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 .

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

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 ) .

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

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 .

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

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 .

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

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 ) .

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

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 .

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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

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

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).


B . ST2 cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . As a control , cells were first solubilized and SF - IL - 17 was added only post lysis . Number of identified peptides ( unique + razor ) and iBAQ intensities for each protein in five independent experiments are shown .

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

B. ST2 cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in five independent experiments are shown.


C . The stoichiometry of IL - 17RSC calculated as the ratio between iBAQ intensities of individual IL - 17RSC components to iBAQ intensity of IL - 17RC . The recruitment of kinases TBK1 and IKKε as compared to related kinases IKKα and IKKβ is significantly enhanced . Mean from five performed MS experiments is shown , the statistical significance was determined using unpaired two ‐ tailed Student ' s t ‐ test .

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

C. The stoichiometry of IL-17RSC calculated as the ratio between iBAQ intensities of individual IL-17RSC components to iBAQ intensity of IL-17RC. The recruitment of kinases TBK1 and IKKε as compared to related kinases IKKα and IKKβ is significantly enhanced. Mean from five performed MS experiments is shown, the statistical significance was determined using unpaired two‐tailed Student's t‐test.


D . ST2 cells were stimulated with IL - 17 ( 500 ng / ml ) , TNF ( 50 ng / ml ) , or IL - 1α ( 50 ng / ml ) for indicated time points and activation of signaling pathways was analyzed by immunoblotting . A representative of two independent experiments is shown .

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

D. ST2 cells were stimulated with IL-17 (500 ng/ml), TNF (50 ng/ml), or IL-1α (50 ng/ml) for indicated time points and activation of signaling pathways was analyzed by immunoblotting. A representative of two independent experiments is shown.


ST2 cells were either left untreated ( A ) followed by stimulation with IL - 17 ( 500 ng / ml ) for 2 hours . Transcription response induced by IL - 17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing . In red are transcripts considered to be significantly changed ( log2 fold change > 1 or < - 1 , - log10 Benjamini - Hochberg adjusted p - value > 2 , based on the analysis of three independent experiments ; tringle is used for transcripts with - log10 adjusted p - value > 100 ) . Names of several significantly upregulated transcripts are indicated .

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

ST2 cells were either left untreated (A) followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated.


ST2 cells were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) for 30 minutes ( B ) , followed by stimulation with IL - 17 ( 500 ng / ml ) for 2 hours . Transcription response induced by IL - 17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing . In red are transcripts considered to be significantly changed ( log2 fold change > 1 or < - 1 , - log10 Benjamini - Hochberg adjusted p - value > 2 , based on the analysis of three independent experiments ; tringle is used for transcripts with - log10 adjusted p - value > 100 ) . Names of several significantly upregulated transcripts are indicated .

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

ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) for 30 minutes (B), followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated.


D - E . Comparison of IL - 17 - induced transcriptional response in presence or absence of TBK1 / IKKε inhibitor . ( D ) Transcripts that are significantly changed in both conditions . ( E ) Transcripts that pass significance threshold for induction only in presence of TBK1 / IKKε inhibitor . Dashed lines indicate significantly upregulated transcripts ( log2 fold change > 1 ) ; red lines separates transcripts that are more upregulated upon IL - 17 stimulation in the presence of TBK1 / IKKε inhibitor as compared to IL - 17 alone .

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

D-E. Comparison of IL-17-induced transcriptional response in presence or absence of TBK1/IKKε inhibitor. (D) Transcripts that are significantly changed in both conditions. (E) Transcripts that pass significance threshold for induction only in presence of TBK1/IKKε inhibitor. Dashed lines indicate significantly upregulated transcripts (log2 fold change > 1); red lines separates transcripts that are more upregulated upon IL-17 stimulation in the presence of TBK1/IKKε inhibitor as compared to IL-17 alone.


F . ST2 cells pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) were left unstimulated or stimulated with IL - 17 ( 500 ng / ml ) for 2 hours and induction of mRNA for selected genes was analyzed by real time PCR . Mean + SEM from five independent experiments is shown , statistical significance was determined using unpaired two ‐ tailed Student ' s t ‐ test .

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

F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) were left unstimulated or stimulated with IL-17 (500 ng/ml) for 2 hours and induction of mRNA for selected genes was analyzed by real time PCR. Mean + SEM from five independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.


G . ST2 cells were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL17 ( 500 ng / ml ) for indicated time points and analyzed by immunoblotting .

O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O

G. ST2 cells were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL17 (500 ng/ml) for indicated time points and analyzed by immunoblotting.


H . ST2 wild type , TBK1 KO , IKKε KO , or cells lacking both kinases ( DKO ) were stimulated with IL - 17 ( 500 ng / ml ) as indicated and analyzed by immunoblotting .

