Figure 5Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors . Control human R00 - 082 chondrocytes were serum starved in the presence of a GRK2 inhibitor , either CMPD101 ( C ) or paroxetine ( P ) , for 24 hours before they were treated with SAG for additional 12 ( A , B ) ( A , B ) Cilia were stained by ARL13B and SMO antibodies , and the intensity of ciliary SMO was analyzed and plotted . Note the impaired SMO accumulation in cilia caused by GRK2 inhibition ( black arrow ) . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cilia are indicated . Scale bar , 1 µm . Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors . Control human R00 - 082 chondrocytes were serum starved in the presence of a GRK2 inhibitor , either CMPD101 ( C ) or paroxetine ( P ) , for 24 hours before they were treated with SAG for additional 24 hours ( C D ) . ( C ) Cell lysates were immunoblotted for GLI3 processing and GLI1 upregulation ; actin levels served as a loading control . GLI3FL and GLI3R , full - length and repressor GLI3 variants , respectively . ( D ) Quantification of GLI3 and GLI1 levels by densitometry . Note the impaired GLI3 processing and no GLI1 upregulation in cells treated with CMPD101 or paroxetine . Mean ± SEM . Mann - Whitney U test ( GLI3 ratio ) and Welch ´ s t - test ( GLI1 levels ) ; number of biological experiments is indicated . Defective SMO cilia accumulation in chondrocytes with downregulated GRK2 . ( E ) Doxycycline ( DOX ) - inducible GRK2 downregulation in control human R00 - 082 chondrocytes , tested by western blot , normalized to actin and plotted below . Mean ± SEM . Welch ´ s t - test ; number of biological experiments is indicated . shScr , scramble shRNA . ( F ) Cells pre - treated with DOX for three days were serum starved , SAG treated , stained and analyzed as in ( A , B ) , and the intensity of ciliary SMO was analyzed and plotted . Note the impaired SMO translocation to cilia caused by GRK2 loss . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cilia are indicated . Scale bar , 1 µm .

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Figure 5Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 12 (A, B) (A,B) Cilia were stained by ARL13B and SMO antibodies, and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO accumulation in cilia caused by GRK2 inhibition (black arrow). Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm.Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 24 hours (C D). (C) Cell lysates were immunoblotted for GLI3 processing and GLI1 upregulation; actin levels served as a loading control. GLI3FL and GLI3R, full-length and repressor GLI3 variants, respectively. (D) Quantification of GLI3 and GLI1 levels by densitometry. Note the impaired GLI3 processing and no GLI1 upregulation in cells treated with CMPD101 or paroxetine. Mean ± SEM. Mann-Whitney U test (GLI3 ratio) and Welch´s t-test (GLI1 levels); number of biological experiments is indicated.Defective SMO cilia accumulation in chondrocytes with downregulated GRK2. (E) Doxycycline (DOX)-inducible GRK2 downregulation in control human R00-082 chondrocytes, tested by western blot, normalized to actin and plotted below. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated. shScr, scramble shRNA.(F) Cells pre-treated with DOX for three days were serum starved, SAG treated, stained and analyzed as in (A,B), and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO translocation to cilia caused by GRK2 loss. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm.


Figure 6 ( A ) Control and GRK2 - / - fibroblasts were transfected with the TOPflash Firefly ( F ) luciferase vector together with a control Renilla ( R ) luciferase vector , and the effect of Wnt3A on TOPflash transcriptional activation was determined by dual - luciferase assay . Note the impaired response to Wnt3A in GRK2 - / - cells . Mean ± SEM . Welch ´ s t - test ; number of biological experiments is indicated . ( B Low phosphorylation ( p ) of Wnt3A co - receptor LRP6 as determined by western blot with pS1490 - and pT1572 - LRP6 specific antibodies , reduced LRP6 and DVL2 expression , and increased steady state phosphorylation of DVL2 . A representative experiment of four is shown . # , nonspecific band . C ) Low phosphorylation ( p ) of Wnt3A co - receptor LRP6 as determined by western blot with pS1490 - and pT1572 - LRP6 specific antibodies , reduced LRP6 and DVL2 expression , and increased steady state phosphorylation of DVL2 . ( C ) Densitometry of western blot results demonstrating decreased levels of LRP6 in GRK2 - / - cells , lower response of GRK2 - / - cells to Wnt3A by LRP6 phosphorylation at S1490 , and tendency towards the similar defect at T1572 . Densitometry also showed less of total DVL2 levels , and increased steady state pDVL2 in GRK2 - / - cells . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated . ( D ) Loss of interaction between Frizzled 4 ( FZD4 ) and ß - Arrestin 2 ( ARRB2 ) in GRK2 - / - fibroblasts . Proximity ligation assay ( PLA ) between expressed FZD4 - GFP and ARRB2 - Flag showed significantly reduced proximity events between the two proteins in GRK2 - / - cells . White dashed lines outline the transfected cells . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cells are indicated . Scale bar , 10 µm .

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Figure 6(A) Control and GRK2-/- fibroblasts were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, and the effect of Wnt3A on TOPflash transcriptional activation was determined by dual-luciferase assay. Note the impaired response to Wnt3A in GRK2-/- cells. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated.(B Low phosphorylation (p) of Wnt3A co-receptor LRP6 as determined by western blot with pS1490- and pT1572-LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. A representative experiment of four is shown. #, nonspecific band.C) Low phosphorylation (p) of Wnt3A co-receptor LRP6 as determined by western blot with pS1490- and pT1572-LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. (C) Densitometry of western blot results demonstrating decreased levels of LRP6 in GRK2-/- cells, lower response of GRK2-/- cells to Wnt3A by LRP6 phosphorylation at S1490, and tendency towards the similar defect at T1572. Densitometry also showed less of total DVL2 levels, and increased steady state pDVL2 in GRK2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.(D) Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in GRK2-/- fibroblasts. Proximity ligation assay (PLA) between expressed FZD4-GFP and ARRB2-Flag showed significantly reduced proximity events between the two proteins in GRK2-/- cells. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bar, 10 µm.


Figure 7 ( A ) Control ( + / + ) and Grk2 - / - NIH3T3 cells were transfected with the TOPflash Firefly ( F ) luciferase vector together with a control Renilla ( R ) luciferase vector , treated with Wnt3A ( 100 ng / ml ) for 24 hours and subjected to dual - luciferase assay . Note the impaired response to Wnt3A in Grk2 - / - cells . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated . ( B ) Control ( + / + ) and Grk2 - / - NIH3T3 cells were treated with 40 ng / ml Wnt3A for 1 hour . Note the lower LRP6 expression and inhibited Wnt3A - induced LRP6 phosphorylation in the Grk2 - / - NIH3T3 cells . Mean ± SEM . Welch ´ s t - test ; number of biological experiments is indicated . Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101 . Control human R00 - 082 chondrocytes ( C ) were treated with 20 µM CMPD101 overnight and then treated with 100 ng / ml Wnt3A for 1 hour . Note the less LRP6 phosphorylation ( pS1490 - and pT1572 - LRP6 ) in cells treated with CMPD101 . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated . Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101 . rat chondrosarcoma ( RCS ) cells ( D ) were treated with 20 µM CMPD101 overnight and then treated with 100 ng / ml Wnt3A for 1 hour . Note the less LRP6 phosphorylation ( pS1490 - and pT1572 - LRP6 ) in cells treated with CMPD101 . Mean ± SEM . Mann - Whitney U test ; number of biological experiments is indicated . Loss of interaction between Frizzled 4 ( FZD4 ) and ß - Arrestin 2 ( ARRB2 ) in Grk2 - / - NIH3T3 cells . ( E ) Proximity ligation assay ( PLA ) between expressed FZD4 - GFP and ARRB2 - Flag showed significantly reduced proximity events between the two proteins in Grk2 - / - cells . White dashed lines outline the transfected cells . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cells are indicated . Scale bars , 10 µm . Loss of interaction between Frizzled 4 ( FZD4 ) and ß - Arrestin 2 ( ARRB2 ) in Grk2 - / - NIH3T3 cells . ( F ) GRK2 addback rescued the interaction . White dashed lines outline the transfected cells . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cells are indicated . Scale bars , 10 µm .

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 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 O O O 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 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 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

Figure 7(A) Control (+/+) and Grk2-/- NIH3T3 cells were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, treated with Wnt3A (100 ng/ml) for 24 hours and subjected to dual-luciferase assay. Note the impaired response to Wnt3A in Grk2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.(B) Control (+/+) and Grk2-/- NIH3T3 cells were treated with 40 ng/ml Wnt3A for 1 hour. Note the lower LRP6 expression and inhibited Wnt3A-induced LRP6 phosphorylation in the Grk2-/- NIH3T3 cells. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated.Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101. Control human R00-082 chondrocytes (C) were treated with 20 µM CMPD101 overnight and then treated with 100 ng/ml Wnt3A for 1 hour. Note the less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with CMPD101. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101. rat chondrosarcoma (RCS) cells (D) were treated with 20 µM CMPD101 overnight and then treated with 100 ng/ml Wnt3A for 1 hour. Note the less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with CMPD101. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in Grk2-/- NIH3T3 cells. (E) Proximity ligation assay (PLA) between expressed FZD4-GFP and ARRB2-Flag showed significantly reduced proximity events between the two proteins in Grk2-/- cells. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bars, 10 µm.Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in Grk2-/- NIH3T3 cells. (F) GRK2 addback rescued the interaction. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bars, 10 µm.


