Figure 3 ( A ) HH / SMO promotes slow - migrating forms of Smaug . Western blot analysis of Cl8 cells that transiently express HA - SmaugWT alone , or together with either SMOWT - GFP or SMOPKA - SD - GFP , in the presence or in absence of HH . GMAP serves as a loading control . Here and in the other panels , the antibodies used are indicated on the left . Here and in ( B , C ) , the black arrows indicate the unphosphorylated form of HA - SmaugWT and the brackets indicate the slower migrating phosphorylated forms of HA - SmaugWT . NT : not transfected . See a phosphatase assay in Fig EV4A . ( B ) Smaug phosphorylation requires its interaction with SMO . Western blot analysis of Cl8 cells expressing HA - SmaugWT , in presence of HH , in combination with SMO ∆ 958 - GFP , SMOPKA - SD - GFP or SMOPKA - SD , ∆ 978 - GFP , as indicated . ( C ) FU controls Smaug phosphorylation . Extracts of Cl8 cells expressing SNAP - Smaug with SMOPKA - SD FU - SD - GFP ( with HH ) in combination with GFP - FUWT , GAP - GFP - FUWT , Myc - FUEE or GAP - null - FUDANA ( as indicated ) were labeled for the SNAP tag before electrophoresis . After quantification of the gels , the percentage ( % ) of phosphorylated Smaug ( % of phosphorylated forms of SNAP - Smaug total amounts of SNAP - Smaug ) was estimated . In C to E , the mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom , and the statistical analysis was done by one tailed bivariate Wilcoxon rank test . See also fig EV4C , D . ( D ) Phosphorylation of Smaug by activated FU requires the co - expression of SMO . Mean values of the percentage of phosphorylated Smaug in extracts of Cl8 cells that express λN - SNAP - Smaug with GAP - GFP - FUWT and SMOWT alone or together ( as indicated ) N = 3 ( billogical triplicates ) . Note that the effect of GAP - GFP - FUWT and SMOWT together is lower than the one observed with GAP - GFP - FUWT and SMOPKA - SD FU - SD - GFP together ( in panel C ) . This could have multiple nonexclusive causes , including the fact that SMOWT is present at much lower levels than SMOPKA - SD FU - SD [ 38 ] and / or the involvement of another kinase . See also Fig EV4B . Note that Smaug does not coIP FU unless SMO is present see Appendix Figure S2 . ( E ) Forcing the interaction of Smaug with activated FU leads to a SMO - independent phosphorylation of Smaug . Mean values of the percentage of phosphorylated Smaug in Cl8 cells coexpressing λN - SNAP - Smaug with GFP - FU , FU - SBR , FUEE - SBR or FUDANA - SBR , with or without HH , as indicated . N = 3 ( biological triplicates ) . See also Fig EV4C and D . Note that the levels of Smaug phosphorylation observed in presence of FUDANA - SBR are not lower than those seen with GFP - FU . This suggests an incomplete inhibition of endogenous FU , which may be due to the trapping of FUDANA - SBR by Smaug . See a representative blot in Fig EV3C and a phosphatase assay in Fig EV4D .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HH/SMO promotes slow-migrating forms of Smaug. Western blot analysis of Cl8 cells that transiently express HA-SmaugWT alone, or together with either SMOWT-GFP or SMOPKA-SD-GFP, in the presence or in absence of HH. GMAP serves as a loading control. Here and in the other panels, the antibodies used are indicated on the left. Here and in (B,C), the black arrows indicate the unphosphorylated form of HA-SmaugWT and the brackets indicate the slower migrating phosphorylated forms of HA-SmaugWT. NT: not transfected. See a phosphatase assay in Fig EV4A.(B) Smaug phosphorylation requires its interaction with SMO. Western blot analysis of Cl8 cells expressing HA-SmaugWT, in presence of HH, in combination with SMO∆958-GFP, SMOPKA-SD-GFP or SMOPKA-SD, ∆978-GFP, as indicated.(C) FU controls Smaug phosphorylation. Extracts of Cl8 cells expressing SNAP-Smaug with SMOPKA-SD FU-SD-GFP (with HH) in combination with GFP-FUWT, GAP-GFP-FUWT, Myc-FUEE or GAP-null-FUDANA (as indicated) were labeled for the SNAP tag before electrophoresis. After quantification of the gels, the percentage (%) of phosphorylated Smaug (% of phosphorylated forms of SNAP-Smaug total amounts of SNAP-Smaug) was estimated. In C to E, the mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom, and the statistical analysis was done by one tailed bivariate Wilcoxon rank test. See also fig EV4C, D.(D) Phosphorylation of Smaug by activated FU requires the co-expression of SMO. Mean values of the percentage of phosphorylated Smaug in extracts of Cl8 cells that express λN-SNAP-Smaug with GAP-GFP-FUWT and SMOWT alone or together (as indicated) N=3 (billogical triplicates). Note that the effect of GAP-GFP-FUWT and SMOWT together is lower than the one observed with GAP-GFP-FUWT and SMOPKA-SD FU-SD-GFP together (in panel C). This could have multiple nonexclusive causes, including the fact that SMOWT is present at much lower levels than SMOPKA-SD FU-SD [38] and/or the involvement of another kinase. See also Fig EV4B. Note that Smaug does not coIP FU unless SMO is present see Appendix Figure S2.(E) Forcing the interaction of Smaug with activated FU leads to a SMO-independent phosphorylation of Smaug. Mean values of the percentage of phosphorylated Smaug in Cl8 cells coexpressing λN-SNAP-Smaug with GFP-FU, FU-SBR, FUEE-SBR or FUDANA-SBR, with or without HH, as indicated. N=3 (biological triplicates). See also Fig EV4C and D. Note that the levels of Smaug phosphorylation observed in presence of FUDANA-SBR are not lower than those seen with GFP-FU. This suggests an incomplete inhibition of endogenous FU, which may be due to the trapping of FUDANA-SBR by Smaug. See a representative blot in Fig EV3C and a phosphatase assay in Fig EV4D.


Figure 4 ( A and C ) Effect of FUEE - SBR on Smaug phosphorylation and levels . Extracts of Cl8 cells transfected with λN - SNAP - smaug ( 25 ng ) and SNAP - GUS - 5BoxB ( 300 ng ) plasmids in absence or with different amounts of FUEE expressing plasmid ( ranging from 15 to 60 μg , as indicated ) were analyzed by electrophoresis before quantification as above . See a reprenstive gel in Fig EV5A . The % of phosphorylated SNAP - Smaug protein ( determined as in Fig 3 ) ( A ) are represented as a function of the levels of FUEE - SBR . The mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom . The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value = 0 . 05 ( * ) . Here CLIP - GUS was used as an internal control . ( B ) Effect of FUEE - SBR on Smaug activity . The relative levels of reporter expression obtained in the same experiment as in ( A - C ) are represented as a function of the % of phosphorylated Smaug . Color of data points correspond to ng of transfected FUEE - SBR in A - C . ( A and C ) Effect of FUEE - SBR on Smaug phosphorylation and levels . Extracts of Cl8 cells transfected with λN - SNAP - smaug ( 25 ng ) and SNAP - GUS - 5BoxB ( 300 ng ) plasmids in absence or with different amounts of FUEE expressing plasmid ( ranging from 15 to 60 μg , as indicated ) were analyzed by electrophoresis before quantification as above . See a reprenstive gel in Fig EV5A . The relative levels of of λN - SNAP - Smaug protein ( C ) ( determined as in Fig 2 ) are represented as a function of the levels of FUEE - SBR . The mean values and ( SD ) for independent biological triplicates ( N = 3 ) are shown in the graph at the bottom . The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value = 0 . 05 ( * ) . Here CLIP - GUS was used as an internal control . ( D ) Effect of FUEE - SBR on Smaug subcellular localization . Representative fluorescent images of Cl8 cells expressing GFP - SmaugWT ( 1 ) , FUEE - SBR - mCherry ( 2 ) , FUDANA - SBR - mCh ( 3 ) or expressing GFP - SmaugWT together with FUEE - SBR - mCherry ( 4 - 4 ' ' ) or GFP - SmaugWT together with FUDANA - SBR - mCh ( 5 - 5 ' ' ) . The merge images in 4 " and 5 " show GFP - SmaugWT in green and FUEE - SBR - mCh or FUDANA - SBR - mCh in red . ( 6 ) In presence of FUEE - SBR , 274 of a total of 294 cotransfected cells show a diffuse localization of Smaug and FU and no foci . In presence of FUDANA - SBR , 100 % of the cotransfected cells ( n = 241 ) show Smaug and FU distributed in bodies and colocalized . 250 ng of plasmid were used for each construct . Scale bar ( shown in D1 , identical for all panels ) :