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

H. ST2 wild type, TBK1 KO, IKKε KO, or cells lacking both kinases (DKO) were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.


I . TBK1 / IKKε DKO cells reconstituted with either wild type or kinase dead mutant ( K38A ) version of both kinases were stimulated with IL - 17 ( 500 ng / ml ) as indicated and analyzed by immunoblotting .

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

I. TBK1/IKKε DKO cells reconstituted with either wild type or kinase dead mutant (K38A) version of both kinases were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.


ST2 wild type or TBK1 / IKKε DKO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( A ) The heatmap shows the row - normalized Z - Score calculated from log2 transformed iBAQ intensities from five independent experiments .

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

ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ intensities from five independent experiments.


ST2 wild type or TBK1 / IKKε DKO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( B ) The ratio between iBAQ intensities of selected IL - 17RSC components to iBAQ intensity of IL - 17RC . Mean + SEM from five independent experiments is shown , statistical significance was determined by two ‐ tailed Mann - Whitney test .

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

ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from five independent experiments is shown, statistical significance was determined by two‐tailed Mann-Whitney test.


C . ST2 wild type or TBK1 / IKKε DKO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) for 15 minutes or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC and samples were analyzed by immunoblotting . ACT1 phosphorylation detected as upper band in WT cells is absent in TBK1 / IKKε DKO cells .

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

C. ST2 wild type or TBK1/IKKε DKO cells were stimulated with SF-IL-17 (500 ng/ml) for 15 minutes or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting. ACT1 phosphorylation detected as upper band in WT cells is absent in TBK1/IKKε DKO cells.


D . Cells were pretreated with TAK1 inhibitor 7 - oxozeanol ( 2 µM ) and / or TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL - 17 ( 500 ng / ml ) for indicated time points and lysates were analyzed by immunoblotting .

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

D. Cells were pretreated with TAK1 inhibitor 7-oxozeanol (2 µM) and/or TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.


E . ST2 wild type or HOIP - deficient cells were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL - 17 ( 500 ng / ml ) as indicated , solubilized , and analyzed by immunoblotting . * indicates unspecific band .

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

E. ST2 wild type or HOIP-deficient cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) as indicated, solubilized, and analyzed by immunoblotting. * indicates unspecific band.


F . ST2 cells pretreated or not with TBK1 / IKKε inhibitor MRT67307 were stimulated with SF - IL - 17 for 15 minutes or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated and analyzed via immunoblotting .

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

F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 were stimulated with SF-IL-17 for 15 minutes or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated and analyzed via immunoblotting.


A . ST2 wild type and ACT1 - deficient cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . As a control , cells were first solubilized and SF - IL - 17 was added only post lysis . Number of identified peptides ( unique + razor ) and iBAQ intensities for each protein in two independent experiments are shown .

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

A. ST2 wild type and ACT1-deficient cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.


B . ST2 wild type , ACT1 KO or TRAF6 KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC and samples were analyzed by immunoblotting .

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

B. ST2 wild type, ACT1 KO or TRAF6 KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting.


C . ST2 cells deficient in TRAF6 reconstituted with TRAF6 ( WT ) or enzymatically inactive TRAF6 ( C70A ) were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . Number of identified peptides ( unique + razor ) and iBAQ intensities for each protein in two independent experiments are shown .

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

C. ST2 cells deficient in TRAF6 reconstituted with TRAF6(WT) or enzymatically inactive TRAF6(C70A) were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.


D . TRAF6 deficient cells reconstituted with TRAF6 ( WT ) , TRAF6 ( C70A ) or empty vector were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated from lysates by anti - Flag immunoprecipitation and analyzed by immunoblotting . Short and long exposure of the same membrane stained for Act1 are shown for lysate and IP samples , respectively .

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

D. TRAF6 deficient cells reconstituted with TRAF6(WT), TRAF6(C70A) or empty vector were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated from lysates by anti-Flag immunoprecipitation and analyzed by immunoblotting. Short and long exposure of the same membrane stained for Act1 are shown for lysate and IP samples, respectively.


TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) and stimulated with IL - 17 ( 500 ng / ml ) for indicated time . ( E ) The activation of signaling pathways analyzed by immunoblotting .

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

TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. (E) The activation of signaling pathways analyzed by immunoblotting.


TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) and stimulated with IL - 17 ( 500 ng / ml ) for indicated time . The induction of mRNA for selected genes analyzed by real time PCR . Mean + SEM from four independent experiments is shown , statistical significance was determined using unpaired two ‐ tailed Student ' s t ‐ test .

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

TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. The induction of mRNA for selected genes analyzed by real time PCR. Mean + SEM from four independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.