Figure 1A - C . Scanning electron microscopy ( SEM ) images of ( A ) nanowires ( VNw ; scale - 5 µm ) and ( B ) crude nanosheets ( VSh ; scale - 200 nm ) , ( C ) ultrathin - nanosheets ( Vs ) , scale - 20 µm .

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Figure 1A-C. Scanning electron microscopy (SEM) images of (A) nanowires (VNw; scale - 5 µm) and (B) crude nanosheets (VSh; scale - 200 nm), (C) ultrathin-nanosheets (Vs), scale - 20 µm.


Figure 2C . Michaelis - Menten plot with the variation of H2O2 ( 0 - 400 µM ) in the presence of Vs ( 20 ng / µL ) , GSH ( 2 mM ) , NADPH ( 0 . 2 mM ) , GR ( 1 . 7 units ) in phosphate buffer ( 100 mM , pH 7 . 4 ) at 25 ° C . D . Michaelis - Menten plot with the variation of GSH ( 0 - 7 mM ) in the presence of Vs ( 20 ng / μL ) , H2O2 ( 200 µM ) , NADPH ( 0 . 2 mM ) , GR ( 1 . 7 units ) in phosphate buffer ( 100 mM , pH 7 . 4 ) at 25 ° C .

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Figure 2C. Michaelis-Menten plot with the variation of H2O2 (0-400 µM) in the presence of Vs (20 ng/µL), GSH (2 mM), NADPH (0.2 mM), GR (1.7 units) in phosphate buffer (100 mM, pH 7.4) at 25°C.D. Michaelis-Menten plot with the variation of GSH (0-7 mM) in the presence of Vs (20 ng/μL), H2O2 (200 µM), NADPH (0.2 mM), GR (1.7 units) in phosphate buffer (100 mM, pH 7.4) at 25°C.


Figure 3G , H . U1 Grx1 - roGFP2 cells were supplemented with BSO ( G ) or GSH ( H ) for 16 h to deplete or replenish GSH , respectively . Following this , cells were treated with Vs for 15 min , exposed to H2O2 , and the ratiometric response was measured .

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Figure 3G, H. U1 Grx1-roGFP2 cells were supplemented with BSO (G) or GSH (H) for 16 h to deplete or replenish GSH, respectively. Following this, cells were treated with Vs for 15 min, exposed to H2O2, and the ratiometric response was measured.


Figure 4E . U1 - Grx1 - roGFP2 cells were serum starved for 30 min in the presence or absence of Vs and sodium selenite ( 0 . 5 nM ) , and the biosensor response was measured . Data were compared to serum starved control cells ( C ) . F . U1 cells were either serum - starved or supplemented with Se ( 0 . 5 nM ) or Vs ( 0 . 62 ng / μL ) and HIV reactivation was measured at 6 h post starvation by gag RT - PCR .

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Figure 4E. U1-Grx1-roGFP2 cells were serum starved for 30 min in the presence or absence of Vs and sodium selenite (0.5 nM), and the biosensor response was measured. Data were compared to serum starved control cells (C).F. U1 cells were either serum-starved or supplemented with Se (0.5 nM) or Vs (0.62 ng/μL) and HIV reactivation was measured at 6 h post starvation by gag RT-PCR.


Figure 5Total RNA isolated from untreated ( UT ) , PMA - treated , Vs - treated and Vs + PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV . ( C Heat map showing functional categories of DEGs under PMA / UT , Vs / UT and PMA + Vs / UT comparisons . mRNA counts were normalised using the internal control β2 microglobulin ( B2M ) , and fold change ( FC ) was calculated using the nSolver 4 . 0 software . Genes showing an absolute FC > 1 . 5 , and P < 0 . 05 were considered as significantly altered . Total RNA isolated from untreated ( UT ) , PMA - treated , Vs - treated and Vs + PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV . D ) Heat map showing functional categories of DEGs under PMA / UT , Vs / UT and PMA + Vs / UT comparisons . mRNA counts were normalised using the internal control β2 microglobulin ( B2M ) , and fold change ( FC ) was calculated using the nSolver 4 . 0 software . Genes showing an absolute FC > 1 . 5 , and P < 0 . 05 were considered as significantly altered .

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Figure 5Total RNA isolated from untreated (UT), PMA-treated, Vs-treated and Vs+PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV. (C Heat map showing functional categories of DEGs under PMA/UT, Vs/UT and PMA+Vs/UT comparisons. mRNA counts were normalised using the internal control β2 microglobulin (B2M), and fold change (FC) was calculated using the nSolver 4.0 software. Genes showing an absolute FC >1.5, and P <0.05 were considered as significantly altered.Total RNA isolated from untreated (UT), PMA-treated, Vs-treated and Vs+PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV. D) Heat map showing functional categories of DEGs under PMA/UT, Vs/UT and PMA+Vs/UT comparisons. mRNA counts were normalised using the internal control β2 microglobulin (B2M), and fold change (FC) was calculated using the nSolver 4.0 software. Genes showing an absolute FC >1.5, and P <0.05 were considered as significantly altered.


Figure 6B - D . A similar assay was performed using Jurkat ( CD4 + T cell line ) and viral replication was assessed by ( B ) gag RT PCR , ( C ) p24 ELISA in the culture supernatant , and ( D ) immunoblotting for p24 ( viral capsid protein ) in the whole cell lysate . F . Primary CD4 + T cells purified from human PBMCs ( 3 healthy donors ) were activated , pre - treated with 25 ng / μL Vs for 15 min , and infected with 0 . 05 moi of HIV - 1 NL - 4 . 3 . Virus released in supernatant was quantified by p24 ELISA . Vs treatment was repeated every 48 h .

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Figure 6B-D. A similar assay was performed using Jurkat (CD4+ T cell line) and viral replication was assessed by (B) gag RT PCR, (C) p24 ELISA in the culture supernatant, and (D) immunoblotting for p24 (viral capsid protein) in the whole cell lysate.F. Primary CD4+ T cells purified from human PBMCs (3 healthy donors) were activated, pre-treated with 25 ng/μL Vs for 15 min, and infected with 0.05 moi of HIV-1 NL-4.3. Virus released in supernatant was quantified by p24 ELISA. Vs treatment was repeated every 48 h.


Figure 7B . Expanded CD4 + T cells from 3 patients were cultured in presence of IL - 2 and ARVs , with and without 25 ng / µL Vs for 21 days . Vs treatment was given for 15 min every 3rd day . HIV transcripts were quantified by RT - qPCR at day 14 , day 21 , and at 24 h post - stimulation of cells cultured for 21 days by prostratin . Limit of detection for RT - qPCR was 3 viral transcripts per million cells . C . At day 21 , cells were stimulated with 1 µM prostratin for 24 h and HIV transcripts were quantified by RT - qPCR . Reduction in viral stimulation in Vs treated samples are represented as percentage values . ND - non determined . D . Aggregate plot for 3 patients from data ( C ) .

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Figure 7B. Expanded CD4+ T cells from 3 patients were cultured in presence of IL-2 and ARVs, with and without 25 ng/µL Vs for 21 days. Vs treatment was given for 15 min every 3rd day. HIV transcripts were quantified by RT-qPCR at day 14, day 21, and at 24 h post-stimulation of cells cultured for 21 days by prostratin. Limit of detection for RT-qPCR was 3 viral transcripts per million cells.C. At day 21, cells were stimulated with 1 µM prostratin for 24 h and HIV transcripts were quantified by RT-qPCR. Reduction in viral stimulation in Vs treated samples are represented as percentage values. ND - non determined. D. Aggregate plot for 3 patients from data (C).


Figure 1A . The expression of EP subtypes in bone marrow myeloid cells and colon tumor myeloid cells . The short black lines indicate individual expressions , the blue and yellow lines indicate the average expression level in each condition . The grey dotted lines represent the overall average between Ptger1 and Ptger3 or Ptger2 and Ptger4 . B . The expression of EP subtypes in CD11b + cells isolated from tumor tissues and spleen of CT26 tumor - bearing mice by magnetic bead separation ( n = 3 ) . D . Representative flow cytometry plots revealing the effects of prostaglandin E2 ( PGE2 ) and EP1 - 4 antagonists on mouse bone marrow monocyte dendritic cell ( DC ) / macrophage differentiation ( n = 3 ) . E . The frequencies of F4 / 80 + CD11C − macrophages and F4 / 80 − CD11C + DCs under varying treatments as analyzed by flow cytometry analysis ( n = 3 ) . G . Representative flow cytometry plots revealing the effects of PGE2 and EP1 - 4 antagonists on mouse bone marrow derived - MDSC differentiation . Upper subpanel : vehicle group and PGE2 group . Lower subpanel : ONO - 8711 ( EP1 antagonist ) group , PF - 04418948 ( EP2 antagonist ) group , L - 798106 ( EP3 antagonist ) group , and E7046 ( EP4 antagonist ) group . ( n = 3 ) . H . The frequencies of Ly6C + Ly6G − mMDSC and Ly6CmidLy6G + PMN - MDSCs under varied treatments as analyzed by flow cytometry analysis ( n = 3 ) .