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

Figure 4(A and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The % of phosphorylated SNAP-Smaug protein (determined as in Fig 3) (A) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control.(B) Effect of FUEE-SBR on Smaug activity. The relative levels of reporter expression obtained in the same experiment as in (A-C) are represented as a function of the % of phosphorylated Smaug. Color of data points correspond to ng of transfected FUEE-SBR in A-C.(A and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The relative levels of of λN-SNAP-Smaug protein (C) (determined as in Fig 2) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control.(D) Effect of FUEE-SBR on Smaug subcellular localization.Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT (1), FUEE-SBR-mCherry (2), FUDANA-SBR-mCh (3) or expressing GFP-SmaugWT together with FUEE-SBR-mCherry (4-4'') or GFP-SmaugWT together with FUDANA-SBR-mCh (5-5''). The merge images in 4" and 5" show GFP-SmaugWT in green and FUEE-SBR-mCh or FUDANA-SBR-mCh in red. (6) In presence of FUEE-SBR, 274 of a total of 294 cotransfected cells show a diffuse localization of Smaug and FU and no foci. In presence of FUDANA-SBR, 100% of the cotransfected cells (n=241) show Smaug and FU distributed in bodies and colocalized. 250 ng of plasmid were used for each construct. Scale bar (shown in D1, identical for all panels): 10μ


Figure 5 ( A ) Effect of FUEE - SBR and HH / SMO on endogeneous Smaug target mRNAs in Cl8 cells . Relative mRNA levels estimated using semi - quantitative RT - PCR for various Smaug ' s targets in Cl8 cells expressing FUEE - SBR ( black ) , FUEE ( dark grey ) , or FUDANA - SBR ( light grey ) were reported with respect to levels of a GFP control . mRNA levels were set to 1 for the GFP control . The list of genes and the sequences of the primers used are shown in Appendix Table S2 and S3 . See also Fig EV5E . Here and in Fig EV5D : RNA encoding mitochondrial proteins are written in blue . N = 3 ( biological triplicates ) and the quantifications were repeated 3 times on each sample . The results are presented as the means ± standard error . The differences between groups were assessed using Student ' s t - tests with Prism V8 . 2 . 1 software . A p value < 0 . 05 was considered to indicate a statistically significant difference ( B ) Effect of HH on endogeneous targets of Smaug in the wing imaginal disc . Relative levels of Smaug ' s targets mRNAs in MS1096 ; UAS - hh or hhts and their respective controls ( as in Fig EV5X ) . N = 3 , with xx disc for each phenotype . ( C ) Effect of smaug mutation on [ fu1 ] wing class distribution . Percent distribution of phenotypic classes ( defined in Appendix Figure S4 ) in fu1 males in presence of zero ( fu1 ) , one ( fu1 ; ; smaug47 / + ) or two copies ( fu1 ; ; smaug47 / smaug47 ) of the smaug47 allele . The wings were double - blindly classified in " weak " and " strong " according to the LV3 - V4 defects ( see Fig EV6B ) . * Chi2 statistical analysis gave a p of 0 , 015 for fu1 and fu1 ; ; smaug47 / + distributions and of 0 , 0024 when comparing fu1 ; ; smaug47 / + to fu1 ; ; smaug47 / smaug47 .

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

Figure 5(A) Effect of FUEE-SBR and HH/SMO on endogeneous Smaug target mRNAs in Cl8 cells. Relative mRNA levels estimated using semi-quantitative RT-PCR for various Smaug's targets in Cl8 cells expressing FUEE-SBR (black) , FUEE (dark grey) , or FUDANA-SBR (light grey) were reported with respect to levels of a GFP control. mRNA levels were set to 1 for the GFP control. The list of genes and the sequences of the primers used are shown in Appendix Table S2 and S3. See also Fig EV5E. Here and in Fig EV5D: RNA encoding mitochondrial proteins are written in blue. N=3 (biological triplicates) and the quantifications were repeated 3 times on each sample.The results are presented as the means ± standard error. The differences between groups were assessed using Student's t-tests with Prism V8.2.1 software. A p value <0.05 was considered to indicate a statistically significant difference(B) Effect of HH on endogeneous targets of Smaug in the wing imaginal disc. Relative levels of Smaug's targets mRNAs in MS1096; UAS-hh or hhts and their respective controls (as in Fig EV5X). N=3, with xx disc for each phenotype.(C) Effect of smaug mutation on [fu1] wing class distribution. Percent distribution of phenotypic classes (defined in Appendix Figure S4) in fu1 males in presence of zero (fu1), one (fu1;;smaug47/+) or two copies (fu1;; smaug47/smaug47) of the smaug47 allele. The wings were double-blindly classified in "weak" and "strong" according to the LV3-V4 defects (see Fig EV6B). *Chi2 statistical analysis gave a p of 0,015 for fu1 and fu1;;smaug47/+ distributions and of 0,0024 when comparing fu1;;smaug47/+ to fu1;; smaug47/smaug47.


Figure 1A . Volcano plot showing the fold change ( y - axis ) versus adjusted ( adj . ) p - value ( x - axis ) of the beta cell transcriptomes between chow and HFD - fed mice ( 16 weeks ) . Genes highlighted in red or green are based on the thresholds of Log2 fold change > 1 and adj . P value < 0 . 01 ( two - tailed paired student ' s t - test ) . Genes with > 10 fold upregulated expression are labeled with their name . Pbk gene expression is indicated by the arrow . RNAseq data was available from https : / / doi . org / 10 . 1371 / journal . pone . 0213299 . t004 . L . Pbk KD in INS - 1 cells suppresses cell growth . Western blot data showed the decreased Pbk expression level in INS - 1 cells with Pbk - targeted shRNA transduction . Cell growth curve was from three independent experimets ( n = 3 ) . * * * P < 0 . 0001 ( Vector : shRNA - # 1 ) , * P = 0 . 0181 ( Vector : shRNA - # 5 ) , ( Two way ANOVA ) . M . The role of ectopic expression of Pbk - WT and Pbk - Mut on INS - 1 cell growth . Western blot data showed the overexpression of V5 - flaged wild type and mutant Pbk in INS - 1 cells . Cell growth curve was from three independent experimets ( n = 3 ) . * * P = 0 . 0069 ( Two way ANOVA ) . ns , not statistically significant difference .

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

Figure 1A.Volcano plot showing the fold change (y-axis) versus adjusted (adj.) p-value (x-axis) of the beta cell transcriptomes between chow and HFD-fed mice (16 weeks). Genes highlighted in red or green are based on the thresholds of Log2 fold change > 1 and adj. P value < 0.01 (two-tailed paired student's t-test). Genes with >10 fold upregulated expression are labeled with their name. Pbk gene expression is indicated by the arrow. RNAseq data was available from https://doi.org/10.1371/journal.pone.0213299.t004.L. Pbk KD in INS-1 cells suppresses cell growth. Western blot data showed the decreased Pbk expression level in INS-1 cells with Pbk-targeted shRNA transduction. Cell growth curve was from three independent experimets (n=3). ***P < 0.0001 (Vector:shRNA-#1), *P = 0.0181 (Vector:shRNA-#5) , (Two way ANOVA). M. The role of ectopic expression of Pbk-WT and Pbk-Mut on INS-1 cell growth. Western blot data showed the overexpression of V5-flaged wild type and mutant Pbk in INS-1 cells. Cell growth curve was from three independent experimets (n=3). **P = 0.0069 (Two way ANOVA). ns, not statistically significant difference.


Figure 2F . Islet number in each pancreas section on 11 - week - old male PbkKI / KI and PbkWT / WT mice ( n = 4 for each group ) . * P = 0 . 0443 ( two - tailed unpaired student ' s t - test ) . G . Beta cell mass comparison between 11 - week - old male PbkKI / KI and PbkWT / WT mice ( n = 4 for each group ) . * * P = 0 . 0015 ( two - tailed unpaired student ' s t - test ) . K , L . Representative images of double immuno - staining for insulin ( green ) and BrdU ( red ) of pancreas from PbkWT / WT ( K ) or PbkKI / KI ( L ) mice with 5 weeks HFD feeding . Islet area was circled with white dashed line . Nuclei were labeled by DAPI ( blue ) . Scale bar : 50 μm .

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Figure 2F. Islet number in each pancreas section on 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). *P = 0.0443 (two-tailed unpaired student's t-test). G. Beta cell mass comparison between 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). **P = 0.0015 (two-tailed unpaired student's t-test). K, L. Representative images of double immuno-staining for insulin (green) and BrdU (red) of pancreas from PbkWT/WT (K) or PbkKI/KI (L) mice with 5 weeks HFD feeding. Islet area was circled with white dashed line. Nuclei were labeled by DAPI (blue). Scale bar: 50 μm.


Figure 3E , F . JunD KD in INS - 1 cells using shRNA upregulated Pbk protein levels ( E ) and mRNA levels ( F ) . qPCR data was from three independent experimets ( n = 3 ) . * * * P = 0 . 0001 ( shRNA - # 1 ) , * * * P = 0 . 0007 ( shRNA - # 2 ) ( two - tailed unpaired student ' s t - test ) . G , H . JunD KD in PIME cells using shRNA increased Pbk protein levels ( G ) and mRNA levels ( H ) . qPCR data was from three independent experimets ( n = 3 ) . * P = 0 . 0236 ( shRNA - # 1 ) , * P = 0 . 0175 ( shRNA - # 2 ) ( two - tailed unpaired student ' s t - test ) . N . JunD overexpression in PIME cells suppressed Pbk protein levels ( M , Lane 2 vs Lane 1 ) , but did not affect Pbk expression in menin - null PIME cells ( M , Lane 4 vs Lane 3 ) .

