( B ) Histograms of the spatial initiation probabilities of processive runs within the first 7 . 2 µm from growing microtubule plus ends in the absence ( black bars ) , or presence of 10 nM EB1 ( red bars ) . 199 and 221 initiation positions were determined for the two conditions ( from three experiments each ) .

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(B) Histograms of the spatial initiation probabilities of processive runs within the first 7.2 µm from growing microtubule plus ends in the absence (black bars), or presence of 10 nM EB1 (red bars). 199 and 221 initiation positions were determined for the two conditions (from three experiments each).


( C ) Averaged fluorescence intensity profiles of GFP - dynein at growing microtubule plus ends for the conditions as in B . Mean values from three separate experiments per condition ( with a total of at least 69 kymographs ) are shown ; error bars are s . e . Experiments were performed at 30 ° C .

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(C) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends for the conditions as in B. Mean values from three separate experiments per condition (with a total of at least 69 kymographs) are shown; error bars are s. e. Experiments were performed at 30°C.


( D ) Histogram of spatial initiation probabilities of DDB on static GMPCPP - microtubules . 294 initiation positions were detemined ( from three experiments ) . Concentrations of DDB components as in B .

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(D) Histogram of spatial initiation probabilities of DDB on static GMPCPP-microtubules. 294 initiation positions were detemined (from three experiments). Concentrations of DDB components as in B.


A , B ) Dual and single colour kymographs showing under certain conditions p150 or dynactin - dependent microtubule plus end tracking of dynein in the absence of EB proteins : ( A ) 10 nM GFP - dynein ( green ) tracking the growing end of a Atto565 - microtubule ( magenta ) in the presence of pig dynactin at an elevated concentration of 80 nM . Experiments in ( A ) and ( B ) were performed in standard assay buffer ( BRB20 supplemented with 50 mM KCl , for details see Methods ) .

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A, B) Dual and single colour kymographs showing under certain conditions p150 or dynactin-dependent microtubule plus end tracking of dynein in the absence of EB proteins: (A) 10 nM GFP-dynein (green) tracking the growing end of a Atto565-microtubule (magenta) in the presence of pig dynactin at an elevated concentration of 80 nM. Experiments in (A) and (B) were performed in standard assay buffer (BRB20 supplemented with 50 mM KCl, for details see Methods).


( A , B ) Dual and single colour kymographs showing under certain conditions p150 or dynactin - dependent microtubule plus end tracking of dynein in the absence of EB proteins : ( B ) 10 nM GFP - dynein ( green ) tracking the plus end of an Atto565 - microtubule ( blue ) in the presence of 10 nM Alexa647 - p150 ( a fragment containing the first 517 amino acids of p150 ; called p150 - N in the text ) ( red ) . Experiments in ( A ) and ( B ) were performed in standard assay buffer ( BRB20 supplemented with 50 mM KCl , for details see Methods ) .

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(A, B) Dual and single colour kymographs showing under certain conditions p150 or dynactin-dependent microtubule plus end tracking of dynein in the absence of EB proteins: (B) 10 nM GFP-dynein (green) tracking the plus end of an Atto565-microtubule (blue) in the presence of 10 nM Alexa647-p150 (a fragment containing the first 517 amino acids of p150; called p150-N in the text) (red). Experiments in (A) and (B) were performed in standard assay buffer (BRB20 supplemented with 50 mM KCl, for details see Methods).


( C , D ) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength : ( C ) In standard assay buffer , 10 nM Alexa647 - p150 ( green ) accumulates at the plus end and on the GMPCPP - stabilised segment of a growing Atto565 - microtubule ( magenta ) .

(C, D) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength: (C) In standard assay buffer, 10 nM Alexa647-p150 (green) accumulates at the plus end and on the GMPCPP-stabilised segment of a growing Atto565-microtubule (magenta).


( C , D ) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength : ( D ) At higher ionic strength ( BRB80 supplemented with 60 mM KCl , see Methods ) , Alexa647 - p150 ( green ) binds only weakly to the microtubule without a detectable plus end preference . Protein concentrations as in C . The Atto565 - tubulin concentration was always 17 . 5 µM tubulin . The temperature was 30 ° C .

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(C, D) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength: (D) At higher ionic strength (BRB80 supplemented with 60 mM KCl, see Methods), Alexa647-p150 (green) binds only weakly to the microtubule without a detectable plus end preference. Protein concentrations as in C. The Atto565-tubulin concentration was always 17.5 µM tubulin. The temperature was 30°C.


( A - C ) Kymographs showing GFP - dynein ( green ) on dynamic Atto647N - microtubules ( magenta ) in the presence of all the regulators at different Lis1 concentrations . Protein concentrations were 10 nM GFP - dynein , 20 nM pig dynactin , 20 nM EB1 , 5 µM BicD2 - N , and ( A ) either no Lis1 , ( B ) 1 μM mCherry - Lis1 , or ( C ) 5 μM mCherry - Lis1 . Together with BicD2 , Lis1 increases the number of processive dynein runs and high concentrations of Lis1 restore plus - end localisation of dynein in the presence of BicD2 - N . Microtubule orientation as indicated .

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(A-C) Kymographs showing GFP-dynein (green) on dynamic Atto647N-microtubules (magenta) in the presence of all the regulators at different Lis1 concentrations. Protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 20 nM EB1, 5 µM BicD2-N, and (A) either no Lis1, (B) 1 μM mCherry-Lis1, or (C) 5 μM mCherry-Lis1. Together with BicD2, Lis1 increases the number of processive dynein runs and high concentrations of Lis1 restore plus-end localisation of dynein in the presence of BicD2-N. Microtubule orientation as indicated.


( D ) Averaged intensity profiles of GFP - dynein at growing microtubule plus ends in the presence of EB1 , dynactin and 5 µM BicD2 - N ( black squares , as in A ) , in the presence of 1 µM mCherry - Lis1 ( blue circles , as in B ) and 5 µM mCherry - Lis1 ( red triangles , as in C ) . Mean values from three separate experiments per condition ( with a total of at least 49 kymographs ) are shown ; error bars are s . e .

(D) Averaged intensity profiles of GFP-dynein at growing microtubule plus ends in the presence of EB1, dynactin and 5 µM BicD2-N (black squares, as in A), in the presence of 1 µM mCherry-Lis1 (blue circles, as in B) and 5 µM mCherry-Lis1 (red triangles, as in C). Mean values from three separate experiments per condition (with a total of at least 49 kymographs) are shown; error bars are s. e.


( E ) The average number of processive DDB runs per µm microtubule length for the three different Lis1 conditions ( as in A , B and , C ) . Error bars are s . d . * p = 0 . 001 , * * p = 0 . 003 , * * * p = 0 . 001 ( unpaired t - test ) . 218 , 969 , and 646 processive DDB events were analysed for the three conditions , respectively ( from two experiments each ) .

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(E) The average number of processive DDB runs per µm microtubule length for the three different Lis1 conditions (as in A, B and, C). Error bars are s. d. *p = 0.001, **p = 0.003, ***p = 0.001 (unpaired t-test). 218, 969, and 646 processive DDB events were analysed for the three conditions, respectively (from two experiments each).


( F ) Bar graphs showing the mean velocity and the mean run length of processive DDB events for the three Lis1 conditions ( as in A , B and C ) . Error bars are the s . e . m . Mean velocities were 0 . 37 ± 0 . 02 µm / s ( A ) , 0 . 38 ± 0 . 03 µm / s ( B ) , and 0 . 35 ± 0 . 03 µm / s ( C ) . Mean run lengths were 3 . 1 ± 0 . 4 µm ( A ) , 3 . 2 ± 0 . 5 µm ( B ) , and 2 . 9 ± 0 . 3 µm ( C ) . For each condition over 300 complexes were analysed from three different data sets .

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(F) Bar graphs showing the mean velocity and the mean run length of processive DDB events for the three Lis1 conditions (as in A, B and C). Error bars are the s.e.m. Mean velocities were 0.37 ± 0.02 µm/s (A), 0.38 ± 0.03 µm/s (B), and 0.35 ± 0.03 µm/s (C). Mean run lengths were 3.1 ± 0.4 µm (A), 3.2 ± 0.5 µm (B), and 2.9 ± 0.3 µm (C). For each condition over 300 complexes were analysed from three different data sets.


( G ) Histogram of spatial initiation probabilities of DDB runs in the presence of EB1 and Lis1 . 381 initiation positions were determined ( from two experiments ) . Experiments were performed at 30 ° C .

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(G) Histogram of spatial initiation probabilities of DDB runs in the presence of EB1 and Lis1. 381 initiation positions were determined (from two experiments). Experiments were performed at 30°C.


( C ) Interaction analysis between hCAP - G and hCAP - H . Bacterial cell lysates co - expressing hCAP - G ( residues 1 - 478 , 554 - 900 ) and hCAP - H ( residues 394 - 515 ) , either wild type ( WT ; lanes 2 , 10 and 13 ) , 3Q ( F463Q , F469Q and F473Q ; lane 3 ) , 5Q ( F463Q , F469Q , F473Q , F501Q and Y503Q ; lane 4 ) , 3A ( F463A , F469A and F473A ; lane 5 ) , 5A ( F463A , F469A , F473A , F501A and Y503A ; lane 6 ) or 2A ( F501A and Y503A ; lane 11 ) , or a C - terminal deletion mutant ( 506 - 514 residues were deleted from 394 - 515 ; lane 14 ) were applied to Ni - NTA agarose resin , and the bound fraction was analyzed by SDS - PAGE . Alternatively , a cell lysate co - expressing mutant hCAP - G ( D647K ) and wild - type hCAP - H was examined ( lane 7 ) . The uninduced cell lysate was also used as a negative control ( lane 9 ) .

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(C) Interaction analysis between hCAP-G and hCAP-H. Bacterial cell lysates co-expressing hCAP-G (residues 1-478, 554-900) and hCAP-H (residues 394-515), either wild type (WT; lanes 2, 10 and 13), 3Q (F463Q, F469Q and F473Q; lane 3), 5Q (F463Q, F469Q, F473Q, F501Q and Y503Q; lane 4), 3A (F463A, F469A and F473A; lane 5), 5A (F463A, F469A, F473A, F501A and Y503A; lane 6) or 2A (F501A and Y503A; lane 11), or a C-terminal deletion mutant (506-514 residues were deleted from 394-515; lane 14) were applied to Ni-NTA agarose resin, and the bound fraction was analyzed by SDS-PAGE. Alternatively, a cell lysate co-expressing mutant hCAP-G (D647K) and wild-type hCAP-H was examined (lane 7). The uninduced cell lysate was also used as a negative control (lane 9).


