Figure 1E ) In vitro tumor T cell co - culture competition assay in IFNGR1 - KO D10 cells containing sgRNAs targeting TSC1 , TSC2 or both . Statistics was done by Kruskal - Wallis test with Dunn ' s post hoc test . Error bars indicate SD of four biological replicates with different T cell donors ( n = 4 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . F ) In vitro tumor T cell co - culture competition assay in multiple melanoma cell lines containing indicated sgRNAs . Statistics was done by Kruskal - Wallis test with Dunn ' s post hoc test . Error bars indicate SD of six biological replicates with different cell lines ( n = 6 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . I ) In vitro T cell co - culture competition assay of sgCtrl and sgTsc2 - expressing lung cancer cell lines . Statistics was done by Mann - Whitney test . Error bars indicate SD of five biological replicates with different T cell donors ( n = 5 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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 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

Figure 1E) In vitro tumor T cell co-culture competition assay in IFNGR1-KO D10 cells containing sgRNAs targeting TSC1, TSC2 or both. Statistics was done by Kruskal-Wallis test with Dunn's post hoc test. Error bars indicate SD of four biological replicates with different T cell donors (n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.F) In vitro tumor T cell co-culture competition assay in multiple melanoma cell lines containing indicated sgRNAs. Statistics was done by Kruskal-Wallis test with Dunn's post hoc test. Error bars indicate SD of six biological replicates with different cell lines (n = 6). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.I) In vitro T cell co-culture competition assay of sgCtrl and sgTsc2-expressing lung cancer cell lines. Statistics was done by Mann-Whitney test. Error bars indicate SD of five biological replicates with different T cell donors (n = 5). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


Figure 2B ) Tumor growth upon adoptive cell transfer with Ctrl ( n = 9 ) or MART - 1 ( n = 8 ) T cells . Data points represent average tumor volume , and error bars represent SEM . D ) Quantification of the in vivo competition assay ( output ) at end point by flow cytometry analysis . Error bars indicate SD . Statistical analysis was performed by Mann - Whitney test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . For this specific experiment , mice that deceased before the analysis ( n = 1 , Ctrl T cell group ) , with failed ACT injection ( n = 1 , MART - 1 T cell group ) or were completely tumor - free after ACT ( n = 1 , MART - 1 T cell group ) were excluded from the analysis .

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Figure 2B) Tumor growth upon adoptive cell transfer with Ctrl (n = 9) or MART-1 (n = 8) T cells. Data points represent average tumor volume, and error bars represent SEM.D) Quantification of the in vivo competition assay (output) at end point by flow cytometry analysis. Error bars indicate SD. Statistical analysis was performed by Mann-Whitney test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. For this specific experiment, mice that deceased before the analysis (n=1, Ctrl T cell group), with failed ACT injection (n=1, MART-1 T cell group) or were completely tumor-free after ACT (n=1, MART-1 T cell group) were excluded from the analysis.


Figure 3A ) Western blot analysis of baseline mTOR signaling in sgCtrl - or sgTsc2 - expressing melanoma and lung cancer cell lines . Representative plot of 2 - 6 independent experiments . All blots shown in this panel were run in parallel . E ) Western blot analysis of sgCtrl - or sgTSC2 - expressing D10 melanoma cells upon MART - 1 T cell challenge for indicated time . Representative of three biological replicates with different T cell donors ( n = 3 ) . F ) In vitro competition assay of sgCtrl - and sgTsc2 - expressing D10 cells co - cultured with MART - 1 T cells in the presence of necroptosis ( Nec1 - s , 20uM ) or apoptosis inhibitors ( Q - VD - Oph , 50uM ) , normalized to inhibitor plus Ctrl T cell - treated groups , showing the net MART - 1 T cell effect . Statistical analysis was performed by one - way ANOVA with Holm - Sidak ' s multiple comparisons test . Error bars indicate SD of three biological replicates with different T cell donors ( n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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 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

Figure 3A) Western blot analysis of baseline mTOR signaling in sgCtrl- or sgTsc2-expressing melanoma and lung cancer cell lines. Representative plot of 2-6 independent experiments. All blots shown in this panel were run in parallel.E) Western blot analysis of sgCtrl- or sgTSC2-expressing D10 melanoma cells upon MART-1 T cell challenge for indicated time. Representative of three biological replicates with different T cell donors (n = 3).F) In vitro competition assay of sgCtrl- and sgTsc2-expressing D10 cells co-cultured with MART-1 T cells in the presence of necroptosis (Nec1-s, 20uM) or apoptosis inhibitors (Q-VD-Oph, 50uM), normalized to inhibitor plus Ctrl T cell-treated groups, showing the net MART-1 T cell effect. Statistical analysis was performed by one-way ANOVA with Holm-Sidak's multiple comparisons test. Error bars indicate SD of three biological replicates with different T cell donors (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


Figure 4A ) Cell viability analysis by CellTiter - Blue assay of sgCtrl - or sgTSC2 - expressing D10 melanoma and A549 lung cancer cells treated with TRAIL at indicated concentrations for three days . Statistical analysis was performed by two - tailed unpaired Student t test . For D10 , error bars represent SD from three independent experiments ( n = 3 ) . For A549 , error bars represent SD of pooled data from two independent experiments with three technical replicates each ( n = 2 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . C ) Flow cytometry analysis of TRAIL - R2 surface expression level in multiple sgTSC2 - expressing tumor cell lines compared to their sgCtrl - expressing controls . Statistical analysis was performed by two - way ANOVA with Sidak ' s multiple comparisons test . Error bars represent SD of three biological replicates ( n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . D ) In vitro competition assay performed by mixing control and tumor cells with ablation for either TSC2 , TRAIL receptors ( TNFRSF10A and TNFRSF10B ) , or both in a co - culture with MART - 1 T cells . Statistical analysis was performed with a one - way ANOVA , followed by a Tukey post - hoc test . Error bars indicate SD of four biological replicates with different T cell donors . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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 O 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

Figure 4A) Cell viability analysis by CellTiter-Blue assay of sgCtrl- or sgTSC2-expressing D10 melanoma and A549 lung cancer cells treated with TRAIL at indicated concentrations for three days. Statistical analysis was performed by two-tailed unpaired Student t test. For D10, error bars represent SD from three independent experiments (n = 3). For A549, error bars represent SD of pooled data from two independent experiments with three technical replicates each (n = 2). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.C) Flow cytometry analysis of TRAIL-R2 surface expression level in multiple sgTSC2- expressing tumor cell lines compared to their sgCtrl-expressing controls. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparisons test. Error bars represent SD of three biological replicates (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.D) In vitro competition assay performed by mixing control and tumor cells with ablation for either TSC2, TRAIL receptors (TNFRSF10A and TNFRSF10B), or both in a co-culture with MART-1 T cells. Statistical analysis was performed with a one-way ANOVA, followed by a Tukey post-hoc test. Error bars indicate SD of four biological replicates with different T cell donors. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


Figure 5B ) TSC2 expression : TRAIL signaling expression ratio for predicting ICB treatment responses in pre - treatment patient samples Combo , anti - PD - 1 plus anti - CTLA - 4 combination treatment . Statistical significance was calculated with Student t test for normally distributed data , or with Mann - Whitney test for non - normally distributed data . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . Partial response and complete response ( PRCR ) are defined as responder ( R ) ; stable disease ( SD ) and progressive disease ( PD ) are defined as non - responder ( NR ) . C ) TSC2 immune challenge signature expression in cohorts of patients after ICB treatment . Early during treatment ( EDT ) , anti - PD - 1 + anti - CTLA - 4 , and anti - PD - 1 alone treatment cohorts . Statistical significance was calculated with Student t test for normally distributed data , or with Mann - Whitney test for non - normally distributed data . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . Partial response and complete response ( PRCR ) are defined as responder ( R ) ; stable disease ( SD ) and progressive disease ( PD ) are defined as non - responder ( NR ) .

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Figure 5B) TSC2 expression: TRAIL signaling expression ratio for predicting ICB treatment responses in pre-treatment patient samples Combo, anti-PD-1 plus anti-CTLA-4 combination treatment. Statistical significance was calculated with Student t test for normally distributed data, or with Mann-Whitney test for non-normally distributed data. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Partial response and complete response (PRCR) are defined as responder (R); stable disease (SD) and progressive disease (PD) are defined as non-responder (NR).C) TSC2 immune challenge signature expression in cohorts of patients after ICB treatment. Early during treatment (EDT), anti-PD-1 + anti-CTLA-4, and anti-PD-1 alone treatment cohorts. Statistical significance was calculated with Student t test for normally distributed data, or with Mann-Whitney test for non-normally distributed data. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Partial response and complete response (PRCR) are defined as responder (R); stable disease (SD) and progressive disease (PD) are defined as non-responder (NR).


Figure 2Ala / Phe substitutions of aa 239 - 244 were introduced in the NCT ectodomain to evaluate the role of this region in the regulation of GSEC processivity . Aβ38 , Aβ40 , and Aβ42 levels present in the conditioned medium collected from KO NCT MEF cells rescued with WT or respective mutant NCT GSECs and transiently expressing with APPC99 were quantified by ELISA . Aβ ( 38 + 40 ) / 42 ratio was calculated to determine GSEC processivity towards APPC99 . DKO PSEN1 / PSEN2 MEFs rescued with the indicated FAD PSEN1 mutant and transduced with APPC99 were used as references . Representative SDS - PAGE / western blot analysis of CHAPSO solubilized membranes from KO NCT MEF cell lines stably expressing WT or mutant NCT subunits . The presence of mature , glycosylated NCT , N - terminal and C - terminal fragments of the endoproteolysed PSEN1 and PEN - 2 , compared to NCT knock - out ( KO ) cells , indicate that WT and mutated NCTs reconstitute GSEC complexes . Arrowheads indicate the position of molecular weight markers . Quantification of the effects of the indicated substitutions of aa 241 , 242 , and 244 in NCT on GSEC processivity of APPC99 , estimated as indicated above by the Aβ ( 38 + 40 ) / 42 ratio . Figure EV1B shows the corresponding Aβ42 / 40 ratios .

