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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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.


Indicated 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 .

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Indicated 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 ) .

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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 .

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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 .

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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 .

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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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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.


Passage - 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 .

Passage-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 .

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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 .

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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 ) .

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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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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.


Ccnd1 + / + 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 .

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Ccnd1+/+ 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 .

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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 .

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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 .

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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 .

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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 .

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.


Ccnd1 + / + 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 .

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Ccnd1+/+ 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 .

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 .

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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 .

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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 .

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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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Isogenic pairs from (E) were grown in serum-proficient media for 24 h, harvested, and probed for total PAR levels via immunoblot.


Ccnd1 + / + 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 ) .

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Ccnd1+/+ 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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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).


Expression 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 .

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Expression 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 .

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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 .

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 .

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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 .

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.


Co - 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 ) .

Co-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 .

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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 .

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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 ) .

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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 .

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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 ) .

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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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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.


Immunoblot 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 ) .

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Immunoblot 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 .

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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 .

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

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 ) .

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

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 ) .

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

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 .

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

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 ) .

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

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 .

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

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 ) .

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

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).


Correlation of gene expression changes upon constitutive transduction of A - 431 cells with either shRNA targeting USP28 ( sh - USP28 # 1 ) or ∆ Np63 ( sh - ∆ Np63 # 2 ) relative to non - targeting control ( sh - NTC ) . The diagonal line reflects a regression build on a linear model . R : Pearsons correlation coefficient , m : slope of the linear regression

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

Correlation of gene expression changes upon constitutive transduction of A-431 cells with either shRNA targeting USP28 (sh-USP28#1) or ∆Np63 (sh-∆Np63#2) relative to non-targeting control (sh-NTC). The diagonal line reflects a regression build on a linear model. R: Pearsons correlation coefficient, m: slope of the linear regression model


Gene set enrichment analysis ( GSEA ) of a gene set of significantly down - regulated genes in sh - ∆ Np63 # 2 transfected A - 431 cells ( " Down - regulated sh - ∆ NP63 " , Appendix Table S1 ) . The gene set was analysed in sh - ∆ Np63 # 2 ( left ) and sh - USP28 # 1 - depleted ( right ) A - 431 cells . ( N ) ES : ( normalised ) enrichment

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

Gene set enrichment analysis (GSEA) of a gene set of significantly down-regulated genes in sh-∆Np63#2 transfected A-431 cells ("Down-regulated sh-∆NP63", Appendix Table S1). The gene set was analysed in sh-∆Np63#2 (left) and sh-USP28#1-depleted (right) A-431 cells. (N)ES: (normalised) enrichment score


Gene set enrichment analysis ( GSEA ) of a gene set of significantly down - regulated genes in sh - USP28 # 1 transfected A - 431 cells ( " Down - regulated sh - USP28 " , Appendix Table S1 ) . The gene set was analysed in shUSP28 # 1 ( left ) and sh - ∆ Np63 # - depleted ( right ) A - 431 cells . ( N ) ES : ( normalised ) enrichment

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

Gene set enrichment analysis (GSEA) of a gene set of significantly down-regulated genes in sh-USP28#1 transfected A-431 cells ("Down-regulated sh-USP28", Appendix Table S1). The gene set was analysed in shUSP28#1 (left) and sh-∆Np63#-depleted (right) A-431 cells. (N)ES: (normalised) enrichment score


Immunoblot of endogenous ΔNp63 and USP28 in A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) or against USP28 and transiently transfected with exogenous ΔNp63 . ACTIN served as loading control . Representative western blot from three independent experiments .

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

Immunoblot of endogenous ΔNp63 and USP28 in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC) or against USP28 and transiently transfected with exogenous ΔNp63. ACTIN served as loading control. Representative western blot from three independent experiments.


Cell growth of A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) or against USP28 and transiently transfected with exogenous ΔNp63 . Total cell number was measured and assessed at indicated time points . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) . p - values were calculated using two - tailed t - test statistical analysis . * p - value < 0 . 05 , * * p - value < 0 .

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

Cell growth of A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC) or against USP28 and transiently transfected with exogenous ΔNp63. Total cell number was measured and assessed at indicated time points. Quantitative graph is represented as mean ± SD of three experiments (n=3). p-values were calculated using two-tailed t-test statistical analysis. *p-value < 0.05, **p-value < 0.01


Relative expression of SCC marker genes KRT5 , 14 and 19 in A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) , two independent shRNA - ΔNp63 , two independent shRNA - USP28 or ΔNp63 in shRNA - USP28 # 1 , normalised to ACTIN . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) .

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

Relative expression of SCC marker genes KRT5, 14 and 19 in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC), two independent shRNA-ΔNp63, two independent shRNA-USP28 or ΔNp63 in shRNA-USP28#1, normalised to ACTIN. Quantitative graph is represented as mean ± SD of three experiments (n=3).


Relative expression of epithelial marker genes KRT10 and GPCR5A in A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) , two independent shRNA - ΔNp63 and shRNA - USP28 # 1 , normalised to ACTIN . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) .

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

Relative expression of epithelial marker genes KRT10 and GPCR5A in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC), two independent shRNA-ΔNp63 and shRNA-USP28#1, normalised to ACTIN. Quantitative graph is represented as mean ± SD of three experiments (n=3).


Genomic signature of primary human lung SCC samples comprising USP28 , ∆ Np63 , KRT5 , KRT14 , KRT19 , DSG3 , MKI67 , PCNA and GPRC5A . Samples were sorted dependent on relative USP28 expression ( high to low ) . n = 553 . Xena UCSC software .

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

Genomic signature of primary human lung SCC samples comprising USP28, ∆Np63, KRT5, KRT14, KRT19, DSG3, MKI67, PCNA and GPRC5A. Samples were sorted dependent on relative USP28 expression (high to low). n=553. Xena UCSC software.


