Figure 4Chk2 inhibition with the small molecule BML - 277 induces transient p53 dynamics with only one pulse after 10 Gy IR . A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and addition of 10 µM BML - 277 by immunofluorescence staining ( see Methods section for details ) are shown as box plots ( see Data Visualization section ) . We quantified promoter activity of BAX ( B , sustained archetype ) and PPM1D ( C , pulsatile archetype ) before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after irradiation with 10 Gy IR and inhibition of the second p53 pulse by Chk2 inhibition . Left panel : The percentage of cells with active TSS , subdivided into populations with strong ( > 75 % of TSS , solid colors ) and weak ( < 75 % of TSS , shaded colors ) activity , is shown as stacked bar graphs , the mean fraction of active promoters is indicated above each bar . Right panel : Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . The fraction of active promoters was reduced at 6 h and 9 h after irradiation ; the transcription rate was not notably affected . Sequential treatment with Nutlin - 3 converts pulsatile p53 dynamics into sustained nuclear levels . A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and sequential treatment with 0 . 75 µM Nutlin - 3 at 2 . 5 h , with 2 . 25 µM at 3 . 5 h and 4 µM at 5 . 5 h post IR based on immunofluorescence staining ( see Methods section for details ) are shown as box plots ( see Data Visualization section ) . We quantified promoter activity of MDM2 ( E , transient archetype ) and CDKN1A ( F , transient archetype ) before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after irradiation with 10 Gy IR and sequential Nutlin - 3 treatment . Left panel : The percentage of cells with active TSS , subdivided into populations with strong ( > 75 % of TSS , solid colors ) and weak ( < 75 % of TSS , shaded colors ) activity , is shown as stacked bar graphs , the mean fraction of active promoters is indicated above each bar . Right panel : Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . The relative fraction of active promoters strongly increased , changing transient to sustained archetypes . The transcription rate increased as well both compared to basal levels and to previous experiments with pulsatile p53 dynamics ( inset , fold change relative to IR alone for each time point ) .

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

Figure 4Chk2 inhibition with the small molecule BML-277 induces transient p53 dynamics with only one pulse after 10 Gy IR . A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and addition of 10 µM BML-277 by immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section).We quantified promoter activity of BAX (B, sustained archetype) and PPM1D (C, pulsatile archetype) before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after irradiation with 10 Gy IR and inhibition of the second p53 pulse by Chk2 inhibition. Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The fraction of active promoters was reduced at 6 h and 9 h after irradiation; the transcription rate was not notably affected.Sequential treatment with Nutlin-3 converts pulsatile p53 dynamics into sustained nuclear levels. A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and sequential treatment with 0.75 µM Nutlin-3 at 2.5 h, with 2.25 µM at 3.5 h and 4 µM at 5.5 h post IR based on immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section).We quantified promoter activity of MDM2 (E, transient archetype) and CDKN1A (F, transient archetype) before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after irradiation with 10 Gy IR and sequential Nutlin-3 treatment. Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The relative fraction of active promoters strongly increased, changing transient to sustained archetypes. The transcription rate increased as well both compared to basal levels and to previous experiments with pulsatile p53 dynamics (inset, fold change relative to IR alone for each time point).


Figure 5Total p53 , p53 acetylated at K382 and K370 as well as GAPDH were measured by Western Blot at indicated time points in the context of different p53 dynamics : pulsing p53 ( 10 Gy IR ) , transient p53 ( 10 Gy IR + BML - 277 , central lanes ) and sustained p53 ( 10 Gy IR + Nutlin - 3 , right lanes ) . See Figure 3 and Methods section for details . The relative change in p53 acetylation at K370 ( light green ) and K382 ( dark green ) was quantified from Western Blot and normalized to the abundance 3 h post IR . Means and propagated standard errors from 3 independent experiments are indicated . Acetylation increased over time in the context of sustained p53 . See also Appendix Fig S12 . The p53 - K370 methylase Smyd2 was down - regulated in a clonal stable A549 cell line expressing a corresponding shRNA . Transcript levels were measured in wild type and knockdown cells by qRT - PCR . Mean levels and standard deviation from technical triplicates are indicated . Promoter activity of CDKN1A ( E ) and MDM2 ( F ) were quantified in Smyd2 knockdown cells before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . Left panel : The percentage of cells with active TSS , subdivided into populations with strong ( > 75 % of TSS , solid colors ) and weak ( < 75 % of TSS , shaded colors ) activity , is shown as stacked bar graphs , the mean fraction of active promoters is indicated above each bar . Right panel : Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . We measured a higher fraction of active promoters upon damage compared to A549 wild type cells ( Fig 3 ) , while transcription rates remained unchanged . The p53 - K382 methylase Set8 was down - regulated in a clonal stable A549 cell line expressing a corresponding shRNA . Transcript levels were measured in wild type and knockdown cells by qRT - PCR . Mean levels and standard deviation from technical triplicates are indicated . Promoter activity of CDKN1A ( E ) and MDM2 ( F ) were quantified in Set8 knock - down cells before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . Left panel : The percentage of cells with active TSS , subdivided into populations with strong ( > 75 % of TSS , solid colors ) and weak ( < 75 % of TSS , shaded colors ) activity , is shown as stacked bar graphs , the mean fraction of active promoters is indicated above each bar . Right panel : Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . We measured a higher fraction of active promoters upon damage compared to A549 wild type cells ( Figure 3 ) , while transcription rates remained unchanged .

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

Figure 5Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western Blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML-277, central lanes) and sustained p53 (10 Gy IR + Nutlin-3, right lanes). See Figure 3 and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western Blot and normalized to the abundance 3 h post IR. Means and propagated standard errors from 3 independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12.The p53-K370 methylase Smyd2 was down-regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild type and knockdown cells by qRT-PCR. Mean levels and standard deviation from technical triplicates are indicated.Promoter activity of CDKN1A (E) and MDM2 (F) were quantified in Smyd2 knockdown cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). We measured a higher fraction of active promoters upon damage compared to A549 wild type cells (Fig 3), while transcription rates remained unchanged.The p53-K382 methylase Set8 was down-regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild type and knockdown cells by qRT-PCR. Mean levels and standard deviation from technical triplicates are indicated.Promoter activity of CDKN1A (E) and MDM2 (F) were quantified in Set8 knock-down cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). We measured a higher fraction of active promoters upon damage compared to A549 wild type cells (Figure 3), while transcription rates remained unchanged.


figf1a , Western blot analysis of yeast cell lysates with polyclonal anti - flag antibody to detect flag - tagged human Beclin 1 expression . Two - hundred micrograms of protein per lane was loaded . SEY6210 , wild - type yeast ; JCY3000 , SEY6210 disrupted in apg6 / vps30 ; ( + ) control , lysates of mammalian cells transfected with flag - beclin 1 . b , Autophagicbody formation in yeast deprived of nitrogen in the presence ( + ) and absence ( - ) of 1 mM PMSF . Arrows denote cells that would be scored as positive in the experiment shown in c . c , Quantitative effects of apg6 / vps30 and beclin 1 transformation on autophagicbody formation in apg6 / vps30 - disrupted yeast in the presence of PMSF . Cells with one or more autophagic bodies within the vacuole were scored as positive ( see arrows in b ) . A minimum of 100 cells was counted for each sample . Results represent the mean ( ± s . e . m . ) percentage of cells with autophagic bodies within the vacuole for triplicate samples . Similar results were obtained in five independent experiments . d , Sorting of vacuolar protein carboxypeptidase Y , ( CPY ) . p2CPY , precursor form ; mCPY , sorted mature form . Mr , relative molecular mass .