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 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 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

Figure 2Ala/Phe substitutions of aa 239-244 were introduced in the NCT ectodomain to evaluate the role of this region in the regulation of GSEC processivity. Aβ38, Aβ40, and Aβ42 levels present in the conditioned medium collected from KO NCT MEF cells rescued with WT or respective mutant NCT GSECs and transiently expressing with APPC99 were quantified by ELISA. Aβ(38+40)/42 ratio was calculated to determine GSEC processivity towards APPC99. DKO PSEN1/PSEN2 MEFs rescued with the indicated FAD PSEN1 mutant and transduced with APPC99 were used as references.Representative SDS-PAGE/western blot analysis of CHAPSO solubilized membranes from KO NCT MEF cell lines stably expressing WT or mutant NCT subunits. The presence of mature, glycosylated NCT, N-terminal and C-terminal fragments of the endoproteolysed PSEN1 and PEN-2, compared to NCT knock-out (KO) cells, indicate that WT and mutated NCTs reconstitute GSEC complexes. Arrowheads indicate the position of molecular weight markers.Quantification of the effects of the indicated substitutions of aa 241, 242, and 244 in NCT on GSEC processivity of APPC99, estimated as indicated above by the Aβ(38+40)/42 ratio. Figure EV1B shows the corresponding Aβ42/40 ratios.


Figure 3 ( Top ) Representative bright field microscopy images show MEF cells stably expressing WT or NCT mutant GSECs after 24h incubation at 37 ° C and 42 ° C . 25µm scale bar is shown . ( Bottom ) Cell viability determined at the indicated time points is not affected by prolonged ( ≥ 24h ) incubation at 42 ° C . Data are presented as mean ± SD of 4 replicates . Relative GSEC - APP / Aβn stabilities were assessed in cell - based GSEC thermoactivity assays , which evaluate changes in GSEC processivity ( Aβ ( 38 + 40 ) / 42 ratio ) upon incubation at 42 ° C for 24h , relative to 37 ° C . Aβ peptides in the conditioned medium collected from the indicated MEF cell lines ( transiently expressing APPC99 ) were quantified and the Aβ ( 38 + 40 ) / 42 ratios determined . The data are shown as mean ± 95 % CI , as % of the WT GSEC cell line incubated at 37 ° C for 24h , N = 6 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine the statistical significance ( F ( DFn , DFd ) : F ( 6 , 46 ) = 100 ) ; * * * * p > 0 . 0001 compared to WT at 37 ° C , # p > 0 . 05 , # # p > 0 . 01 , # # # # p > 0 . 0001 compared to WT at 42 ° C . Note : Aβ profiles ( including Aβ43 levels ) normalized to total Aβ quantified in the cell - based thermoactivity assays with the different mNCT WT / mutants are shown in Figure EV2 .

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Figure 3(Top) Representative bright field microscopy images show MEF cells stably expressing WT or NCT mutant GSECs after 24h incubation at 37°C and 42°C. 25µm scale bar is shown. (Bottom) Cell viability determined at the indicated time points is not affected by prolonged (≥ 24h) incubation at 42°C. Data are presented as mean ± SD of 4 replicates.Relative GSEC-APP/Aβn stabilities were assessed in cell-based GSEC thermoactivity assays, which evaluate changes in GSEC processivity (Aβ(38+40)/42 ratio) upon incubation at 42°C for 24h, relative to 37°C. Aβ peptides in the conditioned medium collected from the indicated MEF cell lines (transiently expressing APPC99) were quantified and the Aβ(38+40)/42 ratios determined. The data are shown as mean ± 95% CI, as % of the WT GSEC cell line incubated at 37°C for 24h, N = 6 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine the statistical significance (F(DFn, DFd): F(6, 46) = 100); ****p > 0.0001 compared to WT at 37°C, #p > 0.05, ##p>0.01, ####p>0.0001 compared to WT at 42°C. Note: Aβ profiles (including Aβ43 levels) normalized to total Aβ quantified in the cell-based thermoactivity assays with the different mNCT WT/mutants are shown in Figure EV2.


Figure 4Aβ profiles ( % contribution of individual Aβ37 , Aβ38 , Aβ40 , and Aβ42 peptides to the total Aβ levels ( Aβ37 + 38 + 40 + 42 ) ) in the conditioned medium collected from WT or mutant ( NCT ) MEF cells lines co - expressing WT or mutant APPC99 substrates were assayed by ELISA . Schematic models of the tested GSEC - APP complexes are shown . To account for the increased production of Aβ37 and Aβ38 at the expense of Aβ40 and Aβ42 in the tested conditions , the ratio of ( ( Aβ 37 / 40 ) + ( Aβ 38 / 42 ) ) / 2 was determined and used to estimate GSEC processivity of APPC99 . All the data are presented as mean ± SD , N ≥ 3 , Unpaired , two - tailed Student ' s t - test was used to determine the statistical significance . * * p > 0 . 01 , * * * * p > 0 . 0001 . Calculated T - and Df values were : T = 11 , 63 ; Df = 16 ( I241 - K28 vs I241E - K28 ) , T = 11 , 37 ; Df = 14 ( I241 - K28 vs I241 - K28E ) , T = 4 , 572 ; Df = 8 ( I241E - K28 vs I241 - K28E ) , T = 4 , 497 ; Df = 5 ( I241 - K28E vs I241K - K28E ) , T = 2 , 561 ; Df = 7 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from WT or mutant (NCT) MEF cells lines co-expressing WT or mutant APPC99 substrates were assayed by ELISA. Schematic models of the tested GSEC-APP complexes are shown.To account for the increased production of Aβ37 and Aβ38 at the expense of Aβ40 and Aβ42 in the tested conditions, the ratio of ((Aβ 37/40) + (Aβ 38/42))/2 was determined and used to estimate GSEC processivity of APPC99. All the data are presented as mean ± SD, N ≥ 3, Unpaired, two-tailed Student's t-test was used to determine the statistical significance. **p > 0.01, ****p > 0.0001. Calculated T- and Df values were: T = 11,63; Df = 16 (I241-K28 vs I241E-K28), T = 11,37; Df = 14 (I241-K28 vs I241-K28E), T = 4,572; Df = 8 (I241E-K28 vs I241-K28E), T = 4,497; Df = 5 (I241-K28E vs I241K-K28E), T = 2,561; Df = 7.


Figure 5Introduction of Cys at position 241 in NCT allows chemical cross - linking of NCT and APPC99 with BMPS . Representative SDS - PAGE / western blot show cross - linked NCT - APPC99 , NCT and APPC99 bands in detergent - extracted membrane proteins from BMPS cross - linker - treated membranes prepared from MEF cell lines expressing the indicated WT or mutant NCT GSEC complexes together with APPC99 or in the absence of the substrate . Orange , black , and white arrows indicate the NCT - APPC99 / Aβ crosslinked , mature NCT , and immature NCT bands , respectively . Arrowheads indicate the position of molecular weight markers . Analysis of the western blot density profiles in panel ( B ) shows that co - migration of NCT ( orange arrow ) with APPC99 shifts the cross - linked band towards a higher molecular weight than the observed for mature NCT ( black arrow ) . Immature NCT is indicated with an open ( white ) arrow . Full - range molecular weight density profiles are shown in Figure EV3C Representative SDS - PAGE / western blot presents NCT - APPC99 , NCT and APPC99 expression in detergent - extracted membranes prepared from MEF cell lines co - expressing the mutant NCT - I241C GSEC complex with the APPC99 - K28C substrate . As negative control , the same sample was supplemented with β - mercaptoethanol ( BME ) to reduce all disulfide bridges . A full - range molecular weight western blot is shown in Figure EV3B . Orange , black , and white arrows indicate the NCT - APPC99 / Aβ crosslinked , mature NCT , and immature NCT bands , respectively . Arrowheads indicate the position of molecular weight markers . Analysis of the integrated density profiles of the respective western blot bands supports the spontaneous formation of a disulfide bond between NCT - I241C andAPPC99 K28C ( mature and immature NCT are indicated with black and white arrows , respectively . The filled orange arrow points at the high molecular weight APPC99 / Aβ density ( crosslinked NCT - APPC99 / Aβ band ) . Note that peak amplitudes for the cross - linked NCT - APPC99 and the mature NCT band were normalized to similar values for clarity purposes . Full - range molecular weight density profiles are shown in Figure EV3D .

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Figure 5Introduction of Cys at position 241 in NCT allows chemical cross-linking of NCT and APPC99 with BMPS. Representative SDS-PAGE/western blot show cross-linked NCT-APPC99, NCT and APPC99 bands in detergent-extracted membrane proteins from BMPS cross-linker-treated membranes prepared from MEF cell lines expressing the indicated WT or mutant NCT GSEC complexes together with APPC99 or in the absence of the substrate. Orange, black, and white arrows indicate the NCT-APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the western blot density profiles in panel (B) shows that co-migration of NCT (orange arrow) with APPC99 shifts the cross-linked band towards a higher molecular weight than the observed for mature NCT (black arrow). Immature NCT is indicated with an open (white) arrow. Full-range molecular weight density profiles are shown in Figure EV3C Representative SDS-PAGE/western blot presents NCT-APPC99, NCT and APPC99 expression in detergent-extracted membranes prepared from MEF cell lines co-expressing the mutant NCT- I241C GSEC complex with the APPC99-K28C substrate. As negative control, the same sample was supplemented with β-mercaptoethanol (BME) to reduce all disulfide bridges. A full-range molecular weight western blot is shown in Figure EV3B. Orange, black, and white arrows indicate the NCT -APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the integrated density profiles of the respective western blot bands supports the spontaneous formation of a disulfide bond between NCT- I241C andAPPC99 K28C (mature and immature NCT are indicated with black and white arrows, respectively. The filled orange arrow points at the high molecular weight APPC99/Aβ density (crosslinked NCT-APPC99/Aβ band). Note that peak amplitudes for the cross-linked NCT-APPC99 and the mature NCT band were normalized to similar values for clarity purposes. Full-range molecular weight density profiles are shown in Figure EV3D.


