Figure 2A . Phosphorylated Y600 of LORE was identified by LC - MS / MS . MBP - tagged LORE - CD was used for in vitro kinase assays . Trypsin digested recombinant proteins were subjected to LC - MS / MS analysis . The doubly - charged peptide with m / z 476 . 24 matches GLLYLHR of LORE with Y600 phosphorylation . Fragment ions retaining charges in the N and C terminus were denoted by " b " and " y " , respectively . pY600 indicated phosphorylated Y600 . B . Y600 mutation reduced LORE kinase activity . MBP - tagged LORE - CD and LORE - Y600F - CD ( Y600 tyrosine was substituted with phenylalanine , F ) were subjected to kinase reactions . The kinase dead mutant LOREK516E served as a negative control . Autophosphorylated LORE was visualized by autoradiography following SDS - PAGE gel electrophoresis . Protein abundance was stained by CBB . C . LORE Y600 is phosphorylated in plants . Two - week - old pLORE : LORE - FLAG complementation plant leaves were subjected to immunoprecipitation ( IP ) with anti - FLAG agarose beads . Y600 specific phosphorylation antibody was used to detect Y600 phosphorylation in LORE . The experiment was repeated three times with similar results . D . 3 - OH - C10 : 0 induced Y600 phosphorylation . Two - week - old pLORE : LORE - FLAG complementation plants were treated with 1 μM 3 - OH - C10 : 0 at indicated times . Plant leaves were subjected to anti - FLAG IP . Phosphorylation level of Y600 was detected by anti - pY600 antibody . The experiment was repeated three times with similar results . E . Y600 mutation abolished the ROS burst triggered by 3 - OH - C10 : 0 . The transgenic plants were treated with 3 - OH - C10 : 0 and ROS burst was measured . WT ( 6 - 1 ) and WT ( 10 - 3 ) are two independent complementation lines with LORE ; Y600F ( 2 - 6 ) and Y600F ( 7 - 1 ) are two independent lines complemented with LOREY600F ; and kinase - dead LOREK516E was transformed to lore mutant as a negative control . The 2 - 3 and 5 - 2 are two independent lines . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) . F . Y600 is required for growth inhibition by 3 - OH - C10 : 0 . The Arabidopsis seedlings were grown on ½ MS containing 2 . 5 μM 3 - OH - C10 : 0 for six days , and the root length was measured . The experiment was repeated three times with similar results . WT ( 6 - 1 ) and Y600 ( 2 - 6 ) lines were used for the assay . Scale bar = 0 . 4 cm . Values are means ± SD ( n = 3 biological replicates ) . ( ANOVA , P < 0 . 01 ) . G . Y600 mutation decreased the disease resistance to Pst DC3000 . Plants were spray - inoculated with Pst DC3000 at a concentration of 1 × 109 cfu / ml , and were subjected to bacterial growth curve analysis at 3 dpi . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) .

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

Figure 2A. Phosphorylated Y600 of LORE was identified by LC-MS/MS. MBP-tagged LORE-CD was used for in vitro kinase assays. Trypsin digested recombinant proteins were subjected to LC-MS/MS analysis. The doubly-charged peptide with m/z 476.24 matches GLLYLHR of LORE with Y600 phosphorylation. Fragment ions retaining charges in the N and C terminus were denoted by "b" and "y", respectively. pY600 indicated phosphorylated Y600.B. Y600 mutation reduced LORE kinase activity. MBP-tagged LORE-CD and LORE-Y600F-CD (Y600 tyrosine was substituted with phenylalanine, F) were subjected to kinase reactions. The kinase dead mutant LOREK516E served as a negative control. Autophosphorylated LORE was visualized by autoradiography following SDS-PAGE gel electrophoresis. Protein abundance was stained by CBB.C. LORE Y600 is phosphorylated in plants. Two-week-old pLORE:LORE-FLAG complementation plant leaves were subjected to immunoprecipitation (IP) with anti-FLAG agarose beads. Y600 specific phosphorylation antibody was used to detect Y600 phosphorylation in LORE. The experiment was repeated three times with similar results.D. 3-OH-C10:0 induced Y600 phosphorylation. Two-week-old pLORE:LORE-FLAG complementation plants were treated with 1 μM 3-OH-C10:0 at indicated times. Plant leaves were subjected to anti-FLAG IP. Phosphorylation level of Y600 was detected by anti-pY600 antibody. The experiment was repeated three times with similar results.E. Y600 mutation abolished the ROS burst triggered by 3-OH-C10:0. The transgenic plants were treated with 3-OH-C10:0 and ROS burst was measured. WT(6-1) and WT(10-3) are two independent complementation lines with LORE; Y600F(2-6) and Y600F(7-1) are two independent lines complemented with LOREY600F; and kinase-dead LOREK516E was transformed to lore mutant as a negative control. The 2-3 and 5-2 are two independent lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).F. Y600 is required for growth inhibition by 3-OH-C10:0. The Arabidopsis seedlings were grown on ½ MS containing 2.5 μM 3-OH-C10:0 for six days, and the root length was measured. The experiment was repeated three times with similar results. WT(6-1) and Y600 (2-6) lines were used for the assay. Scale bar=0.4 cm. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01).G. Y600 mutation decreased the disease resistance to Pst DC3000. Plants were spray-inoculated with Pst DC3000 at a concentration of 1×109 cfu/ml, and were subjected to bacterial growth curve analysis at 3 dpi. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).


Figure 3A . LORE interacts with specific RLCKs ( PBL34 / 35 / 36 ) in yeast . BD - LORE - CD and AD - PBLs were co - transformed to yeast cells and were screened on synthetic dextrose media lacking leucine and tryptophan ( SD / - Leu - Trp ) . The single yeast colonies were serially diluted onto SD / - Leu - Trp and SD / - Leu - Trp - His ( SD media lacking leucine , tryptophan and histidine ) to observe yeast cell growth . 3 - AT ( 20 mM ) was used to inhibit the autoactivation of LORE . Yeast co - transformed with pGADT7 - T + pGBKT7 - 53 ( + ) served as a positive control , and the yeast co - transformed with pGADT7 - T + pGBKT7 - lam ( - ) and AD - PBL39 + BD - LORE served as the negative controls . EV , empty vector . B . LORE interacts with PBLs by MBP pull - down assays . The recombinant GST - LORE - CD and the MBP - PBLs were subjected to MBP pull - down analysis . MBP served as a negative control . The interacting proteins were revealed by anti - GST immunoblotting . The experiment was repeated three times with similar results . C . Co - immunoprecipitation ( Co - IP ) of LORE with PBLs . 35S promoter - driven LORE - T7 and PBLs - FLAG were transiently co - expressed in N . benthamiana by agroinfiltration . Plant leaves were immunoprecipated with anti - FLAG beads and the proteins were immunoblotted by anti - FLAG antibody . Co - IP proteins were immunoblotted by anti - T7 antibody . The experiment was repeated three times with similar results . D . LORE interacts with PBLs , revealed by split - luciferase assays . N . benthamiana leaves were co - infiltrated with Agrobacteria carrying 35S : LORE - nLUC and 35S : cLUC - PBLs . Images of chemiluminescence were obtained by applying 0 . 5 mM luciferin 36 h post infiltration . Similar results were observed in three biological replicates .

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

Figure 3A. LORE interacts with specific RLCKs (PBL34/35/36) in yeast. BD-LORE-CD and AD-PBLs were co-transformed to yeast cells and were screened on synthetic dextrose media lacking leucine and tryptophan (SD/-Leu-Trp). The single yeast colonies were serially diluted onto SD/-Leu-Trp and SD/-Leu-Trp-His (SD media lacking leucine, tryptophan and histidine) to observe yeast cell growth. 3-AT(20 mM) was used to inhibit the autoactivation of LORE. Yeast co-transformed with pGADT7-T+pGBKT7-53(+) served as a positive control, and the yeast co-transformed with pGADT7-T+pGBKT7-lam(-) and AD-PBL39+BD-LORE served as the negative controls. EV, empty vector.B. LORE interacts with PBLs by MBP pull-down assays. The recombinant GST-LORE-CD and the MBP-PBLs were subjected to MBP pull-down analysis. MBP served as a negative control. The interacting proteins were revealed by anti-GST immunoblotting. The experiment was repeated three times with similar results. C. Co-immunoprecipitation (Co-IP) of LORE with PBLs. 35S promoter-driven LORE-T7 and PBLs-FLAG were transiently co-expressed in N. benthamiana by agroinfiltration. Plant leaves were immunoprecipated with anti-FLAG beads and the proteins were immunoblotted by anti-FLAG antibody. Co-IP proteins were immunoblotted by anti-T7 antibody. The experiment was repeated three times with similar results.  D. LORE interacts with PBLs, revealed by split-luciferase assays. N. benthamiana leaves were co-infiltrated with Agrobacteria carrying 35S:LORE-nLUC and 35S:cLUC-PBLs. Images of chemiluminescence were obtained by applying 0.5 mM luciferin 36 h post infiltration. Similar results were observed in three biological replicates.