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

figf1a, Western blot analysis of yeast cell lysates with polyclonal anti-flag antibody to detect flag-tagged human Beclin 1 expression. Two-hundred micrograms of protein per lane was loaded. SEY6210, wild-type yeast; JCY3000, SEY6210 disrupted in apg6/vps30; (+) control, lysates of mammalian cells transfected with flag-beclin 1.b, Autophagicbody formation in yeast deprived of nitrogen in the presence (+) and absence (-) of 1 mM PMSF. Arrows denote cells that would be scored as positive in the experiment shown in c.c, Quantitative effects of apg6/vps30 and beclin 1 transformation on autophagicbody formation in apg6/vps30-disrupted yeast in the presence of PMSF. Cells with one or more autophagic bodies within the vacuole were scored as positive (see arrows in b). A minimum of 100 cells was counted for each sample. Results represent the mean (±s.e.m.) percentage of cells with autophagic bodies within the vacuole for triplicate samples. Similar results were obtained in five independent experiments.d, Sorting of vacuolar protein carboxypeptidase Y, (CPY). p2CPY, precursor form; mCPY, sorted mature form. Mr, relative molecular mass.


figf2a - f , Electron micrographs of MCF7 . control ( clone 38 ) ( a , b ) and MCF7 . beclin1 ( clone 17 ) cells ( c - f ) grown in nutrient - rich media ( a , c ) or subjected to 4 h of serum and amino - acid deprivation ( b , d - f ) . Asterisks in e and f denote autophagic vacuoles that would be counted in the experiment shown in g . Autophagic vacuoles were defined as double - membrane vacuolar structures containing recognizable cytoplasmic contents . Scale bars , 1 µm . g , Quantitative effects of beclin 1 on basal and nutrient - deprivation - induced autophagy of MCF7 cells . Bars indicate mean ( ± s . e . m . ) number of autophagic vacuoles per cell for cells growing in normal media ( black ) , for cells subjected to 4 h of serum and amino - acid deprivation ( grey ) , and for cells pre - treated for 30 min with 10 mM 3 - MA and subjected to 4 h of serum and amino - acid deprivation in the presence of 3 - MA ( open ) . Mean was determined by counting the total number of autophagic vacuoles in each cell for 100 cells per clone per treatment . h , Comparison of the rates of degradation of long - lived proteins in MCF7 . control cells ( squares , clone 38 ) , MCF7 . beclin1 cells ( circles , clone 17 ) and MCF7 . beclin1stop cells ( triangles , clone 70 ) , in EBSS + 10 % serum and complete amino acids ( open symbols , solid lines ) in EBSS alone ( closed symbols , solid lines ) , and in EBSS + 10 mM 3 - MA ( closed symbols , dotted lines ) . Results are mean ( ± s . e . m . ) of triplicate wells . Similar results were obtained in three independent experiments . Results shown with these clones are representative of results obtained with other clones analysed by electron microscopy in g .

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figf2a-f, Electron micrographs of MCF7.control (clone 38) (a, b) and MCF7.beclin1 (clone 17) cells (c- f) grown in nutrient-rich media (a, c) or subjected to 4 h of serum and amino-acid deprivation (b, d-f). Asterisks in e and f denote autophagic vacuoles that would be counted in the experiment shown in g. Autophagic vacuoles were defined as double-membrane vacuolar structures containing recognizable cytoplasmic contents. Scale bars, 1 µm.g, Quantitative effects of beclin 1 on basal and nutrient-deprivation-induced autophagy of MCF7 cells. Bars indicate mean (±s.e.m.) number of autophagic vacuoles per cell for cells growing in normal media (black), for cells subjected to 4 h of serum and amino-acid deprivation (grey), and for cells pre-treated for 30 min with 10 mM 3-MA and subjected to 4 h of serum and amino-acid deprivation in the presence of 3-MA (open). Mean was determined by counting the total number of autophagic vacuoles in each cell for 100 cells per clone per treatment.h, Comparison of the rates of degradation of long-lived proteins in MCF7.control cells (squares, clone 38), MCF7.beclin1 cells (circles, clone 17) and MCF7.beclin1stop cells (triangles, clone 70), in EBSS + 10% serum and complete amino acids (open symbols, solid lines) in EBSS alone (closed symbols, solid lines), and in EBSS + 10 mM 3-MA (closed symbols, dotted lines). Results are mean (±s.e.m.) of triplicate wells. Similar results were obtained in three independent experiments. Results shown with these clones are representative of results obtained with other clones analysed by electron microscopy in g.


figf3a , Western blot analysis of flag - tagged beclin - 1 - transfected MCF7 clones . C , MCF7 . control cells ( clone 38 ) ; St , MCF7 . beclin1stop cells ( clone 70 ) . Mr , relative molecular mass . b , Western blot analysis of flag - tagged beclin1stop - transfected MCF7 clones . C , MCF . control cells ( clone 38 ) ; B , MCF7 . beclin1 cells ( clone 1 ) . c , Photomicrographs of MCF7 . control cells ( left panel , clone 38 ) , MCF7 . beclin1 cells ( middle panel , clone 17 ) and MCF7 . beclin1stop cells ( right panel , clone 70 ) 48 h after seeding at similar densities . d , Proliferation of MCF7 . control cells ( clones 37 and 38 ; closed squares and circles , respectively , solid lines ) , MCF7 . beclin1 cells ( clones 1 , 6 , 17 ; open squares , circles , triangles , respectively , solid lines ) and MCF7 . beclin1stop cells ( clone 70 , closed triangles , dotted line ) . Results represent mean Absorbance ( A ) ( ± s . e . m . ) for six wells ; similar results were obtained in three independent experiments . e , Cell viability of MCF7 clones determined by trypan blue staining . Symbols for each clone are as in df , Clonigenicity in semisolid medium ( soft agar ) of MCF7 clones . Results are mean ( ± s . e . m . ) for pooled triplicate wells from 3 - 4 independent experiments . g , Tumour formation in NCR nude mice injected subcutaneously with MCF7 clones . Numbers on top of bar indicate number of autopsy - confirmed tumours at 8 weeks per number of mice injected with each clone .

