A , B Plates with several layers that are not closely appressed . C - E Large multilayer plates having the size of human metaphase chromatids [ ~ 0 . 6 µm diameter ] ( C ) ; the inset schematically shows the perpendicular orientation of chromatin layers with respect to the chromatid axes proposed in the thin - plate model In other slices ( D , E ) , the multilayer structures shown in ( C ) are more compact and the individual layers are not visible as separate elements . Data information : Scale bars : 50 nm .

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A,B Plates with several layers that are not closely appressed. C-E Large multilayer plates having the size of human metaphase chromatids [~0.6 µm diameter ] (C); the inset schematically shows the perpendicular orientation of chromatin layers with respect to the chromatid axes proposed in the thin-plate model In other slices (D,E), the multilayer structures shown in (C) are more compact and the individual layers are not visible as separate elements. Data information: Scale bars: 50 nm.


A - C Slices showing lateral association between two plates and the corresponding 3D segmentations . D . Edge - to - edge interactions form cylindrical structures . Data information : Scale bars : 50 nm .

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A-C Slices showing lateral association between two plates and the corresponding 3D segmentations. D. Edge-to-edge interactions form cylindrical structures. Data information: Scale bars: 50 nm.


A - C The cation concentrations used are indicated for each experiment ; AU , arbitrary units .

A-C The cation concentrations used are indicated for each experiment; AU, arbitrary units.


A . Spike - binding antibodies in serum . Binding by antibodies of classes M , G , and A is detected with secondary antibodies conjugated to different fluorochromes , and the results are shown on the left , middle , and right plots , respectively . Binding ( geometric mean fluorescence intensity - GMFI - of corresponding secondary antibody ) to TE cells expressing SARS - CoV - 2 spike protein is plotted on the vertical axis , and binding to untransfected cells on the horizontal axis . Each point represents a value from one donor . Samples from donors with no known exposure to SARS - CoV - 2 antigens ( n = 86 ) are plotted with blue circles , recently infected donors ( n = 34 ) with red circles , and vaccinated donors ( n = 14 ) with green diamonds . Red circles with black triangles correspond to donors from whose B cells monoclonal antibodies were isolated ( n = 5 , P values are derived from a two - way analysis of variance followed by Tukey ' s test to compare the specific binding , i . e . , ( binding to spike - expressing cells ) / ( binding to untransfected cells ) between conditions ( convalescent or vaccinated against unexposed ) within each antibody class . The experiment was independently repeated three times , and results shown come from the third replicate .

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A. Spike-binding antibodies in serum. Binding by antibodies of classes M, G, and A is detected with secondary antibodies conjugated to different fluorochromes, and the results are shown on the left, middle, and right plots, respectively. Binding (geometric mean fluorescence intensity - GMFI- of corresponding secondary antibody) to TE cells expressing SARS-CoV-2 spike protein is plotted on the vertical axis, and binding to untransfected cells on the horizontal axis. Each point represents a value from one donor. Samples from donors with no known exposure to SARS-CoV-2 antigens (n = 86) are plotted with blue circles, recently infected donors (n = 34) with red circles, and vaccinated donors (n = 14) with green diamonds. Red circles with black triangles correspond to donors from whose B cells monoclonal antibodies were isolated (n = 5, P values are derived from a two-way analysis of variance followed by Tukey's test to compare the specific binding, i.e., (binding to spike-expressing cells)/(binding to untransfected cells) between conditions (convalescent or vaccinated against unexposed) within each antibody class. The experiment was independently repeated three times, and results shown come from the third replicate.


B . Change in antibody binding following vaccination against SARS - CoV - 2 . Binding of antibodies from sera assessed in the experiment shown in ( A ) , is shown for two samples of serum from each of 7 donors . Data are plotted as in ( A ) with binding to spike - expressing cells on the vertical axis , and to untransfected cells on the horizontal , using blue symbols for samples from before vaccination , and red symbols for samples from 15 - 29 days after the first vaccination ( but before any second vaccination ) . Black lines connect points corresponding to pre / post pairs of samples from each donor . Left plot shows results for IgM and right plot for IgG . C . Comparison of post - vaccination increase in binding to untransfected cells between IgG and IgM . The increase in binding to untransfected TE 0 cells between pre - and post - vaccination samples is shown on the left with blue symbols for IgG and on the right with black symbols for IgM . The horizontal line at y = 0 is the expected result when no increased binding is observed . P value is derived from a two - tailed , Wilcoxon matched pairs signed rank test .

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B. Change in antibody binding following vaccination against SARS-CoV-2. Binding of antibodies from sera assessed in the experiment shown in (A), is shown for two samples of serum from each of 7 donors. Data are plotted as in (A) with binding to spike-expressing cells on the vertical axis, and to untransfected cells on the horizontal, using blue symbols for samples from before vaccination, and red symbols for samples from 15-29 days after the first vaccination (but before any second vaccination). Black lines connect points corresponding to pre/post pairs of samples from each donor. Left plot shows results for IgM and right plot for IgG. C. Comparison of post-vaccination increase in binding to untransfected cells between IgG and IgM. The increase in binding to untransfected TE 0 cells between pre- and post-vaccination samples is shown on the left with blue symbols for IgG and on the right with black symbols for IgM. The horizontal line at y=0 is the expected result when no increased binding is observed. P value is derived from a two-tailed, Wilcoxon matched pairs signed rank test.


D . Comparison of spike - specific IgM , as measured by flow cytometry or ELISA . The left two " FC " are the results of flow cytometry , and the left verical axis shows the ratio of serum IgM binding to spike - expressing cells , vs binding to non - expressing control cells , as plottted in A . The right two columns show spike - RBD - specific IgM as measured by an IgM - capture ELISA , and the right vertical axis shows the optical densities ( OD ) . P values are calculated by two - way analysis of variance , followed by Sidak ' s multiple comparisons test . The entire experiment was repeated three times , and the data shown are derived from the third replicate .

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D. Comparison of spike-specific IgM, as measured by flow cytometry or ELISA. The left two "FC" are the results of flow cytometry, and the left verical axis shows the ratio of serum IgM binding to spike-expressing cells, vs binding to non-expressing control cells, as plottted in A. The right two columns show spike-RBD-specific IgM as measured by an IgM-capture ELISA, and the right vertical axis shows the optical densities (OD). P values are calculated by two-way analysis of variance, followed by Sidak's multiple comparisons test. The entire experiment was repeated three times, and the data shown are derived from the third replicate.


Phenotypes of blood B cells . B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent cells expressing spike - mCherry for 3 hours , then retrieved and labeled with fluorescent antibodies and measured by flow cytometry . ( A ) UMAP algorithm ( 5000 randomly selected cells / sample ) was used to depict the major B cell subsets clustered according to marker expression .

