Figure 4 ( B ) Promoters significantly activated by CasTA2 . 1 using gRNA - H23 ( left ) or gRNA - H22 ( right ) are plotted with basal expression level on x - axis against fold activation by CRISPRa on y - axis . N is the total number of promoters shown . Red box corresponds to highly activated promoters ( fold change > 10 ) . ( C ) Highly activated promoters ( > 10 - fold ) using gRNA - H23 ( left ) or gRNA - H22 ( right ) are plotted . Basal expression levels ( purple lines ) , induced expression level ( orange lines on left or red lines on right , activated with gRNA - H23 or gRNA - H22 , respectively ) and induced fold changes ( gray bars ) are shown . Promoters were first ranked and grouped by basal expression levels . Within each group , promoters were ranked by activation fold changes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (B) Promoters significantly activated by CasTA2.1 using gRNA-H23 (left) or gRNA-H22 (right) are plotted with basal expression level on x-axis against fold activation by CRISPRa on y-axis. N is the total number of promoters shown. Red box corresponds to highly activated promoters (fold change >10).   (C) Highly activated promoters (>10-fold) using gRNA-H23 (left) or gRNA-H22 (right) are plotted. Basal expression levels (purple lines), induced expression level (orange lines on left or red lines on right, activated with gRNA-H23 or gRNA-H22, respectively) and induced fold changes (gray bars) are shown. Promoters were first ranked and grouped by basal expression levels. Within each group, promoters were ranked by activation fold changes.


Figure 5 ( B ) CRISPRi in S . enterica and K . oxytoca using CasTA2 . 1 . ( C ) CRISPRa in S . enterica and K . oxytoca using CasTA1 . 0 ( dCas9 - AsiA _ wt ) , CasTA2 . 1 ( dCas9 - AsiA _ m2 . 1 ) or ancestral strain with basal promoter expression ( none ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (B) CRISPRi in S. enterica and K. oxytoca using CasTA2.1.  (C) CRISPRa in S. enterica and K. oxytoca using CasTA1.0 (dCas9-AsiA_wt), CasTA2.1 (dCas9-AsiA_m2.1) or ancestral strain with basal promoter expression (none).


Figure 1B Expression levels of RBM15 and AS - RBM15 in different blood lineages by meta - analysis of RNA - seq data [ 38 ] : HSC ( hematopoietic stem cells ) , MPP ( multipotent progenitor cells ) , CMP ( common myeloid progenitor cells ) , CLP ( common lymphoid progenitor cells ) , GMP ( Granulocyte - macrophage progenitor cells ) , MEP ( megakaryocyte - erythrocyte progenitor cells ) , MK ( megakaryocyte ) , and EB ( erythroid body ) . C Relative expression levels of AS - RBM15 ( left ) and RBM15 ( right ) in MEG - 01 cells treated with PMA were measured by real - time PCR assays ( n = 4 , mean ± SD ) . SD : standard deviation . D Relative expression levels of AS - RBM15 and RBM15 in CD34 + cells grown in pro - MK medium were measured by real - time PCR . GAPDH was used as an internal control ( n = 3 , mean ± SD ) . Also see Fig EV 1 .

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 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 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

Figure 1B Expression levels of RBM15 and AS-RBM15 in different blood lineages by meta-analysis of RNA-seq data [38]: HSC (hematopoietic stem cells), MPP (multipotent progenitor cells), CMP (common myeloid progenitor cells), CLP (common lymphoid progenitor cells), GMP (Granulocyte-macrophage progenitor cells), MEP (megakaryocyte- erythrocyte progenitor cells), MK (megakaryocyte), and EB (erythroid body).C Relative expression levels of AS-RBM15 (left) and RBM15 (right) in MEG-01 cells treated with PMA were measured by real-time PCR assays (n=4, mean ± SD). SD: standard deviation.D Relative expression levels of AS-RBM15 and RBM15 in CD34+ cells grown in pro-MK medium were measured by real-time PCR. GAPDH was used as an internal control (n=3, mean ± SD). Also see Fig EV 1.


Figure 2A CFU - MK assays with CD34 + cells expressing AS - RBM15 . MK colonies staining for acetylcholinesterase activity were counted ( right ) . P value was calculated using Student ' s t test ( n = 3 , mean ± SD ) . B The number of CD41 + CD42 + mature MK cells was determined by FACS analysis . CD34 + cells expressing sh - AS - RBM15 were grown in pro - MK medium containing thrombopoietin and stem cell factor ( TPO / SCF ) for five days ( n = 4 , mean ± SD ) . D FACS analysis of CD34 + cells grown in pro - MK medium and expressing AS - RBM15 full length or Δ5 or Δ3 . The CD41 + CD42 + cell percentages were analyzed using one way ANOVA , * * P < 0 . 01 , ( n = 4 , mean ± SD ) . E The percentage of CD41 + CD42 + cells in CD34 + cells expressing AS - RBM15 Exon 1 were determined by FACS analysis after these cells were grown in pro - MK medium for five days . P value was calculated by t test , p < 0 . 01 , ( n = 3 , mean ± SD ) .

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 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 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

Figure 2A CFU-MK assays with CD34+ cells expressing AS-RBM15. MK colonies staining for acetylcholinesterase activity were counted (right). P value was calculated using Student's t test (n=3, mean ± SD).B The number of CD41+CD42+ mature MK cells was determined by FACS analysis. CD34+ cells expressing sh-AS-RBM15 were grown in pro-MK medium containing thrombopoietin and stem cell factor (TPO/SCF) for five days (n=4, mean ± SD).D FACS analysis of CD34+ cells grown in pro-MK medium and expressing AS-RBM15 full length or Δ5 or Δ3. The CD41+CD42+ cell percentages were analyzed using one way ANOVA, ** P<0.01, (n=4, mean ± SD).E The percentage of CD41+CD42+ cells in CD34+ cells expressing AS-RBM15 Exon 1 were determined by FACS analysis after these cells were grown in pro-MK medium for five days. P value was calculated by t test, p<0.01, (n=3, mean ± SD).


Figure 3A RBM15 protein levels in leukemia cell lines ( MEG - 01 , K562 , HEL ) were assessed by Western blotting ( WB ) ( left ) ( n = 3 ) . The mRNA levels of RBM15 in cells overexpressing AS - RBM15 were measured by real - time PCR ( right , n = 3 , mean ± SD ) . GAPDH was used as an internal control for WB and PCR . B Protein levels ( left ) and mRNA levels ( right ) of RBM15 in MEG - 01 cells expressing two different shRNAs against AS - RBM15 respectively were measured using WBs and real - time PCR assays , respectively ( n = 3 , mean ± SD ) . C Protein levels ( left ) and mRNA levels ( right ) of RBM15 in MEG - 01 cells expressing truncated AS - RBM15 RNAs ( the schematic as shown on Fig 2C ) were measured using WB and real - time PCR respectively ( n = 3 , mean ± SD ) . D The role of AS - RBM15 in protein translation was assessed by reporter assays . The reporter under control of the CMV promoter contains the 5 ' UTR of RBM15 mRNA ligated to firefly luciferase followed by an IRES element controlling Renilla luciferase as an internal control . The reporter plasmid was cotransfected with plasmids expressing full - length , exon 1 and Δ5 AS - RBM15 respectively . The vector reporter without 5 ' UTR is a negative control . ( n = 3 , mean ± SD ) . P values were calculated by one - way ANOVA test ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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 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 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 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

Figure 3A RBM15 protein levels in leukemia cell lines (MEG-01, K562, HEL) were assessed by Western blotting (WB) (left) (n=3). The mRNA levels of RBM15 in cells overexpressing AS-RBM15 were measured by real-time PCR (right, n=3, mean ± SD). GAPDH was used as an internal control for WB and PCR.B Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing two different shRNAs against AS-RBM15 respectively were measured using WBs and real-time PCR assays, respectively (n=3, mean ± SD).C Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing truncated AS-RBM15 RNAs (the schematic as shown on Fig 2C) were measured using WB and real-time PCR respectively (n=3, mean ± SD).D The role of AS-RBM15 in protein translation was assessed by reporter assays. The reporter under control of the CMV promoter contains the 5'UTR of RBM15 mRNA ligated to firefly luciferase followed by an IRES element controlling Renilla luciferase as an internal control. The reporter plasmid was cotransfected with plasmids expressing full-length, exon 1 and Δ5 AS-RBM15 respectively. The vector reporter without 5'UTR is a negative control. (n=3, mean ± SD). P values were calculated by one-way ANOVA test (*p<0.05, **p<0.01 and ***p<0.001).