( A ) Expression of condensin I subunits in insect cells . The wild - type ( WT ) or IV - 5Q mutant CAP - H subunit was co - expressed with the other four subunits ( GST - SMC4 , SMC2 , CAP - D2 and CAP - G ) in insect cells . Cell lysates were prepared and subjected to SDS - PAGE , followed by immunoblotting with a mixture of antibodies against SMC2 and SMC4 ( left panel ) or against CAP - D2 , - G , and - H ( right panel ) .

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(A) Expression of condensin I subunits in insect cells. The wild-type (WT) or IV-5Q mutant CAP-H subunit was co-expressed with the other four subunits (GST-SMC4, SMC2, CAP-D2 and CAP-G) in insect cells. Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting with a mixture of antibodies against SMC2 and SMC4 (left panel) or against CAP-D2, -G, and -H (right panel).


( B ) Purification of the WT and IV - 5Q mutant condensin I complexes . Protein samples purified through glutathione - affinity chromatography were subjected to SDS - PAGE and analyzed by CBB staining ( left panel ) or immunoblotting with a mixture of antibodies as described above ( middle and right panels ) .

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(B) Purification of the WT and IV-5Q mutant condensin I complexes. Protein samples purified through glutathione-affinity chromatography were subjected to SDS-PAGE and analyzed by CBB staining (left panel) or immunoblotting with a mixture of antibodies as described above (middle and right panels).


( C ) Add - back assay using the WT and mutant condensin I complexes . Xenopus extracts depleted of endogenous condensin complexes were supplemented with the purified complexes ( from top to bottom ; WT , IV - 5Q , ∆ G , ∆ G [ IV - 5Q ] ) . The supplemented extracts were then incubated with sperm nuclei to assemble mitotic chromosomes . The samples were fixed and labeled with an antibody against mSMC4 ( red ) . DNA was counterstained with DAPI ( blue ) . The data from a single representative experiment out of two repeats are shown . In the experiment shown here , multiple images were collected for condensin - depleted extracts supplemented with the WT ( n = 17 ) , IV - 5Q ( n = 22 ) , ∆ G ( n = 20 ) and ∆ G ( IV - 5Q ) ( n = 25 ) complexes . The scale bar represents 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Add-back assay using the WT and mutant condensin I complexes. Xenopus extracts depleted of endogenous condensin complexes were supplemented with the purified complexes (from top to bottom; WT, IV-5Q, ∆G, ∆G[IV-5Q]). The supplemented extracts were then incubated with sperm nuclei to assemble mitotic chromosomes. The samples were fixed and labeled with an antibody against mSMC4 (red). DNA was counterstained with DAPI (blue). The data from a single representative experiment out of two repeats are shown. In the experiment shown here, multiple images were collected for condensin-depleted extracts supplemented with the WT (n = 17), IV-5Q (n = 22), ∆G (n = 20) and ∆G(IV-5Q) (n = 25) complexes. The scale bar represents 10 μm.


( A ) Purification of hCAP - G and hCAP - G - H subcomplexes : wild type ( WT ) , CAP - G K60D / R848E double mutant ( K60D / R848E ) and CAP - G R168E mutant ( R168E ) . Purified protein samples were subjected to SDS - PAGE and analyzed by CBB staining .

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(A) Purification of hCAP-G and hCAP-G-H subcomplexes: wild type (WT), CAP-G K60D/R848E double mutant (K60D/R848E) and CAP-G R168E mutant (R168E). Purified protein samples were subjected to SDS-PAGE and analyzed by CBB staining.


( B ) Double - stranded DNA ( dsDNA ) and single - stranded DNA binding assay of the hCAP - G . 30 - bp dsDNA was incubated with no protein ( lanes 1 ) or increasing amounts of WT hCAP - G ( WT ; lanes 2 - 4 ) . 30 - mer ssDNA was incubated with no protein ( lanes 5 ) or increasing amounts of WT hCAP - G ( WT ; lanes 6 - 8 ) .

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

(B) Double-stranded DNA (dsDNA) and single-stranded DNA binding assay of the hCAP-G. 30-bp dsDNA was incubated with no protein (lanes 1) or increasing amounts of WT hCAP-G (WT; lanes 2-4). 30-mer ssDNA was incubated with no protein (lanes 5) or increasing amounts of WT hCAP-G (WT; lanes 6-8).


( C ) The dsDNA binding assay for the hCAP - G - H subcomplexes . 30 - bp dsDNA was incubated with no protein ( lanes 1 , 5 and 9 ) , increasing amounts of WT hCAP - G - H subcomplex ( WT ; lanes 2 - 4 ) , CAP - G K60D / R848E double mutant ( K60D / R848E ; lanes 6 - 8 ) or CAP - G R168E mutant ( R168E ; lanes 10 - 12 ) .

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(C) The dsDNA binding assay for the hCAP-G-H subcomplexes. 30-bp dsDNA was incubated with no protein (lanes 1, 5 and 9), increasing amounts of WT hCAP-G-H subcomplex (WT; lanes 2-4), CAP-G K60D/R848E double mutant (K60D/R848E; lanes 6-8) or CAP-G R168E mutant (R168E; lanes 10-12).


( D ) The ssDNA binding assay for the hCAP - G - H

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(D) The ssDNA binding assay for the hCAP-G-H subcomplexes


( f ) A549 - ACE2 or control A549 - Venus cells were pretreated with Tubercidin or vehicle ( DMSO ) 3h prior to infection with SARS - CoV - 2 at MOI 0 . 1 . After 24 hours , abundances of SARS - CoV - 2 nucleoprotein ( N ) , ACE2 , Venus and β - actin ( ACTB , loading control ) were visualized using Western blotting . Presented data is representative of 3 independent repeats .

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(f) A549-ACE2 or control A549-Venus cells were pretreated with Tubercidin or vehicle (DMSO) 3h prior to infection with SARS-CoV-2 at MOI 0.1. After 24 hours, abundances of SARS-CoV-2 nucleoprotein (N), ACE2, Venus and β-actin (ACTB, loading control) were visualized using Western blotting. Presented data is representative of 3 independent repeats.


( i ) NHBEs derived from three independent donors were pretreated for 6 h with indicated concentrations of DZNep or vehicle and infected with SARS - CoV - 2 for 24 h . Cells were fixed and the abundance of SARS - CoV - 2 N was quantified by immunofluorescent staining . Shown are vehicle - normalized integrated anti - N fluorescent intensity and cell confluence , error bars represent mean + / - sd of n = 3 donors .

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(i) NHBEs derived from three independent donors were pretreated for 6 h with indicated concentrations of DZNep or vehicle and infected with SARS-CoV-2 for 24 h. Cells were fixed and the abundance of SARS-CoV-2 N was quantified by immunofluorescent staining. Shown are vehicle-normalized integrated anti-N fluorescent intensity and cell confluence, error bars represent mean +/- sd of n=3 donors.


Mass spectrometry based analysis of cells treated with DZNep and infected with SARS - CoV - 2 and SARS - CoV . ( b ) Number of significantly up - or downregulated proteins in indicated comparisons according to ( a ) .

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Mass spectrometry based analysis of cells treated with DZNep and infected with SARS-CoV-2 and SARS-CoV. (b) Number of significantly up- or downregulated proteins in indicated comparisons according to (a).


Mass spectrometry based analysis of cells treated with DZNep and infected with SARS - CoV - 2 and SARS - CoV . ( c ) Donor - normalized LFQ abundances of viral nucleoprotein ( N ) and spike ( S ) in the indicated conditions . Error bars represent mean + / - sd of n = 4 donors ( NHBE ) or n = 4 independently infected A549 - ACE2 cultures . Statistics were calculated using Student ' s two - sided t - test as indicated .

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Mass spectrometry based analysis of cells treated with DZNep and infected with SARS-CoV-2 and SARS-CoV. (c) Donor-normalized LFQ abundances of viral nucleoprotein (N) and spike (S) in the indicated conditions. Error bars represent mean +/- sd of n=4 donors (NHBE) or n=4 independently infected A549-ACE2 cultures. Statistics were calculated using Student's two-sided t-test as indicated.


( d ) Expression patterns according to ( a ) of a selection of genes related to the disease - relevant pathways as annotated .

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(d) Expression patterns according to (a) of a selection of genes related to the disease-relevant pathways as annotated.


( e ) NHBEs ( 6 independent donors ) were pretreated for 6 h with 0 . 75 μM DZNep or vehicle and infected with SARS - CoV or SARS - CoV - 2 at MOI 3 . 24 h later , accumulation of IL - 6 and IP - 10 was measured in the supernatant by ELISA . Donor - wise IL - 6 and IP - 10 secretion , normalized to vehicle treated uninfected controls ( as further described in materials and methods ) is shown .

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(e) NHBEs (6 independent donors) were pretreated for 6 h with 0.75 μM DZNep or vehicle and infected with SARS-CoV or SARS-CoV-2 at MOI 3. 24 h later, accumulation of IL-6 and IP-10 was measured in the supernatant by ELISA. Donor-wise IL-6 and IP-10 secretion, normalized to vehicle treated uninfected controls (as further described in materials and methods) is shown.


C : FACS plots from a representative experiment showing gates for purification of the cell populations . Upper left : GFP + cells in the population of ESCs infected with pGIPZ - shRNA library lentiviruses . Upper right : Isolation of mesodermal ( SSEA1 - Flk1 + Cxcr4 - ) and endodermal ( SSEA1 - Flk1 - Cxcr4 + ) cells . Lower panels : Isolation of D20LS ( Lin - Sca1 + c - Kit - ) , D20LK ( Lin - Sca1 - c - Kit + ) , and D20LSK ( Lin - Sca1 + c - Kit + ) cells .