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

Figure 3E, F. JunD KD in INS-1 cells using shRNA upregulated Pbk protein levels (E) and mRNA levels (F). qPCR data was from three independent experimets (n=3).***P = 0.0001 (shRNA-#1), ***P = 0.0007 (shRNA-#2) (two-tailed unpaired student's t-test). G, H. JunD KD in PIME cells using shRNA increased Pbk protein levels (G) and mRNA levels (H). qPCR data was from three independent experimets (n=3).*P = 0.0236 (shRNA-#1), *P = 0.0175 (shRNA-#2) (two-tailed unpaired student's t-test). N. JunD overexpression in PIME cells suppressed Pbk protein levels (M, Lane 2 vs Lane 1), but did not affect Pbk expression in menin-null PIME cells (M, Lane 4 vs Lane 3).


Figure 4J , K . The effect of T5224 on the binding of JunD , menin , HDAC3 , and acetylated histone3 at the Pbk locus in PIME cells ( J ) or INS - 1 cells ( K ) . Three independent experimets ( n = 3 ) . * P = 0 . 0490 ( J , JunD ) , * P = 0 . 0479 ( J , Menin ) , * P = 0 . 0154 ( J , HDAC3 ) , * * P = 0 . 0029 ( J , Ac - H3 ) , * P = 0 . 0104 ( K , JunD ) , * * P = 0 . 0024 ( K , Menin ) , * * * P = 0 . 0002 ( K , HDAC3 ) , * * * P = 0 . 0001 ( K , Ac - H3 ) , ( two - tailed unpaired student ' s t - test ) . ns , not statistically significant difference .

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Figure 4J, K. The effect of T5224 on the binding of JunD, menin, HDAC3, and acetylated histone3 at the Pbk locus in PIME cells (J) or INS-1 cells (K). Three independent experimets (n=3). *P = 0.0490 (J, JunD), *P = 0.0479 (J, Menin), *P = 0.0154 (J, HDAC3), **P = 0.0029 (J, Ac-H3), *P = 0.0104 (K, JunD), **P = 0.0024 (K, Menin), ***P = 0.0002 (K, HDAC3), ***P = 0.0001 (K, Ac-H3), (two-tailed unpaired student's t-test). ns, not statistically significant difference.


Figure 5F . Mouse primary islets treated with MI - 503 for 3 days were subjected ChIP assay to detect the binding of menin , HDAC3 , JunD , as well as histone acetylation level at the Pbk locus . Three independent experimets ( n = 3 ) . * P = 0 . 0218 ( Menin ) , * P = 0 . 0145 ( HDAC3 ) , * P = 0 . 0184 ( Ac - H3 ) ( two - tailed unpaired student ' s t - test ) . ns , not statistically significant difference .

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Figure 5F. Mouse primary islets treated with MI-503 for 3 days were subjected ChIP assay to detect the binding of menin, HDAC3, JunD, as well as histone acetylation level at the Pbk locus. Three independent experimets (n=3). *P = 0.0218 (Menin), *P = 0.0145 (HDAC3), *P = 0.0184 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.


Figure 6G . MI - 503 increases proliferation of menin - expressing PIME cells but not menin - null PIME cells . Three independent experimets ( n = 3 ) . * * * P < 0 . 0001 ( Two - way ANOVA ) . K - M . 5 days of MI - 503 treatment impacts the expression of Pbk at protein level ( K ) and mRNA level ( L ) , as well as ki67 mRNA levels ( M ) in human primary islets ( donor number is four ) . qPCR data was from three independent experimets ( n = 3 ) . * * P = 0 . 0036 ( L , 10 nM ) , * * P = 0 . 0024 ( L , 50 nM ) , * * P = 0 . 0073 ( L , 100 nM ) , * P = 0 . 0237 ( M , 50 nM ) ( two - tailed unpaired student ' s t - test ) . ns , not statistically significant difference .

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Figure 6G. MI-503 increases proliferation of menin-expressing PIME cells but not menin-null PIME cells. Three independent experimets (n=3). ***P < 0.0001 (Two-way ANOVA).K-M. 5 days of MI-503 treatment impacts the expression of Pbk at protein level (K) and mRNA level (L), as well as ki67 mRNA levels (M) in human primary islets (donor number is four). qPCR data was from three independent experimets (n=3). **P = 0.0036 (L, 10 nM), **P = 0.0024 (L, 50 nM), **P = 0.0073 (L, 100 nM), *P = 0.0237 (M, 50 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference.


Figure 7E - L . Representative images of insulin ( green ) and Ki67 ( red ) double staining on mouse islets with 8 wks MI treatment ( E - H ) , and insulin ( green ) or Pbk ( red ) double staining on mouse islets ( I - L ) . Nuclei were labeled by DAPI ( blue ) . Islet area was circled by white dashed line . Ki67 and Pbk staining are denoted by white arrows . Scale bars : 50 μm .

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Figure 7E-L. Representative images of insulin (green) and Ki67 (red) double staining on mouse islets with 8 wks MI treatment (E-H), and insulin (green) or Pbk (red) double staining on mouse islets (I-L). Nuclei were labeled by DAPI (blue). Islet area was circled by white dashed line. Ki67 and Pbk staining are denoted by white arrows. Scale bars: 50 μm.


Figure 8D , E . Double staining for insulin ( green ) and BrdU ( red ) of mice islets after 12 weeks of vehicle ( D ) or MI ( E ) administration . Islet area was circled by white dashed line . Nuclei were labeled by DAPI ( blue ) . BrdU staining is indicated by arrows . Scale bar : 50 μm . F , G . Quantification of BrdU positive beta cells ( F ) and beta cell mass ( G ) of MI - 463 treated HFD - fed mice or control mice . Five mice for each group , and at least 10 islet images per mouse were analyzed . * * P = 0 . 0059 , * P = 0 . 0103 ( two - tailed unpaired student ' s t - test ) . H , I . Double staining for insulin ( green ) and Pbk ( red ) of mice islets after 12 weeks of vehicle ( H ) or MI ( I ) administration . Islet area was circled by white dashed line . Nuclei were labeled by DAPI ( blue ) . Pbk staining is indicated by arrows . Scale bar : 50 μm . J . Quantification of Pbk staining - positive β - cells . Five mice for each group , and at least 10 islet images per mouse were analyzed . * P = 0 . 0120 ( two - tailed unpaired student ' s t - test ) . K . Quantification of the percentage with pbk and BrdU co - staining cells among total Pbk positive cells in HFD - fed mouse islets . The mean values of five mice from 5 sections for each mouse were presented . O , P . GTT data for MI - 463 or vehicle treated PbkKI / KI or PbkWT / WT mice ( n = 3 for each group ) on 8 weeks HFD ( O ) ( * P = 0 . 0133 ( Two way ANOVA ) , ns , not statistically significant difference . ) and AUC of GTT data ( P ) ( * P = 0 . 0442 ( two - tailed unpaired student ' s t - test ) , ns , not statistically significant difference . ) .

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Figure 8D, E. Double staining for insulin (green) and BrdU (red) of mice islets after 12 weeks of vehicle (D) or MI (E) administration. Islet area was circled by white dashed line. Nuclei were labeled by DAPI (blue). BrdU staining is indicated by arrows. Scale bar:50 μm. F, G. Quantification of BrdU positive beta cells (F) and beta cell mass (G) of MI-463 treated HFD-fed mice or control mice. Five mice for each group, and at least 10 islet images per mouse were analyzed. **P = 0.0059, *P = 0.0103 (two-tailed unpaired student's t-test). H, I. Double staining for insulin (green) and Pbk (red) of mice islets after 12 weeks of vehicle (H) or MI (I) administration. Islet area was circled by white dashed line. Nuclei were labeled by DAPI (blue). Pbk staining is indicated by arrows. Scale bar: 50 μm. J. Quantification of Pbk staining-positive β-cells. Five mice for each group, and at least 10 islet images per mouse were analyzed. *P = 0.0120 (two-tailed unpaired student's t-test). K. Quantification of the percentage with pbk and BrdU co-staining cells among total Pbk positive cells in HFD-fed mouse islets. The mean values of five mice from 5 sections for each mouse were presented.O, P. GTT data for MI-463 or vehicle treated PbkKI/KI or PbkWT/WT mice (n=3 for each group) on 8 weeks HFD (O) (*P = 0.0133 (Two way ANOVA), ns, not statistically significant difference.) and AUC of GTT data (P) (*P = 0.0442 (two-tailed unpaired student's t-test), ns, not statistically significant difference.).


Figure 1 ( A ) SARS - CoV - 2 genomic view showing the distribution of normalized 22nt small RNA reads from Caco - 2 and A549 - ACE2 cells at 24 and 48 hpi . The most abundant small RNAs are marked by the red and blue boxes . For all the experiments shown n = 2 .

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Figure 1(A) SARS-CoV-2 genomic view showing the distribution of normalized 22nt small RNA reads from Caco-2 and A549-ACE2 cells at 24 and 48 hpi. The most abundant small RNAs are marked by the red and blue boxes. For all the experiments shown n=2.