Figure 4A Cytoplasmic extracts from MEG - 01 cell lines stably expressing AS - RBM15 , Exon 1 , or shRNA - 02 against AS - RBM15 were fractionated through sucrose gradients ( 15 % - 60 % ) ( n = 4 ) . Global polyribosome profiles of these cell lines were generated by UV absorbance at 254 nm ( A254 ) . The arrow indicates increasing polyribosome weights . Low molecular weight , LMW ; high molecular weight , HMW . B The distributions of RBM15 mRNA ( top ) , GAPDH mRNA ( middle ) , and AS - RBM15 ( bottom ) were measured by real - time PCR and represented as percentages of total RNA in the fractions ( n = 3 ) . C MS2 pull - down assays : AS - RBM15 full - length ( FL ) and exon 1 were fused to MS2RNA as baits . Plasmids expressing MS2 binding protein ( HA - tagged ) and fusion baits were co - transfected into 293T cells . HA antibody was used to immunoprecipitateHA - MS2 binding protein as shown by a WB on the bottom panel . RBM15 mRNA and MS2 fusions were assessed by real - time PCR ( n = 3 , mean ± SD ) . Fold enrichment was calculated as 2 to the power of [ ( CI . R - Cb . R ) - ( CI . MS2 - Cb . MS2 ) ] . I : input i . e . the RNA in solution before immunoprecipitation , R : RBM15 mRNA , b : bead - bound mRNA , MS2 : RNA levels of MS2 fusions , C : number of cycles in real - time PCR . One - way ANONA for p value : p < 0 . 05 . D WB analysis of the endogenous RBM15 protein levels in 293T cells overexpressing two different doses of AS - RBM15 - MS2 and MS2 ( n = 3 ) . E RBM15 mRNA interacts with AS - RBM15 via RNA - RNA interaction . AS - RBM15 - MS2 - associated RNAs were purified by phenol extraction and measured by reverse transcription and PCR for detection of RBM15 , AS - RBM15 and MS2 under denaturing ( heat ) and non - denaturing conditions ( n = 3 ) . F RNase A protection assays for detecting endogenous association of AS - RBM15 and RBM15 mRNA ( n = 4 ) . RNA was extracted from MEG - 01 cells with phenol and digested with RNase A . cDNA was synthesized after RNase A treatment and detected using AS - RBM15 exon - specific primers as shown in the schematic diagram on top . RT : reverse transcriptase . G MEG - 01 cells expressing AS - RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes . Cells were harvested for WB using antibodies against GAPDH and RBM15 ( n = 2 ) . H The RBM15 5 ' UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS - RBM15 ( n = 4 , mean ± SD ) . P values were calculated by one - way ANOVA test ( * * p < 0 . 01 and * * * p < 0 . 001 ) .

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 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 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 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 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 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 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 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

Figure 4A Cytoplasmic extracts from MEG-01 cell lines stably expressing AS-RBM15, Exon 1, or shRNA-02 against AS-RBM15 were fractionated through sucrose gradients (15%-60%) (n=4). Global polyribosome profiles of these cell lines were generated by UV absorbance at 254 nm (A254). The arrow indicates increasing polyribosome weights. Low molecular weight, LMW; high molecular weight, HMW.B The distributions of RBM15 mRNA (top), GAPDH mRNA (middle), and AS-RBM15 (bottom) were measured by real-time PCR and represented as percentages of total RNA in the fractions (n=3).C MS2 pull-down assays: AS-RBM15 full-length (FL) and exon 1 were fused to MS2RNA as baits. Plasmids expressing MS2 binding protein ( HA-tagged) and fusion baits were co-transfected into 293T cells. HA antibody was used to immunoprecipitateHA-MS2 binding protein as shown by a WB on the bottom panel. RBM15 mRNA and MS2 fusions were assessed by real-time PCR (n=3, mean ± SD). Fold enrichment was calculated as 2 to the power of [(CI.R-Cb.R)-(CI.MS2-Cb.MS2)]. I: input i.e. the RNA in solution before immunoprecipitation, R: RBM15 mRNA, b: bead-bound mRNA, MS2: RNA levels of MS2 fusions, C: number of cycles in real-time PCR. One-way ANONA for p value: p<0.05.D WB analysis of the endogenous RBM15 protein levels in 293T cells overexpressing two different doses of AS-RBM15-MS2 and MS2 (n=3).E RBM15 mRNA interacts with AS-RBM15 via RNA-RNA interaction. AS-RBM15-MS2-associated RNAs were purified by phenol extraction and measured by reverse transcription and PCR for detection of RBM15, AS-RBM15 and MS2 under denaturing (heat) and non-denaturing conditions (n =3).F RNase A protection assays for detecting endogenous association of AS-RBM15 and RBM15 mRNA (n=4). RNA was extracted from MEG-01 cells with phenol and digested with RNase A. cDNA was synthesized after RNase A treatment and detected using AS-RBM15 exon-specific primers as shown in the schematic diagram on top. RT: reverse transcriptase.G MEG-01 cells expressing AS-RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes. Cells were harvested for WB using antibodies against GAPDH and RBM15 (n=2).H The RBM15 5'UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS-RBM15 (n=4, mean ± SD). P values were calculated by one-way ANOVA test (**p<0.01 and ***p<0.001).


Figure 5The subcellular distribution of RBM15 and AS - RBM15 in CD34 + cells grown in TPO - containing medium for 5 days . Cytoplasmic and nuclear RNA levels of AS - RBM15 and RBM15 were normalized to GAPDH ( control for cytoplasmic fraction ) and MALAT1 ( control for nuclear fraction ) , respectively , using the ΔΔCt method ( n = 4 , mean ± SD ) .

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

Figure 5The subcellular distribution of RBM15 and AS-RBM15 in CD34+ cells grown in TPO-containing medium for 5 days. Cytoplasmic and nuclear RNA levels of AS-RBM15 and RBM15 were normalized to GAPDH (control for cytoplasmic fraction) and MALAT1 (control for nuclear fraction), respectively, using the ΔΔCt method (n=4, mean ± SD).


Figure 6A RUNX1 expression levels in human CD34 + cells grown in pro - MK medium in a time course were measured by real - time PCR ( n = 4 , mean ± SD ) . B CD34 + cells were grown in pro - MK medium for 5 days and stained with antibodies against CD41 and CD42 for flow cytometry analysis . The CD41 + CD42 + cell percentages were analyzed using one way ANOVA , * * P < 0 . 01 , ( n = 4 , mean ± SD ) . C The mRNA expression levels of RUNX1 , RBM15 , and AS - RBM15 in RUNX1 knockdown MEG - 01 cells were measured by real - time PCR ( n = 4 , mean ± SD ) . D Chromatin immunoprecipitation using anti - RUNX1 antibody . Immunoprecipitated DNA fragments were measured by real - time PCR using HEL cells expressing shRUNX1 and control ( n = 4 , means ± SD ) . E CD34 + human cord blood cells were transduced with MIG retroviruses co - expressing A / E ( AML1 - ETO , aka RUNX1 - ETO ) and GFP . Around 5 days after transduction , the GFP + CD11b - cells were sorted for real - time PCR ( n = 4 ) . Also see Fig EV6 for RUNX1 - ETO binding profile on RBM15 locus .

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 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 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 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

Figure 6A RUNX1 expression levels in human CD34+ cells grown in pro-MK medium in a time course were measured by real-time PCR (n=4, mean ± SD).B CD34+ cells were grown in pro-MK medium for 5 days and stained with antibodies against CD41 and CD42 for flow cytometry analysis. The CD41+CD42+ cell percentages were analyzed using one way ANOVA, ** P<0.01, (n=4, mean ± SD).C The mRNA expression levels of RUNX1, RBM15, and AS-RBM15 in RUNX1 knockdown MEG-01 cells were measured by real-time PCR (n=4, mean ± SD).D Chromatin immunoprecipitation using anti-RUNX1 antibody. Immunoprecipitated DNA fragments were measured by real-time PCR using HEL cells expressing shRUNX1 and control (n=4, means ± SD).E CD34+ human cord blood cells were transduced with MIG retroviruses co-expressing A/E (AML1-ETO, aka RUNX1-ETO) and GFP. Around 5 days after transduction, the GFP+CD11b- cells were sorted for real-time PCR (n=4). Also see Fig EV6 for RUNX1-ETO binding profile on RBM15 locus.


Figure 1 . ( A ) Vero cell was inoculated with BALF sample , and the CPEs were observed at 4 - dpi . ( B ) RNAs were extracted from the cell culture supernatant , and detected using a commercial kit targeting the ORF 1ab ( red ) and N ( blue ) genes of SARS - CoV - 2 . Testing the convalescent plasma IgG antibody ( C ) using the purified SARS - CoV - 2 particles . The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer , respectively . The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory . The PBS control was serum - free . Testing the SARA - CoV - 2 - RBD specific human monoclonal antibodies ( D ) using the purified SARS - CoV - 2 particles . The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer , respectively . The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory . The PBS control was serum - free . ( E ) Viruses were detected by IFA using the patient ' s plasma , and plasma from a healthy control was used as negative control . Scale bar , 100 μm .