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Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. (A) UMAP algorithm (5000 randomly selected cells/sample) was used to depict the major B cell subsets clustered according to marker expression.


Phenotypes of blood B cells . B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent cells expressing spike - mCherry for 3 hours , then retrieved and labeled with fluorescent antibodies and measured by flow cytometry . FlowSOM - based B cell subpopulations are overlaid as a color dimension , and the colors of the clusters are shown on the left of heatmap ( B ) . B . Heatmap showing mean population expression levels of markers used for UMAP visualization and FlowSOM - clustering . Colors shown in the legend on the left are also used in the UMAP representation in ( A ) .

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Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. FlowSOM-based B cell subpopulations are overlaid as a color dimension, and the colors of the clusters are shown on the left of heatmap (B). B. Heatmap showing mean population expression levels of markers used for UMAP visualization and FlowSOM-clustering. Colors shown in the legend on the left are also used in the UMAP representation in (A).


Phenotypes of blood B cells . B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent cells expressing spike - mCherry for 3 hours , then retrieved and labeled with fluorescent antibodies and measured by flow cytometry . C . Gating strategy to define CD69 - high , mCherry - high , spike - capturing B cells ( i . e . , membrane antigen capture activated cells , MACAC , red box ) . Dot plot shows CD69 and mCherry fluorescence for B cells from one of three donors gated by forward and side scatter , and negative for the dye used to mark the antigen donor cells .

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Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. C. Gating strategy to define CD69-high, mCherry-high, spike-capturing B cells (i.e., membrane antigen capture activated cells, MACAC, red box). Dot plot shows CD69 and mCherry fluorescence for B cells from one of three donors gated by forward and side scatter, and negative for the dye used to mark the antigen donor cells.


Phenotypes of blood B cells . B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent cells expressing spike - mCherry for 3 hours , then retrieved and labeled with fluorescent antibodies and measured by flow cytometry . D . Relative fractions of different B cell subpopulations , from three donors , as shown in A - B , in all B cells compared to within MACAC B cells . P values are based on two - tailed t - tests between the groups . Correlation coefficients ( r ) were calculated from the z - statistic of the Wilcoxon - Mann - Whitney test . A black horizontal line represents the median . Boxplots represent the interquartile range ( IQR ) . Whiskers extend to the farthest data point within a maximum of 1 . 5 × IQR . Every point represents one donor .

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Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. D. Relative fractions of different B cell subpopulations, from three donors, as shown in A-B, in all B cells compared to within MACAC B cells. P values are based on two-tailed t-tests between the groups. Correlation coefficients (r) were calculated from the z-statistic of the Wilcoxon-Mann-Whitney test. A black horizontal line represents the median. Boxplots represent the interquartile range (IQR). Whiskers extend to the farthest data point within a maximum of 1.5× IQR. Every point represents one donor.


E . Gating strategy in MACAC sorting . Single cells are selected based on scatter , antigen - expressing TE spike - mCherry cells excluded on Cell Trace Violet label , and the spike - capturing ( mCherry - high ) , activated ( CD69 - high ) B cells ( population labeled " MACAC " in red on the right - most plot ) are sorted . The middle plot shows B cells that did not adhere to the TE - spike - cherry antigen - expressing cells ( putatively antigen - irrelevant ) , and the right plot those that did ( putatively antigen - recognizing ) . Plots show data from one of five convalescent donors Cells in the MACAC gate were singly distributed into wells of 384 - well plates and cultured for 9 days with IL - 21 and CD40L , and then the single well culture supernatants were screened for anti - spike antibody binding and virus neutralization as described below . F . Results of single well supernatant screening for antibody binding to SARS - CoV - 2 spike protein . Results from 3266 wells from 5 donors are shown for IgM ( left ) , IgG ( middle ) , and IgA ( right ) .

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E. Gating strategy in MACAC sorting. Single cells are selected based on scatter, antigen-expressing TE spike-mCherry cells excluded on Cell Trace Violet label, and the spike-capturing (mCherry-high), activated (CD69-high) B cells (population labeled "MACAC" in red on the right-most plot) are sorted. The middle plot shows B cells that did not adhere to the TE-spike-cherry antigen-expressing cells (putatively antigen-irrelevant), and the right plot those that did (putatively antigen-recognizing). Plots show data from one of five convalescent donors Cells in the MACAC gate were singly distributed into wells of 384-well plates and cultured for 9 days with IL-21 and CD40L, and then the single well culture supernatants were screened for anti-spike antibody binding and virus neutralization as described below. F. Results of single well supernatant screening for antibody binding to SARS-CoV-2 spike protein. Results from 3266 wells from 5 donors are shown for IgM (left), IgG (middle), and IgA (right).


A . Example of neutralization of chimeric VSV * ∆ G - S ∆ 21 by donor - derived IgG , IgM and IgA antibodies . 100 pfu VSV * ∆ G - S ∆ 21 was mixed with antibody at specified concentrations for 1 hour , then added to Vero cells . After 24 hours , the cells were imaged , and infected wells identified by GFP expression , here pseudocolored with GFP - bright shown in black against a white background . Virus neutralization is manifested as reduction in GFP . Images shown here come from wells infected with virus alone ( left column ) , virus pre - incubated with the moderately neutralizing IgG 1I12 ( second and third columns ) , the potent neutralizing IgM 2J17 ( fourth and fitfh columns ) , or with the moderately potent IgA 1B17 ( sixth and seventh columns ) . Each antibody was tested in the range of concentrations shown to the left of the images , from 10000 down to 0 . 1 ng / ml .

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A. Example of neutralization of chimeric VSV*∆G-S∆21 by donor-derived IgG, IgM and IgA antibodies. 100 pfu VSV*∆G-S∆21 was mixed with antibody at specified concentrations for 1 hour, then added to Vero cells. After 24 hours, the cells were imaged, and infected wells identified by GFP expression, here pseudocolored with GFP-bright shown in black against a white background. Virus neutralization is manifested as reduction in GFP. Images shown here come from wells infected with virus alone (left column), virus pre-incubated with the moderately neutralizing IgG 1I12 (second and third columns), the potent neutralizing IgM 2J17 (fourth and fitfh columns), or with the moderately potent IgA 1B17 (sixth and seventh columns). Each antibody was tested in the range of concentrations shown to the left of the images, from 10000 down to 0.1 ng/ml.


