immunoblot . B , C , Mouse endothelial cells ( mHEVc ; B ) and human retinal pigment epithelial cells ( RPE , C ) were challenged with RH T . gondii followed by assessment of Akt phosphorylation . B , C , Mouse endothelial cells ( mHEVc ; B ) and human retinal pigment epithelial cells ( RPE , C ) were challenged with RH T . gondii followed by assessment of Akt phosphorylation . D , HBMEC were challenged with either live or killed T . gondii . Cell lysates were subject to immunoblotting as indicated . E , HBMEC were transiently transfected with a plasmid encoding PH - Akt - GFP . Cells treated with or without LY294002 ( 20 µM ) for 1 h prior to challenge with T . gondii - RFP . Cells were examined by immunofluorescence at 5 min post - challenge to examine accumulation of PH - Akt - GFP around the parasites . F , HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T . gondii . Densitometry data represent means ± SEM of 4 experiments . A vertical line was inserted between densitometry data from control and LY294002 - treated cells to indicate that band densities from infected cells treated with or without LY294002 are compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 . G , HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA . Cells expressing either control siRNA or PI3K p110α siRNA were challenged with T . gondii 48 h after transfection . Cell lysates were subject to immunoblotting as indicated . Densitometry data are shown as above and represent means ± SEM of 3 experiments . Results shown are representative of 3 - 4 independent experiments .

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Figure 1A, Primary HBMEC were challenged with type I (RH) or type II (ME49) strains of T. gondii for the indicated time points. Cell lysates were used to probe for total Akt and phospho-Ser473 Akt by immunoblot.B, C, Mouse endothelial cells (mHEVc; B) and human retinal pigment epithelial cells (RPE, C) were challenged with RH T. gondii followed by assessment of Akt phosphorylation.B, C, Mouse endothelial cells (mHEVc; B) and human retinal pigment epithelial cells (RPE, C) were challenged with RH T. gondii followed by assessment of Akt phosphorylation.D, HBMEC were challenged with either live or killed T. gondii. Cell lysates were subject to immunoblotting as indicated.E, HBMEC were transiently transfected with a plasmid encoding PH-Akt-GFP. Cells treated with or without LY294002 (20 µM) for 1 h prior to challenge with T. gondii-RFP. Cells were examined by immunofluorescence at 5 min post-challenge to examine accumulation of PH-Akt-GFP around the parasites.F, HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T. gondii. Densitometry data represent means ± SEM of 4 experiments. A vertical line was inserted between densitometry data from control and LY294002-treated cells to indicate that band densities from infected cells treated with or without LY294002 are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1.G, HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA. Cells expressing either control siRNA or PI3K p110α siRNA were challenged with T. gondii 48 h after transfection. Cell lysates were subject to immunoblotting as indicated. Densitometry data are shown as above and represent means ± SEM of 3 experiments. Results shown are representative of 3-4 independent experiments.


Figure 2A , HBMEC , mHEVc and human RPE cells were incubated with or without Akt inhibitor IV ( 1 . 25 µM ) for 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy 2 h and 24 h post - challenge . B , HBMEC were transfected with control siRNA or Akt siRNA . Cells were then challenged with T . gondii 48 h after transfection . Monolayers were examined microscopically 24 h post - challenge . C , RAW 264 . 7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy 2 h and 24 h post - challenge . D , mHEVc - LC3 - EGFP cells were incubated with or without Akt inhibitor IVfollowed by challenge with T . gondii - RFP . Monolayers were examined by fluorescence microscopy 5 h post - challenge . Arrowheads indicate accumulation of LC3 around the parasite . E , HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii ( T ) and then processed for electron microscopy at 5 h post - challenge . Images at the bottom represent magnification of the areas within the boxes . Arrow indicates the PVM ; arrowhead indicates the double membrane structure around the vacuole . F , Control or Akt inhibitor IV - treated HBMEC were challenged with T . gondii - YFP . Expression of LAMP - 1 was examined by fluorescent microscopy 8 h post - challenge . Arrowheads indicate accumulation of LAMP - 1 around the parasiteG , H , mHEVc cells were transfected with Beclin 1 siRNA ( G ) , Atg7 siRNA ( H ) or control siRNA . After 48 h , cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy at 24 h . I , mHEVc were treated with or without Akt inhibitor IV and infected with T . gondii . 1 h post infection cells were treated with or without leupeptin plus pepstatin ( Lys inhibitors ) . Monolayers were examined microscopically 24 h post - challenge . J , Mouse microglia were incubated with or without Akt inhibitor IV . 3 - methyl adenine ( 3MA ; 10 mM ) or vehicle were added 2 h post - challenge with T . gondii . Monolayers were examined microscopically 24 h post - challenge . Results are shown as the mean ± SEM and are representative of 3 independent experiments .

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Figure 2A, HBMEC, mHEVc and human RPE cells were incubated with or without Akt inhibitor IV (1.25 µM) for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.B, HBMEC were transfected with control siRNA or Akt siRNA. Cells were then challenged with T. gondii 48 h after transfection. Monolayers were examined microscopically 24 h post-challenge.C, RAW 264.7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.D, mHEVc-LC3-EGFP cells were incubated with or without Akt inhibitor IVfollowed by challenge with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite.E, HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii (T) and then processed for electron microscopy at 5 h post-challenge. Images at the bottom represent magnification of the areas within the boxes. Arrow indicates the PVM; arrowhead indicates the double membrane structure around the vacuole.F, Control or Akt inhibitor IV-treated HBMEC were challenged with T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. Arrowheads indicate accumulation of LAMP-1 around the parasiteG, H, mHEVc cells were transfected with Beclin 1 siRNA (G), Atg7 siRNA (H) or control siRNA. After 48 h, cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 24 h.I, mHEVc were treated with or without Akt inhibitor IV and infected with T. gondii. 1 h post infection cells were treated with or without leupeptin plus pepstatin (Lys inhibitors). Monolayers were examined microscopically 24 h post-challenge.J, Mouse microglia were incubated with or without Akt inhibitor IV. 3-methyl adenine (3MA; 10 mM) or vehicle were added 2 h post-challenge with T. gondii. Monolayers were examined microscopically 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.