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 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 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 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

Figure 1.(A) Vero cell was inoculated with BALF sample, and the CPEs were observed at 4-dpi.(B) RNAs were extracted from the cell culture supernatant, and detected using a commercial kit targeting the ORF 1ab (red) and N (blue) genes of SARS-CoV-2.Testing the convalescent plasma IgG antibody (C) using the purified SARS-CoV-2 particles. The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer, respectively. The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory. The PBS control was serum-free.Testing the SARA-CoV-2-RBD specific human monoclonal antibodies (D) using the purified SARS-CoV-2 particles. The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer, respectively. The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory. The PBS control was serum-free.(E) Viruses were detected by IFA using the patient's plasma, and plasma from a healthy control was used as negative control. Scale bar, 100 μm.


Figure 2 . ( A ) . Image of negative stained SARS - CoV - 2 . Nail - like spikes can be clearly seen . Enlarged view of virion boxed in ( A ) . ( C ) Zoom - in view of a spike boxed in ( B ) . The shape is depicted by red dot line . Length , the diameter of stem and spike ' s head are 23nm , 4nm and 7nm , respectively .

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 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 2.(A). Image of negative stained SARS-CoV-2. Nail-like spikes can be clearly seen.Enlarged view of virion boxed in (A). (C) Zoom-in view of a spike boxed in (B). The shape is depicted by red dot line. Length, the diameter of stem and spike's head are 23nm, 4nm and 7nm, respectively.


Figure 3 . ( A ) and ( B ) . Cryo - EM images of SARS - CoV - 2 . ( C ) . Zoom - in view of the virion showed in ( A ) . Envelope and nucleocapsid are indicated by green and blue respectively , remarkable spikes are indicated by red triangles . ( D ) . Zoom - in views of the two virions showed in ( B ) , remarkable spikes are indicated by red triangles . ( E ) . Zoom - in view of the spike indicated by yellow triangle in ( C ) . The shape is depicted by yellow dot lines . ( F ) . Zoom - in view of the spike indicated by yellow triangle in ( D ) . The shape is depicted by yellow dot lines .

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

Figure 3.(A) and (B). Cryo-EM images of SARS-CoV-2. (C). Zoom-in view of the virion showed in (A). Envelope and nucleocapsid are indicated by green and blue respectively, remarkable spikes are indicated by red triangles. (D). Zoom-in views of the two virions showed in (B), remarkable spikes are indicated by red triangles. (E). Zoom-in view of the spike indicated by yellow triangle in (C). The shape is depicted by yellow dot lines. (F). Zoom-in view of the spike indicated by yellow triangle in (D). The shape is depicted by yellow dot lines.


Figure 1 ( B ) Immunoblotting and quantification of brown preadipocytes ( WT - 1 ) stimulated with insulin at different doses for 5 min ( n = 3 ) . The molecular weight corresponded to the I - Afadin isoform . ( C ) Immunoblotting of mature brown adipocytes ( WT - 1 ) stimulated with insulin ( 100nM ) for 5 min ( n = 4 ) . ( D ) Immunoblotting of immortalized human mature white and brown adipocytes stimulated with insulin for 5 min ( n = 3 ) . ( E ) Immunoblotting and quantification of primary human mature white adipocytes stimulated with insulin for 5 min ( n = 3 ) . ( F ) Immunoblotting and quantification of isolated brown adipose tissue ( BAT ) from male C57BL / 6J mice injected with 5U insulin for 5 min ( n = 4 - 6 ) . ( G ) Quantification of immunoblotting of isolated BAT , subcutaneous white adipose tissue ( sWAT ) and visceral white adipose tissue ( vWAT ) from male C57BL / 6J mice after fasting or re - feeding for 2 / 6 hrs ( n = 6 ) . ( H ) Immunoblotting and quantification of total and Afadin S1795 phosphorylation in adipose tissue depots from 12 - week old C57BL / 6 male mice fed with either chow ( CD ) or high - fat diet ( HFD ) for 6 - weeks injected with vehicle or insulin ( n = 6 ) . ( I ) Immunoblotting and quantification of Afadin S1718 phosphorylation in human Stromal vascular fractions ( SVF ) from lean , obese or Type 2 Diabetic ( T2DM ) subjects stimulated or not with insulin ( n = 6 ) . Representative Western blots are shown . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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 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 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 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 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 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

Figure 1(B) Immunoblotting and quantification of brown preadipocytes (WT-1) stimulated with insulin at different doses for 5 min (n=3). The molecular weight corresponded to the I-Afadin isoform.(C) Immunoblotting of mature brown adipocytes (WT-1) stimulated with insulin (100nM) for 5 min (n=4).(D) Immunoblotting of immortalized human mature white and brown adipocytes stimulated with insulin for 5 min (n=3).(E) Immunoblotting and quantification of primary human mature white adipocytes stimulated with insulin for 5 min (n=3).(F) Immunoblotting and quantification of isolated brown adipose tissue (BAT) from male C57BL/6J mice injected with 5U insulin for 5 min (n=4-6).(G) Quantification of immunoblotting of isolated BAT, subcutaneous white adipose tissue (sWAT) and visceral white adipose tissue (vWAT) from male C57BL/6J mice after fasting or re-feeding for 2/6 hrs (n=6).(H) Immunoblotting and quantification of total and Afadin S1795 phosphorylation in adipose tissue depots from 12-week old C57BL/6 male mice fed with either chow (CD) or high-fat diet (HFD) for 6-weeks injected with vehicle or insulin (n=6).(I) Immunoblotting and quantification of Afadin S1718 phosphorylation in human Stromal vascular fractions (SVF) from lean, obese or Type 2 Diabetic (T2DM) subjects stimulated or not with insulin (n=6). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Figure 2Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . BAT ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . BAT quantification of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . sWAT ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . sWAT ; quantification of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . Liver ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . Liver quantification of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . ( G ) Quantifications of phosphorylated Insulin Receptor β and phosphorylated Akt across the three tissues ( n = 4 - 5 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . ( H ) Quantifications of Total IRβ / tubulin in BAT , sWAT and Liver ( n = 5 - 6 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . ( I ) Insulin - induced lipogenesis in isolated mature adipocytes from CTRL and FAKO ( Mllt4 - / - ) mice ( n = 5 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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 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 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 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 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 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 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 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 2Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (G) Quantifications of phosphorylated Insulin Receptor β and phosphorylated Akt across the three tissues (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (H) Quantifications of Total IRβ/tubulin in BAT, sWAT and Liver (n=5-6). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (I) Insulin-induced lipogenesis in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Figure 3 ( A ) CRISPR / nCas9 - mediated ablation of Afadin protein levels in a clonal cell population termed Afdn - KO . ( B ) Oil red O staining and gene expression of selected adipogenic genes in WT - 1 and Afdn - KO differentiated adipocytes ( n = 3 ) . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . ( C ) Wild - type , Afdn - KO and Afdn - KO with re - expression FLAG - tagged WT I - Afadin or a non - phosphorylable form ( S1795A ) stimulated with vehicle or insulin ( n = 4 ) for Immunoblotting . ( D ) Quantification of insulin - induced Akt phosphorylation from immunoblots . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . ( E ) Real - time measurement and ( F ) quantification of insulin - induced fatty - acid uptake ( n = 4 ) . Representative Western blots are shown . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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 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 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 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 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

Figure 3(A) CRISPR/nCas9-mediated ablation of Afadin protein levels in a clonal cell population termed Afdn-KO.(B) Oil red O staining and gene expression of selected adipogenic genes in WT-1 and Afdn-KO differentiated adipocytes (n=3). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.(C) Wild-type, Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT I-Afadin or a non-phosphorylable form (S1795A) stimulated with vehicle or insulin (n=4) for Immunoblotting.(D) Quantification of insulin-induced Akt phosphorylation from immunoblots. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.(E) Real-time measurement and (F) quantification of insulin-induced fatty-acid uptake (n=4). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Figure 4 ( C ) Acetylated α - tubulin levels in cell lines ( n = 3 ) . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance . ( D ) Acetylated α - tubulin levels in isolated mature adipocytes from CTRL and FAKO ( Mllt4 - / - ) mice ( n = 4 ) . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance . ( E ) IR abundance measured by immunoblotting following 4 hours pre - incubation with the HDAC6 inhibitor ( Tubastatin A ) in differentiated adipocytes exposed to insulin ( 100nM ) stimulation for 15 min ( n = 4 ) . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance . ( F ) Immunolocalization of IR ( green ) and HDAC6 ( red ) in Afdn - KO and Afdn - KO with re - expression FLAG - tagged WT Afadin or S1795A Afadin in mature adipocytes stimulated with insulin . Nuclei were stained with DAPI ( blue ) . Co - localization was detected by the appearance of white pixels where the red and green overlap . Scale bars : 25 µm . Representative images are shown . The ratio of IR co - localized with HDAC6 ( Mandler ' s co - localization coefficient ) was quantified ( 98 - 112 individual cells from two independent experiments ) . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance . ( G ) Immunoprecipitation of IR followed by immunoblotting using an anti - acetyl - Lys antibody in adipocytes lacking Afadin or re - expressing Afadin , with or without insulin stimulation ( n = 1 ) . Representative Western blots are shown . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