B . Neutralization curves from each of the cloned antibodies using plaque reduction assay of VSV * ∆ G - S ∆ 21 . The horizontal axis shows the serial dilution of the antibodies . The vertical axis shows , for each well exposed to antibody , the ratio of infected cell plaque count to the corresponding value from control wells without antibody . Each point is the mean of 9 values pooled from three independent experiments each with triplicate wells . Error bars indicate standard error . Antibodies tested are listed below the figure and include 3 IgM ( black lines and symbols ) , 11 IgG ( blue lines and symbols ) and 6 IgA ( red lines and symbols ) .

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B. Neutralization curves from each of the cloned antibodies using plaque reduction assay of VSV*∆G-S∆21. The horizontal axis shows the serial dilution of the antibodies. The vertical axis shows, for each well exposed to antibody, the ratio of infected cell plaque count to the corresponding value from control wells without antibody. Each point is the mean of 9 values pooled from three independent experiments each with triplicate wells. Error bars indicate standard error. Antibodies tested are listed below the figure and include 3 IgM (black lines and symbols), 11 IgG (blue lines and symbols) and 6 IgA (red lines and symbols).


C . Quantification of results from ( B ) . Area under the curve is plotted for every antibody and compared between different antibody classes by one - way ANOVA followed by Tukey ' s test ( mean from three biological replicates and SEM ) . Area under the curve was chosen as a measure of neutralization capacity , rather than ND50 ( as used in subsequent figures ) , because some antibodies included in this figure exhibited no neutralization at the concentrations tested , precluding the calculation of an ND50 .

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C. Quantification of results from (B). Area under the curve is plotted for every antibody and compared between different antibody classes by one-way ANOVA followed by Tukey's test (mean from three biological replicates and SEM). Area under the curve was chosen as a measure of neutralization capacity, rather than ND50 (as used in subsequent figures), because some antibodies included in this figure exhibited no neutralization at the concentrations tested, precluding the calculation of an ND50.


E . Flow cytometric determination of antibody - dependent complement deposition on cells . Cells expressing SARS - CoV - 2 spike protein and untransfected control cells were incubated with various concentrations of antibody in the presence of fresh human serum ( from a SARS - CoV - 2 unexposed donor ) . Activation of the complement cascade was measured by flow cytometric assessment of complement component C3b deposition on the surface of the cells . Results for the 3 IgM , ( black ) , 11 IgG , ( blue ) , and 6 IgA antibodies ( red ) are shown . P values were calculated by two - way analysis of variance , followed by Tukey ' s test .

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E. Flow cytometric determination of antibody-dependent complement deposition on cells. Cells expressing SARS-CoV-2 spike protein and untransfected control cells were incubated with various concentrations of antibody in the presence of fresh human serum (from a SARS-CoV-2 unexposed donor). Activation of the complement cascade was measured by flow cytometric assessment of complement component C3b deposition on the surface of the cells. Results for the 3 IgM, (black), 11 IgG, (blue), and 6 IgA antibodies (red) are shown. P values were calculated by two-way analysis of variance, followed by Tukey's test.


F . Influence of complement on virus neutralization . A plaque reduction neutralization assay like that shown in Fig 3A - C was conducted with IgM 2J17 in the presence of either fresh , complement - sufficient human serum , or heat inactivated serum . The concentration of 2J17 is shown on the horizontal axis . The vertical axis shows , for each well exposed to antibody , the ratio of infected cell plaque count to the corresponding value from control wells with serum ( either fresh or inactivated ) but without antibody . The points plotted with filled red symbols correspond to the condition with heat inactivated serum , and the open blue symbols results with fresh serum . Each symbol corresponds to the mean of nine wells pooled from three independent experiments , each with triplicate wells . Error bars correspond to standard error . P value was calculated using a paired , two - tailed t - test .

F. Influence of complement on virus neutralization. A plaque reduction neutralization assay like that shown in Fig 3A-C was conducted with IgM 2J17 in the presence of either fresh, complement-sufficient human serum, or heat inactivated serum. The concentration of 2J17 is shown on the horizontal axis. The vertical axis shows, for each well exposed to antibody, the ratio of infected cell plaque count to the corresponding value from control wells with serum (either fresh or inactivated) but without antibody. The points plotted with filled red symbols correspond to the condition with heat inactivated serum, and the open blue symbols results with fresh serum. Each symbol corresponds to the mean of nine wells pooled from three independent experiments, each with triplicate wells. Error bars correspond to standard error. P value was calculated using a paired, two-tailed t-test.


A . The effect of competition on concentration versus binding curves of three spike - specific , class - switched antibodies . Antibodies were expressed either as IgM , as originally isolated ( black symbols and lines ) , or artificially switched to IgG1 ( blue lines and symbols ) . For each antibody , identified by name at the top of each plot , the binding of the antibody alone ( " alone " , open symbols ) , or the competitive binding of the antibody in a mixture of IgM and IgG1 ( " compet . " , filled symbols ) is shown . In the competition scenario , each of the two classes of the antibody were added together at the concentration shown on the horizontal axis . The vertical axis shows the ratio of geometric mean fluorescence of human IgM , or IgG on spike - expressing cells divided by the corresponding signal on spike - non - expressing control cells . Each point shows the mean of 3 values from three independent experiments . Error bars show standard error . P value was calculated by comparing the area under the curve of each antibody class in the " alone " condition with the binding of the " competition " condition by paired , two - tailed t - test .

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A. The effect of competition on concentration versus binding curves of three spike-specific, class-switched antibodies. Antibodies were expressed either as IgM, as originally isolated (black symbols and lines), or artificially switched to IgG1 (blue lines and symbols). For each antibody, identified by name at the top of each plot, the binding of the antibody alone ("alone", open symbols), or the competitive binding of the antibody in a mixture of IgM and IgG1 ("compet.", filled symbols) is shown. In the competition scenario, each of the two classes of the antibody were added together at the concentration shown on the horizontal axis. The vertical axis shows the ratio of geometric mean fluorescence of human IgM, or IgG on spike-expressing cells divided by the corresponding signal on spike-non-expressing control cells. Each point shows the mean of 3 values from three independent experiments. Error bars show standard error. P value was calculated by comparing the area under the curve of each antibody class in the "alone" condition with the binding of the "competition" condition by paired, two-tailed t-test.


B . Virus neutralization by antibodies expressed as IgM or IgG1 . A plaque reduction neutralization assay as described in Fig 3A - B was used to measure neutralization by the three , donor - derived IgM ( black lines and open symbols ) and their artificially class - switched IgG1 equivalents ( blue lines and filled symbols ) . Horizontal axis shows the antibody concentration and the vertical axis shows the level of infection expressed as percentage GFP expression compared to control wells with no antibody added . Each point shows the mean of nine replicate values pooled from three independent experiments , each with triplicate wells . Error bars show standard error . C . Comparison of virus neutralization by antibodies expressed as IgM or IgG1 shown in B . Area under the curve was calculated from each of the three independently performed experiments . P was calculated by paired , two - tailed t - test .