Figure 3A , HBMEC were treated with or without pertussis toxin ( PTx ; 100 ng / mL ) for 4 h prior to challenge with T . gondii . Cell lysates were obtained at 5 , 15 or 30 min post - incubation with T . gondii and used to examine total Akt and phospho - Akt serine 473 by immunoblot . Densitometry data represent means ± SEM of 3 experiments . A vertical line was inserted between densitometry data from control and PTx - treated cells to indicate that band densities from infected cells treated with or without PTx were compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 . HBMEC were also treated with or without LPA ( 10 µM ) in the presence or absence of PTx . Cell lysates were obtained at 5 min and subjected to immunoblottingB , HBMEC were transfected with MyD88 siRNA or control siRNA followed by challenge with T gondii after 48 h . Cell lysates were used to examine total Akt and phospho - serine 473 Akt by immunoblot . Results shown are representative of 3 independent experiments .

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Figure 3A, HBMEC were treated with or without pertussis toxin (PTx; 100 ng/mL) for 4 h prior to challenge with T. gondii. Cell lysates were obtained at 5, 15 or 30 min post-incubation with T. gondii and used to examine total Akt and phospho-Akt serine 473 by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and PTx-treated cells to indicate that band densities from infected cells treated with or without PTx were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. HBMEC were also treated with or without LPA (10 µM) in the presence or absence of PTx. Cell lysates were obtained at 5 min and subjected to immunoblottingB, HBMEC were transfected with MyD88 siRNA or control siRNA followed by challenge with T gondii after 48 h. Cell lysates were used to examine total Akt and phospho-serine 473 Akt by immunoblot. Results shown are representative of 3 independent experiments.


Figure 4A , HBMEC cells were challenged with RH T . gondii and cell lysates were obtained to probe for total EGFR , phospho - tyrosine 1068 EGFR and phospho - tyrosine 1148 EGFR by immunoblot . B , HBMEC were challenged with live vs killed tachyzoites . Total EGFR and phospho - tyrosine 1068 EGFR expression was examined by immunoblot . C , D , Human RPE cells ( C ) and mouse microglia ( D ) were challenged with RH T . gondii and total EGFR and phospho - tyrosine 1068 EGFR expression was examined by immunoblot . Results shown are representative of 3 independent experiments .

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Figure 4A, HBMEC cells were challenged with RH T. gondii and cell lysates were obtained to probe for total EGFR, phospho-tyrosine 1068 EGFR and phospho- tyrosine 1148 EGFR by immunoblot.B, HBMEC were challenged with live vs killed tachyzoites. Total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot.C, D, Human RPE cells (C) and mouse microglia (D) were challenged with RH T. gondii and total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot. Results shown are representative of 3 independent experiments.


Figure 5A , mHEVc cells were transfected with control siRNA or EGFR siRNA . Expression of EGFR and actin were assessed by immunoblot 48 h post - transfection . mHEVc cells expressing either control siRNA or EGFR siRNA were challenged with T . gondii . Cell lysates were used to examine the expression total Akt or phospho - Ser473 Akt by immunoblot . B , Mouse microglia were treated with or without AG1478 ( 1 µM ) 1 h prior to challenge with T . gondii . Cell lysates were used to examine the expression total Akt or phospho - Ser473 Akt by immunoblot . Densitometry data represent means ± SEM of 3 experiments . A vertical line was inserted between densitometry data from control siRNA and EGFR siRNA or control and AG1478 - treated cells to indicate that band densities from infected control cells or infected cells subjected to EGFR blockade were compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 . Results shown are representative of 3 independent experiments .

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Figure 5A, mHEVc cells were transfected with control siRNA or EGFR siRNA. Expression of EGFR and actin were assessed by immunoblot 48 h post-transfection. mHEVc cells expressing either control siRNA or EGFR siRNA were challenged with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot.B, Mouse microglia were treated with or without AG1478 (1 µM) 1 h prior to challenge with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control siRNA and EGFR siRNA or control and AG1478-treated cells to indicate that band densities from infected control cells or infected cells subjected to EGFR blockade were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.


Figure 6A , HBMEC were incubated with AG1478 ( 1 µM ) 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy at 2 and 24 h . B , Human RPE cells transfected with either EGFR or control siRNA were challenged with T . gondii followed by examination of monolayers by light microscopy at 24 h . C , Mouse bone marrow - derived macrophages were incubated with AG1478 and challenged with T . gondii . Monolayers were examined by light microscopy at 2 and 24 h . D , Parental CHO and CHO - EGFR cells were challenged with RH T . gondii . Monolayers were examined microscopically 2 h or 24 h post - challenge . E , mHEVc - LC3 - EGFP cells transfected with control siRNA or EGFR siRNA were challenged with T . gondii - RFP . Monolayers were examined by fluorescence microscopy 5 h post - challenge to determine the percentage of endothelial cells with LC3 accumulation around the parasite . F , HBMEC cells treated with or without AG1478 were challenged with either T . gondii - YFP . Expression of LAMP - 1 was examined by fluorescent microscopy 8 h post - challenge . The percentages of endothelial cells with LAMP - 1 accumulation of around the parasite were determined . G , H , mHEVc cells transfected with Beclin1 siRNA ( G ) or Atg7 siRNA ( H ) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T . gondii . Monolayers were examined by light microscopy 24 h post - challenge . Results are shown as the mean ± SEM and are representative of 3 independent experiments .

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Figure 6A, HBMEC were incubated with AG1478 (1 µM) 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.B, Human RPE cells transfected with either EGFR or control siRNA were challenged with T. gondii followed by examination of monolayers by light microscopy at 24 h.C, Mouse bone marrow-derived macrophages were incubated with AG1478 and challenged with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.D, Parental CHO and CHO-EGFR cells were challenged with RH T. gondii. Monolayers were examined microscopically 2 h or 24 h post-challenge.E, mHEVc-LC3-EGFP cells transfected with control siRNA or EGFR siRNA were challenged with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge to determine the percentage of endothelial cells with LC3 accumulation around the parasite.F, HBMEC cells treated with or without AG1478 were challenged with either T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. The percentages of endothelial cells with LAMP-1 accumulation of around the parasite were determined.G, H, mHEVc cells transfected with Beclin1 siRNA (G) or Atg7 siRNA (H) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T. gondii. Monolayers were examined by light microscopy 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.