Figure 4 A . ROS burst in pbl34 / 35 / 36 triple mutant , induced by 3 - OH - C10 : 0 . Two - week - old seedlings were treated with 1 μM 3 - OH - C10 : 0 , and the ROS burst was measured . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) . B . PTI marker gene expression was reduced in pbl34 / 35 / 36 triple mutant in response to 3 - OH - C10 : 0 . Treatment is same as in A . Samples were collected for RT - qPCR at 4 h post infiltration . Values are means ± SD ( n = 3 biological replicates ) . ( Student ' s t - test , * * P < 0 . 01 ) . C . pbl34 / 35 / 36 triple mutant displayed decreased callose depositions in response to 3 - OH - C10 : 0 treatment . Two - week - old seedlings were sprayed with 1 μM 3 - OH - C10 : 0 . The leaves were sampled for callose deposition assays 12 hrs later . The stained callose was counted for 1 mm2 of the leaves . Mock is solvent control . Scale bar = 100μm . Values are means ± SD ( n = 6 leaves ) . ( Student ' s t - test , * * P < 0 . 01 ) . D . Root length of pbl34 / 35 / 36 triple mutant treated with 3 - OH - C10 : 0 . The Arabidopsis seedlings were grown on ½ MS containing 2 . 5 μM 3 - OH - C10 : 0 for six days , and the root length was measured . Scale bar = 0 . 4cm . Values are means ± SD ( n = 3 biological replicates ) . ( ANOVA , P < 0 . 01 ) . E . Disease phenotype of pbl34 / 35 double , pbl34 / 35 / 36 triple mutants and PBL34 overexpression plants . 35S : PBL34 - FLAG was introduced to the Col - 0 to generate the overexpression lines . The homozygous transgenic plants were used for the assays . The plants were sprayed with Pst DC3000 at a concentration of 1 × 109 cfu / ml . Plants were subjected to bacterial growth curve analysis at 3 dpi . OE - 3 # and OE - 11 # are two independent transgenic lines . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) . F . pbl34 / 35 / 36 triple mutant phenotype was rescued by PBL34 . PBL34 - HA was introduced to the triple mutant driven by its native promoter . Four - week - old homozygous seedlings were inoculated with 5 × 104 cfu / ml Pst DC3000 , and the leaves were subjected to bacterial growth curve analysis at 3 dpi . CM2 - 5 and CM7 - 3 are two independent transgenic lines . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) .

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

Figure 4 A. ROS burst in pbl34/35/36 triple mutant, induced by 3-OH-C10:0. Two-week-old seedlings were treated with 1 μM 3-OH-C10:0, and the ROS burst was measured. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).  B. PTI marker gene expression was reduced in pbl34/35/36 triple mutant in response to 3-OH-C10:0. Treatment is same as in A. Samples were collected for RT-qPCR at 4 h post infiltration. Values are means ± SD (n=3 biological replicates). (Student's t-test, **P< 0.01).  C. pbl34/35/36 triple mutant displayed decreased callose depositions in response to 3-OH-C10:0 treatment. Two-week-old seedlings were sprayed with 1 μM 3-OH-C10:0. The leaves were sampled for callose deposition assays 12 hrs later. The stained callose was counted for 1 mm2 of the leaves. Mock is solvent control. Scale bar=100μm.Values are means ± SD (n=6 leaves). (Student's t-test, **P< 0.01).  D. Root length of pbl34/35/36 triple mutant treated with 3-OH-C10:0. The Arabidopsis seedlings were grown on ½ MS containing 2.5 μM 3-OH-C10:0 for six days, and the root length was measured. Scale bar=0.4cm. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01).  E. Disease phenotype of pbl34/35 double, pbl34/35/36 triple mutants and PBL34 overexpression plants. 35S:PBL34-FLAG was introduced to the Col-0 to generate the overexpression lines. The homozygous transgenic plants were used for the assays. The plants were sprayed with Pst DC3000 at a concentration of 1×109 cfu/ml. Plants were subjected to bacterial growth curve analysis at 3 dpi. OE-3# and OE-11# are two independent transgenic lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).  F. pbl34/35/36 triple mutant phenotype was rescued by PBL34. PBL34-HA was introduced to the triple mutant driven by its native promoter. Four-week-old homozygous seedlings were inoculated with 5×104 cfu/ml Pst DC3000 , and the leaves were subjected to bacterial growth curve analysis at 3 dpi. CM2-5 and CM7-3 are two independent transgenic lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).


Figure 5 A . PBL34 was phosphorylated by LORE in vitro . Purified recombinant proteins were used for in vitro kinase assays . The kinase dead mutant MBP - PBL34 * ( PBL K180 was substituted to E , predicted by https : / / www . uniprot . org / uniprot / ) , was used as the substrate of GST - LORE - CD or its kinase dead variant , GST - LORE - CD * ( LOREK516E ) . MBP served as a negative control . Incorporation of isotope labeled phosphates was visualized by 32P autoradiography . The CBB staining showing protein abundance . B . Mutation of PBL34 T306 / T310 significantly reduced its phosphorylation by LORE . Recombinant kinase dead PBL34 * and PBL34 * - 2A ( T306 / T310 was mutated to A ) were incubated with LORE - CD . Phosphorylation of PBL34 was visualized by 32P autoradiography . Protein abundance was revealed by CBB staining . C . Mutation at Y600 diminished trans - phosphorylation activity of LORE . Recombinant LORE - CD and LOREY600F - CD were used for the trans - phosphorylation assays . Phosphorylation of PBL34 was visualized by 32P autoradiography . Protein abundance was revealed by CBB staining . D . PBL34 phosphorylation is induced by 3 - OH - C10 : 0 . The protoplast of Col - 0 was transformed with 35S : PBL34 - FLAG and 35S : PBL34 - 2A - FLAG , respectively . Sixteen hours later , the protoplasts were treated with1 μM 3 - OH - C10 : 0 . Phosphorylated proteins were immunoprecipitated and detected by anti - pS / T antibody . The experiment was repeated three times with similar results . E . 3 - OH - C10 : 0 - induced PBL34 phosphorylation depends on LORE . The 35S : PBL34 - FLAG was introduced to Col - 0 and lore , respectively . Four - week - old homozygous transgenic plants were treated with 1 μM 3 - OH - C10 : 0 . Phosphorylated proteins were immunoprecipitated and detected by anti - pS / T antibody . The experiment was repeated three times with similar results . F . PBL34 dissociated from LORE upon 3 - OH - C10 : 0 activation . The 35S : LORE - FLAG plants were transformed with native promoter driven PBL34 ( pPBL34 : PBL34 - HA ) . 100 nM flg22 or 1 μM 3 - OH - C10 : 0 was sprayed onto plant leaves , and the leaves were sampled for assays15 min later . The experiment was repeated three times with similar results . G . Phosphorylation of PBL34K180E did not disassociate from LORE . The 35S : LORE - FLAG protoplasts were transfected with 35S : PBL34K180E - HA . 100nM flg22 or 1 μM 3 - OH - C10 : 0 was used to treat the protoplasts . The protoplasts were sampled 15 min later . The experiment was repeated three times with similar results . H . PBL34 rescues pbl34 / 35 / 36 triple mutant ROS burst in response to 3 - OH - C10 : 0 . Treatment was same as in ( E ) . pPBL34 : PBL34 - HA was introduced to pbl34 / 35 / 36 triple mutant , and the homozygous transgenic line was used for ROS burst assays . Values are means ± SD ( n = 6 biological replicates ) . ( ANOVA , P < 0 . 01 ) . I . PTI marker gene expression in PBL34 complementation lines . The PTI marker genes FRK1 and NHL10 expression was examined by RT - qPCR in Col - 0 , pbl34 / 35 / 36 , and CM - PBL34 - 2A lines after 3 - OH - C10 : 0 treatments for 4 hrs . Values are means ± SD ( n = 6 ; ANOVA ; p < 0 . 01 ) . J . PBL34 phosphorylation at T306 / T310 is essential for disease resistance . pbl34 / 35 / 36 triple mutants were complemented with PBL34 or PBL34 - 2A driven by its native promoter . The homozygous plants were pretreated with 3 - OH - C10 : 0 ( C10 ) 1 d before pathogen inoculation . The plants were inoculated with 2 × 105 cfu / ml Pst DC3000 , and the bacterial titer was analyzed at 3 dpi . Mock is solvent control . Letters indicate the difference from Col - 0 . Values are means ± SD ( n = 6 ; ANOVA ; p < 0 . 01 ) .