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B. Virus neutralization by antibodies expressed as IgM or IgG1. A plaque reduction neutralization assay as described in Fig 3A-B was used to measure neutralization by the three, donor-derived IgM (black lines and open symbols) and their artificially class-switched IgG1 equivalents (blue lines and filled symbols). Horizontal axis shows the antibody concentration and the vertical axis shows the level of infection expressed as percentage GFP expression compared to control wells with no antibody added. Each point shows the mean of nine replicate values pooled from three independent experiments, each with triplicate wells. Error bars show standard error. C. Comparison of virus neutralization by antibodies expressed as IgM or IgG1 shown in B. Area under the curve was calculated from each of the three independently performed experiments. P was calculated by paired, two-tailed t-test.


Virus neutralization by antibodies artificially class switched from IgA or IgG1 to IgM . A plaque reduction neutralization assay as described in Fig 3A - B was used to measure neutralization by the two donor - derived IgA ( pink and light blue lines ) and one donor - derived IgG ( bordeaux lines ) . The antibodies expressed in their original classes ( IgA or IgG ) are plotted with filled symbols of the same colors as the lines , and their artificially class - switched IgM equivalents are plotted with open symbols . Horizontal axis shows the antibody concentration and the vertical axis shows the level of infection expressed as percentage GFP expression compared to control wells with no antibody added . Each point shows the mean of nine replicate values pooled from three independent experiments , each with triplicate wells . ND50 was calculated from each of the three independently performed experiments . P was calculated by paired , two - tailed t - test for each antibody pair .

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Virus neutralization by antibodies artificially class switched from IgA or IgG1 to IgM. A plaque reduction neutralization assay as described in Fig 3A-B was used to measure neutralization by the two donor-derived IgA (pink and light blue lines) and one donor-derived IgG (bordeaux lines). The antibodies expressed in their original classes (IgA or IgG) are plotted with filled symbols of the same colors as the lines, and their artificially class-switched IgM equivalents are plotted with open symbols. Horizontal axis shows the antibody concentration and the vertical axis shows the level of infection expressed as percentage GFP expression compared to control wells with no antibody added. Each point shows the mean of nine replicate values pooled from three independent experiments, each with triplicate wells. ND50 was calculated from each of the three independently performed experiments. P was calculated by paired, two-tailed t-test for each antibody pair.


Affinities of donor - derived spike - binding IgG1 ( n = 8 ) and donor - derived IgM ( n = 3 ) expressed as IgG1 , measured by surface plasmon resonance . P value was calculated by unpaired two - tailed t - test .

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

Affinities of donor-derived spike-binding IgG1 (n = 8) and donor-derived IgM (n = 3) expressed as IgG1, measured by surface plasmon resonance. P value was calculated by unpaired two-tailed t-test.


A . Heatmap of antibody binding to spike protein subdomains ( complete spike extracellular domain " spike " , S1 , S2 , RBD and NTD in ELISA . Color gradient shown on right represents optical density at 450 nm ( OD ) . Values are mean OD from two independent experiments . Analogous ELISA results for 3 IgM and their IgG1 - class - switched

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Heatmap of antibody binding to spike protein subdomains (complete spike extracellular domain "spike", S1, S2, RBD and NTD in ELISA. Color gradient shown on right represents optical density at 450 nm (OD). Values are mean OD from two independent experiments. Analogous ELISA results for 3 IgM and their IgG1-class-switched derivatives


C . Flow - cytometric epitope binning of monoclonal IgM , utilizing competitive displacement of IgG by IgM with identical variable region Cells expressing SARS - CoV - 2 spike protein and untransfected control cells were incubated with each of 3 neutralizing IgM antibodies artificially switched to IgG1 , either alone , or mixed with one of the 3 antibodies expressed as IgM . The binding of each IgG1 alone is shown in the last column of the heatmap , and in competition with each other IgM in columns 1 - 3 . Color shade represents the specific IgG binding ( ratio of GMFI on TE spike - mCherry cells to TE 0 cells ) . Values are mean GMFI ratios from three independent experiments . When IgM binds the same epitope as the IgG ( automatically the case when the source antibody is the same ) , the IgM will displace the IgG , resulting in a reduction in the IgG signal . Asterisks mark those combinations of antibodies with a statistically significant decrease in binding of the IgG1 in the presence of IgM , compared to the IgG alone across 3 independent experiments , ( * p < 0 . 05 , two - way analysis of variance , followed by Tukey ' s test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 epitope binning of monoclonal IgM, utilizing competitive displacement of IgG by IgM with identical variable region Cells expressing SARS-CoV-2 spike protein and untransfected control cells were incubated with each of 3 neutralizing IgM antibodies artificially switched to IgG1, either alone, or mixed with one of the 3 antibodies expressed as IgM. The binding of each IgG1 alone is shown in the last column of the heatmap, and in competition with each other IgM in columns 1-3. Color shade represents the specific IgG binding (ratio of GMFI on TE spike-mCherry cells to TE 0 cells). Values are mean GMFI ratios from three independent experiments. When IgM binds the same epitope as the IgG (automatically the case when the source antibody is the same), the IgM will displace the IgG, resulting in a reduction in the IgG signal. Asterisks mark those combinations of antibodies with a statistically significant decrease in binding of the IgG1 in the presence of IgM, compared to the IgG alone across 3 independent experiments, (* p<0.05, two-way analysis of variance, followed by Tukey's test).


( D ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated ; All scale bars are 20 μm . n = 3 biological replicates .

O O O O O O B-MEASURED_VAR 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

(D) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated; All scale bars are 20 μm. n=3 biological replicates.


( G ) Liver tissues were obtained from the indicated mice and then examined using transmission electron microscopy ( Arrowheads indicate mitochondria ) ; Scale bars are 1 μm and 5 μm . n = 3 biological replicates .

O O O O O O O O O O O O O O O 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) Liver tissues were obtained from the indicated mice and then examined using transmission electron microscopy (Arrowheads indicate mitochondria); Scale bars are 1 μm and 5 μm. n = 3 biological replicates.


( B ) Gpx4 protein was measured in liver of WT and PPARα - / - mice fed blank solvent or GW7647 ; n = 4 biological replicates .

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

(B) Gpx4 protein was measured in liver of WT and PPARα-/- mice fed blank solvent or GW7647; n = 4 biological replicates.


( E ) Chromatin immunoprecipitation assays were performed on soluble formaldehyde - crosslinked chromatin isolated from untreated and GW7647 - treated WT or PPARα - / - livers with polyclonal anti - PPARα antibodies ( anti - PPARα ) or control IgG . The final DNA extraction was polymerase chain reaction - amplified with a primer pair that covered the sequence in intron 3 of Gpx4 .