GSEA of consensus squamous cancer marker genes ( se Appendix Table S2 ) in A - 431 cells stably transduced with sh - ∆ Np63 # 2 or sh - NTC . ( N ) ES : ( normalised ) enrichment score GSEA of consensus squamous cancer marker genes in A - 431 cells stably transduced with sh - USP28 # 1 or sh - NTC . ( N ) ES : ( normalised ) enrichment

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

GSEA of consensus squamous cancer marker genes (se Appendix Table S2) in A-431 cells stably transduced with sh-∆Np63#2 or sh-NTC. (N)ES: (normalised) enrichment score GSEA of consensus squamous cancer marker genes in A-431 cells stably transduced with sh-USP28#1 or sh-NTC. (N)ES: (normalised) enrichment score


Correlation of mRNA expression of consensus squamous cancer marker genes and TP63 in lung SCC and non - transformed lung tissue ( Normal ) . R : Spearmans correlation coefficient . n = 836 . Gepia software . Correlation of mRNA expression of consensus squamous cancer marker genes and USP28 in lung SCC and non - transformed lung tissue ( Normal ) . R : Spearmans correlation coefficient . N = 836 . Gepia software .

Correlation of mRNA expression of consensus squamous cancer marker genes and TP63 in lung SCC and non-transformed lung tissue (Normal). R: Spearmans correlation coefficient. n=836. Gepia software. Correlation of mRNA expression of consensus squamous cancer marker genes and USP28 in lung SCC and non-transformed lung tissue (Normal). R: Spearmans correlation coefficient. N=836. Gepia software.


Representative haematoxylin and eosin ( H & E ) staining of tumour bearing animals 12 weeks post intratracheal infection . Boxes indicate highlighted tumour areas in ( C ) ( a , b , a ' and b ' ) . Scale bar = 2000µm ; nKPL = 6 and nKPLU = 5 . H = heart .

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

Representative haematoxylin and eosin (H&E) staining of tumour bearing animals 12 weeks post intratracheal infection. Boxes indicate highlighted tumour areas in (C) (a, b, a' and b'). Scale bar = 2000µm; nKPL= 6 and nKPLU = 5. H=heart.


Representative IHC staining for ADC ( TTF - 1 ) and SCC ( KRT5 and ∆ Np63 ) marker expression as well as Usp28 abundance in KPL ( n = 6 ) and KPLU ( n = 5 ) lung tumours . Scale bar : 20µm .

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

Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 abundance in KPL (n=6) and KPLU (n=5) lung tumours. Scale bar: 20µm.


Quantification of % tumour area ( normalised to total lung area ) in KPL ( n = 6 ) and KPLU ( n = 5 ) animals . Quantification of lung tumour numbers in KPL ( n = 6 ) and KPLU ( n = 5 ) animals .

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

Quantification of % tumour area (normalised to total lung area) in KPL (n=6) and KPLU (n=5) animals. Quantification of lung tumour numbers in KPL (n=6) and KPLU (n=5) animals.


Immunoblot of endogenous LKB1 , USP28 and ∆ Np63 in KPL and KPLU tumours . ( VINCULIN as loading control ) . Western blot shown are representative of three independent experiments ( n = 3 ) . Representative immunoblot of endogenous NOTCH1 , c - MYC and c - JUN in KPL and KPLU ( ACTIN as a control ) . n =

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

Immunoblot of endogenous LKB1, USP28 and ∆Np63 in KPL and KPLU tumours. (VINCULIN as loading control). Western blot shown are representative of three independent experiments (n=3). Representative immunoblot of endogenous NOTCH1, c-MYC and c-JUN in KPL and KPLU (ACTIN as a control). n=3


Kaplan - Meier survival curves comparing KPL ( n = 6 ) and KPLU ( n = 5 ) animals upon AAV intratracheal infection . The p - value was calculated using a log - rank test .

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

Kaplan-Meier survival curves comparing KPL (n=6) and KPLU (n=5) animals upon AAV intratracheal infection. The p-value was calculated using a log-rank test.


Representative qPCR of SCC and ADC marker expression in two independent KP lung tumour clones , resulting in KPADC and KPSCC ; ( ACTIN served as loading control ) . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) .

Representative qPCR of SCC and ADC marker expression in two independent KP lung tumour clones, resulting in KPADC and KPSCC; (ACTIN served as loading control). Quantitative graph is represented as mean ± SD of three experiments (n=3).


Immunoblot against endogenous USP28 in the murine KPSCC cell line upon targeting with two independent shRNA sequences . ACTIN served as loading control .

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

Immunoblot against endogenous USP28 in the murine KPSCC cell line upon targeting with two independent shRNA sequences. ACTIN served as loading control.


Representative haematoxylin and eosin ( H & E ) images of tumour bearing animals 8 weeks post intratracheal transplantation of 2 × 105 cells / animal . KPSCC sh - NTC ( n = 3 ) ; KPSCC sh - USP28 ( n = 3 ) , Scale bar =

Representative haematoxylin and eosin (H&E) images of tumour bearing animals 8 weeks post intratracheal transplantation of 2 × 105 cells/animal. KPSCC sh-NTC (n=3); KPSCC sh-USP28 (n=3), Scale bar = 5000µm


Quantification of % tumour area ( top , normalised to total lung area ) and lung tumour numbers ( bottom ) on KPSCC sh - NTC ( n = 6 ) and KPSCC sh - USP28 ( n = 6 ) animals .

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

Quantification of % tumour area (top, normalised to total lung area) and lung tumour numbers (bottom) on KPSCC sh-NTC (n=6) and KPSCC sh-USP28 (n=6) animals.


Representative IHC staining for ADC ( TTF - 1 ) and SCC ( KRT5 and ∆ Np63 ) marker expression as well as Usp28 and GFP abundance in KPSCC sh - NTC ( n = 3 ) and KPSCC sh - USP28 ( n = 3 ) lung tumours . Scale bar = 50

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

Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 and GFP abundance in KPSCC sh-NTC (n=3) and KPSCC sh-USP28 (n=3) lung tumours. Scale bar =50 µm


Analysis of occurring TP63 genetic alterations in lung squamous ( LUSC ) , cervical ( CESC ) , oesophagus ( ESCA ) , head and neck ( HNSC ) and pancreatic ( PAAD ) tumours . Cbioportal .

Analysis of occurring TP63 genetic alterations in lung squamous (LUSC), cervical (CESC), oesophagus (ESCA), head and neck (HNSC) and pancreatic (PAAD) tumours. Cbioportal.


Expression of TP63 ( left ) and USP28 ( right ) in human Lung ( n = 498 ) , Cervix ( n = 254 ) , Oesophagus ( n = 96 ) and HNSC ( n = 522 ) SCC tumours and normal non - transformed tissue ( nLung = 338 nCervix = 3 , nOesophagus = 11 and nHNSC = 44 ) . 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 . p - values were calculated using two - tailed t - test statistical analysis . Xena UCSC software .