Figure 2 ( C ) Percentage of conservation along the nucleotide positions in the ORF7a among 4 , 055 , 609 sequenced SARS - CoV - 2 genomes . The first 70 nt are shown in red and shows a higher percentage of conservation compared to the rest of the sequence of ORF7a . Boxplot shows the distribution of conservation percentage for each nucleotide either in the first 70 nt or 71 - 366 nt of ORF7a among 4 , 055 , 609 sequenced SARS - CoV - 2 genomes . Box plots display median ( line ) , first and third quartiles ( box ) , and 5th / 95th percentile value ( whiskers ) . Each dot represents the outliers . Two - tailed P values were calculated using the student ' s t - test .

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Figure 2(C) Percentage of conservation along the nucleotide positions in the ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. The first 70 nt are shown in red and shows a higher percentage of conservation compared to the rest of the sequence of ORF7a. Boxplot shows the distribution of conservation percentage for each nucleotide either in the first 70 nt or 71-366 nt of ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. Box plots display median (line), first and third quartiles (box), and 5th /95th percentile value (whiskers). Each dot represents the outliers. Two-tailed P values were calculated using the student's t-test.


Figure 3 ( A ) Log2 fold changes of the levels of hsa - miR - let - 7a , CoV2 - miR - O7a . 1 and COV2 - miR - O7a . 2 in DICER1 knockdown SARS - CoV - 2 - infected A549 - ACE2 cells compared to control cells analyzed by stem - loop RT - qPCR . Results from three independent replicates are shown . ( B ) In vitro kinetics of DICER1 mediated cleavage of pre - CoV2 - miR - O7a stem - loop precursor , pre - CoV2 - miR - O7a with mutations that prevent the formation of stem - loop structure and pre - hsa - miR21 . Folded conformations for the three precursor molecules are shown . Fold change in the production of mature CoV2 - miR - O7a . 2 is shown in presence of DICER1 compared to control reaction ( no DICER1 ) as measure by stem - loop RT - qPCR . Bars and error bars represent the average and standard deviation from three independent experiments . ( C ) Expression levels of the virus - derived miRNAs as a percentage of the viral genome . Absolute quantification of virus - derived miRNAs and viral genome from infected A549 - ACE2 cells was performed using two spike - in ( see methods ) . Line represents the average and individual dots represent data from two independent experiments . ( D ) copy number per cell of hsa - miR - let - 7a , CoV2 - miR - O7a . 1 and CoV2 - miR - O7a . 2 in infected A549 - ACE2 cells quantified using a synthetic small RNA spike - in standard curve by stem - loop RT - qPCR . Levels of hsa - miR - let - 7a were normalized for the percentage of infected cells . Bars and error bars represent the average and standard deviation from at least three independent experiments . ( E ) Loading of hsa - miR - let - 7a , CoV2 - miR - O7a . 1 and COV2 - miR - O7a . 2 into AGOs as measured by stem - loop RT - qPCR and analyzed as a percentage of input from the immunoprecipitates ( IPs ) of either pan - AGO IP or control IgG IP from infected A549 - ACE2 cells . Levels of miRNA were normalized for the percentage of infected cells .

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

Figure 3(A) Log2 fold changes of the levels of hsa-miR-let-7a, CoV2-miR-O7a.1 and COV2-miR-O7a.2 in DICER1 knockdown SARS-CoV-2-infected A549-ACE2 cells compared to control cells analyzed by stem-loop RT-qPCR. Results from three independent replicates are shown.(B) In vitro kinetics of DICER1 mediated cleavage of pre-CoV2-miR-O7a stem-loop precursor, pre-CoV2-miR-O7a with mutations that prevent the formation of stem-loop structure and pre-hsa-miR21. Folded conformations for the three precursor molecules are shown. Fold change in the production of mature CoV2-miR-O7a.2 is shown in presence of DICER1 compared to control reaction (no DICER1) as measure by stem-loop RT-qPCR. Bars and error bars represent the average and standard deviation from three independent experiments.(C) Expression levels of the virus-derived miRNAs as a percentage of the viral genome. Absolute quantification of virus-derived miRNAs and viral genome from infected A549-ACE2 cells was performed using two spike-in (see methods). Line represents the average and individual dots represent data from two independent experiments.(D) copy number per cell of hsa-miR-let-7a, CoV2-miR-O7a.1 and CoV2-miR-O7a.2 in infected A549-ACE2 cells quantified using a synthetic small RNA spike-in standard curve by stem-loop RT-qPCR. Levels of hsa-miR-let-7a were normalized for the percentage of infected cells. Bars and error bars represent the average and standard deviation from at least three independent experiments.(E) Loading of hsa-miR-let-7a, CoV2-miR-O7a.1 and COV2-miR-O7a.2 into AGOs as measured by stem-loop RT-qPCR and analyzed as a percentage of input from the immunoprecipitates (IPs) of either pan-AGO IP or control IgG IP from infected A549-ACE2 cells. Levels of miRNA were normalized for the percentage of infected cells.


Figure 4 ( B ) Relative firefly luciferase normalized to Renilla luciferase of pmirGLO Dual - Luciferase miRNA Target Expression Vector with either CoV2 - miR - O7a . 2 target site or CoV2 - miR - O7a . 2 site with mutations in seed region at 24 h on co - transfection with CoV2 - miR - O7a . 2 or control miRNA mimics . Activity on transfecting control miRNA mimic was considered 100 % . Bars and error bars represent the average and standard deviation from three independent experiments . ( D ) Kinetics of BATF2 and LAMP3 mRNA levels in non - induced and interferon IFN - α - induced ( for 8 , 16 , and 24 h ) A549 - ACE2 cells transfected with either control or CoV2 - miR - O7a . 2 mimics . Normalized read abundances in transcript per million ( TPM ) are shown for two individual experiments ( n = 2 ) . ( E ) Genomic view of the human BATF2 gene showing normalized RNA - seq reads from non - induced and IFN - α - induced ( for 8 h ) A549 - ACE2 cells transfected with either control , CoV2 - miR - O7a . 1 or CoV2 - miR - O7a . 2 mimics . The complementary site of CoV2 - miR - O7a to BATF2 3 ´ UTR is shown .

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Figure 4(B) Relative firefly luciferase normalized to Renilla luciferase of pmirGLO Dual-Luciferase miRNA Target Expression Vector with either CoV2-miR-O7a.2 target site or CoV2-miR-O7a.2 site with mutations in seed region at 24 h on co-transfection with CoV2-miR-O7a.2 or control miRNA mimics. Activity on transfecting control miRNA mimic was considered 100 %. Bars and error bars represent the average and standard deviation from three independent experiments.(D) Kinetics of BATF2 and LAMP3 mRNA levels in non-induced and interferon IFN-α-induced (for 8, 16, and 24 h) A549-ACE2 cells transfected with either control or CoV2-miR-O7a.2 mimics. Normalized read abundances in transcript per million (TPM) are shown for two individual experiments (n=2).(E) Genomic view of the human BATF2 gene showing normalized RNA-seq reads from non-induced and IFN-α-induced (for 8 h) A549-ACE2 cells transfected with either control, CoV2-miR-O7a.1 or CoV2-miR-O7a.2 mimics. The complementary site of CoV2-miR-O7a to BATF2 3´UTR is shown.


Figure 5 ( A , B ) Volcano plots showing the log2 fold change and corresponding significance levels of ISGs upon 8 hours of IFN - α treatment in A549 - ACE2 cells transfected with CoV2 - miR - O7a . 1 ( A ) or CoV2 - miR - O7a . 2 ( B ) compared to control mimic . Significantly downregulated genes are marked in red , and upregulated genes in blue . The orange horizontal line indicates two - tailed P = 0 . 05 . n = 2 , dashed lines represent + 1 and - 1 log2 fold change . ( C ) Log2 fold change of ISGs at 8 , 16 , 24 hours of IFN - α treatment and categorized based on CoV2 - miR - O7a . 2 target sites 8mer , 7mer - m8 , 7mer - A1 , and no seed as shown in the schematic . The mean and standard error of the mean is shown . Number of ISGs with 8mer site n = 8 , 7mer - m8 n = 32 , 7mer - A1 n = 26 and no seed n = 92 . The two - tailed p values were calculated using the Mann - Whitney - Wilcoxon test . ISGs were calculated as all the upregulated genes ( ≥ 3 - fold ; padj < 0 . 05 ) in two replicates of IFN - induced versus non - induced conditions in all time points .

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 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

Figure 5(A, B) Volcano plots showing the log2 fold change and corresponding significance levels of ISGs upon 8 hours of IFN-α treatment in A549-ACE2 cells transfected with CoV2-miR-O7a.1 (A) or CoV2-miR-O7a.2 (B) compared to control mimic. Significantly downregulated genes are marked in red, and upregulated genes in blue. The orange horizontal line indicates two-tailed P = 0.05. n=2, dashed lines represent +1 and -1 log2 fold change.(C) Log2 fold change of ISGs at 8, 16, 24 hours of IFN-α treatment and categorized based on CoV2-miR-O7a.2 target sites 8mer, 7mer-m8, 7mer-A1, and no seed as shown in the schematic. The mean and standard error of the mean is shown. Number of ISGs with 8mer site n=8, 7mer-m8 n= 32, 7mer-A1 n=26 and no seed n= 92. The two-tailed p values were calculated using the Mann-Whitney-Wilcoxon test. ISGs were calculated as all the upregulated genes (≥ 3-fold; padj < 0.05) in two replicates of IFN-induced versus non-induced conditions in all time points.