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

(E) Chromatin immunoprecipitation assays were performed on soluble formaldehyde-crosslinked chromatin isolated from untreated and GW7647-treated WT or PPARα-/- livers with polyclonal anti-PPARα antibodies (anti-PPARα) or control IgG. The final DNA extraction was polymerase chain reaction-amplified with a primer pair that covered the sequence in intron 3 of Gpx4.


( B ) Liver sections were obtained from the indicated mice and stained with probe1 . All scale bars are 20 μm . n = 3 biological replicates .

O O O O O 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) Liver sections were obtained from the indicated mice and stained with probe1. All scale bars are 20 μm. n = 3 biological replicates.


( D ) TRF protein was measured in liver of WT and PPARα - / - mice fed blank solvent or GW7647 . n = 4 biological replicates .

(D) TRF protein was measured in liver of WT and PPARα-/- mice fed blank solvent or GW7647. n = 4 biological replicates.


( E ) Liver tissue was obtained from the indicated mice and then examined using transmission electron microscopy ( Arrowheads indicate mitochondria ) . Scale bars are 1 μm and 5 μm . n = 4 biological replicates .

(E) Liver tissue was obtained from the indicated mice and then examined using transmission electron microscopy (Arrowheads indicate mitochondria). Scale bars are 1 μm and 5 μm. n = 4 biological replicates.


( J ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated . All scale bars are 20 μm . n = 3 biological replicates .

(J) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. All scale bars are 20 μm. n = 3 biological replicates.


( E ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated . n = 3 biological replicates . All scale bars are 20 μm .

(E) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. n = 3 biological replicates. All scale bars are 20 μm.


( J ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated . n = 3 biological replicates . All scale bars are 20 μm .

(J) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. n = 3 biological replicates. All scale bars are 20 μm.


( D ) HaloTag pulse - chase analysis in WT MEF monitoring fluorescent Halo - AATNNN during up to 6 h of chase in the presence of BafA1 . The fluorescent JF646 - Halo ligand and the polymer - specific 2C1 antibody stains are shown . ( E ) Same as ( D ) for fluorescent Halo - ATZNNN . ( F ) Quantification of accumulation of fluorescent Halo - AATNNN and Halo - ATZNNN within LAMP1 - positive endolysosomes . Mean ± SEM , n = 20 , 11 , 10 , 13 for Halo - AATNNN , n = 18 , 12 , 12 , 13 for Halo - ATZNNN . Two - way ANOVA and Sidak ' s multiple comparison test , ns P > 0 . 05 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HaloTag pulse-chase analysis in WT MEF monitoring fluorescent Halo-AATNNN during up to 6 h of chase in the presence of BafA1. The fluorescent JF646-Halo ligand and the polymer-specific 2C1 antibody stains are shown. (E) Same as (D) for fluorescent Halo-ATZNNN. (F) Quantification of accumulation of fluorescent Halo-AATNNN and Halo-ATZNNN within LAMP1-positive endolysosomes. Mean ± SEM, n=20, 11, 10, 13 for Halo-AATNNN, n= 18, 12, 12, 13 for Halo-ATZNNN. Two-way ANOVA and Sidak's multiple comparison test, ns P>0.05, *** P<0.001.


A Confocal laser scanning microscopy analyses in cells treated with BafA1 . Total HA - ATZNNN ( red ) or HA - ATZNNN polymers ( magenta ) in LAMP1 - positive endolysosome ( green ) in WT MEF . ( C ) Same as ( A ) in Cnx - KO MEF . ( D ) Same as ( A ) in WT MEF exposed to kifunensine ( KIF ) . ( E ) Same as ( A ) in WT MEF exposed to Castanospermine ( CST ) . ( F ) Same as ( A ) in sCNX MEF . ( G ) Same as ( A ) in Uggt1 - KO MEF .

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

A Confocal laser scanning microscopy analyses in cells treated with BafA1. Total HA-ATZNNN (red) or HA-ATZNNN polymers (magenta) in LAMP1-positive endolysosome (green) in WT MEF. (C) Same as (A) in Cnx-KO MEF. (D) Same as (A) in WT MEF exposed to kifunensine (KIF). (E) Same as (A) in WT MEF exposed to Castanospermine (CST). (F) Same as (A) in sCNX MEF. (G) Same as (A) in Uggt1-KO MEF.


( J ) Halo - ATZNNN in Uggt1 - KO MEF mock - transfected ( empty pcDNA3 plasmid ) . ( K , L ) ( K ) Same as ( J ) , with a plasmid for expression of active or ( L ) inactive UGGT1 . ( M ) Quantification of Halo - ATZNNN - positive endolysosomes ( mean , n = 22 , 10 , 15 cells for panels J - L , respectively ) . One - way ANOVA and Dunnett ' s multiple comparison test , ns P > 0 . 05 , * * * * P < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Halo-ATZNNN in Uggt1-KO MEF mock-transfected (empty pcDNA3 plasmid). (K, L) (K) Same as (J), with a plasmid for expression of active or (L) inactive UGGT1. (M) Quantification of Halo-ATZNNN-positive endolysosomes (mean, n=22, 10, 15 cells for panels J-L, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001).


( B - I ) Confocal laser scanning microscopy analyses ( as in Fig . 2 ) in WT MEF treated with BafA1 . ( J , K ) ( J ) Quantification of ATZxxx - positive endolysosomes and ( K ) of ATZxxx polymers - positive endolysosomes ( mean , n = 18 , 30 , 24 , 14 , 27 , 22 , 27 , 18 cells for HA stain and n = 12 , 14 , 14 , 11 , 14 , 14 , 17 , 11 cells for 2C1 stain of panels B - I , respectively ) . One - way ANOVA and Dunnett ' s multiple comparison test , ns P > 0 . 05 , * * * * P < 0 . 0001 .

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

(B-I) Confocal laser scanning microscopy analyses (as in Fig. 2) in WT MEF treated with BafA1. (J, K) (J) Quantification of ATZxxx-positive endolysosomes and (K) of ATZxxx polymers-positive endolysosomes (mean, n= 18, 30, 24, 14, 27, 22, 27, 18 cells for HA stain and n= 12, 14, 14, 11, 14, 14, 17, 11 cells for 2C1 stain of panels B-I, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001.


