The mepM gene was expressed under the control of TIS1 or TIS2 ( translation initiation ) and of PrhaBAD ( the L - rhamnose - inducible promoter of vector pHV30 ) . Growth of BW25113 ( ycbB , relA ' ) ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg / ml , IPTG 40 µM ( induction of ycbB ) and L - arabinose 1 % ( induction of relA ' ) . ( A ) For TIS2 , growth around paper disks containing 1 or 2 mg of L - rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance . In contrast , a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer . ( B ) The experiment was repeated with bacteria pre - exposed to L - rhamnose that were harvested at the vicinity of the disk containing 2 mg of L - rhamnose . Expression of ceftriaxone resistance remained dependent on the presence of L - rhamnose indicating that the requirement for MepM is not transient . The diameter of the growth zones is larger in panel ( B ) than in panel ( A ) as expected from the fact that bacteria in the inoculum used in ( B ) had been grown in the presence of the inducer and already contained MepM . In ( A ) growth is only possible after diffusion of L - rhamnose in the medium prior to the action of ceftriaxone . At a distance from the disk , diffusion was not sufficiently rapid to observe resistance .

O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

The mepM gene was expressed under the control of TIS1 or TIS2 (translation initiation) and of PrhaBAD (the L-rhamnose-inducible promoter of vector pHV30). Growth of BW25113(ycbB, relA') ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg/ml, IPTG 40 µM (induction of ycbB) and L-arabinose 1% (induction of relA'). (A) For TIS2, growth around paper disks containing 1 or 2 mg of L-rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance. In contrast, a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer. (B) The experiment was repeated with bacteria pre-exposed to L-rhamnose that were harvested at the vicinity of the disk containing 2 mg of L-rhamnose. Expression of ceftriaxone resistance remained dependent on the presence of L-rhamnose indicating that the requirement for MepM is not transient. The diameter of the growth zones is larger in panel (B) than in panel (A) as expected from the fact that bacteria in the inoculum used in (B) had been grown in the presence of the inducer and already contained MepM. In (A) growth is only possible after diffusion of L-rhamnose in the medium prior to the action of ceftriaxone. At a distance from the disk, diffusion was not sufficiently rapid to observe resistance.


( A ) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase and digested by lysozyme ( upper panel ) or by lysozyme and 5 µM MepM ( lower panel ) . Absorbance was monitored at 205 nm ( mAU , milli - absorbance unit ) .

O O O B-EXP_ASSAY O O B-SUBCELLULAR O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

(A) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase and digested by lysozyme (upper panel) or by lysozyme and 5 µM MepM (lower panel). Absorbance was monitored at 205 nm (mAU, milli-absorbance unit).


Functional complementation of the mepM deletion in BW25113 ( ycbB , relA ' ) ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal . Induction of endopeptidase ( ED ) genes was performed with 0 . 2 % L - rhamnose in the presence or absence of 8 µg / ml ceftriaxone . BHI agar plates contained 40 µM IPTG and 1 % L - arabinose for induction of ycbB and relA ' , respectively .

O O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O

Functional complementation of the mepM deletion in BW25113(ycbB, relA') ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal. Induction of endopeptidase (ED) genes was performed with 0.2% L-rhamnose in the presence or absence of 8 µg/ml ceftriaxone. BHI agar plates contained 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively.


( B ) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53 ( mepM ) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter . In this assay , functional replacement of MepM is detected based on growth in media containing 0 . 2 % or 1 % L - arabinose for expression of the endopeptidase gene carried by vector pHV7 , while by - passing the requirement for induction of the mepM copy of pHV53 by L - rhamnose . Complementation was observed with both concentrations of inducer for mepH , mepS , and pbpG . Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L - arabinose ( 1 % ) was lethal . The right panel presents the growth control performed in the presence of 0 . 2 % L - rhamnose for induction of the mepM copy carried by pHV53 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O 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-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O

(B) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53(mepM) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter. In this assay, functional replacement of MepM is detected based on growth in media containing 0.2% or 1% L-arabinose for expression of the endopeptidase gene carried by vector pHV7, while by-passing the requirement for induction of the mepM copy of pHV53 by L-rhamnose. Complementation was observed with both concentrations of inducer for mepH, mepS, and pbpG. Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L-arabinose (1%) was lethal. The right panel presents the growth control performed in the presence of 0.2% L-rhamnose for induction of the mepM copy carried by pHV53.


( B ) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63 ( ycbB ) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter . Induction of ycbB and of endopeptidase ( ED ) genes was performed with 0 . 2 % L - arabinose and 1 % L - rhamnose , respectively . BHI agar plates contained chloramphenicol ( 20 µg / ml ) to counter - select loss of pHV63 ( ycbB ) .

O O O O O O O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O O 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-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O

(B) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).


( A ) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63 ( ycbB ) was performed with the pHV30 vector or recombinant plasmids encoding mepA , mepK , mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter . ( B ) The complementation assay was repeated for BW25113 Δ7EDs pHV63 ( ycbB ) , which was obtained by deletion of all chromosomal endopeptidase genes except mepM . Induction of ycbB and of endopeptidase ( ED ) genes was performed with 0 . 2 % L - arabinose and 1 % L - rhamnose , respectively . BHI agar plates contained chloramphenicol ( 20 µg / ml ) to counter - select loss of pHV63 ( ycbB ) .

O O O O O O O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O

(A) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding mepA, mepK, mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter. (B) The complementation assay was repeated for BW25113 Δ7EDs pHV63(ycbB), which was obtained by deletion of all chromosomal endopeptidase genes except mepM. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).


( A ) Growth curves of ( i ) BW25113 ( ycbB , relA ' ) and its ΔsltY derivative ; and ( ii ) strain M1 and its derivatives ( M1 . 1 to 1 . 4 ) harboring mutations in sltY . The growth medium contained ( i ) 40 µM IPTG , 1 % L - arabinose , and 8 µg / ml ceftriaxone for BW25113 ( ycbB , relA ' ) and its ΔsltY derivative or ( ii ) 50 µM IPTG and 16 µg / ml ampicillin for M1 and its derivatives .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O 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-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O

(A) Growth curves of (i) BW25113(ycbB, relA') and its ΔsltY derivative; and (ii) strain M1 and its derivatives (M1.1 to 1.4) harboring mutations in sltY. The growth medium contained (i) 40 µM IPTG, 1% L-arabinose, and 8 µg/ml ceftriaxone for BW25113(ycbB, relA') and its ΔsltY derivative or (ii) 50 µM IPTG and 16 µg/ml ampicillin for M1 and its derivatives.


a , Pancreatic sections stained with Atg7 , LC3 and p62 showing Atg7 − / − next to Atg7 + / + regions after Pdx - Cre - mediated recombination . Right - side images are cropped and magnified regions ( boxed ) from left - side images . The magnification is × 2 . 5 .

O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O 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, Pancreatic sections stained with Atg7, LC3 and p62 showing Atg7−/− next to Atg7+/+ regions after Pdx-Cre-mediated recombination. Right-side images are cropped and magnified regions (boxed) from left-side images. The magnification is × 2.5.


b , Haematoxylin and eosin ( HE ) , p53 and caspase - 3 staining of exocrine tissue in Atg7 + / + and Atg7 − / − pancreases .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O

b, Haematoxylin and eosin (HE), p53 and caspase-3 staining of exocrine tissue in Atg7+/+ and Atg7−/− pancreases.


c , Quantification ( median , s . e . m . ) of p53 and caspase - 3 activation in Atg7 - deficient acinar tissue .

O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE O

c, Quantification (median, s.e.m.) of p53 and caspase-3 activation in Atg7-deficient acinar tissue.


d , Kaplan - Meier analysis comparing overall survival of mice either wild type ( blue ) or Atg7 − / − ( red ) in the pancreas . Mantel - Cox test was used for statistics . ' n / a ' , 14 / 16 mice were killed > 500 days when completely healthy .