Figure 6Aβ profiles ( % contribution of individual Aβ37 , Aβ38 , Aβ40 , and Aβ42 peptides to the total Aβ levels ( Aβ37 + 38 + 40 + 42 ) ) in the conditioned medium collected from vehicle ( DMSO ) or GSM - treated ( 1 µM final concentration ) MEF cells co - expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA . Data generated using 0 . 3 µM GSM - treated MEF cells is shown in Figure EV4 . Data are presented as mean ± SD , N ≥ 3 independent experiments . Aβ 37 / 40 and Aβ 38 / 42 ratios were calculated to evaluate the efficiency of the GSEC - mediated cleavage of APPC99 ( Figure EV4 ) and statistical analysis ( P values ) ( two - way ANOVA with Sidak ' s post hoc test ) for the effect of GSMs on WT and mutant complexes is presented ( response to GSM normalized to the corresponding vehicle condition ) .

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Figure 6Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from vehicle (DMSO) or GSM-treated (1 µM final concentration) MEF cells co-expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA. Data generated using 0.3 µM GSM-treated MEF cells is shown in Figure EV4. Data are presented as mean ± SD, N ≥ 3 independent experiments.Aβ 37/40 and Aβ 38/42 ratios were calculated to evaluate the efficiency of the GSEC-mediated cleavage of APPC99 (Figure EV4) and statistical analysis (P values) (two-way ANOVA with Sidak's post hoc test) for the effect of GSMs on WT and mutant complexes is presented (response to GSM normalized to the corresponding vehicle condition).


Figure 1B C99 - Bpa constructs remain essentially cleavage - competent . Numbers denote the relative γ - secretase cleavage efficiencies of C99 - Bpa substrates as calculated from the AICD / C99 - Bpa ratios relative to that of WT C99 , which was set to 1 . Inefficiently cleaved substrates ( cleavage efficiency below 20 % ) were mostly found for Bpa substitutions in extramembraneous domains . Specificity of substrate cleavage was confirmed by inhibition of AICD formation in the presence of L - 685 , 458 . Residues of the TMD are highlighted in orange here and elsewhere where appropriate . C Values obtained in ( B ) were additionally plotted . Yellow line indicates the cleavage efficiency of WT C99 , which was set to 1 . ECD , extracellular domain ; ICD , intracellular domain .

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Figure 1B C99-Bpa constructs remain essentially cleavage-competent. Numbers denote the relative γ-secretase cleavage efficiencies of C99-Bpa substrates as calculated from the AICD/C99-Bpa ratios relative to that of WT C99, which was set to 1. Inefficiently cleaved substrates (cleavage efficiency below 20%) were mostly found for Bpa substitutions in extramembraneous domains. Specificity of substrate cleavage was confirmed by inhibition of AICD formation in the presence of L-685,458. Residues of the TMD are highlighted in orange here and elsewhere where appropriate.C Values obtained in (B) were additionally plotted. Yellow line indicates the cleavage efficiency of WT C99, which was set to 1. ECD, extracellular domain; ICD, intracellular domain.


Figure 2B Identification of C99 interaction sites with γ - secretase . C99 - Bpa substrates were irradiated with UV light in the presence of CHAPSO - solubilized γ - secretase . Crosslinked substrates were captured by Ni - NTA affinity pulldown and bound subunits were identified by a ~ 10 kDa molecular weight increase compared to the input . Results are shown for the major substrate crosslinking residues . Crosslink formation was not observed in the absence of UV irradiation or in the presence of Triton X - 100 , which dissociates the γ - secretase complex , proving crosslink specificity . Bracket indicates the molecular weight range of putative C99 - APH - 1 crosslink bands . APH - 1aL , long splice variant of APH - 1a . C Excess amounts of parental C99 WT substrate compete crosslinking of C99 - Bpa substrates to γ - secretase . Single and double asterisks indicate antibody crossreactivities to C99 monomer and dimer , respectively .

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Figure 2B Identification of C99 interaction sites with γ-secretase. C99-Bpa substrates were irradiated with UV light in the presence of CHAPSO-solubilized γ-secretase. Crosslinked substrates were captured by Ni-NTA affinity pulldown and bound subunits were identified by a ~10 kDa molecular weight increase compared to the input. Results are shown for the major substrate crosslinking residues. Crosslink formation was not observed in the absence of UV irradiation or in the presence of Triton X-100, which dissociates the γ-secretase complex, proving crosslink specificity. Bracket indicates the molecular weight range of putative C99-APH-1 crosslink bands. APH-1aL, long splice variant of APH-1a.C Excess amounts of parental C99 WT substrate compete crosslinking of C99-Bpa substrates to γ-secretase. Single and double asterisks indicate antibody crossreactivities to C99 monomer and dimer, respectively.


Figure 3Quantitative crosslinking analysis revealed V44 , L49 , M51 and L52 as major interactions sites of C99 with γ - secretase with high crosslinking efficiencies ranging from ~ 2 - 8 % of input . While the PS1 NTF was bound by C99 over nearly the whole range of residues , only very few residues bound to PS1 CTF . Several residues in the substrate N - terminal extracellular domain bound to NCT . PEN - 2 - binding residues clustered in the membrane - flanking extracellular and intracellular domains of C99 .

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Figure 3Quantitative crosslinking analysis revealed V44, L49, M51 and L52 as major interactions sites of C99 with γ-secretase with high crosslinking efficiencies ranging from ~2-8% of input. While the PS1 NTF was bound by C99 over nearly the whole range of residues, only very few residues bound to PS1 CTF. Several residues in the substrate N-terminal extracellular domain bound to NCT. PEN-2-binding residues clustered in the membrane-flanking extracellular and intracellular domains of C99.


Figure 4A Inhibition of substrate crosslinking by the GSIs identifies substrate residues binding in the active site . L , L - 685 , 458 ; D , DAPT . B Preventing access to the active site by GSIs identifies substrate residues interacting with the exosites by their increased labeling intensity .

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Figure 4A Inhibition of substrate crosslinking by the GSIs identifies substrate residues binding in the active site. L, L-685,458; D, DAPT.B Preventing access to the active site by GSIs identifies substrate residues interacting with the exosites by their increased labeling intensity.


Figure 5A PS1 FAD mutations display little or no effect on substrate interactions at the exosites . Asterisks indicate antibody crossreactivities to C99 monomer . B PS1 FAD mutations alter substrate binding to the PS1 NTF in the γ - secretase cleavage domain . The weak PS1 A246E FAD mutation alters substrate interactions predominantly at V49 and L52 , whereas the highly pathogenic PS1 L166P FAD mutation causes more broad changes over the whole cleavage domain . Arrows denote changes in binding of individual residues . Corresponding quantitation of substrate binding is shown below the immunoblots . Bars denote the mean ± S . E . ( n = 3 ) . Yellow line indicates level of substrate binding to PS1 WT , which was set to 1 . Blue and red bars indicate increases or decreases in binding , respectively . C Both mutants decrease crosslinking to the PS1 CTF .

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Figure 5A PS1 FAD mutations display little or no effect on substrate interactions at the exosites. Asterisks indicate antibody crossreactivities to C99 monomer.B PS1 FAD mutations alter substrate binding to the PS1 NTF in the γ-secretase cleavage domain. The weak PS1 A246E FAD mutation alters substrate interactions predominantly at V49 and L52, whereas the highly pathogenic PS1 L166P FAD mutation causes more broad changes over the whole cleavage domain. Arrows denote changes in binding of individual residues. Corresponding quantitation of substrate binding is shown below the immunoblots. Bars denote the mean ± S.E. (n = 3). Yellow line indicates level of substrate binding to PS1 WT, which was set to 1. Blue and red bars indicate increases or decreases in binding, respectively.C Both mutants decrease crosslinking to the PS1 CTF.


Figure 6B Substrates bound to exosites in the PS1 NTF at 4 ° C can be cleaved in the 37 ° C chase incubation in a L - 685 , 458 - dependent manner . Note that , although many of these substrates crosslinked only weakly at the exosites , their cleavage in the 37 ° C chase proved that these reflected functional substrate - binding states . C Substrates crosslinking in the exosites to NCT and PEN - 2 cannot be cleaved . D , E Substrates can be cleaved when crosslinked to PS1 NTF but not when crosslinked to NCT ( D ) or PEN - 2 ( E ) . F Excess amounts of Notch1 substrate N102 - FmH compete with exosite - binding of C99 . See Fig EV4 for competition of C99 binding in the active site region .

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Figure 6B Substrates bound to exosites in the PS1 NTF at 4°C can be cleaved in the 37°C chase incubation in a L-685,458-dependent manner. Note that, although many of these substrates crosslinked only weakly at the exosites, their cleavage in the 37°C chase proved that these reflected functional substrate-binding states.C Substrates crosslinking in the exosites to NCT and PEN-2 cannot be cleaved.D, E Substrates can be cleaved when crosslinked to PS1 NTF but not when crosslinked to NCT (D) or PEN-2 (E).F Excess amounts of Notch1 substrate N102-FmH compete with exosite-binding of C99. See Fig EV4 for competition of C99 binding in the active site region.