( C ) Co - immunoprecipitation of ATZXXX ( Bait , lower panel ) with CNX ( upper panel ) and BiP ( middle panel ) . Protein content in the total cell extracts ( TCE ) is shown . ( D ) Quantification of ( C ) for CNX association ( n = 3 ) . Unpaired two - tailed t - test , * * P < 0 . 01 * * * P < 0 . 001 , * * * * P < 0 . 0001 . ( E ) Quantification of ( C ) for BiP association ( n = 3 for ATZNNN and ATZQQN ; n = 4 for ATZQNQ and ATZQQQ ) . Unpaired two - tailed t - test , * P < 0 . 005 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation of ATZXXX (Bait, lower panel) with CNX (upper panel) and BiP (middle panel). Protein content in the total cell extracts (TCE) is shown. (D) Quantification of (C) for CNX association (n=3). Unpaired two-tailed t-test, ** P<0.01*** P<0.001, **** P<0.0001. (E) Quantification of (C) for BiP association (n=3 for ATZNNN and ATZQQN; n=4 for ATZQNQ and ATZQQQ). Unpaired two-tailed t-test, * P<0.005, ** P<0.01, *** P<0.001, **** P<0.0001.


( G ) Confocal laser scanning microscopy in Flp - In 3T3 cells stably expressing ATZNNN in the absence of BafA1 ( to allow clearance of misfolded model proteins delivered within endolysosomes ) . Co - localization with Calnexin ( CNX ) . ( H ) Same as ( G ) for BiP co - localization . ( I ) Same as ( G ) for ATZQQQ . ( J ) Same as ( H ) for ATZQQQ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal laser scanning microscopy in Flp-In 3T3 cells stably expressing ATZNNN in the absence of BafA1 (to allow clearance of misfolded model proteins delivered within endolysosomes). Co-localization with Calnexin (CNX). (H) Same as (G) for BiP co-localization. (I) Same as (G) for ATZQQQ. (J) Same as (H) for ATZQQQ.


( K ) HaloTag pulse - chase analysis in MEF monitoring fluorescent Halo - ATZNNN during up to 24 h . ( L ) Same as ( K ) for fluorescent Halo - ATZQQQ . Arrowheads show select clusters of JF646 - labeled Halo - ATZQQQ . Arrowheads show co - localization Halo - ATZQQQ : BiP .

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

(K) HaloTag pulse-chase analysis in MEF monitoring fluorescent Halo-ATZNNN during up to 24 h. (L) Same as (K) for fluorescent Halo-ATZQQQ. Arrowheads show select clusters of JF646-labeled Halo-ATZQQQ. Arrowheads show co-localization Halo-ATZQQQ:BiP.


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 .

O O O O O O O O O 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) 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 ) .

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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 ) .

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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 ) .

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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 .

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 .

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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.


( A ) Quantification of CXCR4 + cells by flow cytometry and Sox17 + cells by immunostaining at day 7 following differentiation in the presence or absence of 3 µM CH . Average and standard deviation ( SD ) of eight independent experiments .

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(A) Quantification of CXCR4+ cells by flow cytometry and Sox17+ cells by immunostaining at day 7 following differentiation in the presence or absence of 3 µM CH. Average and standard deviation (SD) of eight independent experiments.


( B ) Gene expression profiling by RT - PCR at day 7 differentiation . Average and SD of three independent experiments .

(B) Gene expression profiling by RT-PCR at day 7 differentiation. Average and SD of three independent experiments.


( C ) Immunostaining for Ecadherin ( left ) and FoxA2 ( right ) at day 7 of differentiation in presence of 3 µM CH .

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(C) Immunostaining for Ecadherin (left) and FoxA2 (right) at day 7 of differentiation in presence of 3 µM CH.


( D ) Quantification of CXCR4 + by flow cytometry over a seven - day differentiation time course in the presence of 3 µM CH . Average and SD of three independent experiments .

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(D) Quantification of CXCR4+ by flow cytometry over a seven-day differentiation time course in the presence of 3 µM CH. Average and SD of three independent experiments.


( E ) Immunostaining for Sox17 at day 4 to day 7 of differentiation in the continuous presence or absence of 3 µM CH . Scale bars = 200

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(E) Immunostaining for Sox17 at day 4 to day 7 of differentiation in the continuous presence or absence of 3 µM CH. Scale bars = 200 µM


( A ) Assay of Tcf7l1 mRNA by RT - PCR for the first 4 days of differentiation in the presence or absence of 3 μM CH . Average and SD of three independent experiments . Day2 * p = 0 . 048 , Day3 * p = 0 . 036 .

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(A) Assay of Tcf7l1 mRNA by RT-PCR for the first 4 days of differentiation in the presence or absence of 3 μM CH. Average and SD of three independent experiments. Day2 *p= 0.048, Day3 *p=0.036.


B ) Immunoblot for Tcf7l1 at day 3 of differentiation in the presence ( + ) or absence ( - ) of 3 μM CH .

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B) Immunoblot for Tcf7l1 at day 3 of differentiation in the presence (+) or absence (-) of 3 μM CH.


( C ) Assay of FoxA2 mRNA by RT - PCR for the first 3 days of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments . * * p = 5x10 - 7 .

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(C) Assay of FoxA2 mRNA by RT-PCR for the first 3 days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. **p=5x10-7.


( D ) ChiP - seq data from a compendium of transcription factor ChIP - seq analyses ( Sánchez - Castillo et al , 2015 ) . Gene tracks represent binding of Tcf7l1 at the FoxA2 , Nodal and Eomesodermin ( Eomes ) gene locus . Red lines indicate the regions analyzed by ChIP PCR for Tcf7l1 .

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(D) ChiP-seq data from a compendium of transcription factor ChIP-seq analyses (Sánchez-Castillo et al, 2015). Gene tracks represent binding of Tcf7l1 at the FoxA2, Nodal and Eomesodermin (Eomes) gene locus. Red lines indicate the regions analyzed by ChIP PCR for Tcf7l1.


( E ) ChIP for Tcf7l1 followed by qPCR for the regions indicated in Fig . 2D . Analysis was performed in wild - type ES cells in 2i culture conditions . Average and SD of three independent experiments . Klf2 * p = 0 . 025 , FoxA2 * p = 0 . 050 , Eomes * p = 0 . 023 .

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(E) ChIP for Tcf7l1 followed by qPCR for the regions indicated in Fig. 2D. Analysis was performed in wild-type ES cells in 2i culture conditions. Average and SD of three independent experiments. Klf2 *p=0.025, FoxA2 *p=0.050, Eomes *p=0.023.


( F ) ChIP for Tcf7l1 followed by qPCR . Analysis was performed in wild - type ES cells on day 3 of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments . FoxA2 * p = 0 . 049 .

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(F) ChIP for Tcf7l1 followed by qPCR. Analysis was performed in wild-type ES cells on day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. FoxA2 *p = 0.049.