O O O O O O O O B-EXP_ASSAY O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O

d, Kaplan-Meier analysis comparing overall survival of mice either wild type (blue) or Atg7−/− (red) in the pancreas. Mantel-Cox test was used for statistics. 'n/a', 14/16 mice were killed >500 days when completely healthy.


e , Biochemical analysis of endocrine function in moribund Atg7 − / − or age - matched healthy control mice . Detailed information about the mice is tabulated . A Mann - Whitney U - test was used for statistics . Scale bars : 50 µm . * * P 0 . 01 and * * * P 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O 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, Biochemical analysis of endocrine function in moribund Atg7−/− or age-matched healthy control mice. Detailed information about the mice is tabulated. A Mann-Whitney U-test was used for statistics. Scale bars: 50 µm. **P 0.01 and ***P 0.001.


a , Alcian blue / PAS ( AB / PAS ) staining in pancreases from 150 - day - old Atg7 - proficient or Atg7 - deficient KrasG12D / + mice ( wild - type mice with expression of KrasG12D in their pancreases ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O B-ORGANISM O O O O O O B-TISSUE O O

a, Alcian blue/PAS (AB/PAS) staining in pancreases from 150-day-old Atg7-proficient or Atg7-deficient KrasG12D/+ mice (wild-type mice with expression of KrasG12D in their pancreases).


b , Quantification of PanIN from mice of the indicated genotype and age . Fold increase and male / female ratios are shown . Box plots express the first ( Q1 ) and third ( Q3 ) quartiles set by the upper and lower limits of the box , whereas the median is shown by the horizontal line inside the box . The whiskers extend upward and downward to the highest or lowest observation within the upper ( Q3 + 1 . 5 × the interquartile range ) and lower ( Q1 − 1 . 5 × interquartile range ) limits . Values outside the upper and lower limits are ' outliers ' and are shown by individual symbols .

O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PanIN from mice of the indicated genotype and age. Fold increase and male/female ratios are shown. Box plots express the first (Q1) and third (Q3) quartiles set by the upper and lower limits of the box, whereas the median is shown by the horizontal line inside the box. The whiskers extend upward and downward to the highest or lowest observation within the upper (Q3 + 1.5 × the interquartile range) and lower (Q1 − 1.5 × interquartile range) limits. Values outside the upper and lower limits are 'outliers' and are shown by individual symbols.


c , d , Kaplan - Meier analysis comparing overall survival ( c ) or PDAC - free survival ( d ) of Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / + mice .

O O O O O O O O O O B-EXP_ASSAY O O O O B-TISSUE O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-ORGANISM O

c, d, Kaplan-Meier analysis comparing overall survival (c) or PDAC-free survival (d) of Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ mice.


c , d , Kaplan - Meier analysis comparing overall survival ( c ) or PDAC - free survival ( d ) of Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / +

O O O O O O O O O O B-EXP_ASSAY O O O O B-TISSUE O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O

c, d, Kaplan-Meier analysis comparing overall survival (c) or PDAC-free survival (d) of Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ mice


e , PanIN grading of pancreases from 150 - day - old Atg7 + / + or Atg7 − / − Pdx - Cre KrasG12D / + mice . Values are mean and error bars are s . d . b , e , A Mann - Whitney U - test was used for statistics .

O O B-TISSUE O O B-TISSUE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-ORGANISM O O O O 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, PanIN grading of pancreases from 150-day-old Atg7+/+ or Atg7−/− Pdx-Cre KrasG12D/+ mice. Values are mean and error bars are s.d. b, e, A Mann-Whitney U-test was used for statistics.


f , Representative p53 staining from 150 - day - old mice of both genotypes .

O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O

f, Representative p53 staining from 150-day-old mice of both genotypes.


g , PDAC - free survival of autophagy - proficient ( blue ) and Atg5 - deficient ( red ) KrasG12D / + mice . d , g , Median survival ± s . d . and male / female ( m / f ) ratio are provided . ' n / a ' , no animals succumbed to PDAC . A log - rank test ( Mantel - Cox ) was used for statistics . a , f , Scale bars : 500 µm ( a ) , 50 µm ( f ) . * P 0 . 05 , * * P 0 . 01 ; NS , not significant .

O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

g, PDAC-free survival of autophagy-proficient (blue) and Atg5-deficient (red) KrasG12D/+ mice. d, g, Median survival ± s.d. and male/female (m/f) ratio are provided. 'n/a', no animals succumbed to PDAC. A log-rank test (Mantel-Cox) was used for statistics. a, f, Scale bars: 500 µm (a), 50 µm (f). *P  0.05, **P  0.01; NS, not significant.


a , Kaplan - Meier analysis comparing PDAC - free survival in Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / + p53 − / − mice .

O O O O O O O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O

a, Kaplan-Meier analysis comparing PDAC-free survival in Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ p53−/− mice.


b , Pancreatic tumours of indicated genotypes stained for Atg7 , LC3 and p62 . Insets are magnified crops from the same images to show specifics of LC3 and p62 staining . The magnification is × 4 .

O O B-TISSUE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

b, Pancreatic tumours of indicated genotypes stained for Atg7, LC3 and p62. Insets are magnified crops from the same images to show specifics of LC3 and p62 staining. The magnification is × 4.


c , Tumour incidence and tumour weight ( median , s . e . m . ) in mice from indicated genotypes , killed at 21 , 29 and 38 days of age . Numbers in the incidence diagram represent tumour - bearing mice versus all mice . Significance was assessed with Fisher ' s exact test ( incidence ) and Mann - Whitney U - test ( weight ) .

O O O O O O O O O O O O O O O O O O B-ORGANISM 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-ORGANISM O O B-ORGANISM 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, Tumour incidence and tumour weight (median, s.e.m.) in mice from indicated genotypes, killed at 21, 29 and 38 days of age. Numbers in the incidence diagram represent tumour-bearing mice versus all mice. Significance was assessed with Fisher's exact test (incidence) and Mann-Whitney U-test (weight).


d , Representative haematoxylin and eosin stainings of pancreases from indicated time points and genotypes .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O

d, Representative haematoxylin and eosin stainings of pancreases from indicated time points and genotypes.


e , Kaplan - Meier analysis comparing PDAC - free survival of Atg5 + / + ( blue ) or Atg5 − / − ( green ) KrasG12D / + p53 − / − mice .

e, Kaplan-Meier analysis comparing PDAC-free survival of Atg5+/+ (blue) or Atg5−/− (green) KrasG12D/+ p53−/− mice.


f , Kaplan - Meier analysis comparing PDAC - free survival of KrasG12D / + p53 − / − mice that were chloroquine - treated ( CQ , red ) , vehicle - treated ( PBS , black ) or untreated ( blue ) . a , e , f , The blue curve in all Kaplan - Meier plots represents the same colony of mice ( KrasG12D / + , p53 − / − , Atg7 + / + , Atg5 + / + ) . A Mantel - Cox test was used for statistics . b , d , Scale bars : 100 µm ( b ) , 200 µm ( d ) . * P 0 . 05 , * * P 0 . 01 .

O O O O O O O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-ORGANISM O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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, Kaplan-Meier analysis comparing PDAC-free survival of KrasG12D/+ p53−/− mice that were chloroquine-treated (CQ, red), vehicle-treated (PBS, black) or untreated (blue). a, e, f, The blue curve in all Kaplan-Meier plots represents the same colony of mice (KrasG12D/+, p53−/−, Atg7+/+, Atg5+/+). A Mantel-Cox test was used for statistics. b, d, Scale bars: 100 µm (b), 200 µm (d). *P 0.05, **P 0.01.


a , LC3 staining of representative pancreatic tumours from mice of indicated genotypes . Scale bars : 50 µm .