Figure 2okp1 - R164C suppressed the temperature - sensitive growth defect of cbf1 ∆ cse4 - R37A . Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC . The original okp1 - R164C isolate from the suppressor screen is indicated as " UV mutagenesis " . Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC . The original okp1 - R164C isolate from the suppressor screen is indicated as " UV mutagenesis " . okp1 - R164C suppressed the growth defect of cse4 - R37A with chl4 ∆ . okp1 - R164C was unable to suppress the lethality of cse4 - R37A with ame1 - 4 . Tetrad dissection of a genetic cross between a cse4 - R37A okp1 - R164C and an ame1 - 4 strain is shown . The four spores from individual asci are aligned in vertical rows . okp1 - R164C suppressed the maintenance defect of cse4 - R37A for plasmids lacking the CDEI sequence of CEN6 ( CEN ∆ CDEI , at 37ºC ) . Error bars give SD of at least three independent transformants . * p = 0 . 03 . The okp1 mutations I45T , S94T and E208V suppressed the temperature - sensitive growth defect of cbf1 ∆ cse4 - R37A . Strains with the indicated okp1 alleles on a plasmid ( derivatives of AEY5584 )

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Figure 2okp1-R164C suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC. The original okp1-R164C isolate from the suppressor screen is indicated as "UV mutagenesis".Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC. The original okp1-R164C isolate from the suppressor screen is indicated as "UV mutagenesis". okp1-R164C suppressed the growth defect of cse4-R37A with chl4∆.okp1-R164C was unable to suppress the lethality of cse4-R37A with ame1-4. Tetrad dissection of a genetic cross between a cse4-R37A okp1-R164C and an ame1-4 strain is shown. The four spores from individual asci are aligned in vertical rows.okp1-R164C suppressed the maintenance defect of cse4-R37A for plasmids lacking the CDEI sequence of CEN6 (CEN ∆CDEI, at 37ºC). Error bars give SD of at least three independent transformants. * p = 0.03.The okp1 mutations I45T, S94T and E208V suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Strains with the indicated okp1 alleles on a plasmid (derivatives of AEY5584)


Figure 3okp1 - R164C restored binding of Okp1 to centromeric sequences . ChIP of 9xmyc - tagged Okp1 was performed in strains with the indicated genotypes that were grown at 37ºC for 4 hours before ChIP . Enrichment of CEN4 and POL1 ( as a control ) relative to input is given . Below , Western blot analysis of the amounts of 9xmyc - Okp1 and histone H2B ( loading control ) in whole cell extracts . okp1 - R164C restored binding of Ame1 to centromeric sequences . ChIP of 9xmyc - tagged Ame1 was performed as in ( A ) . Below , Western blot analysis of the amounts of 9xmyc - Ame1 and H2B as in ( A ) . okp1 - R164C restored binding of Mtw1 to centromeric sequences . ChIP of 9xmyc - Mtw1 and Western blots ( right ) okp1 - R164C improved in vivo association of Okp1 with Cse4 - R37A . Co - immunoprecipitation of Cse4 - R37A and Okp1 was carried out in cells carrying cse4 - R37A with ( + ) or without ( - ) 3xHA - tag and OKP1 or okp1 - R164C with ( + ) or without ( - ) 9xmyc - tag ( AEY5040 , AEY5972 , AEY5973 , AEY6589 , AEY6592 ) . Cells were grown at 23ºC or were shifted to 37ºC for 5 hours prior to harvesting . Cse4 - R37A was immunoprecipitated using an α - HA antibody , and the presence of Cse4 - R37A and Okp1 or Okp1 - R164C in the immunoprecipitate was tested by Western blotting with α - HA and α - myc antibody , respectively . Left , inputs ( the two bands indicate Okp1 and a shorter degradation product ) ; right , α - HA immunoprecipitates .

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Figure 3okp1-R164C restored binding of Okp1 to centromeric sequences. ChIP of 9xmyc-tagged Okp1 was performed in strains with the indicated genotypes that were grown at 37ºC for 4 hours before ChIP. Enrichment of CEN4 and POL1 (as a control) relative to input is given. Below, Western blot analysis of the amounts of 9xmyc-Okp1 and histone H2B (loading control) in whole cell extracts.okp1-R164C restored binding of Ame1 to centromeric sequences. ChIP of 9xmyc-tagged Ame1 was performed as in (A). Below, Western blot analysis of the amounts of 9xmyc-Ame1 and H2B as in (A).okp1-R164C restored binding of Mtw1 to centromeric sequences. ChIP of 9xmyc-Mtw1 and Western blots (right)okp1-R164C improved in vivo association of Okp1 with Cse4-R37A. Co-immunoprecipitation of Cse4-R37A and Okp1 was carried out in cells carrying cse4-R37A with (+) or without (-) 3xHA-tag and OKP1 or okp1-R164C with (+) or without (-) 9xmyc-tag (AEY5040, AEY5972, AEY5973, AEY6589, AEY6592). Cells were grown at 23ºC or were shifted to 37ºC for 5 hours prior to harvesting. Cse4-R37A was immunoprecipitated using an α-HA antibody, and the presence of Cse4-R37A and Okp1 or Okp1-R164C in the immunoprecipitate was tested by Western blotting with α-HA and α-myc antibody, respectively. Left, inputs (the two bands indicate Okp1 and a shorter degradation product); right, α-HA immunoprecipitates.


Figure 4Cse4 ( 21 - 129 , Cse4N ) and Okp1 / Ame1 were expressed and purified separately from bacteria and co - incubated before separation by analytical gel filtration . Top , representative size - exclusion chromatography ( SEC ) chromatogram with absorbance measurements at 280 nanometers ( nm ) is shown . Below , image of Coomassie Blue - stained SDS - PAGE gels with fractions from principal SEC peaks of runs with Okp1 / Ame1 alone ( top ) , Cse4N alone ( middle ) and Okp1 / Ame1 pre - incubated with Cse4N before SEC ( bottom ) . Molecular weight standards are shown in kilodalton ( kDa ) . The ame1 mutations S59P , N159H , S275N and a C - terminal truncation after aa 273 ( ame1 - 273 * ) suppressed the growth defect of cbf1 ∆ cse4 - R37A . cbf1 ∆ cse4 - R37A ame1 ∆ strains carrying the indicated plasmid - borne ame1

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Figure 4Cse4(21-129, Cse4N) and Okp1/ Ame1 were expressed and purified separately from bacteria and co-incubated before separation by analytical gel filtration. Top, representative size-exclusion chromatography (SEC) chromatogram with absorbance measurements at 280 nanometers (nm) is shown. Below, image of Coomassie Blue-stained SDS-PAGE gels with fractions from principal SEC peaks of runs with Okp1/ Ame1 alone (top), Cse4N alone (middle) and Okp1/ Ame1 pre-incubated with Cse4N before SEC (bottom). Molecular weight standards are shown in kilodalton (kDa).The ame1 mutations S59P, N159H, S275N and a C-terminal truncation after aa 273 (ame1-273*) suppressed the growth defect of cbf1∆ cse4-R37A. cbf1∆ cse4-R37A ame1∆ strains carrying the indicated plasmid-borne ame1 alleles


Figure 5Cse4 - K49Ac and - R37Me are increased in S - phase of the cell cycle . Cells carrying 3xHA - tagged Cse4 were used to immunoprecipitate Cse4 with an α - Cse4 - K49Ac ( left , top ) and an α - HA antibody ( left , bottom ) . To detect Cse4 - R37 methylation , 3xHA - tagged Cse4 was immunoprecipitated with an α - Cse4 - R37Me2a ( right , top ) ( Samel et al . , 2012 ) and an α - HA antibody ( right , bottom ) . Western blots were probed for the presence of Cse4 with the α - HA antibody . G1 , treatment with α - factor ; S , arrest with hydroxyurea ; G2 / M , arrest in nocodazole ; asynch , asynchronous culture . Mimicking the unacetylated state of Cse4 - K49 ( cse4 - K49R ) partially suppressed the temperature - sensitivity of cbf1 ∆ cse4 - R37A . cbf1 ∆ cse4 ∆ strains carrying the indicated cse4 alleles on a plasmid were spotted on full medium and growncse4 - K49R suppressed the lethality of cse4 - R37A with ctf19 ∆ . Tetrad dissection of a ctf19 ∆ strain crossed with cse4 - R37A ( left ) and cse4 - R37A K49R ( right ) . RepresentationAcetylation of Cse4 - K49 was reduced in the absence of Gcn5 and the SAGA complex , but not in the absence of the HAT Sas2 . Cse4 was precipitated as in ( A ) from strains wild - type ( wt ) or gcn5 ∆ ( top ) or sas2 ∆ strains ( bottom ) carrying HA - tagged Cse4 . w / o , control immunoprecipitation without antibody . Cse4 - K49Ac depended on the SAGA components Ada2 and Spt7 . Cse4 - K49Ac was determined as in ( A , left ) with cells carrying ada2 ∆ or spt7 ∆ .