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figf3a, Western blot analysis of flag-tagged beclin-1-transfected MCF7 clones. C, MCF7.control cells (clone 38); St, MCF7.beclin1stop cells (clone 70). Mr, relative molecular mass.b, Western blot analysis of flag-tagged beclin1stop-transfected MCF7 clones. C, MCF.control cells (clone 38); B, MCF7.beclin1 cells (clone 1).c, Photomicrographs of MCF7.control cells (left panel, clone 38), MCF7. beclin1 cells (middle panel, clone 17) and MCF7.beclin1stop cells (right panel, clone 70) 48 h after seeding at similar densities.d, Proliferation of MCF7.control cells (clones 37 and 38; closed squares and circles, respectively, solid lines), MCF7.beclin1 cells (clones 1, 6, 17; open squares, circles, triangles, respectively, solid lines) and MCF7.beclin1stop cells (clone 70, closed triangles, dotted line). Results represent mean Absorbance (A) (±s.e.m.) for six wells; similar results were obtained in three independent experiments.e, Cell viability of MCF7 clones determined by trypan blue staining. Symbols for each clone are as in df, Clonigenicity in semisolid medium (soft agar) of MCF7 clones. Results are mean (±s.e.m.) for pooled triplicate wells from 3-4 independent experiments.g, Tumour formation in NCR nude mice injected subcutaneously with MCF7 clones. Numbers on top of bar indicate number of autopsy-confirmed tumours at 8 weeks per number of mice injected with each clone.


figf4a , Western blot analysis of human breast carcinoma cell lines . Results are from 8 representative cell lines out of a total of 11 analysed . b , Western blot analysis of matched normal breast and breast carcinoma tissue from patients with sporadic invasive breast carcinoma . Results are from 7 representative cases out of a total of 17 analysed . N , normal breast tissue ; T , tumour tissue ; Mr , relative molecular mass . c , Immunohistochemical analysis of paraffin - embedded sections of breast tissue from patient 441 shown in b . Adjacent sections are stained with pre - immune 843 rabbit serum ( left column ) , 843 anti - Beclin 1serum ( middle column ) and anti - cytokeratin ( right column ) . Top row shows low power view of region of breast containing tumour ( T ) and normal mammary epithelial duct ( N ) . Middle row shows higher power view of normal mammary epithelial duct . Bottom row shows higher power view of tumour . Scale bars , 1 mm .

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figf4a, Western blot analysis of human breast carcinoma cell lines. Results are from 8 representative cell lines out of a total of 11 analysed.b, Western blot analysis of matched normal breast and breast carcinoma tissue from patients with sporadic invasive breast carcinoma. Results are from 7 representative cases out of a total of 17 analysed. N, normal breast tissue; T, tumour tissue; Mr, relative molecular mass.c, Immunohistochemical analysis of paraffin-embedded sections of breast tissue from patient 441 shown in b. Adjacent sections are stained with pre-immune 843 rabbit serum (left column), 843 anti-Beclin 1serum (middle column) and anti-cytokeratin (right column). Top row shows low power view of region of breast containing tumour (T) and normal mammary epithelial duct (N). Middle row shows higher power view of normal mammary epithelial duct. Bottom row shows higher power view of tumour. Scale bars, 1 mm.


Figure 1 ( A ) Western blot detection of p - CHK2 Thr68 , CHK2 , p62 and LC3 in H1299 cells in normal medium ( HBSS ' ' - ' ' ) or after 1 hr HBSS starvation ( HBSS ' ' + ' ' ) pretreated with or without NAC ( 3mM ) . ( B ) Western blot detection of p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 ( 500μM ) treatment . ( C ) Western blot detection of p62 and LC3 in H1299 cells cotransfected with the indicated shRNA and the indicated plasmid and cultured in normal medium or after H2O2 ( 500μM ) treatment . CHK2 NTm , a shRNA nontargetable mutant CHK2 rescue plasmid . ( D ) Autophagic flux is shown by representative confocal microscopic images of 293 cells stably - expressing GFP - mCherry - LC3 transfected with the indicated shRNA following HBSS starvation and H2O2 ( 500μM ) treatment for 3h . Scale bar , 10 μm . ( E ) Quantitation of autophagosomal ( yellow ) and autolysosomal ( red ) LC3 puncta following HBSS starvation and H2O2 ( 500μM ) treatment for 1h ( n = 30 ) . Data are presented as mean ± s . e . m . from 3 independent experiments ; * * P < 0 . 01 ( Student ' s t - test ) . ( F ) Representative electron microscopic image of autophagic vesicles or autophagosomes in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 ( 500μM ) . Scale bars , 500 nm . The red arrows indicate double - membraned autophagic structures . ( G ) Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 ( 500μM ) . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ; * * P < 0 . 01 ( Student ' s t - test ) .

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

Figure 1(A) Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3 in H1299 cells in normal medium (HBSS ''-'') or after 1 hr HBSS starvation (HBSS ''+'') pretreated with or without NAC (3mM).(B) Western blot detection of p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment.(C) Western blot detection of p62 and LC3 in H1299 cells cotransfected with the indicated shRNA and the indicated plasmid and cultured in normal medium or after H2O2 (500μM) treatment. CHK2 NTm, a shRNA nontargetable mutant CHK2 rescue plasmid.(D) Autophagic flux is shown by representative confocal microscopic images of 293 cells stably-expressing GFP-mCherry-LC3 transfected with the indicated shRNA following HBSS starvation and H2O2 (500μM) treatment for 3h. Scale bar, 10 μm.(E) Quantitation of autophagosomal (yellow) and autolysosomal (red) LC3 puncta following HBSS starvation and H2O2 (500μM) treatment for 1h (n=30). Data are presented as mean±s.e.m. from 3 independent experiments; **P < 0.01 (Student's t-test).(F) Representative electron microscopic image of autophagic vesicles or autophagosomes in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Scale bars, 500 nm. The red arrows indicate double-membraned autophagic structures.(G) Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Data are presented as mean±s.e.m. from 3 independent experiments; *P < 0.05; **P < 0.01 (Student's t-test).


Figure 2 ( A ) Immunoprecipitation assays testing the physical interaction between CHK2 and proteins encoded by autophagy - related genes in HCT116 cells . Lysates were extracted for immunoprecipitation with CHK2 - specific antibody or control IgG , followed by probing with antibodies specific for Ulk1 , Atg5 , Beclin1 , Atg7 or LC3 . ( B ) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions ( t = 0 ) and after HBSS starvation in the cytoplasmic fractions . ( C ) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions ( t = 0 ) and after H2O2 ( 500μM ) stimulation in the cytoplasmic fractions . ( D ) HCT116 cells were pretreated with NAC in RPMI 1640 complete medium for 4 h and then cultured for 1 hr in HBSS starvation . The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control . HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr and then cultured for 1 hr in HBSS starvation ( E ) The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control . HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr or H2O2 ( 500μM ) stimulation ( F ) . The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control . HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were untreated ( G ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody . HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were treated with HBSS starvation ( H ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody . HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were treated with H2O2 ( 500μM ) stimulation ( I ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody . ( J ) Western blot detection of p62 and LC3 in H1299 cells transfected with indicated plasmid in normal medium or after H2O2 ( 500 μM ) cultured for 3 hr . ( K ) H1299 cells were pretreated with CHK2 inhibitorⅡ in complete medium for 4 h and then cultured for 3 hr in H2O2 ( 500μM ) stimulation . Western blot detection of p - CHK2 Thr68 , CHK2 , p62 and LC3 .

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

Figure 2(A) Immunoprecipitation assays testing the physical interaction between CHK2 and proteins encoded by autophagy-related genes in HCT116 cells. Lysates were extracted for immunoprecipitation with CHK2-specific antibody or control IgG, followed by probing with antibodies specific for Ulk1, Atg5, Beclin1, Atg7 or LC3.(B) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t=0) and after HBSS starvation in the cytoplasmic fractions.(C) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t=0) and after H2O2 (500μM) stimulation in the cytoplasmic fractions.(D) HCT116 cells were pretreated with NAC in RPMI 1640 complete medium for 4 h and then cultured for 1 hr in HBSS starvation. The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr and then cultured for 1 hr in HBSS starvation (E) The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr or H2O2 (500μM) stimulation (F). The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were untreated (G) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were treated with HBSS starvation (H) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were treated with H2O2 (500μM) stimulation (I) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.(J) Western blot detection of p62 and LC3 in H1299 cells transfected with indicated plasmid in normal medium or after H2O2 (500 μM) cultured for 3 hr.(K) H1299 cells were pretreated with CHK2 inhibitorⅡ in complete medium for 4 h and then cultured for 3 hr in H2O2 (500μM) stimulation. Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3.