O O B-GENEPROD O O O B-TISSUE O O B-ORGANISM O O O O O O O O O O

a, LC3 staining of representative pancreatic tumours from mice of indicated genotypes. Scale bars: 50 µm.


b , Western blotting for LC3 , p53 and Atg7 in cell lines derived from individual tumours which developed in the absence or presence of p53 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O

b, Western blotting for LC3, p53 and Atg7 in cell lines derived from individual tumours which developed in the absence or presence of p53.


c , d , Average oxygen consumption rate ( OCR , c ) and extracellular acidification rate ( ECAR ,

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR,


c , d , Average oxygen consumption rate ( OCR , c ) and extracellular acidification rate ( ECAR , d ) of eight Atg7 + / + and seven Atg7 − / − cell lines revealed increased ECAR in autophagy - deficient cells . Error bars are s . e . m .

O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-GENEPROD 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, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR, d) of eight Atg7+/+ and seven Atg7−/− cell lines revealed increased ECAR in autophagy-deficient cells. Error bars are s.e.m.


e , Treatment with 2 - deocyglucose ( 2 - DG ) reduces ECAR and abrogates the difference between cell lines from Atg7 + / + and Atg7 − / − tumours . Values are the average of three biological replicates . Error bars are s . e . m .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

e, Treatment with 2-deocyglucose (2-DG) reduces ECAR and abrogates the difference between cell lines from Atg7+/+ and Atg7−/− tumours. Values are the average of three biological replicates. Error bars are s.e.m.


, g , LC - MS analysis revealed increased glucose consumption from medium ( f ) and extracellular lactate accumulation ( g ) in Atg7 - null cell lines . Values are the average of eight ( Atg7 + / + ) and seven ( Atg7 − / − ) biological replicates .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O

, g, LC-MS analysis revealed increased glucose consumption from medium (f) and extracellular lactate accumulation (g) in Atg7-null cell lines. Values are the average of eight (Atg7+/+) and seven (Atg7−/−) biological replicates.


h , LC - MS analysis shows increased accumulation of 2 - DG and its metabolite 2 - DG - 6 - phosphate in Atg7 - deficient cell lines . Values are averages of five biological replicates for each genotype .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O

h, LC-MS analysis shows increased accumulation of 2-DG and its metabolite 2-DG-6-phosphate in Atg7-deficient cell lines. Values are averages of five biological replicates for each genotype.


i , Background levels of 18F - FDG uptake in brain and heart can be seen in sagittal and coronal views ( middle and right panels , respectively ) of wild - type Atg7 mice ( top row ) . In contrast , intense 18F - FDG uptake is seen in pancreatic tumours of Atg7 deficient mice ( bottom row ) .

O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O B-GENEPROD O B-ORGANISM O O O O O

i, Background levels of 18F-FDG uptake in brain and heart can be seen in sagittal and coronal views (middle and right panels, respectively) of wild-type Atg7 mice (top row). In contrast, intense 18F-FDG uptake is seen in pancreatic tumours of Atg7 deficient mice (bottom row).


j , LC - MS of intracellular glycolytic and pentose phosphate pathway intermediates shows enhanced accumulation in Atg7 − / − tumour cell lines . Values are average of eight ( Atg7 + / + ) and six ( Atg7 − / − ) biological replicates . f - h , j , Error bars are s . e . m . a , f - h , j , A Mann - Whitney U - test was used to ascertain significance . * P 0 . 05 , * * P 0 . 01 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

j, LC-MS of intracellular glycolytic and pentose phosphate pathway intermediates shows enhanced accumulation in Atg7−/− tumour cell lines. Values are average of eight (Atg7+/+) and six (Atg7−/−) biological replicates. f-h, j, Error bars are s.e.m. a, f-h, j, A Mann-Whitney U-test was used to ascertain significance. *P  0.05, **P  0.01.


C Subcellular fractionation of the WT and NLS - deficient FolSrpk1 - GFP proteins . N , nucleus ; C , cytosol . The amount of nuclear FolSrpk1 - GFP was set as 1 . Data information : For C , each gel shown is a representative experiment carried out at least three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and Coomassie brilliant blue ( CBB ) staining .

O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-SUBCELLULAR B-ORGANISM O B-GENEPROD O O O O O 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-ORGANISM B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

C Subcellular fractionation of the WT and NLS-deficient FolSrpk1-GFP proteins. N, nucleus; C, cytosol. The amount of nuclear FolSrpk1-GFP was set as 1. Data information: For C, each gel shown is a representative experiment carried out at least three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and Coomassie brilliant blue (CBB) staining.


D Quantitative measurement of H2O2 in tomato roots after infection by the WT and FolSrpk1p : : FolSrpk1 - GFP strains at 0 - 5 days post incubation ( dpi ) . Data are presented as mean ± SD . The presence of different letters above the mean values of three replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) .

O O O O B-SMALL_MOLECULE O B-ORGANISM B-TISSUE O O O O O O B-ORGANISM O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantitative measurement of H2O2 in tomato roots after infection by the WT and FolSrpk1p::FolSrpk1-GFP strains at 0-5 days post incubation (dpi). Data are presented as mean ± SD. The presence of different letters above the mean values of three replicates indicates a significant difference between different samples (P < 0.05, ANOVA).


E Subcellular fractionation of FolSrpk1 - GFP during the early infection stage . Total proteins were extracted from tomato roots at 0 ( conidia ) , 1 , 3 and 5 dpi . Equal amount of total , nuclear and cytoplasm proteins was separated by SDS - PAGE , and FolSrpk1 was detected using anti - GFP . The amount of total and nuclear FolSrpk1 - GFP at 0 dpi was each set as 1 . Data information : For E , each gel shown is a representative experiment carried out at least three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and Coomassie brilliant blue ( CBB ) staining .

O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR B-ORGANISM O B-GENEPROD O O O O O O O O O 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-ORGANISM B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

E Subcellular fractionation of FolSrpk1-GFP during the early infection stage. Total proteins were extracted from tomato roots at 0 (conidia), 1, 3 and 5 dpi. Equal amount of total, nuclear and cytoplasm proteins was separated by SDS-PAGE, and FolSrpk1 was detected using anti-GFP. The amount of total and nuclear FolSrpk1-GFP at 0 dpi was each set as 1. Data information: For E, each gel shown is a representative experiment carried out at least three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and Coomassie brilliant blue (CBB) staining.


F Association of FolSrpk1 with FolAha1 under ROS stress . After treatment with 10 mM H2O2 for 0 , 1 and 2 h , proteins pulled down with GFP - Trap beads were probed with anti - GFP and anti - Flag ( top ) . Input proteins were shown by Western blotting with anti - GFP and anti - FLAG antibodies ( bottom ) . The amount of immunoprecipitated FolSrpk1 - Flag and FolAha1 - GFP at 0 h was each set as 1 . Data information : For , F each gel shown is a representative experiment carried out at least three times .

O O O B-ORGANISM O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O 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 Association of FolSrpk1 with FolAha1 under ROS stress. After treatment with 10 mM H2O2 for 0, 1 and 2 h, proteins pulled down with GFP-Trap beads were probed with anti-GFP and anti-Flag (top). Input proteins were shown by Western blotting with anti-GFP and anti-FLAG antibodies (bottom). The amount of immunoprecipitated FolSrpk1-Flag and FolAha1-GFP at 0 h was each set as 1. Data information: For , F each gel shown is a representative experiment carried out at least three times.


G Fluorescence microscopy analysis of FolSrpk1 - GFP in mycelia of the FolSrpk1p : : FolSrpk1 - GFP strain treated with 10 mM H2O2 for 0 , 1 and 2 h . Arrows indicate the nucleus . Scale bars = 5 µm .

O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O B-GENEPROD O B-SUBCELLULAR O O B-ORGANISM O O B-ORGANISM O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O

G Fluorescence microscopy analysis of FolSrpk1-GFP in mycelia of the FolSrpk1p::FolSrpk1-GFP strain treated with 10 mM H2O2 for 0, 1 and 2 h. Arrows indicate the nucleus. Scale bars = 5 µm.