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

Expression of TP63 (left) and USP28 (right) in human Lung (n=498), Cervix (n=254), Oesophagus (n=96) and HNSC (n=522) SCC tumours and normal non-transformed tissue (nLung=338 nCervix=3, nOesophagus=11 and nHNSC=44). 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. p-values were calculated using two-tailed t-test statistical analysis. Xena UCSC software.


Correlation of mRNA expression of KRT14 / KRT7 and TP63 in ADC and SCC tumours for Lung ( nADC = 513 and nSCC = 498 ) , Cervix ( nADC = 47 and nSCC = 254 ) and Oesophagus ( nADC = 89 and nSCC = 96 ) . Blue dots : ADC ; Red dots : SCC ; R = Spearmans correlation coefficient ; m : Slope . Xena UCSC .

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

Correlation of mRNA expression of KRT14 / KRT7 and TP63 in ADC and SCC tumours for Lung (nADC=513 and nSCC=498), Cervix (nADC=47 and nSCC=254) and Oesophagus (nADC=89 and nSCC=96). Blue dots: ADC; Red dots: SCC; R= Spearmans correlation coefficient; m: Slope. Xena UCSC.


Immunoblot of control ( sh - NTC ) and two independent shRNA targeting USP28 ( sh - USP28 # 1 and # 2 ) for ∆ Np63 , KRT14 and USP28 protein abundance in H1299 , LUDLU - 1adh , HELA , SiHa , Ca Ski , PANC - 1 and BXPC - 3 ( ACTIN as loading control ) .

Immunoblot of control (sh-NTC) and two independent shRNA targeting USP28 (sh-USP28#1 and #2) for ∆Np63, KRT14 and USP28 protein abundance in H1299, LUDLU-1adh, HELA, SiHa, Ca Ski, PANC-1 and BXPC-3 (ACTIN as loading control).


Cells were seeded at equal cell density and counted after five days , Brigth field images of control or sh - USP28 # 2 infected H1299 , LUDLU - 1adh , Hela , CaSKI , PANC - 1 and BXPC - 3 cells before quantification . Scale bar = 30µm Relative number of H1299 , LUDLU - 1adh , Hela , CaSKI , SiHa , PANC - 1 and BXPC - 3 sh - USP28 # 2 cells compared with sh - NTC control cells . p - values were calculated using two - tailed t - test statistical analysis . SiHa * = Notably , the human SCC cell line SiHa was negative for ΔNp63 .

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

Cells were seeded at equal cell density and counted after five days, Brigth field images of control or sh-USP28#2 infected H1299, LUDLU-1adh, Hela, CaSKI, PANC-1 and BXPC-3 cells before quantification. Scale bar = 30µm Relative number of H1299, LUDLU-1adh, Hela, CaSKI, SiHa, PANC-1 and BXPC-3 sh-USP28#2 cells compared with sh-NTC control cells. p-values were calculated using two-tailed t-test statistical analysis. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63.


Immunoblot of endogenous USP28 in A - 431 cells upon treatment with warheads ( WH ) and either DMSO or indicated concentrations of AZ1 ( USP28 Inh . ) for 24 hours . USP28 Upper band : Active USP28 ; USP28 lower band : Inactive USP28 , VINCULIN as loading control .

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

Immunoblot of endogenous USP28 in A-431 cells upon treatment with warheads (WH) and either DMSO or indicated concentrations of AZ1 (USP28 Inh.) for 24 hours. USP28 Upper band: Active USP28; USP28 lower band: Inactive USP28, VINCULIN as loading control.


Immunoblot of endogenous USP28 , ∆ Np63 and KRT14 in A - 431 cells treated for 24 hours with either DMSO or indicated concentrations of AZ1 . VINCULIN served as loading control . TP63 and USP28 half maximal inhibitory protein abundance ( IC50 ) was calculated . Graphs are represented as mean ± SD ( n = 3 ) .

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

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control. TP63 and USP28 half maximal inhibitory protein abundance (IC50) was calculated. Graphs are represented as mean ± SD (n=3).


TUBE pulldown of endogenous ubiquitylated ∆ Np63 in A - 431 cells upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours . VINCULIN served as loading control for relative Ubiquitination .

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

TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours. VINCULIN served as loading control for relative Ubiquitination.


Immunoblot of endogenous USP28 , ∆ Np63 and KRT14 in A - 431 cells treated for 18 hours with either DMSO or 15µM AZ1 followed by 6 hours of treatment with 20µM proteasomal inhibitor MG - 132 ( 20µM ) or DMSO . VINCULIN served as loading control .

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

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 18 hours with either DMSO or 15µM AZ1 followed by 6 hours of treatment with 20µM proteasomal inhibitor MG-132 (20µM) or DMSO. VINCULIN served as loading control.


A - 431 cells were seeded at equal cell density and cultured in the presence of either DMSO , 1µM , 10µM or 30µM AZ1 for 48h . Cells were quantified with the Operetta imaging system using Hoechst staining . 50 % growth inhibition ( GI50 ) was calculated . Scale bar = 250µm . n = 30 fields

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

A-431 cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. Scale bar = 250µm. n= 30 fields analysed


Immunoblot of endogenous USP28 , ∆ Np63 and KRT14 in LUDLU - 1adh , H1299 , Ca Ski , Hela and SiHa cells treated for 24 hours with either DMSO or indicated concentrations of AZ1 . VINCULIN served as loading

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

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in LUDLU-1adh, H1299, Ca Ski, Hela and SiHa cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control


LUDLU - 1ADH , H1299 , Ca Ski , Hela and SiHa cells were seeded at equal cell density and cultured in the presence of either DMSO , 1µM , 10µM or 30µM AZ1 for 48h . Scale bar for LUDLU - 1adh and H1299 = 500µm . Scale bar for Ca Ski , Hela and SiHa = 250µm . SiHa * = Notably , the human SCC cell line SiHa was negative for ΔNp63 . Number of cells were quantified with the Operetta imaging system using Hoechst staining . 50 % growth inhibition ( GI50 ) was calculated . n = 30 fields

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

LUDLU-1ADH, H1299, Ca Ski, Hela and SiHa cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Scale bar for LUDLU-1adh and H1299 = 500µm. Scale bar for Ca Ski, Hela and SiHa = 250µm. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63. Number of cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. n=30 fields analysed


Immunofluorescence ( IF ) staining against endogenous ∆ Np63 in KP and KPL cell lines ( blue : DAPI [ staining of nuclei ] and red / magenta : ∆ Np63 ) , Scale bar = 32µm .