G . ST2 cells wild type or NEMO KO were stimulated with IL - 17 ( 500 ng / ml ) for indicated time points , solubilized and analyzed by immunoblotting .

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

G. ST2 cells wild type or NEMO KO were stimulated with IL-17 (500 ng/ml) for indicated time points, solubilized and analyzed by immunoblotting.


ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC . Samples were analyzed by immunoblotting ( H ) .

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

ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC. Samples were analyzed by immunoblotting (H).


ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 or were left unstimulated and IL - 17 was added post lysis . results from four independent experiments were quantified by densitometry and mean + SEM from four independent experiments is shown ( I ) .

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

ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 or were left unstimulated and IL-17 was added post lysis. results from four independent experiments were quantified by densitometry and mean + SEM from four independent experiments is shown (I).


ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( A ) The heatmap shows the row - normalized Z - Score calculated from log2 transformed iBAQ values from two independent experiments .

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ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ values from two independent experiments.


ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( B ) The ratio between iBAQ intensities of selected IL - 17RSC components to iBAQ intensity of IL - 17RC . Mean + SEM from two independent experiments is shown .

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

ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from two independent experiments is shown.


ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC ( C ) and analyzed by immunoblotting .

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ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC (C) and analyzed by immunoblotting.


ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were tested for activation of signaling pathways ( D ) and analyzed by immunoblotting .

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ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were tested for activation of signaling pathways (D) and analyzed by immunoblotting.


E . ST2 cells were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) or IKKα / IKKβ inhibitor TPCA1 ( 10 µM ) , stimulated with IL - 17 ( 500 ng / ml ) as indicated and analyzed by immunoblotting .

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E. ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) or IKKα/IKKβ inhibitor TPCA1 (10 µM), stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.


F . ST2 wild type or NEMO KO cells were stimulated with TNF ( 50 ng / ml ) for indicated time points and lysates were analyzed by immunoblotting .

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F. ST2 wild type or NEMO KO cells were stimulated with TNF (50 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.


B . Analysis of ACT1 structure using three different algorithms predicting disordered regions . Detected phosphorylation sites are all in the disordered region of the protein .

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

B. Analysis of ACT1 structure using three different algorithms predicting disordered regions. Detected phosphorylation sites are all in the disordered region of the protein.


ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho - sites mutated to alanines ( 9ST mut ) . Cells were stimulated with IL - 17 ( 500 ng / ml ) as indicated and lysates were analyzed by immunoblotting . * indicates nonspecific band .

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ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.


E . ACT1 KO cells expressing either ACT1 ( WT ) or ACT1 ( 9ST mut ) were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated via anti - Flag immunoprecipitation and analyzed by immunoblotting .

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E. ACT1 KO cells expressing either ACT1(WT) or ACT1(9ST mut) were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated via anti-Flag immunoprecipitation and analyzed by immunoblotting.


F . ACT1 KO cells were reconstituted with either ACT1 ( WT ) or ACT1 ( Δ20 - 380 ) . Cells were stimulated with IL - 17 ( 500 ng / ml ) as indicated and lysates were analyzed by immunoblotting . * indicates nonspecific band .

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F. ACT1 KO cells were reconstituted with either ACT1(WT) or ACT1(Δ20-380). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.


G . Analysis of iBAQ intensity of ACT1 normalized to iBAQ intensity of IL - 17RC . Data are composite of all 14 MS analyses of IL - 17RSC from wild type ST2 cells performed in this study . Mean + SEM is shown .

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

G. Analysis of iBAQ intensity of ACT1 normalized to iBAQ intensity of IL-17RC. Data are composite of all 14 MS analyses of IL-17RSC from wild type ST2 cells performed in this study. Mean + SEM is shown.


B , single optical plane in the HPuHG of wildtype mice and Alk1EndoKO mice showing dramatic vascular defect in Alk1EndoKO mice . Severely tangled and tortuous vasculatures were outlined in white dotted rectangles .

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B, single optical plane in the HPuHG of wildtype mice and Alk1EndoKO mice showing dramatic vascular defect in Alk1EndoKO mice. Severely tangled and tortuous vasculatures were outlined in white dotted rectangles.


C , 8 μm skin section of Cre - control ( CT ) and Alk1EndoKO mice stained for Cdh5 ( red ) , Ki67 ( green ) , Erg ( white ) , and Dapi ( blue ) . Examples of Cdh5 / Ki67 double positive cells were enlarged in the white dotted inset for visualization purposes .

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C, 8 μm skin section of Cre- control (CT) and Alk1EndoKO mice stained for Cdh5 (red), Ki67 (green), Erg (white), and Dapi (blue). Examples of Cdh5/Ki67 double positive cells were enlarged in the white dotted inset for visualization purposes.