Figure 6 ( A ) Heat map showing log2 fold change of expression of ISGs in A549 - ACE2 cells across 8 , 16 and 24 h time points upon IFN - α treatment compared to non - treated controls . The common upregulated genes ( ≥ 3 - fold ; padj < 0 . 05 ) in IFN - induced versus non - induced conditions at all time points were categorized as ISGs .

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Figure 6(A) Heat map showing log2 fold change of expression of ISGs in A549-ACE2 cells across 8, 16 and 24 h time points upon IFN-α treatment compared to non-treated controls. The common upregulated genes (≥ 3-fold; padj < 0.05) in IFN-induced versus non-induced conditions at all time points were categorized as ISGs.


Figure 7 ( A ) Expression levels of CoV2 - miR - O7a . 1 , CoV2 - miR - O7a . 2 and a 22 nt region from the ORF6 of the viral genome that does not produce detectable levels of small RNAs ( Control ORF6 ) analyzed by stem - loop RT - qPCR from nasopharyngeal swabs of patients tested positive for COVID - 19 or another seasonal human coronavirus ( HCoV ) . Relative expression to hsa - miR - let - 7a is shown . Ct values in parenthesis refer to the Ct value for the detection of viral genome in patient swab samples .

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Figure 7(A) Expression levels of CoV2-miR-O7a.1, CoV2-miR-O7a.2 and a 22 nt region from the ORF6 of the viral genome that does not produce detectable levels of small RNAs (Control ORF6) analyzed by stem-loop RT-qPCR from nasopharyngeal swabs of patients tested positive for COVID-19 or another seasonal human coronavirus (HCoV). Relative expression to hsa-miR-let-7a is shown. Ct values in parenthesis refer to the Ct value for the detection of viral genome in patient swab samples.


Figure 1C - F Division pattern of seam cells at the postembryonic stage in hermaphrodites . Seam cells V1 - V4 and V6 divide symmetrically at the L2 stage ( highlighted in red ) , thus doubling in number . ( G , H ) dcr ‐ 1 young adults have an increased number of seam cells ( shown in brackets ) and gaps in the alae ( between arrows ) . ( I , J ) atg ‐ 2 mutations suppress the retarded heterochronic defect in dcr ‐ 1mutants . Complete alae ( arrow ) are formed in dcr ‐ 1 ; atg ‐ 2 double mutants . K - M Confocal images showing expression of hbl ‐ 1 : : gfp : : hbl ‐ 1 reporter in VNC in wild type ( K ) , atg ‐ 2 ( L ) and epg ‐ 6 ( M ) L3 larvae . N Distribution of relative fluorescence intensity of HBL ‐ 1 : : GFP in 50 unit areas in VNC in the indicated strains . O - Q Confocal images showing the expression of the col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) reporter in wild type ( O ) , atg ‐ 2 ( P ) and atg ‐ 3 ( Q ) late L4 larvae . R Distribution of relative fluorescence intensity of col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in 50 unit areas of hypodermal cells in the indicated strains . S , T Distribution of relative fluorescence intensity of hbl ‐ 1 : : gfp in VNC and col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in hypodermal cells in 50 unit areas in wild type , let ‐ 363 ( RNAi ) or starvation ‐ treated animals . Black lines in ( N , R - T ) represent the average fluorescence intensity of the reporter in each strain . P values are determined by Student ' s two ‐ tailed unpaired t ‐ test in ( N , R - T ) . ( C , E , G , I ) Scale bar , 20 μm ; ( D , F , H , J - M , O - Q ) scale bar , 10 μm . GFP , green fluorescent protein ; miRISC , miRNA ‐ induced silencing complex ; RNAi , RNA ‐ mediated interference ; UTR , untranslated region ; VNC , ventral nerve cord .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1C-F Division pattern of seam cells at the postembryonic stage in hermaphrodites. Seam cells V1-V4 and V6 divide symmetrically at the L2 stage (highlighted in red), thus doubling in number.(G,H) dcr‐1 young adults have an increased number of seam cells (shown in brackets) and gaps in the alae (between arrows).(I,J) atg‐2 mutations suppress the retarded heterochronic defect in dcr‐1mutants. Complete alae (arrow) are formed in dcr‐1; atg‐2 double mutants.K-M Confocal images showing expression of hbl‐1::gfp::hbl‐1 reporter in VNC in wild type (K), atg‐2 (L) and epg‐6 (M) L3 larvae.N Distribution of relative fluorescence intensity of HBL‐1::GFP in 50 unit areas in VNC in the indicated strains.O-Q Confocal images showing the expression of the col‐10::gfp::lin‐41(3′UTR) reporter in wild type (O), atg‐2 (P) and atg‐3 (Q) late L4 larvae.R Distribution of relative fluorescence intensity of col‐10::gfp::lin‐41(3′UTR) in 50 unit areas of hypodermal cells in the indicated strains.S,T Distribution of relative fluorescence intensity of hbl‐1::gfp in VNC and col‐10::gfp::lin‐41(3′UTR) in hypodermal cells in 50 unit areas in wild type, let‐363(RNAi) or starvation‐treated animals. Black lines in (N,R-T) represent the average fluorescence intensity of the reporter in each strain. P values are determined by Student's two‐tailed unpaired t‐test in (N,R-T). (C,E,G,I) Scale bar, 20 μm; (D,F,H,J-M,O-Q) scale bar, 10  μm. GFP, green fluorescent protein; miRISC, miRNA‐induced silencing complex; RNAi, RNA‐mediated interference; UTR, untranslated region; VNC, ventral nerve cord.


Figure 2A - C Wild ‐ type and atg ‐ 2 hermaphrodites have a single vulva ( arrow ) . ( B ) let ‐ 60 ( n1046gf ) mutants show a Muv phenotype . Black arrows indicate pseudovulvae . D Loss of autophagy activity suppresses the Muv phenotype in let ‐ 60 ( n1046 ) mutants . The number of adult animals examined was : wild type ( n = 450 ) , let ‐ 60 ( n = 406 ) , epg ‐ 6 ; let ‐ 60 ( n = 444 ) , let ‐ 60 ; atg ‐ 2 ( n = 803 ) , epg ‐ 5 ; let ‐ 60 ( n = 672 ) , epg ‐ 5 ; let ‐ 60 ; ain ‐ 1 ( ku322 ) ( n = 635 ) . P values are determined by a two ‐ tailed χ2 test . E , F lsy ‐ 6 mutants show defects in ASEL fate specification and do not express the ASEL ‐ specific reporter lim ‐ 6pro : : GFP . G Expression of lim ‐ 6pro : : GFP in various strains . aCompared with lsy ‐ 6 ( ot150 ) . bCompared with lsy ‐ 6 ( ot150 ) ; ain ‐ 1 ( ku322 ) . P values are determined by a two ‐ tailed χ2 test .

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

Figure 2A-C Wild‐type and atg‐2 hermaphrodites have a single vulva (arrow). (B) let‐60(n1046gf) mutants show a Muv phenotype. Black arrows indicate pseudovulvae.D Loss of autophagy activity suppresses the Muv phenotype in let‐60(n1046) mutants. The number of adult animals examined was: wild type (n=450), let‐60 (n=406), epg‐6; let‐60 (n=444), let‐60; atg‐2 (n=803), epg‐5; let‐60 (n=672), epg‐5; let‐60; ain‐1(ku322) (n=635). P values are determined by a two‐tailed χ2 test.E,F lsy‐6 mutants show defects in ASEL fate specification and do not express the ASEL‐specific reporter lim‐6pro::GFP.G Expression of lim‐6pro::GFP in various strains. aCompared with lsy‐6(ot150).bCompared with lsy‐6(ot150); ain‐1(ku322). P values are determined by a two‐tailed χ2 test.


Figure 3AIN ‐ 1 : : GFP is weakly and diffusely expressed in the cytoplasm during embryogenesis in wild ‐ type animals . ( A ) Nomarski image of the embryo shown in ( B ) . Scale bar , 10 μm for whole embryo . C . elegans embryos remain the same size during embryogenesis and loss of autophagy activity has no effect on the embryo size . Thus , scale bars for embryos are only shown once in each figure . ( C ) AIN ‐ 1 : : GFP forms a large number of aggregates in epg ‐ 6 mutant embryos . ( A - C ) are confocal images . ( D ) Western blotting shows that endogenous AIN ‐ 1 protein levels are dramatically elevated in autophagy mutant embryos compared with wild type . ( E ) No AIN ‐ 1 : : GFP aggregates are formed in sqst ‐ 1 mutant embryos . ( F ) AIN ‐ 1 : : GFP accumulates into numerous aggregates in epg ‐ 7 mutant embryos . ( G , H ) Number of AIN ‐ 1 : : GFP ( G ) and ALG ‐ 2 : : GFP ( H ) aggregates per focal plane in the indicated strains . Error bars indicate the s . d . of four examined embryos . ( I , J ) ALG ‐ 2 : : GFP is diffusely localized in the cytoplasm in wild ‐ type embryos . ( I ) Nomarski image of the embryo shown in ( J ) . ( K ) ALG ‐ 2 : : GFP forms a few aggregates in epg ‐ 6 mutant embryos . ( L ) ALG ‐ 2 : : GFP forms a large number of aggregates in epg ‐ 6 mutant embryos under stress conditions . GFP , green fluorescent protein ; s . d . , standard deviation .