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

Figure 4(C) Acetylated α-tubulin levels in cell lines (n=3). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.(D) Acetylated α-tubulin levels in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=4). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.(E) IR abundance measured by immunoblotting following 4 hours pre-incubation with the HDAC6 inhibitor (Tubastatin A) in differentiated adipocytes exposed to insulin (100nM) stimulation for 15 min (n=4). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.(F) Immunolocalization of IR (green) and HDAC6 (red) in Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT Afadin or S1795A Afadin in mature adipocytes stimulated with insulin. Nuclei were stained with DAPI (blue). Co-localization was detected by the appearance of white pixels where the red and green overlap. Scale bars: 25 µm. Representative images are shown. The ratio of IR co-localized with HDAC6 (Mandler's co-localization coefficient) was quantified (98-112 individual cells from two independent experiments). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.(G) Immunoprecipitation of IR followed by immunoblotting using an anti-acetyl-Lys antibody in adipocytes lacking Afadin or re-expressing Afadin, with or without insulin stimulation (n=1). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


Figure 5Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( A ) Body weight . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( B ) Body composition . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( C ) Sirius Red staining of representative embedded BAT , sWAT and vWAT from mice on HFD and quantification of adipocyte areas ( ~ 1000 adipocytes / genotype ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( D ) IPGTT after 12 weeks of diet and AUC . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( E ) ITT after 13 weeks of diet and AUC Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( F ) normalized to basal insulin . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance . Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( G ) Plasma insulin levels 5 min after an IPGTT performed after 18 weeks of diet . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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 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 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 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 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 O O O O O O O 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 5Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (A) Body weight. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (B) Body composition. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (C) Sirius Red staining of representative embedded BAT, sWAT and vWAT from mice on HFD and quantification of adipocyte areas (~1000 adipocytes/genotype). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (D) IPGTT after 12 weeks of diet and AUC. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (E) ITT after 13 weeks of diet and AUC Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.Seven-week old CTRL and FAKO (Mllt4-/-) mice were given high-fat diet (HFD) for 16 weeks (n=5-6). (F) normalized to basal insulin. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (G) Plasma insulin levels 5 min after an IPGTT performed after 18 weeks of diet. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Figure 1 ( A qRT - PCR for different ISGs relative to Rps29 using epidermal RNA from 8 week - old ( A ) K5 - R1 / R2 and control ( CTRL ) mice . qRT - PCR for different ISGs relative to Rps29 using epidermal RNA from 5 day - or 9 day - old ( B ) K5 - R1 / R2 and control ( CTRL ) mice . ( C ) Confocal microscopy images of epidermal sheets from back skin of K5 - R1 / R2 and CTRL mice stained for IRF7 and K14 , counterstained with DAPI . Arrows denote nuclear IRF7 ( red ) in basal and suprabasal keratinocytes of K5 - R1 / R2 mice . The strong red staining of the stratum corneum ( sc ) is unspecific background and is more pronounced in K5 - R1 / R2 mice due to the increased thickness of this layer . ( D ) qRT - PCR for different ISGs , Ifnb , and Ifnl3 relative to Rps29 using RNA from freshly isolated , non - cultured keratinocytes of K5 - R1 / R2 and control ( CTRL ) mice at P7 . ( E ) qRT - PCR for ISGs relative to Rps29 using RNA from primary keratinocytes of neonatal CTRL and K5 - R1 / R2 mice . ( F ) qRT - PCR for ISGs and Cre relative to Rps29 using RNA from primary keratinocytes of neonatal K5 - Cre and wild - type ( WT ) mice . ( G ) Western blot analysis of total ( L ) and nuclear ( N ) lysates from immortalized ( imm . ) keratinocytes of CTRL and K5 - R1 / R2 mice for IRF1 , IRF7 , IRF9 , Lamin A / C ( nuclear marker , loading control ) and GAPDH ( cytosolic marker , loading control ) . ( H ) qRT - PCR for RSAD2 and ISG15 relative to RPLP0 using RNA from HaCaT keratinocytes treated for 48 h with the FGFR inhibitor BGJ398 ( 3 . 5 μM ) or vehicle in the presence of serum .

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 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 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 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 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 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

Figure 1(A qRT-PCR for different ISGs relative to Rps29 using epidermal RNA from 8 week-old (A) K5-R1/R2 and control (CTRL) mice.qRT-PCR for different ISGs relative to Rps29 using epidermal RNA from 5 day- or 9 day-old (B) K5-R1/R2 and control (CTRL) mice.(C) Confocal microscopy images of epidermal sheets from back skin of K5-R1/R2 and CTRL mice stained for IRF7 and K14, counterstained with DAPI. Arrows denote nuclear IRF7 (red) in basal and suprabasal keratinocytes of K5-R1/R2 mice. The strong red staining of the stratum corneum (sc) is unspecific background and is more pronounced in K5-R1/R2 mice due to the increased thickness of this layer.(D) qRT-PCR for different ISGs, Ifnb, and Ifnl3 relative to Rps29 using RNA from freshly isolated, non-cultured keratinocytes of K5-R1/R2 and control (CTRL) mice at P7.(E) qRT-PCR for ISGs relative to Rps29 using RNA from primary keratinocytes of neonatal CTRL and K5-R1/R2 mice.(F) qRT-PCR for ISGs and Cre relative to Rps29 using RNA from primary keratinocytes of neonatal K5-Cre and wild-type (WT) mice.(G) Western blot analysis of total (L) and nuclear (N) lysates from immortalized (imm.) keratinocytes of CTRL and K5-R1/R2 mice for IRF1, IRF7, IRF9, Lamin A/C (nuclear marker, loading control) and GAPDH (cytosolic marker, loading control).(H) qRT-PCR for RSAD2 and ISG15 relative to RPLP0 using RNA from HaCaT keratinocytes treated for 48 h with the FGFR inhibitor BGJ398 (3.5 μM) or vehicle in the presence of serum.


Figure 2 ( A ) Growth factor - starved primary keratinocytes from WT mice were treated for 3 or 6 h with FGF7 ( 10 ng / ml ) or vehicle ( CTRL ) . RNA samples were analyzed by qRT - PCR for the indicated ISGs relative to Rps29 . ( B ) Serum - starved HaCaT keratinocytes were treated for 6 h with FGF7 or FGF10 ( each 10 ng / ml ) . RNA samples were analyzed by qRT - PCR for IRF7 and RSAD2 relative to RPLP0 . ( C ) Serum - starved HaCaT keratinocytes were treated for 16 h with FGF7 ( 10 ng / ml ) . Protein lysates were analyzed by Western blot for IRF1 , IRF3 , IRF9 and GAPDH . ( D ) RNA samples from untreated , serum - starved primary keratinocytes from WT and Ifnar1 - KO mice were analyzed by qRT - PCR for Irf7 , Rsad2 , Oasl2 and Ifnar1 relative to Rps29 . ( E ) Growth factor - starved primary keratinocytes from neonatal WT , Ifnar1 - KO or Ifnar1 / Ifnlr1 - KO mice were treated for 24 h with FGF7 ( 10 ng / ml ) . RNA samples were analyzed by qRT - PCR for Irf7 relative to Rps29 . ( F ) HaCaT keratinocytes were transiently transfected with an ISRE - Luciferase reporter construct and starved 24 h after transfection for 9 h . They were then incubated in the presence or absence of FGF7 ( 10 ng / ml ) for 20 h . Lysates were analyzed using a DualGlo Luciferase Assay . HaCaT keratinocytes were pre - treated for 2 h with the proteasome inhibitors MG132 ( 10 μm ( G ) followed by a 20h treatment with FGF7 ( 10 ng / ml ) or vehicle . RNA samples were analyzed by qRT - PCR for IRF7 and IRF1 relative to RPLP0 . HaCaT keratinocytes were pre - treated for 2 h with epoxomicin ( 100 nM ) ( H ) , followed by a 20h treatment with FGF7 ( 10 ng / ml ) or vehicle . RNA samples were analyzed by qRT - PCR for IRF7 and IRF1 relative to RPLP0 .