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

Figure 5 A. PBL34 was phosphorylated by LORE in vitro. Purified recombinant proteins were used for in vitro kinase assays. The kinase dead mutant MBP-PBL34* (PBL K180 was substituted to E, predicted by https://www.uniprot.org/uniprot/), was used as the substrate of GST-LORE-CD or its kinase dead variant, GST-LORE-CD* (LOREK516E). MBP served as a negative control. Incorporation of isotope labeled phosphates was visualized by 32P autoradiography. The CBB staining showing protein abundance.  B. Mutation of PBL34 T306/T310 significantly reduced its phosphorylation by LORE. Recombinant kinase dead PBL34* and PBL34*-2A (T306/T310 was mutated to A) were incubated with LORE-CD. Phosphorylation of PBL34 was visualized by 32P autoradiography. Protein abundance was revealed by CBB staining.  C. Mutation at Y600 diminished trans-phosphorylation activity of LORE. Recombinant LORE-CD and LOREY600F-CD were used for the trans-phosphorylation assays. Phosphorylation of PBL34 was visualized by 32P autoradiography. Protein abundance was revealed by CBB staining.  D. PBL34 phosphorylation is induced by 3-OH-C10:0. The protoplast of Col-0 was transformed with 35S:PBL34-FLAG and 35S:PBL34-2A-FLAG, respectively. Sixteen hours later, the protoplasts were treated with1 μM 3-OH-C10:0. Phosphorylated proteins were immunoprecipitated and detected by anti-pS/T antibody. The experiment was repeated three times with similar results.  E. 3-OH-C10:0-induced PBL34 phosphorylation depends on LORE. The 35S:PBL34-FLAG was introduced to Col-0 and lore, respectively. Four-week-old homozygous transgenic plants were treated with 1 μM 3-OH-C10:0. Phosphorylated proteins were immunoprecipitated and detected by anti-pS/T antibody. The experiment was repeated three times with similar results.  F. PBL34 dissociated from LORE upon 3-OH-C10:0 activation. The 35S:LORE-FLAG plants were transformed with native promoter driven PBL34 (pPBL34:PBL34-HA). 100 nM flg22 or 1 μM 3-OH-C10:0 was sprayed onto plant leaves, and the leaves were sampled for assays15 min later. The experiment was repeated three times with similar results.  G. Phosphorylation of PBL34K180E did not disassociate from LORE. The 35S:LORE-FLAG protoplasts were transfected with 35S:PBL34K180E-HA. 100nM flg22 or 1 μM 3-OH-C10:0 was used to treat the protoplasts. The protoplasts were sampled 15 min later. The experiment was repeated three times with similar results.  H. PBL34 rescues pbl34/35/36 triple mutant ROS burst in response to 3-OH-C10:0. Treatment was same as in (E). pPBL34:PBL34-HA was introduced to pbl34/35/36 triple mutant, and the homozygous transgenic line was used for ROS burst assays. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).  I. PTI marker gene expression in PBL34 complementation lines. The PTI marker genes FRK1 and NHL10 expression was examined by RT-qPCR in Col-0, pbl34/35/36, and CM-PBL34-2A lines after 3-OH-C10:0 treatments for 4 hrs. Values are means ± SD (n=6; ANOVA; p < 0.01).  J. PBL34 phosphorylation at T306/T310 is essential for disease resistance. pbl34/35/36 triple mutants were complemented with PBL34 or PBL34-2A driven by its native promoter. The homozygous plants were pretreated with 3-OH-C10:0(C10) 1 d before pathogen inoculation. The plants were inoculated with 2×105 cfu/ml Pst DC3000, and the bacterial titer was analyzed at 3 dpi. Mock is solvent control. Letters indicate the difference from Col-0. Values are means ± SD (n=6; ANOVA; p < 0.01).


Figure 6 A . LORE interacts with HopAO1 by MBP pull - down assays . The recombinant GST - HopAO1 and the MBP - LORE - CD were subjected to MBP pull - down analysis . MBP and GST proteins served as the negative controls . The interacting proteins were revealed by anti - GST immunoblotting . The experiment was repeated three times with similar results . B . LORE interacts with HopAO1 by Co - IP assays . 35S promoter - driven LORE - T7 and HopAO1 - FLAG were transiently co - expressed in N . benthamiana by agroinfiltration . Plant leaves were immunoprecipated with anti - FLAG beads and immunoblotted by anti - FLAG . Coimmunoprecipitated proteins were immunoblotted by anti - T7 . The experiment was repeated three times with similar results . C . HopAO1 dephosphorylates LORE Y600 in vitro . The recombinant GST - HopAO1 / HopAO1CS and GST - LORE - CD were subjected to in vitro phosphatase assays . Phosphorylation level of Y600 was detected by anti - pY600 immunoblotting . The experiment was repeated three times with similar results . D . HopAO1 dephosphorylates LORE Y600 in N . benthamiana . LORE - FLAG was transiently co - expressed with GUS - T7 , HopAO1 - T7 or HopAO1CS - T7 in N . benthamiana leaves mediated by Agrobacteria . The protein was immunoprecipitated and probed by anti - pY600 antibody . The experiment was repeated at least three times with similar results . E . HopAO1 suppresses 3 - OH - C10 : 0 - triggered ROS burst . HopAO1 was ectopically expressed in Col - 0 driven by estradiol - inducible promoter . 100μM estradiol was sprayed on Est : HopAO1 plants , and the leaf disc was used for ROS burst assays 12 hrs later . Values are means ± SD ( n = 6 biological replicates ) . F . HopAO1 inhibits 3 - OH - C10 : 0 - induced disease resistance . The Est : HopAO1 plants were pretreated with 1 μM 3 - OH - C10 : 0 ( C10 ) and100 μM estradiol , and then the plants were inoculated with 5 × 104 cfu / ml Pst DC3000 24 hrs later . The bacterial titer was analyzed at 3 dpi . Values are means ± SD ( n = 6 ; ANOVA ; p < 0 . 01

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

Figure 6 A. LORE interacts with HopAO1 by MBP pull-down assays. The recombinant GST-HopAO1 and the MBP-LORE-CD were subjected to MBP pull-down analysis. MBP and GST proteins served as the negative controls. The interacting proteins were revealed by anti-GST immunoblotting. The experiment was repeated three times with similar results.  B. LORE interacts with HopAO1 by Co-IP assays. 35S promoter-driven LORE-T7 and HopAO1-FLAG were transiently co-expressed in N. benthamiana by agroinfiltration. Plant leaves were immunoprecipated with anti-FLAG beads and immunoblotted by anti-FLAG. Coimmunoprecipitated proteins were immunoblotted by anti-T7. The experiment was repeated three times with similar results.  C. HopAO1 dephosphorylates LORE Y600 in vitro. The recombinant GST-HopAO1/HopAO1CS and GST-LORE-CD were subjected to in vitro phosphatase assays. Phosphorylation level of Y600 was detected by anti-pY600 immunoblotting. The experiment was repeated three times with similar results.  D. HopAO1 dephosphorylates LORE Y600 in N. benthamiana. LORE-FLAG was transiently co-expressed with GUS-T7, HopAO1-T7 or HopAO1CS-T7 in N. benthamiana leaves mediated by Agrobacteria. The protein was immunoprecipitated and probed by anti-pY600 antibody. The experiment was repeated at least three times with similar results.   E. HopAO1 suppresses 3-OH-C10:0-triggered ROS burst. HopAO1 was ectopically expressed in Col-0 driven by estradiol-inducible promoter. 100μM estradiol was sprayed on Est:HopAO1 plants, and the leaf disc was used for ROS burst assays 12 hrs later. Values are means ± SD (n=6 biological replicates).  F. HopAO1 inhibits 3-OH-C10:0-induced disease resistance. The Est:HopAO1 plants were pretreated with 1 μM 3-OH-C10:0 (C10) and100 μM estradiol, and then the plants were inoculated with 5×104 cfu/ml Pst DC3000 24 hrs later. The bacterial titer was analyzed at 3 dpi. Values are means ± SD (n=6; ANOVA; p < 0.01


Figure 1 ( A to D ) 10 - to 11 - week - old mice were treated with vehicle control or AT845 at the vector doses indicated and followed for 12 weeks ( N = 7 or 8 per cohort ) . ( A ) Vector copy number , ( B ) GAA levels , ( C ) GAA activity , and ( D ) glycogen content in cardiac and skeletal muscles . ( C to D ) Statistical analysis : two - way ANOVA , Dunnett ' s test . Data are presented as box - and - whisker plots with Tukey whiskers that show minimum , median , and maximum . Asterisks ( * ) indicate significant differences compared with untreated Gaa - / - mice ( black ) or wild - type mice ( red ) . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 . See also Appendix Table S1 .

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

Figure 1(A to D) 10- to 11-week-old mice were treated with vehicle control or AT845 at the vector doses indicated and followed for 12 weeks (N = 7 or 8 per cohort). (A) Vector copy number, (B) GAA levels, (C) GAA activity, and (D) glycogen content in cardiac and skeletal muscles. (C to D) Statistical analysis: two-way ANOVA, Dunnett's test. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum. Asterisks (*) indicate significant differences compared with untreated Gaa-/- mice (black) or wild-type mice (red). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P <0.0001. See also Appendix Table S1.


Figure 2Representative images of H & E ( top ) and PAS ( bottom ) staining of the quadriceps in wild - type and Gaa - / - mice untreated or treated with escalating doses of AT845 ( 3 × 1013 , 1 × 1014 , and 3 × 1014 vg / kg ) . Scale bar , 80 µm . ( See also Appendix Table S1 )

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

Figure 2Representative images of H&E (top) and PAS (bottom) staining of the quadriceps in wild-type and Gaa-/- mice untreated or treated with escalating doses of AT845 (3×1013, 1×1014, and 3×1014 vg/kg). Scale bar, 80 µm. (See also Appendix Table S1)


Figure 3A - D ( A , B ) Body weight curves and ( C , D ) grip response - ability to grip onto an inverted wire screen for a 60 - second period - in wild - type and Gaa - / - mice untreated or treated with escalating doses of AT845 ( 3 × 1013 , 1 × 1014 , and 3 × 1014 vg / kg ) . All groups had n = 9 at all timepoints , except the 6 week timepoint ( females ) and the 12 week timepoint ( males and females ) for the Gaa - / - mice , which had n = 8 per group . Female mice dosed with 3 × 1013 , 1 × 1014 , and 3 × 1014 vg / kg had a 22 % , 11 % , and 0 % failure rate at week 12 , respectively ; in comparison , the female control WT mice had a 22 % failure rate and the control Gaa - / - mice had a 38 % failure . Male mice dosed with 3 × 1013 , 1 × 1014 , and 3 × 1014 vg / kg had a 56 % , 33 % , and 11 % failure rate at week 12 , respectively ; in comparison , the male control WT mice had a 11 % failure rate and the control Gaa - / - mice had a 100 % failure . Bars represent median . Data show a dose - dependent improvement in grip response following treatment with AT845 . Statistical analysis : two - way ANOVA , Dunnett ' s test . Asterisks ( * ) indicate significant differences compared with untreated Gaa - / - mice at each respective time point . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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