Immunofluorescence (IF) staining against endogenous ∆Np63 in KP and KPL cell lines (blue: DAPI [staining of nuclei] and red/magenta: ∆Np63), Scale bar = 32µm.


GFP Fluorescent images of lungs treated with either PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . Tumour cells are positive for GFP ( green arrows ) . n = 3 Quantification of % tumour area ( normalised to total lung area ) in control ( n = 5 ) , 125 mg / kg AZ1 ( n = 4 ) and 375 mg / kg AZ1 ( n = 4 ) treated animals .

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GFP Fluorescent images of lungs treated with either PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Tumour cells are positive for GFP (green arrows). n=3 Quantification of % tumour area (normalised to total lung area) in control (n=5), 125 mg/kg AZ1 (n=4) and 375 mg/kg AZ1 (n=4) treated animals.


Representative staining of H & E and IHC for USP28 and ∆ Np63 of SCC tumours from mice treated with PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . Boxes indicate highlighted tumour areas . First line Scale bars = 500µm ; Lower scale bars = 20µm . n = 3 ∆ Np63 staining intensity of lung tumours treated with PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . nControl = 33712 cells ; nAZ1 125mg / kg = 7382 cells ; nAZ1 375mg / kg = 4967 cells . Cells were analysed from three independent animals .

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

Representative staining of H&E and IHC for USP28 and ∆Np63 of SCC tumours from mice treated with PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Boxes indicate highlighted tumour areas. First line Scale bars = 500µm; Lower scale bars = 20µm. n=3   ∆Np63 staining intensity of lung tumours treated with PBS/DMSO/0,1%Tween80 or indicated concentrations of AZ1. nControl = 33712 cells ; nAZ1 125mg/kg = 7382 cells; nAZ1 375mg/kg = 4967 cells. Cells were analysed from three independent animals.


Kaplan Meier survival curve of N2 , odr - 3 ( n2150 ) and odr - 3 ( n2046 ) worms on partial lawn of P . aeruginosa at 25 ° C . Kaplan Meier survival curve of N2 , odr - 3 ( n2150 ) and odr - 3 ( n2046 ) worms on full lawn of P . aeruginosa at 25 ° C .

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

Kaplan Meier survival curve of N2, odr-3(n2150) and odr-3(n2046) worms on partial lawn of P. aeruginosa at 25°C.   Kaplan Meier survival curve of N2, odr-3(n2150) and odr-3(n2046)   worms on full lawn of P. aeruginosa at 25°C.


Chemotactic response of N2 worms to varying dilutions of 1 - undecene . n ≥ 3 assays . Error bars indicate SEM .

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

Chemotactic response of N2 worms to varying dilutions of 1-undecene. n ≥ 3 assays. Error bars indicate SEM.


( i ) Schematic of setup used to record the movement of 5 worms each under control and 1 - undecene odor exposure . ( ii ) Movement trajectories of N2 worms on E . coli OP50 lawn exposed to 1 - undecene odor for 30 min and 60 min , each color represents the trajectory of single worm . Scale bar = 10 mm .

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

(i) Schematic of setup used to record the movement of 5 worms each under control and 1-undecene odor exposure. (ii) Movement trajectories of N2 worms on E. coli OP50 lawn exposed to 1-undecene odor for 30 min and 60 min, each color represents the trajectory of single worm. Scale bar = 10 mm.


Average calcium responses of AWB : : GCaMP3 worms exposed to 1 - undecene at indicated concentrations . Each worm was recorded for 180 s . Worms were under stimulus between 11 s - 130 s , window shown in grey , and stimulus withdrawal beyond 130 s . Shaded regions around the curves represent SEM . n = 3 . Two dashed box represents 10 s time window after stimulus addition and stimulus removal respectively .

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

Average calcium responses of AWB::GCaMP3 worms exposed to 1-undecene at indicated concentrations. Each worm was recorded for 180 s. Worms were under stimulus between 11 s-130 s, window shown in grey, and stimulus withdrawal beyond 130 s. Shaded regions around the curves represent SEM. n = 3. Two dashed box represents 10 s time window after stimulus addition and stimulus removal respectively.


Real time PCR analysis of irg - 1 , irg - 2 and irg - 3 genes in N2 , odr - 3 ( n2150 ) and odr - 3 ( n2046 ) worms exposed to 1 - undecene odor upon respective naive worms . n = 3 . * P ≤ 0 . 05 , * * P ≤ 0 . 01 as determined by two - tailed unpaired t - test . Error bars indicate SEM . Real time PCR analysis of irg - 1 , irg - 2 and irg - 3 genes in N2 , lim - 4 ( ky403 ) and lim - 4 ( yz12 ) worms exposed to 1 - undecene odor upon respective naive worms . n = 3 . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 as determined by two - tailed unpaired t - test . Error bars indicate SEM .

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Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2, odr-3(n2150) and odr-3(n2046) worms exposed to 1-undecene odor upon respective naive worms. n = 3. * P ≤ 0.05, ** P ≤ 0.01 as determined by two-tailed unpaired t-test. Error bars indicate SEM. Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2, lim-4(ky403) and lim-4(yz12) worms exposed to 1-undecene odor upon respective naive worms. n = 3. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 as determined by two-tailed unpaired t-test. Error bars indicate SEM.


Kaplan Meier survival curve on the full lawn of P . aeruginosa for N2 ( naive ) worms and N2 worms pre exposed to 1 - undecene odor . Survival assay was performed at 20 ° C .

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Kaplan Meier survival curve on the full lawn of P. aeruginosa for N2 (naive) worms and N2 worms pre exposed to 1-undecene odor. Survival assay was performed at 20°C.