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C: FACS plots from a representative experiment showing gates for purification of the cell populations. Upper left: GFP+ cells in the population of ESCs infected with pGIPZ-shRNA library lentiviruses. Upper right: Isolation of mesodermal (SSEA1-Flk1+Cxcr4-) and endodermal (SSEA1-Flk1-Cxcr4+) cells. Lower panels: Isolation of D20LS (Lin-Sca1+c-Kit-), D20LK (Lin-Sca1-c-Kit+), and D20LSK (Lin-Sca1+c-Kit+) cells.


A : mRNA analysis of expression of endothelial ( Tie2 , CD31 , Cdh5 ) , epithelial ( Cdh1 ) , and early hematopoietic ( Scl , CD41 ) marker genes in the indicated sorted cell populations . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test . * p < 0 . 05 .

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A: mRNA analysis of expression of endothelial (Tie2, CD31, Cdh5), epithelial (Cdh1), and early hematopoietic (Scl, CD41) marker genes in the indicated sorted cell populations. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05.


B : Confirmation of the purity of sorted populations by qRT - PCR : c - Kit is highly expressed only in the c - Kit + ( D20LSK and D20LK ) populations , and Sca1 is highly expressed only in the in Sca1 + ( D20LSK and D20LS ) populations . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test . * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Confirmation of the purity of sorted populations by qRT-PCR: c-Kit is highly expressed only in the c-Kit+ (D20LSK and D20LK) populations, and Sca1 is highly expressed only in the in Sca1+ (D20LSK and D20LS) populations. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05.


C : qRT - PCR analysis of Gata1 , Gata2 , Lyz2 , and Scl expression in the sorted cell populations on day 20 . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test . * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: qRT-PCR analysis of Gata1, Gata2, Lyz2, and Scl expression in the sorted cell populations on day 20. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05.


D : FACS analysis of ESC - derived cells confirming that D20LS , D20LK , and D20LSK cell populations express the early hematopoietic marker CD41 and the pan - hematopoietic marker CD45 .

O O O O O O O O O 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: FACS analysis of ESC-derived cells confirming that D20LS, D20LK, and D20LSK cell populations express the early hematopoietic marker CD41 and the pan-hematopoietic marker CD45.


A : Clustering and enrichment analyses of shRNA reads in D6C ( endoderm ) , D6F ( HM / E ) , D20LS , D20LK , and D20LSK ( HSPC ) populations compared with ESCs . Specifically enriched / depleted shRNAs are grouped according to their target gene function ( I - X ) . Color coding bar is shown below .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Clustering and enrichment analyses of shRNA reads in D6C (endoderm), D6F (HM/E), D20LS, D20LK, and D20LSK (HSPC) populations compared with ESCs. Specifically enriched/depleted shRNAs are grouped according to their target gene function (I-X). Color coding bar is shown below.


B : Heat map of shRNA reads in D6C and D6F populations compared with ESCs . shRNAs specifically depleted in D6C and D6F are labeled as Groups XI and XII , respectively .

B: Heat map of shRNA reads in D6C and D6F populations compared with ESCs. shRNAs specifically depleted in D6C and D6F are labeled as Groups XI and XII, respectively.


E : Heat map showing depletion of shRNAs targeting Cdc and Cdk genes in all day 20 samples .

E: Heat map showing depletion of shRNAs targeting Cdc and Cdk genes in all day 20 samples.


A : The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Esrra , S100a8 , or Hcfc2 and induced to form EBs . The day 6 EB cells were analyzed by flow cytometry to measure the Mesoderm - Endothelium cells ( Flk1 + / Cxcr4 - ) and Endoderm cells ( Cxcr4 + / Flk1 - ) . The percentages of Mesoderm - Endothelium cells ( Flk1 + / Cxcr4 - ) were decreased on day 6 with shRNA knockdown of Group XII genes Esrra , S100a8 , and Hcfc2 compared to control as quantified by flow cytometric analysis . B : Knockdown of Esrra , S100a8 , and Hcfc2 showed that the ratios of percentage of Mesoderm - Endothelium population vs Endoderm population were decreased as measured by Flk1 + / Cxcr4 - vs . Flk1 - / Cxcr4 + on day 6 . The data were generated based on the quantification of flow cytometry analyses . Error bars represent standard deviation ( SD ) , n = 2 . Student ' s t - test , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Esrra, S100a8, or Hcfc2 and induced to form EBs. The day 6 EB cells were analyzed by flow cytometry to measure the Mesoderm-Endothelium cells (Flk1+/Cxcr4-) and Endoderm cells (Cxcr4+/Flk1-). The percentages of Mesoderm-Endothelium cells (Flk1+/Cxcr4-) were decreased on day 6 with shRNA knockdown of Group XII genes Esrra, S100a8, and Hcfc2 compared to control as quantified by flow cytometric analysis.B: Knockdown of Esrra, S100a8, and Hcfc2 showed that the ratios of percentage of Mesoderm-Endothelium population vs Endoderm population were decreased as measured by Flk1+/Cxcr4- vs. Flk1-/Cxcr4+ on day 6. The data were generated based on the quantification of flow cytometry analyses. Error bars represent standard deviation (SD), n=2. Student's t-test, *p<0.05.


C : The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Atp5g3 , Bub1b , Eci1 , or Dis3l and induced to form EBs . The day 6 EB cells were analyzed by flow cytometry to measure the Mesoderm - Endothelium cells ( Flk1 + / Cxcr4 - ) and Endoderm cells ( Cxcr4 + / Flk1 - ) . Flow cytometric analysis showed that Endoderm cells ( Cxcr4 + / Flk1 - ) were deceased while Mesoderm - Endothelium cells ( Flk1 + / Cxcr4 - ) were slightly increased in the cells with shRNA knockdown of Group XI genes Atp5g3 , Bub1b , Eci1 , and Dis3l compared to control . D : Knockdown of Atp5g3 , Bub1b , Eci1 , or Dis3l led to increase of Mesoderm - Endothelium / Endoderm population ratio as quantified based on the flow cytometry data . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Atp5g3, Bub1b, Eci1, or Dis3l and induced to form EBs. The day 6 EB cells were analyzed by flow cytometry to measure the Mesoderm-Endothelium cells (Flk1+/Cxcr4-) and Endoderm cells (Cxcr4+/Flk1-). Flow cytometric analysis showed that Endoderm cells (Cxcr4+/Flk1-) were deceased while Mesoderm-Endothelium cells (Flk1+/Cxcr4-) were slightly increased in the cells with shRNA knockdown of Group XI genes Atp5g3, Bub1b, Eci1, and Dis3l compared to control.D: Knockdown of Atp5g3, Bub1b, Eci1, or Dis3l led to increase of Mesoderm-Endothelium/Endoderm population ratio as quantified based on the flow cytometry data. Error bars represent standard deviation (SD), n=3. Student's t-test, *p<0.05.


E : The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Wbp5 , Rbm26 , Gdpd4 , and Nrxn1 and differentiated towards HSPCs . The differentiated cells on day 20 were analyzed by flow cytometry . Silencing of group IV genes including Wbp5 , Rbm26 , Gdpd4 , and Nrxn1 decreased overall LK , LS , and LSK population on day 20 . F : Quantification of LK + LSK + LS cell fractions with Wbp5 , Rbm26 , Gdpd4 , and Nrxn1 silencing based on flow cytometric analyses . Error bars represent standard deviation ( SD ) , n = 2 . Student ' s t - test , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Wbp5, Rbm26, Gdpd4, and Nrxn1 and differentiated towards HSPCs. The differentiated cells on day 20 were analyzed by flow cytometry. Silencing of group IV genes including Wbp5, Rbm26, Gdpd4, and Nrxn1 decreased overall LK, LS, and LSK population on day 20.F: Quantification of LK+LSK+LS cell fractions with Wbp5, Rbm26, Gdpd4, and Nrxn1 silencing based on flow cytometric analyses. Error bars represent standard deviation (SD), n=2. Student's t-test, *p<0.05.


G : The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Ap2a1 , Mettl22 , Lrsam1 , or Hal and differentiated towards HSPCs . The differentiated cells on day 20 were analyzed by flow cytometry . Flow cytometric analysis showed notable reduction of LSK population on day 20 with Group X genes Ap2a1 , Mettl22 , Lrsam1 , or Hal knockdown . H : Quantification of flow cytometric analyses showed knockdown of Ap2a1 , Mettl22 , Lrsam1 , and Hal significantly impaired generation of LSK population on day 20 in vitro compared to control . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Ap2a1, Mettl22, Lrsam1, or Hal and differentiated towards HSPCs. The differentiated cells on day 20 were analyzed by flow cytometry. Flow cytometric analysis showed notable reduction of LSK population on day 20 with Group X genes Ap2a1, Mettl22, Lrsam1, or Hal knockdown.H: Quantification of flow cytometric analyses showed knockdown of Ap2a1, Mettl22, Lrsam1, and Hal significantly impaired generation of LSK population on day 20 in vitro compared to control. Error bars represent standard deviation (SD), n=3. Student's t-test, *p<0.05.


B : Heat map showing bona fide HSC regulators in Group IV target genes .

B: Heat map showing bona fide HSC regulators in Group IV target genes.


C : Heat map showing the most depleted shRNAs in pair - wise comparisons between the D20LSK and D20LK populations .

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C: Heat map showing the most depleted shRNAs in pair-wise comparisons between the D20LSK and D20LK populations.


D : Validation of the involvement of Group X target genes Lrsam1 , Ap2a1 , Mettl22 , and Hal in zebrafish hematopoiesis by MO - mediated knockdown . Whole - mount in situ hybridization of Runx1 and c - Myb expression is shown at 26 hpf and 30hpf , respectively . Prdm16 knockdown serves as a positive control . Note that nonspecific staining for Runx1 and c - Myb was largely unaffected by targeted MO knockdown , whereas HSC markers Runx1 and c - Myb staining were notably reduced in discrete cells in the ventral region of the dorsal aorta ( arrows ) . Scale bar = 100 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Validation of the involvement of Group X target genes Lrsam1, Ap2a1, Mettl22, and Hal in zebrafish hematopoiesis by MO-mediated knockdown. Whole-mount in situ hybridization of Runx1 and c-Myb expression is shown at 26 hpf and 30hpf, respectively. Prdm16 knockdown serves as a positive control. Note that nonspecific staining for Runx1 and c-Myb was largely unaffected by targeted MO knockdown, whereas HSC markers Runx1 and c-Myb staining were notably reduced in discrete cells in the ventral region of the dorsal aorta (arrows). Scale bar = 100 µm.