Figure 7HBMEC were treated with or without GM6001 ( 10 µM ) for 1 h prior to challenge with T . gondii ( A ) or with Pertussis Toxin ( PTx ; 100 ng / ml ) for 4 h prior to parasite challenge ( B ) . Cell lysates were used to examine total EGFR and phospho - tyrosine 1068 EGFR by immunoblot . Densitometry data represent means ± SEM of 3 experiments . A vertical line was inserted between densitometry data from control and GM6001 - or PTX - treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 . Results shown are representative of 3 independent experiments . HBMEC were treated with or without GM6001 ( 10 µM ) for 1 h prior to challenge with T . gondii ( A ) or with Pertussis Toxin ( PTx ; 100 ng / ml ) for 4 h prior to parasite challenge ( B ) . Cell lysates were used to examine total EGFR and phospho - tyrosine 1068 EGFR by immunoblot . Densitometry data represent means ± SEM of 3 experiments . A vertical line was inserted between densitometry data from control and GM6001 - or PTX - treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 . Results shown are representative of 3 independent experiments .

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Figure 7HBMEC were treated with or without GM6001 (10 µM) for 1 h prior to challenge with T. gondii (A) or with Pertussis Toxin (PTx; 100 ng/ml) for 4 h prior to parasite challenge (B). Cell lysates were used to examine total EGFR and phospho-tyrosine 1068 EGFR by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and GM6001- or PTX-treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.HBMEC were treated with or without GM6001 (10 µM) for 1 h prior to challenge with T. gondii (A) or with Pertussis Toxin (PTx; 100 ng/ml) for 4 h prior to parasite challenge (B). Cell lysates were used to examine total EGFR and phospho-tyrosine 1068 EGFR by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and GM6001- or PTX-treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.


Figure 8A , B , C HBMEC were challenged with ΔHx ( WT ) , MIC1 ko , MIC3 ko , MIC1 - 3 ko T . gondii at MOIs that yielded similar percentages of infected cells ( A ) . Cell lysates were obtained and used to examine total EGFR and phospho - tyrosine 1068 EGFR ( B ) or total Akt and phospho - Ser 473 Akt ( C ) by immunoblot . HBMEC were incubated with 10 nM of recombinant MIC4 , MIC3 , M2AP or MIC6 for 15 minutes followed by examination of total EGFR and phospho - tyrosine 1068 EGFR ( D ) or total Akt and phospho - Ser473 ( E ) by immunoblot . Densitometry data represent means ± SEM of 4 experiments . Band densities from infected cells or cells treated with MICs were compared to bands from uninfected or untreated cells ( Un ) , which were given an arbitrary number of 1 . Results shown are representative of 4 independent experiments .

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Figure 8A, B, C HBMEC were challenged with ΔHx (WT), MIC1 ko, MIC3 ko, MIC1-3 ko T. gondii at MOIs that yielded similar percentages of infected cells (A).Cell lysates were obtained and used to examine total EGFR and phospho-tyrosine 1068 EGFR (B) or total Akt and phospho-Ser 473 Akt (C) by immunoblot.HBMEC were incubated with 10 nM of recombinant MIC4, MIC3, M2AP or MIC6 for 15 minutes followed by examination of total EGFR and phospho-tyrosine 1068 EGFR (D) or total Akt and phospho-Ser473 (E) by immunoblot. Densitometry data represent means ± SEM of 4 experiments. Band densities from infected cells or cells treated with MICs were compared to bands from uninfected or untreated cells (Un), which were given an arbitrary number of 1. Results shown are representative of 4 independent experiments.


Figure 9A , hmCD40 mHEVc expressing LC3 - EGFP were treated with or without CD154 followed by challenge with T . gondii - RFP in the presence or absence of EGF ( 50 ng / ml ) or AG1478 ( 1 µM ) . LC3 accumulation around T . gondii was assessed by immunofluorescence . B , mHEVc - LC3 - EGFP cells were infected with T . gondii - T . gondii . Cells were treated with vehicle or rapamycin ( 1 µM ) 2 h after challenge with T . gondii in the presence or absent of EGF or AG1478 . Recruitment of LC3 around the parasite was examined at 5 h after challenge with T . gondii . . C , hmCD40 mHEVc - LC3 - EGFP cells were treated with or without CD154 followed by challenged with WT , MIC1 ko , MIC1 ko + MIC1 , MIC3 ko , MIC3 ko + MIC3 , MIC1 - 3 ko , MIC1 - 3 ko + MIC1 - 3 tachyzoites . LC3 accumulation around the parasite was examined by fluorescence microscopy 5 h post - challenge . D , hmCD40 mHEVc - LC3 - EGFP treated with or without CD154 were challenged with WT , MIC1 ko , MIC1 ko + MIC1 , MIC3 ko , MIC3 ko + MIC3 , MIC1 - 3 ko or MIC1 - 3 ko + MIC1 - 3 tachyzoites . Parasite load was examined 24 h post - challenge . E , HBMEC cells were challenged with WT , MIC1 ko , MIC3 ko , MIC1 - 3 ko tachyzoites followed by treatment with vehicle or rapamycin . Parasite load was examined 24 h post - challenge . F , HBMEC treated with or without CD154 or IFN - γ / TNF - α were challenged with WT , MIC1 ko , MIC3 ko , MIC1 - 3 ko tachyzoites . Monolayers were examined microscopically at 24 h post - challenge . G , hmCD40 mHEVc cells treated with or without CD154 were infected with either WT or MIC1 - 3 ko tachyzoites in the presence of complete medium ( CM ) alone or medium plus MIC3 , MIC4 , M2AP or MIC6 ( all 10 nM ) . Parasite load was determined microscopically 24 h post challenge . Results shown are representative of 3 - 4 independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9A, hmCD40 mHEVc expressing LC3-EGFP were treated with or without CD154 followed by challenge with T. gondii-RFP in the presence or absence of EGF (50 ng/ml) or AG1478 (1 µM). LC3 accumulation around T. gondii was assessed by immunofluorescence. B, mHEVc-LC3-EGFP cells were infected with T. gondii-T. gondii. Cells were treated with vehicle or rapamycin (1 µM) 2 h after challenge with T. gondii in the presence or absent of EGF or AG1478. Recruitment of LC3 around the parasite was examined at 5 h after challenge with T. gondii.. C, hmCD40 mHEVc-LC3-EGFP cells were treated with or without CD154 followed by challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko, MIC1-3 ko+MIC1-3 tachyzoites. LC3 accumulation around the parasite was examined by fluorescence microscopy 5 h post-challenge. D, hmCD40 mHEVc-LC3-EGFP treated with or without CD154 were challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko or MIC1-3 ko+MIC1-3 tachyzoites. Parasite load was examined 24 h post-challenge.E, HBMEC cells were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites followed by treatment with vehicle or rapamycin. Parasite load was examined 24 h post-challenge. F, HBMEC treated with or without CD154 or IFN-γ/TNF-α were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites. Monolayers were examined microscopically at 24 h post-challenge.G, hmCD40 mHEVc cells treated with or without CD154 were infected with either WT or MIC1-3 ko tachyzoites in the presence of complete medium (CM) alone or medium plus MIC3, MIC4, M2AP or MIC6 (all 10 nM). Parasite load was determined microscopically 24 h post challenge. Results shown are representative of 3-4 independent experiments.