B . Quantification of PLAs between BRCA2 and DPP9 in control HeLa WT cells treated with non - targeting siRNA ( siNT ) or silenced with the indicated oligos . 300 ng / mL MMC was added for 24 h . Each dot represents the number of PLA events in a single cell , from two to seven biological replicates ( siNT - MMC ( n = 6 ) , siNT + MMC ( n = 7 ) , siDPP9 - MMC ( n = 4 ) , siDPP9 + MMC ( n = 5 ) , siBRCA2 - MMC ( n = 2 ) , siBRCA2 + MMC ( n = 3 ) , siFLNA - MMC ( n = 5 ) , siFLNA + MMC ( n = 6 ) , NgtCntrl - MMC ( n = 3 ) , NgtCntrl + MMC ( n = 6 ) ) . The number of foci are shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * = p ≤ 0 . 05 , * * * * = p ≤ 0 . 0001 ) . C . Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa WT cells . Exposure of cells to MMC triggers more PLA events ( white ) . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _

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

B. Quantification of PLAs between BRCA2 and DPP9 in control HeLa WT cells treated with non-targeting siRNA (siNT) or silenced with the indicated oligos. 300 ng / mL MMC was added for 24 h. Each dot represents the number of PLA events in a single cell, from two to seven biological replicates (siNT-MMC (n = 6), siNT+MMC (n = 7), siDPP9-MMC (n = 4), siDPP9+MMC (n = 5), siBRCA2-MMC (n = 2), siBRCA2+MMC (n = 3), siFLNA-MMC (n = 5), siFLNA+MMC (n = 6), NgtCntrl-MMC (n = 3), NgtCntrl+MMC (n = 6)). The number of foci are shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, **** = p ≤ 0.0001). C. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa WT cells. Exposure of cells to MMC triggers more PLA events (white). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071


D . Quantification of PLAs between BRCA2 and DPP9 in asynchronous HeLa WT cells or in cells in S - Phase . Each dot represents the number of PLA events in a single cell , from three biological replicates . Cells were synchronised with a double thymidine block and released for 3 h before addition of NCS ( 250 ng / mL for 30 min ) . Cells were sampled prior to NCS treatment , after NCS treatment and with 1 . 5 h and 3 h of recovery after NCS treatment . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , * * * * = p ≤ 0 . 0001 ) . E . Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa cells . Exposure of cells to NCS triggers more PLA events ( white ) , which increase in time . Cells in S - Phase show a greater fold - increase in the number of PLA events in comparison to asynchronous cells . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Antibodies as in ( C ) .

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

D. Quantification of PLAs between BRCA2 and DPP9 in asynchronous HeLa WT cells or in cells in S-Phase. Each dot represents the number of PLA events in a single cell, from three biological replicates. Cells were synchronised with a double thymidine block and released for 3 h before addition of NCS (250 ng / mL for 30 min). Cells were sampled prior to NCS treatment, after NCS treatment and with 1.5 h and 3 h of recovery after NCS treatment. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.0001). E. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa cells. Exposure of cells to NCS triggers more PLA events (white), which increase in time. Cells in S-Phase show a greater fold-increase in the number of PLA events in comparison to asynchronous cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Antibodies as in (C).


F . Co - immunoprecipitation assays showing binding of BRCA2 and DPP9 - SWT . HEK293 DPP9 KO + DPP9WT cells , were treated with 1µg / mL Dox ( 24 h ) to induce the expression of DPP9 - FLAG . DNA damage was induced with 300 nM MMC treatment for 24h . Control cells do not express DPP9 ( - Dox ) . Bound proteins were eluted with a FLAG peptide and analysed by Western Blotting ( anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 731947 , anti - FLAG RRID : AB _ 262044 ) .

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

F. Co-immunoprecipitation assays showing binding of BRCA2 and DPP9-SWT. HEK293 DPP9 KO + DPP9WT cells, were treated with 1µg/mL Dox (24 h) to induce the expression of DPP9-FLAG. DNA damage was induced with 300 nM MMC treatment for 24h. Control cells do not express DPP9 (- Dox). Bound proteins were eluted with a FLAG peptide and analysed by Western Blotting (anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-FLAG RRID:AB_262044).


B . Pull - down assay showing direct binding of purified recombinant DPP9 to a BRCA2 N - terminal1 - 40 fragment immobilized on HA - beads . The DPP9 inhibitors 1G244 or SLRFLYEG compete with BRCA21 - 40HA for interaction with DPP9 . Representative data of three technical replicates is shown . Anti - HA : RRID : AB _ 2565334 , anti - DPP9 : RRID : AB _ 2889071 .

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

B. Pull-down assay showing direct binding of purified recombinant DPP9 to a BRCA2 N-terminal1-40 fragment immobilized on HA-beads. The DPP9 inhibitors 1G244 or SLRFLYEG compete with BRCA21-40HA for interaction with DPP9. Representative data of three technical replicates is shown. Anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.


C . Quantification of PLAs showing fewer MMC - induced DPP9 - BRCA2 PLA events in HeLa cells treated with 10µM 1G244 . Each dot represents the number of PLA events in a single cell , from three biological replicates . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * = p ≤ 0 . 001 , * * * * = p ≤ 0 . 0001 ) . D . Representative images of DPP9 - BRCA2 PLA in the presence of 1G244 - a competitive inhibitor of DPP9 . Control cells were mock treated with DMSO . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _

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

C. Quantification of PLAs showing fewer MMC-induced DPP9-BRCA2 PLA events in HeLa cells treated with 10µM 1G244. Each dot represents the number of PLA events in a single cell, from three biological replicates. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (*** = p ≤ 0.001, **** = p ≤ 0.0001). D. Representative images of DPP9-BRCA2 PLA in the presence of 1G244 - a competitive inhibitor of DPP9. Control cells were mock treated with DMSO. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071


F . Pull - down assay showing a competition between DPP9 and PALB2 for binding to the BRCA2 N - terminus . While each protein can bind to the BRCA2 N - terminal peptide , in the presence of DPP9 , the PALB2 - BRCA2 binding is negatively affected . Representative data of three technical replicates is shown . Anti - PALB2 : RRID : AB _ 890607 , anti - HA : RRID : AB _ 2565334 , anti - DPP9 : RRID : AB _ 2889071 .

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

F. Pull-down assay showing a competition between DPP9 and PALB2 for binding to the BRCA2 N-terminus. While each protein can bind to the BRCA2 N-terminal peptide, in the presence of DPP9, the PALB2-BRCA2 binding is negatively affected. Representative data of three technical replicates is shown. Anti-PALB2: RRID:AB_890607, anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.