A : Representative flow cytometric analysis showed that CD34 + cells were significantly decreased at 5dpi with knockdown of AP2A1 , METTL22 , LRSAM1 , or HAL . The human cord blood CD34 + cells were transduced with pLKO - shRNAs targeting human AP2A1 , METTL22 , LRSAM1 , or HAL and cultured for 5 days . Then , the flow cytometric analysis was performed to examine the percentage of CD34 + cells in culture . B : Quantification of CD34 + cells at 5dpi with knockdown of AP2A1 , METTL22 , LRSAM1 , and HALshowed significant decrease of CD34 + cells compared to control . Error bars represent standard deviation ( SD ) from duplicates , * p < 0 . 05 .

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

A: Representative flow cytometric analysis showed that CD34+cells were significantly decreased at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, or HAL. The human cord blood CD34+cells were transduced with pLKO-shRNAs targeting human AP2A1, METTL22, LRSAM1, or HAL and cultured for 5 days. Then, the flow cytometric analysis was performed to examine the percentage of CD34+cells in culture.B: Quantification of CD34+cells at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HALshowed significant decrease of CD34+ cells compared to control. Error bars represent standard deviation (SD) from duplicates, *p<0.05.


C : Flow cytometric analysis showed CD34 + / CD90 + cells were strikingly decreased at 5dpi with knockdown of AP2A1 , METTL22 , LRSAM1 , and HAL . D : Quantification of CD34 + CD90 + cells at 5dpi with knockdown of AP2A1 , METTL22 , LRSAM1 , and HALshowed significant decrease of CD34 + CD90 + cells compared to control . Error bars represent standard deviation ( SD ) from duplicates , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Flow cytometric analysis showed CD34+/CD90+cells were strikingly decreased at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HAL.D: Quantification of CD34+CD90+cells at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HALshowed significant decrease of CD34+CD90+cells compared to control. Error bars represent standard deviation (SD) from duplicates, *p<0.05.


The ERMIT assay relies on the signaling properties of IRE1 , one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER . The assay can be applied to heterodimerization or homodimerization events . Western blot showing the expression of AGR2 dimers ( D ) and monomers ( M ) in HEK293T subjected to DSP - mediated cross - linking and that were previously transfected with either a control siRNA ( siCTL ) or a siRNA targeting AGR2 ( siAGR2 ) for 24 h ( C ) Reduced or non - reduced samples were resolved by SDS - PAGE and immunoblotted using anti - AGR2 , anti - ERK1 antibodies ( for loading control ) .

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

The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. Western blot showing the expression of AGR2 dimers (D) and monomers (M) in HEK293T subjected to DSP-mediated cross-linking and that were previously transfected with either a control siRNA (siCTL) or a siRNA targeting AGR2 (siAGR2) for 24 h (C) Reduced or non-reduced samples were resolved by SDS-PAGE and immunoblotted using anti-AGR2, anti-ERK1 antibodies (for loading control).


The ERMIT assay relies on the signaling properties of IRE1 , one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER . The assay can be applied to heterodimerization or homodimerization events . Western blot showing the expression of AGR2 dimers ( D ) and monomers ( M ) in HEK293T subjected to DSP - mediated cross - linking and that were previously transfected with either a control siRNA ( siCTL ) or treated or not with tunicamycin ( Tun ) prior to cross - linking ( D ) . Reduced or non - reduced samples were resolved by SDS - PAGE and immunoblotted using anti - AGR2 anti - calnexin ( CANX ) antibodies ( for loading control ) .

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

The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. Western blot showing the expression of AGR2 dimers (D) and monomers (M) in HEK293T subjected to DSP-mediated cross-linking and that were previously transfected with either a control siRNA (siCTL) or treated or not with tunicamycin (Tun) prior to cross-linking (D). Reduced or non-reduced samples were resolved by SDS-PAGE and immunoblotted using anti-AGR2 anti-calnexin (CANX) antibodies (for loading control).


The ERMIT assay relies on the signaling properties of IRE1 , one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER . The assay can be applied to heterodimerization or homodimerization events . F ) AGR2 dimerization was monitored with ERMIT ( wild - type ( WT ) ) . The luminescence signal was abrogated when using the kinase dead ( KD ) constructs and by the constructs exhibiting mutations in the dimerization domain ( E60A or C81S or double mutant ( DM ) ) . The graph represents average signal normalized on reporter protein expression ± SD ( n = 5 ; * * : WT / KD ( p = 0 . 002 ) , E60 / WT ( p = 0 . 0034 ) , E60 / KD ( p = 0 . 001 ) , C81 / KD ( p = 0 . 004 ) , DM / WT ( p = 0 . 0021 ) and DM / KD ( p = 0 . 0017 ) , respectively ; * : C81 / WT ( p = 0 . 0098 ) ) .

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

The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. F) AGR2 dimerization was monitored with ERMIT (wild-type (WT)). The luminescence signal was abrogated when using the kinase dead (KD) constructs and by the constructs exhibiting mutations in the dimerization domain (E60A or C81S or double mutant (DM)). The graph represents average signal normalized on reporter protein expression ± SD (n=5; **: WT/KD (p=0.002), E60/WT (p=0.0034), E60/KD (p=0.001), C81/KD (p=0.004), DM/WT (p=0.0021) and DM/KD (p=0.0017), respectively; *: C81/WT (p=0.0098)).


The ERMIT assay relies on the signaling properties of IRE1 , one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER . The assay can be applied to heterodimerization or homodimerization events . G ) Cells expressing various bait and prey constructs and the XBP1 splicing reporter or the XBP1 splicing reporter alone were exposed to increasing concentrations of DTT . The ERMIT signals obtained with both baits were then normalized to that of XBP1s to obtain results independent of the activation of endogenous IRE1 by the use of chemical ER stressors ( n = 4 ) .

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

The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. G) Cells expressing various bait and prey constructs and the XBP1 splicing reporter or the XBP1 splicing reporter alone were exposed to increasing concentrations of DTT. The ERMIT signals obtained with both baits were then normalized to that of XBP1s to obtain results independent of the activation of endogenous IRE1 by the use of chemical ER stressors (n = 4).


B ) Volcano plot representing the statistical analysis of the data from the screen , where the y axis represents statistical criteria and the x axis the fold change in luminescence units .

B) Volcano plot representing the statistical analysis of the data from the screen, where the y axis represents statistical criteria and the x axis the fold change in luminescence units.


A ) Co - immunoprecipitation of AGR2 with TMED2 under basal and tunicamycin induced ER stress in intestinal epithelial cells treated in situ . * indicates Immunoglobulin heavy and light chains .

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

A) Co-immunoprecipitation of AGR2 with TMED2 under basal and tunicamycin induced ER stress in intestinal epithelial cells treated in situ. *indicates Immunoglobulin heavy and light chains.


C ) Changes in AGR2 dimer and monomer ratio in TMED2 overexpressing cells . DSP - stabilized AGR2 was analyzed under non - reducing ( top blot ) or reducing conditions ( bottom blot ) . D = dimeric , M = monomeric AGR2 .

O O O O O O O O O O O O O O O O O O O O O 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) Changes in AGR2 dimer and monomer ratio in TMED2 overexpressing cells. DSP-stabilized AGR2 was analyzed under non-reducing (top blot) or reducing conditions (bottom blot). D=dimeric, M=monomeric AGR2.


E ) : Co - immunoprecipitation of AGR2 wild - type ( WT , left panel ) or AGR2 AA mutant ( right panel ) with TMED2 in HEK293T cells . Immunoprecipitation was performed with mouse anti - Flag antibody to pull - down the ectopic protein tag .

O O O O O O O O O O O O O O O O O O O O O O O O O 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): Co-immunoprecipitation of AGR2 wild-type (WT, left panel) or AGR2 AA mutant (right panel) with TMED2 in HEK293T cells. Immunoprecipitation was performed with mouse anti-Flag antibody to pull-down the ectopic protein tag.


F ) Western blot analysis ( left panel ) and quantification ( right panel ) of AGR2 intracellular expression upon TMED2 overexpression . Data are representative of 4 independent experiments . Tubulin ( TUB ) was used as a loading control .

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

F) Western blot analysis (left panel) and quantification (right panel) of AGR2 intracellular expression upon TMED2 overexpression. Data are representative of 4 independent experiments. Tubulin (TUB) was used as a loading control.


G ) HEK293T cells were transfected with control ( ev ) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT ( n = 3 ) .

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

G) HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT (n=3).


H ) Western blot analysis ( left panel ) and quantification ( right panel ) of AGR2 intracellular expression in total cell lysate ( TCL ) upon TMED2 silencing . Data are representative of 4 independent experiments . Tubulin ( TUB ) was used as a loading control .

H) Western blot analysis (left panel) and quantification (right panel) of AGR2 intracellular expression in total cell lysate (TCL) upon TMED2 silencing. Data are representative of 4 independent experiments. Tubulin (TUB) was used as a loading control.


I ) HEK293T cells were silencing for TMED2 and then used in the ERMIT assay with AGR2 WT ( n = 3 ) .

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

I) HEK293T cells were silencing for TMED2 and then used in the ERMIT assay with AGR2 WT (n=3).


A ) The ERMIT - based validation of the complex formation between AGR2 and LYPD3 or CD59 , but not OS9 . The monomeric AGR2 E60A was used as bait , while LYPD3 , CD59 and OS9 were used as prays ( n = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O 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) The ERMIT-based validation of the complex formation between AGR2 and LYPD3 or CD59, but not OS9. The monomeric AGR2 E60A was used as bait, while LYPD3, CD59 and OS9 were used as prays (n=6).


B - C ) Expression and subcellular localization of CD59 - GFP WT and C94S .

B-C) Expression and subcellular localization of CD59-GFP WT and C94S.


D and E ) Co - immunoprecipitation of CD59 - GFP WT or CD59 C94S mutant with AGR2 WT ( D ) or AGR2 AA mutant ( E ) in HEK293T cells .