( A ) Assay of FoxA2 mRNA by RT - PCR in Tcf7l1 null cells on day 1 to 4 of differentiation without CH . Average and SD of three independent experiments .

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(A) Assay of FoxA2 mRNA by RT-PCR in Tcf7l1 null cells on day 1 to 4 of differentiation without CH. Average and SD of three independent experiments.


( B ) Gene expression analysis of endodermal associated genes by RT - PCR in control wild - type and Tcf7l1 null cells on day 4 of differentiation in the presence and absence of 3 µM CH . Average and SD of three independent experiments .

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(B) Gene expression analysis of endodermal associated genes by RT-PCR in control wild-type and Tcf7l1 null cells on day 4 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.


( C ) FoxA2 ( green ) and Oct4 ( red ) immunostaining of Tcf7l1 null ES cells on day 4 of differentiation in the presence or absence of 3 µM CH . DAPI staining in blue .

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(C) FoxA2 (green) and Oct4 (red) immunostaining of Tcf7l1 null ES cells on day 4 of differentiation in the presence or absence of 3 µM CH. DAPI staining in blue.


( D ) Assay of CXCR4 , Hex and Sox17 mRNA by RT - PCR in control wild - type and Tcf7l1 null cells on day 6 of differentiation in the presence and absence of 3 µM CH . Average and SD of three independent experiments .

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(D) Assay of CXCR4, Hex and Sox17 mRNA by RT-PCR in control wild-type and Tcf7l1 null cells on day 6 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.


( A ) Assay of FoxA2 mRNA by RT - PCR in FoxA2 - Tet ES cell lines 48 hr after treatment with the indicated dose of DOX . Average and SD of two independent experiments from 2 independent lines .

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(A) Assay of FoxA2 mRNA by RT-PCR in FoxA2-Tet ES cell lines 48 hr after treatment with the indicated dose of DOX. Average and SD of two independent experiments from 2 independent lines.


B ) FoxA2 immunostaining of FoxA2 - Tet ES cells 48 hr after DOX treatment . Left , no DOX control . Middle and right , 10ng / ml DOX . DAPI staining shown in blue . Right panel shows middle image without DAPI signal .

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B) FoxA2 immunostaining of FoxA2-Tet ES cells 48 hr after DOX treatment. Left, no DOX control. Middle and right, 10ng/ml DOX. DAPI staining shown in blue. Right panel shows middle image without DAPI signal.


( C ) Assay of FoxA2 and CXCR4 mRNA by RT - PCR on day 3 of differentiation with DOX at the indicated dose . Average and SD of two experiments from two independent cell lines .

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(C) Assay of FoxA2 and CXCR4 mRNA by RT-PCR on day 3 of differentiation with DOX at the indicated dose. Average and SD of two experiments from two independent cell lines.


( D ) Assay of Sox17 mRNA by RT - PCR on day 7 of differentiation with addition of 3 µM CH and / or 10 ng / ml DOX at the indicated time points ( in days ) . Average and SD of two independent experiments from two independent cell lines .

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(D) Assay of Sox17 mRNA by RT-PCR on day 7 of differentiation with addition of 3 µM CH and/or 10 ng/ml DOX at the indicated time points (in days). Average and SD of two independent experiments from two independent cell lines.


( E ) Sox17 immunostaining of FoxA2 - Tet ES cells at day 7 of differentiation in the presence or absence of 10 ng / ml DOX and the presence and absence of 3 µM CH as indicated . Far right panel shows image to its left without DAPI .

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(E) Sox17 immunostaining of FoxA2-Tet ES cells at day 7 of differentiation in the presence or absence of 10 ng/ml DOX and the presence and absence of 3 µM CH as indicated. Far right panel shows image to its left without DAPI.


( A ) TOPFlash reporter activity following differentiation for 3 days in 3 µM CH or 100 ng / ml ( W100 ) or 200 ng / ml ( W200 ) recombinant Wnt3a plus 500 ng / ml recombinant RSpondin1 . Relative luciferase units are normalised to FOPflash . Average and SD of three independent experiments .

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(A) TOPFlash reporter activity following differentiation for 3 days in 3 µM CH or 100 ng/ml (W100) or 200 ng/ml (W200) recombinant Wnt3a plus 500 ng/ml recombinant RSpondin1. Relative luciferase units are normalised to FOPflash. Average and SD of three independent experiments.


( B ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR at day 2 of differentiation in the presence or absence of 3 µM CH or recombinant Wnt3a ( 200 ng / ml plus 500 ng / ml RSpondin1 ( WR ) ) . Average and SD of three independent experiments . * * p < 0 . 01 .

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(B) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR at day 2 of differentiation in the presence or absence of 3 µM CH or recombinant Wnt3a (200 ng/ml plus 500 ng/ml RSpondin1(WR)). Average and SD of three independent experiments. ** p<0.01.


( C ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR in Ctnnb1 null cells at day 1 and 3 of differentiation in the absence or presence of 3 µM CH . Average and SD of three independent experiments .

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(C) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in Ctnnb1 null cells at day 1 and 3 of differentiation in the absence or presence of 3 µM CH. Average and SD of three independent experiments.


( D ) Immunoblot for Tcf7l1 in Ctnnb1 null cells during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH . Results from two independent , clonally - derived Ctnnb1 null ES cell lines are shown . Tubulin was used as a loading control .

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(D) Immunoblot for Tcf7l1 in Ctnnb1 null cells during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH. Results from two independent, clonally-derived Ctnnb1 null ES cell lines are shown. Tubulin was used as a loading control.


( A ) Assay of cMyc mRNA by RT - PCR in wild - type ES cells at day 1 to 3 of differentiation . Average and SD of three independent experiments .

(A) Assay of cMyc mRNA by RT-PCR in wild-type ES cells at day 1 to 3 of differentiation. Average and SD of three independent experiments.


( B ) Immunoblot for total cMyc and phospho Ser62 ( pSer62 ) cMyc during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH .

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(B) Immunoblot for total cMyc and phospho Ser62 (pSer62) cMyc during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH.


C , D ) Immunoblot for the destabilized form of cMyc protein ( pThr58 ) during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH in wild - type and Ctnnb1 null cells respectively .

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C,D) Immunoblot for the destabilized form of cMyc protein (pThr58) during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH in wild-type and Ctnnb1 null cells respectively.


( E ) Assay of Tcf7l1 mRNA by RT - PCR in wild - type cells at day 2 of differentiation in the presence or absence of a small molecule inhibitor of cMyc ( cMyc Inhibitor II ; Myci ) . Treatment with Myc inhibitor 10058 - F4 ( Sigma ) generated similar results ( data not shown ) . Average and SD of three independent experiments . * * p = 0 . 0002 .