A - B . Representative Western Blots and accompanying graph from more than three biological replicates show that MMC ( 300 nM , 24 h ) induces a rapid turnover of endogenous BRCA2 in HeLa WT cells , which is less pronounced in HeLa WT cells treated with the proteasome inhibitor MG132 ( 100µM ) , in HeLa DPP9 KD cells and in cells transiently silenced for DPP9 ( siDPP9 ) . RAD51 stability is not altered in HeLa DPP9 KD cells . Vinculin is a loading control for BRCA2 , Tubulin is a loading control for RAD51 . Shown images originate from one representative cycloheximide ( CHX ) chase assay . The ratios of BRCA2 to Vinculin and RAD51 to Tubulin are defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test . ( * = p ≤ 0 . 03 ) . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 731947 , anti - Vinculin : RRID : AB _ 477629 , anti - Tubulin : RRID : AB _ 628412 , anti - RAD51 : RRID : AB _ 1142428 .

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

A-B. Representative Western Blots and accompanying graph from more than three biological replicates show that MMC (300 nM, 24 h) induces a rapid turnover of endogenous BRCA2 in HeLa WT cells, which is less pronounced in HeLa WT cells treated with the proteasome inhibitor MG132 (100µM), in HeLa DPP9 KD cells and in cells transiently silenced for DPP9 (siDPP9). RAD51 stability is not altered in HeLa DPP9 KD cells. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin and RAD51 to Tubulin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test. (*= p ≤ 0.03). Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-Vinculin: RRID:AB_477629, anti-Tubulin: RRID:AB_628412, anti-RAD51: RRID:AB_1142428.


C - D . Representative Western Blots and graph summarizing results of more than three biological replicates . CHX chase assays show that the MMC - induced degradation of BRCA2 is less pronounced in HEK293 DPP9 KO cells as well as in HEK293 DPP9 KO + DPP9 - SS729A cells overexpressing an inactive DPP9 - S mutant . Expression of DPP9 was induced ( + Dox , 1µg / ml ) simultaneously with MMC ( 300 nM ) , 24 h . Cells that were overexpressing the active variant ( DPP9 - SWT ) show similar levels of MMC - induced BRCA2 degradation . RAD51 stability is not altered by MMC and is similar in all cell lines . Vinculin is a loading control for BRCA2 , Tubulin is a loading control for RAD51 . Shown images originate from one representative cycloheximide ( CHX ) chase assay . The ratios of BRCA2 to Vinculin are defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test ( * = p ≤ 0 . 03 , * * = p ≤ 0 . 002 ) . Antibodies as described in ( A - B ) .

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

C-D. Representative Western Blots and graph summarizing results of more than three biological replicates. CHX chase assays show that the MMC-induced degradation of BRCA2 is less pronounced in HEK293 DPP9 KO cells as well as in HEK293 DPP9 KO + DPP9-SS729A cells overexpressing an inactive DPP9-S mutant. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. Cells that were overexpressing the active variant (DPP9-SWT) show similar levels of MMC-induced BRCA2 degradation. RAD51 stability is not altered by MMC and is similar in all cell lines. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03, **= p ≤ 0.002). Antibodies as described in (A-B).


F - G . Representative Western Blots and graphs summarizing CHX assays from three biological replicates showing that the BRCA2 ∆ MP3 - 1000 truncation mutant is degraded at a higher rate compared to the untruncated BRCA21 - 1000 . Cells were co - transfected with a GFP expressing plasmid , as transfection and loading control . Control cells were transfected with GFP only ( U . T ) . Image originates from one representative experiment . BRCA2 - FLAG signals are related to the transfection and loading control GFP . BRCA2 levels in relation to GFP were defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test ( * = p ≤ 0 . 03 ) . Anti - FLAG : RRID : AB _ 262044 , anti - GFP : RRID : AB _ 641123 .

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

F-G. Representative Western Blots and graphs summarizing CHX assays from three biological replicates showing that the BRCA2∆MP3-1000 truncation mutant is degraded at a higher rate compared to the untruncated BRCA21-1000. Cells were co-transfected with a GFP expressing plasmid, as transfection and loading control. Control cells were transfected with GFP only (U.T). Image originates from one representative experiment. BRCA2-FLAG signals are related to the transfection and loading control GFP. BRCA2 levels in relation to GFP were defined as 100 % at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03). Anti-FLAG: RRID:AB_262044, anti-GFP: RRID:AB_641123.


A . Representative immunofluorescence images of γH2AX signals ( white , RRID : AB _ 309864 ) in gki MEF DPP9S729A , showing more γH2AX in gki MEF DPP9S729A cells following removal of Neocarzinostatin ( NCS ) , quantification in ( B ) . MEF DPP9S729A and control MEF cells were treated with 250 ng / mL NCS for 30 min and allowed to recover for the indicated time - points . γH2AX signals at time 0 , reflect 30 min of NCS and no recovery time . Nuclei are shown in blue ( DAPI ) . Scale bar 10 µm . B . Quantification of γH2AX in gki MEF DPP9S729A and control MEF cells as described in ( A ) . Signals from more than 1700 cells were quantified per condition per experiment . Mean ± SEM from six biological replicates , each in technical duplicates . Data were analysed by an unpaired two - way ANOVA with Sidak ' s multiple comparison test ( * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 ) .

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A. Representative immunofluorescence images of γH2AX signals (white, RRID:AB_309864) in gki MEF DPP9S729A, showing more γH2AX in gki MEF DPP9S729A cells following removal of Neocarzinostatin (NCS), quantification in (B). MEF DPP9S729A and control MEF cells were treated with 250 ng/mL NCS for 30 min and allowed to recover for the indicated time-points. γH2AX signals at time 0, reflect 30 min of NCS and no recovery time. Nuclei are shown in blue (DAPI). Scale bar 10 µm. B. Quantification of γH2AX in gki MEF DPP9S729A and control MEF cells as described in (A). Signals from more than 1700 cells were quantified per condition per experiment. Mean ± SEM from six biological replicates, each in technical duplicates. Data were analysed by an unpaired two-way ANOVA with Sidak's multiple comparison test (* = p ≤ 0.05, ** = p ≤ 0.01).


C . Representative PLA images of BRCA2 - PALB2 PLA experiments showing more PLA events ( white ) in HeLa cells silenced for DPP9 ( siDPP9 ) with respect to cells treated with non - targeting siRNA ( siNT ) . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - PALB2 : RRID : AB _ 890607 , anti - BRCA2 : RRID : AB _ 2259370 .