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Figure 5Cse4-K49Ac and -R37Me are increased in S-phase of the cell cycle. Cells carrying 3xHA-tagged Cse4 were used to immunoprecipitate Cse4 with an α-Cse4-K49Ac (left, top) and an α-HA antibody (left, bottom). To detect Cse4-R37 methylation, 3xHA-tagged Cse4 was immunoprecipitated with an α-Cse4-R37Me2a (right, top) (Samel et al., 2012) and an α-HA antibody (right, bottom). Western blots were probed for the presence of Cse4 with the α-HA antibody. G1, treatment with α-factor; S, arrest with hydroxyurea; G2/ M, arrest in nocodazole; asynch, asynchronous culture.Mimicking the unacetylated state of Cse4-K49 (cse4-K49R) partially suppressed the temperature-sensitivity of cbf1∆ cse4-R37A. cbf1∆ cse4∆ strains carrying the indicated cse4 alleles on a plasmid were spotted on full medium and growncse4-K49R suppressed the lethality of cse4-R37A with ctf19∆. Tetrad dissection of a ctf19∆ strain crossed with cse4-R37A (left) and cse4-R37A K49R (right). RepresentationAcetylation of Cse4-K49 was reduced in the absence of Gcn5 and the SAGA complex, but not in the absence of the HAT Sas2. Cse4 was precipitated as in (A) from strains wild-type (wt) or gcn5∆ (top) or sas2∆ strains (bottom) carrying HA-tagged Cse4. w/o, control immunoprecipitation without antibody.Cse4-K49Ac depended on the SAGA components Ada2 and Spt7. Cse4-K49Ac was determined as in (A, left) with cells carrying ada2∆ or spt7∆.


Figure 6A synthetic 78 - aa Cse4 peptide ( aa 33 - 110 ) binds to Okp1 / Ame1 in vitro . Microscale thermophoresis ( MST ) measurement of binding of fluorescently labeled Cse4 ( 33 - 110 ) to Okp1 / Ame1 is shown . Normalized fluorescence , from which the fluorescence of unbound Okp1 / Ame1 was subtracted ( ∆ F norm ) , is plotted against Cse4 ( 33 - 110 ) concentration ( logarithmic scale ) . Mean values ± SD ( n = 3 ) are shown . Plot of MST measuring the competition of fluorescently labelled Cse4 ( 33 - 110 ) bound to Okp1 / Ame1 ( black ) ( or Okp1 - R164C / Ame1 , red ) with unlabelled Cse4 ( 33 - 87 ) that is unmodified or monomethylated on R37 ( R37Me ) . Normalized fluorescence from which the fluorescence of unbound Cse4 ( 33 - 110 ) was subtracted ( ∆ F norm ) is plotted against peptide concentration . Mean values ± standard deviation ( n = 3 ) are given . Control , normalized fluorescence of Cse4 ( 33 - 110 ) alone at increasing concentrations .

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Figure 6A synthetic 78-aa Cse4 peptide (aa 33 - 110) binds to Okp1/ Ame1 in vitro. Microscale thermophoresis (MST) measurement of binding of fluorescently labeled Cse4 (33 - 110) to Okp1/ Ame1 is shown. Normalized fluorescence, from which the fluorescence of unbound Okp1/ Ame1 was subtracted (∆F norm), is plotted against Cse4(33-110) concentration (logarithmic scale). Mean values ± SD (n = 3) are shown.Plot of MST measuring the competition of fluorescently labelled Cse4 (33 - 110) bound to Okp1/ Ame1 (black) (or Okp1-R164C/ Ame1, red) with unlabelled Cse4 (33 - 87) that is unmodified or monomethylated on R37 (R37Me). Normalized fluorescence from which the fluorescence of unbound Cse4 (33 - 110) was subtracted (∆F norm) is plotted against peptide concentration. Mean values ± standard deviation (n = 3) are given. Control, normalized fluorescence of Cse4 (33 - 110) alone at increasing concentrations.


Figure 1A . Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease ( MNase ) for three types of DNA wrapped around the CENP - A nucleosome ( red ) and the H3 nucleosome ( green ) . For α - satellite DNA with initial size of 171 bp , size ranges ( 141 - 171 ) corresponding to DNA lengths above NCP ( nucleosome core particle ) is presented . Data are presented as mean ( SD ) for each time point based on three independent experiments .

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Figure 1A. Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease (MNase) for three types of DNA wrapped around the CENP-A nucleosome (red) and the H3 nucleosome (green). For α-satellite DNA with initial size of 171 bp, size ranges (141-171) corresponding to DNA lengths above NCP (nucleosome core particle) is presented. Data are presented as mean (SD) for each time point based on three independent experiments.


Figure 2B . Native PAGE gel stained with Commassie blue showing complexes formed between CENP - A or H3 nucleosome and CENP - CCR . Lane 1 : CENP - A nucleosome , Lanes 2 - 4 : Increasing amounts of CENP - CCR are added to CENP - A nucleosome . Generation of a sharp band with slower mobility indicates formation of a specific CENP - A / CENP - CCR complex . Lane 5 : H3 nucleosome . Lanes 6 - 8 : Increasing amounts of CENP - CCR are added to H3 nucleosome . Smear on the gel indicates formation of non - specific CENP - A / CENP - CCR complexes . C . Same experiment as in ( B ) using CENP - Cmotif . Lane 1 . CENP - A nucleosome . Lanes 2 - 4 . Increasing amounts of CENP - Cmotif are added to CENP - A nucleosome . Upon binding CENP - Cmotif , CENP - A nucleosome migrates slower through the gel . Note only modest change in mobility due to small size of CENP - Cmotif , comparing to CENP - CCR in ( B ) . Lane 5 . H3 nucleosome . Lanes 6 - 8 . Increasing amounts of CENP - Cmotif are added to H3 nucleosome . Smear on the gel indicates formation of non - specific CENP - A / CENP - Cmotif complexes . D . Native gel showing CENP - CCR competing out CENP - Cmotif bound to CENP - A nucleosome . Lane 1 : CENP - A nucleosome , Lane 2 : CENP - A / CENP - Cmotif complex . Lane 3 - 6 : Increasing amounts of CENP - CCR are added to the pre - formed CENP - A / CENP - Cmotif complex . Formation of slower migrating bands indicates that longer CENP - CCR is replacing shorter CENP - Cmotif bound to the CENP - A nucleosome . E . Native gel showing the inability of CENP - Cmotif to compete out CENP - CCR bound to CENP - A nucleosome . Lane 1 : CENP - A nucleosome . Lane 2 and 6 : CENP - CCR / CENP - A nucleosome complex . Lanes 3 - 5 : Increasing amounts of CENP - Cmotif are added to the pre - formed CENP - A / CENP - CCR complex . Formation of smear at high amounts of CENP - Cmotif added , indicates that CENP - Cmotif , at high concentrations , non - specifically binds CENP - A / CENP - CCR complex rather than replacing bound CENP - CCR . Lane 7 - 9 : Increasing amounts of CENP - CCR - short are added to the pre - formed CENP - A / CENP - CCR complex . Formation of bands with higher mobility indicates that smaller CENP - CCR - short is effectively replacing bigger CENP - CCR bound to CENP - A nucleosome .

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Figure 2B. Native PAGE gel stained with Commassie blue showing complexes formed between CENP-A or H3 nucleosome and CENP-CCR. Lane 1: CENP-A nucleosome, Lanes 2-4: Increasing amounts of CENP-CCR are added to CENP-A nucleosome. Generation of a sharp band with slower mobility indicates formation of a specific CENP-A/CENP-CCR complex. Lane 5: H3 nucleosome. Lanes 6-8: Increasing amounts of CENP-CCR are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-CCR complexes. C. Same experiment as in (B) using CENP-Cmotif. Lane 1. CENP-A nucleosome. Lanes 2-4. Increasing amounts of CENP-Cmotif are added to CENP-A nucleosome. Upon binding CENP-Cmotif, CENP-A nucleosome migrates slower through the gel. Note only modest change in mobility due to small size of CENP-Cmotif, comparing to CENP-CCR in (B). Lane 5. H3 nucleosome. Lanes 6-8. Increasing amounts of CENP-Cmotif are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-Cmotif complexes. D. Native gel showing CENP-CCR competing out CENP-Cmotif bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome, Lane 2: CENP-A/CENP-Cmotif complex. Lane 3-6: Increasing amounts of CENP-CCR are added to the pre-formed CENP-A/CENP-Cmotif complex. Formation of slower migrating bands indicates that longer CENP-CCR is replacing shorter CENP-Cmotif bound to the CENP-A nucleosome.E. Native gel showing the inability of CENP-Cmotif to compete out CENP-CCR bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome. Lane 2 and 6: CENP-CCR/CENP-A nucleosome complex. Lanes 3-5: Increasing amounts of CENP-Cmotif are added to the pre-formed CENP-A/CENP-CCR complex. Formation of smear at high amounts of CENP-Cmotif added, indicates that CENP-Cmotif, at high concentrations, non-specifically binds CENP-A/CENP-CCR complex rather than replacing bound CENP-CCR. Lane 7-9: Increasing amounts of CENP-CCR-short are added to the pre-formed CENP-A/CENP-CCR complex. Formation of bands with higher mobility indicates that smaller CENP-CCR-short is effectively replacing bigger CENP-CCR bound to CENP-A nucleosome.


Figure 3G . Native gels showing the impaired ability of mutated CENP - CCR to form complexes with the CENP - A nucleosome . The molar ratio of CENP - CCR / CENP - A nucleosomes is shown above each lane .

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Figure 3G. Native gels showing the impaired ability of mutated CENP-CCR to form complexes with the CENP-A nucleosome. The molar ratio of CENP-CCR/CENP-A nucleosomes is shown above each lane.


Figure 4A . Graph showing the relative abundance of undigested DNA ( 145 bp ) as a function of time during digestion with micrococcal nuclease ( MN - ase ) for the CENP - A nucleosome ( red ) , the CENP - A / CENP - CCR complex ( blue ) and the CENP - A / CENP - Cmotif complex ( black ) . D . Same type of data as in ( A ) for the CENP - A nucleosome assembled with tailless H2A ( H2A , 1 - 109 ) alone ( pink ) and in complex with CENP - CCR ( light blue ) .