D - G , Error bars represent standard deviation . Each bar represents one individual mouse , and each dot represents measurement from one image . N = 3 mice per group , and n = 9 - 16 images per mouse . Restricted maximum likelihood ( REML ) analysis with unbounded variance components was used as statistical test and individual mouse was treated as random effect to take into the account of mouse - to - mouse variability . D , Quantification of number of ECs in HPuHG area from C . E , Percentage of proliferative ECs ( percentage of Ki67 + Cdh5 + cells in total Cdh5 + cells ) in HPuHG from C . F , Percentage of proliferative ECs ( percentage of Ki67 + / Cdh5 + cells in total skin Cdh5 + cells ) from C G , Percentage of proliferative ECs ( percentage of BrdU + Cdh5 + cells in total Cdh5 + cells ) in total skin from PD17 - 24 BrdU - pulsed CT and Alk1EndoKO

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

D-G, Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice per group, and n = 9-16 images per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used as statistical test and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability. D, Quantification of number of ECs in HPuHG area from C. E, Percentage of proliferative ECs (percentage of Ki67+ Cdh5+ cells in total Cdh5+ cells) in HPuHG from C. F, Percentage of proliferative ECs (percentage of Ki67+/Cdh5+ cells in total skin Cdh5+ cells) from C G, Percentage of proliferative ECs (percentage of BrdU+ Cdh5+ cells in total Cdh5+ cells) in total skin from PD17-24 BrdU-pulsed CT and Alk1EndoKO mice


A - C , Immunofluorescence staining images of 12 μm skin section from PD17 induced PD20 telogen CT , PD25 early anagen CT , and PD25 telogen Alk1EndoKO mice stained for Endomucin ( EMCN , red ) , CD31 ( green ) , and Dapi ( blue ) . HPuHG region was outlined in yellow dotted lines . D - F , Error bars represent standard deviation . Each bar represents one individual mouse , and each dot represents measurement from one image . N = 3 mice per group , and n = 8 - 9 images per mouse . Restricted maximum likelihood ( REML ) analysis with unbounded variance components was used to calculate p - values and individual mouse was treated as random effect to take into the account of mouse - to - mouse variability . D , Quantification of EMCN + / CD31 + area from images like those in A - C , shown as percentage of BV in the HPuHG . E , Quantification of absolute EMCN + area ( e . g . BV area ) in HPuHG in images like those in A - C . F , Quantification of EMCN - / CD31 + area from images like those in A - C as LV area in the HPuHG .

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

A-C, Immunofluorescence staining images of 12 μm skin section from PD17 induced PD20 telogen CT, PD25 early anagen CT, and PD25 telogen Alk1EndoKO mice stained for Endomucin (EMCN, red), CD31 (green), and Dapi (blue). HPuHG region was outlined in yellow dotted lines. D-F, Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice per group, and n = 8-9 images per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used to calculate p-values and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability. D, Quantification of EMCN+/CD31+ area from images like those in A-C, shown as percentage of BV in the HPuHG. E, Quantification of absolute EMCN+ area (e.g. BV area) in HPuHG in images like those in A-C. F, Quantification of EMCN-/CD31+ area from images like those in A-C as LV area in the HPuHG.


G - J , Maximum projection through optical Z - stacks of 60 μm skin sections from mice TM - induced at PD17 and sacrificed at PD20 telogen CT , PD25 early anagen CT , PD32 Anagen CT , and PD25 telogen Alk1EndoKO mice stained for VEGFR3 ( red ) , LYVE1 ( green ) , and Dapi ( blue ) . Lymphatic capillaries were outlined in white dotted lines . K , Quantification of lymphatic capillary caliber from images like those in G - J in PD20 telogen CT , PD25 early anagen CT , and PD25 telogen Alk1EndoKO mice Error bars represent standard deviation . Each bar represents one individual mouse , and each dot represents measurement from one lymphatic capillary . N = 3 mice per group , and n = 9 - 25 measurements per mouse . Restricted maximum likelihood ( REML ) analysis with unbounded variance components was used to calculate p - values and individual mouse was treated as random effect to take into the account of mouse - to - mouse variability .

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

G-J, Maximum projection through optical Z-stacks of 60 μm skin sections from mice TM-induced at PD17 and sacrificed at PD20 telogen CT, PD25 early anagen CT, PD32 Anagen CT, and PD25 telogen Alk1EndoKO mice stained for VEGFR3 (red), LYVE1 (green), and Dapi (blue). Lymphatic capillaries were outlined in white dotted lines. K, Quantification of lymphatic capillary caliber from images like those in G-J in PD20 telogen CT, PD25 early anagen CT, and PD25 telogen Alk1EndoKO mice Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one lymphatic capillary. N = 3 mice per group, and n = 9-25 measurements per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used to calculate p-values and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability.