Figure 3 ( A ) The indicated recombinant fragments of GST - Beclin1 were incubated with recombinant CHK2 in the presence of 32P - labeled ATP for in vitro kinase assays . MBP was used as a positive control . Ponceau staining indicates the expression of GST - Beclin 1 fragments for in vitro kinase assays . The asterisks in the blot indicate the domain of phosphorylated Beclin 1 . ( C ) In vitro kinase assays to test the ability of recombinant CHK2 to phosphorylate recombinant GST - Beclin 1 - WT , S9093A ( AA ) , S90A and S93A protein . Reactions were analyzed by SDS - PAGE followed by autoradiography . The asterisks in the blot indicate the phosphorylation of Beclin 1 mutants . Western blot detection of p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or after 1 hr HBSS starvation ( D ) Western blot detection of p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or H2O2 ( 500μM ) stimulation ( E ) . Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or after HBSS starvation ( F ) Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or H2O2 ( 500μM ) stimulation ( G ) . ( H ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells pretreated with NAC for 4 h and then cultured for 1 hr in HBSS starvation .

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

Figure 3(A) The indicated recombinant fragments of GST-Beclin1 were incubated with recombinant CHK2 in the presence of 32P-labeled ATP for in vitro kinase assays. MBP was used as a positive control. Ponceau staining indicates the expression of GST-Beclin 1 fragments for in vitro kinase assays. The asterisks in the blot indicate the domain of phosphorylated Beclin 1.(C) In vitro kinase assays to test the ability of recombinant CHK2 to phosphorylate recombinant GST-Beclin 1-WT, S9093A (AA), S90A and S93A protein. Reactions were analyzed by SDS-PAGE followed by autoradiography. The asterisks in the blot indicate the phosphorylation of Beclin 1 mutants.Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or after 1 hr HBSS starvation (D)Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or H2O2 (500μM) stimulation (E).Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or after HBSS starvation (F)Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or H2O2 (500μM) stimulation (G).(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells pretreated with NAC for 4 h and then cultured for 1 hr in HBSS starvation.


Figure 4 ( A ) The effect of Beclin 1 Ser90 / 93 phosphorylation on the interaction between Beclin 1 and Bcl - 2 . GST - Beclin1 WT , AA mutant and DD mutant ( bait ) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with flag - Bcl - 2 ( preys ) . ( B ) Immunoprecipitation of Bcl - 2 with Flag - Beclin 1 WT , AA mutant and DD mutant in HCT116 cells in normal medium or after H2O2 ( 500μM ) treatment for 1h . ( C - D ) Autophagy levels were determined in Beclin 1 - depleted H1299 cells with reconstituted expression of Beclin 1 WT , AA mutant or DD mutant in normal medium or after H2O2 ( 500μM ) treatment for 3h in the presence ( C ) or absence ( D ) of CHK2 . ( E ) Representative electron microscopic image of an autophagosome ( arrow , left panel , AP ) and an autolysosome ( arrow , right panel , AL ) in H1299 Beclin 1 WT cells treated with H2O2 ( 500μM ) for 6 hr . Scale bars , 500 nm . Electron microscopic quantification of autophagosomes ( AP ) and autolysosomes ( AL ) in H1299 cells with the indicated plasmids cultured for 6 hr in normal or H2O2 ( 500μM ) treatment . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 001 compared to Beclin 1 WT ; # P < 0 . 001 compared to Beclin 1 WT treated with H2O2 ; & P < 0 . 001 compared to Beclin 1 WT treated with H2O2 in the absence of CHK2 . ( F ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 , Beclin 1 , p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 ( 500μM ) treatment for 3h .

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

Figure 4(A) The effect of Beclin 1 Ser90/93 phosphorylation on the interaction between Beclin 1 and Bcl-2. GST-Beclin1 WT, AA mutant and DD mutant (bait) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with flag-Bcl-2 (preys).(B) Immunoprecipitation of Bcl-2 with Flag-Beclin 1 WT, AA mutant and DD mutant in HCT116 cells in normal medium or after H2O2 (500μM) treatment for 1h.(C-D) Autophagy levels were determined in Beclin 1-depleted H1299 cells with reconstituted expression of Beclin 1 WT, AA mutant or DD mutant in normal medium or after H2O2 (500μM) treatment for 3h in the presence (C) or absence (D) of CHK2.(E) Representative electron microscopic image of an autophagosome (arrow, left panel, AP) and an autolysosome (arrow, right panel, AL) in H1299 Beclin 1 WT cells treated with H2O2 (500μM) for 6 hr. Scale bars, 500 nm. Electron microscopic quantification of autophagosomes (AP) and autolysosomes (AL) in H1299 cells with the indicated plasmids cultured for 6 hr in normal or H2O2 (500μM) treatment. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.001 compared to Beclin 1 WT; #P < 0.001 compared to Beclin 1 WT treated with H2O2; &P < 0.001 compared to Beclin 1 WT treated with H2O2 in the absence of CHK2.(F) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93, Beclin 1, p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment for 3h.


Figure 5 ( A ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , AMPKα , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation . ( B ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia . ( C ) Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation . ( D ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia . ( E ) P - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells , and the cells were subjected to hypoxia with or without pretreatment with NAC . ( F ) P - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , AMPKα , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells , and the cells were subjected to glucose starvation with or without pretreatment with NAC . ( G ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , and AMPKα in H1299 cells in normal medium or after H2O2 ( 25μM ) stimulation . ( H ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 and AMPKα in H1299 cells in normal medium or after 2DG ( 5mM ) and oligomycin ( 2 . 5μM ) treatment .

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

Figure 5(A) Western blot detection of p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.(B) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia.(C) Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.(D) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia.(E) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to hypoxia with or without pretreatment with NAC.(F) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to glucose starvation with or without pretreatment with NAC.(G) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, and AMPKα in H1299 cells in normal medium or after H2O2 (25μM) stimulation.(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172 and AMPKα in H1299 cells in normal medium or after 2DG (5mM) and oligomycin (2.5μM) treatment.


Figure 6Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 12hr glucose starvation ( Glu - ) Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 36hr hypoxia ( B ) . ( C ) Western blot detection of CHK2 , HSP60 , TOM20 and TIM23 in H1299 cells transfected with the indicated shRNA in normal medium or after12 hr glucose starvation ( Glu - ) or 36 hr hypoxia . ( D - E ) Transmission electron microscopy of indicated H1299 cells cultured in glucose starvation ( Glu - ) medium or hypoxia . The yellow asterisks mark mitochondria . The red arrows indicate autophagic structures . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * * * P < 0 . 001 compared to shNC treated with H2O2 or hypoxia ( Mann - Whitney test ) ; Scale bar , 1 μm . ( F ) Representative confocal images of the shift in the fluorescence emission of Keima from 458 to 561 nm . Scale bar , 10 μm . The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation ( G ) All results are from three independent experiments . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ( Mann - Whitney test ) . The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or 48 hr hypoxia ( H ) . All results are from three independent experiments . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ( Mann - Whitney test ) .