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Figure 1A. The expression of EP subtypes in bone marrow myeloid cells and colon tumor myeloid cells. The short black lines indicate individual expressions, the blue and yellow lines indicate the average expression level in each condition. The grey dotted lines represent the overall average between Ptger1 and Ptger3 or Ptger2 and Ptger4.B. The expression of EP subtypes in CD11b+ cells isolated from tumor tissues and spleen of CT26 tumor-bearing mice by magnetic bead separation (n=3).D. Representative flow cytometry plots revealing the effects of prostaglandin E2 (PGE2) and EP1-4 antagonists on mouse bone marrow monocyte dendritic cell (DC)/macrophage differentiation (n = 3). E. The frequencies of F4/80+CD11C− macrophages and F4/80−CD11C+ DCs under varying treatments as analyzed by flow cytometry analysis (n = 3). G. Representative flow cytometry plots revealing the effects of PGE2 and EP1-4 antagonists on mouse bone marrow derived-MDSC differentiation. Upper subpanel: vehicle group and PGE2 group. Lower subpanel: ONO-8711 (EP1 antagonist) group, PF-04418948 (EP2 antagonist) group, L-798106 (EP3 antagonist) group, and E7046 (EP4 antagonist) group. (n = 3). H. The frequencies of Ly6C+Ly6G− mMDSC and Ly6CmidLy6G+ PMN-MDSCs under varied treatments as analyzed by flow cytometry analysis (n = 3).


Figure 2B . Dose - effect curve of TP - 16 in GloSensor ™ cAMP assay in EP4 - expressing 293 cells ( n = 3 ) . C . Dose - effect curves of TP - 16 in PGE2 - induced calcium flux assay ( n = 3 ) . D . The Schild plot of PGE2 in the presence of varying concentrations of TP - 16 . TP - 16 shifted the dose - response curve of PGE2 - induced intracellular cAMP levels in a dose - dependent manner ( n = 3 ) . E . The pA2 value and slope of the Schild plot .

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Figure 2B. Dose-effect curve of TP-16 in GloSensor™ cAMP assay in EP4- expressing 293 cells (n=3).C. Dose-effect curves of TP-16 in PGE2-induced calcium flux assay (n=3).D. The Schild plot of PGE2 in the presence of varying concentrations of TP-16. TP-16 shifted the dose-response curve of PGE2-induced intracellular cAMP levels in a dose-dependent manner (n=3). E. The pA2 value and slope of the Schild plot.


Figure 3B . The antitumor activities of E7046 ( 150 mg / kg ) and TP - 16 ( 37 . 5 , 75 , and150 mg / kg ) in CT26 tumor - bearing BALB / c mice ( n = 11 per group ) . Tumor growth curves in the CT26 model ( left panel ) . Bar graphs show the individual changes in tumor growth relative to after and before drug administration ( right panel ) . Tumor volumes reached 100 - 200 mm3 on day 7 . C . Representative graph showing that the antitumor activity of TP - 16 is lost in CT26 tumors engrafted into BALB / c nude mice ( left panel ) ( n = 8 ) . Bar graphs show the individual changes in tumor growth relative to after and before drug administration ( right panel ) . Tumor volumes reached 100 - 200 mm3 on day 7 . D . Single - cell suspensions of tumor tissues from CT26 tumor - bearing BALB / c mice treated with vehicle or TP - 16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis . Representative flow cytometry and dot plots of intratumoral frequencies of CD8 + T cells ( n = 5 ) . E . Growth curves ( left panel ) and relative change after 18 days treatment ( right panel ) in colon cancer ( MC38 ) xenograft tumors in C57BL / 6 mice ( n = 7 ) . Tumor volumes reached 100 - 200 mm3 on day 7 . F . Single - cell suspensions of tumor tissues from MC38 tumor - bearing C57BL / 6 mice treated with vehicle or TP - 16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis . Representative flow cytometry and dot plots of intratumoral frequencies of CD8 + T cells ( n = 5 ) . G . The representative bioluminescence images ( left panel ) and quantitative growth curves ( right panel ) of an orthotopic colorectal cancer mouse model , CT26 - Luc model ( n = 4 ) .

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Figure 3B. The antitumor activities of E7046 (150 mg/kg) and TP-16 (37.5, 75, and150 mg/kg) in CT26 tumor-bearing BALB/c mice (n = 11 per group). Tumor growth curves in the CT26 model (left panel). Bar graphs show the individual changes in tumor growth relative to after and before drug administration (right panel). Tumor volumes reached 100-200 mm3 on day 7.C. Representative graph showing that the antitumor activity of TP-16 is lost in CT26 tumors engrafted into BALB/c nude mice (left panel) (n = 8). Bar graphs show the individual changes in tumor growth relative to after and before drug administration (right panel). Tumor volumes reached 100-200 mm3 on day 7.D. Single-cell suspensions of tumor tissues from CT26 tumor-bearing BALB/c mice treated with vehicle or TP-16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis. Representative flow cytometry and dot plots of intratumoral frequencies of CD8+ T cells (n = 5).E. Growth curves (left panel) and relative change after 18 days treatment (right panel) in colon cancer (MC38) xenograft tumors in C57BL/6 mice (n = 7). Tumor volumes reached 100-200 mm3 on day 7.F. Single-cell suspensions of tumor tissues from MC38 tumor-bearing C57BL/6 mice treated with vehicle or TP-16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis. Representative flow cytometry and dot plots of intratumoral frequencies of CD8+ T cells (n = 5).G. The representative bioluminescence images (left panel) and quantitative growth curves (right panel) of an orthotopic colorectal cancer mouse model, CT26-Luc model (n = 4).


Figure 4A , B . Tumors from CT26 tumor - bearing BALB / c mice treated with vehicle or TP - 16 for 2 weeks were harvested , and tumor single - cell suspensions were analyzed for tumor - associated myeloid cells by flow cytometry analysis . Representative graphs and quantification of F4 / 80 + MHC - II + immunosuppressive myeloid cells ( IMCs ) - M1 macrophages ( A ) and F4 / 80 + CD206 + IMCs - M2 macrophages ( B ) gated on CD45 + CD11b + myeloid cells ( n = 5 ) . C . Representative flow cytometry analysis and quantification of Ly6C + Ly6G - IMCs - monocytic myeloid - derived suppressor cell ( mMDSC ) and Ly6CmidLy6G + IMCs - PMN - MDSC frequencies gated on CD45 + CD11b + myeloid cells ( n = 5 ) . Relative expression of selected M1 , M2 markers on flow - sorted M1 macrophages ( D ) , M2 macrophages ( E ) from CT26 tumors . Data are normalized to β - actin expression levels ( n = 5 ) . Relative expression of selected MDSC markers on flow - sorted mMDSCs ( F ) , and PMN - MDSCs ( G ) from CT26 tumors . Data are normalized to β - actin expression levels ( n = 5 ) . H - K . Representative flow analysis and quantification of CD8 + T cell activation markers : Granzyme B ( H ) , IFN - γ ( I ) , TNF - α ( J ) , and PD - 1 ( K ) ( n = 5 ) .

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Figure 4A, B. Tumors from CT26 tumor-bearing BALB/c mice treated with vehicle or TP-16 for 2 weeks were harvested, and tumor single-cell suspensions were analyzed for tumor-associated myeloid cells by flow cytometry analysis. Representative graphs and quantification of F4/80+MHC-II+ immunosuppressive myeloid cells (IMCs)-M1 macrophages (A) and F4/80+CD206+ IMCs-M2 macrophages (B) gated on CD45+CD11b+ myeloid cells (n = 5).C. Representative flow cytometry analysis and quantification of Ly6C+Ly6G- IMCs-monocytic myeloid-derived suppressor cell (mMDSC) and Ly6CmidLy6G+ IMCs-PMN-MDSC frequencies gated on CD45+CD11b+ myeloid cells (n = 5).Relative expression of selected M1, M2 markers on flow-sorted M1 macrophages (D), M2 macrophages (E) from CT26 tumors. Data are normalized to β-actin expression levels (n = 5).Relative expression of selected MDSC markers on flow-sorted mMDSCs (F), and PMN-MDSCs (G) from CT26 tumors. Data are normalized to β-actin expression levels (n = 5).H-K. Representative flow analysis and quantification of CD8+ T cell activation markers: Granzyme B (H), IFN-γ (I), TNF-α (J), and PD-1 (K) (n = 5).