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

Figure 3AIN‐1::GFP is weakly and diffusely expressed in the cytoplasm during embryogenesis in wild‐type animals. (A) Nomarski image of the embryo shown in (B). Scale bar, 10 μm for whole embryo. C. elegans embryos remain the same size during embryogenesis and loss of autophagy activity has no effect on the embryo size. Thus, scale bars for embryos are only shown once in each figure. (C) AIN‐1::GFP forms a large number of aggregates in epg‐6 mutant embryos. (A-C) are confocal images.(D) Western blotting shows that endogenous AIN‐1 protein levels are dramatically elevated in autophagy mutant embryos compared with wild type.(E) No AIN‐1::GFP aggregates are formed in sqst‐1 mutant embryos.(F) AIN‐1::GFP accumulates into numerous aggregates in epg‐7 mutant embryos.(G,H) Number of AIN‐1::GFP (G) and ALG‐2::GFP (H) aggregates per focal plane in the indicated strains. Error bars indicate the s.d. of four examined embryos.(I,J) ALG‐2::GFP is diffusely localized in the cytoplasm in wild‐type embryos. (I) Nomarski image of the embryo shown in (J).(K) ALG‐2::GFP forms a few aggregates in epg‐6 mutant embryos. (L) ALG‐2::GFP forms a large number of aggregates in epg‐6 mutant embryos under stress conditions. GFP, green fluorescent protein; s.d., standard deviation.


Figure 4 ( A - D ) Both AIN ‐ 1 : : GFP and SQST ‐ 1 , detected by anti ‐ SQST ‐ 1 antibody , are very weakly expressed and diffusely localized in wild ‐ type embryos . ( E - H ) AIN ‐ 1 : : GFP aggregates colocalize with SQST ‐ 1 aggregates in atg ‐ 3 mutant embryos . Scale bars , 10 μm for insets that show a magnified view . ( I - L ) AIN ‐ 1 : : GFP aggregates are separable from PGL granules , detected by anti ‐ SEPA ‐ 1 , in atg ‐ 3 mutant embryos . ( M - P ) Colocalization of AIN ‐ 1 : : GFP aggregates with LGG ‐ 1 puncta in epg ‐ 6 mutant embryos . ( Q ) % of AIN ‐ 1 : : GFP aggregates colocalized with SQST ‐ 1 aggregates , SEPA ‐ 1 aggregates and LGG ‐ 1 puncta in autophagy mutants . Error bars indicate the s . d . of four examined embryos . ( R ) A co ‐ IP assay shows that AIN ‐ 1 associates with LGG ‐ 1 in vivo . ( S ) Direct interaction between AIN ‐ 1 and LGG ‐ 1 in an in vitro pull ‐ down assay . 5 % of the His fusion proteins used for pull ‐ down are shown as input . Corresponding Ponceau ‐ stained blots show the amount of proteins used for pull ‐ down . ( T ) A fragment of AIN ‐ 1 directly binds to LGG ‐ 1 in an in vitro pull ‐ down assay .

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

Figure 4(A-D) Both AIN‐1::GFP and SQST‐1, detected by anti‐SQST‐1 antibody, are very weakly expressed and diffusely localized in wild‐type embryos.(E-H) AIN‐1::GFP aggregates colocalize with SQST‐1 aggregates in atg‐3 mutant embryos. Scale bars, 10 μm for insets that show a magnified view.(I-L) AIN‐1::GFP aggregates are separable from PGL granules, detected by anti‐SEPA‐1, in atg‐3 mutant embryos.(M-P) Colocalization of AIN‐1::GFP aggregates with LGG‐1 puncta in epg‐6 mutant embryos.(Q) % of AIN‐1::GFP aggregates colocalized with SQST‐1 aggregates, SEPA‐1 aggregates and LGG‐1 puncta in autophagy mutants. Error bars indicate the s.d. of four examined embryos.(R) A co‐IP assay shows that AIN‐1 associates with LGG‐1 in vivo.(S) Direct interaction between AIN‐1 and LGG‐1 in an in vitro pull‐down assay. 5% of the His fusion proteins used for pull‐down are shown as input. Corresponding Ponceau‐stained blots show the amount of proteins used for pull‐down.(T) A fragment of AIN‐1 directly binds to LGG‐1 in an in vitro pull‐down assay.


Figure 1 ( B ) Representative images of GFAP + Sox2 + BrdU + RGLs , GFAP - Sox2 + BrdU + progenitors , Tbr2 + BrdU + IPCs in the DG . Scale bar = 20 μm . ( C ) Quantification of GFAP + Sox2 + BrdU + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 022 ) , GFAP - Sox2 + BrdU + progenitors ( WT vs . KO , Student ' s t - test , p = 0 . 0046 ) , Tbr2 + BrdU + IPCs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) in the DG of WT and KO mice . WT , n = 4 mice ; KO , n = 6 mice . ( D ) Representative images of GFAP + Mcm2 + RGLs , Tbr2 + Mcm2 + IPCs , DCX + Mcm2 + NBs in the DG . Scale bar = 20 μm . ( E ) Quantification of GFAP + Mcm2 + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 016 ) , Tbr2 + Mcm2 + IPCs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) , DCX + Mcm2 + NBs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) in the DG of WT and KO mice . WT , n = 3 mice ; KO , n = 4 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

Figure 1(B) Representative images of GFAP+Sox2+BrdU+ RGLs, GFAP-Sox2+BrdU+ progenitors, Tbr2+BrdU+ IPCs in the DG. Scale bar = 20 μm. (C) Quantification of GFAP+Sox2+BrdU+ RGLs (WT vs. KO, Student's t-test, p = 0.022), GFAP-Sox2+BrdU+ progenitors (WT vs. KO, Student's t-test, p = 0.0046), Tbr2+BrdU+ IPCs (WT vs. KO, Student's t-test, p = 0.003) in the DG of WT and KO mice. WT, n = 4 mice; KO, n = 6 mice. (D) Representative images of GFAP+Mcm2+ RGLs, Tbr2+Mcm2+ IPCs, DCX+Mcm2+ NBs in the DG. Scale bar = 20 μm. (E) Quantification of GFAP+Mcm2+ RGLs (WT vs. KO, Student's t-test, p = 0.016), Tbr2+Mcm2+ IPCs (WT vs. KO, Student's t-test, p = 0.003), DCX+Mcm2+ NBs (WT vs. KO, Student's t-test, p = 0.003) in the DG of WT and KO mice. WT, n = 3 mice; KO, n = 4 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


Figure 2 ( B ) Representative images used for fate mapping of tdT + cells in the DG by co - stained with GFAP ( RGL ) , Tbr2 ( IPC ) , DCX ( IN ) , NeuN ( MN ) . Scale bar = 20 μm . ( C ) Quantification of composition of tdT + cells ( RGL : WT vs . KO , Student ' s t - test , p = 0 . 026 ; IPC : WT vs . KO , Student ' s t - test , p = 0 . 028 ; IN : WT vs . KO , Student ' s t - test , p = 0 . 049 ; N : WT vs . KO , Student ' s t - test , p = 0 . 048 ) in the DG of WT and cKO mice . WT , n = 3 mice ; cKO , n = 3 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 ( H ) Representative images of BrdU - retaining RGLs ( tdT + BrdU + ) in the DG . Scale bar = 20 μm . ( I ) Quantification of BrdU - retaining RGLs ( BrdU + tdT + GFAP + , WT vs . cKO , Student ' s t - test , p = 0 . 009 ) in the DG of WT and cKO mice . WT , n = 4 mice ; cKO , n = 3 mice .

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

Figure 2(B) Representative images used for fate mapping of tdT+ cells in the DG by co-stained with GFAP (RGL), Tbr2 (IPC), DCX (IN), NeuN (MN). Scale bar = 20 μm.(C) Quantification of composition of tdT+ cells (RGL: WT vs. KO, Student's t-test, p = 0.026; IPC: WT vs. KO, Student's t-test, p = 0.028; IN: WT vs. KO, Student's t-test, p = 0.049; N: WT vs. KO, Student's t-test, p = 0.048) in the DG of WT and cKO mice. WT, n = 3 mice; cKO, n = 3 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01(H) Representative images of BrdU-retaining RGLs (tdT+BrdU+) in the DG. Scale bar = 20 μm. (I) Quantification of BrdU-retaining RGLs (BrdU+tdT+GFAP+, WT vs. cKO, Student's t-test, p = 0.009) in the DG of WT and cKO mice. WT, n = 4 mice; cKO, n = 3 mice.