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 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 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 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 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 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

Figure 2(A) Growth factor-starved primary keratinocytes from WT mice were treated for 3 or 6 h with FGF7 (10 ng/ml) or vehicle (CTRL). RNA samples were analyzed by qRT-PCR for the indicated ISGs relative to Rps29.(B) Serum-starved HaCaT keratinocytes were treated for 6 h with FGF7 or FGF10 (each 10 ng/ml). RNA samples were analyzed by qRT-PCR for IRF7 and RSAD2 relative to RPLP0.(C) Serum-starved HaCaT keratinocytes were treated for 16 h with FGF7 (10 ng/ml). Protein lysates were analyzed by Western blot for IRF1, IRF3, IRF9 and GAPDH.(D) RNA samples from untreated, serum-starved primary keratinocytes from WT and Ifnar1-KO mice were analyzed by qRT-PCR for Irf7, Rsad2, Oasl2 and Ifnar1 relative to Rps29.(E) Growth factor-starved primary keratinocytes from neonatal WT, Ifnar1-KO or Ifnar1/Ifnlr1-KO mice were treated for 24 h with FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for Irf7 relative to Rps29.(F) HaCaT keratinocytes were transiently transfected with an ISRE-Luciferase reporter construct and starved 24 h after transfection for 9 h. They were then incubated in the presence or absence of FGF7 (10 ng/ml) for 20 h. Lysates were analyzed using a DualGlo Luciferase Assay.HaCaT keratinocytes were pre-treated for 2 h with the proteasome inhibitors MG132 (10 μm (G) followed by a 20h treatment with FGF7 (10 ng/ml) or vehicle. RNA samples were analyzed by qRT-PCR for IRF7 and IRF1 relative to RPLP0.HaCaT keratinocytes were pre-treated for 2 h with epoxomicin (100 nM) (H), followed by a 20h treatment with FGF7 (10 ng/ml) or vehicle. RNA samples were analyzed by qRT-PCR for IRF7 and IRF1 relative to RPLP0.


Figure 3Serum - starved HaCaT keratinocytes were treated with FGF7 ( 10 ng / ml ( A and / or IFN - α ( 500 - 1000 U / ml ) for 12 - 16 h . RNA was analyzed by qRT - PCR for ISGs relative to RPLP0 ( ASerum - starved HaCaT keratinocytes were treated with FGF7 different concentrations as indicated ( B ) ) and / or IFN - α ( 500 - 1000 U / ml ) for 12 - 16 h . RNA was analyzed by qRT - PCR for ISGs relative to RPLP0Serum - starved HaCaT keratinocytes were treated with FGF7 ( 10 ng / ml and / or IFN - α ( 500 - 1000 U / ml ) for 12 - 16 h . protein samples by Western blot for total and pSTAT1 ( Y701 and S727 ) , IRF1 , IRF9 and RSAD2 and GAPDH ( C ) . The vertical line in the RSAD2 blot indicates a place where the gel was broken . Densitometric quantification of the different blots ( N = 3 ) is shown on the right . ( D ) Human primary keratinocytes were starved and treated for 12 h with FGF7 ( 10 ng / ml ) and / or IFN - α ( 1000 U / ml ) . RNA samples were analyzed by qRT - PCR for ISGs relative to RPLP0 .

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 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 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 3Serum-starved HaCaT keratinocytes were treated with FGF7 (10 ng/ml (A and/or IFN-α (500-1000 U/ml) for 12-16 h. RNA was analyzed by qRT-PCR for ISGs relative to RPLP0 (ASerum-starved HaCaT keratinocytes were treated with FGF7 different concentrations as indicated (B)) and/or IFN-α (500-1000 U/ml) for 12-16 h. RNA was analyzed by qRT-PCR for ISGs relative to RPLP0Serum-starved HaCaT keratinocytes were treated with FGF7 (10 ng/ml and/or IFN-α (500-1000 U/ml) for 12-16 h. protein samples by Western blot for total and pSTAT1 (Y701 and S727), IRF1, IRF9 and RSAD2 and GAPDH (C). The vertical line in the RSAD2 blot indicates a place where the gel was broken. Densitometric quantification of the different blots (N=3) is shown on the right.(D) Human primary keratinocytes were starved and treated for 12 h with FGF7 (10 ng/ml) and/or IFN-α (1000 U/ml). RNA samples were analyzed by qRT-PCR for ISGs relative to RPLP0.


Figure 4 ( A ) Serum - starved HaCaT cells were treated for 24 h with poly ( I : C ) ( 5 μg / ml ) in the presence or absence of FGF7 ( 10 ng / ml ) . RNA samples were analyzed by qRT - PCR for IFNL1 , IFNB , RSAD2 , IRF1 , IRF7 , CGAS , TLR3 or DDX58 , relative to RPLP0 . ( B ) Serum - starved HaCaT cells were treated for 18 h with poly ( I : C ) ( 5 μg / ml ) in the presence or absence of FGF7 ( 10 ng / ml ) . Protein lysates were analyzed by Western blot for total and pSTAT1 ( Y701 and S727 ) , total and pSTAT2 ( Y690 ) , IRF1 , IRF3 , pIRF3 ( S396 ) , IRF9 , RIG - 1 , RSAD2 and GAPDH .

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 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

Figure 4(A) Serum-starved HaCaT cells were treated for 24 h with poly(I:C) (5 μg/ml) in the presence or absence of FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for IFNL1, IFNB, RSAD2, IRF1, IRF7, CGAS, TLR3 or DDX58, relative to RPLP0.(B) Serum-starved HaCaT cells were treated for 18 h with poly(I:C) (5 μg/ml) in the presence or absence of FGF7 (10 ng/ml). Protein lysates were analyzed by Western blot for total and pSTAT1 (Y701 and S727), total and pSTAT2 (Y690), IRF1, IRF3, pIRF3 (S396), IRF9, RIG-1, RSAD2 and GAPDH.


Figure 5Serum - starved HaCaT cells were infected with HSV - 1 ( MOI = 0 . 5 ) in the presence or absence of FGF7 ( 10 ng / ml ) or IFN - α ( 1000 U / ml ) . Viral load was determined 24 h post infection ( hpi ) by qPCR for the HSV - 1 Glyc - B gene relative to the human β - actin gene ( ACTB ) ( A ) Serum - starved HaCaT cells were infected with HSV - 1 ( MOI = 0 . 5 ) in the presence or absence of FGF7 ( 10 ng / ml ) or IFN - α ( 1000 U / ml ) . Viral load was determined 24 h post infection ( hpi ) by HSV - 1 Glyc - D immunofluorescence ( red ) and DAPI counterstaining ( blue ) ( B ) . Transmission microscopy images of HaCaT cells 48 h post infection ( hpi ) . Serum - starved HaCaT cells ( D ) were infected with HSV - 1 ( MOI = 0 . 5 ) in the presence or absence of FGF7 at the indicated concentrations . Virus load was determined by qPCR for HSV - 1 Glyc - B relative to ACTB 24 hpi . human primary keratinocytes ( HPK , from two donors ) ( E ) were infected with HSV - 1 ( MOI = 0 . 5 ) in the presence or absence of FGF7 at the indicated concentrations . Virus load was determined by qPCR for HSV - 1 Glyc - B relative to ACTB 24 hpi . ( F ) Serum - starved HaCaT cells were infected with HSV - 1 ( MOI = 0 . 5 ) in the presence or absence of FGF7 , FGF10 or FGF2 ( 10 ng / ml ) . DNA samples were analyzed by qPCR for Glyc - D DNA relative to ACTB and protein lysates were analyzed by Western blot for Glyc - D and vinculin 10 hpi . ( G ) Epidermal sheets from tail skin , infected ex vivo with HSV - 1 ( MOI = 2 ) + / - FGF7 ( 10 ng / ml ) , were stained 48 hpi for Glyc - D ( red ) using DAPI as counterstain ( blue ) . The infected area ( red staining ) was measured using Image J software . Data are expressed as A . U . = arbitrary unit . Background staining of hairs was subtracted manually . ( H ) Serum - starved HaCaT cells were infected with HSV - 1 in the presence or absence of FGF7 ( 10 ng / ml ) or IFN - α ( 1000 U / ml ) . Viral load was determined 24 hpi by qPCR for the HSV - 1 Glyc - B gene relative to ACTB or by Western blot for the viral protein Glyc - D and GAPDH . ( I ) Serum - starved HaCaT cells were infected with HSV - 1 ( MOI = 0 . 5 ) . Four hours hpi , infected cells were washed and incubated in fresh serum - free medium containing FGF7 ( 10 ng / ml ) . Viral load was measured 8 h later by qPCR for Glyc - B relative to ACTB . ( J ) Serum - starved HaCaT keratinocytes were treated for 6 h with FGF7 ( 10 ng / ml ) . RNA samples were analyzed by qRT - PCR for NECT1 ( encoding nectin - 1 ) relative to RPLP0 .