Figure 3A-D (A, B) Body weight curves and (C, D) grip response - ability to grip onto an inverted wire screen for a 60-second period - in wild-type and Gaa-/- mice untreated or treated with escalating doses of AT845 (3×1013, 1×1014, and 3×1014 vg/kg). All groups had n=9 at all timepoints, except the 6 week timepoint (females) and the 12 week timepoint (males and females) for the Gaa-/- mice, which had n=8 per group. Female mice dosed with 3×1013, 1×1014, and 3×1014 vg/kg had a 22%, 11%, and 0% failure rate at week 12, respectively; in comparison, the female control WT mice had a 22% failure rate and the control Gaa-/- mice had a 38% failure. Male mice dosed with 3×1013, 1×1014, and 3×1014 vg/kg had a 56%, 33%, and 11% failure rate at week 12, respectively; in comparison, the male control WT mice had a 11% failure rate and the control Gaa-/- mice had a 100% failure. Bars represent median. Data show a dose-dependent improvement in grip response following treatment with AT845. Statistical analysis: two-way ANOVA, Dunnett's test. Asterisks (*) indicate significant differences compared with untreated Gaa-/- mice at each respective time point. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


Figure 4A , B Equal numbers of male and female cynomolgus monkeys were treated with vehicle control ( n = 4 ) or escalating doses AT845 ( n = 6 ) and followed for approximately 12 weeks . ( A ) Vector copy number and ( B ) GAA activity in heart , quadriceps , triceps , diaphragm , and liver . Statistical analysis : mixed - effects analysis with Dunnett ' s test . Data are presented as box - and - whisker plots with Tukey whiskers that show minimum , median , and maximum values . Asterisks ( * ) indicate significant differences compared with vehicle control NHPs . * , P < 0 . 05 ; * * , P < 0 . 01 .

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

Figure 4A, B Equal numbers of male and female cynomolgus monkeys were treated with vehicle control (n=4) or escalating doses AT845 (n=6) and followed for approximately 12 weeks. (A) Vector copy number and (B) GAA activity in heart, quadriceps, triceps, diaphragm, and liver. Statistical analysis: mixed-effects analysis with Dunnett's test. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum values. Asterisks (*) indicate significant differences compared with vehicle control NHPs. *, P<0.05; **, P<0.01.


Figure 5A - C Male cynomolgus monkeys were treated with vehicle control ( n = 2 ) , AT845 ( n = 3 ) at 6 × 1013 , 2 × 1014 , or 5 × 1014 vg / kg , or Cyno - AT845 ( n = 3 ) at 2 × 1014 vg / kg and followed for approximately 12 weeks . ( A ) Anti - GAA antibody titer , ( B ) cardiac troponin I ( cTnI ) , and ( C ) N - terminal pro b - type natriuretic peptide ( BNP ) . Bars show mean and SEM .

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

Figure 5A-C Male cynomolgus monkeys were treated with vehicle control (n=2), AT845 (n=3) at 6×1013, 2×1014, or 5×1014 vg/kg, or Cyno-AT845 (n=3) at 2×1014 vg/kg and followed for approximately 12 weeks. (A) Anti-GAA antibody titer, (B) cardiac troponin I (cTnI), and (C) N-terminal pro b-type natriuretic peptide (BNP). Bars show mean and SEM.


Figure 6A , B Male cynomolgus monkeys were treated with vehicle control ( n = 2 ) , AT845 ( n = 3 ) , or Cyno - AT845 ( n = 3 ) at 2 × 1014 vg / kg and followed for approximately 12 weeks . GAA activity in ( A ) heart , quadriceps , triceps , and diaphragm , and ( B ) liver , brain , spinal cord , and testes . Data are presented as box - and - whisker plots with Tukey whiskers that show minimum , median , and maximum values .

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

Figure 6A, B Male cynomolgus monkeys were treated with vehicle control (n=2), AT845 (n=3), or Cyno-AT845 (n=3) at 2×1014 vg/kg and followed for approximately 12 weeks. GAA activity in (A) heart, quadriceps, triceps, and diaphragm, and (B) liver, brain, spinal cord, and testes. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum values.


Figure 7A , B Western blot analysis of heart and quadriceps lysates using anti - GAA detects both the precursor GAA ( 110 kDa ) and the mature lysosomal GAA ( 70 to 76 kDa ) forms in muscle of NHPs treated with ( A ) AT845 ( n = 3 ) or ( B ) Cyno - AT845 ( n = 3 ) at 2 × 1014 vg / kg . Each tissue was analyzed in triplicate , and each respective symbol indicates use of a vehicle control heart tissue ( ^ ) or a vehicle control quadriceps tissue ( * ) . ( See also Appendix Table S3 )

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

Figure 7A, B Western blot analysis of heart and quadriceps lysates using anti-GAA detects both the precursor GAA (110 kDa) and the mature lysosomal GAA (70 to 76 kDa) forms in muscle of NHPs treated with (A) AT845 (n=3) or (B) Cyno-AT845 (n=3) at 2×1014 vg/kg. Each tissue was analyzed in triplicate, and each respective symbol indicates use of a vehicle control heart tissue (^) or a vehicle control quadriceps tissue (*). (See also Appendix Table S3)


Figure 1Impaired wound closure in αSMA - TK mice . ( A ) IHC for αSMA + cells in wound tissue sections at day 6 post - wounding . Representative images are shown . Scale bar , 50 µm . Black boxes denote zoomed regions displayed in bottom panels . ( B ) Quantification of αSMA + cells at day 6 post - wounding . WT , N = 6 ; TK , N = 5 biological replicates . Two - tailed non - parametric Mann - Whitney test performed . ( F ) Expression levels of ACTA2 in normal and diabetic human wound tissue . RNA - seq reads are derived from ( Davis et al . , 2020 , Data ref : Davis et al , 2020 ) . Normal , N = 3 ; diabetic , N = 4 biological replicates . Data are presented as a box and whisker plot of min to max values . Unpaired t - test performed . ( J - O ) Impaired granulation tissue formation and angiogenesis in αSMA - TK mice . ( J ) Representative H & E images of wound tissue sections at day 17 post - wounding . Granulation areas are marked by black lines , triangles ( open : incomplete closure , filled : closed wounds ) denote the epithelial tongues . Epi , epidermis ; derm , dermis regions denoted . Scale bar : 200 μm . ( K ) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control ( set to 100 % ) . NA ( not assessed ) reflects the lack of granulation in αSMA - TK mice . WT , N = 5 ; TK , N = 4 biological replicates . ( L ) Immunofluorescent staining for CD31 ( red ) and nuclei ( blue ) on wound tissue sections at day 17 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( M ) Quantification of CD31 + cells per visual field normalized to WT mice . WT , N = 3 ; TK , N = 5 biological replicates . ( N ) IHC for hypoxyprobe ( brown ) on wound tissue sections at day 17 post - wounding . Black boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( O ) Quantification of hypoxyprobe area per visual field normalized to WT mice . WT , N = 5 ; TK , N = 6 biological replicates .

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

Figure 1Impaired wound closure in αSMA-TK mice. (A) IHC for αSMA+ cells in wound tissue sections at day 6 post-wounding. Representative images are shown. Scale bar, 50 µm. Black boxes denote zoomed regions displayed in bottom panels. (B) Quantification of αSMA+ cells at day 6 post-wounding. WT, N=6; TK, N=5 biological replicates. Two-tailed non-parametric Mann-Whitney test performed.(F) Expression levels of ACTA2 in normal and diabetic human wound tissue. RNA-seq reads are derived from (Davis et al., 2020, Data ref: Davis et al, 2020). Normal, N=3; diabetic, N=4 biological replicates. Data are presented as a box and whisker plot of min to max values. Unpaired t-test performed.(J-O) Impaired granulation tissue formation and angiogenesis in αSMA-TK mice. (J) Representative H&E images of wound tissue sections at day 17 post-wounding. Granulation areas are marked by black lines, triangles (open: incomplete closure, filled: closed wounds) denote the epithelial tongues. Epi, epidermis; derm, dermis regions denoted. Scale bar: 200 μm. (K) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control (set to 100%). NA (not assessed) reflects the lack of granulation in αSMA-TK mice. WT, N=5; TK, N=4 biological replicates. (L) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (M) Quantification of CD31+ cells per visual field normalized to WT mice. WT, N=3; TK, N=5 biological replicates. (N) IHC for hypoxyprobe (brown) on wound tissue sections at day 17 post-wounding. Black boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (O) Quantification of hypoxyprobe area per visual field normalized to WT mice. WT, N=5; TK, N=6 biological replicates.