Figure 1a , Flow chart of an in vivo screen to identify active DUBs by using biotin - Ub - VME pull - down assay ( left panel ) , in which the USP8 peptide was identified by mass - spectrometry analysis ( right panel ) . Silver staining and immunoblot ( middle panel ) of precipitants pulled down by biotin - Ub - VME probe of MDA - MB - 231 cells . b , Experimental procedure in vivo : flow chart and pictures of an in vivo screen to identify DUBs that promote breast cancer metastasis in mice ( left panel ) . Low metastatic MDA - MB - 231 - Luciferase / GFP breast cancer cells were infected with viral vectors expressing DUB cDNAs and subsequently intracardially injected into nude mice . The mice were monitored for 4 weeks by in vivo BLI : of the 30 mice injected with cells expressing DUB cDNA pool ; 3 had metastases in the brain and bone ( 3 / 30 ) , 4 had metastases on the back and in the bone ( 4 / 30 ) , and 9 had micro - metastasis ( 9 / 30 ) ; the 30 mice injected with control cells showed no detectable metastasis ( 30 / 30 ) . Right panel : hints of the identified DUBs were shown . c , The expression distribution of USP8 gene in breast cancer tumor tissues ( n = 1097 ) and normal tissues ( n = 572 ) , where the horizontal axis represents different groups of samples , the vertical axis represents the gene expression distribution ( left panel ) . The box - and - whisker plots represent the medians ( middle line ) , first quartiles ( lower bound line ) , third quartiles ( upper bound line ) , and the ± 1 . 5 × interquartile ranges ( whisker lines ) . Kaplan - Meier curves showing the metastasis - free survival of individuals was negatively correlated with USP8 expression by log - rank test ( right panel ) . d , MCF10A - RAS cells infected with control ( Co . sh ) or USP8 shRNA lentivirus ( # 1 and # 2 ) were analyzed in a tumor sphere assay . The number of the mammospheres with diameter > 60 μm was quantified . The graph shows the number ( No . ) of tumor spheres per 5 × 103 cells seeded . Scale bar 50 μme , MCF10A - RAS cells infected with control ( Co . sh ) or USP8 shRNA lentivirus ( # 1 and # 2 ) were subcutaneously injected into nude mice at the indicated numbers . Mean of tumor volumes at week 5 ( n = 10 mice for each group ) . f , Microscopy pictures of representative images of zebrafish tail fin injected with control or USP8 stably depleted - MDA - MB - 231 cells at 5 days post injection , n = 20 for each group . g , The final invasive areas of zebrafish embryos displaying invasion and experimental metastasis at 5 dpi ( left panel ) ; Percentage of embryos injected with control or USP8 stably depleted MDA - MB - 231 cells ( # 1 and # 2 ) displaying metastasis at 1 , 3 and 5 days post injection ( right panel ) . h , Bioluminescent images of three representative mice from each group ( n = 6 ) at week 6 injected into left heart ventricle with control or MDA - MB - 231 cells stably depleted of endogenous USP8 with two independent shRNA ( # 1 and # 2 ) . i , BLI signal ( left panel ) , number of bone metastasis of every mouse ( middle panel ) and the percentage of bone metastasis free mice in each experimental group ( n = 6 mice per group ) followed in time as in h ( right panel ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1a, Flow chart of an in vivo screen to identify active DUBs by using biotin-Ub-VME pull-down assay (left panel), in which the USP8 peptide was identified by mass-spectrometry analysis (right panel). Silver staining and immunoblot (middle panel) of precipitants pulled down by biotin-Ub-VME probe of MDA-MB-231 cells.b, Experimental procedure in vivo: flow chart and pictures of an in vivo screen to identify DUBs that promote breast cancer metastasis in mice (left panel). Low metastatic MDA-MB-231-Luciferase/GFP breast cancer cells were infected with viral vectors expressing DUB cDNAs and subsequently intracardially injected into nude mice. The mice were monitored for 4 weeks by in vivo BLI: of the 30 mice injected with cells expressing DUB cDNA pool; 3 had metastases in the brain and bone (3/30), 4 had metastases on the back and in the bone (4/30), and 9 had micro-metastasis (9/30); the 30 mice injected with control cells showed no detectable metastasis (30/30). Right panel: hints of the identified DUBs were shown.c, The expression distribution of USP8 gene in breast cancer tumor tissues (n = 1097) and normal tissues (n = 572), where the horizontal axis represents different groups of samples, the vertical axis represents the gene expression distribution (left panel). The box-and-whisker plots represent the medians (middle line), first quartiles (lower bound line), third quartiles (upper bound line), and the ±1.5× interquartile ranges (whisker lines). Kaplan-Meier curves showing the metastasis-free survival of individuals was negatively correlated with USP8 expression by log-rank test (right panel).d, MCF10A-RAS cells infected with control (Co.sh) or USP8 shRNA lentivirus (# 1 and # 2) were analyzed in a tumor sphere assay. The number of the mammospheres with diameter > 60 μm was quantified. The graph shows the number (No.) of tumor spheres per 5 × 103 cells seeded. Scale bar 50 μme, MCF10A-RAS cells infected with control (Co.sh) or USP8 shRNA lentivirus (# 1 and # 2) were subcutaneously injected into nude mice at the indicated numbers. Mean of tumor volumes at week 5 (n = 10 mice for each group).f, Microscopy pictures of representative images of zebrafish tail fin injected with control or USP8 stably depleted-MDA-MB-231 cells at 5 days post injection, n = 20 for each group. g, The final invasive areas of zebrafish embryos displaying invasion and experimental metastasis at 5 dpi (left panel); Percentage of embryos injected with control or USP8 stably depleted MDA-MB-231 cells (# 1 and # 2) displaying metastasis at 1, 3 and 5 days post injection (right panel).h, Bioluminescent images of three representative mice from each group (n = 6) at week 6 injected into left heart ventricle with control or MDA-MB-231 cells stably depleted of endogenous USP8 with two independent shRNA (# 1 and # 2). i, BLI signal (left panel), number of bone metastasis of every mouse (middle panel) and the percentage of bone metastasis free mice in each experimental group (n = 6 mice per group) followed in time as in h (right panel).