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 B-PANEL_START 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 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 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 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

Figure 5Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml). Viral load was determined 24 h post infection (hpi) by qPCR for the HSV-1 Glyc-B gene relative to the human β-actin gene (ACTB) (A)Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml). Viral load was determined 24 h post infection (hpi) by HSV-1 Glyc-D immunofluorescence (red) and DAPI counterstaining (blue) (B).Transmission microscopy images of HaCaT cells 48 h post infection (hpi).Serum-starved HaCaT cells (D) were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7 at the indicated concentrations. Virus load was determined by qPCR for HSV-1 Glyc-B relative to ACTB 24 hpi.human primary keratinocytes (HPK, from two donors) (E) were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7 at the indicated concentrations. Virus load was determined by qPCR for HSV-1 Glyc-B relative to ACTB 24 hpi.(F) Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7, FGF10 or FGF2 (10 ng/ml). DNA samples were analyzed by qPCR for Glyc-D DNA relative to ACTB and protein lysates were analyzed by Western blot for Glyc-D and vinculin 10 hpi.(G) Epidermal sheets from tail skin, infected ex vivo with HSV-1 (MOI = 2) +/- FGF7 (10 ng/ml), were stained 48 hpi for Glyc-D (red) using DAPI as counterstain (blue). The infected area (red staining) was measured using Image J software. Data are expressed as A.U. = arbitrary unit. Background staining of hairs was subtracted manually.(H) Serum-starved HaCaT cells were infected with HSV-1 in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml). Viral load was determined 24 hpi by qPCR for the HSV-1 Glyc-B gene relative to ACTB or by Western blot for the viral protein Glyc-D and GAPDH.(I) Serum-starved HaCaT cells were infected with HSV-1 (MOI = 0.5). Four hours hpi, infected cells were washed and incubated in fresh serum-free medium containing FGF7 (10 ng/ml). Viral load was measured 8 h later by qPCR for Glyc-B relative to ACTB.(J) Serum-starved HaCaT keratinocytes were treated for 6 h with FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for NECT1 (encoding nectin-1) relative to RPLP0.


Figure 6Serum - starved HaCaT cells were infected with HSV - 1 ( MOI = 0 . 5 ) + / - FGF7 ( 10 ng / ml ) , added at the indicated time points 12 h after infection , RNA samples were analyzed by qRT - PCR for RSAD2 , IRF7 and IFNL2 relative to RPLP0 ( B ) , Serum - starved HaCaT cells were infected with HSV - 1 ( MOI = 0 . 5 ) + / - FGF7 ( 10 ng / ml ) , added at the indicated time points 12 h after infection , , DNA samples were analyzed by qPCR for Glyc - B DNA relative to ACTB ( C ) , Serum - starved HaCaT cells were infected with HSV - 1 ( MOI = 0 . 5 ) + / - FGF7 ( 10 ng / ml ) , added at the indicated time points D ) shows mean Glyc - B DNA and RSAD2 , IRF7 and IFNL2 mRNA levels ( based on results shown in Fig . 6B and C ) in virus - infected cells treated for different periods with FGF7 . Serum - starved HaCaT cells were infected with HSV - 1 ( MOI = 0 . 5 ) + / - FGF7 ( 10 ng / ml ) , added at the indicated time points 12 h after infection , protein lysates were analyzed by Western blot for Glyc - D , total and phosphorylated STAT1 ( Y701 ) , STAT2 and IRF3 , for IRF1 , IRF7 , IRF9 and RSAD2 and for GAPDH or vinculin ( loading controls ) ( E ) . Quantification of the Glyc - D , RSAD2 , IRF1 , IRF7 , IRF9 and STAT2 band intensity relative to GAPDH is shown on the right hand side . ( F ) HaCaT cells were infected with LCMV in the presence or absence of FGF7 ( 10 ng / ml ) or IFN - α ( 1000 U / ml ) and analyzed for LCMV nucleoprotein ( NP ) by flow cytometry 24 hpi . Arbitrary units are shown . HaCaT cells were infected with ZIKV strains 976 ( Uganda isolate ) ( MOI = 0 . 1 ) or PF13 / 251013 - 18 ( French Polynesia isolate ) ( MOI = 20 ) and treated 2 hpi with FGF7 ( 20 ng / ml ) or vehicle ( CTRL ) . Immunofluorescence staining for the ZIKV envelope protein and quantification of the percentage of cells expressing the ZIKV envelope protein ( G ) 48 hpi is shown . HaCaT cells were infected with ZIKV strains 976 ( Uganda isolate ) ( MOI = 0 . 1 ) or PF13 / 251013 - 18 ( French Polynesia isolate ) ( MOI = 20 ) and treated 2 hpi with FGF7 ( 20 ng / ml ) or vehicle ( CTRL ) . qRT - PCR analysis of RNA samples for ZIKV RNA ( H ) 48 hpi is shown . ( I , J ) HaCaT cells were infected with the ZIKV PF13 / 251013 - 18 ( MOI = 20 ) in the presence or absence of FGF7 ( 20 ng / ml ) . RNA samples were analyzed by qRT - PCR for OAS1 and MxA relative to RPLP0 72 hpi .

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 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 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 O 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

Figure 6Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) +/- FGF7 (10 ng/ml), added at the indicated time points 12 h after infection, RNA samples were analyzed by qRT-PCR for RSAD2, IRF7 and IFNL2 relative to RPLP0 (B),Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) +/- FGF7 (10 ng/ml), added at the indicated time points 12 h after infection, , DNA samples were analyzed by qPCR for Glyc-B DNA relative to ACTB (C),Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) +/- FGF7 (10 ng/ml), added at the indicated time points D) shows mean Glyc-B DNA and RSAD2, IRF7 and IFNL2 mRNA levels (based on results shown in Fig. 6B and C) in virus-infected cells treated for different periods with FGF7.Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) +/- FGF7 (10 ng/ml), added at the indicated time points 12 h after infection, protein lysates were analyzed by Western blot for Glyc-D, total and phosphorylated STAT1 (Y701), STAT2 and IRF3, for IRF1, IRF7, IRF9 and RSAD2 and for GAPDH or vinculin (loading controls) (E). Quantification of the Glyc-D, RSAD2, IRF1, IRF7, IRF9 and STAT2 band intensity relative to GAPDH is shown on the right hand side.(F) HaCaT cells were infected with LCMV in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml) and analyzed for LCMV nucleoprotein (NP) by flow cytometry 24 hpi. Arbitrary units are shown.HaCaT cells were infected with ZIKV strains 976 (Uganda isolate) (MOI = 0.1) or PF13/251013-18 (French Polynesia isolate) (MOI = 20) and treated 2 hpi with FGF7 (20 ng/ml) or vehicle (CTRL). Immunofluorescence staining for the ZIKV envelope protein and quantification of the percentage of cells expressing the ZIKV envelope protein (G) 48 hpi is shown.HaCaT cells were infected with ZIKV strains 976 (Uganda isolate) (MOI = 0.1) or PF13/251013-18 (French Polynesia isolate) (MOI = 20) and treated 2 hpi with FGF7 (20 ng/ml) or vehicle (CTRL). qRT-PCR analysis of RNA samples for ZIKV RNA (H) 48 hpi is shown.(I, J) HaCaT cells were infected with the ZIKV PF13/251013-18 (MOI = 20) in the presence or absence of FGF7 (20 ng/ml). RNA samples were analyzed by qRT-PCR for OAS1 and MxA relative to RPLP0 72 hpi.


Figure 7 ( A ) Serum - starved HaCaT cells were infected with HSV - 1 ( MOI = 0 . 5 ) in the presence or absence of FGF7 ( 10 ng / ml ) and the FGFR kinase inhibitors AZD4547 ( 1 μM ) or BGJ398 ( 3 . 5 μM ) . DNA and protein lysates were analyzed by qPCR for Glyc - B relative to ACTB or by Western blot for Glyc - D and GAPDH , respectively , 16 hpi . HaCaT cells were cultured in DMEM / 10 % FCS ( B ) infected with HSV - 1 ( MOI = 0 . 5 ) in the presence or absence of AZD45347 ( 1 μM ) and / or BGJ398 ( 3 . 5 μM ) . Viral load was determined by qPCR for Glyc - D relative to ACTB ( B ) HaCaT cells were cultured in DMEM / 5 % FCS ( C ) and infected with HSV - 1 ( MOI = 0 . 5 ) in the presence or absence of AZD45347 ( 1 μM ) and / or BGJ398 ( 3 . 5 μM ) . Viral load was determined by measurement of viral titers ( plaque forming units ( PFU ) ) 14 hpi ( C ) . ( D , E ) Adult C57BL / 6 wild - type ( D ) or K5 - R1 / R2 mice and respective controls ( E ) were infected subcutaneously with HSV - 1 ( MOI = 10 ) . RNA from infected skin was analyzed by qRT - PCR for Fgf7 relative to Rps29 ( D ) and DNA was analyzed by qPCR for ICP0 , normalized to the host gene Tbx15 48 hpi ( E ) .