Figure 2 ( D ) Representative FACS plots of digested skin from bone marrow ( BM ) transplanted αSMA - RFP mice 17 days post - wounding . ( E ) FACS quantification of αSMA - RFP + cells in wounds of BM transplanted mice . αSMA - RFP with αSMA - RFP BM , N = 2 ; WT with αSMA - RFP BM , N = 3 ; αSMA - RFP with WT BM , N = 4 biological replicates . One - way ANOVA with Tukey ' s multiple comparison test performed comparing WT with αSMA - RFP BM and αSMA - RFP with WT BM to αSMA - RFP with αSMA - RFP BM . ( ( H - J ) Multispectral imaging of changes in fibroblast subsets during wound repair . ( H ) Representative images ( 20x magnification ) of orthogonal wound tissue sections at day 6 post - wounding displaying αSMA , FAP , and FSP1 overlaid with DAPI in the wound bed . Subcutaneous ( subq ) , dermis ( derm ) , epidermis ( epi ) , and scar regions are denoted . Individual αSMA , FAP , and FSP1 channel images overlaid with DAPI . Scale bar , 50 µm . ( I ) Analysis of multiplex immunostaining of full thickness skin wounds performed at indicated time points . Data are presented as the fractional area of each image positive for the indicated markers and negative for CD31 . Day 0 , N = 2 ; Day 6 , N = 4 ; Day 9 , N = 3 ; Day 14 , N = 3 ; Day 21 , N = 3 ; Day 32 , N = 4 biological replicates . One - way ANOVA with Dunnett ' s multiple comparison test performed . ( J ) Percent overlap of αSMA with FAP and FSP1 throughout wound healing . αSMA + denotes cells positive for αSMA but not FAP or FSP1 . Day 0 , N = 2 ; Day 6 , N = 4 ; Day 9 , N = 3 ; Day 14 , N = 3 ; Day 21 , N = 3 ; Day 32 , N = 4 biological replicates . ( L ) Representative H & E images of wound tissue sections at day 17 post - wounding . Granulation areas are marked by black lines . Scale bar , 200 μm . ( M ) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control . WT , N = 3 ; FAP - TK , N = 3 ; WT , N = 17 , FSP1 - TK , N = 9 biological replicates . ( N ) Immunofluorescent staining for CD31 ( red ) and nuclei on wound tissue sections at day 17 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( O ) Quantification of CD31 + cells per visual field normalized to WT mice . WT , N = 4 ; FAP - TK , N = 4 ; WT , N = 3 ; FSP1 - TK , N = 3 biological replicates . ( P ) IHC for hypoxyprobe ( brown ) on wound tissue sections at day 17 post - wounding . Black boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( Q ) Quantification of hypoxyprobe area per visual field normalized to WT mice . WT , N = 3 ; FAP - TK , N = 3 ; WT , N = 3 ; FSP1 - TK , N = 4 biological replicates . ( R )

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

Figure 2(D) Representative FACS plots of digested skin from bone marrow (BM) transplanted αSMA-RFP mice 17 days post-wounding. (E) FACS quantification of αSMA-RFP+ cells in wounds of BM transplanted mice. αSMA-RFP with αSMA-RFP BM, N=2; WT with αSMA -RFP BM, N=3; αSMA-RFP with WT BM, N=4 biological replicates. One-way ANOVA with Tukey's multiple comparison test performed comparing WT with αSMA-RFP BM and αSMA-RFP with WT BM to αSMA-RFP with αSMA-RFP BM. ((H-J) Multispectral imaging of changes in fibroblast subsets during wound repair. (H) Representative images (20x magnification) of orthogonal wound tissue sections at day 6 post-wounding displaying αSMA, FAP, and FSP1 overlaid with DAPI in the wound bed. Subcutaneous (subq), dermis (derm), epidermis (epi), and scar regions are denoted. Individual αSMA, FAP, and FSP1 channel images overlaid with DAPI. Scale bar, 50 µm. (I) Analysis of multiplex immunostaining of full thickness skin wounds performed at indicated time points. Data are presented as the fractional area of each image positive for the indicated markers and negative for CD31. Day 0, N=2; Day 6, N=4; Day 9, N=3; Day 14, N=3; Day 21, N=3; Day 32, N=4 biological replicates. One-way ANOVA with Dunnett's multiple comparison test performed. (J) Percent overlap of αSMA with FAP and FSP1 throughout wound healing. αSMA+ denotes cells positive for αSMA but not FAP or FSP1. Day 0, N=2; Day 6, N=4; Day 9, N=3; Day 14, N=3; Day 21, N=3; Day 32, N=4 biological replicates.(L) Representative H&E images of wound tissue sections at day 17 post-wounding. Granulation areas are marked by black lines. Scale bar, 200 μm. (M) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control. WT, N=3; FAP-TK, N=3; WT, N=17, FSP1-TK, N=9 biological replicates. (N) Immunofluorescent staining for CD31 (red) and nuclei on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (O) Quantification of CD31+ cells per visual field normalized to WT mice. WT, N=4; FAP-TK, N=4; WT, N=3; FSP1-TK, N=3 biological replicates. (P) IHC for hypoxyprobe (brown) on wound tissue sections at day 17 post-wounding. Black boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (Q) Quantification of hypoxyprobe area per visual field normalized to WT mice. WT, N=3; FAP-TK, N=3; WT, N=3; FSP1-TK, N=4 biological replicates. (R) W


Figure 3 ( C ) Enrichment plot of genes in αSMA C1 ( left panel ) . Violin plots for genes enriched in αSMA C1 ( right panels ) . ( D ) Enrichment plot of genes in αSMA C2 ( left panel ) . Violin plots for genes enriched in αSMA C2 ( right panels ) . ( E ) Enrichment plot of genes in αSMA C3 ( left panel ) . Violin plots of enriched genes in the αSMA C3 cluster ( right panels ) . Raw scRNA - seq data are derived from ( Haensel et al . , 2020 , Data ref : Haensel et al 2020 ) .

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

Figure 3(C) Enrichment plot of genes in αSMA C1 (left panel). Violin plots for genes enriched in αSMA C1 (right panels). (D) Enrichment plot of genes in αSMA C2 (left panel). Violin plots for genes enriched in αSMA C2 (right panels). (E) Enrichment plot of genes in αSMA C3 (left panel). Violin plots of enriched genes in the αSMA C3 cluster (right panels). Raw scRNA-seq data are derived from (Haensel et al., 2020, Data ref: Haensel et al 2020). Da


Figure 4 ( A ) IHC for collagen I on wound tissue sections at day 17 post - wounding ( brown stain ) . Black boxes denote zoomed regions depicted in bottom panels . Scale bar : 50 μm . ( B ) Quantification of collagen I positive area normalized to the average area in WT controls . WT , N = 5 ; TK , N = 5 biological replicates . ( C ) Polarized light images of picrosirius red stained wound tissues at day 17 post - wounding . Red : collagen fibers . White boxes denote zoomed regions depicted in bottom panels . Scale bar , 50 µm . ( D ) Quantification of collagen fiber ( picrosirius red ) percent area normalized to the average area in WT controls . WT , N = 3 ; TK , N = 3 biological replicates . ( E - ( E - H ) Reduced collagen deposition in Col1α1cKO wounds . ( E ) IHC for collagen I on wound tissue sections at day 14 post - wounding ( brown stain ) . Black boxes denote zoomed regions depicted in bottom panels . Scale bar : 25 μm . ( F ) Quantification of collagen I positive area normalized to the average area in WT controls . WT , N = 7 ; Col1α1cKO , N = 4 biological replicates . ( G ) Polarized light images of picrosirius red stained wound tissues at day 14 post - wounding . Red : collagen fibers . White boxes denote zoomed regions depicted in bottom panels . Scale bar , 50 µm . ( H ) Quantification of collagen fiber percent area and fiber width normalized to the average area and width , respectively , in WT controls . WT , N = 4 ; Col1α1cKO , N = 5 biological replicates . ( J - M ) Reepithelization and vascularization in WT and Col1α1cKO mice . ( J ) Representative images of keratin 5 ( Krt5 , red ) stained wounds at day 14 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 500 µm . ( K ) Quantification of epidermal thickness . WT , N = 6 ; Col1α1cKO , N = 5 biological replicates . ( L ) Immunofluorescent staining for CD31 ( red ) and nuclei ( blue ) on wound tissue sections at day 14 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 20 μm . ( M ) Quantification of CD31 + area per visual field normalized to WT mice . WT , N = 6 ; Col1α1cKO , N = 5 biological replicates .

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

Figure 4(A) IHC for collagen I on wound tissue sections at day 17 post-wounding (brown stain). Black boxes denote zoomed regions depicted in bottom panels. Scale bar: 50 μm. (B) Quantification of collagen I positive area normalized to the average area in WT controls. WT, N=5; TK, N=5 biological replicates. (C) Polarized light images of picrosirius red stained wound tissues at day 17 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in bottom panels. Scale bar, 50 µm. (D) Quantification of collagen fiber (picrosirius red) percent area normalized to the average area in WT controls. WT, N=3; TK, N=3 biological replicates. (E-(E-H) Reduced collagen deposition in Col1α1cKO wounds. (E) IHC for collagen I on wound tissue sections at day 14 post-wounding (brown stain). Black boxes denote zoomed regions depicted in bottom panels. Scale bar: 25 μm. (F) Quantification of collagen I positive area normalized to the average area in WT controls. WT, N=7; Col1α1cKO, N=4 biological replicates. (G) Polarized light images of picrosirius red stained wound tissues at day 14 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in bottom panels. Scale bar, 50 µm. (H) Quantification of collagen fiber percent area and fiber width normalized to the average area and width, respectively, in WT controls. WT, N=4; Col1α1cKO, N=5 biological replicates.(J-M) Reepithelization and vascularization in WT and Col1α1cKO mice. (J) Representative images of keratin 5 (Krt5, red) stained wounds at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 500 µm. (K) Quantification of epidermal thickness. WT, N=6; Col1α1cKO, N=5 biological replicates. (L) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 20 μm. (M) Quantification of CD31+ area per visual field normalized to WT mice. WT, N=6; Col1α1cKO, N=5 biological replicates.