B , Immunofluorescence image of 12 μm skin section from mice treated as shown in A , stained for VEGFR3 ( green ) and Dapi ( blue ) . C , Quantification of tdTomato + / VEGFR3 + area from images like those in B , shows high induction efficiency . Error bars represent standard deviation . Each bar represents one individual mouse , and each dot represents measurement from one image . N = 3 mice , and n = 7 - 8 images per mouse .

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

B, Immunofluorescence image of 12 μm skin section from mice treated as shown in A, stained for VEGFR3 (green) and Dapi (blue). C, Quantification of tdTomato+/VEGFR3+ area from images like those in B, shows high induction efficiency. Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice, and n = 7-8 images per mouse.


F , Quantification of lymphatic capillary caliber from images like those in D in CT and Alk1LymphKO mice Error bars represent standard deviation . Each bar represents one individual mouse , and each dot represents measurement of one lymphatic capillary . N = 3 mice per group , and n = 13 - 16 measurements per mouse . Restricted maximum likelihood ( REML ) analysis with unbounded variance components was used as statistical test and individual mouse was treated as random effect to take into the account of mouse - to - mouse variability .

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

F, Quantification of lymphatic capillary caliber from images like those in D in CT and Alk1LymphKO mice Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement of one lymphatic capillary. N = 3 mice per group, and n = 13-16 measurements per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used as statistical test and individual mouse was treated as random effect to take into the account of mouse-to-mouse variability.


G , Immunofluorescence images of 12 μm skin section of PD17 induced PD25 CT and Alk1LymphKO mice stained for Ki67 ( red ) , Itga6 ( green ) and Dapi ( blue ) showing representative hair follicle . Hair follicles were outlined with yellow dotted lines . Note Ki67 + cells in the hair germ ( HG ) area indicative of HFSC activation . Telogen hair follicle is defined as no Ki67 in the hair germ . Anagen I is defined as ≤ 7 Ki67 + cells in the unenlarged hair germ . Anagen II is defined as > 7 Ki67 + cells in the hair germ or enlarged hair germ with changed shape that starts to enclose the DP . Experiments were repeated at least 2 times with at least 3 skin sections / each mouse analyzed with N = 6 / 4 mice / group . See Table 1 for quantification detail .

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

G, Immunofluorescence images of 12 μm skin section of PD17 induced PD25 CT and Alk1LymphKO mice stained for Ki67 (red), Itga6 (green) and Dapi (blue) showing representative hair follicle. Hair follicles were outlined with yellow dotted lines. Note Ki67+ cells in the hair germ (HG) area indicative of HFSC activation. Telogen hair follicle is defined as no Ki67 in the hair germ. Anagen I is defined as ≤7 Ki67+ cells in the unenlarged hair germ. Anagen II is defined as >7 Ki67+ cells in the hair germ or enlarged hair germ with changed shape that starts to enclose the DP. Experiments were repeated at least 2 times with at least 3 skin sections/each mouse analyzed with N = 6/4 mice/group. See Table 1 for quantification detail.


B , Immunofluorescence image of 12 μm skin section from CT and Alk1EndoKO mice used for RNA - seq stained for Ki67 ( red ) and Dapi ( blue ) show that HFSCs are in quiescence .

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

B, Immunofluorescence image of 12 μm skin section from CT and Alk1EndoKO mice used for RNA-seq stained for Ki67 (red) and Dapi (blue) show that HFSCs are in quiescence.


C , Images of 20 μm skin section of PD20 and PD25 BMP4 - LacZ ; Alk1f / f ; Cdh5 - CreERT2 negative , injected with tamoxifen TM at PD17 ( labelled as CT ) , and PD20 / PD25 Cdh5CreERT2 ; Alk1f / f ; BMP4 - LacZ mice ( labelled as Alk1EndoKO ) TM injected at PD17 , all stained for CD31 ( IHC method , brown ) and X - gal ( blue ) . Hair follicles ( HF ) were outlined with black dotted lines . Arrows point to X - Gal + vascular structure in HPuHG area .

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

C, Images of 20 μm skin section of PD20 and PD25 BMP4-LacZ; Alk1f/f; Cdh5-CreERT2 negative, injected with tamoxifen TM at PD17 (labelled as CT), and PD20/PD25 Cdh5CreERT2; Alk1f/f; BMP4-LacZ mice (labelled as Alk1EndoKO) TM injected at PD17, all stained for CD31 (IHC method, brown) and X-gal (blue). Hair follicles (HF) were outlined with black dotted lines. Arrows point to X-Gal+ vascular structure in HPuHG area.


G - J , Immunofluorescence images of 12 μm skin section of PD17 induced stained for ( Top panel ) Ki67 ( red ) , Itga6 ( green ) , and Dapi ( blue ) and ( Bottom panel ) CD31 ( red ) and Dapi ( blue ) . HPuHG region was outlined with white dotted lines .