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

Figure 6Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 12hr glucose starvation (Glu-)Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 36hr hypoxia (B).(C) Western blot detection of CHK2, HSP60, TOM20 and TIM23 in H1299 cells transfected with the indicated shRNA in normal medium or after12 hr glucose starvation (Glu-) or 36 hr hypoxia.(D-E) Transmission electron microscopy of indicated H1299 cells cultured in glucose starvation (Glu-) medium or hypoxia. The yellow asterisks mark mitochondria. The red arrows indicate autophagic structures. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ***P < 0.001 compared to shNC treated with H2O2 or hypoxia (Mann-Whitney test); Scale bar, 1 μm.(F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 to 561 nm. Scale bar, 10 μm.The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation (G) All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or 48 hr hypoxia (H). All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).


Figure 7H1299 cells transfected with the indicated shRNA in normal medium or after 12h glucose starvation ( A ) Representative FACS analysis of apoptosis . Data are presented as mean ± s . e . m . from 3 independent experiments ; * p < 0 . 05 compared to NC glucose starvation ( A ) ( Student ' s t - test ) . H1299 cells transfected with the indicated shRNA in normal medium or 48h hypoxia stimulation ( B ) . Representative FACS analysis of apoptosis . Data are presented as mean ± s . e . m . from 3 independent experiments * * p < 0 . 01 compared to NC hypoxia ( B ) ( Student ' s t - test ) . H1299 cells transfected with the indicated plasmids in normal medium or after 12h glucose starvation ( C ) Representative FACS analysis of apoptosis . Results from three independent experiments are presented as a histogram . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 compared to Beclin 1 S9093A ( AA ) treated with H2O2 or hypoxia ( Mann - Whitney test ) . H1299 cells transfected with the indicated plasmids in normal medium or 48h hypoxia stimulation ( D ) . Representative FACS analysis of apoptosis . Results from three independent experiments are presented as a histogram . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 compared to Beclin 1 S9093A ( AA ) treated with H2O2 or hypoxia ( Mann - Whitney test ) . ( E ) CHK2 + / + and CHK2 - / - mice were subjected to MCAO for 1 hr and reperfusion for 12 h . Contralateral ( C ) and ipsilateral ( I ) tissues of the mouse brain were coronally sectioned and stained with 2 % TTC . ( F ) The infarct volume was determined by measuring infarct size relative to normal in the slice . ( n = 5 mice ) . Data are presented as mean ± s . e . m . ; * * P < 0 . 01 ( Student ' s t - test ) . ( G - J ) Immunoblots for p62 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in the cortical extracts from ischemia and reperfusion - treated CHK2 + / + and CHK2 - / - mice . Quantification of p62 , p - Beclin 1 Ser90 and p - Beclin 1 Ser93 protein levels ( n = 3 mice ) . Data are presented as mean ± s . e . m . ; * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) .

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

Figure 7H1299 cells transfected with the indicated shRNA in normal medium or after 12h glucose starvation (A) Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments; *p < 0.05 compared to NC glucose starvation (A) (Student's t-test).H1299 cells transfected with the indicated shRNA in normal medium or 48h hypoxia stimulation (B). Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments **p < 0.01 compared to NC hypoxia (B) (Student's t-test).H1299 cells transfected with the indicated plasmids in normal medium or after 12h glucose starvation (C) Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).H1299 cells transfected with the indicated plasmids in normal medium or 48h hypoxia stimulation (D). Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).(E) CHK2+/+ and CHK2-/- mice were subjected to MCAO for 1 hr and reperfusion for 12 h. Contralateral (C) and ipsilateral (I) tissues of the mouse brain were coronally sectioned and stained with 2 % TTC.(F) The infarct volume was determined by measuring infarct size relative to normal in the slice. (n = 5 mice). Data are presented as mean ± s.e.m.; **P < 0.01 (Student's t-test).(G-J) Immunoblots for p62, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in the cortical extracts from ischemia and reperfusion-treated CHK2+/+ and CHK2-/- mice. Quantification of p62, p-Beclin 1 Ser90 and p-Beclin 1 Ser93 protein levels (n = 3 mice). Data are presented as mean ± s.e.m.; *P < 0.05, **P < 0.01 (Student's t-test).


Figure 1Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours . Subsequently , they were pre - treated overnight with either type I or type III IFNs ( 1 ng / mL IFN - λ1 and 10 U / mL IFN - α2 , respectively ) before infection with Sendai virus ( SeV ) and influenza A virus ( IAV , strain PR8 ) for 6 hours as indicated . Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2 , IFNB1 , and IFNL1 . ISG response measured by ISG15 and RSAD2 . Each colour corresponds to a donor ; 9 different donors were analysed . The dashed line represents the detection limit . An ordinary one - way - ANOVA test was used for the statistical analysis : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 . Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours . Subsequently , they were pre - treated overnight with either type I or type III IFNs ( 1 ng / mL IFN - λ1 and 10 U / mL IFN - α2 , respectively ) before infection with Sendai virus ( SeV ) and influenza A virus ( IAV , strain PR8 ) for 6 hours as indicated . The column labelled c shows control samples collected directly after purification . Quantification of the presence of SeV or IAV . Each colour corresponds to a donor ; 9 different donors were analysed . The dashed line represents the detection limit . An ordinary one - way - ANOVA test was used for the statistical analysis : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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Figure 1Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. ISG response measured by ISG15 and RSAD2. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. The column labelled c shows control samples collected directly after purification. Quantification of the presence of SeV or IAV. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


Figure 2Alveolar macrophages from different donors were purified from BAL fluid and pre - treated with either Poly ( I : C ) or IFN - λ1 ( + ; 1 ng / mL , or + + ; 10 ng / mL ) before infection with SARS - CoV - 2 . ( A ) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2 , IFNB1 , and IFNL1 ( Donors marked in orange and blue were not quantifiable in all test conditions ) . ( B ) ISG response measured by ISG15 , RSAD2 , and IFIT1 . Alveolar macrophages from different donors were purified from BAL fluid and pre - treated with either Poly ( I : C ) or IFN - λ1 ( + ; 1 ng / mL , or + + ; 10 ng / mL ) before infection with SARS - CoV - 2 . ( C ) Quantification of the presence of SARS - CoV - 2 .

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Figure 2Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (A) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1 (Donors marked in orange and blue were not quantifiable in all test conditions). (B) ISG response measured by ISG15, RSAD2, and IFIT1.Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (C) Quantification of the presence of SARS-CoV-2.


Figure 3Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( B ) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2 , IFNB1 , and IFNL1 . ( C ) ISG response measured by ISG15 , RSAD2 , and IFIT1 ( Donors marked in green and red were not quantifiable in all test conditions ) . Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( D ) Quantification of the presence of SARS - CoV - 2 . Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( E ) Western blots were performed using samples from two donors . ( A - D ) Each colour corresponds to a donor ; 5 different donors were analysed . The dashed line represents the detection limit . An ordinary one - way - ANOVA test was used for statistical analysis : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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

Figure 3Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (B) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. (C) ISG response measured by ISG15, RSAD2, and IFIT1 (Donors marked in green and red were not quantifiable in all test conditions).Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (D) Quantification of the presence of SARS-CoV-2.Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (E) Western blots were performed using samples from two donors. (A-D) Each colour corresponds to a donor; 5 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


Figure 4Alveolar macrophages from different donors were purified from BAL and treated with SARS - CoV - 2 for either 2 or 24 hours . ( A ) The amount of SARS - CoV - 2 nucleocapsid phosphoprotein was quantified using LC - MS / MS . ( B ) After subtraction of the background ( unchallenged AMs ) , the ratio between the amount of SARS - CoV - 2 nucleocapsid phosphoprotein at 24 and 2 hours was calculated . Each colour corresponds to a donor ; 3 different donors were analyzed . An ordinary one - way - ANOVA test was used for statistical analysis : ns , not significant ; * , P ≤ 0 . 05 .