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Figure 4A. Graph showing the relative abundance of undigested DNA (145 bp) as a function of time during digestion with micrococcal nuclease (MN-ase) for the CENP-A nucleosome (red), the CENP-A/CENP-CCR complex (blue) and the CENP-A/CENP-Cmotif complex (black).D. Same type of data as in (A) for the CENP-A nucleosome assembled with tailless H2A (H2A, 1-109) alone (pink) and in complex with CENP-CCR (light blue).


Figure 1Top : Schematic of primer pairs designed to discriminate between wild - type and knock - in alleles . Bottom : Representative genotyping of Ccnd1 + / + , Ccnd1 + / KI , and Ccnd1KI / KI mice validating specificity of the primer pairs and somatic insertion of the targeting construct . PCR analysis of transcript b expression in organs harvested from Ccnd1 + / + and Ccnd1KI / KI mice , demonstrating production of transcript b specifically in KI mice . Gapdh serves as a control ( NTC , Non - template control ) . Immunoblot from parallel samples in ( C ) utilizing antisera specific to the 33 amino acids generated by human CCND1 intron 4 . Lamin B serves as a control . Arrow indicates the cyclin D1b band .

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Figure 1Top: Schematic of primer pairs designed to discriminate between wild-type and knock-in alleles. Bottom: Representative genotyping of Ccnd1+/+, Ccnd1+/KI, and Ccnd1KI/KI mice validating specificity of the primer pairs and somatic insertion of the targeting construct.PCR analysis of transcript b expression in organs harvested from Ccnd1+/+ and Ccnd1KI/KI mice, demonstrating production of transcript b specifically in KI mice. Gapdh serves as a control (NTC, Non-template control).Immunoblot from parallel samples in (C) utilizing antisera specific to the 33 amino acids generated by human CCND1 intron 4. Lamin B serves as a control. Arrow indicates the cyclin D1b band.


Figure 2Neonates from 2 independent litters of Ccnd1 + / KI × Ccnd1 + / KI crosses were sacrificed at birth and genotyped . Mice were organized by genotype and total size measured . Mass of neonates was calculated at birth and average mass quantified ( n = at least 4 mice per group ) . Mice were weighed weekly , and growth is plotted as the average mass of each genotype / week ( left : male , right : female , n > 5 per gender and genotype ) . Mice were held by the tails and analyzed for the leg clasping phenotype . Representative images of Ccnd1 + / + and Ccnd1KI / KI age - matched mice are shown ( n > 10 for each group ) . Top : Individual whole - cell lysates were generated from the eyes of mice of each genotype and analyzed for expression of + / + cyclin D1 ( cyclin D1a ) and KI / KI cyclin D1b via immunoblot using antisera specific to each isoform . Bottom : Representative H & amp ; amp ; E staining of retinal tissue from Ccnd1 + / + and Ccnd1KI / KI mice showing individual layers of the retina . The ganglion cell layer and inner plexiform layer ( G + IPL ) , inner nuclear layer ( INL ) , and outer nuclear layer ( ONL ) were quantified ( right ) . Images were taken at 400 × magnification .

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Figure 2Neonates from 2 independent litters of Ccnd1+/KI × Ccnd1+/KI crosses were sacrificed at birth and genotyped. Mice were organized by genotype and total size measured.Mass of neonates was calculated at birth and average mass quantified (n = at least 4 mice per group).Mice were weighed weekly, and growth is plotted as the average mass of each genotype/week (left: male, right: female, n > 5 per gender and genotype).Mice were held by the tails and analyzed for the leg clasping phenotype. Representative images of Ccnd1+/+ and Ccnd1KI/KI age-matched mice are shown (n > 10 for each group).Top: Individual whole-cell lysates were generated from the eyes of mice of each genotype and analyzed for expression of +/+ cyclin D1 (cyclin D1a) and KI/KI cyclin D1b via immunoblot using antisera specific to each isoform. Bottom: Representative H&amp;E staining of retinal tissue from Ccnd1+/+ and Ccnd1KI/KI mice showing individual layers of the retina. The ganglion cell layer and inner plexiform layer (G + IPL), inner nuclear layer (INL), and outer nuclear layer (ONL) were quantified (right). Images were taken at 400× magnification.


Figure 3Indicated passage - matched MAF lines were grown to 70 % confluency and then harvested for RNA and protein extraction . Top : qPCR analysis of cyclin D1 transcript using primer pairs common to both transcript a and transcript b . Bottom : Immunoblot of cyclin D1 levels in Ccnd1 + / + and Ccnd1KI / KMAF lines using an antibody common to both isoforms . Percentage of tumor - free mice post - injection of 1 million cells mixed with Matrigel ( 1 : 1 ratio ) into the flanks of nude mice over a period of 10 months ( n = 10 per genotype ) . Indicated tumors were harvested 3 weeks after detectable tumor formation , fixed in formalin , and stained with H & amp ; amp ; E . Tumors were analyzed for features of malignancy by a board - certified veterinary pathologist . Arrows indicate the specific tumor - associated features noted . Top panels were taken at 40 × magnification , and lower panels were taken at 400 × magnification . Ccnd1 + / + or Ccnd1KI / KI MAF lines were stably transfected with either cyclin D1a ( + / + ) , cyclin D1b ( KI / KI ) or vector control constructs . After selection with puromycin , individual lines were assessed for the induction of cyclin D1a in the + / + line and cyclin D1b in KI / KI lines via immunoblot . One million cells from the indicated stable cell lines were injected subcutaneously into the flanks of nude mice and monitored for tumor formation over a period of 365 days . Indicated time points represent time of palpable tumor detection and are plotted as % of tumor - free mice over time . Sections from KI / KI vec and KI / KI - D1b tumors were stained for the proliferative marker Ki67 . Three random fields from each tumor were quantified for Ki67 positivity , and are plotted as the mean ratio of Ki67 - positive cells / total cell number for each individual tumor .

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Figure 3Indicated passage-matched MAF lines were grown to 70% confluency and then harvested for RNA and protein extraction. Top: qPCR analysis of cyclin D1 transcript using primer pairs common to both transcript a and transcript b. Bottom: Immunoblot of cyclin D1 levels in Ccnd1+/+ and Ccnd1KI/KMAF lines using an antibody common to both isoforms.Percentage of tumor-free mice post-injection of 1 million cells mixed with Matrigel (1:1 ratio) into the flanks of nude mice over a period of 10 months (n = 10 per genotype).Indicated tumors were harvested 3 weeks after detectable tumor formation, fixed in formalin, and stained with H&amp;E. Tumors were analyzed for features of malignancy by a board-certified veterinary pathologist. Arrows indicate the specific tumor-associated features noted. Top panels were taken at 40× magnification, and lower panels were taken at 400× magnification.Ccnd1+/+ or Ccnd1KI/KI MAF lines were stably transfected with either cyclin D1a (+/+), cyclin D1b (KI/KI) or vector control constructs. After selection with puromycin, individual lines were assessed for the induction of cyclin D1a in the +/+ line and cyclin D1b in KI/KI lines via immunoblot.One million cells from the indicated stable cell lines were injected subcutaneously into the flanks of nude mice and monitored for tumor formation over a period of 365 days. Indicated time points represent time of palpable tumor detection and are plotted as % of tumor-free mice over time.Sections from KI/KI vec and KI/KI-D1b tumors were stained for the proliferative marker Ki67. Three random fields from each tumor were quantified for Ki67 positivity, and are plotted as the mean ratio of Ki67-positive cells/total cell number for each individual tumor.


Figure 4Passage - matched Ccnd1 + / + or Ccnd1KI / KI MAF lines were infected with lentivirus containing vector control or h - Ras constructs . Cells were selected with puromycin for 14 days and assayed for β - galactosidase activity . DAPI serves as a control for cell number . Images were taken at 200 × magnification with insets taken at 400 × magnification . The indicated cell lines were grown in complete media and assayed for h - Ras expression via immunoblot . Lamin B serves as a loading control . Cells were plated in soft agar and allowed to grow for a period of 3 weeks . Plates were then washed , fixed , and stained with 0 . 01 % crystal violet . Colonies greater than 50 μm were counted and are plotted ( top ) . Representative images of colony growth are shown for each cell line . Images were taken at 40 × with insets taken at 200 × magnification . Indicated MAF lines were injected subcutaneously into the flanks of nude mice as in ( A ) , and tumor incidence is plotted as % of tumor - free mice over time ( n = 10 ) . Indicated tumors were harvested , fixed , and stained for the proliferative marker mKi67 . Three random fields from each tumor were counted for mKi67 positivity , and are plotted as a % of total cell number , n = 5 ( right : representative images ) . Images were taken at 400 × magnification .

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Figure 4Passage-matched Ccnd1+/+ or Ccnd1KI/KI MAF lines were infected with lentivirus containing vector control or h-Ras constructs. Cells were selected with puromycin for 14 days and assayed for β-galactosidase activity. DAPI serves as a control for cell number. Images were taken at 200× magnification with insets taken at 400× magnification.The indicated cell lines were grown in complete media and assayed for h-Ras expression via immunoblot. Lamin B serves as a loading control.Cells were plated in soft agar and allowed to grow for a period of 3 weeks. Plates were then washed, fixed, and stained with 0.01% crystal violet. Colonies greater than 50 μm were counted and are plotted (top). Representative images of colony growth are shown for each cell line. Images were taken at 40× with insets taken at 200× magnification.Indicated MAF lines were injected subcutaneously into the flanks of nude mice as in (A), and tumor incidence is plotted as % of tumor-free mice over time (n = 10).Indicated tumors were harvested, fixed, and stained for the proliferative marker mKi67. Three random fields from each tumor were counted for mKi67 positivity, and are plotted as a % of total cell number, n = 5 (right: representative images). Images were taken at 400× magnification.