Figure 5M - CSF induced mouse bone marrow derived macrophages ( BMMs ) were stimulated with 50 ng / mL mouse recombinant IFN - γ ( A ) in the absence or presence of PGE2 ± varying concentrations of TP - 16 for 12 hr . The mRNA levels of selected M1 markers ( Cxcl10 and Tnfa ) were measured by q - PCR . Data are normalized to β - actin expression levels ( n = 3 ) . B . M - CSF induced mouse bone marrow derived macrophages ( BMMs ) were stimulated with 20 ng / mL mouse recombinant IL - 4 ( B ) in the absence or presence of PGE2 ± varying concentrations of TP - 16 for 12 hr . The mRNA levels of selected M2 markers ( Arg - 1 and Ym - 1 ) were measured by q - PCR . Data are normalized to β - actin expression levels ( n = 3 ) . C . Bone marrow cells were cultured with 40 ng / mL GM - CSF / IL - 6 or co - stimulated with PGE2 ± varying concentrations of TP - 16 for 6 days . Representative quantification of Ly6C + Ly6G − monocytic myeloid - derived suppressor cells ( mMDSCs ) and Ly6CmidLy6G + PMN - MDSCs ( n = 3 ) . D . Bone marrow cells were treated with GM - CSF / IL - 6 for 6 days to induce MDSCs and thereafter , with PGE2 ± varying concentrations of TP - 16 for 12 hr . The mRNA levels of selected MDSC markers ( Arg - 1 , Ptgs2 , Il - 4 , and Il - 10 ) were measured by qPCR . Data are normalized to β - actin expression levels ( n = 3 ) . E , F . In vitro T cell suppression activity of CT26 - tumor infiltrating CD11b + myeloid cells collected at 2 weeks post - treatment with vehicle or 75 mg / kg TP - 16 . CD8 + T cells were labeled with CFSE and then stimulated with CD3 / CD28 antibodies for 3 days in the absence or presence of tumor - infiltrating CD11b + myeloid cells from vehicle - or 75 mg / kg TP - 16 - treated CT26 tumors . Representative flow cytometry histograms ( E ) and percentages ( F ) of proliferating CD8 + T cells when plated in a ratio of 2 : 1 , 1 : 1 , and 1 : 2 CD8 + T cells to CD11b + myeloid cells ( n = 3 ) .

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 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 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 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 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

Figure 5M-CSF induced mouse bone marrow derived macrophages (BMMs) were stimulated with 50 ng/mL mouse recombinant IFN-γ (A) in the absence or presence of PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected M1 markers (Cxcl10 and Tnfa) were measured by q-PCR. Data are normalized to β-actin expression levels (n=3).B. M-CSF induced mouse bone marrow derived macrophages (BMMs) were stimulated with 20 ng/mL mouse recombinant IL-4 (B) in the absence or presence of PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected M2 markers (Arg-1 and Ym-1) were measured by q-PCR. Data are normalized to β-actin expression levels (n=3).C. Bone marrow cells were cultured with 40 ng/mL GM-CSF/IL-6 or co-stimulated with PGE2 ± varying concentrations of TP-16 for 6 days. Representative quantification of Ly6C+Ly6G− monocytic myeloid-derived suppressor cells (mMDSCs) and Ly6CmidLy6G+ PMN-MDSCs (n = 3).D. Bone marrow cells were treated with GM-CSF/IL-6 for 6 days to induce MDSCs and thereafter, with PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected MDSC markers (Arg-1, Ptgs2, Il-4, and Il-10) were measured by qPCR. Data are normalized to β-actin expression levels (n=3).E, F. In vitro T cell suppression activity of CT26-tumor infiltrating CD11b+ myeloid cells collected at 2 weeks post-treatment with vehicle or 75 mg/kg TP-16. CD8+ T cells were labeled with CFSE and then stimulated with CD3/CD28 antibodies for 3 days in the absence or presence of tumor-infiltrating CD11b+ myeloid cells from vehicle- or 75 mg/kg TP-16-treated CT26 tumors. Representative flow cytometry histograms (E) and percentages (F) of proliferating CD8+ T cells when plated in a ratio of 2:1, 1:1, and 1:2 CD8+ T cells to CD11b+ myeloid cells (n = 3).


Figure 6A . The growth of CT26 tumor volume when treated with vehicle , TP - 16 ( 75 mg / kg , p . o . , daily ) , anti - PD - 1 antibody ( 50 μg , i . p . , twice weekly ) or their combination ( left ) and percent change in tumor volumes between days 0 and 20 ( right ) ( n = 12 ) . B . BALB / c mice with subcutaneous CT26 tumors were treated with vehicle , TP - 16 ( 75 mg / kg , p . o . , daily ) , PD - 1 antibody ( 50 μg , i . p . , twice weekly ) or combined therapies for 2 weeks . Animal survival ( time to tumor burden reaching 2000 mm3 ) was analyzed by the Kaplan - Meier method using GraphPad Prism ( n = 10 ) . P values were calculated using the Log - rank test . C . Representative immunofluorescence staining images of tumor sections from CT26 tumor - bearing mice stained for CD8 and DAPI . Scale bars , 50 μm . D . Representative immunostaining images of tumor sections from CT26 tumor - bearing mice stained for p - STAT3 , p - AKT and PD - L1 . Scale bars , 100 μm . F . The heatmap of T cell activation biomarkers for RNA - seq analysis . The unit of heatmap scale is expression value after centering and scaling by genes .

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Figure 6A. The growth of CT26 tumor volume when treated with vehicle, TP-16 (75 mg/kg, p.o., daily), anti-PD-1 antibody (50 μg, i.p., twice weekly) or their combination (left) and percent change in tumor volumes between days 0 and 20 (right) (n=12).B. BALB/c mice with subcutaneous CT26 tumors were treated with vehicle, TP-16 (75 mg/kg, p.o., daily), PD-1 antibody (50 μg, i.p., twice weekly) or combined therapies for 2 weeks. Animal survival (time to tumor burden reaching 2000 mm3) was analyzed by the Kaplan-Meier method using GraphPad Prism (n=10). P values were calculated using the Log-rank test.C. Representative immunofluorescence staining images of tumor sections from CT26 tumor-bearing mice stained for CD8 and DAPI. Scale bars, 50 μm. D. Representative immunostaining images of tumor sections from CT26 tumor-bearing mice stained for p-STAT3, p-AKT and PD-L1. Scale bars, 100 μm. F. The heatmap of T cell activation biomarkers for RNA-seq analysis. The unit of heatmap scale is expression value after centering and scaling by genes.


Figure 7Representative resected colons ( B ) after 5 weeks of indicated treatment ( n = 7 ) . total number of colon tumors per mouse ( C , left ) and tumor size distribution ( C , right ) , and colon length ( D ) after 5 weeks of indicated treatment ( n = 7 ) . E . Representative hematoxylin and eosin ( H & E ) staining and immunostaining for CD8 , p - STAT3 , p - AKT , and ARG - 1 of tumors treated with vehicle , TP - 16 , anti - PD - 1 antibody , or TP - 16 and anti - PD - 1 combined . Scale bars , 500 μm ( left images ) , 50 μm ( right images ) . F . Quantitative polymerase chain reaction ( q - PCR ) analysis of the mRNA level of genes associated with immunosuppression ( n = 6 ) .

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Figure 7Representative resected colons (B) after 5 weeks of indicated treatment (n=7).total number of colon tumors per mouse (C, left) and tumor size distribution (C, right), and colon length (D) after 5 weeks of indicated treatment (n=7).E. Representative hematoxylin and eosin (H&E) staining and immunostaining for CD8, p-STAT3, p-AKT, and ARG-1 of tumors treated with vehicle, TP-16, anti-PD-1 antibody, or TP-16 and anti-PD-1 combined. Scale bars, 500 μm (left images), 50 μm (right images).F. Quantitative polymerase chain reaction (q-PCR) analysis of the mRNA level of genes associated with immunosuppression (n = 6).


Fig 1HeLa cells were uninfected ( ' control ' ) or infected with B . abortus ( ' B . abortus ' ) . Cell lysates were collected at the indicated time points and analyzed by Western blotting . ( A ) Representative immunoblots for pIRE1 , spliced - XBP1 , and GAPDH . GAPDH was used for normalization . The intensity of the bands was quantified using the MultiGauge software . ( B , C ) Relative protein levels of pIRE1 ( B ) and spliced - XBP1 ( C ) in uninfected control ( open circles ) and Brucella - infected ( solid circles ) cells . The protein levels at time 0 hr were assigned the value 1 . Data are means ± SD from three independent experiments . ( D ) Representative immunoblots for pPERK , cleaved - ATF6 , and GAPDH , and relative protein levels of pPERK and cleaved - ATF6 in control ( open bars ) and Brucella - infected ( solid bars ) cells . GAPDH was used for normalization . The intensity of the bands was quantified using the MultiGauge software , and the results are shown in the bar graphs . As a positive control for activation of PERK or ATF6 , HeLa cells were treated with 5 μg / ml of tunicamycin for 8hr ( ' Tm ' ) . The protein levels at time 0 hr were assigned the value 1 . Data are means ± SD from three independent experiments . ( E ) Representative immunoblots for Sar1 , Sec23 , Sec24D , and GAPDH , and relative protein levels of Sar1 , Sec23 , and Sec24D in control ( open bars ) and Brucella - infected ( solid bars ) cells . GAPDH was used for normalization . The intensity of the bands was quantified using the MultiGauge software , and the results are shown in the bar graphs . The protein levels in control cells were assigned the value 1 . Data are means ± SD from three independent experiments . * : p < 0 . 05 ; * * : p < 0 . 01 .