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 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 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

Figure 7(A) Serum-starved HaCaT cells were infected with HSV-1 (MOI = 0.5) in the presence or absence of FGF7 (10 ng/ml) and the FGFR kinase inhibitors AZD4547 (1 μM) or BGJ398 (3.5 μM). DNA and protein lysates were analyzed by qPCR for Glyc-B relative to ACTB or by Western blot for Glyc-D and GAPDH, respectively, 16 hpi.HaCaT cells were cultured in DMEM/10% FCS (B) infected with HSV-1 (MOI=0.5) in the presence or absence of AZD45347 (1 μM) and/or BGJ398 (3.5 μM). Viral load was determined by qPCR for Glyc-D relative to ACTB (B)HaCaT cells were cultured in DMEM/5% FCS (C) and infected with HSV-1 (MOI=0.5) in the presence or absence of AZD45347 (1 μM) and/or BGJ398 (3.5 μM). Viral load was determined by measurement of viral titers (plaque forming units (PFU)) 14 hpi (C).(D, E) Adult C57BL/6 wild-type (D) or K5-R1/R2 mice and respective controls (E) were infected subcutaneously with HSV-1 (MOI = 10). RNA from infected skin was analyzed by qRT-PCR for Fgf7 relative to Rps29 (D) and DNA was analyzed by qPCR for ICP0, normalized to the host gene Tbx15 48 hpi (E).


Figure 1 ( C ) Kaplan - Meier plot of PFS stratifying the patients according to high or low diversity index . N = 69 ( 33 above the cut - off , 36 below ) . ( D ) As in ( C ) but plotting OS . N = 72 ( 34 above the cut - off , 38 below ) . ( G ) Baseline plasma FKN concentrations in responders ( R , n = 26 ) , progressors ( PR , n = 62 ) , hyperprogressors ( HPR , n = 3 ) , age - matched healthy donors ( H , n = 32 ) and patients that were not eligible for immunotherapy ( NO IT , n = 28 ) . Sample sizes represent biological replicates . Error bars are shown ( standard deviations , SD ) , and relevant statistical comparisons by Mann - Whitney U tests are shown within the graphs . ( J ) Kaplan - Meier plot of PFS stratifying the patients according to the FKN cut - off value identified in the ROC curve ( n = 42 ) . The associated P value is shown . ( K ) As in ( J ) but plotting OS . n = 46 ( 17 above the cut - off , 29 below ) .

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 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 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 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

Figure 1(C) Kaplan-Meier plot of PFS stratifying the patients according to high or low diversity index. N=69 (33 above the cut-off, 36 below).(D) As in (C) but plotting OS. N= 72 (34 above the cut-off, 38 below).(G) Baseline plasma FKN concentrations in responders (R, n=26), progressors (PR, n=62), hyperprogressors (HPR, n=3), age-matched healthy donors (H, n=32) and patients that were not eligible for immunotherapy (NO IT, n=28). Sample sizes represent biological replicates. Error bars are shown (standard deviations, SD), and relevant statistical comparisons by Mann-Whitney U tests are shown within the graphs.(J) Kaplan-Meier plot of PFS stratifying the patients according to the FKN cut-off value identified in the ROC curve (n=42). The associated P value is shown. (K) As in (J) but plotting OS. n= 46 (17 above the cut-off, 29 below).


Figure 2 ( A ) Secreted FKN in cell cultures of a collection of 10 LUAD and LUSC cancer cell lines quantified by ELISA . Results are presented as means ± SD . Samples were assayed in duplicates from a pool of three independent replicates . Statistical differences among groups were analyzed by ANOVA followed by Tukey ' s tests . ( B ) Tumor growth curves of 3LL - tumor bearing mice treated with recombinant FKN ( rFKN ) , the CX3CR1 inhibitor AZD8797 , a non - relevant protein ( IgG2a ) or saline buffer as vehicle control ( WT ) . Arrows indicate the time of intraperitoneal injections of rFKN ( blue ) and AZD8797 ( black ) . Data are expressed as means ± SD ( n = 6 mice per group ) . Statistical comparisons by ANOVA followed by Tukey ´ s pairwise comparison tests are provided . ( G ) Kaplan - Meier survival plots . Differences between the control group ( WT ) and the FKN group were evaluated by a two - sided log - rank test .

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 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

Figure 2(A) Secreted FKN in cell cultures of a collection of 10 LUAD and LUSC cancer cell lines quantified by ELISA. Results are presented as means ± SD. Samples were assayed in duplicates from a pool of three independent replicates. Statistical differences among groups were analyzed by ANOVA followed by Tukey's tests.(B) Tumor growth curves of 3LL-tumor bearing mice treated with recombinant FKN (rFKN), the CX3CR1 inhibitor AZD8797, a non-relevant protein (IgG2a) or saline buffer as vehicle control (WT). Arrows indicate the time of intraperitoneal injections of rFKN (blue) and AZD8797 (black). Data are expressed as means ± SD (n=6 mice per group). Statistical comparisons by ANOVA followed by Tukey´s pairwise comparison tests are provided.(G) Kaplan-Meier survival plots. Differences between the control group (WT) and the FKN group were evaluated by a two-sided log-rank test.


Figure 3 ( A ) FKN plasma concentration at day 15 in the indicated groups of tumor - bearing mice . Data are expressed as mean ± SD from a pool of 6 mice / group . Statistical comparisons were performed by ANOVA and Tukey ´ s pairwise comparison tests . ( D ) Percentage of the indicated peripheral immune cell populations at day 15 after injection of groups of mice ( n = 6 mice per group ) with 3LL cells overexpressing the indicated myeloid - regulating cytokines . The relevant immune populations were quantified as percentages of total leukocytes ( CD45 + cells ) , specifying DCs ( CD11c ) , macrophages ( F4 / 80 ) , Ly6C + monocytes and Ly6C - monocytes , granulocytes ( Ly6G ) , B cells ( CD19 ) , NKs ( NK1 . 1 ) , CD4 T cells ( CD3 CD4 ) and CD8 T cells ( CD3 CD8 ) . Relevant statistical comparisons were performed by Wilcoxon ´ s test followed by pair - wise comparisons of relevance by Mann - Whitney U test . Box and whisker plots indicate : median ( central line ) , 25th to 75th percentiles ( box ) and minimum to maximum values ( whiskers ) .

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 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

Figure 3(A) FKN plasma concentration at day 15 in the indicated groups of tumor-bearing mice. Data are expressed as mean ± SD from a pool of 6 mice/group. Statistical comparisons were performed by ANOVA and Tukey´s pairwise comparison tests.(D) Percentage of the indicated peripheral immune cell populations at day 15 after injection of groups of mice (n=6 mice per group) with 3LL cells overexpressing the indicated myeloid-regulating cytokines. The relevant immune populations were quantified as percentages of total leukocytes (CD45+ cells), specifying DCs (CD11c), macrophages (F4/80), Ly6C+ monocytes and Ly6C- monocytes, granulocytes (Ly6G), B cells (CD19), NKs (NK1.1), CD4 T cells (CD3 CD4) and CD8 T cells (CD3 CD8). Relevant statistical comparisons were performed by Wilcoxon´s test followed by pair-wise comparisons of relevance by Mann-Whitney U test. Box and whisker plots indicate: median (central line), 25th to 75th percentiles (box) and minimum to maximum values (whiskers).


Figure 4 ( C ) Kaplan - Meier survival plots of the indicated groups of mice . Survival differences between the control group ( WT + αPD - 1 ) and FKN + αPD - 1 were evaluated by a two - sided log - rank test . ( D ) Percentage of splenic Ly6C + monocytes , ( E ) Ly6G - monocytes , ( F ) Ly6G + neutrophils , ( G ) PD - 1 + T cells ( CD3 + ) , Data are presented as mean ± SD ( n = 6 mice per group ) .

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 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

Figure 4(C) Kaplan-Meier survival plots of the indicated groups of mice. Survival differences between the control group (WT + αPD-1) and FKN + αPD-1 were evaluated by a two-sided log-rank test.(D) Percentage of splenic Ly6C+ monocytes, (E) Ly6G- monocytes, (F) Ly6G+ neutrophils, (G) PD-1+ T cells (CD3+), Data are presented as mean ± SD (n=6 mice per group).