Figure 5Ccnd1 + / + and Ccnd1KI / KIMAF lines were arrested in G1 , S , and G2 / M phases of the cell cycle and expression of cyclin D1 isoforms determined by immunoblot . cyclin A2 and cyclin B1 serve as phase - specific cell cycle controls . Cells were plated in serum - proficient media and protein lysates generated from each genetic line . Individual D1 cyclins were immunoblotted to verify genetic identity along with other essential cell cycle components . Lamin B serves as a control . Indicated cells lines were immunoprecipitated for CDK4 and analyzed for their ability to incorporate 32P - ATP into full - length RB substrate . Radioactive counts were normalized to CDK4 activity of + / + cell lines and adjusted for efficiency of CDK4 pull down as determined by densitometry . Data is representative of three independent biological replicates . Indicated cell lines were plated in serum - proficient ( 10 % ) or serum - deficient ( 1 % ) media and total cell number counted at 24 , 48 , and 72 h in biological triplicate . Ccnd1 + / + and Ccnd1KI / KI MAF lines were transfected with a validated pool of siRNA directed against the N - terminus of the murine cyclin D1 transcript or control siRNA in full serum . 48 h post - transfection cells were harvested and analyzed for biochemical markers of cell cycle kinetics via immunoblot . GAPDH and Lamin B serve as controls . Cells were treated as ( E ) , and incubated in the indicated serum concentration for 48 h . Cells were treated with BrdU for 1 h prior to harvesting and then stained for BrdU incorporation . Three random fields from each of three biological triplicates were counted for BrdU incorporation via immunofluorescence and data is represented as percent positive / total cell number .

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Figure 5Ccnd1+/+ and Ccnd1KI/KIMAF lines were arrested in G1, S, and G2/M phases of the cell cycle and expression of cyclin D1 isoforms determined by immunoblot. cyclin A2 and cyclin B1 serve as phase-specific cell cycle controls.Cells were plated in serum-proficient media and protein lysates generated from each genetic line. Individual D1 cyclins were immunoblotted to verify genetic identity along with other essential cell cycle components. Lamin B serves as a control.Indicated cells lines were immunoprecipitated for CDK4 and analyzed for their ability to incorporate 32P-ATP into full-length RB substrate. Radioactive counts were normalized to CDK4 activity of +/+ cell lines and adjusted for efficiency of CDK4 pull down as determined by densitometry. Data is representative of three independent biological replicates.Indicated cell lines were plated in serum-proficient (10%) or serum-deficient (1%) media and total cell number counted at 24, 48, and 72 h in biological triplicate.Ccnd1+/+ and Ccnd1KI/KI MAF lines were transfected with a validated pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or control siRNA in full serum. 48 h post-transfection cells were harvested and analyzed for biochemical markers of cell cycle kinetics via immunoblot. GAPDH and Lamin B serve as controls.Cells were treated as (E), and incubated in the indicated serum concentration for 48 h. Cells were treated with BrdU for 1 h prior to harvesting and then stained for BrdU incorporation. Three random fields from each of three biological triplicates were counted for BrdU incorporation via immunofluorescence and data is represented as percent positive/total cell number.


Figure 6Ccnd1 + / + and Ccnd1KI / KI cells were transfected with a pool of siRNA directed against the N - terminus of the murine cyclin D1 transcript or scramble control for 48 h ( as in Figure5 ) . Cells were then fixed and stained for markers of double - strand breaks ( p - H2AX and 53BP1 , 400 × objective ) . Total number of foci for each cell was quantified and represented as the % of cells with > 10 foci / cell ( p - H2AX ) or % of cells with > 2 foci / cell ( 53BP1 ) . Error bars represent ± SEM . Ccnd1 + / + and Ccnd1KI / KIMAF lines were grown in serum - proficient media for 24 h and probed for expression of auto - modified PARP1 via immunoblot . Gapdh and cyclin D1b serve as controls . Cells were plated as in ( B ) and treated with 0 . 5 μM etoposide for 3 h . Cells were harvested and then probed for markers of DNA damage via immunoblot . Lamin B serves as a control . Indicated MAF lines were treated as in ( A ) and harvested 48 h post - transfection . Cells were analyzed for total PAR levels via immunoblot . cyclin D1 levels serve as siRNA validation controls . cyclin D1b expression was induced in the prostate cancer cell line LNCaP ( previously described ( Augello et al , 2014 ) ) and stained for p - H2AX and 53BP1 foci as in ( A ) via immunofluorescence ( 400 × objective ) . Total number of foci / cell is reported for LNCaP vector control and cyclin D1b - expressing isogenic pairs in biological triplicate . Isogenic pairs from ( E ) were grown in serum - proficient media for 24 h , harvested , and probed for total PAR levels via immunoblot .

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Figure 6Ccnd1+/+ and Ccnd1KI/KI cells were transfected with a pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or scramble control for 48 h (as in Figure5). Cells were then fixed and stained for markers of double-strand breaks (p-H2AX and 53BP1, 400× objective). Total number of foci for each cell was quantified and represented as the % of cells with > 10 foci/cell (p-H2AX) or % of cells with > 2 foci/cell (53BP1). Error bars represent ± SEM.Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h and probed for expression of auto-modified PARP1 via immunoblot. Gapdh and cyclin D1b serve as controls.Cells were plated as in (B) and treated with 0.5 μM etoposide for 3 h. Cells were harvested and then probed for markers of DNA damage via immunoblot. Lamin B serves as a control.Indicated MAF lines were treated as in (A) and harvested 48 h post-transfection. Cells were analyzed for total PAR levels via immunoblot. cyclin D1 levels serve as siRNA validation controls.cyclin D1b expression was induced in the prostate cancer cell line LNCaP (previously described (Augello et al, 2014)) and stained for p-H2AX and 53BP1 foci as in (A) via immunofluorescence (400× objective). Total number of foci/cell is reported for LNCaP vector control and cyclin D1b-expressing isogenic pairs in biological triplicate.Isogenic pairs from (E) were grown in serum-proficient media for 24 h, harvested, and probed for total PAR levels via immunoblot.


Figure 7Ccnd1 + / + and Ccnd1KI / KIMAF lines were grown in serum - proficient media for 24 h . Cells were then treated with control DMSO or 2 . 5 μM of the PARP inhibitor ABT - 888 . One hour post - treatment , the indicated lines were treated with 5 Gy of radiation . Cells were then allowed to recover for 48 h , after which BrdU was added for 1 h , and then harvested for bivariate flow cytometry . Representative traces for each condition are shown ( left ) and BrdU incorporation of biological triplicates was quantified ( right ) . Cells were plated and treated as in ( A ) . Forty - eight hours post - treatment , cells were fixed and stained for markers of senescence ( β - galactosidase activity ) . Cells positive for the staining ( blue ) were quantified for each condition ( 400 × magnification ) and reported as a percentage of the total population ( right ) . Plates treated in parallel with both IR and ABT - 888 were harvested at 48 h via 1 × trypsin and re - plated in serum - proficient media . Cells were allowed to grow for 96 h and were then stained with crystal violet ( 4 × objective , with inset at 200 × magnification , boxes highlight area of magnified images ) .

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Figure 7Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h. Cells were then treated with control DMSO or 2.5 μM of the PARP inhibitor ABT-888. One hour post-treatment, the indicated lines were treated with 5 Gy of radiation. Cells were then allowed to recover for 48 h, after which BrdU was added for 1 h, and then harvested for bivariate flow cytometry. Representative traces for each condition are shown (left) and BrdU incorporation of biological triplicates was quantified (right).Cells were plated and treated as in (A). Forty-eight hours post-treatment, cells were fixed and stained for markers of senescence (β-galactosidase activity). Cells positive for the staining (blue) were quantified for each condition (400× magnification) and reported as a percentage of the total population (right). Plates treated in parallel with both IR and ABT-888 were harvested at 48 h via 1× trypsin and re-plated in serum-proficient media. Cells were allowed to grow for 96 h and were then stained with crystal violet (4× objective, with inset at 200× magnification, boxes highlight area of magnified images).


Figure 1Expression of USP28 ( left ) and TP63 ( right ) in human lung squamous cell carcinomas ( SCC , n = 498 ) , adenocarcinomas ( ADC , n = 513 ) and normal non - transformed tissue ( normal SCC = 338 , normal ADC = 348 ) . Xena UCSC software . In box plots , the centre line reflects the median , the cross represents the mean and the upper and lower box limits indicates the first and third quartile . Whiskers extend 1 . 5x the IQR and outliers are marked as dots . Correlation of mRNA expression of USP28 and TP63 in lung SCC ( left , n = 498 ) , ADC ( right , n = 513 ) and normal , non - transformed tissue ( normal SCC = 338 , normal ADC = 348 ) . R : Spearmans correlation coefficient ; m = Slope . Xena UCSC software . IHC analysis of USP28 and ∆ Np63 protein abundance in lung cancer and non - transformed human samples ( n = 300 ) . The staining intensity was quantified in arbitrary units from 0 up to 3 by three independent pathologists . In box plots , the centre line reflects the median , the cross represents the mean and the upper and lower box limits indicates the first and third quartile . Whiskers extend 1 . 5x the IQR and outliers are marked as dots . p - values were calculated using two - tailed t - test . Kaplan - Meier estimator of NSCLC patients stratified by USP28 ( left , n = 1145 ) and TP63 ( right , n = 1926 ) expression . p - values were calculated using log - rank test . HR : hazard ratio . Kmplot software . Kaplan - Meier estimator of lung SCC patients stratified by USP28 expression ( n = 271 ) . The p - value was calculated using a logrank test . HR : hazard ratio . Kmplot software .