I Subcellular fractionation analysis of FolSrpk1 - GFP in mycelia of the FolSrpk1p : : FolSrpk1 - GFP strain treated with 10 mM H2O2 for 0 , 1 and 2 h . The amount of nuclear FolSrpk1 - GFP at 0 h was set as 1 . Data information : For I , each gel shown is a representative experiment carried out at least three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and Coomassie brilliant blue ( CBB ) staining .

O O O O O B-ORGANISM O B-GENEPROD O B-SUBCELLULAR O O B-ORGANISM O O B-ORGANISM O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR B-ORGANISM O B-GENEPROD O O O O O O O O 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-ORGANISM B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

I Subcellular fractionation analysis of FolSrpk1-GFP in mycelia of the FolSrpk1p::FolSrpk1-GFP strain treated with 10 mM H2O2 for 0, 1 and 2 h. The amount of nuclear FolSrpk1-GFP at 0 h was set as 1. Data information: For I, each gel shown is a representative experiment carried out at least three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and Coomassie brilliant blue (CBB) staining.


Co - IP ( C ) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1 . The amount of immunoprecipitated FolSrpk1 - Flag and FolAha1 - GFP in the first line was each set as 1 . Data information : For C , each gel shown is a representative experiment carried out at least three times .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O B-ORGANISM O O O O B-EXP_ASSAY B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Co-IP (C) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1. The amount of immunoprecipitated FolSrpk1-Flag and FolAha1-GFP in the first line was each set as 1. Data information: For C, each gel shown is a representative experiment carried out at least three times.


Y2H ( D ) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1 .

B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O B-ORGANISM O

Y2H (D) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1.


F Fluorescence microscopy of FolSrpk1 - GFP and its mutant forms . Arrows indicate the nucleus . Bars = 5 µm . G Quantification of FolSrpk1 - GFP distribution in F . The calculation was from three independent replicates with three hyphae per replicate and three cells per hyphae . Different letters indicate statistically significant differences according to one - way ANOVA ( P < 0 . 05 ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescence microscopy of FolSrpk1-GFP and its mutant forms. Arrows indicate the nucleus. Bars = 5 µm. G Quantification of FolSrpk1-GFP distribution in F. The calculation was from three independent replicates with three hyphae per replicate and three cells per hyphae. Different letters indicate statistically significant differences according to one-way ANOVA (P < 0.05). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


H Subcellular fractionation of FolSrpk1 - GFP and its mutant forms . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and CBB staining . The amount of nuclear FolSrpk1 - GFP was set as 1 . Data information : For H , , each gel shown is a representative experiment carried out at least three times .

O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

H Subcellular fractionation of FolSrpk1-GFP and its mutant forms. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and CBB staining. The amount of nuclear FolSrpk1-GFP was set as 1. Data information: For H, , each gel shown is a representative experiment carried out at least three times.


I K304 acetylation of WT and mutant FolSrpk1 proteins in the indicated strains after treatment with 10 mM H2O2 for 0 , 1 and 2 h . The amount of acetylated and total FolSrpk1 - GFP at 0 h was each set as 1 . Data information : For each gel shown is a representative experiment carried out at least three times .

O O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

I K304 acetylation of WT and mutant FolSrpk1 proteins in the indicated strains after treatment with 10 mM H2O2 for 0, 1 and 2 h. The amount of acetylated and total FolSrpk1-GFP at 0 h was each set as 1. Data information: For each gel shown is a representative experiment carried out at least three times.


A K304 acetylation ( top ) and amount ( middle ) of FolSrpk1 - GFP and FolSrpk1K304Q - GFP in WT and FolArd1 - OE strains treated with 10 mM H2O2 for 2 h . The over - expressed FolArd1 - Flag was detected by anti - Flag ( bottom ) . The amount of acetylated and total FolSrpk1 in the WT was each set as 1 . Data information : each gel shown is a representative experiment carried out three times .

O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-ORGANISM 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 K304 acetylation (top) and amount (middle) of FolSrpk1-GFP and FolSrpk1K304Q-GFP in WT and FolArd1-OE strains treated with 10 mM H2O2 for 2 h. The over-expressed FolArd1-Flag was detected by anti-Flag (bottom). The amount of acetylated and total FolSrpk1 in the WT was each set as 1. Data information: each gel shown is a representative experiment carried out three times.


C FolArd1 directly acetylates FolSrpk1 in vitro . Purified GST - FolSrpk1 or its K304Q isoform ( 50 ng ) was incubated with or without 50 ng of purified FolArd1 and then analyzed by immunoblotting using anti - K304ac or anti - GST antibody . Anti - GST immunoblotting was used to show equal loading . The amount of acetylated and total GST - FolSrpk1 in the first lane was each set as 1 . Data information : each gel shown is a representative experiment carried out three times .

O B-ORGANISM O B-EXP_ASSAY B-ORGANISM O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-ORGANISM O 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 FolArd1 directly acetylates FolSrpk1 in vitro. Purified GST-FolSrpk1 or its K304Q isoform (50 ng) was incubated with or without 50 ng of purified FolArd1 and then analyzed by immunoblotting using anti-K304ac or anti-GST antibody. Anti-GST immunoblotting was used to show equal loading. The amount of acetylated and total GST-FolSrpk1 in the first lane was each set as 1. Data information: each gel shown is a representative experiment carried out three times.


E Subcellular fractionation analysis of FolSrpk1 - GFP in WT , ΔFolSir2 , and FolSir2 - OE strains . The amount of nuclear FolSrpk1 - GFP in the WT was set as 1 . Data information : each gel shown is a representative experiment carried out three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and CBB staining .

O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O B-SUBCELLULAR B-ORGANISM O B-GENEPROD O O O 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-ORGANISM B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O

E Subcellular fractionation analysis of FolSrpk1-GFP in WT, ΔFolSir2, and FolSir2-OE strains. The amount of nuclear FolSrpk1-GFP in the WT was set as 1. Data information: each gel shown is a representative experiment carried out three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and CBB staining.


G , H Expression profile of FolArd1 ( G ) and FolSir2 ( H ) during the infection stage . Total RNA extracted from host roots at 0 ( conidia ) and 1 - 5 dpi was reverse transcribed . The expression levels were normalized to that of the Fol actin gene . Data are presented as mean ± SD . The presence of different letters above the mean values of three replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) .

O O O B-EXP_ASSAY O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H Expression profile of FolArd1 (G) and FolSir2 (H) during the infection stage. Total RNA extracted from host roots at 0 (conidia) and 1-5 dpi was reverse transcribed. The expression levels were normalized to that of the Fol actin gene. Data are presented as mean ± SD. The presence of different letters above the mean values of three replicates indicates a significant difference between different samples (P < 0.05, ANOVA).


A Relative expression level of six antioxidant enzymes during the infection stage . The experiments were carried out as in Fig 3 . Data are presented as mean ± SD . The presence of different letters above the mean values of three biological replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative expression level of six antioxidant enzymes during the infection stage. The experiments were carried out as in Fig 3. Data are presented as mean ± SD. The presence of different letters above the mean values of three biological replicates indicates a significant difference between different samples (P < 0.05, ANOVA).


B Secretion of the FOXG _ 17180 - GFP , FOXG _ 15294 - GFP and FOXG _ 13788 - GFP proteins . The proteins extracted from mycelia ( M ) and culture supernatants ( S ) were analyzed by Western blotting with anti - GFP and anti - Actin . Each gel shown is a representative experiment carried out three times . The loading controls were Fol Actin and CBB staining .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O

B Secretion of the FOXG_17180-GFP, FOXG_15294-GFP and FOXG_13788-GFP proteins. The proteins extracted from mycelia (M) and culture supernatants (S) were analyzed by Western blotting with anti-GFP and anti-Actin. Each gel shown is a representative experiment carried out three times. The loading controls were Fol Actin and CBB staining.