Figure 3 ( B ) Representative images of Nestin - GFP + GFAP + Mcm2 + RGLs in the DG . Scale bar = 50 μm . ( C ) Quantification of GFAP + Nestin - GFP + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 001 ) in the DG of WT and KO mice at 6 months of age . WT , n = 5 mice ; KO , n = 4 mice . ( D ) Percentage of GFAP + Nestin - GFP + Mcm2 + activated RGLs among total GFAP + Nestin - GFP + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 0013 ) in the DG of WT and KO mice at 6 months of age . WT , n = 5 mice ; KO , n = 4 mice . Data information : Data are presented as the mean ± SEM . * * , p < 0 . 01 , * * * , p < 0 . 001 . ( I ) Representative images of NeuN + BrdU + adult - born neurons in the DG of 6 month - old mice . Scale bar = 50 μm . ( J ) Quantification of NeuN + BrdU + adult - born neurons ( WT vs . KO , Student ' s t - test , p = 0 . 0052 ) in the DG of WT and KO mice at 7 months of age . WT , n = 4 mice ; KO , n = 3 mice . Data information : Data are presented as the mean ± SEM . * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

Figure 3(B) Representative images of Nestin-GFP+GFAP+Mcm2+ RGLs in the DG. Scale bar = 50 μm. (C) Quantification of GFAP+Nestin-GFP+ RGLs (WT vs. KO, Student's t-test, p = 0.001) in the DG of WT and KO mice at 6 months of age. WT, n = 5 mice; KO, n = 4 mice. (D) Percentage of GFAP+Nestin-GFP+Mcm2+ activated RGLs among total GFAP+Nestin-GFP+ RGLs (WT vs. KO, Student's t-test, p = 0.0013) in the DG of WT and KO mice at 6 months of age. WT, n = 5 mice; KO, n = 4 mice. Data information: Data are presented as the mean ± SEM. **, p < 0.01, ***, p < 0.001.(I) Representative images of NeuN+BrdU+ adult-born neurons in the DG of 6 month-old mice. Scale bar = 50 μm. (J) Quantification of NeuN+BrdU+ adult-born neurons (WT vs. KO, Student's t-test, p = 0.0052) in the DG of WT and KO mice at 7 months of age. WT, n = 4 mice; KO, n = 3 mice. Data information: Data are presented as the mean ± SEM. **, p < 0.01, ***, p < 0.001.


Figure 4 ( A ) Representative images of aNPCs in vitro isolated from WT and KO mice and followed by a BrdU pulse labeling . Scale bar = 50 μm . ( B ) Percentage of BrdU + aNPCs among total aNPCs isolated from WT or KO mice ( WT vs . KO , Student ' s t - test , p = 0 . 0075 ) . WT , n = 3 independent experiments ; KO , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

Figure 4(A) Representative images of aNPCs in vitro isolated from WT and KO mice and followed by a BrdU pulse labeling. Scale bar = 50 μm. (B) Percentage of BrdU+ aNPCs among total aNPCs isolated from WT or KO mice (WT vs. KO, Student's t-test, p = 0.0075). WT, n = 3 independent experiments; KO, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; **, p < 0.01, ***, p < 0.001.


Figure 5 ( A ) DARTS analysis indicated HK1 as a molecular target of L - arginine in aNPCs . The aNPC lysates treated with L - arginine , digested with pronase and then analyzed by Western blot with anti - HK1 and GAPDH antibodies . ( B ) Quantification of HK1 expression level in aNPCs lysates treated with L - arginine and followed by digesting with pronase ( One - way ANOVA , F ( 3 , 8 ) = 3 . 61 , p = 0 . 043 ) . n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 . ( I ) Co - IP of HK1 with VDAC1 in vehicle or L - arginine - treated cells . ( J ) Quantification of the co - IP efficacy of HK1 and VDAC interaction in vehicle or L - arginine - treated cells ( IP : HK1 , vehicle vs . L - arginine , Student ' s t - test , p < 0 . 001 ; IP : VDAC1 , vehicle vs . L - arginine , Student ' s t - test , p = 0 . 0078 ) . Vehicle , n = 3 independent experiments ; L - arginine , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

Figure 5(A) DARTS analysis indicated HK1 as a molecular target of L-arginine in aNPCs. The aNPC lysates treated with L-arginine, digested with pronase and then analyzed by Western blot with anti-HK1 and GAPDH antibodies. (B) Quantification of HK1 expression level in aNPCs lysates treated with L-arginine and followed by digesting with pronase (One-way ANOVA, F(3, 8)= 3.61, p = 0.043). n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.(I) Co-IP of HK1 with VDAC1 in vehicle or L-arginine-treated cells. (J) Quantification of the co-IP efficacy of HK1 and VDAC interaction in vehicle or L-arginine-treated cells (IP: HK1, vehicle vs. L-arginine, Student's t-test, p < 0.001; IP: VDAC1, vehicle vs. L-arginine, Student's t-test, p = 0.0078). Vehicle, n = 3 independent experiments; L-arginine, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


Figure 6 ( C - E ) Quantification of glucose ( C , WT vs . KO , Student ' s t - test , p < 0 . 001 ) and glutamine ( E , WT vs . KO , Student ' s t - test , p = 0 . 94 ) consumption and lactate production ( D , WT vs . KO , Student ' s t - test , p = 0 . 019 ) in WT - and KO - aNPCs . WT , n = 3 independent experiments ; KO , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 * * * , p < 0 . 001 . ( J ) Representative profile of mitochondria stress test showing the OCR of WT - or KO - aNPCs . WT , n = 4 independent experiments , KO , n = 4 independent experiments .

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

Figure 6(C-E) Quantification of glucose (C, WT vs. KO, Student's t-test, p < 0.001) and glutamine (E, WT vs. KO, Student's t-test, p = 0.94) consumption and lactate production (D, WT vs. KO, Student's t-test, p = 0.019) in WT- and KO-aNPCs. WT, n = 3 independent experiments; KO, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05 ***, p < 0.001.(J) Representative profile of mitochondria stress test showing the OCR of WT- or KO-aNPCs. WT, n = 4 independent experiments, KO, n = 4 independent experiments.


Figure 7 ( C ) Quantification of Nestin - GFP + GFAP + RGLs in the DG of WT and KO mice after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 99 ) . WT + Vehicle , n = 4 mice ; WT + IACS , n = 4 mice ; KO + Vehicle , n = 4 mice , KO + IACS , n = 3 mice . ( D ) Quantification of Nestin - GFP + GFAP + BrdU + activated RGLs in the DG of WT and KO mice after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p < 0 . 001 ) . WT + Vehicle , n = 3 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 3 mice , KO + IACS , n = 3 mice . ( E ) Quantification of Nestin - GFP + GFAP - BrdU + progenitors ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 028 ) in the DG of WT and KO mice after vehicle or IACS treatment . WT + Vehicle , n = 3 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 3 mice , KO + IACS , n = 3 mice . ( F ) Quantification of Nestin - GFP + Tbr2 + BrdU + IPCs ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 02 ) in the DG of WT and KO mice after vehicle or IACS treatment . WT + Vehicle , n = 4 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 4 mice , KO + IACS , n = 4 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 . ( H ) Representative images of NeuN + BrdU + adult - born neurons in the DG . Scale bar = 20 μm . ( I ) Quantification of NeuN + BrdU + adult - born neurons in the DG of WT and KO mice at P90 after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p < 0 . 001 ) . WT + Vehicle , n = 5 mice ; WT + IACS , n = 5 mice ; KO + Vehicle , n = 5 mice , KO + IACS , n = 5 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

Figure 7(C) Quantification of Nestin-GFP+GFAP+ RGLs in the DG of WT and KO mice after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.99). WT + Vehicle, n = 4 mice; WT + IACS, n = 4 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 3 mice. (D) Quantification of Nestin-GFP+GFAP+BrdU+ activated RGLs in the DG of WT and KO mice after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice. (E) Quantification of Nestin-GFP+GFAP-BrdU+ progenitors (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.028) in the DG of WT and KO mice after vehicle or IACS treatment. WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice. (F) Quantification of Nestin-GFP+Tbr2+BrdU+ IPCs (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.02) in the DG of WT and KO mice after vehicle or IACS treatment. WT + Vehicle, n = 4 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 4 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.(H) Representative images of NeuN+BrdU+ adult-born neurons in the DG. Scale bar = 20 μm. (I) Quantification of NeuN+BrdU+ adult-born neurons in the DG of WT and KO mice at P90 after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 5 mice; WT + IACS, n = 5 mice; KO + Vehicle, n = 5 mice, KO + IACS, n = 5 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


Figure 2 ( a ) Species relative abundance as a function of time for all pairwise communities . Experimental measurements and model fits based on T3 are represented as data points and lines , respectively . In each subplot , time and species relative abundance are displayed on the x and y - axis , respectively . Stars denote datasets with a sum of mean squared errors greater than 0 . 15 . Error bars represent 1 s . d . from the mean of at least three biological replicates . ( b ) Temporal changes in species relative abundance of a selected set of pairwise assemblages inoculated at 5 % species A , 95 % species B or 95 % species A , 5 % species B based on OD600 values . Time and relative abundance are represented on the x and y - axis , respectively . Data points and lines represent experimental measurements and model fits to T3 , respectively . Error bars represent 1 s . d . from the mean of at least three biological replicates . Stars denote datasets with a sum of mean squared errors greater than 0 . 15 . ( c ) Inferred inter - species interaction coefficients for the gLV model trained on T3 . Gray and green edges denote negative ( αij < 0 ) and positive ( αij > 0 ) interaction coefficients . The edge width and node size represent the magnitude of the inter - species interaction coefficient and steady - state monospecies abundance ( xe = - μiαii - 1 ) , respectively . To highlight significant interactions , inter - species interaction coefficients with a magnitude less than 1e - 5 were not displayed .