O O O O O O O O O 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 and E) Co-immunoprecipitation of CD59-GFP WT or CD59 C94S mutant with AGR2 WT (D) or AGR2 AA mutant (E) in HEK293T cells.


F - I ) FACS analysis of CD59 WT ( F and G ) or CD59 C94S ( H and I ) total ( F and H ) and membrane ( G and I ) expression in HEK293T cells silenced for AGR2 ( siAGR2 ) and TMED2 ( siTMED2 ) when compared to control ( siGL2 ) cells ( F and H ) or silenced for TMED2 ( siTMED2 ) and overexpressing AGR2 WT or AGR2 AA mutant ( G and I ) . Data are representative of three to four independent experiments . Membranous CD59 expression was detected with anti - GFP APC . ns : non - statistically significant ; for F ( * ) : p = 0 . 0385 , ( * * * ) : p = 0 . 0003 ( left panel ) and p = 0 . 0002 ( right panel ) , respectively ; for G ( * ) : p = 0 . 0334 , ( * * ) p = 0 . 0036 and ( * * * ) p = 0 . 0003 ; for H ( * ) : p = 0 . 0171 and ( * * ) : p = 0 . 0084 ; and for I ( * * ) p = 0 . 0086 ( left panel ) and p = 0 . 0076 ( right panel ) , respectively .

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F-I) FACS analysis of CD59 WT (F and G) or CD59 C94S (H and I) total (F and H) and membrane (G and I) expression in HEK293T cells silenced for AGR2 (siAGR2) and TMED2 (siTMED2) when compared to control (siGL2) cells (F and H) or silenced for TMED2 (siTMED2) and overexpressing AGR2 WT or AGR2 AA mutant (G and I). Data are representative of three to four independent experiments. Membranous CD59 expression was detected with anti-GFP APC. ns: non-statistically significant; for F (*): p=0.0385, (***): p=0.0003 (left panel) and p=0.0002 (right panel), respectively; for G (*): p=0.0334, (**) p=0.0036 and (***) p=0.0003; for H (*): p=0.0171 and (**): p=0.0084; and for I (**) p=0.0086 (left panel) and p=0.0076 (right panel), respectively.


A ) Formation of GFP - LC3 autophagic puncta in TMED2 overexpressing HEK239T cells as monitored using confocal microscopy ( right panel ) . DAPI was used for nuclear staining visualization . Percentage of GFP - LC3 puncta in control ( CTL ) and TMED2 overexpressing HEK293T cells as quantified from three independent experiments by counting 240 GFP - LC3 positive cells for each condition ( left panel ) . ( * * ) : p = 0 . 0032 .

A) Formation of GFP-LC3 autophagic puncta in TMED2 overexpressing HEK239T cells as monitored using confocal microscopy (right panel). DAPI was used for nuclear staining visualization. Percentage of GFP-LC3 puncta in control (CTL) and TMED2 overexpressing HEK293T cells as quantified from three independent experiments by counting 240 GFP-LC3 positive cells for each condition (left panel). (**): p=0.0032.


B ) Western blot detection ( left panel ) and quantification ( right panel ) of LC3 level in control and TMED2 overexpressing HEK293T cells treated or not with 50 µM chloroquine for 2 hours . Actin ( ACT ) served as a loading control . ( * ) : p = 0 . 0498 .

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B) Western blot detection (left panel) and quantification (right panel) of LC3 level in control and TMED2 overexpressing HEK293T cells treated or not with 50 µM chloroquine for 2 hours. Actin (ACT) served as a loading control. (*): p=0.0498.


C ) Western blot analysis of AGR2 expression in control and TMED2 overexpressing HEK293T cells upon autophagy inhibition with 20 µM and 10 µM chloroquine treatment . Actin ( ACT ) served as a loading control .

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C) Western blot analysis of AGR2 expression in control and TMED2 overexpressing HEK293T cells upon autophagy inhibition with 20 µM and 10 µM chloroquine treatment. Actin (ACT) served as a loading control.


D ) HEK293T cells were transfected with control ( ev ) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT the presence of gradual amounts of chloroquine .

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D) HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT the presence of gradual amounts of chloroquine.


E ) Western blot analysis of secreted AGR2 upon TMED2 overexpression . eAGR2 , extracellular AGR2 ; iAGE2 , intracellular AGR2 .

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E) Western blot analysis of secreted AGR2 upon TMED2 overexpression. eAGR2, extracellular AGR2; iAGE2, intracellular AGR2.


F ) Representative pictures of extracellular vesicles ( EVs ) heterogeneity purified from conditioned media of HEK293T cells transfected with control ( ev ) or TMED2 plasmid and analyzed by cryo - electron microscopy . Right upper panel : western blot analysis of CD63 in total medium ( left ) and in the purified extracellular vesicles fraction purified from media conditioned by control and TMED2 overexpressing cells ( middle ) and western blot analysis of AGR2 levels ( right ) in extracellular vesicles enriched from culture media conditioned by control and TMED2 overexpressing cells .

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F) Representative pictures of extracellular vesicles (EVs) heterogeneity purified from conditioned media of HEK293T cells transfected with control (ev) or TMED2 plasmid and analyzed by cryo-electron microscopy. Right upper panel: western blot analysis of CD63 in total medium (left) and in the purified extracellular vesicles fraction purified from media conditioned by control and TMED2 overexpressing cells (middle) and western blot analysis of AGR2 levels (right) in extracellular vesicles enriched from culture media conditioned by control and TMED2 overexpressing cells.


G ) Western blot analysis of secreted AGR2 upon TMED2 silencing eAGR2 , extracellular AGR2 ; iAGE2 , intracellular AGR2 . H ) Quantification of extracellular AGR2 level ( eAGR2 ) in cells silenced for TMED2 ( siTMED2 ) , compared to control ( siGL2 ) . Data are representative of three independent experiments and are presented as extracellular - to - intracellular AGR2 ratio . ( * * ) : p = 0 . 0029 .

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G) Western blot analysis of secreted AGR2 upon TMED2 silencing eAGR2, extracellular AGR2; iAGE2, intracellular AGR2. H) Quantification of extracellular AGR2 level (eAGR2) in cells silenced for TMED2 (siTMED2), compared to control (siGL2). Data are representative of three independent experiments and are presented as extracellular-to-intracellular AGR2 ratio. (**): p=0.0029.


I ) Secretion of AGR2 wild - type ( wt ) , E60A and ∆ 45 mutant as determined by Western blot . eAGR2 , extracellular AGR2 ; iAGE2 , intracellular AGR2 .

I) Secretion of AGR2 wild-type (wt), E60A and ∆45 mutant as determined by Western blot. eAGR2, extracellular AGR2; iAGE2, intracellular AGR2.


A ) Representative histological H & E staining of sections of the proximal colon and ileum of WT and TMED2 hypomorph mice .

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A) Representative histological H&E staining of sections of the proximal colon and ileum of WT and TMED2 hypomorph mice.


B ) Immunohistological analysis of AGR2 and MUC2 expression in the proximal colon of WT and TMED2 hypomorph mice .

B) Immunohistological analysis of AGR2 and MUC2 expression in the proximal colon of WT and TMED2 hypomorph mice.


C ) Semi - quantitative analysis of AGR2 and MUC2 expression in the proximal colon of WT ( blank bars ) and TMED2 hypomorph mice ( black bars ) ( n = 6 ) .

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C) Semi-quantitative analysis of AGR2 and MUC2 expression in the proximal colon of WT (blank bars) and TMED2 hypomorph mice (black bars) (n=6).


D ) Freshly isolated PBMCs were placed in Boyden chambers towards media conditioned by cells overexpressing AGR2 WT , E60A , ∆ 45 or AGR2 AA mutants and incubated for 24 h . Migrating cells were then characterized and quantified by flow cytometry using FCS / SSC parameters or CD14 monocyte marker . ( * * ) : p = 0 . 0016 .

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D) Freshly isolated PBMCs were placed in Boyden chambers towards media conditioned by cells overexpressing AGR2 WT, E60A, ∆45 or AGR2 AA mutants and incubated for 24 h. Migrating cells were then characterized and quantified by flow cytometry using FCS/SSC parameters or CD14 monocyte marker. (**): p=0.0016.


E and F ) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells overexpressing TMED2 and either AGR2 WT ( E ) or AGR2 AA mutant ( F ) and incubated for 24 h . Migrating cells were then characterized and quantified For E ( * ) : p = 0 . 0461 ( AGR2WT / TMED2 ) and 0 . 018 ( AGR2WT / TMED2 + blocking Ab ) respectively ; ( * * ) : p = 0 . 0053 and ( * * * ) : p = 0 . 0048 ; for F ( * * * ) : p = 0 . 0007 .

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E and F) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells overexpressing TMED2 and either AGR2 WT (E) or AGR2 AA mutant (F) and incubated for 24 h. Migrating cells were then characterized and quantified For E (*): p=0.0461 (AGR2WT/TMED2) and 0.018 (AGR2WT/TMED2 + blocking Ab) respectively; (**): p=0.0053 and (***): p=0.0048; for F (***): p=0.0007.


G ) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells silenced for TMED2 and incubated for 24 h . Migrating cells were then characterized and quantified ( * ) : p = 0 . 0149 .

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G) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells silenced for TMED2 and incubated for 24 h. Migrating cells were then characterized and quantified (*): p=0.0149.


H ) Freshly isolated PBMC were placed in Boyden chambers towards decreased doses of recombinant AGR2 and incubated for 24 h . CCL2 cytokine was used as positive control for monocyte migration . ns : non - statistically significant , ( * ) : p = 0 . 0171 , ( * * ) : p = 0 . 0084 , ( * * * ) : p = 0 . 0003 .

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H) Freshly isolated PBMC were placed in Boyden chambers towards decreased doses of recombinant AGR2 and incubated for 24 h. CCL2 cytokine was used as positive control for monocyte migration. ns: non-statistically significant, (*): p=0.0171, (**): p=0.0084, (***): p=0.0003.


I ) Impact of AGR2 blocking antibodies on AGR2 - mediated monocytes migration was tested using Boyden chambers as described above . The concentrations of recombinant human AGR2 was of 200 ng / ml and decreasing amounts of antibodies were used from 20 µg to 1 µg . The non - relevant antibody ( Isotype ) was used at the maximal dose of 20 µg . Data are representative of three independent experiments . ns : non - statistically significant , ( * ) : p = 0 . 0165 ( Ab = 20μg / ml ) , 0 . 0223 ( Ab = 10μg / ml ) and 0 . 0493 ( Ab = 5μg / ml ) , respectively .