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Fig 1HeLa cells were uninfected ('control') or infected with B. abortus ('B. abortus'). Cell lysates were collected at the indicated time points and analyzed by Western blotting. (A) Representative immunoblots for pIRE1, spliced-XBP1, and GAPDH. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software. (B, C) Relative protein levels of pIRE1 (B) and spliced-XBP1 (C) in uninfected control (open circles) and Brucella-infected (solid circles) cells. The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.(D) Representative immunoblots for pPERK, cleaved-ATF6, and GAPDH, and relative protein levels of pPERK and cleaved-ATF6 in control (open bars) and Brucella-infected (solid bars) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. As a positive control for activation of PERK or ATF6, HeLa cells were treated with 5 μg/ml of tunicamycin for 8hr ('Tm'). The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.(E) Representative immunoblots for Sar1, Sec23, Sec24D, and GAPDH, and relative protein levels of Sar1, Sec23, and Sec24D in control (open bars) and Brucella-infected (solid bars) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. The protein levels in control cells were assigned the value 1. Data are means ± SD from three independent experiments. *: p<0.05; **: p<0.01.


Fig 2 ( A ) Representative immunoblots showing co - immunoprecipitation with pIRE1 . After 5 hr of Tm treatment , immunoprecipitation was performed on HeLa cell lysates with an anti - pIRE1 antibody ( lanes labeled ' pIRE1 ' ) or control anti - rabbit IgG ( lanes labeled ' IgG ' ) , and the immunoprecipitates were analyzed by Western blotting with a panel of antibodies against molecules involved in the ER - Golgi vesicular transport pathways or against ER - ( Sec61α , HSP47 , and calnexin ) , ERGIC - ( ERGIC53 ) and cis - Golgi - ( GM130 ) resident proteins . ( B ) Representative immunoblot showing the co - immunoprecipitation of pIRE1 with Yip1A . Following 5 hr of Tm treatment , immunoprecipitation was performed on HeLa cell lysates with an anti - Yip1A antibody ( lane labeled ' Yip1A ' ) or control anti - rabbit IgG ( lane labeled ' IgG ' ) , and the immunoprecipitates were analyzed by Western blotting with an anti - pIRE1 antibody . ( C ) Representative confocal micrographs of HeLa cells double - stained for pIRE1 ( green ) and Yip1A ( blue ) after 5 hr of Tm treatment . Insets are magnifications of the boxed areas on the main image . Co - localized proteins were detected as large foci . Scale bars are 10 μm . ( D ) Representative confocal micrographs of HeLa cells triple - stained for pIRE1 ( green ) , Sec31 ( red ) , and Yip1A ( blue ) after 5 hr of Tm treatment . Insets are magnifications of the boxed areas on the main image . Co - localized proteins were identified as large , bright foci ( arrows ) . Scale bars are 10 μm .

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 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 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

Fig 2(A) Representative immunoblots showing co-immunoprecipitation with pIRE1. After 5 hr of Tm treatment, immunoprecipitation was performed on HeLa cell lysates with an anti-pIRE1 antibody (lanes labeled 'pIRE1') or control anti-rabbit IgG (lanes labeled 'IgG'), and the immunoprecipitates were analyzed by Western blotting with a panel of antibodies against molecules involved in the ER-Golgi vesicular transport pathways or against ER- (Sec61α, HSP47, and calnexin), ERGIC- (ERGIC53) and cis-Golgi- (GM130) resident proteins.(B) Representative immunoblot showing the co-immunoprecipitation of pIRE1 with Yip1A. Following 5 hr of Tm treatment, immunoprecipitation was performed on HeLa cell lysates with an anti-Yip1A antibody (lane labeled 'Yip1A') or control anti-rabbit IgG (lane labeled 'IgG'), and the immunoprecipitates were analyzed by Western blotting with an anti-pIRE1 antibody.(C) Representative confocal micrographs of HeLa cells double-stained for pIRE1 (green) and Yip1A (blue) after 5 hr of Tm treatment. Insets are magnifications of the boxed areas on the main image. Co-localized proteins were detected as large foci. Scale bars are 10 μm.(D) Representative confocal micrographs of HeLa cells triple-stained for pIRE1 (green), Sec31 (red), and Yip1A (blue) after 5 hr of Tm treatment. Insets are magnifications of the boxed areas on the main image. Co-localized proteins were identified as large, bright foci (arrows). Scale bars are 10 μm.


Fig 3HeLa cells were transfected with each siRNA for 24 hr , and then treated with Tm to induce the UPR . Cell lysates were prepared at the indicated time points and analyzed by Western blotting . ( A ) Representative immunoblots for pIRE1 , spliced - XBP1 , and β - tubulin . β - tubulin was used for normalization . The intensity of the bands was quantified using the MultiGauge software . ( B - D ) Relative protein levels of pIRE1 ( B ) , relative mRNA levels of spliced - XBP1 ( C ) , and relative protein levels of spliced - XBP1 ( C ) in control ( solid circles ) and Yip1A - knockdown ( open circles ) cells . The protein or mRNA levels in control cells at the beginning of the Tm treatment were assigned the value 1 . Data are means ± SD from three independent experiments . ( E ) Representative immunoblots for Sar1 , Sec23 , Sec24D , and GAPDH , and relative protein levels of Sar1 , Sec23 , and Sec24D in control ( solid circles ) , Yip1A - knockdown ( open circles ) , and IRE1 - knockdown ( solid gray circles ) cells . GAPDH was used for normalization . The intensity of the bands was quantified using the MultiGauge software , and the results are shown in the line graphs . The protein levels in control cells at the beginning of the Tm treatment were assigned the value 1 . Data are means ± SD from three independent experiments . ( F ) Representative confocal micrographs of control ( upper panels ) or Yip1A - knockdown ( lower panels ) cells during the Tm treatment . Fixed cells at the indicated time points were stained for pIRE1 . Cells are outlined with white dashed lines . Scale bars are 10 μm . The numbers of pIRE1 foci per cell were counted , and shown in the line graph . Data are means ± SD ( N = 30 ) . ( G ) Representative immunoblot for pIRE1 after native PAGE , showing two high - order complexes of pIRE1 ( pIRE1 - I and pIRE1 - II ) . Lane ' S ' represents lysate from HeLa cells transfected with control scramble siRNA , and lane ' Y ' represents lysate from HeLa cells transfected with Yip1A siRNA . Numbers on the left - hand side correspond to the standard molecular weight . The intensity of the bands was quantified by using the MultiGauge software , and the results are shown in the bar graphs . Protein levels in control cells at the beginning of the Tm treatment were assigned the value 1 . Data are means ± SD from three independent experiments . * * : 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 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 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 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

Fig 3HeLa cells were transfected with each siRNA for 24 hr, and then treated with Tm to induce the UPR. Cell lysates were prepared at the indicated time points and analyzed by Western blotting. (A) Representative immunoblots for pIRE1, spliced-XBP1, and β-tubulin. β-tubulin was used for normalization. The intensity of the bands was quantified using the MultiGauge software.(B-D) Relative protein levels of pIRE1 (B), relative mRNA levels of spliced-XBP1 (C), and relative protein levels of spliced-XBP1 (C) in control (solid circles) and Yip1A-knockdown (open circles) cells. The protein or mRNA levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments.(E) Representative immunoblots for Sar1, Sec23, Sec24D, and GAPDH, and relative protein levels of Sar1, Sec23, and Sec24D in control (solid circles), Yip1A-knockdown (open circles), and IRE1-knockdown (solid gray circles) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the line graphs. The protein levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments.(F) Representative confocal micrographs of control (upper panels) or Yip1A-knockdown (lower panels) cells during the Tm treatment. Fixed cells at the indicated time points were stained for pIRE1. Cells are outlined with white dashed lines. Scale bars are 10 μm. The numbers of pIRE1 foci per cell were counted, and shown in the line graph. Data are means ± SD (N = 30).(G) Representative immunoblot for pIRE1 after native PAGE, showing two high-order complexes of pIRE1 (pIRE1-I and pIRE1-II). Lane 'S' represents lysate from HeLa cells transfected with control scramble siRNA, and lane 'Y' represents lysate from HeLa cells transfected with Yip1A siRNA. Numbers on the left-hand side correspond to the standard molecular weight. The intensity of the bands was quantified by using the MultiGauge software, and the results are shown in the bar graphs. Protein levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments. **: p<0.01.