F qRT - PCR analysis of Fol EF - 1α transcript levels in tomato roots 14 days after infection by the indicated strains . The expression of tomato RCE1 was used as a control to ensure the use of equal amounts of RNA for RT - PCR . Data information : For F , the presence of different letters above the mean values of four replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qRT-PCR analysis of Fol EF-1α transcript levels in tomato roots 14 days after infection by the indicated strains. The expression of tomato RCE1 was used as a control to ensure the use of equal amounts of RNA for RT-PCR. Data information: For F, the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


B Relative growth reduction of the indicated strains representing the reduction in colony diameters between normal and stress conditions normalized to colony diameters under normal conditions ( four biological replicates ) . Data information : For B , the presence of different letters above the mean values of four replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative growth reduction of the indicated strains representing the reduction in colony diameters between normal and stress conditions normalized to colony diameters under normal conditions (four biological replicates). Data information: For B, the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


C Determination of H2O2 content in tomato roots infected by the indicted strains at 0 - 5 dpi . The experiments were carried out as in Fig 1 . Data are presented as mean ± SD of three biological replicates .

O O O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE O O O O O O O O O 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 Determination of H2O2 content in tomato roots infected by the indicted strains at 0-5 dpi. The experiments were carried out as in Fig 1. Data are presented as mean ± SD of three biological replicates.


F qRT - PCR analysis of Fol EF - 1α transcript levels in tomato roots 14 days after infection by the indicated strains . The experiments ( four biological replicates ) Data information : For F , the presence of different letters above the mean values of four replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qRT-PCR analysis of Fol EF-1α transcript levels in tomato roots 14 days after infection by the indicated strains. The experiments (four biological replicates) Data information: For F, the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


A Subcellular fractionation of BcSrpk1 - GFP in WT and BcSrpk1mutant strains . The amount of nuclear BcSrpk1 - GFP was set as 1 . Data information : , each gel shown is a representative experiment carried out three times . The fractionation controls were histone H1 ( nucleus ) , Actin ( cytosol ) and CBB staining .

O O O O B-ORGANISM O B-GENEPROD O O O B-ORGANISM O O O O O B-SUBCELLULAR B-ORGANISM O B-GENEPROD O 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-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O

A Subcellular fractionation of BcSrpk1-GFP in WT and BcSrpk1mutant strains. The amount of nuclear BcSrpk1-GFP was set as 1. Data information: , each gel shown is a representative experiment carried out three times. The fractionation controls were histone H1 (nucleus), Actin (cytosol) and CBB staining.


B Co - IP analysis revealing the interaction of BcAha1 with BcSrpk1 and its mutant forms . Data information : each gel shown is a representative experiment carried out three times .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O

B Co-IP analysis revealing the interaction of BcAha1 with BcSrpk1 and its mutant forms. Data information: each gel shown is a representative experiment carried out three times.


C K367 acetylation of BcSrpk1 during the early infection stage . Total proteins were extracted from tomato leaves at 0 ( spores ) , 12 , 24 and 36 hpi . Eluted proteins bound to the anti - GFP beads were probed by the anti - K304ac and anti - GFP antibodies . The amount of acetylated and total BcSrpk1 at 0 hpi was each set as 1 . Data information : each gel shown is a representative experiment carried out three times .

O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O B-ORGANISM 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 K367 acetylation of BcSrpk1 during the early infection stage. Total proteins were extracted from tomato leaves at 0 (spores), 12, 24 and 36 hpi. Eluted proteins bound to the anti-GFP beads were probed by the anti-K304ac and anti-GFP antibodies. The amount of acetylated and total BcSrpk1 at 0 hpi was each set as 1. Data information: each gel shown is a representative experiment carried out three times.


F Relative density of DAB staining in E ( four biological replicates ) . Data information : For F the presence of different letters above the mean values of four replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative density of DAB staining in E (four biological replicates). Data information: For F the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


Two top - ranked SplashRNA - designed shRNAmirs targeting human Nurd complex components and a control shRNAmir targeting human CD4 were cloned into both miR - AB and miR - E lentiviral vectors with Venus as reporter . The lentiviruses packaged by these constructs were used for transduction of multiple human cell lines . RNAi efficiency was determined by western blot ( WB ) analysis of target protein levels in the lysates of the transduced cells . - actin served as loading control .

O O O O O O O O O B-ORGANISM B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-ORGANISM B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O

Two top-ranked SplashRNA-designed shRNAmirs targeting human Nurd complex components and a control shRNAmir targeting human CD4 were cloned into both miR-AB and miR-E lentiviral vectors with Venus as reporter. The lentiviruses packaged by these constructs were used for transduction of multiple human cell lines. RNAi efficiency was determined by western blot (WB) analysis of target protein levels in the lysates of the transduced cells. -actin served as loading control.


The RNAi efficiency of Pten . 1524 and Pten . 932 shRNAmir mediated by miR - AB vs . miR - E was tested in NIH - 3T3 cells in the context of < 30 % transduction efficiency ( GFP as reporter ) . Representative western blots of Pten protein expression of the FACS - sorted GFP + cells from two biological replicates were shown . β - actin served as loading control .

O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

The RNAi efficiency of Pten.1524 and Pten.932 shRNAmir mediated by miR-AB vs. miR-E was tested in NIH-3T3 cells in the context of <30% transduction efficiency (GFP as reporter). Representative western blots of Pten protein expression of the FACS-sorted GFP+ cells from two biological replicates were shown. β-actin served as loading control.


Two top - ranked SplashRNA - designed shRNAmirs targeting human or mouse surface protein FAS were cloned into Venus - expressing miR - AB lentiviral vectors which harbor different promoters to drive the shRNAmir expression . The packaged lentiviruses were used to infect the indicated human or mouse cell lines . A shRNAmir targeting human or mouse CD4 driven under hCMV promoter was used as control . After > 96h of infection , FAS protein levels in the transduced Venus - positive cells were determined by surface staining and shown in histograms ( top panels ) and quantified by MFI of APC ( bottom panels ) .

O O O O O O O O O B-ORGANISM O B-ORGANISM O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O B-ORGANISM O O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

Two top-ranked SplashRNA-designed shRNAmirs targeting human or mouse surface protein FAS were cloned into Venus-expressing miR-AB lentiviral vectors which harbor different promoters to drive the shRNAmir expression. The packaged lentiviruses were used to infect the indicated human or mouse cell lines. A shRNAmir targeting human or mouse CD4 driven under hCMV promoter was used as control. After >96h of infection, FAS protein levels in the transduced Venus-positive cells were determined by surface staining and shown in histograms (top panels) and quantified by MFI of APC (bottom panels).


293T cells were co - transduced with four miR - AB lentiviruses at 1 : 1 ratio , which carries a neutral shRNAmir targeting CD4 or CD19 , with Azurite , GFP , Ametrine or mOrange as reporter respectively . Cells were cultured for 22 days and fluorescence positive cells were quantified at the time points as indicated . Representative contour FACS plots cells on day 4 and day 22 shown ( top panels ) . Cell numbers of untransduced , single fluorescence positive cells and quadruple positive cells were quantified and normalized to untransduced cells on day 4 , and plotted over time ( bottom panel ) . Data are shown as mean ± s . d . and n = 4 . NS indicates no significant difference ( P > 0 . 05 , two - tailed unpaired Student ' s t - test ) .

B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O 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-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

293T cells were co-transduced with four miR-AB lentiviruses at 1:1 ratio, which carries a neutral shRNAmir targeting CD4 or CD19, with Azurite, GFP, Ametrine or mOrange as reporter respectively. Cells were cultured for 22 days and fluorescence positive cells were quantified at the time points as indicated. Representative contour FACS plots cells on day 4 and day 22 shown (top panels). Cell numbers of untransduced, single fluorescence positive cells and quadruple positive cells were quantified and normalized to untransduced cells on day 4, and plotted over time (bottom panel). Data are shown as mean ± s.d. and n=4. NS indicates no significant difference (P>0.05, two-tailed unpaired Student's t-test).