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I) Impact of AGR2 blocking antibodies on AGR2-mediated monocytes migration was tested using Boyden chambers as described above. The concentrations of recombinant human AGR2 was of 200 ng/ml and decreasing amounts of antibodies were used from 20 µg to 1 µg. The non-relevant antibody (Isotype) was used at the maximal dose of 20 µg. Data are representative of three independent experiments. ns: non-statistically significant, (*): p=0.0165 (Ab=20μg/ml), 0.0223 (Ab=10μg/ml) and 0.0493 (Ab=5μg/ml), respectively.


A ) Selective enrichment of AGR2 modulators in colonic Crohn ' s disease ( CD ) . Percentage of tested genes with altered expression in colonic biopsies from patients with Ulcerative Colitis ( UC , n = 8 ) and colonic CD ( CC , n = 15 ) and in ileal biopsies from patients with ileo - colonic CD ( IC , n = 9 ) .

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A) Selective enrichment of AGR2 modulators in colonic Crohn's disease (CD). Percentage of tested genes with altered expression in colonic biopsies from patients with Ulcerative Colitis (UC, n=8) and colonic CD (CC, n=15) and in ileal biopsies from patients with ileo-colonic CD (IC, n=9).


B ) Hierarchical clustering of the AGR2 modulator genes significantly deregulated in non - inflamed colonic mucosa from patients with Crohn ' s Disease ( CD ) versus healthy controls ( C ) ( dChip software t - Test )

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B) Hierarchical clustering of the AGR2 modulator genes significantly deregulated in non-inflamed colonic mucosa from patients with Crohn's Disease (CD) versus healthy controls (C) (dChip software t-Test)


C ) TMED2 mRNA expression in healthy controls ( CT ) , colonic Crohn ' s disease ( CC ) and ulcerative colitis ( UC ) .

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C) TMED2 mRNA expression in healthy controls (CT), colonic Crohn's disease (CC) and ulcerative colitis (UC).


D ) Representative immunohistological analysis of TMED2 in non - inflamed colonic biopsies from healthy controls and patients with active CD .

D) Representative immunohistological analysis of TMED2 in non-inflamed colonic biopsies from healthy controls and patients with active CD.


E ) Representative immunohistological analysis of CD163 ( scavenger receptor present in macrophages ) , AGR2 , and TMED2 in non - inflamed colonic biopsies from healthy controls and patients with active or quiescent CD .

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E) Representative immunohistological analysis of CD163 (scavenger receptor present in macrophages), AGR2, and TMED2 in non-inflamed colonic biopsies from healthy controls and patients with active or quiescent CD.


F ) Semi - quantitative analysis of CD163 , AGR2 , and TMED2 immunostaining in colonic mucosa from healthy controls ( C ) and patients with quiescent ( Q ) or active ( A ) CD . Three random sections from each patient of the four groups were scored . Final IHC scores ( AxB range to 0 - 9 ) combine the percentage of positively labelled cells . A : % of IHC positive labeled cells - 0 ( 0 % ) , 1 ( < 30 % ) , 2 ( 30 to 60 % ) , 3 ( > 60 % ) ) and the intensity of the reaction product . B : 0 ( no reaction ) , 1 ( weak ) , 2 ( mild ) , 3 ( strong ) in most of the examined fields . All values are mean ± S . E . M .

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F) Semi-quantitative analysis of CD163, AGR2, and TMED2 immunostaining in colonic mucosa from healthy controls (C) and patients with quiescent (Q) or active (A) CD. Three random sections from each patient of the four groups were scored. Final IHC scores (AxB range to 0-9) combine the percentage of positively labelled cells. A: % of IHC positive labeled cells - 0 (0%), 1 (<30%), 2 (30 to 60%), 3 (>60%)) and the intensity of the reaction product. B: 0 (no reaction), 1 (weak), 2 (mild), 3 (strong) in most of the examined fields. All values are mean ± S.E.M.


B . Analyses of Hog1 phosphorylation by immunoblotting with anti - phospho - p38 ( Hog1 - P ) and anti - Hog1 ( total Hog1 ) antibodies . Cells of the indicated genotypes were stimulated with the indicated concentrations of NaCl for the indicated time . Strains used are TM257 , KT207 and KY594 - 1 .

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B. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Cells of the indicated genotypes were stimulated with the indicated concentrations of NaCl for the indicated time. Strains used are TM257, KT207 and KY594-1.


C . Analyses of Hog1 phosphorylation by Phos - tag band - shift assay . Yeast strain KY594 - 1 was stimulated with the indicated concentrations of NaCl for 5 min . The percentages of phosphorylated Hog1 [ Hog1 - P ( % ) ] were calculated as explained in Materials and Methods , and are shown beneath the panel . Data information : Representative results from three independent experiments .

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C. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strain KY594-1 was stimulated with the indicated concentrations of NaCl for 5 min. The percentages of phosphorylated Hog1 [Hog1-P (%)] were calculated as explained in Materials and Methods, and are shown beneath the panel. Data information: Representative results from three independent experiments.


D . Analyses of Hog1 phosphorylation by immunoblotting with anti - phospho - p38 ( Hog1 - P ) and anti - Hog1 ( total Hog1 ) antibodies . Yeast strain KT219 was transformed with the indicated STE11 mutant gene carried by a single - copy plasmid that is expressed from the STE11 promoter : vec , vector ; WT , wild - type ; DDD , S281D / S285D / T286D . Cells were incubated with ( + ) or without ( - ) 1 M NaCl for 5 min .

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D. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Yeast strain KT219 was transformed with the indicated STE11 mutant gene carried by a single-copy plasmid that is expressed from the STE11 promoter: vec, vector; WT, wild-type; DDD, S281D/S285D/T286D. Cells were incubated with (+) or without (-) 1 M NaCl for 5 min.


E - H . Analyses of Hog1 phosphorylation by Phos - tag band - shift assay . Yeast strains ( E ) KY603 - 3 ; ( F ) TM142 ; ( G ) TM257 ; and ( H ) FP54 were stimulated with the indicated concentrations of NaCl for 5 min . Data information : Representative results from three independent experiments .

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E-H. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strains (E) KY603-3; (F) TM142; (G) TM257; and (H) FP54 were stimulated with the indicated concentrations of NaCl for 5 min. Data information: Representative results from three independent experiments.


I . Comparison of the NaCl dose - responses of Hog1 activation by various strains . Phos - tag band - shift assays were independently repeated three times , and average values were plotted . Data information : ( I ) Error bars are SEM ( n = 3 ) .

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I. Comparison of the NaCl dose-responses of Hog1 activation by various strains. Phos-tag band-shift assays were independently repeated three times, and average values were plotted. Data information: (I) Error bars are SEM (n=3).


A - C . Immunoblot analyses of Hog1 phosphorylation . Yeast strains ( A ) KT235 ; ( B ) KT209 ; and ( C ) KT234 were stimulated with the indicated concentrations of NaCl for 10 min , and phosphorylated Hog1 ( Hog1 - P ) and total Hog1 in cell lysates were detected by immunoblotting . The relevant genotypes of the strains are indicated in the top row of each panel , and are schematically shown in the diagrams at left . The second row from the top indicates the genes carried by a single - copy plasmid that are expressed from their own promoters . vec , vector ; WT , wild - type .

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A-C. Immunoblot analyses of Hog1 phosphorylation. Yeast strains (A) KT235; (B) KT209; and (C) KT234 were stimulated with the indicated concentrations of NaCl for 10 min, and phosphorylated Hog1 (Hog1-P) and total Hog1 in cell lysates were detected by immunoblotting. The relevant genotypes of the strains are indicated in the top row of each panel, and are schematically shown in the diagrams at left. The second row from the top indicates the genes carried by a single-copy plasmid that are expressed from their own promoters. vec, vector; WT, wild-type.


D . Phos - tag band - shift analyses of Hog1 phosphorylation . The yeast strain KT234 carrying the single - copy expression plasmid YCplac22I ' - Pbs2 S514D / T518D was stimulated with the indicated concentrations of NaCl for 5 min .

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D. Phos-tag band-shift analyses of Hog1 phosphorylation. The yeast strain KT234 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was stimulated with the indicated concentrations of NaCl for 5 min.


E . Comparison of the NaCl dose - responses of Hog1 activation by constitutively - active Ste11 - Q301P and constitutively - active Pbs2 - DD . Phos - tag band - shift assays were independently repeated three times , and the average values were plotted . Data information : ( E ) Error bars are SEM ( n = 3 ) .

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E. Comparison of the NaCl dose-responses of Hog1 activation by constitutively-active Ste11-Q301P and constitutively-active Pbs2-DD. Phos-tag band-shift assays were independently repeated three times, and the average values were plotted. Data information: (E) Error bars are SEM (n=3).


C . Immunoblot analyses of Hog1 phosphorylation . The yeast strain KT235 was transformed with pRS416 - FLAG - Hog1 ( WT ) or its indicated deletion derivatives . FLAG - Hog1 was immunoprecipitated ( IP ) , and immunoblotted ( IB ) with anti - phospho - p38 ( for Hog1 - P ; upper panel ) or anti - FLAG ( for total FLAG - Hog1 ; lower panel )

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C. Immunoblot analyses of Hog1 phosphorylation. The yeast strain KT235 was transformed with pRS416-FLAG-Hog1 (WT) or its indicated deletion derivatives. FLAG-Hog1 was immunoprecipitated (IP), and immunoblotted (IB) with anti-phospho-p38 (for Hog1-P; upper panel) or anti-FLAG (for total FLAG-Hog1; lower panel)


D - G . Phos - tag band - shift assay of Hog1 phosphorylation . Yeast strain ( D - E ) KT235 or ( F - G ) KT290 carrying the single - copy expression plasmid YCplac22I ' - Pbs2 S514D / T518D was transformed with either pRS416 - Hog1 ( WT ) or its indicated mutant derivatives , and was treated with the indicated concentrations of NaCl for 5 min . ( D ) and ( F ) show typical results , and ( E ) and ( G ) summarizes the averages of three independent experiments . Data information : ( E , G Error bars are SEM ( n = 3 ) .