Figure 5 ( C ) IF for β1 integrin ( red ) and αSMA ( green ) on wound tissue sections at day 17 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar : 20 μm . ( F - K ) Reepithelization , granulation tissue formation , and vascularization in WT and Itgb1cKO mice . ( F ) Representative images of keratin 5 ( Krt5 , red ) stained wounds at day 14 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 500 µm . ( G ) Quantification of epidermal thickness . WT , N = 4 ; Itgb1cKO , N = 6 biological replicates . ( H ) Immunofluorescent staining for CD31 ( red ) and nuclei ( blue ) on wound tissue sections at day 14 post - wounding . White boxes denote zoomed regions depicted in right panels . Scale bar , 20 μm . ( I ) Quantification of CD31 + area per visual field normalized to WT mice . WT , N = 6 ; Itgb1cKO , N = 6 biological replicates . ( J ) Polarized light images of picrosirius red stained wound tissues at day 14 post - wounding . Red : collagen fibers . White boxes denote zoomed regions depicted in right panels . Scale bar , 50 µm . ( K ) Quantification of collagen fiber percent area normalized to the average area in WT controls . WT , N = 6 ; Itgb1cKO , N = 6 biological replicates . ( L ) Quantification of collagen fiber width normalized to the average width in WT controls . WT , N = 6 ; Itgb1cKO , N = 6 biological replicates .

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

Figure 5(C) IF for β1 integrin (red) and αSMA (green) on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar: 20 μm.(F-K) Reepithelization, granulation tissue formation, and vascularization in WT and Itgb1cKO mice. (F) Representative images of keratin 5 (Krt5, red) stained wounds at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 500 µm. (G) Quantification of epidermal thickness. WT, N=4; Itgb1cKO, N=6 biological replicates. (H) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 20 μm. (I) Quantification of CD31+ area per visual field normalized to WT mice. WT, N=6; Itgb1cKO, N=6 biological replicates. (J) Polarized light images of picrosirius red stained wound tissues at day 14 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in right panels. Scale bar, 50 µm. (K) Quantification of collagen fiber percent area normalized to the average area in WT controls. WT, N=6; Itgb1cKO, N=6 biological replicates. (L) Quantification of collagen fiber width normalized to the average width in WT controls. WT, N=6; Itgb1cKO, N=6 biological replicates. D


Figure 1AGS cells were infected with H . pylori or treated with 20 ng / ml LTα1β2 for the indicated times . Total cell lysates were analyzed by immunoblotting using the indicated antibodies . AGS cells were infected with H . pylori P1 wild ‑ type or isogenic ∆ HP0857 ( mutated in the gmhA gene ) strains . Total cell lysates were analyzed by immunoblotting using the indicated antibodies . AGS cells were treated with 200 nM synthetic ADP ‑ heptose for the indicated times . Total cell lysates were analyzed by immunoblotting using the indicated antibodies . AGS cells were pre ‑ incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by H . pylori infection or addition of ADP - heptose ( 200 nM ) . Total cell lysates were analyzed by immunoblotting using the indicated antibodies . CagA demonstrated a similar infection rate .

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

Figure 1AGS cells were infected with H. pylori or treated with 20 ng/ml LTα1β2 for the indicated times. Total cell lysates were analyzed by immunoblotting using the indicated antibodies.AGS cells were infected with H. pylori P1 wild‑type or isogenic ∆HP0857 (mutated in the gmhA gene) strains. Total cell lysates were analyzed by immunoblotting using the indicated antibodies.AGS cells were treated with 200 nM synthetic ADP‑heptose for the indicated times. Total cell lysates were analyzed by immunoblotting using the indicated antibodies.AGS cells were pre‑incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by H. pylori infection or addition of ADP-heptose (200 nM). Total cell lysates were analyzed by immunoblotting using the indicated antibodies. CagA demonstrated a similar infection rate.


Figure 2Parental AGS , ALPK1 ‑ KO and TIFA ‑ KO cells were infected with H . pylori for the times shown . Total cell lysates were analyzed by immunoblotting using the indicated antibodies . CagA demonstrated a similar infection rate of the different cells . Asterisk denotes an unspecific band and the arrow indicates the band corresponding to phosphorylated p100 . Total RNA was isolated from ALPK1 ‑ KO cells and analyzed using quantitative RT ‑ PCR for the ALPK1 transcript . Data shown depict the average of triplicate determinations normalized to the RPL13A housekeeping gene . Error bars denote RQ ± RQmin / RQmax . TIFA - KO cells were transfected with His ‑ tagged recombinant TIFA protein 24 h prior to H . pylori infection . Co ‑ IP with an anti ‑ His antibody was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies . Asterisk denotes an unspecific band and the arrow indicates the TRAF2 band . AGS and ALPK1 - KO cells were infected with H . pylori for the times shown . Co ‑ IP with an anti ‑ TRAF6 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies . AGS and TIFA - KO cells were infected with H . pylori for the times shown . Co ‑ IP with an anti ‑ TRAF6 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies . AGS and TIFA - KO cells were treated with 30 ng / ml IL - 1β for the times shown . Co ‑ IP with an anti ‑ TRAF6 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies .

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

Figure 2Parental AGS, ALPK1‑KO and TIFA‑KO cells were infected with H. pylori for the times shown. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. CagA demonstrated a similar infection rate of the different cells. Asterisk denotes an unspecific band and the arrow indicates the band corresponding to phosphorylated p100.Total RNA was isolated from ALPK1‑KO cells and analyzed using quantitative RT‑PCR for the ALPK1 transcript. Data shown depict the average of triplicate determinations normalized to the RPL13A housekeeping gene. Error bars denote RQ ± RQmin/RQmax.TIFA-KO cells were transfected with His‑tagged recombinant TIFA protein 24 h prior to H. pylori infection. Co‑IP with an anti‑His antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies. Asterisk denotes an unspecific band and the arrow indicates the TRAF2 band.AGS and ALPK1-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TRAF6 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.AGS and TIFA-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TRAF6 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.AGS and TIFA-KO cells were treated with 30 ng/ml IL-1β for the times shown. Co‑IP with an anti‑TRAF6 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


Figure 3TIFA - KO cells were transfected with His ‑ tagged recombinant TIFA protein 24 h prior to H . pylori infection or treatment with 30 ng / ml LTα1β2 . Co ‑ IP with an anti ‑ His antibody was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies . AGS cells were pre ‑ incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by addition of ADP - heptose ( 200 nM ) . Co ‑ IP with an anti ‑ TIFA antibody was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies . Asterisk indicates an unspecific band and the arrow indicates the TRAF3 band . AGS and ALPK1 - KO cells were infected with H . pylori for the times shown . Co ‑ IP with an anti ‑ TIFA antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies . AGS and TIFA - KO cells were infected with H . pylori for the times shown . Co ‑ IP with an anti ‑ TRAF2 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies . AGS cells were transfected with siRNA against cIAP1 . Co ‑ IP with an anti ‑ TIFA antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies . TIFA - KO cells were transfected with empty vector plasmid ( pCMV6 ) , wild ‑ type human ( phTIFA ) , phosphorylation ‑ defective mutant ( pT9A ) or TRAF6 binding ‑ defective mutant ( pE178A ) FLAG ‑ tagged TIFA plasmids . Total cell lysates were analyzed by immunoblotting using the indicated antibodies . TIFA - KO cells were transfected with empty vector plasmid ( pCMV6 ) , wild ‑ type human ( phTIFA ) , phosphorylation ‑ defective mutant ( pT9A ) or TRAF6 binding ‑ defective mutant ( pE178A ) FLAG ‑ tagged TIFA plasmids . Total cell lysates were separated by native gel electrophoresis under non - reducing conditions .

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

Figure 3TIFA-KO cells were transfected with His‑tagged recombinant TIFA protein 24 h prior to H. pylori infection or treatment with 30 ng/ml LTα1β2. Co‑IP with an anti‑His antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.AGS cells were pre‑incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by addition of ADP-heptose (200 nM). Co‑IP with an anti‑TIFA antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies. Asterisk indicates an unspecific band and the arrow indicates the TRAF3 band.AGS and ALPK1-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TIFA antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.AGS and TIFA-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TRAF2 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.AGS cells were transfected with siRNA against cIAP1. Co‑IP with an anti‑TIFA antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.TIFA-KO cells were transfected with empty vector plasmid (pCMV6), wild‑type human (phTIFA), phosphorylation‑defective mutant (pT9A) or TRAF6 binding‑defective mutant (pE178A) FLAG‑tagged TIFA plasmids. Total cell lysates were analyzed by immunoblotting using the indicated antibodies.TIFA-KO cells were transfected with empty vector plasmid (pCMV6), wild‑type human (phTIFA), phosphorylation‑defective mutant (pT9A) or TRAF6 binding‑defective mutant (pE178A) FLAG‑tagged TIFA plasmids. Total cell lysates were separated by native gel electrophoresis under non-reducing conditions.


Figure 4In TIFA ‑ KO cells transiently expressing TIFA ‑ tdTomato , co - localization of TRAF6 ( A ) , TRAF2 ( B ) , cIAP1 ( C ) and TRAF3 ( D ) with TIFA - tdTomato , which formed TIFAsomes upon infection with H . pylori P1 wild ‑ type strain , were detected by immunofluorescence staining . The nuclei were counterstained with DAPI . Scale bar = 10 µm . TIFA ‑ KO cells transiently expressing TIFA ‑ tdTomato were infected with H . pylori P1 strain mutated in the gmhA gene ( ∆ HP0857 ) . Scale bar = 10 µm .