Fig 4HeLa cells were transfected with each siRNA for 24 hr , and then treated with Tm for 5 hr to induce the UPR . ( A , B ) Representative confocal micrographs of control ( left - hand panels ) , Yip1A - knockdown ( middle panels ) , and IRE1 - knockdown ( right - had panels ) cells after 0 hr or 5 hr of Tm treatment ( A ) . The ER structure was visualized with an anti - calnexin antibody . Large vacuoles were observed in control cells ( arrowheads ) , but not in Yip1A - or IRE1 - knockdown cells . Scale bars are 10 μm . The percentage of cells with vacuoles was counted , and is shown in the bar graph ( B ) . Data are means ± SD from three independent experiments ( N = 100 ) . * * : p < 0 . 01 . ( C , D ) Representative confocal micrographs of control , Atg9 - knockdown , WIPI1 - knockdown , and DFCP1 - knockdown cells after 0 hr or 5 hr of Tm treatment ( C ) . The ER structure was visualized with an anti - calnexin antibody . Large vacuoles were observed in control and DFCP1 - knockdown cells ( arrowheads ) , but rarely seen in Atg9 - or WIPI1 - knockdown cells . Scale bars are 10 μm . The percentage of cells with vacuoles was counted , and is shown in the bar graph ( D ) . Data are means ± SD from three independent experiments ( N = 100 ) . * * : 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 O O O O O O 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

Fig 4HeLa cells were transfected with each siRNA for 24 hr, and then treated with Tm for 5 hr to induce the UPR. (A, B) Representative confocal micrographs of control (left-hand panels), Yip1A-knockdown (middle panels), and IRE1-knockdown (right-had panels) cells after 0 hr or 5 hr of Tm treatment (A). The ER structure was visualized with an anti-calnexin antibody. Large vacuoles were observed in control cells (arrowheads), but not in Yip1A- or IRE1-knockdown cells. Scale bars are 10 μm. The percentage of cells with vacuoles was counted, and is shown in the bar graph (B). Data are means ± SD from three independent experiments (N = 100). **: p<0.01.(C, D) Representative confocal micrographs of control, Atg9-knockdown, WIPI1-knockdown, and DFCP1-knockdown cells after 0 hr or 5 hr of Tm treatment (C). The ER structure was visualized with an anti-calnexin antibody. Large vacuoles were observed in control and DFCP1-knockdown cells (arrowheads), but rarely seen in Atg9- or WIPI1-knockdown cells. Scale bars are 10 μm. The percentage of cells with vacuoles was counted, and is shown in the bar graph (D). Data are means ± SD from three independent experiments (N = 100). **: p<0.01.


Fig 5HeLa cells were infected with B . abortus , and then transfected with each siRNA at 1 hr p . i . Cell lysates were collected at the indicated time points , and analyzed by Western blotting . ( A ) Representative immunoblots for pIRE1 , spliced - XBP1 , and GAPDH . GAPDH was used for normalization . The intensity of the bands was quantified using the MultiGauge software ( B - D ) Relative protein levels of pIRE1 ( B ) and spliced - XBP1 ( C ) , and relative mRNA levels of spliced - XBP1 ( D ) in control ( solid circles ) and Yip1A - knockdown ( open circles ) cells . The protein levels at time 0 hr were assigned the value 1 . Data are means ± SD from three independent experiments . ( E ) Representative immunoblots for pPERK , cleaved - ATF6 , and GAPDH , and relative protein levels of pPERK and cleaved - ATF6 in control ( solid bars ) and Yip1A - knockdown ( open bars ) cells . GAPDH was used for normalization . The intensity of the bands was quantified using the MultiGauge software , and the results are shown in the bar graphs . As a positive control for activation of PERK or ATF6 , HeLa cells were treated with 5 μg / ml tunicamycin for 8 hr ( ' Tm ' ) . The protein levels at time 0 hr were assigned the value 1 . Data are means ± SD from three independent experiments . . ( F ) Representative immunoblots for Sar1 , Sec23 , Sec24D , and GAPDH , and relative protein levels of Sar1 , Sec23 , and Sec24D in control ( solid bars ) and Yip1A - knockdown ( open bars ) cells at 24 hr p . i . GAPDH was used for normalization . The intensity of the bands was quantified using the MultiGauge software , and the results are shown in the bar graphs . The protein levels in control cells were assigned the value 1 . Data are means ± SD from three independent experiments . * : p < 0 . 05 ; * * : p < 0 . 01 . ( G ) Representative immunoblots for Sar1 , Sec23 , Sec24D , and GAPDH , and relative protein levels of Sar1 , Sec23 , and Sec24D in control ( solid bars ) and IRE1 - knockdown ( open bars ) cells at 24 hr p . i . GAPDH was used for normalization . The intensity of the bands was quantified using the MultiGauge software , and the results are shown in the bar graphs . The protein levels in control cells were assigned the value 1 . Data are means ± SD from three independent experiments . * : 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Fig 5HeLa cells were infected with B. abortus, and then transfected with each siRNA at 1 hr p.i. Cell lysates were collected at the indicated time points, and analyzed by Western blotting. (A) Representative immunoblots for pIRE1, spliced-XBP1, and GAPDH. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software(B-D) Relative protein levels of pIRE1 (B) and spliced-XBP1 (C), and relative mRNA levels of spliced-XBP1 (D) in control (solid circles) and Yip1A-knockdown (open circles) cells. The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.(E) Representative immunoblots for pPERK, cleaved-ATF6, and GAPDH, and relative protein levels of pPERK and cleaved-ATF6 in control (solid bars) and Yip1A-knockdown (open bars) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. As a positive control for activation of PERK or ATF6, HeLa cells were treated with 5 μg/ml tunicamycin for 8 hr ('Tm'). The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.. (F) Representative immunoblots for Sar1, Sec23, Sec24D, and GAPDH, and relative protein levels of Sar1, Sec23, and Sec24D in control (solid bars) and Yip1A-knockdown (open bars) cells at 24 hr p.i. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. The protein levels in control cells were assigned the value 1. Data are means ± SD from three independent experiments. *: p<0.05; **: p<0.01.(G) Representative immunoblots for Sar1, Sec23, Sec24D, and GAPDH, and relative protein levels of Sar1, Sec23, and Sec24D in control (solid bars) and IRE1-knockdown (open bars) cells at 24 hr p.i. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. The protein levels in control cells were assigned the value 1. Data are means ± SD from three independent experiments. *: p<0.05; **: p<0.01.


Fig 6HeLa cells were infected with B . abortus , and then transfected with each siRNA at 1 hr p . i . ( A ) Intracellular growth of B . abortus within control ( solid bars ) , Yip1A - knockdown ( open bars ) , and IRE1 - knockdown ( solid gray bars ) cells . CFUs were enumerated at the indicated time points . Data are means ± SD from three independent experiments . * : p < 0 . 05 ; * * : p < 0 . 01 . ( B , C ) Representative confocal micrographs of control ( left - hand panel ) and Yip1A - knockdown ( right - hand panel ) cells at 24 hr p . i . ( B ) . Fixed cells were double - stained for Yip1A ( green ) and B . abortus ( red ) . The infected cells are outlined with white dashed lines . Scale bars are 10 μm . The knockdown efficiency of Yip1A in infected cells was evaluated by quantifying the intensity of immunofluorescence staining for Yip1A , and the result is shown in the bar graph ( C ) . ( D , E ) Representative confocal micrographs of control ( left - hand panel ) and IRE1 - knockdown ( right - hand panel ) cells at 24 hr p . i . ( D ) . Fixed cells were double - stained for IRE1 ( green ) and B . abortus ( red ) . The infected cells are outlined with white dashed lines . Scale bars are 10 μm . The knockdown efficiency of IRE1 in infected cells was evaluated by quantifying the intensity of immunofluorescence staining for IRE1 , and the result is shown in the bar graph ( E ) . ( F , G ) Representative confocal micrographs of control ( left - hand panels ) , Yip1A - knockdown ( middle panels ) , and IRE1 - knockdown ( right - hand panels ) cells at 8 hr , 16 hr , and 24 hr p . i . ( F ) . Fixed cells were stained for B . abortus . The infected cells are outlined with white dashed lines . Scale bars are 10 μm . To assess the replication efficiency , the percentage of infected cells with fewer than ten B . abortus was determined at 24hr p . i . , and the result is shown in the bar graph ( G ) .

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 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 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 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

Fig 6HeLa cells were infected with B. abortus, and then transfected with each siRNA at 1 hr p.i. (A) Intracellular growth of B. abortus within control (solid bars), Yip1A-knockdown (open bars), and IRE1-knockdown (solid gray bars) cells. CFUs were enumerated at the indicated time points. Data are means ± SD from three independent experiments. *: p<0.05; **: p<0.01.(B, C) Representative confocal micrographs of control (left-hand panel) and Yip1A-knockdown (right-hand panel) cells at 24 hr p.i. (B). Fixed cells were double-stained for Yip1A (green) and B. abortus (red). The infected cells are outlined with white dashed lines. Scale bars are 10 μm. The knockdown efficiency of Yip1A in infected cells was evaluated by quantifying the intensity of immunofluorescence staining for Yip1A, and the result is shown in the bar graph (C).(D, E) Representative confocal micrographs of control (left-hand panel) and IRE1-knockdown (right-hand panel) cells at 24 hr p.i. (D). Fixed cells were double-stained for IRE1 (green) and B. abortus (red). The infected cells are outlined with white dashed lines. Scale bars are 10 μm. The knockdown efficiency of IRE1 in infected cells was evaluated by quantifying the intensity of immunofluorescence staining for IRE1, and the result is shown in the bar graph (E).(F, G) Representative confocal micrographs of control (left-hand panels), Yip1A-knockdown (middle panels), and IRE1-knockdown (right-hand panels) cells at 8 hr, 16 hr, and 24 hr p.i. (F). Fixed cells were stained for B. abortus. The infected cells are outlined with white dashed lines. Scale bars are 10 μm. To assess the replication efficiency, the percentage of infected cells with fewer than ten B. abortus was determined at 24hr p.i., and the result is shown in the bar graph (G).