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Figure 4Alveolar macrophages from different donors were purified from BAL and treated with SARS-CoV-2 for either 2 or 24 hours. (A) The amount of SARS-CoV-2 nucleocapsid phosphoprotein was quantified using LC-MS/MS. (B) After subtraction of the background (unchallenged AMs), the ratio between the amount of SARS-CoV-2 nucleocapsid phosphoprotein at 24 and 2 hours was calculated. Each colour corresponds to a donor; 3 different donors were analyzed. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05.


Figure 5 ( A ) Total RNA was harvested from AMs and primary human airway epithelial cells ( HAE ) and subjected to qPCR for quantification of the ACE2 receptor . Each colour corresponds to a donor . The dashed line represents the detection limit . ( B ) The presence of nucleoprotein mRNA of SARS - CoV - 2 in AMs and primary HAE was also quantified after SARS - CoV - 2 treatment . Each colour corresponds to a donor . An unpaired t - test test was used for statistical analysis : * * * * , P ≤ 0 . 0001 . ( C ) Primer efficiency was tested using serial dilutions of positive samples using qPCR . The Ct values were plotted on a logarithmic scale along with the corresponding dilutions before performing a linear regression in order to calculate the slope of the trend line through the data points . This analysis revealed an amplification factor of 1 . 92 , 1 . 91 , and 1 . 85 for the SARS - CoV - 2 , IAV , and SeV primer set , respectively . The different colours correspond to different primer sets .

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

Figure 5(A) Total RNA was harvested from AMs and primary human airway epithelial cells (HAE) and subjected to qPCR for quantification of the ACE2 receptor. Each colour corresponds to a donor. The dashed line represents the detection limit.(B) The presence of nucleoprotein mRNA of SARS-CoV-2 in AMs and primary HAE was also quantified after SARS-CoV-2 treatment. Each colour corresponds to a donor. An unpaired t-test test was used for statistical analysis: ****, P≤0.0001.(C) Primer efficiency was tested using serial dilutions of positive samples using qPCR. The Ct values were plotted on a logarithmic scale along with the corresponding dilutions before performing a linear regression in order to calculate the slope of the trend line through the data points. This analysis revealed an amplification factor of 1.92, 1.91, and 1.85 for the SARS-CoV-2, IAV, and SeV primer set, respectively. The different colours correspond to different primer sets.


Figure 1 ( b ) Volcano plots of up ( red ) and down ( blue ) regulated genes ( compared to healthy control samples ) in S . Typhi and S . Paratyphi A positive individuals ( Nepal and Oxford ) . Black numbers indicate the up - and down - regulated genes compared to healthy controls . ( d - e ) Scatter plots of BTMs enriched ( p > 0 . 05 ) in blood - culture positive samples in Nepal ( y - axis ) versus Oxford ( x - axis ) for typhoid fever ( d ) and paratyphoid fever ( e ) . For further details on BTMs refer to reference ( Chaussabel et al . , 2014 ) .

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Figure 1(b) Volcano plots of up (red) and down (blue) regulated genes (compared to healthy control samples) in S. Typhi and S. Paratyphi A positive individuals (Nepal and Oxford). Black numbers indicate the up- and down-regulated genes compared to healthy controls.(d-e) Scatter plots of BTMs enriched (p>0.05) in blood-culture positive samples in Nepal (y-axis) versus Oxford (x-axis) for typhoid fever (d) and paratyphoid fever (e). For further details on BTMs refer to reference (Chaussabel et al., 2014).


Figure 2 ( a ) Single - sample GSEA Normalised Enrichment Scores ( NES ) of IFN and DC BTMs of individuals with blood - culture confirmed enteric fever in Nepal and Oxford . ( b ) Heatmap of the 500 most variably expressed genes in samples of the Nepali cohort . Bar graph on top of the heatmap shows temperature of each individual at the time of sampling . Three samples labelled as ' 03NP - CONT ' are samples that grew bacterial contaminants and were thus excluded from the entire analysis .

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Figure 2(a) Single-sample GSEA Normalised Enrichment Scores (NES) of IFN and DC BTMs of individuals with blood-culture confirmed enteric fever in Nepal and Oxford.(b) Heatmap of the 500 most variably expressed genes in samples of the Nepali cohort. Bar graph on top of the heatmap shows temperature of each individual at the time of sampling. Three samples labelled as '03NP-CONT' are samples that grew bacterial contaminants and were thus excluded from the entire analysis.


Figure 4 ( d ) Combined expression score for samples based on the 5 - gene signature for samples in the discovery cohort ( top ) and validation cohort ( bottom ) . Ox . CTRL : Oxford controls ( D0 ) ; CTRL : Nepali control samples . PTB : pulmonary TB ; DENV : Dengue samples ; bsPf : blood - stage P . falciparum ; SPT : S . Paratyphi A ; ST : S . Typhi . ST and SPT samples are derived from the challenge models as well as from Nepal .

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Figure 4(d) Combined expression score for samples based on the 5-gene signature for samples in the discovery cohort (top) and validation cohort (bottom). Ox.CTRL: Oxford controls (D0); CTRL: Nepali control samples. PTB: pulmonary TB; DENV: Dengue samples; bsPf: blood-stage P. falciparum; SPT: S. Paratyphi A; ST: S. Typhi. ST and SPT samples are derived from the challenge models as well as from Nepal.


Figure 5 ( b ) Dot plot of prediction probability of being class EF versus the expression score calculated on the bases of the 5 - gene signature ( based on gene array data ) . ( c ) qPCR gene expression scores of the 5 - gene signature ( ∆ ∆ CT over PPIA ) for CTRLs , 03NP - sEF , 03NP - SPT and 03NP - ST samples from Nepal . Yellow diamonds in the 03NP - sEF category represent the 9 patients classified as EF based on the random forest algorithm . ( d ) qPCR expression values ( ∆ ∆ Ct over PPIA ) of the 5 - gene signature in control samples ( Oxford and Nepal ) , S . Paratyphi A ( 03NP - SPT ) or S . Typhi ( 03NP - ST ) in Nepal , samples at day 7 after challenge of participants who stayed well following challenge with S . Typhi ( nD7 ) , or typhoid diagnosis after challenge ( TD ) in the Vi - TCV study ( e ) Combined qPCR expression score of the 5 - gene signature . Black arrows indicate outlier samples . ( f ) Temperature and CRP for samples of which data was available ( CRP was only measured in the Oxford CHIM ) . D0 = pre - challenge baseline Vi - TCV study ; nD7 = day 7 samples of participants who stayed well following challenge ( Vi - TCV study ) ; SPT = S . Paratyphi A ( 03NP ) ; ST = S . Typhi ( 03NP ) ; TD = Typhoid diagnosis ( Vi - TCV study ) . ( g ) Spearman ' s rank correlation of the 5 - gene combined expression score and ( left ) temperature ( only nD7 and TD samples from the Oxford CHIM - Vi - TCV and SPT and ST cases from Nepal at presentation to hospital were included ) and ( right ) CRP ( CRP was only available for Oxford CHIM - Vi - TCV samples and we excluded D0 baseline measures ) .