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D-G. Phos-tag band-shift assay of Hog1 phosphorylation. Yeast strain (D-E) KT235 or (F-G) KT290 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was transformed with either pRS416-Hog1 (WT) or its indicated mutant derivatives, and was treated with the indicated concentrations of NaCl for 5 min. (D) and (F) show typical results, and (E) and (G) summarizes the averages of three independent experiments. Data information: (E, G Error bars are SEM (n=3).


H . Phos - tag band - shift assay of Hog1 phosphorylation . The yeast strain FP4 was transformed with the single - copy expression plasmid pRS416 - Hog1 ( WT ) or pRS416 - Hog1 - ΔL16 , and was treated with the indicated concentrations of NaCl for 5 min . The averages of three independent experiments are shown . Data information : Error bars are SEM ( n = 3 ) .

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H. Phos-tag band-shift assay of Hog1 phosphorylation. The yeast strain FP4 was transformed with the single-copy expression plasmid pRS416-Hog1 (WT) or pRS416-Hog1-ΔL16, and was treated with the indicated concentrations of NaCl for 5 min. The averages of three independent experiments are shown. Data information: Error bars are SEM (n=3).


A . Phos - tag band - shift analyses of osmostress - induced Hog1 phosphorylation . The yeast strain FP4 ( hog1Δ ) was transformed with the single - copy expression plasmid pRS416 - Hog1 - ΔL16 , and was stimulated with the indicated concentrations of NaCl for 5 min . B . Averages of three independent experiments from A were plotted . Results for Hog1 - WT ( from Fig 1I ) are included for comparison . Data information : Representative results from three independent experiments . Error bars are SEM ( n = 3 ) .

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A. Phos-tag band-shift analyses of osmostress-induced Hog1 phosphorylation. The yeast strain FP4 (hog1Δ) was transformed with the single-copy expression plasmid pRS416-Hog1-ΔL16, and was stimulated with the indicated concentrations of NaCl for 5 min. B. Averages of three independent experiments from A were plotted. Results for Hog1-WT (from Fig 1I) are included for comparison. Data information: Representative results from three independent experiments. Error bars are SEM (n=3).


C - D . the yeast strain KT259 ( ste11Δ hog1Δ ) was used . Data information : Representative results from three independent experiments . Error bars are SEM ( n = 3 ) .

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C-D. the yeast strain KT259 (ste11Δ hog1Δ) was used. Data information: Representative results from three independent experiments. Error bars are SEM (n=3).


E - F . the yeast strain KY523 ( ssk2 / 22Δ hog1Δ ) was used . Data information : Representative results from three independent experiments . Error bars are SEM ( n = 3 ) .

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E-F. the yeast strain KY523 (ssk2/22Δ hog1Δ) was used. Data information: Representative results from three independent experiments. Error bars are SEM (n=3).


Detection of Pbs2 phosphorylation at S514 The yeast strains KT003 ( pbs2Δ ) , KT005 ( ste11Δ pbs2Δ ) , TM280 ( ssk2 / 22Δ pbs2Δ ) and KT043 ( ste11Δ ssk2 / 22Δ pbs2Δ ) were transformed with YCplac22I ' - Pbs2 - HA , and were treated with the indicated concentrations of NaCl for 5 min . Pbs2 - HA was immunoprecipitated from cell extract , and phosphorylated Pbs2 was analyzed by ( A ) Phos - tag band - shift assay In ( A ) , the positions of Pbs2 - HA phosphorylated ( S514 - P ) and unphosphorylated ( S514 - OH ) at S514 are indicated .

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Detection of Pbs2 phosphorylation at S514 The yeast strains KT003 (pbs2Δ), KT005 (ste11Δ pbs2Δ), TM280 (ssk2/22Δ pbs2Δ) and KT043 (ste11Δ ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extract, and phosphorylated Pbs2 was analyzed by (A) Phos-tag band-shift assay In (A), the positions of Pbs2-HA phosphorylated (S514-P) and unphosphorylated (S514-OH) at S514 are indicated.


Detection of Pbs2 phosphorylation at T518 . The yeast strains KT003 ( pbs2Δ ) , KT005 ( ste11Δ pbs2Δ ) , TM280 ( ssk2 / 22Δ pbs2Δ ) and KT043 ( ste11Δ ssk2 / 22Δ pbs2Δ ) were transformed with YCplac22I ' - Pbs2 - HA , and were treated with the indicated concentrations of NaCl for 5 min . Pbs2 - HA was immunoprecipitated from cell extract , and phosphorylated Pbs2 was analyzed by ( B ) anti - phospho - T518 immunoblotting .

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Detection of Pbs2 phosphorylation at T518. The yeast strains KT003 (pbs2Δ), KT005 (ste11Δ pbs2Δ), TM280 (ssk2/22Δ pbs2Δ) and KT043 (ste11Δ ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extract, and phosphorylated Pbs2 was analyzed by (B) anti-phospho-T518 immunoblotting.


C - F . NaCl dose - response analyses of Pbs2 phosphorylation . ( C - D ) KT005 ( ste11Δ pbs2Δ ) and ( E - F ) TM280 ( ssk2 / 22Δ pbs2Δ ) were transformed with YCplac22I ' - Pbs2 - HA , and were treated with the indicated concentrations of NaCl for 5 min . ( C and E ) S514 phosphorylation was analyzed using the Phos - tag band - shift assay . PC ; Positive Control . ( D and F ) T518 phosphorylation was analyzed using anti - phospho - T518 immunoblotting . G - H . Average values of three independent experiments from ( C - D ) and ( E - F ) , respectively , were plotted . AU , arbitrary unit . Data information : ( C - F ) Representative results from three independent experiments . ( G - H ) Error bars are SEM ( n = 3 ) .

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C-F. NaCl dose-response analyses of Pbs2 phosphorylation. (C-D) KT005 (ste11Δ pbs2Δ) and (E-F) TM280 (ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. (C and E) S514 phosphorylation was analyzed using the Phos-tag band-shift assay. PC; Positive Control. (D and F) T518 phosphorylation was analyzed using anti-phospho-T518 immunoblotting. G-H. Average values of three independent experiments from (C-D) and (E-F), respectively, were plotted. AU, arbitrary unit. Data information: (C-F) Representative results from three independent experiments. (G-H) Error bars are SEM (n=3).


I . Detection of di - phosphorylated Pbs2 . KT005 ( ste11Δ pbs2Δ ) transformed with YCplac22I ' - Pbs2 - HA was treated with the indicated concentrations of NaCl for 5 min . Pbs2 - HA was immunoprecipitated from cell extracts , and subjected to Phos - tag SDS - PAGE . Blots of these gels were probed with ( upper panel ) anti - phospho - T518 or ( lower panel ) anti - HA . J . Same as in ( I ) , except that the yeast strain KT003 ( pbs2Δ ) was used .

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

I. Detection of di-phosphorylated Pbs2. KT005 (ste11Δ pbs2Δ) transformed with YCplac22I'-Pbs2-HA was treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extracts, and subjected to Phos-tag SDS-PAGE. Blots of these gels were probed with (upper panel) anti-phospho-T518 or (lower panel) anti-HA. J. Same as in (I), except that the yeast strain KT003 (pbs2Δ) was used.


B . Phos - tag band - shift analyses of osmostress - induced Hog1 phosphorylation . KT005 ( ste11Δ pbs2Δ ) was transformed with YCplac22I ' - Pbs2 - HA ( WT ) or its indicated mutant derivatives . Cells were stimulated with ( + ) or without ( - ) 0 . 6 M NaCl for 5 min .

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B. Phos-tag band-shift analyses of osmostress-induced Hog1 phosphorylation. KT005 (ste11Δ pbs2Δ) was transformed with YCplac22I'-Pbs2 -HA (WT) or its indicated mutant derivatives. Cells were stimulated with (+) or without (-) 0.6 M NaCl for 5 min.


C - F . Phos - tag band - shift analyses of Hog1 phosphorylation . KT291 ( ste11Δ pbs2Δ hog1Δ ) was transformed with ( C and D ) pRS416 - Hog1 or ( E and F ) pRS416 - Hog1 - ΔL16 , together with ( C and E ) YCplac22I ' - Pbs2 - S514A - HA or ( D and F ) YCplac22I ' - Pbs2 - T518A - HA . Cells were stimulated with the indicated concentrations of NaCl for 5 min . G - H . Average values of three independent experiments from ( C and E ) and ( D and F ) , respectively , are plotted . Data information : ( C - F ) Representative results from three independent experiments . ( G - H ) Error bars are SEM ( n = 3 ) .

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C-F. Phos-tag band-shift analyses of Hog1 phosphorylation. KT291 (ste11Δ pbs2Δ hog1Δ) was transformed with (C and D) pRS416-Hog1 or (E and F) pRS416-Hog1-ΔL16, together with (C and E) YCplac22I'-Pbs2-S514A-HA or (D and F) YCplac22I'-Pbs2-T518A-HA. Cells were stimulated with the indicated concentrations of NaCl for 5 min. G-H. Average values of three independent experiments from (C and E) and (D and F), respectively, are plotted. Data information: (C-F) Representative results from three independent experiments. (G-H) Error bars are SEM (n=3).


A Phos - tag band - shift assay of Hog1 phosphorylation . KT235 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - Q301P ) was transformed with pRS416 - Hog1 ( WT ) or its indicated mutant derivatives . Cell extracts were prepared from fresh cultures without applying osmostress . For each HOG1 mutant plasmid , three independent cultures were assayed . Data information : Error bars are SEM ( n = 3 ) .

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A Phos-tag band-shift assay of Hog1 phosphorylation. KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P) was transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives. Cell extracts were prepared from fresh cultures without applying osmostress. For each HOG1 mutant plasmid, three independent cultures were assayed. Data information: Error bars are SEM (n=3).


B . the yeast strains KT260 ( ssk2 / 22Δ ste11Δ hog1Δ ) , KT235 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - Q301P ) , and KT248 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ pbs2Δ ) carrying YCplac22I ' - Pbs2 - DD , were used . Data information : Error bars are SEM ( n = 3 ) .