Figure 5 ( D ) Kaplan - Meier survival plot of the indicated treatment groups . Survival differences between the control group ( WT ) and FKN were evaluated by two - sided log - rank test . ( E - J ) ( E ) Percentage of splenic NKs ( NK1 . 1 + ) expressing KLRG1 , ( F ) TIGIT and ( G ) TIM3 . ( H ) Percentage of splenic T cells ( CD3 + ) expressing KLRG1 , ( I ) TIGIT and ( J ) TIM3 . Data are presented as means ± SD ( n = 6 mice per group ) . Comparisons among groups were performed by ANOVA and Tukey ´ s pairwise comparison tests .

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 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

Figure 5(D) Kaplan-Meier survival plot of the indicated treatment groups. Survival differences between the control group (WT) and FKN were evaluated by two-sided log-rank test.(E-J) (E) Percentage of splenic NKs (NK1.1+) expressing KLRG1, (F) TIGIT and (G) TIM3. (H) Percentage of splenic T cells (CD3+) expressing KLRG1, (I) TIGIT and (J) TIM3. Data are presented as means ± SD (n=6 mice per group). Comparisons among groups were performed by ANOVA and Tukey´s pairwise comparison tests.


Figure 1Flow cytometric analysis of splenocytes of mice immunized with cInsulin or control immunization on day 26 . Left and middle panel showing germinal center ( GC ) B cells ( GL - 7 + ) pre - gated on B cells ( CD19 + B220 + ) . Right panel showing insulin - reactive GC B cells pre - gated on GL - 7 + cells in a histogram . Data are representative of two independent experiments with n = 4 / group .

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

Figure 1Flow cytometric analysis of splenocytes of mice immunized with cInsulin or control immunization on day 26. Left and middle panel showing germinal center (GC) B cells (GL-7+) pre-gated on B cells (CD19+ B220+). Right panel showing insulin-reactive GC B cells pre-gated on GL-7+ cells in a histogram. Data are representative of two independent experiments with n=4/group.


Figure 2Coomassie stained SDS - page showing purified serum IgG of cInsulin immunized ( red ) and CI mice ( grey ) under reducing ( + β - ME ) , and non - reducing conditions . Representative of two independent experiments . Blood glucose levels of intravenously ( i . v . ) injected wild - type mice at indicated hours post injection . 20 µg of purified serum IgG from cInsulin immunized mice ( red , n = 6 ) or CI mice ( grey , n = 5 ) was used for injection . Dots represent individual mice . Mean ± SD , statistical significance was calculated by using repeated measure ANOVA test , * * * 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 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

Figure 2Coomassie stained SDS-page showing purified serum IgG of cInsulin immunized (red) and CI mice (grey) under reducing (+ β-ME), and non-reducing conditions. Representative of two independent experiments.Blood glucose levels of intravenously (i.v.) injected wild-type mice at indicated hours post injection. 20 µg of purified serum IgG from cInsulin immunized mice (red, n=6) or CI mice (grey, n=5) was used for injection. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, ***P < 0.001, ****P < 0.0001.


Figure 4Serum anti - Insulin - immunoglobulin titers of mice immunized with InsA - peptide ( red : 0 : 1 , n = 10 and green : 100 : 1 , n = 5 ) and control - immunization ( CI : CpG only , grey , n = 3 ) measured by ELISA at indicated days ( coating : Insulin ) . Dots represent individual mice . Mean ± SD , statistical significance was calculated by using Kruskal - Wallis test , * P < 0 . 05 , * * * P < 0 . 001 . Western blot analysis of insulin - specific serum IgG derived from InsA - peptide immunized mice . Top panel ( green ) : 100 : 1 serum , lower panel ( red ) : 0 : 1 serum ( sInsA : cInsA ) . Serum of mice was used as primary antibody and detection was done with anti - mouse - IgG - HRP . Black filled arrow : Proinsulin ( 12 kD ) , Black non - filled arrow : Insulin ( 6 kD ) , β - actin ( 42 kD , loading control ) . Representative of two independent experiments . Blood glucose levels of InsA - peptide immunized ( red : 0 : 1 , n = 5 for E and n = 10 for F ; green : 100 : 1 , n = 5 and n = 4 for F , d30 , d32 ) and CI ( grey , n = 5 for E and n = 10 for F ) mice were assessed at indicated days . Dots represent individual mice . Mean ± SD , statistical significance was calculated by using Kruskal - Wallis test ( E ) and repeated measure ANOVA test ( F ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Urine glucose levels of InsA peptide immunized ( red : n = 10 for d28 , d32 and n = 8 for d30 ; green : n = 5 for d28 , n = 4 for d30 , n = 3 for d32 ) and CI ( grey , n = 3 ) mice were monitored at indicated days post immunization . Dots represent individual mice . For 100 : 1 : representative data are shown for two independent experiments with total n = 10 . Mean ± SD , statistical significance was calculated by using repeated measure ANOVA test , * * * P < 0 . 001 .

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 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 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 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 4Serum anti-Insulin-immunoglobulin titers of mice immunized with InsA-peptide (red: 0:1, n=10 and green: 100:1, n=5) and control-immunization (CI: CpG only, grey, n=3) measured by ELISA at indicated days (coating: Insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test, *P < 0.05, ***P < 0.001.Western blot analysis of insulin-specific serum IgG derived from InsA-peptide immunized mice. Top panel (green): 100:1 serum, lower panel (red): 0:1 serum (sInsA:cInsA). Serum of mice was used as primary antibody and detection was done with anti-mouse-IgG-HRP. Black filled arrow: Proinsulin (12 kD), Black non-filled arrow: Insulin (6 kD), β-actin (42 kD, loading control). Representative of two independent experiments.Blood glucose levels of InsA-peptide immunized (red: 0:1, n=5 for E and n=10 for F; green: 100:1, n=5 and n=4 for F, d30, d32) and CI (grey, n=5 for E and n=10 for F) mice were assessed at indicated days. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test (E) and repeated measure ANOVA test (F), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Urine glucose levels of InsA peptide immunized (red: n=10 for d28, d32 and n=8 for d30; green: n=5 for d28, n=4 for d30, n=3 for d32) and CI (grey, n=3) mice were monitored at indicated days post immunization. Dots represent individual mice. For 100:1: representative data are shown for two independent experiments with total n=10. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, ***P < 0.001.


Figure 5 ( C , F ) Urine glucose levels of InsA peptide immunized ( n = 3 ) and CI ( n = 3 ) mice were monitored at indicated days post immunization . Dots represent individual mice . Mean ± SD , statistical significance was calculated by using repeated measure ANOVA test , * P < 0 . 05 . ( D , E ) Serum anti - Insulin - immunoglobulin titers of mice immunized with InsA - peptide ( day 59 , day 78 : n = 3 , day 49 : n = 7 ) and control - immunization ( CI : CpG only , n = 3 ) measured by ELISA at indicated days ( coating : Insulin ) . Dots represent individual mice . Mean ± SD , statistical significance was calculated by using Mann - Whitney - U test ( D ) and repeated measure ANOVA test ( E ) . * P < 0 . 05 , * * P < 0 . 01 .

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 5(C, F) Urine glucose levels of InsA peptide immunized (n=3) and CI (n=3) mice were monitored at indicated days post immunization. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, *P < 0.05. (D, E) Serum anti-Insulin-immunoglobulin titers of mice immunized with InsA-peptide (day 59, day 78: n=3, day 49: n=7) and control-immunization (CI: CpG only, n=3) measured by ELISA at indicated days (coating: Insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test (D) and repeated measure ANOVA test (E). *P < 0.05, **P < 0.01.


Figure 6Anti - Insulin affinity of PR - IgM ( blue ) , primary IgM ( red ) and isotype control ( black ) measured by bio - layer interferometry . IgM binding to insulin was acquired in pm and used to calculate the dissociation constant shown in the graph ( Kd = 1 / Ka ) . Graph is showing antigen - antibody association phase . Data are representative for three independent experiments . Blood glucose levels of intravenously injected mice with purified IgM from control immunization ( CI , n = 5 ) , complex InsA peptide ( cInsA ) immunization day 7 ( total IgM d7 , n = 4 ) and day 85 ( total IgM d85 , n = 5 ) . Mean ± SD , statistical significance was calculated by using repeated measure ANOVA test showing comparison of red to black line , * * 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 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

Figure 6Anti-Insulin affinity of PR-IgM (blue), primary IgM (red) and isotype control (black) measured by bio-layer interferometry. IgM binding to insulin was acquired in pm and used to calculate the dissociation constant shown in the graph (Kd = 1/Ka). Graph is showing antigen-antibody association phase. Data are representative for three independent experiments.Blood glucose levels of intravenously injected mice with purified IgM from control immunization (CI, n=5), complex InsA peptide (cInsA) immunization day 7 (total IgM d7, n=4) and day 85 (total IgM d85, n=5). Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test showing comparison of red to black line, **P < 0.01, ***P < 0.001, ****P < 0.0001.