CD8 + T cells were activated for 16h and then co - transduced with miR - AB retroviruses at 1 : 1 ratio , which carries a SplashRNA - designed shRNAmir , targeting surface protein CD127 , CD90 , CD44 or CD8 , with Azurite , GFP , Ametrine or mCherry2 as fluorescent reporters respectively for 4h . miR - AB retroviruses expressing CD19 - specific shRNAmirs with same fluorescent reporters were used as controls . Surface staining was performed 72h after transduction . FACS data were shown as overlaid histograms of control shRNAmirs ( shCD19s ) and target shRNAmirs ( shCD127 , shCD90 , shCD44 or shCD8 ) . Cells were categorized as sixteen populations expressing single , double , triple or quadruple fluorescent proteins or not ( none ) and fluorescent reporter - positive plots were highlighted with thick frames and white background . Each population ' s surface staining profile was shown on the right . The data was representative of two biologically independent experiments .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O

CD8+ T cells were activated for 16h and then co-transduced with miR-AB retroviruses at 1:1 ratio, which carries a SplashRNA-designed shRNAmir, targeting surface protein CD127, CD90, CD44 or CD8, with Azurite, GFP, Ametrine or mCherry2 as fluorescent reporters respectively for 4h. miR-AB retroviruses expressing CD19-specific shRNAmirs with same fluorescent reporters were used as controls. Surface staining was performed 72h after transduction. FACS data were shown as overlaid histograms of control shRNAmirs (shCD19s) and target shRNAmirs (shCD127, shCD90, shCD44 or shCD8). Cells were categorized as sixteen populations expressing single, double, triple or quadruple fluorescent proteins or not (none) and fluorescent reporter-positive plots were highlighted with thick frames and white background. Each population's surface staining profile was shown on the right. The data was representative of two biologically independent experiments.


A ) ( left ) t - sne plot from scRNAseq data of human organoids and cluster assignment of colonic cell populations . Red color corresponds to undifferentiated cells , light green corresponds to surface colonocytes ( enterocytes ) . t - sne plot for selected genes from single cell RNAseq of human colon epithelial cells revealing expression of ACE2 specifically in differentiated surface colonocytes expressing KRT20 and AQP8 , which also express the IFN - γ receptor IFNGR2 . B ) Immunofluorescence staining for ACE2 ( red ) and KRT20 ( green ) of human colon tissue section indicating ACE2 expression in surface colonocytes . Scale bar : 50 µm .

O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM B-TISSUE O O O B-GENEPROD O O O O O O O O O O O

A) (left) t-sne plot from scRNAseq data of human organoids and cluster assignment of colonic cell populations. Red color corresponds to undifferentiated cells, light green corresponds to surface colonocytes (enterocytes). t-sne plot for selected genes from single cell RNAseq of human colon epithelial cells revealing expression of ACE2 specifically in differentiated surface colonocytes expressing KRT20 and AQP8, which also express the IFN-γ receptor IFNGR2. B) Immunofluorescence staining for ACE2 (red) and KRT20 (green) of human colon tissue section indicating ACE2 expression in surface colonocytes. Scale bar: 50 µm.


C ) Human colon organoids cultured in full medium ( FM ) condition ( upper panel ) or FM treated with IFN - γ ( lower panel ) . Left : bright field images of 7 - day cultured human colon organoids , scale bar : 100 µm . Right : Immunofluorescence for KRT20 ( green ; note increased expression upon IFN - γ treatment ) . Scale bar : 25 µm .

O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

C) Human colon organoids cultured in full medium (FM) condition (upper panel) or FM treated with IFN-γ (lower panel). Left: bright field images of 7-day cultured human colon organoids, scale bar: 100 µm. Right: Immunofluorescence for KRT20 (green; note increased expression upon IFN-γ treatment). Scale bar: 25 µm.


D ) Human colon organoids cultured in basic medium ( upper panel ) or additionally treated with IFN - γ ( lower panel ) . Left : bright field images of organoids that were cultured for 4 days in basic medium and in addition for 3 days with IFN - γ ( lower panel ) scale bar : 100 µm . Middle : Immunofluorescence labelling for KRT20 ( green ) Right : Immunofluorescence labelling for E - cadherin ( green ) to visualize epithelial cell boundaries ; insets with higher magnification indicate elongation of epithelial cells that are grown in basic medium and treated with IFN - γ . Scale bar : 25 µm .

O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

D) Human colon organoids cultured in basic medium (upper panel) or additionally treated with IFN-γ (lower panel). Left: bright field images of organoids that were cultured for 4 days in basic medium and in addition for 3 days with IFN-γ (lower panel) scale bar: 100 µm. Middle: Immunofluorescence labelling for KRT20 (green) Right: Immunofluorescence labelling for E-cadherin (green) to visualize epithelial cell boundaries; insets with higher magnification indicate elongation of epithelial cells that are grown in basic medium and treated with IFN-γ. Scale bar: 25 µm.


Comparison of E ) KRT20 , F ) MUC2 , G ) CHGA , H ) LGR5 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN - γ ( n = 3 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of E) KRT20, F) MUC2, G) CHGA, H) LGR5 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN-γ (n=3). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test


ACE2 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN - γ ( n = 3 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ACE2 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN-γ (n=3). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test


J ) Immunofluorescence staining for ACE2 ( red ) and KRT20 ( green ) of organoids grown in full medium ( upper ) or basic medium ( lower ) and additionally treated with IFN - γ ( right ) , indicating expression of ACE2 in differentiated enterocytes . White arrows point to ACE2 . Scale bar : 10

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O

J) Immunofluorescence staining for ACE2 (red) and KRT20 (green) of organoids grown in full medium (upper) or basic medium (lower) and additionally treated with IFN-γ (right), indicating expression of ACE2 in differentiated enterocytes. White arrows point to ACE2. Scale bar: 10 µm


A ) Immunofluorescence for N - protein ( green ) 48 h after SARS - CoV - 2 infection of human colon organoids cultured in full medium ( upper ) , full medium + IFN - γ ( upper right ) , basic medium ( lower left ) and basic medium + IFN - γ ( lower right ) . Yellow arrows point to N - protein positive cells . Scale bar : 25 µm .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM B-TISSUE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

A) Immunofluorescence for N-protein (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Yellow arrows point to N-protein positive cells. Scale bar: 25 µm.


B ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium vs . basic medium organoids 48 h after infection and normalized to GAPDH ( n = 6 ) . C ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium vs . full medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 9 ) . D ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in basic medium vs . basic medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 4 ) . E ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium + IFN - γ vs . basic medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 4 ) . ( B - E ) Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by Student ' s t - test

O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. basic medium organoids 48 h after infection and normalized to GAPDH (n=6). C) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. full medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=9). D) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in basic medium vs. basic medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4). E) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium + IFN-γ vs. basic medium+ IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4). (B-E) Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by Student's t-test


F ) qPCR data comparing the relative virus load of SARS - CoV - 2 measured by viral genome quantity normalized to GAPDH at 24 and 48 h after infection in full medium ( left ) and basic medium ( right ) untreated or treated with IFN - γ , indicating increase in virus load in differentiated and IFN - γ treated conditions ( n = 8 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR data comparing the relative virus load of SARS-CoV-2 measured by viral genome quantity normalized to GAPDH at 24 and 48 h after infection in full medium (left) and basic medium (right) untreated or treated with IFN-γ, indicating increase in virus load in differentiated and IFN-γ treated conditions (n=8). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test


G ) Electron microscopy imaging of organoids grown in basic medium , pretreated with IFN - γ for 3 days and infected for 48 h with SARS - CoV - 2 . Upper : Overview showing large virus - loaded vesicles in an epithelial cell . Scale bar : 500nm . Lower : Disintegrated organoid cell containing high virus load , scale bar : 2 . 5µm , higher magnification to visualize virus particles , scale bar : 100nm .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

G) Electron microscopy imaging of organoids grown in basic medium, pretreated with IFN-γ for 3 days and infected for 48 h with SARS-CoV-2. Upper: Overview showing large virus-loaded vesicles in an epithelial cell. Scale bar: 500nm. Lower: Disintegrated organoid cell containing high virus load, scale bar: 2.5µm, higher magnification to visualize virus particles, scale bar: 100nm.