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

G-J, Immunofluorescence images of 12 μm skin section of PD17 induced stained for (Top panel) Ki67 (red), Itga6 (green), and Dapi (blue) and (Bottom panel) CD31 (red) and Dapi (blue). HPuHG region was outlined with white dotted lines.


K , Quantification of hair follicle percentage of PD17 - induced PD25 CT , Alk1EndoKO , Alk1 BMP4dKO , and BMP4EndoKO at telogen , anagen I , and anagen II stages . Telogen hair follicle is defined as no Ki67 in the hair germ . Anagen I is defined as ≤ 7 Ki67 + cells in the unenlarged hair germ . Anagen II is defined as > 7 Ki67 + cells in the hair germ or enlarged hair germ with changed shape that starts to enclose the DP . P - value was generated from Chi - square test . See Table 1 for quantification detail .

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

K, Quantification of hair follicle percentage of PD17-induced PD25 CT, Alk1EndoKO, Alk1 BMP4dKO, and BMP4EndoKO at telogen, anagen I, and anagen II stages. Telogen hair follicle is defined as no Ki67 in the hair germ. Anagen I is defined as ≤7 Ki67+ cells in the unenlarged hair germ. Anagen II is defined as >7 Ki67+ cells in the hair germ or enlarged hair germ with changed shape that starts to enclose the DP. P-value was generated from Chi-square test. See Table 1 for quantification detail.


( A ) Laser injured DRG axons showing growth cone regeneration ( upper panels ) and regenerative failure ( lower panels ) . Arrows as indicated .

O O O O O B-TISSUE B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O

(A) Laser injured DRG axons showing growth cone regeneration (upper panels) and regenerative failure (lower panels). Arrows as indicated.


( B ) Upper graph shows percentage of regenerating axons of DIV 1 - 2 adult DRG neurons in the presence of p110 inhibitors , 2 h after injury . Lower graph shows time taken to regenerate a new growth cone . Inhibitors were added 1hr before axons were injured , and maintained throughout : LY294002 , 20 μM . A66 5 μM . XL - 147 5 μM . TGX221 500 nM . IC - 87114 10μM . Idelalisib 500 nM . Upper graph data are shown as the mean + / - SEM . P values indicate statistical significance analysed by Fishers exact ( upper graph ) or Kruskal - Wallis test ( lower graph ) .

O O O O O O O O O B-SUBCELLULAR O O O O O O B-TISSUE B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Upper graph shows percentage of regenerating axons of DIV 1-2 adult DRG neurons in the presence of p110 inhibitors, 2 h after injury. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: LY294002, 20 μM. A66 5 μM. XL-147 5 μM. TGX221 500 nM. IC-87114 10μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance analysed by Fishers exact (upper graph) or Kruskal-Wallis test (lower graph).


( C ) Examples of static vs . growing uninjured axons treated with p110α or δ inhibitors as indicated . Both arrows in bottom right image indicate growing axons .

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(C) Examples of static vs. growing uninjured axons treated with p110α or δ inhibitors as indicated. Both arrows in bottom right image indicate growing axons.


( D ) Percentage of uninjured , growing axons in the presence of p110 inhibitors . Inhibitors were added as for ( B ) . Lower graph shows length grown in 2 h . Data are shown as the mean + / - SEM . P values indicate significance measured by Fishers exact test ( upper graph ) or ANOVA with Tukey ' s post - hoc analysis ( lower graph ) .

O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Percentage of uninjured, growing axons in the presence of p110 inhibitors. Inhibitors were added as for (B). Lower graph shows length grown in 2 h. Data are shown as the mean +/- SEM. P values indicate significance measured by Fishers exact test (upper graph) or ANOVA with Tukey's post-hoc analysis (lower graph).


( E ) Adult DRG neurons in microfluidic compartmental chambers . Cell bodies on the left side , axons extending through microchannels on the right . Lower panels are schematics .

O O O O B-TISSUE B-CELL_TYPE O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O

(E) Adult DRG neurons in microfluidic compartmental chambers. Cell bodies on the left side, axons extending through microchannels on the right. Lower panels are schematics.


( F ) Upper graph shows percentage of regenerating axons in microfluidic chambers . p110α or δ inhibitors were applied to either soma or axons . Lower graph shows time taken to regenerate a new growth cone . Inhibitors were added 1hr before axons were injured , and maintained throughout : A66 5 μM . Idelalisib 500 nM . Upper graph data are shown as the mean + / - SEM . P values indicate statistical significance as measured by Fishers exact test ( upper graph ) or by Kruskal - Wallis test ( lower graph ) .