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Figure 1Expression of USP28 (left) and TP63 (right) in human lung squamous cell carcinomas (SCC, n=498), adenocarcinomas (ADC, n=513) and normal non-transformed tissue (normal SCC=338, normal ADC=348). Xena UCSC software. In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR and outliers are marked as dots.Correlation of mRNA expression of USP28 and TP63 in lung SCC (left, n=498), ADC (right, n=513) and normal, non-transformed tissue (normal SCC=338, normal ADC=348). R: Spearmans correlation coefficient; m=Slope. Xena UCSC software.IHC analysis of USP28 and ∆Np63 protein abundance in lung cancer and non-transformed human samples (n=300). The staining intensity was quantified in arbitrary units from 0 up to 3 by three independent pathologists. In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR and outliers are marked as dots. p-values were calculated using two-tailed t-test.Kaplan-Meier estimator of NSCLC patients stratified by USP28 (left, n=1145) and TP63 (right, n=1926) expression. p-values were calculated using log-rank test. HR: hazard ratio. Kmplot software.Kaplan-Meier estimator of lung SCC patients stratified by USP28 expression (n=271). The p-value was calculated using a logrank test. HR: hazard ratio. Kmplot software.


Figure 2Co - immunoprecipitation of exogenous HA - USP28 and FLAG - ΔNp63 in HEK293 cells . Either HA - USP28 or FLAG - ΔNp63 were precipitated and blotted against FLAG - ΔNp63 or HA - USP28 . The input corresponds to 10 % of the total protein amount used for the IP ( ACTIN as loading control ) . Ni - NTA His - ubiquitin pulldown in control transfected or HA - USP28 overexpressing HEK293 cells , followed by immunoblot against exogenous ΔNp63 . The input corresponds to 10 % of the total protein amount used for the pull down . Relative ubiquitination of the representative immunoblot was calculated using undefined for normalization . Ni - NTA His - ubiquitin pulldown K48 or K63 in control and HA - USP28 overexpressing HEK293 cells , followed by immunoblot against exogenous ΔNp63 . The input corresponds to 10 % of the total protein amount used for the pulldown . Relative ubiquitination of the representative immunoblot was calculated using VINCULIN for normalization . Co - immunoprecipitation of exogenous FLAG - USP28 C171A and FLAG - ΔNp63 in HEK293 cells . ΔNp63 was precipitated and blotted against FLAG - USP28 or ΔNp63 . The input corresponds to 10 % of the total protein amount used for the IP ( ACTIN as loading control ) . Ni - NTA His - ubiquitin pulldown in control , FLAG - USP28 or FLAG - USP28 C171A transfected HEK293 cells , followed by immunoblot against exogenous ΔNp63 . The input corresponds to 10 % of the total protein amount used for the pulldown . Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization . CHX chase assay ( 100ug / ml ) of control , FLAG - USP28 or FLAG - USP28 C171A transfected HEK293 cells for indicated time points . Representative immunoblot analysis of FLAG ( USP28 ) and ∆ Np63 as well quantification of relative protein abundance ( ACTIN as loading control ) . Immunoblot of USP28 and ∆ Np63 in transfected HEK293 cells upon treatment with either DMSO or indicated concentrations of PR - 619 for 24 hours . Relative protein abundance was calculated ACTIN as loading control .

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Figure 2Co-immunoprecipitation of exogenous HA-USP28 and FLAG-ΔNp63 in HEK293 cells. Either HA-USP28 or FLAG-ΔNp63 were precipitated and blotted against FLAG-ΔNp63 or HA-USP28. The input corresponds to 10% of the total protein amount used for the IP (ACTIN as loading control).Ni-NTA His-ubiquitin pulldown in control transfected or HA-USP28 overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pull down. Relative ubiquitination of the representative immunoblot was calculated using undefined for normalization.Ni-NTA His-ubiquitin pulldown K48 or K63 in control and HA-USP28 overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using VINCULIN for normalization.Co-immunoprecipitation of exogenous FLAG-USP28 C171A and FLAG-ΔNp63 in HEK293 cells. ΔNp63 was precipitated and blotted against FLAG-USP28 or ΔNp63. The input corresponds to 10% of the total protein amount used for the IP (ACTIN as loading control).Ni-NTA His-ubiquitin pulldown in control, FLAG-USP28 or FLAG-USP28 C171A transfected HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.CHX chase assay (100ug/ml) of control, FLAG-USP28 or FLAG-USP28 C171A transfected HEK293 cells for indicated time points. Representative immunoblot analysis of FLAG (USP28) and ∆Np63 as well quantification of relative protein abundance (ACTIN as loading control).Immunoblot of USP28 and ∆Np63 in transfected HEK293 cells upon treatment with either DMSO or indicated concentrations of PR-619 for 24 hours. Relative protein abundance was calculated ACTIN as loading control.


Figure 3Immunoblot of endogenous USP25 , USP28 and ∆ Np63 immunoprecipitated ( IP ) from A - 431 cells and co - precipitated ∆ Np63 or USP28 . Beads were coupled to specific USP25 , USP28 and ∆ Np63 antibodies or non - specific rabbit IgG as control . The input corresponds to 10 % of the total protein amount used for the IP ( TUBULIN as loading control ) . Inducible depletion of USP28 in A - 431 upon treatment with doxycycline ( 1µg / ml ) for 96h , western blot ( left , VINCULIN as loading control ) and qPCR analysis of USP28 and ∆ Np63 expression relative to ACTIN ( right ) was performed . Tandem ubiquitin binding entity ( TUBE ) pulldown of endogenous ubiquitylated ∆ Np63 in A - 431 cells upon DOX depletion of USP28 . Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization . Cycloheximide ( CHX ) chase assay ( 100µg / ml ) of control or inducible sh - USP28 A - 431 cell line ( EtOH or 1µg / ml dox ) for indicated time points . Representative immunoblot ( left , VINCULIN as loading control ) of USP28 and ∆ Np63 and quantification of relative protein abundance ( right ) . Doxycycline induced murine USP28 overexpression ( EtOH or 1ug / ml dox for 96 hours ) in A - 431 cells followed by immunoblot ( VINCULIN as loading control ) and qPCR analysis of USP28 and ∆ Np63 . For qPCR , human USP28 and murine USP28 ( mUSP28 ) primers were used . Relative mRNA was calculated using ∆ ∆ Ct analysis for human USP28 and ∆ Ct for mUSP28 ( ACTIN as housekeeping ) . TUBE pulldown of endogenous ubiquitylated ∆ Np63 in A - 431 cells upon overexpression of mUSP28 for 96 hours ( EtOH or 1µg / ml dox ) . Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization . CHX chase assay ( 100µg / ml ) of control or inducible mUSP28 overexpressing A - 431 cell line ( EtOH or 1µg / ml dox ) for indicated time points . Representative immunoblot analysis of USP28 and ∆ Np63 as well quantification of relative protein abundance ( VINCULIN as loading control ) . Immunoblot of control ( sh - NTC ) and two independent shRNA targeting USP28 ( sh - USP28 # 1 and # 2 ) for ∆ Np63 and USP28 protein abundance in LUDLU - 1adh ( VINCULIN as loading control ) , followed by qPCR analysis of USP28 and ∆ Np63 expression relative to ACTIN . Doxycycline induced mUSP28 overexpression ( EtOH or 1µg / ml dox for 96 hours ) in LUDLU - 1adh cells followed by immunoblot ( VINCULIN as loading control ) and qPCR analysis of USP28 , mUSP28 and ∆ Np63 . Relative mRNA was calculated using ∆ ∆ Ct analysis for human USP28 and ∆ Ct for mUSP28 ( ACTIN as housekeeping for the analysis ) .

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 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 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 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 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 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 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 3Immunoblot of endogenous USP25, USP28 and ∆Np63 immunoprecipitated (IP) from A-431 cells and co-precipitated ∆Np63 or USP28. Beads were coupled to specific USP25, USP28 and ∆Np63 antibodies or non-specific rabbit IgG as control. The input corresponds to 10% of the total protein amount used for the IP (TUBULIN as loading control).Inducible depletion of USP28 in A-431 upon treatment with doxycycline (1µg/ml) for 96h, western blot (left, VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN (right) was performed.Tandem ubiquitin binding entity (TUBE) pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon DOX depletion of USP28. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.Cycloheximide (CHX) chase assay (100µg/ml) of control or inducible sh-USP28 A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot (left, VINCULIN as loading control) of USP28 and ∆Np63 and quantification of relative protein abundance (right).Doxycycline induced murine USP28 overexpression (EtOH or 1ug/ml dox for 96 hours) in A-431 cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63. For qPCR, human USP28 and murine USP28 (mUSP28) primers were used. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping).TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon overexpression of mUSP28 for 96 hours (EtOH or 1µg/ml dox). Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.CHX chase assay (100µg/ml) of control or inducible mUSP28 overexpressing A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot analysis of USP28 and ∆Np63 as well quantification of relative protein abundance (VINCULIN as loading control).Immunoblot of control (sh-NTC) and two independent shRNA targeting USP28 (sh-USP28#1 and #2) for ∆Np63 and USP28 protein abundance in LUDLU-1adh (VINCULIN as loading control), followed by qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN.Doxycycline induced mUSP28 overexpression (EtOH or 1µg/ml dox for 96 hours) in LUDLU-1adh cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28, mUSP28 and ∆Np63. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping for the analysis).