H ) Immunofluorescence for cleaved - caspase3 ( red ) and E - cadherin ( green ) 48 h after SARS - CoV - 2 infection of human colon organoids cultured in full medium ( upper ) , full medium + IFN - γ ( upper right ) , basic medium ( lower left ) and basic medium + IFN - γ ( lower right ) . Scale bar : 50 µm . ( below ) Quantification of apoptotic cells , indicating increased cell death in the differentiated and IFN - γ treated condition after SARS - CoV - 2 infection ( n = 7 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM B-TISSUE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H) Immunofluorescence for cleaved-caspase3 (red) and E-cadherin (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Scale bar: 50 µm. (below) Quantification of apoptotic cells, indicating increased cell death in the differentiated and IFN-γ treated condition after SARS-CoV-2 infection (n=7). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test


C ) Analysis of discordance and concordance of transcriptomic responses ( DISCO ) between untreated vs . SARS - CoV - 2 infected and untreated vs IFN - γ treated organoids . The 50 most regulated genes are highlighted ; grey : discordant and black : concordant .

O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O

C) Analysis of discordance and concordance of transcriptomic responses (DISCO) between untreated vs. SARS-CoV-2 infected and untreated vs IFN-γ treated organoids. The 50 most regulated genes are highlighted; grey: discordant and black: concordant.


D ) Relative expression of selected enterocyte - specific genes upon SARS - CoV - 2 infection , normalized to GAPDH . Differences in gene expression were assessed using the linear model ' lmFit ' and ' makeContrasts ' in limma .

O O O B-EXP_ASSAY O O B-CELL_TYPE O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

D) Relative expression of selected enterocyte-specific genes upon SARS-CoV-2 infection, normalized to GAPDH. Differences in gene expression were assessed using the linear model 'lmFit' and 'makeContrasts' in limma.


E ) Immunofluorescence for KRT20 ( green ) in human colon organoids cultured in basic medium and either non - infected ( left ) or 48 h after SARS - CoV - 2 infection ( right ) , indicating a strong increase in KRT20 expression after viral infection . Scale bar : 50 µm . Quantification of fluorescence intensity below ( n = 8 ) . Data are presented as mean + / - SD , * * * * : p ≤ 0 . 0001 , as determined by Student ' s t -

O O B-EXP_ASSAY O B-GENEPROD O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence for KRT20 (green) in human colon organoids cultured in basic medium and either non-infected (left) or 48 h after SARS-CoV-2 infection (right), indicating a strong increase in KRT20 expression after viral infection. Scale bar: 50 µm. Quantification of fluorescence intensity below (n=8). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-test


F ) qPCR data displaying the increase of INF - γ target genes ( CXCL11 , IFIT3 ) ( upper ) and enterocyte marker genes ( KRT20 , ACE2 ) ( lower ) after SARS - CoV - 2 infection , validating the results from the microarray analysis ( n = 3 ) . Data are presented as mean + / - SD , * * * * : p ≤ 0 . 0001 , as determined by Student ' s t -

O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O 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) qPCR data displaying the increase of INF-γ target genes (CXCL11, IFIT3) (upper) and enterocyte marker genes (KRT20, ACE2) (lower) after SARS-CoV-2 infection, validating the results from the microarray analysis (n=3). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-test


( A ) Wild - type primary B cells were treated with 5 μg / mL of anti - kappa antibody ( a - k chain , top ) or 1 µM LatrunculinA ( LatA , bottom ) . Intracellular calcium flux was measured by flow cytometry in the presence of Src - inhibitor ( PP2 ) , Syk - inhibitor ( BAY 613606 ) or vehicle control .

O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

(A) Wild-type primary B cells were treated with 5 μg/mL of anti-kappa antibody (a-k chain, top) or 1 µM LatrunculinA (LatA, bottom). Intracellular calcium flux was measured by flow cytometry in the presence of Src-inhibitor (PP2), Syk-inhibitor (BAY 613606) or vehicle control.


( B ) Wild - type primary B cells were treated with vehicle control ( - ) , 5 μg / mL anti - kappa antibody or 1 µM LatA for the indicated time . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - Lyn ( Y507 ) and actin as loading control . The intensity of phosphorylated proteins , normalized to actin , was referred to the respective unstimulated sample , set as 1 .

O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

(B) Wild-type primary B cells were treated with vehicle control (-), 5 μg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-Lyn (Y507) and actin as loading control. The intensity of phosphorylated proteins, normalized to actin, was referred to the respective unstimulated sample, set as 1.


( C and D ) Wild - type primary B cells were treated with vehicle control ( - ) , 5 µg / mL anti - kappa antibody or 1 µM LatA for the indicated time . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - Syk ( pSyk ) , phospho - CD19 ( pCD19 ) , phospho - Akt ( pAkt ) , phospho - ERK ( pERK ) and total ERK as loading control . The intensity of phosphorylated proteins , normalized to ERK , was referred to the respective unstimulated sample , set as 1 .

O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

(C and D) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-Syk (pSyk), phospho-CD19 (pCD19), phospho-Akt (pAkt), phospho-ERK (pERK) and total ERK as loading control. The intensity of phosphorylated proteins, normalized to ERK, was referred to the respective unstimulated sample, set as 1.


( E and F ) Wild - type primary B cells were treated with vehicle control ( - ) , 5 µg / mL anti - kappa antibody or 1 µM LatA for the indicated time . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - PLCγ2 ( pPLCγ2 ) and total ERK as loading control . The intensity of phosphorylated proteins , normalized to ERK , was referred to the respective unstimulated sample , set as 1 .

O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

(E and F) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-PLCγ2 (pPLCγ2) and total ERK as loading control. The intensity of phosphorylated proteins, normalized to ERK, was referred to the respective unstimulated sample, set as 1.


( G and H ) Wild - type primary B cells were treated with vehicle control ( - ) , 5 µg / mL anti - kappa antibody or 1 µM LatA for the indicated time . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - CD22 ( pCD22 ) , phospho - SHP1 ( pSHP1 ) , and CD22 or total ERK as loading controls . The intensity of phosphorylated proteins , normalized to CD22 or ERK , was referred to the respective unstimulated sample , set as 1 .

O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O

(G and H) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD22 (pCD22), phospho-SHP1 (pSHP1), and CD22 or total ERK as loading controls. The intensity of phosphorylated proteins, normalized to CD22 or ERK, was referred to the respective unstimulated sample, set as 1.


( I ) Wild - type and Cd22 - / - primary B cells were treated with 5 µg / mL anti - kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry .

O O O O O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O

(I) Wild-type and Cd22-/- primary B cells were treated with 5 µg/mL anti-kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry.


( J ) Wild - type and Cd22Y2 , 5 , 6F primary B cells were treated with 5 µg / mL anti - kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry .

O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O

(J) Wild-type and Cd22Y2,5,6F primary B cells were treated with 5 µg/mL anti-kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry.


( K and L ) Wild - type , Cd22 - / - and Cd22Y2 , 5 , 6F primary B cells were treated with vehicle control ( - ) or 1 µM LatA for the indicated time . Cells were lysed and analysed by SDS - PAGE followed by immunoblotting with phospho - CD19 ( pCD19 ) , phospho - Akt ( pAkt ) , phospho - ERK ( pERK ) and actin as loading control . The intensity of phosphorylated proteins , normalized to actin , was referred to the unstimulated sample of the wild type cells , set as 1 .