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

(F) Upper graph shows percentage of regenerating axons in microfluidic chambers. p110α or δ inhibitors were applied to either soma or axons. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: A66 5 μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance as measured by Fishers exact test (upper graph) or by Kruskal-Wallis test (lower graph).


( A ) DIV 3 cortical neurons immunolabelled for PIP3 . Spectrum heatmap shows fluorescence intensity . ( B ) DIV 8 cortical neuron transfected with GFP and immunolabelled for PIP3 . Spectrum heatmap shows fluorescence intensity . ( C ) DIV 16 cortical neuron transfected GFP and immunolabelled for PIP3 . Spectrum heatmap shows fluorescence intensity . ( D ) Somatic PIP3 quantification in the soma at increasing days in vitro . Data are shown as the mean + / - SEM . P values are significance measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 neurons . ( E ) Growth cone PIP3 quantification at increasing days in vitro . Data are shown as the mean + / - SEM . P values indicate significance measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 growth cones .

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

(A) DIV 3 cortical neurons immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (B) DIV 8 cortical neuron transfected with GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (C) DIV 16 cortical neuron transfected GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (D) Somatic PIP3 quantification in the soma at increasing days in vitro. Data are shown as the mean +/- SEM. P values are significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons. (E) Growth cone PIP3 quantification at increasing days in vitro. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones.


( A ) PIP3 immunofluorescence in the soma of DIV 16 cortical neurons expressing p110 isoforms and GFP . Spectrum heatmap shows fluorescence intensity . ( B ) PIP3 immunofluorescence at the axonal growth cone of DIV 16 neurons expressing p110 isoforms and GFP . Spectrum heatmap shows fluorescence intensity . ( C ) Quantification of PIP3 immunofluorescence in the soma . Data are shown as the mean + / - SEM . P values indicate significance as measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 neurons . ( D ) Graph showing PIP3 quantification of immunofluorescence at the axon growth cone . Data are shown as the mean + / - SEM . P values indicate significance measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 growth cones .

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

(A) PIP3 immunofluorescence in the soma of DIV 16 cortical neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity. (B) PIP3 immunofluorescence at the axonal growth cone of DIV 16 neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity. (C) Quantification of PIP3 immunofluorescence in the soma. Data are shown as the mean +/- SEM. P values indicate significance as measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons. (D) Graph showing PIP3 quantification of immunofluorescence at the axon growth cone. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones.


( A ) DIV 4 cortical neurons expressing p110 isoforms and GFP . Arrow marks the axon tip . ( B ) Quantification of axon length . n = 3 experiments , 45 neurons per group . ( C ) Quantification of dendrite length . n = 3 experiments , 45 neurons per group . ( D ) Quantification of the axon : dendrite length ratio . n = 3 experiments , 45 neurons per group .

O O O O O B-TISSUE B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR O O O O O O O O B-CELL_TYPE O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-CELL_TYPE O O O

(A) DIV 4 cortical neurons expressing p110 isoforms and GFP. Arrow marks the axon tip. (B) Quantification of axon length. n = 3 experiments, 45 neurons per group. (C) Quantification of dendrite length. n = 3 experiments, 45 neurons per group. (D) Quantification of the axon:dendrite length ratio. n = 3 experiments, 45 neurons per group.


( E ) DIV 14 cortical neurons expressing p110 isoforms and GFP , immunolabelled for pS6 . ( F ) Sholl analysis of branches . n = 60 neurons per group . Data are shown as the mean + / - SEM . P values indicate significance measured by repeated measure ANOVA with Bonferonni ' s post - hoc test . ( G ) Quantification of the total neurite length . n = 3 experiments , 60 neurons per group . ( H ) Quantification of soma area . n = 3 experiments , 60 neurons per group . ( I ) Quantification of the pS6 immunofluorescence . n = 3 experiments , 60 neurons per group .

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

(E) DIV 14 cortical neurons expressing p110 isoforms and GFP, immunolabelled for pS6. (F) Sholl analysis of branches. n = 60 neurons per group. Data are shown as the mean +/- SEM. P values indicate significance measured by repeated measure ANOVA with Bonferonni's post-hoc test. (G) Quantification of the total neurite length. n = 3 experiments, 60 neurons per group. (H) Quantification of soma area. n = 3 experiments, 60 neurons per group. (I) Quantification of the pS6 immunofluorescence. n = 3 experiments, 60 neurons per group.