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

Figure 2(a) Species relative abundance as a function of time for all pairwise communities. Experimental measurements and model fits based on T3 are represented as data points and lines, respectively. In each subplot, time and species relative abundance are displayed on the x and y-axis, respectively. Stars denote datasets with a sum of mean squared errors greater than 0.15. Error bars represent 1 s.d. from the mean of at least three biological replicates. (b) Temporal changes in species relative abundance of a selected set of pairwise assemblages inoculated at 5% species A, 95% species B or 95% species A, 5% species B based on OD600 values. Time and relative abundance are represented on the x and y-axis, respectively. Data points and lines represent experimental measurements and model fits to T3, respectively. Error bars represent 1 s.d. from the mean of at least three biological replicates. Stars denote datasets with a sum of mean squared errors greater than 0.15. (c) Inferred inter-species interaction coefficients for the gLV model trained on T3. Gray and green edges denote negative (αij < 0) and positive (αij > 0) interaction coefficients. The edge width and node size represent the magnitude of the inter-species interaction coefficient and steady-state monospecies abundance (xe = -μiαii-1), respectively. To highlight significant interactions, inter-species interaction coefficients with a magnitude less than 1e-5 were not displayed.


Figure 3 ( a ) Stacked bar plots of all 11 and 12 - member ( full ) multi - species communities . The text above each subplot denotes the absent organism in the community . Time and relative abundance are represented on the x and y - axis of each subplot , respectively . Colors represent different organisms in the community . ( b ) Difference in community assembly for single - species dropout communities . Difference in community assembly was computed as $ \ sum _ { i = 1 , \ } ^ { 11 } { \ frac { 1 } { 7 } \ sum _ { j = 1 } ^ { 7 } { ( { \ widehat { v } } _ { ji , FULL } - v _ { ji , X } ) } } ^ { 2 } $ where i and j represent species and time points , respectively . X represents a single - species dropout community lacking the organism X . v ̂ denotes the renormalized relative abundance of the shared set of species in the full ( 12 - member ) and single - species dropout community . Here $ { \ widehat { v } } _ { i } = \ \ frac { v _ { i } } { \ sum _ { i \ \ in C } ^ { } v _ { i } } $ where C denotes the set of 11 shared organisms in the full and single - species dropout community . Data points represent biological replicates ( n = 6 , circles ) and the mean of biological replicates ( blue squares ) , respectively . ( c ) Box plot of the Pearson correlation coefficient for three model training sets ( left ) including T1 : monospecies ( M ) ; T2 : M and pairwise 1 ( PW1 ) ; and T3 : M , PW1 and pairwise 2 ( PW2 ) . On each box , the red line represents the median , the edges of the box are the 25th and 75th percentiles , the whiskers extend to the most extreme data points and the outliers are plotted as red crosses . ( d ) Box plot of the sum of mean squared errors for the model predictions of the time - resolved measurements of multi - species communities trained on T1 , T2 or T3 training sets . On each box , the red line represents the median , the edges of the box are the 25th and 75th percentiles , the whiskers extend to the most extreme data points and the outliers are plotted as red crosses . ( e ) Comparison between model prediction based on T1 - T4 and experimental data for each organism in the full community as a function of time . On each subplot , the x and y - axis represent time and relative abundance of each species , respectively . Data points ( red ) and lines denote experimental data and model predictions based on T1 - T4 . Species names are displayed in the upper left corner of each subplot . Error bars represent 1 s . d . from the mean of six biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stacked bar plots of all 11 and 12-member (full) multi-species communities. The text above each subplot denotes the absent organism in the community. Time and relative abundance are represented on the x and y-axis of each subplot, respectively. Colors represent different organisms in the community. (b) Difference in community assembly for single-species dropout communities. Difference in community assembly was computed as $\sum_{i = 1,\ }^{11}{\frac{1}{7}\sum_{j = 1}^{7}{({\widehat{v}}_{ji,FULL} - v_{ji,X})}}^{2}$ where i and j represent species and time points, respectively. X represents a single-species dropout community lacking the organism X. v̂ denotes the renormalized relative abundance of the shared set of species in the full (12-member) and single-species dropout community. Here ${\widehat{v}}_{i} = \ \frac{v_{i}}{\sum_{i\ \in C}^{}v_{i}}$ where C denotes the set of 11 shared organisms in the full and single-species dropout community. Data points represent biological replicates (n = 6, circles) and the mean of biological replicates (blue squares), respectively. (c) Box plot of the Pearson correlation coefficient for three model training sets (left) including T1: monospecies (M); T2: M and pairwise 1 (PW1); and T3: M, PW1 and pairwise 2 (PW2). On each box, the red line represents the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points and the outliers are plotted as red crosses. (d) Box plot of the sum of mean squared errors for the model predictions of the time-resolved measurements of multi-species communities trained on T1, T2 or T3 training sets. On each box, the red line represents the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points and the outliers are plotted as red crosses. (e) Comparison between model prediction based on T1-T4 and experimental data for each organism in the full community as a function of time. On each subplot, the x and y-axis represent time and relative abundance of each species, respectively. Data points (red) and lines denote experimental data and model predictions based on T1-T4. Species names are displayed in the upper left corner of each subplot. Error bars represent 1 s.d. from the mean of six biological replicates.


Figure 5 ( a ) Bipartite network of species ( left ) and metabolites ( right ) for metabolites that decreased by at least two - fold compared to the abundance of each metabolite at the beginning of the experiment . Colors represent modules containing many overlapping interactions in the network . Network partitioning into modules was performed using BiMat ( Flores Metabolites in bold were depleted and secreted by distinct organisms . ( b ) Bipartite network of species ( left ) and metabolites ( right ) for metabolites that increased in abundance by at least two - fold compared to the beginning of the experiment . Metabolites highlighted in bold were depleted or secreted by different species . Colors represent modules containing many overlapping interactions in the network . Network partitioning into modules was performed using BiMat ( Flores ( c ) Scatter plot of the number of consumed metabolites that decreased by at least two - fold compared to the beginning of the experiment vs . the OD600 value of the monospecies culture at the corresponding time point . Error bars represent 1 s . d . from the mean of three biological replicates .

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

Figure 5(a) Bipartite network of species (left) and metabolites (right) for metabolites that decreased by at least two-fold compared to the abundance of each metabolite at the beginning of the experiment. Colors represent modules containing many overlapping interactions in the network. Network partitioning into modules was performed using BiMat(Flores Metabolites in bold were depleted and secreted by distinct organisms. (b) Bipartite network of species (left) and metabolites (right) for metabolites that increased in abundance by at least two-fold compared to the beginning of the experiment. Metabolites highlighted in bold were depleted or secreted by different species. Colors represent modules containing many overlapping interactions in the network. Network partitioning into modules was performed using BiMat(Flores(c) Scatter plot of the number of consumed metabolites that decreased by at least two-fold compared to the beginning of the experiment vs. the OD600 value of the monospecies culture at the corresponding time point. Error bars represent 1 s.d. from the mean of three biological replicates.


Figure 1RNA - Seq analysis of : different time points of primary CD4 + T - cells infected in vitro with HIV - 1 or mock infected Expression levels were normalized as Log2 fold change in infected vs matched mock - infected controls For ( A ) data were analyzed by Fisher test ( number of donors = 3 biological replicates ) . Boxplots in ( A ) depict median and 25 - 75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1 . 5 * IQR ( inter - quartile range ) of the hinge . Data beyond the end of the whiskers are outliers and plotted as points . For each time point , dots illustrate the pathway enrichment analysis of genes up - regulated in infected vs matched mock - infected controls . Dots are color - coded based on the enrichment q - values and their size indicates the fraction of differentially expressed genes ( DEGs ) in the pathway ; grey dots are not statistically significantRNA - Seq analysis of PBMCs of macaques infected with SIVmac239 before and after suppression of viremia with ART Expression levels were normalized as Log2 fold change or as enrichment score in ART - treated vs ART - naïve animals ( B ) . ( B ) data were analyzed by Gene Set Enrichment Analysis ( GSEA , number of animals = 8 ) . Symbols in the GSEA enrichment plots represent the position of the gene set members in the transcriptome ranked by differential expression between ART - naïve and ART - treated time points . Red color indicates leading edge genes . All analyses were conducted on the genes of the pathway GO cellular response to oxidative stress ( 184 genes ; GO : 0034599 ) .

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 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

Figure 1RNA-Seq analysis of: different time points of primary CD4+ T-cells infected in vitro with HIV-1 or mock infected Expression levels were normalized as Log2 fold change in infected vs matched mock-infected controls For (A) data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in (A) depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots illustrate the pathway enrichment analysis of genes up-regulated in infected vs matched mock-infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed genes (DEGs) in the pathway; grey dots are not statistically significantRNA-Seq analysis of PBMCs of macaques infected with SIVmac239 before and after suppression of viremia with ART Expression levels were normalized as Log2 fold change or as enrichment score in ART-treated vs ART-naïve animals (B). (B) data were analyzed by Gene Set Enrichment Analysis (GSEA, number of animals =8). Symbols in the GSEA enrichment plots represent the position of the gene set members in the transcriptome ranked by differential expression between ART-naïve and ART-treated time points. Red color indicates leading edge genes. All analyses were conducted on the genes of the pathway GO cellular response to oxidative stress (184 genes; GO:0034599).