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C. Representative PLA images of BRCA2-PALB2 PLA experiments showing more PLA events (white) in HeLa cells silenced for DPP9 (siDPP9) with respect to cells treated with non-targeting siRNA (siNT). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-PALB2: RRID:AB_890607, anti-BRCA2: RRID:AB_2259370.


D . Quantification of PLAs experiments showing more BRCA2 - PALB2 PLA events in cells silenced for DPP9 ( siDPP9 ) in comparison to non - targeting controls ( siNT ) . Each dot represents the number of PLA events in a single cell , from four biological replicates . The technical control samples ( NgtCntrl ) omitted the BRCA2 antibody . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) .

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D. Quantification of PLAs experiments showing more BRCA2-PALB2 PLA events in cells silenced for DPP9 (siDPP9) in comparison to non-targeting controls (siNT). Each dot represents the number of PLA events in a single cell, from four biological replicates. The technical control samples (NgtCntrl) omitted the BRCA2 antibody. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).


E . Representative PLA images of γH2Ax - BRCA2 PLA experiment showing a reduction in the number of PLA events ( white ) between γH2Ax and BRCA2 in HeLa DPP9 KD cells . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - γH2Ax : RRID : AB _ 2118009 , anti - BRCA2 : RRID : AB _ 2259370 F . Quantification of PLAs showing fewer MMC - induced γH2Ax - BRCA2 PLA events in HeLa DPP9 KD cells , in comparison to HeLa WT cells . Each dot represents the number of PLA events in a single cell , from three biological replicates . The technical control samples ( NgtCntrl ) omitted the γH2Ax antibody . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) .

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

E. Representative PLA images of γH2Ax-BRCA2 PLA experiment showing a reduction in the number of PLA events (white) between γH2Ax and BRCA2 in HeLa DPP9 KD cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-γH2Ax: RRID:AB_2118009, anti-BRCA2: RRID:AB_2259370 F. Quantification of PLAs showing fewer MMC-induced γH2Ax-BRCA2 PLA events in HeLa DPP9 KD cells, in comparison to HeLa WT cells. Each dot represents the number of PLA events in a single cell, from three biological replicates. The technical control samples (NgtCntrl) omitted the γH2Ax antibody. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).


A . Representative immunofluorescence images showing that re - expression of DPP9 - SWT leads to an increase in the number of RAD51 foci formed following exposure to MMC in HEK293 DPP9 KO + DPP9 - SWT cells . Expression of DPP9 was induced ( + Dox , 1µg / ml ) simultaneously with MMC ( 300 nM ) , 24 h . RAD51 foci are shown in white , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 . B . Graph showing the number of RAD51 foci following induction of DPP9 - SWT expression , compared to uninduced HEK293 DPP9 KO + DPP9WT cells ( - Dox ) . Induction of HEK293 DPP9 KO + DPP9S729A for expression of DPP9 - SS729A did not result in more RAD51 foci . Each dot represents the number of RAD51 foci in a single cell , from three biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) .

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

A. Representative immunofluorescence images showing that re-expression of DPP9-SWT leads to an increase in the number of RAD51 foci formed following exposure to MMC in HEK293 DPP9 KO + DPP9-SWT cells. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428. B. Graph showing the number of RAD51 foci following induction of DPP9-SWT expression, compared to uninduced HEK293 DPP9 KO + DPP9WT cells (-Dox). Induction of HEK293 DPP9 KO + DPP9S729A for expression of DPP9-SS729A did not result in more RAD51 foci. Each dot represents the number of RAD51 foci in a single cell, from three biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).


Representative images ( C , and summarizing graph ( D showing the number of RAD51 foci per nucleus in HeLa WT cells ( C , D ) Where stated , cells were treated with control siRNA ( siNT ) or silenced for BRCA2 , and transiently transfected with the BRCA21 ‑ 3418 or BRCA23 - 3418 constructs . Both BRCA2 constructs can rescue the RAD51 foci formation phenotype to the control levels in HeLa WT cells ( C , D ) . Each dot represents the number of RAD51 foci in a single cell , from three ( D ) biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) . RAD51 foci are shown in white , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 ; ; anti - BRCA2 : RRID : AB _ 2259370 .

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

Representative images (C, and summarizing graph (D showing the number of RAD51 foci per nucleus in HeLa WT cells (C, D) Where stated, cells were treated with control siRNA (siNT) or silenced for BRCA2, and transiently transfected with the BRCA21‑3418 or BRCA23-3418 constructs. Both BRCA2 constructs can rescue the RAD51 foci formation phenotype to the control levels in HeLa WT cells (C, D). Each dot represents the number of RAD51 foci in a single cell, from three (D) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; ; anti-BRCA2: RRID:AB_2259370.


Representative images , E ) and summarizing graph F ) showing the number of RAD51 foci per nucleus in HeLa DPP9 KD cells ( E , F ) . In HeLa DPP9 KD cells significantly more RAD51 foci were present in cells transfected with BRCA2 ∆ MP3 - 3418 compared to BRCA2 silenced cells and cells expressing the untruncated BRCA21 - 3418 ( E , F ) . Each dot represents the number of RAD51 foci in a single cell , from four ( F ) biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) . RAD51 foci are shown in white , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 ; anti - BRCA2 : RRID : AB _ 2259370 .

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

Representative images , E) and summarizing graph F) showing the number of RAD51 foci per nucleus in HeLa DPP9 KD cells (E, F). In HeLa DPP9 KD cells significantly more RAD51 foci were present in cells transfected with BRCA2∆MP3-3418 compared to BRCA2 silenced cells and cells expressing the untruncated BRCA21-3418 (E, F). Each dot represents the number of RAD51 foci in a single cell, from four (F) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; anti-BRCA2: RRID:AB_2259370.


a , Phase partitioning of Apg8 . Wild - type and Δapg4Δapg8 cells producing Apg8FG and Δapg7 cells were subjected to phase partitioning ( see Methods ) .

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a, Phase partitioning of Apg8. Wild-type and Δapg4Δapg8 cells producing Apg8FG and Δapg7 cells were subjected to phase partitioning (see Methods).


b , MALDI mass spectrum of His6 - Apg8 . Purified His6 - Apg8 ( predominantly Apg8FG - X and a small amount of Apg8FG ) was extracted with 0 . 4 % β - octylglycoside from a polyacrylamide gel and desalted with ZipTipC4 ( Millipore ) before mass measurement . The principal MH + ion signal at m / z 15 , 003 is shifted in mass by about + 635 from the MH + value corresponding to His6 - Apg8FG .