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Figure 5(b) Dot plot of prediction probability of being class EF versus the expression score calculated on the bases of the 5-gene signature (based on gene array data).(c) qPCR gene expression scores of the 5-gene signature (∆∆CT over PPIA) for CTRLs, 03NP-sEF, 03NP-SPT and 03NP-ST samples from Nepal. Yellow diamonds in the 03NP-sEF category represent the 9 patients classified as EF based on the random forest algorithm.(d) qPCR expression values (∆∆Ct over PPIA) of the 5-gene signature in control samples (Oxford and Nepal), S. Paratyphi A (03NP-SPT) or S. Typhi (03NP-ST) in Nepal, samples at day 7 after challenge of participants who stayed well following challenge with S. Typhi (nD7), or typhoid diagnosis after challenge (TD) in the Vi-TCV study(e) Combined qPCR expression score of the 5-gene signature. Black arrows indicate outlier samples.(f) Temperature and CRP for samples of which data was available (CRP was only measured in the Oxford CHIM). D0 = pre-challenge baseline Vi-TCV study; nD7 = day 7 samples of participants who stayed well following challenge (Vi-TCV study); SPT = S. Paratyphi A (03NP); ST = S. Typhi (03NP); TD = Typhoid diagnosis (Vi-TCV study). (g) Spearman's rank correlation of the 5-gene combined expression score and (left) temperature (only nD7 and TD samples from the Oxford CHIM - Vi-TCV and SPT and ST cases from Nepal at presentation to hospital were included) and (right) CRP (CRP was only available for Oxford CHIM - Vi-TCV samples and we excluded D0 baseline measures).


Figure 1IHC images for collagen The inserts in panel B represent the hematoxylin ( nucleus ) & eosin ( cytoplasm ) staining for the same tissue area of the collagen staining In all cases n = 4 control mice , and ≥ 3 mice for 2mg and 5 mg ) The quantification for each staining is shown in the histogram in the right sideIHC images fo aSMA , CD68 , and CD204 For aSMA , CD68 , and CD204 the percentage of positive cells is relative to the control condition . In all cases n = 4 control mice , and ≥ 3 mice for 2mg and 5 mg ) Scale bar : 100 mm . The quantification for each staining is shown in the histogram in the right

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Figure 1IHC images for collagen The inserts in panel B represent the hematoxylin (nucleus) & eosin (cytoplasm) staining for the same tissue area of the collagen staining In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg) The quantification for each staining is shown in the histogram in the right sideIHC images fo aSMA, CD68, and CD204 For aSMA, CD68, and CD204 the percentage of positive cells is relative to the control condition. In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg) Scale bar: 100 mm. The quantification for each staining is shown in the histogram in the right side


Figure 2Representative traces tracking bead displacements Histogram shows relative bead displacement for the first and last pulse , n = 27 control and n = 37 tamoxifenQuantification of PSCs stiffnessHistogram shows percentage of gel contraction , n > 10 gels per conditionRepresentative mechanosensing trace of PSC treated with tamoxifen and Estrogen receptor ( ER antagonists Histogram shows relative bead displacement for the first and last pulse , n > 20 for all conditionsRepresentative mechanosensing trace of PSC treated with tamoxifen an G coupled - protein estrogen receptor ( GPER ) antagonists Histogram shows relative bead displacement for the first and last pulse , n > 20 for all conditionsHistogram shows percentage of gel contraction by PSCs treated with tamoxifen in the presence of indicated antagonistHistogram shows percentage of gel contraction after treatment with indicated agonist , n > 10 gels per

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Figure 2Representative traces tracking bead displacements Histogram shows relative bead displacement for the first and last pulse, n=27 control and n=37 tamoxifenQuantification of PSCs stiffnessHistogram shows percentage of gel contraction, n>10 gels per conditionRepresentative mechanosensing trace of PSC treated with tamoxifen and Estrogen receptor (ER antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditionsRepresentative mechanosensing trace of PSC treated with tamoxifen an G coupled-protein estrogen receptor (GPER) antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditionsHistogram shows percentage of gel contraction by PSCs treated with tamoxifen in the presence of indicated antagonistHistogram shows percentage of gel contraction after treatment with indicated agonist, n>10 gels per conditio


Figure 3Young " s modulus for matrices previously remodeled by PSCs ; control n = 126 and tamoxifen n = 145 . Lines and error bars indicate mean ± s . e . m . Four experimental replRepresentative images of H & E staining showing AsPC1 cancer cell invasions in matrices previously remodeled by control PSCs ( top ) or tamoxifen treated PSCs ( bottom ) , scale bar 50 μm AsPC1 invasion quantification , the central box represents values from the lower to upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum value , n = 30 control and n = 40

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Figure 3Young"s modulus for matrices previously remodeled by PSCs; control n=126 and tamoxifen n=145. Lines and error bars indicate mean±s.e.m. Four experimental replRepresentative images of H&E staining showing AsPC1 cancer cell invasions in matrices previously remodeled by control PSCs (top) or tamoxifen treated PSCs (bottom), scale bar 50 μm AsPC1 invasion quantification, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value, n=30 control and n=40 tamoxifen


Figure 4Immunofluorescence images of PSCs stained for YAPQuantification of the nuclear / cytoplasm YAP in PSCs ( four experimental replicates ) qPCR mRNA levels for YAP target genes connective tissue grow factor ( CTGF ) and Ankyrin Repeat Domain 1 ( ANRKD1 ) , ( three experimental replicates ) Western blot bands for YAP , pS127 YAP and total protein . ( E ) Quantification of YAP and pYAP Ser127 normalised to total protein , expressed relative to unstimulated control ( n = 8 ) IHC images for YAP staining in KPC mice pancreatic tissues ( control and treated with 2 and 5 mg tamoxifen ) , YAP staining is brown . Scale bar : 100 μmQuantification of YAP positive nucleus . n = 5 control mice , and ≥ 3 mice for 2mg and 5

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Figure 4Immunofluorescence images of PSCs stained for YAPQuantification of the nuclear/cytoplasm YAP in PSCs (four experimental replicates)qPCR mRNA levels for YAP target genes connective tissue grow factor (CTGF) and Ankyrin Repeat Domain 1 (ANRKD1), (three experimental replicates)Western blot bands for YAP, pS127 YAP and total protein. (E) Quantification of YAP and pYAP Ser127 normalised to total protein, expressed relative to unstimulated control (n=8)IHC images for YAP staining in KPC mice pancreatic tissues (control and treated with 2 and 5 mg tamoxifen), YAP staining is brown. Scale bar: 100 μmQuantification of YAP positive nucleus. n=5 control mice, and ≥3 mice for 2mg and 5 mg


Figure 5IF images for total ( MLC - 2 ) and phosphorylated ( pMLC - 2 ) in PSCs , scale bar is 20 μmQuantification of staining in A . Each data point represents a cell Western blot for total and phosphorylated mlc - 2Quantification of total and active RhoA , expressed as percentage of the total RhoA in the control conditionIHC images for phosphorylated mlc - 2 in mice pancreatic tissues ( n = 5 control , n = 5 2mg , and n = 3 5mg ) . Scale bar : 100 μm Quantification of staining in