( A ) Axotomised DIV 14 - 16 rat cortical neurons expressing p110δ and GFP or GFP alone . Axons were cut > 1000 μm from the soma imaged for 14 h . Black arrows mark the cut site , white arrows marks the axon tip . See also Movies EV7 and EV8 . ( B ) Percentage of regenerating axons 14 h after laser axotomy . Numbers on bars are injured axons per group , from 4 experiments . ( C ) Quantification of axon regeneration length 14 h post - axotomy . n = 9 axons for GFP , 9 for p110α , 25 for p110αH1047R and 27 for p110δ from 4 experiments . ( D ) Quantification of time to start of regeneration . n = 9 axons for GFP , 9 for p110α , 25 for p110αH1047R and 27 for p110δ from 4 experiments .

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

(A) Axotomised DIV 14-16 rat cortical neurons expressing p110δ and GFP or GFP alone. Axons were cut >1000 μm from the soma imaged for 14 h. Black arrows mark the cut site, white arrows marks the axon tip. See also Movies EV7 and EV8. (B) Percentage of regenerating axons 14 h after laser axotomy. Numbers on bars are injured axons per group, from 4 experiments. (C) Quantification of axon regeneration length 14 h post-axotomy. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments. (D) Quantification of time to start of regeneration. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments.


( E ) Axotomised human embryonic stem cell neurons p110δ and GFP or GFP alone . Black arrow marks the cut site , white arrows mark the axon tip . ( F ) Percentage of regenerating axons of human hESC neurons , Numbers on bars are injured axons per group , from 5 experiments . ( G ) Quantification of axon regeneration length . n = 22 axons for GFP , 44 for p110δ , from 5 experiments . Data are shown as the mean + / - SEM . Data were analysed by two tailed Student ' s T - test . ( H ) Quantification of time to start of regeneration . n = 22 axons for GFP , 44 for p110δ , from 5 experiments . Data are shown as the mean + / - SEM . Data were analysed by two tailed Student ' s T - test .

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

(E) Axotomised human embryonic stem cell neurons p110δ and GFP or GFP alone. Black arrow marks the cut site, white arrows mark the axon tip. (F) Percentage of regenerating axons of human hESC neurons, Numbers on bars are injured axons per group, from 5 experiments. (G) Quantification of axon regeneration length. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test. (H) Quantification of time to start of regeneration. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test.


( A ) Percentage of regenerating axons after laser axotomy of DIV 14 - 17 rat cortical neurons expressing either GFP ( control ) or GFP and p110δ in the presence of the indicated inhibitors , 14h after laser injury . Numbers on bars are injured axons per group .

O O O O O O B-SUBCELLULAR O O O O O O O O B-ORGANISM B-TISSUE B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O

(A) Percentage of regenerating axons after laser axotomy of DIV 14-17 rat cortical neurons expressing either GFP (control) or GFP and p110δ in the presence of the indicated inhibitors, 14h after laser injury. Numbers on bars are injured axons per group.


( B ) Single confocal section through a DIV 16 neuron expressing p110δ and GFP ( upper panels ) . Green colour is GFP , red is overexpressed p110δ , detected with anti - p110δ . Lower panels show p110δ and GFP in a distal axon section , and at the growth cone . Arrow indicates the axon .

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

(B) Single confocal section through a DIV 16 neuron expressing p110δ and GFP (upper panels). Green colour is GFP, red is overexpressed p110δ, detected with anti-p110δ. Lower panels show p110δ and GFP in a distal axon section, and at the growth cone. Arrow indicates the axon.


( C ) Active ARF and total ARF6 ( red ) in axons of DIV 16 rat cortical neurons expressing GFP or GFP and p110δ , as indicated . Arrows indicate axons . ( D ) Quantification of mean axonal ARF activity and total ( mean ) ARF6 in DIV 16 neurons expressing GFP or GFP and p110δ n = 60 axons for each condition .

O O O O B-GENEPROD O O B-GENEPROD O O O O B-SUBCELLULAR O O O B-ORGANISM B-TISSUE B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O

(C) Active ARF and total ARF6 (red) in axons of DIV 16 rat cortical neurons expressing GFP or GFP and p110δ, as indicated. Arrows indicate axons. (D) Quantification of mean axonal ARF activity and total (mean) ARF6 in DIV 16 neurons expressing GFP or GFP and p110δ n=60 axons for each condition.


( E ) Kymographs showing dynamics of α9 integrin - GFP in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . ( F ) Quantification of α9 integrin - GFP dynamics in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n numbers are axon sections analysed for each condition . ( G ) Quantification of total α9 integrin - GFP number in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n = 43 for control , 47 for co - transfected with p110δ . n numbers are axon sections analysed for each condition .

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

(E) Kymographs showing dynamics of α9 integrin-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. (F) Quantification of α9 integrin-GFP dynamics in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n numbers are axon sections analysed for each condition. (G) Quantification of total α9 integrin-GFP number in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n=43 for control, 47 for co-transfected with p110δ. n numbers are axon sections analysed for each condition.