Fig 7 ( A ) Representative electron micrographs of Brucella - infected control ( left - hand panel ) and Yip1A - knockdown ( right - hand panel ) cells at 24 hr p . i . Insets are magnifications of the boxed areas on the main image , showing the typical forms of BCVs . In control cells , BCVs can be seen in the form of ER - derived membrane - bound compartments ( inset in left - hand panel , defined as ' I ' ) . Note the presence of ribosomes on the membrane ( arrowheads ) . In Yip1A - knockdown cells , the bacteria were not sequestered into such compartments ( inset in right - hand panel , defined as ' II ' ) . Scale bars are 2 μm . ( B ) Representative electron micrograph of Brucella - infected IRE1 - knockdown cells at 24 hr p . i . Inset is a magnification of the boxed area in the main image , showing that the bacteria were not sequestered into ER - derived membrane . Scale bar is 2 μm . ) The percentages of the two forms of BCVs ( I and II ) present in control and Yip1A - knockdown cells at 24 hr p . i . The total numbers of BCVs analyzed were 67 for control cells and 37 for Yip1A - knockdown cells . ( D , E ) Representative confocal micrographs of control ( upper panels ) , Yip1A - knockdown ( middle panels ) , and IRE1 - knockdown ( lower panels ) cells double - stained for Lamp2 ( a marker for late endosomes / lysosomes ; green ) and B . abortus ( red ) at 24 hr p . i . BCVs co - localized with Lamp2 are indicated by arrowheads . The infected cells are outlined with white dashed lines . Scale bars are 10 μm . The percentage of Lamp2 - positive BCVs was determined , and is shown in the line graph ( E ) . ( D , E ) Representative confocal micrographs of control ( upper panels ) , Yip1A - knockdown ( middle panels ) , and IRE1 - knockdown ( lower panels ) cells double - stained for Lamp2 ( a marker for late endosomes / lysosomes ; green ) and B . abortus ( red ) at 24 hr p . i . BCVs co - localized with Lamp2 are indicated by arrowheads . The infected cells are outlined with white dashed lines . Scale bars are 10 μm . The percentage of Lamp2 - positive BCVs was determined , and is shown in the line graph ( E ) . . ( F ) Magnification of the electron micrograph of the Brucella - infected control cell in ( A ) , showing large vacuoles studded locally with ribosomes ( arrows ) . ( G - I ) Representative confocal micrographs of Brucella - infected control cells at 24 hr p . i . triple - stained for HA - Sec61α ( a marker for rough ER ; green ) , B . abortus ( red ) , and Lamp2 ( a marker for endosomes / lysosomes ; blue ) ( G ) . An expression construct for HA - Sec61α was co - transfected with scramble siRNA . The infected cell is outlined with white dashed lines . Scale bars are 10 μm . Magnifications of the boxed areas ' a ' and ' b ' are shown in ( H ) and ( I ) , respectively . Large vacuoles adjacent to replicating bacteria were stained for both Sec61α and Lamp2 ( H ) . B . abortus was co - stained with Sec61α but not with Lamp2 ( I ) .

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 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 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 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 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 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 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

Fig 7(A) Representative electron micrographs of Brucella-infected control (left-hand panel) and Yip1A-knockdown (right-hand panel) cells at 24 hr p.i. Insets are magnifications of the boxed areas on the main image, showing the typical forms of BCVs. In control cells, BCVs can be seen in the form of ER-derived membrane-bound compartments (inset in left-hand panel, defined as 'I'). Note the presence of ribosomes on the membrane (arrowheads). In Yip1A-knockdown cells, the bacteria were not sequestered into such compartments (inset in right-hand panel, defined as 'II'). Scale bars are 2 μm.(B) Representative electron micrograph of Brucella-infected IRE1-knockdown cells at 24 hr p.i. Inset is a magnification of the boxed area in the main image, showing that the bacteria were not sequestered into ER-derived membrane. Scale bar is 2 μm.) The percentages of the two forms of BCVs (I and II) present in control and Yip1A-knockdown cells at 24 hr p.i. The total numbers of BCVs analyzed were 67 for control cells and 37 for Yip1A-knockdown cells.(D, E) Representative confocal micrographs of control (upper panels), Yip1A-knockdown (middle panels), and IRE1-knockdown (lower panels) cells double-stained for Lamp2 (a marker for late endosomes/lysosomes; green) and B. abortus (red) at 24 hr p.i. BCVs co-localized with Lamp2 are indicated by arrowheads. The infected cells are outlined with white dashed lines. Scale bars are 10 μm. The percentage of Lamp2-positive BCVs was determined, and is shown in the line graph (E).(D, E) Representative confocal micrographs of control (upper panels), Yip1A-knockdown (middle panels), and IRE1-knockdown (lower panels) cells double-stained for Lamp2 (a marker for late endosomes/lysosomes; green) and B. abortus (red) at 24 hr p.i. BCVs co-localized with Lamp2 are indicated by arrowheads. The infected cells are outlined with white dashed lines. Scale bars are 10 μm. The percentage of Lamp2-positive BCVs was determined, and is shown in the line graph (E).. (F) Magnification of the electron micrograph of the Brucella-infected control cell in (A), showing large vacuoles studded locally with ribosomes (arrows).(G-I) Representative confocal micrographs of Brucella-infected control cells at 24 hr p.i. triple-stained for HA-Sec61α (a marker for rough ER; green), B. abortus (red), and Lamp2 (a marker for endosomes/lysosomes; blue) (G). An expression construct for HA-Sec61α was co-transfected with scramble siRNA. The infected cell is outlined with white dashed lines. Scale bars are 10 μm. Magnifications of the boxed areas 'a' and 'b' are shown in (H) and (I), respectively. Large vacuoles adjacent to replicating bacteria were stained for both Sec61α and Lamp2 (H). B. abortus was co-stained with Sec61α but not with Lamp2 (I).


Fig 8HeLa cells were infected with B . abortus , and then transfected with each siRNA at 1 hr p . i . ( A ) Intracellular growth of B . abortus within control ( blue bars ) , Atg9 - knockdown ( red bars ) , WIPI1 - knockdown ( green bars ) , and DFCP1 - knockdown ( purple bars ) cells . CFUs were counted at the indicated time points after infection . Data are means ± SD from three independent experiments . * * : p < 0 . 01 . ( B , C ) Representative confocal micrographs of control ( top row ) , Atg9 - knockdown ( second row ) , WIPI1 - knockdown ( third row ) , and DFCP1 - knockdown ( bottom row ) cells double - stained for Lamp2 ( green ) and B . abortus ( red ) at 24 hr p . i . BCVs co - localized with Lamp2 are indicated by arrowheads . The infected cells are outlined with white dashed lines . Scale bars are 10 μm . The percentage of Lamp2 - positive BCVs was determined , and is shown in the line graph ( C ) .

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 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

Fig 8HeLa cells were infected with B. abortus, and then transfected with each siRNA at 1 hr p.i. (A) Intracellular growth of B. abortus within control (blue bars), Atg9-knockdown (red bars), WIPI1-knockdown (green bars), and DFCP1-knockdown (purple bars) cells. CFUs were counted at the indicated time points after infection. Data are means ± SD from three independent experiments. **: p<0.01.(B, C) Representative confocal micrographs of control (top row), Atg9-knockdown (second row), WIPI1-knockdown (third row), and DFCP1-knockdown (bottom row) cells double-stained for Lamp2 (green) and B. abortus (red) at 24 hr p.i. BCVs co-localized with Lamp2 are indicated by arrowheads. The infected cells are outlined with white dashed lines. Scale bars are 10 μm. The percentage of Lamp2-positive BCVs was determined, and is shown in the line graph (C).