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

Figure 4In TIFA‑KO cells transiently expressing TIFA‑tdTomato, co-localization of TRAF6 (A), TRAF2 (B), cIAP1 (C) and TRAF3 (D) with TIFA-tdTomato, which formed TIFAsomes upon infection with H. pylori P1 wild‑type strain, were detected by immunofluorescence staining. The nuclei were counterstained with DAPI. Scale bar = 10 µm. TIFA‑KO cells transiently expressing TIFA‑tdTomato were infected with H. pylori P1 strain mutated in the gmhA gene (∆HP0857). Scale bar = 10 µm.


Figure 1 ( B - F ) Basal views through living SCs , with dashed white circles approximating the outline of a single SC , and acidic compartments marked by the vital dye LysoTracker ® Red ( magenta ) . In merge images , a single non - acidic ( C , D , F ) and acidic ( E ) compartment containing intraluminal vesicles ( ILVs ) is boxed and magnified in the right panel ( Zoom ) . ILVs appear as membrane - delineated vesicles , using super - resolution 3D - structured illumination ( 3D - SIM ) microscopy for the brighter overexpressed GFP - tagged constructs ( yellow ; C and F ) . However , ILVs appear only as puncta , using lower resolution wide - field microscopy for the fainter endogenously expressed YFP - tagged Rab GTPases ( yellow ; D and E ) . ( B ) Wide - field fluorescence image , including differential interference contrast ( DIC ) , of SC expressing a GFP - tagged version of human CD63 ( CD63 - GFP ) . CD63 - GFP expression is apparent on the limiting membranes of non - acidic compartments and their ILVs , and also on the limiting membranes of the enlarged acidic compartments . Most large non - acidic compartments are Rab11 - positive ( D ) and contain dense - core granules , which have a ' fried egg ' appearance with DIC ( arrowheads ) ( Corrigan et al . , 2014 ; Redhai et al . , 2016 ) . ( C ) 3D - SIM image of CD63 - GFP - expressing SC . Arrow highlights CD63 - GFP - marked ILVs ( Zoom ) . Many more ILVs are apparent in non - acidic compartments in a complete Z - stack of a non - acidic compartment ( Movie EV1 ) . ( D ) Wide - field fluorescence image of an SC expressing a YFP - Rab11 gene trap . YFP - Rab11 marks the limiting membranes of most non - acidic compartments and internal puncta ( arrow in Zoom ) , but not the surface of acidic compartments ( Appendix Fig S1B ) . ( E ) Wide - field fluorescence image of SC expressing a YFP - Rab7 gene trap . YFP - Rab7 marks the limiting membranes of acidic compartments and internal puncta ( arrow in Zoom ) . Enlarged acidic compartments are also present in adjacent main cells . ( F ) 3D - SIM image of SC expressing a GFP - tagged version of Breathless ( Btl - GFP ) . Btl - GFP marks the limiting membranes of non - acidic compartments and their ILVs ( arrow in Zoom ) , but not the surface of acidic compartments ( Appendix Fig S1C ) . Images from six - day - old male flies shifted to 29 ° C at eclosion . This induces GAL4 / UAS - dependent SC transgene expression in ( B ) , ( C ) and ( F ) . The genotypes of flies carrying multiple transgenes are : w ; P [ w + , UAS - CD63 - GFP ] P [ w + , tub - GAL80ts ] / + ; dsx - GAL4 / + ( B , C ) ; w ; P [ w + , tub - GAL80ts ] / + ; dsx - GAL4 / P [ w + , UAS - btl - GFP ] ( F ) .

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

Figure 1(B-F) Basal views through living SCs, with dashed white circles approximating the outline of a single SC, and acidic compartments marked by the vital dye LysoTracker® Red (magenta). In merge images, a single non-acidic (C, D, F) and acidic (E) compartment containing intraluminal vesicles (ILVs) is boxed and magnified in the right panel (Zoom). ILVs appear as membrane-delineated vesicles, using super-resolution 3D-structured illumination (3D-SIM) microscopy for the brighter overexpressed GFP-tagged constructs (yellow; C and F). However, ILVs appear only as puncta, using lower resolution wide-field microscopy for the fainter endogenously expressed YFP-tagged Rab GTPases (yellow; D and E). (B) Wide-field fluorescence image, including differential interference contrast (DIC), of SC expressing a GFP-tagged version of human CD63 (CD63-GFP). CD63-GFP expression is apparent on the limiting membranes of non-acidic compartments and their ILVs, and also on the limiting membranes of the enlarged acidic compartments. Most large non-acidic compartments are Rab11-positive (D) and contain dense-core granules, which have a 'fried egg' appearance with DIC (arrowheads) (Corrigan et al., 2014; Redhai et al., 2016). (C) 3D-SIM image of CD63-GFP-expressing SC. Arrow highlights CD63-GFP-marked ILVs (Zoom). Many more ILVs are apparent in non-acidic compartments in a complete Z-stack of a non-acidic compartment (Movie EV1). (D) Wide-field fluorescence image of an SC expressing a YFP-Rab11 gene trap. YFP-Rab11 marks the limiting membranes of most non-acidic compartments and internal puncta (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1B). (E) Wide-field fluorescence image of SC expressing a YFP-Rab7 gene trap. YFP-Rab7 marks the limiting membranes of acidic compartments and internal puncta (arrow in Zoom). Enlarged acidic compartments are also present in adjacent main cells. (F) 3D-SIM image of SC expressing a GFP-tagged version of Breathless (Btl-GFP). Btl-GFP marks the limiting membranes of non-acidic compartments and their ILVs (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1C). Images from six-day-old male flies shifted to 29°C at eclosion. This induces GAL4/UAS-dependent SC transgene expression in (B), (C) and (F). The genotypes of flies carrying multiple transgenes are: w; P[w+, UAS-CD63-GFP] P[w+, tub-GAL80ts]/+; dsx-GAL4/+ (B, C); w; P[w+, tub-GAL80ts]/+; dsx-GAL4/P[w+, UAS-btl-GFP] (F).


Figure 2 ( A - D ) Wide - field fluorescence images of basal views through living SCs expressing a GFP - tagged form of Btl - GFP ( yellow ) in non - acidic compartments , with SC outline approximated by dashed white circles . Acidic compartments are marked by LysoTracker Red ® ( magenta ) . Boxed non - acidic compartments in merge images are magnified in A - D , Zoom . On the right , lower magnification confocal transverse images of fixed accessory gland ( AG ) lumens are shown with dotted lines indicating the basal side of the AG epithelial layer , which contains several fluorescent SCs ( highlighted by arrows ) . ( A ) SC with no RNAi construct expressed ( control ) and AG lumen from same genotype . Btl - GFP - positive compartments containing fluorescent ILVs ( in Btl - GFP panel ) , ILV membranes inside compartments ( in Zoom panel ) and secreted fluorescent puncta ( AG lumen panel ) are marked by arrowheads . ( B ) SC also expressing RNAi construct targeting ESCRT - 0 subunit , Stam , and AG lumen from same genotype . Btl - GFP - positive ILVs ( Zoom ) and secreted puncta ( AG lumen ) are strongly reduced . ( C ) SC also expressing RNAi construct targeting ESCRT - I subunit , Vps28 , and AG lumen from same genotype . Btl - GFP - positive ILVs ( Zoom ) and secreted puncta ( AG lumen ) are strongly reduced . ( D ) SC also expressing RNAi construct targeting ESCRT - III subunit , shrb , and AG lumen from same genotype . Btl - GFP - positive ILVs ( Zoom ) and secreted puncta are strongly reduced . Genotypes are : w ; P [ w + , tub - GAL80ts ] / + ; dsx - GAL4 / P [ w + , UAS - btl - GFP ] with no knockdown construct ( A ) , UAS - Stam - RNAi ( HMS01429 ; B ) , UAS - Vps28 - RNAi ( v31894 ; C ) or UAS - shrb - RNAi ( v106823 ; D ) . ( E ) Bar chart showing the number of large ( diameter greater than one micrometre ) non - acidic Btl - GFP - positive compartments per SC . Data from 39 SCs ( three per gland ) are shown . ( F ) Bar chart showing percentage of these large Btl - GFP compartments containing Btl - GFP - positive ILVs in control and ESCRT knockdown SCs . Data from 39 SCs ( three per gland ) are shown . ( G ) Bar chart showing the total number of Btl - GFP fluorescent puncta in ten Z - planes from the AG lumen following ESCRT knockdown in SCs , compared to controls without knockdown . Data from at least 17 AG lumens per condition are shown .