B. the yeast strains KT260 (ssk2/22Δ ste11Δ hog1Δ), KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P), and KT248 (ΔS/O/H/M ssk2/22Δ hog1Δ pbs2Δ) carrying YCplac22I'-Pbs2-DD, were used. Data information: Error bars are SEM (n=3).


C . KT250 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - WT ) and KT235 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ STE11 - Q301P ) were transformed with pRS416 - Hog1 ( WT ) or pRS416 - Hog1 - N149H D162G ( N149H D162G ) together with pRS414 - 8xCRE - lacZ . Expression of the Hog1 reporter gene 8xCRE - lacZ in the absence of osmostress was assayed . Data information : ( C ) Error bars are SEM ( n = 4 ) .

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C. KT250 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-WT) and KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P) were transformed with pRS416-Hog1 (WT) or pRS416-Hog1-N149H D162G (N149H D162G) together with pRS414-8xCRE-lacZ. Expression of the Hog1 reporter gene 8xCRE-lacZ in the absence of osmostress was assayed. Data information: (C) Error bars are SEM (n=4).


D . KT248 ( ΔS / O / H / M ssk2 / 22Δ hog1Δ pbs2Δ ) was transformed with pRS416 - Hog1 ( WT ) or its indicated mutant derivatives together with pRS414 - 8xCRE - lacZ . Expression of the Hog1 reporter gene 8xCRE - lacZ in the absence of osmostress was assayed . Data information : Error bars are SEM ( n = 3 ) .

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D. KT248 (ΔS/O/H/M ssk2/22Δ hog1Δ pbs2Δ) was transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives together with pRS414-8xCRE-lacZ. Expression of the Hog1 reporter gene 8xCRE-lacZ in the absence of osmostress was assayed. Data information: Error bars are SEM (n=3).


E - F . Phos - tag band - shift assay of Hog1 phosphorylation . Yeast strains of the indicated genotypes ( shown below the graph ) were transformed with pRS416 - Hog1 ( WT ) or its indicated mutant derivatives ( shown inside the graph ) . Strain used : ( E ) FP4 , KT259 , KT260 , KT292 , and KY523 ; and ( F ) KY523 , KT293 , KT294 , and KT295 . N / H , N149H ; D / G , D162G . Data information : Error bars are SEM ( n = 3 ) .

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E-F. Phos-tag band-shift assay of Hog1 phosphorylation. Yeast strains of the indicated genotypes (shown below the graph) were transformed with pRS416-Hog1 (WT) or its indicated mutant derivatives (shown inside the graph). Strain used: (E) FP4, KT259, KT260, KT292, and KY523; and (F) KY523, KT293, KT294, and KT295. N/H, N149H; D/G, D162G. Data information: Error bars are SEM (n=3).


A . KT299 ( MATa hkr1Δ msb2Δ ssk2 / 22Δ ) was exposed to the indicated concentrations of α - factor for 15 min in the absence of osmostress . Phosphorylation of Fus3 and Kss1 was detected by immunoblotting .

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A. KT299 (MATa hkr1Δ msb2Δ ssk2/22Δ) was exposed to the indicated concentrations of α-factor for 15 min in the absence of osmostress. Phosphorylation of Fus3 and Kss1 was detected by immunoblotting.


B . KT299 ( MATa ssk2 / 22Δ hkr1Δ msb2Δ ) was exposed to the indicated concentrations of α - factor for 15 min in the presence or absence of 0 . 8 M NaCl , and Hog1 phosphorylation was determined using the Phos - tag band - shift assay . Data information : ( B ) Representative results from three independent experiments .

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B. KT299 (MATa ssk2/22Δ hkr1Δ msb2Δ) was exposed to the indicated concentrations of α-factor for 15 min in the presence or absence of 0.8 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Data information: (B) Representative results from three independent experiments.


C . Average values of four independent experiments were plotted . Data information : Error bars are SEM ( C ) n =

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C. Average values of four independent experiments were plotted. Data information: Error bars are SEM (C) n=4


D . HM06 - 1 ( MATa ΔS / O / H / M ssk2 / 22Δ ) was exposed to the indicated concentrations ( log scale ) of α - factor for 15 min in the presence or absence of 1 . 0 M NaCl , and Hog1 phosphorylation was determined using the Phos - tag band - shift assay . Average values of three or more independent experiments were plotted . Data information : Error bars are SEM : ( D ) , n = 3 or

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D. HM06-1 (MATa ΔS/O/H/M ssk2/22Δ) was exposed to the indicated concentrations (log scale) of α-factor for 15 min in the presence or absence of 1.0 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average values of three or more independent experiments were plotted. Data information: Error bars are SEM: (D), n=3 or more


E . KT306 ( MATa hkr1Δ msb2Δ ssk2 / 22Δ hog1Δ ) was transformed with pRS416 - Hog1 ( WT ) or pRS416 - Hog1 - N149H D162G ( N / H D / G ) , and was exposed to 10 μM α - factor for the indicated time in the absence of osmostress , and Hog1 phosphorylation was determined using the Phos - tag band - shift assay . Average of three independent experiments are plotted . Data information : Error bars are SEM ( E ) n = 3 .

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E. KT306 (MATa hkr1Δ msb2Δ ssk2/22Δ hog1Δ) was transformed with pRS416-Hog1 (WT) or pRS416-Hog1-N149H D162G (N/H D/G), and was exposed to 10 μM α-factor for the indicated time in the absence of osmostress, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average of three independent experiments are plotted. Data information: Error bars are SEM (E) n=3.


G . Typical FRET ( YFP / CFP ratio ) images showing p38 activation . HeLa cells carrying the p38 reporter PerKy - p38 ( Tomida et al . , 2015 ) were stably transfected with an expression vector for the indicated p38α mutant proteins . FRET analysis was performed as described in Materials and Methods . H . Distribution of p38 activity in individual cells from sets ( a ) - ( d ) in ( G ) . Data information : ( G ) Scale bars : 20 μm . ( H ) Statistics , Student ' s two - tailed t - test .

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G. Typical FRET (YFP/CFP ratio) images showing p38 activation. HeLa cells carrying the p38 reporter PerKy-p38 (Tomida et al., 2015) were stably transfected with an expression vector for the indicated p38α mutant proteins. FRET analysis was performed as described in Materials and Methods. H. Distribution of p38 activity in individual cells from sets (a) - (d) in (G). Data information: (G) Scale bars: 20 μm. (H) Statistics, Student's two-tailed t-test.


A . An example of the time - course experiments for the osmostress - induced Hog1 phosphorylation . The yeast strain KY603 - 3 ( ΔS / O / H / M ssk2 / 22Δ STE11 - Q301P ) was stimulated with 1 . 0 M NaCl for the indicated times , and the percentage of phosphorylated Hog1 ( Hog1 - P ) was determined using a Phos - tag band - shift assay .

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A. An example of the time-course experiments for the osmostress-induced Hog1 phosphorylation. The yeast strain KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P) was stimulated with 1.0 M NaCl for the indicated times, and the percentage of phosphorylated Hog1 (Hog1-P) was determined using a Phos-tag band-shift assay.


Compilations of the time - courses of osmostress - induced Hog1 activation in ( B ) KY603 - 3 ( ΔS / O / H / M ssk2 / 22Δ STE11 - Q301P ) , For clarity , time - course curves are shown in two panels for lower and higher ranges of NaCl concentrations . Data information : For each data point , n = 1 or more .

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Compilations of the time-courses of osmostress-induced Hog1 activation in (B) KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P), For clarity, time-course curves are shown in two panels for lower and higher ranges of NaCl concentrations. Data information: For each data point, n=1 or more.


Compilations of the time - courses of osmostress - induced Hog1 activation in ( C ) TM257 ( ssk2 / 22Δ ) , and ( D ) TM142 ( wild - type ) . For clarity , time - course curves are shown in two panels for lower and higher ranges of NaCl concentrations . The color chart below ( D ) indicates the concentrations of NaCl used . Data information : For each data point , n = 1 or more .

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Compilations of the time-courses of osmostress-induced Hog1 activation in (C) TM257 (ssk2/22Δ), and (D) TM142 (wild-type). For clarity, time-course curves are shown in two panels for lower and higher ranges of NaCl concentrations. The color chart below (D) indicates the concentrations of NaCl used. Data information: For each data point, n=1 or more.


E . Effects of Hog1 kinase activity and osmostress - induced glycerol accumulation on the time - courses of Hog1 activation . Cells were stimulated with 0 . 4 M NaCl ( left panel ) or 1 . 6 M NaCl ( right panel ) for the indicated times , and the percentage of phosphorylated Hog1 was determined using Phos - tag band - shift assay . Strains used were : TM142 ( WT ) , KT254 ( gpd1Δ gpd2Δ stl1Δ ) , and TM232 ( hog1Δ ) carrying pRS416 - HOG1 - K52S K53N . K / S K / N , K52S K53N ; gpd1 / 2 , gpd1Δ gpd2Δ . Data information : ( E ) Error bars are SEM ( n = 3 or more ) .

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E. Effects of Hog1 kinase activity and osmostress-induced glycerol accumulation on the time-courses of Hog1 activation. Cells were stimulated with 0.4 M NaCl (left panel) or 1.6 M NaCl (right panel) for the indicated times, and the percentage of phosphorylated Hog1 was determined using Phos-tag band-shift assay. Strains used were: TM142 (WT), KT254 (gpd1Δ gpd2Δ stl1Δ), and TM232 (hog1Δ) carrying pRS416-HOG1-K52S K53N. K/S K/N, K52S K53N; gpd1/2, gpd1Δ gpd2Δ. Data information: (E) Error bars are SEM (n=3 or more).


RT ‐ PCR performed on cDNA from RNA isolated from splenic B cells derived from Tam ‐ treated mb1 ‐ CreERT2 ( left lane ) or mb1 ‐ CreERT2 ; Sykfl / flmice ( right lane ) ; the Syk ‐ and Hprt ‐ specific amplification products were identified by agarose gel electrophoresis with Hprt used as an endogenous loading control .

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RT‐PCR performed on cDNA from RNA isolated from splenic B cells derived from Tam‐treated mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/flmice (right lane); the Syk‐ and Hprt‐specific amplification products were identified by agarose gel electrophoresis with Hprt used as an endogenous loading control.