Figure 2a , Heatmap of USP8 - correlated genes in a comparison of breast cancer patients with USP8 - high ( n = 38 ) versus with USP8 - low ( n = 39 ) . b , Pre - ranked gene set enrichment analysis ( GSEA ) in USP8 - high ( n = 38 ) versus USP8 - low ( n = 39 ) breast cancer patients . c , Experimental procedure in vivo : MDA - MB - 231 cells were intracardially injected into nude mice ( n = 10 ) . qRT - PCR analysis of bone metastatic cells showing the correlations between USP8 and Snai1 or Snai2 ( Slug ) in cells isolated from mutiple metastatic nodules ( n = 21 ) from mice . d , 3D spheroid invasion assay of MCF10A - Ras cells showing USP8 wt increased TGF - β - induced invasion . Relative invasion was quantified and shown in the right panel . e , MCF10A - Ras cells stably expressing control vector ( Co . vec ) , USP8 wt or USP8 cs were injected into the blood circulation of 48 - hpf zebrafish embryos . SB431542 ( 5 μM ) was added to the zebrafish environment . Representative images of zebrafish at 5 dpi ( left panel ) ; the experimental metastatic areas ( middle panel ) and percentage of embryos showing metastasis ( right panel ) are shown , n = 20 . f , Diagram of DUB cDNA screening data of TGF - β - induced SMAD3 / SMAD4 - dependent CAGA12 - Luc transcriptional reporter in HEK293T cells . The X - and Y - axes are the relative luciferase activity in two replicates . g , CAGA12 - Luc transcriptional response of HEK293T cells transfected with USP8 wt / cs or shUSP8 ( # 1 and # 2 ) as indicated and treated with TGF - β ( 1 ng / ml ) overnight . h , Heatmap of TGF - β target genes by qRT - PCR analysis in control or MCF10A - RAS cells stably depleting USP8 ( # 1 and # 2 shRNA ) or expressing USP8 wt / cs and treated with or without TGF - β ( 2 . 5 ng / ml ) for 8 hrs . Appendix Table S2 for details . i , Immunoblot analysis of total cell lysates , immunoprecipitates derived from control and USP8 stably depleted ( # 1 and # 2 shRNA ) ( left panel ) and USP8 wt / cs over - expressed ( right panel ) MDA - MB - 231 cells treated with TGF - β ( 2 . 5 ng / ml ) for indicated time points . j , Immunofluorescence and 4 , 6 - diamidino - 2 - phenylindole ( DAPI ) staining of HaCaT cells infected with control or USP8 wt / cs and treated with TGF - β ( 2 . 5 ng / ml ) and SB431542 ( 10 μM ) for 72 hrs . Scale bar , 20 μm . k , Immunoblot analysis of cell lysate of control and USP8 stably depleted - MCF10AR - RAS ( MII ) cells treated with TGF - β ( 5 ng / ml ) for 48 hrs ( left ) ; Immunoblot analysis of cell lysate of control and USP8 wt or USP8 cs stably expressed - MCF10AR - RAS ( MII ) cells treated with TGF - β ( 5 ng / ml ) or SB431542 ( 10 μM ) for 48 hrs ( right ) .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2a, Heatmap of USP8-correlated genes in a comparison of breast cancer patients with USP8-high (n = 38) versus with USP8-low (n = 39).b, Pre-ranked gene set enrichment analysis (GSEA) in USP8-high (n = 38) versus USP8-low (n = 39) breast cancer patients.c, Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice (n = 10). qRT-PCR analysis of bone metastatic cells showing the correlations between USP8 and Snai1 or Snai2 (Slug) in cells isolated from mutiple metastatic nodules (n = 21) from mice.d, 3D spheroid invasion assay of MCF10A-Ras cells showing USP8 wt increased TGF-β-induced invasion. Relative invasion was quantified and shown in the right panel.e, MCF10A-Ras cells stably expressing control vector (Co.vec), USP8 wt or USP8 cs were injected into the blood circulation of 48-hpf zebrafish embryos. SB431542 (5 μM) was added to the zebrafish environment. Representative images of zebrafish at 5 dpi (left panel); the experimental metastatic areas (middle panel) and percentage of embryos showing metastasis (right panel) are shown, n = 20.f, Diagram of DUB cDNA screening data of TGF-β-induced SMAD3/ SMAD4-dependent CAGA12-Luc transcriptional reporter in HEK293T cells. The X- and Y-axes are the relative luciferase activity in two replicates.g, CAGA12-Luc transcriptional response of HEK293T cells transfected with USP8 wt/cs or shUSP8 (#1 and #2) as indicated and treated with TGF-β (1 ng/ml) overnight.h, Heatmap of TGF-β target genes by qRT-PCR analysis in control or MCF10A-RAS cells stably depleting USP8 (#1 and #2 shRNA) or expressing USP8 wt/cs and treated with or without TGF-β (2.5 ng/ml) for 8 hrs. Appendix Table S2 for details.i, Immunoblot analysis of total cell lysates, immunoprecipitates derived from control and USP8 stably depleted (#1 and #2 shRNA) (left panel) and USP8 wt/cs over-expressed (right panel) MDA-MB-231 cells treated with TGF-β (2.5 ng/ml) for indicated time points.j, Immunofluorescence and 4, 6-diamidino-2-phenylindole (DAPI) staining of HaCaT cells infected with control or USP8 wt/cs and treated with TGF-β (2.5 ng/ml) and SB431542 (10 μM) for 72 hrs. Scale bar, 20 μm.k, Immunoblot analysis of cell lysate of control and USP8 stably depleted-MCF10AR-RAS (MII) cells treated with TGF-β (5 ng/ml) for 48 hrs (left); Immunoblot analysis of cell lysate of control and USP8 wt or USP8 cs stably expressed-MCF10AR-RAS (MII) cells treated with TGF-β (5 ng/ml) or SB431542 (10 μM) for 48 hrs (right).