Figure 1a , Ultrasonic vocalizations ( USV ) in isolated miR379 - 410 ko mouse pups emitted on postnatal day P3 , P6 , P9 and P12 . Left panel : total number of ultrasonic vocalizations ( USV ) in isolated miR379 - 410 wt and ko mouse pups emitted on average ( P 3 , 6 , 9 and 12 ) when isolated from the mother , wt n = 13 ( male n = 9 , female n = 4 ) , ko n = 20 ( male n = 11 , female n = 9 ) , t31 = 2 . 274 , * p = 0 . 0301 , unpaired Student ' s t - test . Right panel : developmental course for total number of USV , wt n = 13 ( male n = 9 , female n = 4 ) , ko n = 20 ( male n = 11 , female n = 9 ) , ( development : F3 , 87 = 25 . 463 , p < 0 . 001 ; genotype : F1 , 29 = 4 . 979 , p = 0 . 03 ; development x genotype : F3 , 87 = 1 . 403 , p = 0 . 22 , rm - ANOVA ) ; P 9 : t31 = 2 . 096 , * p = 0 . 044 ; unpaired Student ` s t - test . b , Reciprocal social interaction in juvenile miR379 - 410 wt - and ko mice pairs ( P23 ) . Aberrant time spending in social interaction activity in miR379 - 410 ko - pairs compared to their control wt littermate pairs . Left panel : wt - pairs male n = 13 , ko - pairs male n = 10 , t21 = 2 . 433 , * p = 0 . 0240 , unpaired Student ' s t - test . Middle panel : wt - pairs female n = 15 , ko - pairs female n = 13 , t26 = 1 . 873 , ns p = 0 . 0723 , unpaired Student ' s t - test . Right panel : pooled data of wt - pairs n = 28 ( male n = 13 , female n = 15 ) , ko - pairs n = 23 ( male n = 10 , female n = 13 ) , t49 = 2 . 969 , * * p = 0 . 005 ; unpaired Student ` s t - test . c , Emitted USVs during social interaction activity in genotype - matched pairs in juvenile mice ( P23 ) . Left panel : wt - pairs male n = 13 , ko - pairs male n = 15 , t21 = 2 . 704 , * p = 0 . 0133 , unpaired Student ' s t - test . Middle panel : wt - pairs female n = 15 , ko - pairs female n = 13 , t26 = 2 . 869 , * * p = 0 . 0081 , unpaired Student ' s t - test . Right panel : pooled data of wt - pairs n = 28 ( male n = 13 , female n = 15 ) , ko - pairs n = 23 ( male n = 10 , female n = 13 ) , t49 = 3 . 992 , * * * p < 0 . 001 ; unpaired Student ` s t - test . d , Social preference index in adolescent mice - defined as the ratio of time sniffing a stranger mouse vs . an object - is shown . Left panel : wt male n = 26 , ko male n = 26 , t50 = 1 . 075 , ns p = 0 . 2876 , unpaired Student ' s t - test . Middle panel : wt female n = 27 , ko female n = 30 , t55 = 2 . 017 , * p = 0 . 0486 , unpaired Student ' s t - test . Right panel : pooled data of wt n = 53 ( male n = 26 , female n = 27 ) , ko n = 56 ( male n = 26 , female n = 30 ) , t107 = 2 . 283 , * p = 0 . 0244 ; unpaired Student ` s t - test .

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 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 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 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

Figure 1a, Ultrasonic vocalizations (USV) in isolated miR379-410 ko mouse pups emitted on postnatal day P3, P6, P9 and P12. Left panel: total number of ultrasonic vocalizations (USV) in isolated miR379-410 wt and ko mouse pups emitted on average (P 3, 6, 9 and 12) when isolated from the mother, wt n=13 (male n=9, female n=4), ko n=20 (male n=11, female n=9), t31=2.274, *p=0.0301, unpaired Student's t-test. Right panel: developmental course for total number of USV, wt n=13 (male n=9, female n=4), ko n=20 (male n=11, female n=9), (development: F3,87=25.463, p<0.001; genotype: F1,29=4.979, p=0.03; development x genotype: F3,87=1.403, p= 0.22, rm-ANOVA); P 9: t31=2.096, *p=0.044; unpaired Student`s t-test.b, Reciprocal social interaction in juvenile miR379-410 wt- and ko mice pairs (P23). Aberrant time spending in social interaction activity in miR379-410 ko-pairs compared to their control wt littermate pairs. Left panel: wt-pairs male n=13, ko-pairs male n=10, t21=2.433, *p=0.0240, unpaired Student's t-test. Middle panel: wt-pairs female n=15, ko-pairs female n=13, t26=1.873, ns p=0.0723, unpaired Student's t-test. Right panel: pooled data of wt-pairs n=28 (male n=13, female n=15), ko-pairs n=23 (male n=10, female n=13), t49=2.969, **p=0.005; unpaired Student`s t-test.c, Emitted USVs during social interaction activity in genotype-matched pairs in juvenile mice (P23). Left panel: wt-pairs male n=13, ko-pairs male n=15, t21=2.704, *p=0.0133, unpaired Student's t-test. Middle panel: wt-pairs female n=15, ko-pairs female n=13, t26=2.869, **p=0.0081, unpaired Student's t-test. Right panel: pooled data of wt-pairs n=28 (male n=13, female n=15), ko-pairs n=23 (male n=10, female n=13), t49=3.992, ***p<0.001; unpaired Student`s t-test.d, Social preference index in adolescent mice - defined as the ratio of time sniffing a stranger mouse vs. an object - is shown. Left panel: wt male n=26, ko male n=26, t50=1.075, ns p=0.2876, unpaired Student's t-test. Middle panel: wt female n=27, ko female n=30, t55=2.017, *p=0.0486, unpaired Student's t-test. Right panel: pooled data of wt n=53 (male n=26, female n=27), ko n=56 (male n=26, female n=30), t107=2.283, *p=0.0244; unpaired Student`s t-test.


Figure 2A , B Single - molecule FISH ( smFISH ) on mouse hippocampal neurons . ( A ) Representative pictures of smFISH on BDNF - treated WT ( top ) and KO ( bottom ) mouse hippocampal neurons ( DIV6 ) using a probe directed against the Mirg transcript ( green ) . ( Middle ) Hoechst ( blue ) . ( Right ) MAP2 immunostaining ( red ) . Pictures show a zoom - in on the cell body , since the signal detected in WT neurons was restricted to the nuclei ( scale bar : 5μm ) . ( B ) Representative pictures of smFISH on mouse wild type hippocampal neuron cultures ( DIV6 ; basal ) using a probe specific for the Mirg transcript ( green ) . ( Middle ) Hoechst ( blue ) . ( Right ) MAP2 immunostaining ( red ) . Top : overview , scale bar : 10μm ; bottom : zoom - in , scale bar : 5μm . C , D , Patch - clamp electrophysiological recordings in cultured mouse hippocampal neurons ( DIV 8 - 10 ) isolated from wt or miR379 - 410 ko animals ( n = 3 per group ) . ( C ) Cumulative distribution ( left panel , D = 0 . 25 , p < 0 . 0001 , KS - test ) and mean ( right panel , t16 = 2 . 736 , * p = 0 . 0146 , unpaired Student ` s t - test ) of mEPSC inter - event intervals ; wt n = 9 , ko n = 9 cells analyzed . ( D ) Cumulative distribution ( left panel , D = 0 . 34741 , p < 0 . 0001 , KS - test ) and mean ( right panel , t18 = 2 . 357 , * p = 0 . 0299 , unpaired Student ` s t - test ) of mEPSC amplitudes ; wt n = 10 , ko n = 10 cells analyzed . e , Representative confocal images of CA1 pyramidal neuron apical dendrites from adult Thy1 - GFP / miR379 - 410 wt and Thy1 - GFP / miR379 - 410 ko mouse tissue . Scale bar : 10 µm . f , Spine density in CA1 pyramidal neurons of adult male Thy1 - GFP / miR379 - 410 wt and ko mice ; wt n = 5 , ko n = 6 animals . Mean of basal and apical neurons per mouse are shown , t9 = 3 . 662 , * * p = 0 . 0052 , unpaired Student ` s t - test . g , Spine volume in CA1 pyramidal neurons of adult Thy1 - GFP / miR379 - 410 wt and ko mice ; wt n = 5 , ko n = 6 animals . Mean of basal and apical neurons per mouse are shown , t9 = 2 . 265 , * p = 0 . 0497 , unpaired Student ` s t - test .

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 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 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

Figure 2A, B Single-molecule FISH (smFISH) on mouse hippocampal neurons. (A) Representative pictures of smFISH on BDNF-treated WT (top) and KO (bottom) mouse hippocampal neurons (DIV6) using a probe directed against the Mirg transcript (green). (Middle) Hoechst (blue). (Right) MAP2 immunostaining (red). Pictures show a zoom-in on the cell body, since the signal detected in WT neurons was restricted to the nuclei (scale bar: 5μm). (B) Representative pictures of smFISH on mouse wild type hippocampal neuron cultures (DIV6; basal) using a probe specific for the Mirg transcript (green). (Middle) Hoechst (blue). (Right) MAP2 immunostaining (red). Top: overview, scale bar: 10μm; bottom: zoom-in, scale bar: 5μm.C, D, Patch-clamp electrophysiological recordings in cultured mouse hippocampal neurons (DIV 8-10) isolated from wt or miR379-410 ko animals (n=3 per group). (C) Cumulative distribution (left panel, D=0.25, p<0.0001, KS-test) and mean (right panel, t16=2.736, *p=0.0146, unpaired Student`s t-test) of mEPSC inter-event intervals; wt n=9, ko n=9 cells analyzed. (D) Cumulative distribution (left panel, D=0.34741, p<0.0001, KS-test) and mean (right panel, t18=2.357, *p=0.0299, unpaired Student`s t-test) of mEPSC amplitudes; wt n=10, ko n=10 cells analyzed.e, Representative confocal images of CA1 pyramidal neuron apical dendrites from adult Thy1-GFP/miR379-410 wt and Thy1-GFP/miR379-410 ko mouse tissue. Scale bar: 10 µm. f, Spine density in CA1 pyramidal neurons of adult male Thy1-GFP/miR379-410 wt and ko mice; wt n=5, ko n=6 animals. Mean of basal and apical neurons per mouse are shown, t9=3.662, **p=0.0052, unpaired Student`s t-test. g, Spine volume in CA1 pyramidal neurons of adult Thy1-GFP/miR379-410 wt and ko mice; wt n=5, ko n=6 animals. Mean of basal and apical neurons per mouse are shown, t9=2.265, *p=0.0497, unpaired Student`s t-test.