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

Figure 2(A-D) Wide-field fluorescence images of basal views through living SCs expressing a GFP-tagged form of Btl-GFP (yellow) in non-acidic compartments, with SC outline approximated by dashed white circles. Acidic compartments are marked by LysoTracker Red® (magenta). Boxed non-acidic compartments in merge images are magnified in A-D, Zoom. On the right, lower magnification confocal transverse images of fixed accessory gland (AG) lumens are shown with dotted lines indicating the basal side of the AG epithelial layer, which contains several fluorescent SCs (highlighted by arrows). (A) SC with no RNAi construct expressed (control) and AG lumen from same genotype. Btl-GFP-positive compartments containing fluorescent ILVs (in Btl-GFP panel), ILV membranes inside compartments (in Zoom panel) and secreted fluorescent puncta (AG lumen panel) are marked by arrowheads. (B) SC also expressing RNAi construct targeting ESCRT-0 subunit, Stam, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta (AG lumen) are strongly reduced. (C) SC also expressing RNAi construct targeting ESCRT-I subunit, Vps28, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta (AG lumen) are strongly reduced. (D) SC also expressing RNAi construct targeting ESCRT-III subunit, shrb, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta are strongly reduced. Genotypes are: w; P[w+, tub-GAL80ts]/+; dsx-GAL4/P[w+, UAS-btl-GFP] with no knockdown construct (A), UAS-Stam-RNAi (HMS01429; B), UAS-Vps28-RNAi (v31894; C) or UAS-shrb-RNAi (v106823; D).(E) Bar chart showing the number of large (diameter greater than one micrometre) non-acidic Btl-GFP-positive compartments per SC. Data from 39 SCs (three per gland) are shown.(F) Bar chart showing percentage of these large Btl-GFP compartments containing Btl-GFP-positive ILVs in control and ESCRT knockdown SCs. Data from 39 SCs (three per gland) are shown.(G) Bar chart showing the total number of Btl-GFP fluorescent puncta in ten Z-planes from the AG lumen following ESCRT knockdown in SCs, compared to controls without knockdown. Data from at least 17 AG lumens per condition are shown.


Figure 3 ( A - D ) Confocal images of fixed HCT116 cells , with boxed regions enlarged to the right . DAPI ( grey ) marks nucleus . Rab11a antibody is isoform - specific except in ( F ) . ( A ) Rab11a ( yellow ) is located in compartments distinct from the late endosomal and lysosomal marker , LAMP2 ( magenta ) . ( B ) CD63 ( magenta ) predominantly co - localises with the late endosomal and lysosomal marker , LAMP1 ( yellow ) . ( C ) Rab11a ( yellow ) is located in compartments distinct from CD63 ( magenta ) under glutamine - replete conditions . ( D ) Rab11a ( yellow ) is located in compartments distinct from CD63 ( magenta ) under glutamine - depleted conditions . ( E ) Super - resolution 3D - SIM image of fixed HCT116 cell expressing GFP - Rab11a ( yellow ) , and stained with CD63 ( magenta ) . DAPI ( grey ) marks nucleus . Boxed Rab11a - positive compartments , which frequently cluster , are magnified in Merge Zoom . This panel is further magnified in Merge Zoom x 2 , revealing GFP - Rab11a ( arrows in right panel ) inside compartments . ( F ) Wide - field fluorescence image of fixed HCT116 cells , stained with Rab11a ( yellow ) and Rab7 ( magenta ) , expressing constitutively active GFP - tagged Rab5 ( GFP - Rab5CA ; cyan ) , which stalls endosomal maturation and produces enlarged Rab5 - positive endosomes . One of these is boxed in the Merge and magnified in Merge Zoom , revealing internal puncta marked by Rab11a ( arrows ) and Rab7 ( arrowheads ) and limiting membrane subdomains of Rab11a ( yellow arrowhead ) and Rab7 ( magenta arrowhead ) . DAPI ( grey ) marks nuclei .

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

Figure 3(A-D) Confocal images of fixed HCT116 cells, with boxed regions enlarged to the right. DAPI (grey) marks nucleus. Rab11a antibody is isoform-specific except in (F). (A) Rab11a (yellow) is located in compartments distinct from the late endosomal and lysosomal marker, LAMP2 (magenta). (B) CD63 (magenta) predominantly co-localises with the late endosomal and lysosomal marker, LAMP1 (yellow). (C) Rab11a (yellow) is located in compartments distinct from CD63 (magenta) under glutamine-replete conditions. (D) Rab11a (yellow) is located in compartments distinct from CD63 (magenta) under glutamine-depleted conditions.(E) Super-resolution 3D-SIM image of fixed HCT116 cell expressing GFP-Rab11a (yellow), and stained with CD63 (magenta). DAPI (grey) marks nucleus. Boxed Rab11a-positive compartments, which frequently cluster, are magnified in Merge Zoom. This panel is further magnified in Merge Zoom x 2, revealing GFP-Rab11a (arrows in right panel) inside compartments.(F) Wide-field fluorescence image of fixed HCT116 cells, stained with Rab11a (yellow) and Rab7 (magenta), expressing constitutively active GFP-tagged Rab5 (GFP-Rab5CA; cyan), which stalls endosomal maturation and produces enlarged Rab5-positive endosomes. One of these is boxed in the Merge and magnified in Merge Zoom, revealing internal puncta marked by Rab11a (arrows) and Rab7 (arrowheads) and limiting membrane subdomains of Rab11a (yellow arrowhead) and Rab7 (magenta arrowhead). DAPI (grey) marks nuclei.


Figure 4 ( A ) Western blot analysis of putative exosome markers in lysates from HCT116 cells cultured in glutamine - replete ( 2 . 00 mM ) and glutamine - depleted ( 0 . 15 mM ) medium for 24 h . Gel was loaded with equal amounts of protein ( total cell lysate protein was reduced by 19 ± 4 % after glutamine depletion ) . The activity of mTORC1 was assessed via phosphorylation of S6 and 4E - BP1 , using phospho - specific antibodies and a pan - 4E - BP1 antibody , where the hyper - phosphorylated form of 4E - BP1 produced by mTORC1 gives the slowest migrating γ band . Bar chart shows the abundance of putative exosome proteins relative to tubulin in these lysates . Western blot analyses of EV preparations . ( B ) EVs isolated by ultracentrifugation ( UC ) of medium from HCT116 cells cultured in glutamine - replete and - depleted conditions for 24 h , and loaded according to cell lysate protein levels to compare secretion on a per cell basis , as shown in bar chart from four biological replicates . Western blot analyses of EV preparations . ( C ) EV preparation isolated by size - exclusion chromatography ( SEC ) from glutamine - depleted HCT116 cells and then separated by high - resolution iodixanol - PBS density centrifugation . Note that exosomes ( exos ) and microvesicles ( MVs ) are found in only partially overlapping fractions and the pattern of Rab11a separation mirrors that of exosome markers ( primarily 1 . 064 - 1 . 085 g / ml ) . Western blot analyses of EV preparations . ( D ) EVs isolated by UC under glutamine - depleted conditions as in ( B ) and then subjected to Proteinase K ( Prot K ) digestion in the presence or absence of Triton ® X - 100 ( Triton - X ) . Only membrane - associated CD81 is digested in the absence of Triton ® X - 100 , while CD63 is resistant to digestion , even in the presence of Triton ® X - 100 . Western blot analyses of EV preparations . EVs isolated under glutamine - depleted conditions were immunocaptured with anti - CD63 antibodies coupled to magnetic beads . This method only pulls down a fraction of CD63 , so the protein from pull - down of eight times the input is loaded for comparison . Approximately the same low levels of Rab11a are captured by control IgG and anti - CD63 beads . Western blot analyses of EV preparations . ( F ) EVs isolated under glutamine - depleted conditions were immunocaptured with anti - CD81 antibodies coupled to magnetic beads . All or most CD63 and CD81 appears to be pulled down , but less than 10 % of Rab11a and Cav - 1 is captured . NTA analysis suggests about 10 % of all particles are pulled down with this approach .

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

Figure 4(A) Western blot analysis of putative exosome markers in lysates from HCT116 cells cultured in glutamine-replete (2.00 mM) and glutamine-depleted (0.15 mM) medium for 24 h. Gel was loaded with equal amounts of protein (total cell lysate protein was reduced by 19 ± 4 % after glutamine depletion). The activity of mTORC1 was assessed via phosphorylation of S6 and 4E-BP1, using phospho-specific antibodies and a pan-4E-BP1 antibody, where the hyper-phosphorylated form of 4E-BP1 produced by mTORC1 gives the slowest migrating γ band. Bar chart shows the abundance of putative exosome proteins relative to tubulin in these lysates.Western blot analyses of EV preparations. (B) EVs isolated by ultracentrifugation (UC) of medium from HCT116 cells cultured in glutamine-replete and -depleted conditions for 24 h, and loaded according to cell lysate protein levels to compare secretion on a per cell basis, as shown in bar chart from four biological replicates.Western blot analyses of EV preparations. (C) EV preparation isolated by size-exclusion chromatography (SEC) from glutamine-depleted HCT116 cells and then separated by high-resolution iodixanol-PBS density centrifugation. Note that exosomes (exos) and microvesicles (MVs) are found in only partially overlapping fractions and the pattern of Rab11a separation mirrors that of exosome markers (primarily 1.064 - 1.085 g/ml).Western blot analyses of EV preparations. (D) EVs isolated by UC under glutamine-depleted conditions as in (B) and then subjected to Proteinase K (Prot K) digestion in the presence or absence of Triton® X-100 (Triton-X). Only membrane-associated CD81 is digested in the absence of Triton® X-100, while CD63 is resistant to digestion, even in the presence of Triton® X-100.Western blot analyses of EV preparations. EVs isolated under glutamine-depleted conditions were immunocaptured with anti-CD63 antibodies coupled to magnetic beads. This method only pulls down a fraction of CD63, so the protein from pull-down of eight times the input is loaded for comparison. Approximately the same low levels of Rab11a are captured by control IgG and anti-CD63 beads.Western blot analyses of EV preparations. (F) EVs isolated under glutamine-depleted conditions were immunocaptured with anti-CD81 antibodies coupled to magnetic beads. All or most CD63 and CD81 appears to be pulled down, but less than 10% of Rab11a and Cav-1 is captured. NTA analysis suggests about 10% of all particles are pulled down with this approach.