Figure 3a , Immunofluorescence and DAPI staining of HeLa cells transfected with TβRII - HA and Flag - USP8 . Scale bar , 10 μm . b , USP8 interacts with TβRII . Purified prokaryotically expressed USP8 and recombinant GST - TβRII - ICD were incubated in vitro and immunoprecipitated with anti - USP8 antibody ; immunoprecipitates were then immunoblotted for TβRII - ICD and USP8 ( top panel ) ; Immunoblot analysis of total cell lysate derived from MDA - MB - 231 cells treated with TGF - β as indicated and immunoprecipitated with control IgG ( ns ) or anti - TβRII antibody ( bottom panel ) . c , Immunoblot analysis of anti - TβRII immunoprecipitates from HEK293T cells stably expressing HA - Ub and in vitro incubated with purified USP8 wt / cs . d , IB of Nickle - pull down precipitants derived from His - Ub stably expressed HEK293T cells incubated with purified USP8 protein as indicated time points . e , IB of TCL and immunoprecipitants derived from HEK293T cells transfected with indicated plasmids and treat with or without MG132 ( 10 μM ) for 4 h . f , IB of TCL and immunoprecipitants derived from HA - Ub stably expressed HEK293T cells transfected with Myc - USP8 wt / cs ( left panel ) or infected with control ( Co . sh ) or USP8 shRNA lentivirus ( # 1 and # 2 ) ( right panel ) and treated with TGF - β ( 5 ng / ml ) as indicated . g , IB of HeLa cells stably expressing TβRII - HA and transfected with control empty vector ( Co . vector ) and Flag - USP8 wt / cs plasmids and treated with CHX ( 20 μg / ml ) for indicated time points . h , Immunoblot analysis of MCF10A - RAS cells stably expressing USP8 wt / cs or USP8 shRNA ( # 1 and # 2 ) . i , Immunoblot analysis of biotinylated TβRII in MDA - MB - 231 cells ectopically expressing USP8 wt / cs and treated with TGF - β ( 5 ng / ml ) as indicated ( left panel ) . Quantification of the band intensities is shown in right panel . Band intensity was normalized to the t = 0 controls ( right panel ) . n = 2 technical replicates . j , Immunoblot analysis of biotinylated TβRII in MDA - MB - 231 cells infected with lentivirus encoding control shRNA ( Co . sh ) , USP8 shRNA ( # 1 and # 2 ) and treated with TGF - β ( 5 ng / ml ) as indicated ( left panel ) . Quantification of the band intensities is shown in right panel . Band intensity was normalized to the t = 0 controls ( right panel ) . l , Expression of USP8 of normal tissues ( n = 11 ) and breast cancer patients ( n = 110 ) . m , Scatter - plot showing the positive correlation of USP8 , TβRII and p - SMAD2 expression in patients ( n = 110 ) . H score of every tissue samples of USP8 , TβRII and p - SMAD2 was calculated . n , Percentage of specimens displaying low or high USP8 expression compared to the expression levels of TβRII and P - SMAD2 in human breast cancer tissue ( n = 110 ) . p values from Student ' s t tests were indicated . o , GEPIA analysis curves showing the metastasis - free survival of breast cancer patients was significantly correlated with USP8 expression by log - rank test in the TCGA and GTEx datasets ( http : / / gepia . cancer - pku . cn / ) . Cutoff - high = 67 % , cutoff - low = 25 % .