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Figure 5IF images for total (MLC-2) and phosphorylated (pMLC-2) in PSCs, scale bar is 20 μmQuantification of staining in A. Each data point represents a cell Western blot for total and phosphorylated mlc-2Quantification of total and active RhoA, expressed as percentage of the total RhoA in the control conditionIHC images for phosphorylated mlc-2 in mice pancreatic tissues (n=5 control, n=5 2mg, and n=3 5mg). Scale bar: 100 μm Quantification of staining in E


Figure 1B Light transmittance of tissue increased more rapidly with FEC than with CLARITY . Each datapoint plotted is the mean from three neighboring transmittance readings ( hence resulting in 10 datapoints from the 30 measurements / brain , technical replicates ) ( One - way ANOVA * * * p < 0 . 001 , * * * * p < 0 . 0001 ) ( n = 3 per group , biological replicates ) . C Comparison of mouse brains cleared by FEC ( 6 hrs ) and passive ( 3 days ) . Passive clearing was incomplete even after 3 days . D After whole - brain electrophoretic staining with polythiophenes , sagittal 500 µm slices were cut and plaques were counted . By passively re - staining the same slice , no additional plaques were detected ( " FEC + passive " ) . Focal shifts after slice reprocessing account for the slight differences between the images . One biological replicate . Scale bars : 100

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Figure 1B Light transmittance of tissue increased more rapidly with FEC than with CLARITY. Each datapoint plotted is the mean from three neighboring transmittance readings (hence resulting in 10 datapoints from the 30 measurements/brain, technical replicates) (One-way ANOVA *** p<0.001, **** p<0.0001) (n = 3 per group, biological replicates).C Comparison of mouse brains cleared by FEC (6 hrs) and passive (3 days). Passive clearing was incomplete even after 3 days.D After whole-brain electrophoretic staining with polythiophenes, sagittal 500 µm slices were cut and plaques were counted. By passively re-staining the same slice, no additional plaques were detected ("FEC + passive"). Focal shifts after slice reprocessing account for the slight differences between the images. One biological replicate. Scale bars: 100 µm


Figure 4A , B The distinct significant effect of each of the strongest treatments on plaque size cluster , NB360 5 - month and LIN5044 14 - month , as well as their overlap , using either ( A ) neuroanatomical boundaries , or ( B ) SAVs .

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Figure 4A, B The distinct significant effect of each of the strongest treatments on plaque size cluster, NB360 5-month and LIN5044 14-month, as well as their overlap, using either (A) neuroanatomical boundaries, or (B) SAVs.


Figure 1A . Tunicamycin ( Tn , 2 µg / ml ) increased Nox4 protein levels in H9c2 cells . Tubulin was used as a loading control . n = 4 - 6 / group . * , significant compared to baseline . B . Thapsigargin ( Tp , 1 µg / ml ) increased Nox4 protein levels in H9c2 cells . n = 4 - 6 / group . * , significant compared to baseline . C . Effect of Nox4 on the unfolded protein response . Nox4 was depleted in H9c2 cells by shRNA - mediated knockdown ( Ad . shNox4 ) or cells were treated with a control adenovirus ( Ad . Ctl ) . In cells with Nox4 knockdown , tunicamycin treatment resulted in lower increases in protein levels of the ER chaperones Grp94 , Grp78 and calreticulin than in conrol cells . Nuclear protein levels of ATF4 were substantially lower in Nox4 - depleted cells than control cells but the levels of cleaved ATF6 ( ATF6c ) were similar . Histone was used as a loading control . The relative mRNA levels of Xbp1 - s ( a readout of IRE1 signaling ) were unaltered after Nox4 knockdown . Mean data are shown in Appendix Fig S1E . Similar results were obtained with an independent siRNA approach ( Appendix Fig S2A ) . D . Effect of adenoviral - mediated overexpression of Nox4 ( Ad . Nox4 ) or a control β - galactosidase protein ( Ad . β - Gal ) on tunicamycin responses of H9c2 cells . Nox4 enhanced the increase in cellular ER chaperones and nuclear ATF4 levels but did not affect tunicamycin - induced changes in nuclear ATF6c levels and caused minor reduction in Xbp1s mRNA levels . Mean data are shown in Appendix Fig S2B . E , F . Effect of Nox4 knockdown or overexpression , respectively , on the tunicamycin - induced changes in mRNA levels of ATF4 target genes . n = 4 / group . Psat1 , phosphoserine aminotransferase ; Phgdh , 3 - phosphoglycerate dehydrogenase ; Asns , asparagine synthetase ; Slc6a9 , glycine transporter 1 . G . Effect of ATF4 silencing with two different siRNAs on Nox4 protein levels in tunicamycin - treated H9c2 cells . Scrambled siRNAs were used as a control ( Ctl ) . Representative immunoblots shown to the top ( captions at bottom of bar graphs refer also to the immunoblots ) ; tubulin was used as a loading control . n = 4 / group . * , significant compared to baseline ; # , significant comparing siATF4 versus corresponding siCtl . H . Effect of ATF4 overexpression on Nox4 mRNA and protein levels . n = 3 / group .

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Figure 1A. Tunicamycin (Tn, 2 µg/ml) increased Nox4 protein levels in H9c2 cells. Tubulin was used as a loading control. n=4-6/group. *, significant compared to baseline.B. Thapsigargin (Tp, 1 µg/ml) increased Nox4 protein levels in H9c2 cells. n=4-6/group. *, significant compared to baseline.C. Effect of Nox4 on the unfolded protein response. Nox4 was depleted in H9c2 cells by shRNA-mediated knockdown (Ad.shNox4) or cells were treated with a control adenovirus (Ad.Ctl). In cells with Nox4 knockdown, tunicamycin treatment resulted in lower increases in protein levels of the ER chaperones Grp94, Grp78 and calreticulin than in conrol cells. Nuclear protein levels of ATF4 were substantially lower in Nox4-depleted cells than control cells but the levels of cleaved ATF6 (ATF6c) were similar. Histone was used as a loading control. The relative mRNA levels of Xbp1-s (a readout of IRE1 signaling) were unaltered after Nox4 knockdown. Mean data are shown in Appendix Fig S1E. Similar results were obtained with an independent siRNA approach (Appendix Fig S2A).D. Effect of adenoviral-mediated overexpression of Nox4 (Ad.Nox4) or a control β-galactosidase protein (Ad.β-Gal) on tunicamycin responses of H9c2 cells. Nox4 enhanced the increase in cellular ER chaperones and nuclear ATF4 levels but did not affect tunicamycin-induced changes in nuclear ATF6c levels and caused minor reduction in Xbp1s mRNA levels. Mean data are shown in Appendix Fig S2B.E,F. Effect of Nox4 knockdown or overexpression, respectively, on the tunicamycin-induced changes in mRNA levels of ATF4 target genes. n=4/group. Psat1, phosphoserine aminotransferase; Phgdh, 3-phosphoglycerate dehydrogenase; Asns, asparagine synthetase; Slc6a9, glycine transporter 1.G. Effect of ATF4 silencing with two different siRNAs on Nox4 protein levels in tunicamycin-treated H9c2 cells. Scrambled siRNAs were used as a control (Ctl). Representative immunoblots shown to the top (captions at bottom of bar graphs refer also to the immunoblots); tubulin was used as a loading control. n=4/group. *, significant compared to baseline; #, significant comparing siATF4 versus corresponding siCtl.H. Effect of ATF4 overexpression on Nox4 mRNA and protein levels. n=3/group.