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(E) Assay of Tcf7l1 mRNA by RT-PCR in wild-type cells at day 2 of differentiation in the presence or absence of a small molecule inhibitor of cMyc (cMyc Inhibitor II; Myci). Treatment with Myc inhibitor 10058-F4 (Sigma) generated similar results (data not shown). Average and SD of three independent experiments. **p=0.0002.


( F ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR in cMyc null / nMyc heterozygous cells ( C - / - ; N + / - ) and floxed parental cells ( cMycfl / fl ; nMycfl / fl ( control ) ) at day 3 of differentiation in the presence or absence of 3 µM CH . Average and SD of four or six independent experiments . Tcf7l1 * p = 0 . 01 , FoxA2 * p = 0 . 034 .

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(F) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in cMyc null/nMyc heterozygous cells (C-/-;N+/-) and floxed parental cells (cMycfl/fl;nMycfl/fl (control)) at day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of four or six independent experiments. Tcf7l1 *p=0.01, FoxA2 *p=0.034.


( G ) FoxA2 immunostaining of control and cMyc null / nMyc heterozygous ( cMyc - / - ; nMyc + / - ) cells at day 3 of differentiation in the presence of 3 µM CH .

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(G) FoxA2 immunostaining of control and cMyc null/nMyc heterozygous (cMyc-/-;nMyc+/-) cells at day 3 of differentiation in the presence of 3 µM CH.


( H ) Assay of cMyc and Tcf7l1 mRNA by RT - PCR in cMycT58A inducible cell lines ( icMycT58A ) 24 hr after DOX treatment . Average of 2 independent experiments .

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(H) Assay of cMyc and Tcf7l1 mRNA by RT-PCR in cMycT58A inducible cell lines (icMycT58A) 24 hr after DOX treatment. Average of 2 independent experiments.


( I ) Assay of Tcf7l1 mRNA by RT - PCR in icMycT58A clone 1 cell line at day 3 of differentiation in the absence of 3 µM CH following cMyc induction with 10 ng / ml DOX . Average and SD of 3 independent experiments . * * p = 0 . 007 .

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(I) Assay of Tcf7l1 mRNA by RT-PCR in icMycT58A clone 1 cell line at day 3 of differentiation in the absence of 3 µM CH following cMyc induction with 10 ng/ml DOX. Average and SD of 3 independent experiments. **p=0.007.


( J ) Immunoblot for cMyc and Tcf7l1 in icMycT58A clonal cell line 1 48 hr after DOX induction at 10ng / ml .

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(J) Immunoblot for cMyc and Tcf7l1 in icMycT58A clonal cell line 1 48 hr after DOX induction at 10ng/ml.


( B ) ChIP for cMyc performed in wild - type ES cells differentiated for 2 days in the absence or presence of 3 µM CH . qPCR was carried out for the regions indicated in Fig . 7a . Average and SD for five independent experiments , * p = 0 . 028 .

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(B) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the absence or presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, *p=0.028.


( C ) ChIP for cMyc performed in wild - type ES cells differentiated for 2 days in the presence of 3 µM CH plus or minus Myc inhibitor 10058 - F4 . Average and SD for three independent experiments , * p = 0 . 026 .

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(C) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the presence of 3 µM CH plus or minus Myc inhibitor 10058-F4. Average and SD for three independent experiments, *p=0.026.


( D ) Assay for Miz1 mRNA by RT - PCR for the first three days of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments .

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(D) Assay for Miz1 mRNA by RT-PCR for the first three days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments.


( E ) ChIP for Miz1 or IgG control performed in E14 ES cells differentiated for 2 days in presence of 3 µM CH . qPCR was carried out for the regions indicated in Fig . 7a . Average and SD for five independent experiments , * * p = 0 . 0015 .

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(E) ChIP for Miz1 or IgG control performed in E14 ES cells differentiated for 2 days in presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, **p=0.0015.


WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation . Cilia were stained with Arl13B ( green ) while the basal bodies were marked with C - Nap1 ( red ) . DNA was stained with DAPI . Cilia in the magenta box are shown enlarged on the right picture

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WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation. Cilia were stained with Arl13B (green) while the basal bodies were marked with C-Nap1 (red). DNA was stained with DAPI. Cilia in the magenta box are shown enlarged on the right picture


WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation . Cilia were stained with Arl13B ( green ) while the basal bodies were marked with C - Nap1 ( red ) . DNA was stained with DAPI Percent of ciliated cells , as well as the length of cilia , was quantified from A . Cilia length was measured by a semi - automated ImageJ macro as described in methods . Data points are the average of three independent experiments with N = 150 cilia for each experiment and condition . Error bars represent the mean ± SD . * * * * P ≤ 0 . 0001 . High means 80 - 90 % and low 40 - 50 % confluency of RPE1

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WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation. Cilia were stained with Arl13B (green) while the basal bodies were marked with C-Nap1 (red). DNA was stained with DAPI Percent of ciliated cells, as well as the length of cilia, was quantified from A. Cilia length was measured by a semi-automated ImageJ macro as described in methods. Data points are the average of three independent experiments with N = 150 cilia for each experiment and condition. Error bars represent the mean ± SD. **** P≤ 0.0001. High means 80-90% and low 40-50% confluency of RPE1 cells


Elongated cilia phenotype was confirmed by siRNA - mediated knockdown of hCDC14A . Three independent experiments , N = 150 cilia for each experiment and condition . Mean ± SD . * * * * P ≤ 0 . 0001

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Elongated cilia phenotype was confirmed by siRNA-mediated knockdown of hCDC14A. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. **** P≤ 0.0001


hCDC14A - GFP and hCDC14AC278S - GFP under control of the TetON promoter were stably integrated into RPE1 cells . hCDC14A - GFP and hCDC14AC278S - GFP localized to the C - Nap1 marked basal body upon Dox induction . In addition , expression of hCDC14A - GFP but not the phosphatase dead hCDC14AC278S - GFP mutant inhibited cilia formation . Ciliated cells were quantified 48 h after serum starvation with and without Dox addition . Three independent experiments ; N = 150 cilia for each experiment and condition . Mean ± SD . * * * P ≤ 0 . 001 ; ns : not significant

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hCDC14A-GFP and hCDC14AC278S-GFP under control of the TetON promoter were stably integrated into RPE1 cells. hCDC14A-GFP and hCDC14AC278S-GFP localized to the C-Nap1 marked basal body upon Dox induction. In addition, expression of hCDC14A-GFP but not the phosphatase dead hCDC14AC278S-GFP mutant inhibited cilia formation. Ciliated cells were quantified 48 h after serum starvation with and without Dox addition. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. *** P≤ 0.001; ns: not significant