O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3a, Immunofluorescence and DAPI staining of HeLa cells transfected with TβRII-HA and Flag-USP8. Scale bar, 10 μm.b, USP8 interacts with TβRII. Purified prokaryotically expressed USP8 and recombinant GST-TβRII-ICD were incubated in vitro and immunoprecipitated with anti-USP8 antibody; immunoprecipitates were then immunoblotted for TβRII-ICD and USP8 (top panel); Immunoblot analysis of total cell lysate derived from MDA-MB-231 cells treated with TGF-β as indicated and immunoprecipitated with control IgG (ns) or anti-TβRII antibody (bottom panel).c, Immunoblot analysis of anti-TβRII immunoprecipitates from HEK293T cells stably expressing HA-Ub and in vitro incubated with purified USP8 wt/cs.d, IB of Nickle-pull down precipitants derived from His-Ub stably expressed HEK293T cells incubated with purified USP8 protein as indicated time points.e, IB of TCL and immunoprecipitants derived from HEK293T cells transfected with indicated plasmids and treat with or without MG132 (10 μM) for 4 h.f, IB of TCL and immunoprecipitants derived from HA-Ub stably expressed HEK293T cells transfected with Myc-USP8 wt/cs (left panel) or infected with control (Co.sh) or USP8 shRNA lentivirus (#1 and #2) (right panel) and treated with TGF-β (5 ng/ml) as indicated.g, IB of HeLa cells stably expressing TβRII-HA and transfected with control empty vector (Co.vector) and Flag-USP8 wt/cs plasmids and treated with CHX (20 μg/ml) for indicated time points.h, Immunoblot analysis of MCF10A-RAS cells stably expressing USP8 wt/cs or USP8 shRNA (#1 and #2).i, Immunoblot analysis of biotinylated TβRII in MDA-MB-231 cells ectopically expressing USP8 wt/cs and treated with TGF-β (5 ng/ml) as indicated (left panel). Quantification of the band intensities is shown in right panel. Band intensity was normalized to the t=0 controls (right panel). n = 2 technical replicates.j, Immunoblot analysis of biotinylated TβRII in MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh), USP8 shRNA (# 1 and # 2) and treated with TGF-β (5 ng/ml) as indicated (left panel). Quantification of the band intensities is shown in right panel. Band intensity was normalized to the t=0 controls (right panel).l, Expression of USP8 of normal tissues (n = 11) and breast cancer patients (n = 110).m, Scatter-plot showing the positive correlation of USP8, TβRII and p-SMAD2 expression in patients (n = 110). H score of every tissue samples of USP8, TβRII and p-SMAD2 was calculated.n, Percentage of specimens displaying low or high USP8 expression compared to the expression levels of TβRII and P-SMAD2 in human breast cancer tissue (n = 110). p values from Student's t tests were indicated.o, GEPIA analysis curves showing the metastasis-free survival of breast cancer patients was significantly correlated with USP8 expression by log-rank test in the TCGA and GTEx datasets (http://gepia.cancer-pku.cn/). Cutoff-high = 67%, cutoff-low = 25%.


Figure 4a , Comparison of the pre - treatment levels of USP8 mRNA in patient - derived tissue samples by qRT - PCR ( left panel ) , enzyme activity of USP8 in patient - derived tissue samples by Ub - VME ( middle panel ) and TβRII + crEVs level in the plasma from breast cancer patients by ELISA ( right panel ) between breast cancer patients with or without clinical response to PTX . R , responders ; n = 16 ; NR , non - responders ; n = 14 . b , Pearson correlation of the USP8 mRNA or USP8 enzyme activity in patient - derived tissue samples with the EV - TβRII levels in the plasma from breast cancer patients without clinical response to PTX as in a ( NR , n = 14 ) . c , Objective response rate ( ORR ) for patients as in a ( n = 30 ) with high ( > 0 . 95 ng / ml ) and low ( < 0 . 95 ng / ml ) pre - treatment levels of circulating EV - TβRII . d , Pearson correlation of the percentage of IFN - γ + of CD8 + cells to the TβRII + crEV level in the plasma from breast cancer patients without clinical response to PTX as in a ( NR , n = 14 ) . e , TβRII + crEVs ( left panel ) , the USP8 enzyme activity ( midde panel ) and USP8 mRNA ( right panel ) from breast cancer patients with or without clinical response to combined therapy using anti - PD - L1 ( Atezolizumab ) and paclitaxel . R , responders ; n = 10 ( left panel ) , n = 5 ( middle and right panels , only 5 patient - derived tissue samples obtained ) . NR , non - responders ; n = 10 ( left panel ) , n = 6 ( middle and right panels , only 5 patient - derived tissue samples obtained ) . Experimental procedure in vivo : MDA - MB - 231 cells were intracardially injected into nude mice . Bioluminescent imaging ( BLI ) of representative mice from each group at week 6 injected with control ( n = 9 ) or MDA - MB - 231 cells stably ovexpressed with USP8 - WT ( n = 7 ) or USP8 - CS ( n = 9 ) . BLI images are shown ( f ( left panel ) ) . BLI signal of each mice ( f ( middle panel ) ) and number of bone metastasis ( f ( right panel ) ) of every mouseExperimental procedure in vivo : MDA - MB - 231 cells were intracardially injected into nude mice . mice from each group at week 6 injected with control ( n = 9 ) or MDA - MB - 231 cells stably ovexpressed with USP8 - WT ( n = 7 ) or USP8 - CS ( n = 9 ) . the percentage of bone metastasis free mice survival in each experimental group followed in time ( g ) . Experimental procedure in vivo : MDA - MB - 231 cells were intracardially injected into nude mice . mice from each group at week 6 injected with control ( n = 9 ) or MDA - MB - 231 cells stably ovexpressed with USP8 - WT ( n = 7 ) or USP8 - CS ( n = 9 ) . Fold change of circulating EV - TβRII by ELISA analysis in plasma samples of mice in each experimental group at week 6 ( h ) . nude mice were tail vein - injected with 4T07 - Luc cells ( 5 × 105 cells per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs and tumors were grown for 2 weeks , followed by the administration of doxycycline ( n = 6 for each group ) Normalized BLI signals ( left panel ) and representative mice ( right panel ) from each group at the indicated times ( j ) . nude mice were tail vein - injected with 4T07 - Luc cells ( 5 × 105 cells per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs and tumors were grown for 2 weeks , followed by the administration of doxycycline ( n = 6 for each group ) Immunoblot analysis of the circulating EVs from plasma in mice ( k ) . nude mice were tail vein - injected with 4T07 - Luc cells ( 5 × 105 cells per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs and tumors were grown for 2 weeks , followed by the administration of doxycycline ( n = 6 for each group ) ELISA analysis of circulating exosomal TβRII level in plasma samples of mice at the indicated times ( l ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4a, Comparison of the pre-treatment levels of USP8 mRNA in patient-derived tissue samples by qRT-PCR (left panel), enzyme activity of USP8 in patient-derived tissue samples by Ub-VME (middle panel) and TβRII+ crEVs level in the plasma from breast cancer patients by ELISA (right panel) between breast cancer patients with or without clinical response to PTX. R, responders; n = 16; NR, non-responders; n = 14.b, Pearson correlation of the USP8 mRNA or USP8 enzyme activity in patient-derived tissue samples with the EV-TβRII levels in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).c, Objective response rate (ORR) for patients as in a (n = 30) with high (>0.95 ng/ml) and low (<0.95 ng/ml) pre-treatment levels of circulating EV-TβRII.d, Pearson correlation of the percentage of IFN-γ+ of CD8+ cells to the TβRII+ crEV level in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).e, TβRII+ crEVs (left panel), the USP8 enzyme activity (midde panel) and USP8 mRNA (right panel) from breast cancer patients with or without clinical response to combined therapy using anti-PD-L1 (Atezolizumab) and paclitaxel. R, responders; n = 10 (left panel), n = 5 (middle and right panels, only 5 patient-derived tissue samples obtained). NR, non-responders; n = 10 (left panel), n = 6 (middle and right panels, only 5 patient-derived tissue samples obtained).Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. Bioluminescent imaging (BLI) of representative mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). BLI images are shown (f (left panel)). BLI signal of each mice (f (middle panel)) and number of bone metastasis (f (right panel)) of every mouseExperimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). the percentage of bone metastasis free mice survival in each experimental group followed in time (g).Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). Fold change of circulating EV-TβRII by ELISA analysis in plasma samples of mice in each experimental group at week 6 (h).nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) Normalized BLI signals (left panel) and representative mice (right panel) from each group at the indicated times (j).nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) Immunoblot analysis of the circulating EVs from plasma in mice (k).nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) ELISA analysis of circulating exosomal TβRII level in plasma samples of mice at the indicated times (l).