O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

(K and L) Wild-type, Cd22-/- and Cd22Y2,5,6F primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD19 (pCD19), phospho-Akt (pAkt), phospho-ERK (pERK) and actin as loading control. The intensity of phosphorylated proteins, normalized to actin, was referred to the unstimulated sample of the wild type cells, set as 1.


( A ) Determining the number and density of CD22 , CD19 , IgM and IgD on the surface of wild - type primary B cells . Cells were stained with PE - conjugated antibody against CD22 ( blue profile ) , CD19 ( red profile ) , IgM ( red profile ) or IgD ( red profile ) and their fluorescence profiles compared to those of standard beads ( grey profiles ) , which each possess a well - defined number of binding sites ( st0 = blank , st1 - st4 = increasing number of binding sites ) against the primary antibody . For IgM , where the range of expression is large , the low and high 25 percentiles were calculated as in Mattila et al . 2013 and shown in brackets . Data are representative of three independent experiments .

O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O 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) Determining the number and density of CD22, CD19, IgM and IgD on the surface of wild-type primary B cells. Cells were stained with PE-conjugated antibody against CD22 (blue profile), CD19 (red profile), IgM (red profile) or IgD (red profile) and their fluorescence profiles compared to those of standard beads (grey profiles), which each possess a well-defined number of binding sites (st0=blank, st1-st4=increasing number of binding sites) against the primary antibody. For IgM, where the range of expression is large, the low and high 25 percentiles were calculated as in Mattila et al. 2013 and shown in brackets.Data are representative of three independent experiments.


( B - E ) Dual - colour SIM analysis of CD22 , IgM , IgD and CD19 . Wild - type primary B cells were fixed , stained with Atto633 or 488 - conjugated antibody against CD22 , IgM or IgD and Alexa647 - conjugated antibody against CD19 and settled onto nonstimulatory coverslips . Cells were then embedded in agarose , imaged with SIM and analysed . ( B ) SIM images of CD22 , IgM , IgD and CD19 . ( C ) Cross - correlation function . ( D ) Pearson correlation coefficient . ( E ) Cluster size calculated from autocorrelation analysis .

O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O 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-E) Dual-colour SIM analysis of CD22, IgM, IgD and CD19. Wild-type primary B cells were fixed, stained with Atto633 or 488-conjugated antibody against CD22, IgM or IgD and Alexa647-conjugated antibody against CD19 and settled onto nonstimulatory coverslips. Cells were then embedded in agarose, imaged with SIM and analysed.(B) SIM images of CD22, IgM, IgD and CD19.(C) Cross-correlation function.(D) Pearson correlation coefficient.(E) Cluster size calculated from autocorrelation analysis.


Figure 3 . CD22 is highly clustered on the surface of resting B cells ( A - C ) dSTORM analysis of CD22 , CD19 , IgM and IgD . Wild - type primary B cells were stained with Alexa647 - conjugated antibody against CD22 , Cy5 - conjugated antibody against CD19 or IgD , or Cy5 - conjugated Fab fragments against IgM and settled onto nonstimulatory coverslips . Cells were then fixed , imaged with dSTORM and analysed . ( A ) 2D ( top and middle ) and pseudo 3D ( bottom ) dSTORM images were reconstructed from single molecule localizations . The white dashed square is shown in magnification ( middle ) and pseudo 3D ( bottom ) . ( B and C ) Quantification of the distribution of CD22 , CD19 , IgM and IgD with H function and Hopkins index . Error bars ( Hopkins index ) and thin lines ( H function ) denote mean ± SEM . Data are pooled from at least three experiments per receptor . * P < 0 . 001 , * * P < 0 . 0001 ( Student ' s t - test ) .

O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. CD22 is highly clustered on the surface of resting B cells(A-C) dSTORM analysis of CD22, CD19, IgM and IgD. Wild-type primary B cells were stained with Alexa647-conjugated antibody against CD22, Cy5-conjugated antibody against CD19 or IgD, or Cy5-conjugated Fab fragments against IgM and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(A) 2D (top and middle) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in magnification (middle) and pseudo 3D (bottom).(B and C) Quantification of the distribution of CD22, CD19, IgM and IgD with H function and Hopkins index. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM. Data are pooled from at least three experiments per receptor.*P < 0.001, **P < 0.0001 (Student's t-test).


( A - C ) Single - particle - tracking of CD22 , CD19 , IgM and IgD . Wild - type primary B cells were stained with Atto633 - conjugated Fab fragments against CD22 , CD19 , IgM and IgD , settled onto nonstimulatory coverslips and imaged . Single particle trajectories were then analysed . ( A ) Trajectories of CD22 showing diffusion of single particles over 6 s . ( B ) Magnified region from ( A ) traces one particle ( current position indicated with white dashed circle , trajectory in red ) over 6 s . ( C ) Diffusion coefficients of 300 representative CD22 , CD19 , IgM and IgD particles . Bars indicate the median . Data are pooled from at least three experiments per receptor . * P < 0 . 0001 ( Student ' s t - test ) .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O 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-C) Single-particle-tracking of CD22, CD19, IgM and IgD. Wild-type primary B cells were stained with Atto633-conjugated Fab fragments against CD22, CD19, IgM and IgD, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(A) Trajectories of CD22 showing diffusion of single particles over 6 s.(B) Magnified region from (A) traces one particle (current position indicated with white dashed circle, trajectory in red) over 6 s.(C) Diffusion coefficients of 300 representative CD22, CD19, IgM and IgD particles. Bars indicate the median. Data are pooled from at least three experiments per receptor. *P < 0.0001 (Student's t-test).


( D - F ) Single - particle - tracking of CD22 in primary LifeAct - GFP - expressing B cells . Cells were labelled with Atto633 - conjugated Fab fragments against CD22 , settled onto nonstimulatory coverslips and imaged . Single particle trajectories were then analysed . ( D ) Trajectories of CD22 ( left ) showing diffusion of single particles over 6 s and overlaid with a still image of F - actin intensity ( middle ) . Areas of high F - actin density ( grey line boundaries ) with trajectories of CD22 classified as being inside ( red ) and outside ( blue ) of F - actin rich areas . ( E ) Diffusion coefficients of CD22 particles from ( D ) whose trajectories were classed as inside ( in ) or outside ( out ) of actin rich areas . The overall diffusion is also shown ( all ) . Bars and numbers are the median . Data are pooled from two different experiments . * P < 0 . 0001 ( Student ' s t - test ) . ( F ) Correlation analysis . For each position in each CD22 track , the actin intensity was measured and compared to the same number of random positions on the same cell . Values from different cells were normalized so that the average intensity in F - actin rich regions corresponds to a value of one and the average for F - actin low regions to zero .

O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

(D-F) Single-particle-tracking of CD22 in primary LifeAct-GFP-expressing B cells. Cells were labelled with Atto633-conjugated Fab fragments against CD22, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(D) Trajectories of CD22 (left) showing diffusion of single particles over 6 s and overlaid with a still image of F-actin intensity (middle). Areas of high F-actin density (grey line boundaries) with trajectories of CD22 classified as being inside (red) and outside (blue) of F-actin rich areas.(E) Diffusion coefficients of CD22 particles from (D) whose trajectories were classed as inside (in) or outside (out) of actin rich areas. The overall diffusion is also shown (all). Bars and numbers are the median. Data are pooled from two different experiments. *P < 0.0001 (Student's t-test).(F) Correlation analysis. For each position in each CD22 track, the actin intensity was measured and compared to the same number of random positions on the same cell. Values from different cells were normalized so that the average intensity in F-actin rich regions corresponds to a value of one and the average for F-actin low regions to zero.