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b, MALDI mass spectrum of His6-Apg8. Purified His6-Apg8 (predominantly Apg8FG-X and a small amount of Apg8FG) was extracted with 0.4% β-octylglycoside from a polyacrylamide gel and desalted with ZipTipC4(Millipore) before mass measurement. The principal MH+ ion signal at m/z 15,003 is shifted in mass by about +635 from the MH+ value corresponding to His6-Apg8FG.


c , MS / MS spectra of the C - terminal peptides of Apg8FG - X ( top ) and Apg8FG ( bottom ) . C - terminal fragments were generated from Apg8FG - X and Apg8FG ( see Methods ) . Each fragment that was observed only as non - 18O - labelled signals was subjected to MS / MS . The MS / MS spectra , deconvoluted with MaxEnt3 , were interpreted by SeqMS software26 . Arrows show the sequences from the N and C terminus based on y ″ m and bl ions , respectively ( where m and l denote positions counted from the C and N terminus ) that were produced by cleavage of peptide bonds during MS / MS . Amino acids shown in three - letter code denote immonium ions . The nomenclature of these ions is as in previous work28 .

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

c, MS/MS spectra of the C-terminal peptides of Apg8FG-X (top) and Apg8FG (bottom). C-terminal fragments were generated from Apg8FG-X and Apg8FG (see Methods). Each fragment that was observed only as non-18O-labelled signals was subjected to MS/MS. The MS/MS spectra, deconvoluted with MaxEnt3, were interpreted by SeqMS software26. Arrows show the sequences from the N and C terminus based on y″m and bl ions, respectively (where m and l denote positions counted from the C and N terminus) that were produced by cleavage of peptide bonds during MS/MS. Amino acids shown in three-letter code denote immonium ions. The nomenclature of these ions is as in previous work28.


d , Thioester formation between Apg8 and Apg7 . Cell extracts of Δapg7Δapg8 cells expressing Apg7 - Myc and / or HA - Apg8 from 2µ plasmids were immunoprecipitated with anti - HA antibody , and immunoblotted ( IB ) with anti - Myc and anti - HA antibody . DTT - , no DTT ; DTT + , 100 mM DTT ; asterisk , Apg8 - Apg7 conjugate .

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d, Thioester formation between Apg8 and Apg7. Cell extracts of Δapg7Δapg8 cells expressing Apg7-Myc and/or HA-Apg8 from 2µ plasmids were immunoprecipitated with anti-HA antibody, and immunoblotted (IB) with anti-Myc and anti-HA antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg7 conjugate.


b , Interaction between Apg8 and Apg7 . Δapg7Δapg8 cells were transformed with APG7 - Myc and / or APG8 on 2µ plasmids . Cell lysates were immunoprecipitated with anti - Myc antibody , and immunoblotted with anti - Myc or anti - Apg8 antibody . Apg8 co - immunoprecipitated with Apg7 - Myc .

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b, Interaction between Apg8 and Apg7. Δapg7Δapg8 cells were transformed with APG7-Myc and/or APG8 on 2µ plasmids. Cell lysates were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc or anti-Apg8 antibody. Apg8 co-immunoprecipitated with Apg7-Myc.


c , Effect of Apg8 C - terminal processing on the Apg8 - Apg7 interaction . Δapg4 cells co - expressing Apg7 - Myc and HA - Apg8 ( FGR , FG and F ) from 2µ plasmids . FGR , wild - type Apg8 ; FG , Apg8 lacking C - terminal arginine ; F , Apg8 lacking C - terminal glycine and arginine . Cell lysates were immunoprecipitated with anti - HA antibody , and immunoblotted with anti - HA and anti - Myc antibody .

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c, Effect of Apg8 C-terminal processing on the Apg8-Apg7 interaction. Δapg4 cells co-expressing Apg7-Myc and HA-Apg8 (FGR, FG and F) from 2µ plasmids. FGR, wild-type Apg8; FG, Apg8 lacking C-terminal arginine; F, Apg8 lacking C-terminal glycine and arginine. Cell lysates were immunoprecipitated with anti-HA antibody, and immunoblotted with anti-HA and anti-Myc antibody.


b , Interaction between Apg8 and Apg3 . Lysates of Δapg3 cells expressing APG3 and / or Myc - APG8 from 2µ plasmids were immunoprecipitated with anti - Apg3 antibody , and immunoblotted with anti - Apg3 and anti - Myc antibody .

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b, Interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing APG3 and/or Myc-APG8 from 2µ plasmids were immunoprecipitated with anti-Apg3 antibody, and immunoblotted with anti-Apg3 and anti-Myc antibody.


c , Effect of mutated Cys 234 in Apg3 on the interaction between Apg8 and Apg3 . Lysates of Δapg3 cells expressing Myc - APG8 and either wild - type ( wt ) APG3 or mutant APG3 ( C234S and C234A ) from 2µ plasmids were immunoprecipitated with anti - Myc antibody , and immunoblotted with anti - Myc and anti - Apg3 antibodies .

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c, Effect of mutated Cys 234 in Apg3 on the interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing Myc-APG8 and either wild-type (wt) APG3 or mutant APG3 (C234S and C234A) from 2µ plasmids were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc and anti-Apg3 antibodies.


d , Thioester formation between Apg8 and Apg3 . Δapg3 , Δapg7 and Δapg10 cells were transformed with both Myc - APG8 and Apg3 , or only Apg3 , on 2µ plasmids . Cell extracts were immunoprecipitated with anti - Myc antibody , and subjected to immunoblot ( IB ) using anti - Myc and anti - Apg3 antibody . DTT - , no DTT ; DTT + , 100 mM DTT ; asterisk , Apg8 - Apg3 conjugate ; diamond , possible Myc - Apg8 dimer .

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d, Thioester formation between Apg8 and Apg3. Δapg3, Δapg7 and Δapg10 cells were transformed with both Myc-APG8 and Apg3, or only Apg3, on 2µ plasmids. Cell extracts were immunoprecipitated with anti-Myc antibody, and subjected to immunoblot (IB) using anti-Myc and anti-Apg3 antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg3 conjugate; diamond, possible Myc-Apg8 dimer.