Figure 5a , Gene signatures indicating the Golgi - associated vesicle biogenesis - and membrane trafficking - related genes are significantly enriched in USP8 - high ( n = 38 ) patients versus USP8 - low ( n = 39 ) expressing patients ; shown by pre - ranked gene - set enrichment analysis ( GSEA ) . b , Heatmap shows the up - regulated genes upon USP8 over - expression and the down - regulated genes upon USP8 depletion when compared with control MDA - MB - 231 cells by RNA - seq analysis ( left panel ) . qRT - PCR analysis of control or MDA - MB - 231 cells stably over - expressing USP8 ( USP8 OE ) or USP8 shRNA ( right panel ) . e , FACS analysis of EVs from 4T1 and MDA - MB - 231 cells infected with lentivirus encoding control shRNA ( Co . sh ) or USP8 shRNA . f , Immunoblot analysis of EVs from MDA - MB - 231 cells infected with lentivirus encoding control shRNA ( Co . sh ) or USP8 shRNA ( # 1 & # 2 ) . g , Immunoblot analysis of the EVs derived from MDA - MB - 231 cells ectopically expressing USP8 wt / cs . h , Experimental procedure in vivo : BALB / c mice were nipple injected with 4T1 - Luc cells ( 5 × 105 cells per mouse ) expressing control shRNA or doxycycline - inducible shRNA targeting USP8 ( shUSP8 ) and tumors were grown for 3 weeks , followed by the administration of doxycycline for 3 weeks ( n = 6 for each group ) . i , Quantification of the percentage of TβRII + crEVs in plasma samples from mice at day 24 . j , Immunoblot analysis of tumor and the TEVs ( left panel ) and quantification of the percentage of TβRII + EVs ( right panel ) in plasma samples from mice at day 42 . k , Normalized photon ﬂux at the indicated times . l , l , Representative BLI signals of primary tumor in each group at day 42 after implantation of 4T1 cells ( 2 × 105 cells per mouse , n = 6 per group ) . m , Normalized BLI signals ( left panel ) and bioluminescent images ( right panel ) of the lung metastasis nodules . Scale bars , 2 mm . n , Percentage of CD8 + T cells of tumor - draining lymph nodes ( TDLN ) at day 42 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 5a, Gene signatures indicating the Golgi-associated vesicle biogenesis- and membrane trafficking-related genes are significantly enriched in USP8-high (n = 38) patients versus USP8-low (n = 39) expressing patients; shown by pre-ranked gene-set enrichment analysis (GSEA).b, Heatmap shows the up-regulated genes upon USP8 over-expression and the down-regulated genes upon USP8 depletion when compared with control MDA-MB-231 cells by RNA-seq analysis (left panel). qRT-PCR analysis of control or MDA-MB-231 cells stably over-expressing USP8 (USP8 OE) or USP8 shRNA (right panel).e, FACS analysis of EVs from 4T1 and MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh) or USP8 shRNA.f, Immunoblot analysis of EVs from MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh) or USP8 shRNA (#1 & #2).g, Immunoblot analysis of the EVs derived from MDA-MB-231 cells ectopically expressing USP8 wt/cs.h, Experimental procedure in vivo: BALB/c mice were nipple injected with 4T1-Luc cells (5 × 105 cells per mouse) expressing control shRNA or doxycycline-inducible shRNA targeting USP8 (shUSP8) and tumors were grown for 3 weeks, followed by the administration of doxycycline for 3 weeks (n = 6 for each group). i, Quantification of the percentage of TβRII+ crEVs in plasma samples from mice at day 24. j, Immunoblot analysis of tumor and the TEVs (left panel) and quantification of the percentage of TβRII+ EVs (right panel) in plasma samples from mice at day 42. k, Normalized photon ﬂux at the indicated times. l,l, Representative BLI signals of primary tumor in each group at day 42 after implantation of 4T1 cells (2 × 105 cells per mouse, n = 6 per group).m, Normalized BLI signals (left panel) and bioluminescent images (right panel) of the lung metastasis nodules. Scale bars, 2 mm.n, Percentage of CD8+ T cells of tumor-draining lymph nodes (TDLN) at day 42.