EMBO/soda-nel huggingface dataset:
RBPJ/CBF1 interacts with L3MBTL3/MBT1 to promote repression of Notch signaling via histone demethylase KDM1A/LSD1 (2017)
Figure 5

Figure 5. L3MBTL3 recruits KDM1A at RBPJ-bound Notch-responsive elements to repress Notch target genes.

A The RBPJ/KDM1A interaction is indirect and occurs via L3MBTL3. IP of HA-KDM1A in the presence of overexpressed V5-L3MBTL3 or V5-L3MBTL3-Δ(1-64) in L3MBTL3 KO U87-MG cells. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to overexpress the L3MBTL3 proteins. The experiment was independently replicated twice.

B KDM1A occupancy at the proximal Notch-responsive elements of Notch target genes is L3MBTL3-dependent. ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of duplicate experiments measured twice each.

C KDM1A occupancy at the proximal Notch-responsive elements of Notch target genes is dependent on L3MBTL3 and both its RBPJ interaction and KDM1A interaction domains. ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87-MG cells upon overexpression of L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM). Control: empty vector. Shown are means ±s.d. of duplicate experiments measured twice each.

D L3MBTL3, but neither L3MBTL3-Δ(1-64) nor L3MBTL3-Δ(SAM), leads to decreasing H3K4me2 at the proximal Notch-responsive element of HES1. ChIP analysis of H3K4me2 at the proximal Notch-responsive element of HES1 upon overexpression of LacZ control (Control), L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM) in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of duplicate experiments measured twice each. P values were estimated via a one-way ANOVA on log-transformed data.

E L3MBTL3, but neither L3MBTL3-Δ(1-64) nor L3MBTL3-Δ(SAM), represses HES1. Expression analysis of HES1 upon overexpression of LacZ control (Control), L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM) mutants in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of triplicate experiments. P values were estimated via a one-way ANOVA on log-transformed data. NS: Not Significant. WB: Western blot; IP: immuno-precipitation. We note that in the context of this experiment, i.e., in the absence of endogenous L3MBTL3, the overexpression of L3MBTL3-Δ(1-64) does not result in the increased expression of HES1, contrasting with the result obtained in Fig 4G, i.e., in the presence of endogenous L3MBTL3. Indeed, as expected, the dominant negative effect of L3MBTL3-(1-64) on endogenous WT L3MBTL3's ability to repress the expression of Notch target genes can only be observed when WT L3MBTL3 is expressed.

Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).
RBPJ/CBF1 interacts with L3MBTL3/MBT1 to promote repression of Notch signaling via histone demethylase KDM1A/LSD1 (2017)
Figure 6

Figure 6. The interaction between RBPJ/Su(H)/lag-1 and L3MBTL3/dL(3)mbt/lin-61 is evolutionarily conserved.

A GST pulldown showing that dL(3)mbt, the Drosophila homolog of L3MBTL3, directly interacts with Su(H), the Drosophila homolog of RBPJ. In vitro transcribed and translated dL(3)mbt or dNotch ICD (dNotch ICD fragment containing the RAM domain and ANK repeats), as positive control, was incubated with bacterially purified GST-Su(H) or GST alone pre-bound to GSH beads. Proteins were resolved via SDS-PAGE and signals were acquired via X-ray exposure. The experiment was independently replicated four times.

B dL(3)mbt and Su(H) co-localize genome-wide. Venn diagram showing the genome-wide co-localization of dL(3)mbt and Su(H).

C Snapshot showing the co-localization of dL(3)mbt and Su(H) at the dNotch (N) locus.

D In the wing imaginal disc, dL(3)mbt overexpression in the dorso-ventral (D-V) boundary results in the downregulation of the Notch target gene cut. Wing discs expressing UAS-GFP (top panels) or UAS-HA-dL(3)mbt;UAS-GFP (bottom panels) under the vg-Gal4 driver at 25°C were stained for cut and HA. GFP marks the vg-Gal4 expression domain. Insets below each panel show a closer view of the D-V boundary with yellow arrows marking the regions where HA-dL(3)mbt is expressed and cut is downregulated. At least 20 discs for each genotype were analyzed. Representative images are shown. Scale bars: 100μm.

E The vg-Gal4-driven HA-dL(3)mbt overexpression causes a serrated wing (wing notching) phenotype. Flies expressing either UAS-GFP or UAS-HA-dL(3)mbt;UAS-GFP under vg-Gal4 were reared to adulthood at 25°C. P values were estimated by comparing the proportions via a two-proportion Z-test. Scale bars: 200μm.

F Functional interaction between lag-1/RBPJ and lin-61/L3MBTL3 during C. elegans vulva development. Proportion of animals (n ≥100) displaying a protruding vulva (Pvl) phenotype after RNAi treatment for two generations. Worms were grown at 25°C. Shown are means ±s.d of duplicate experiments. EV: Empty Vector control.
Genome‐wide siRNA screen reveals amino acid starvation‐induced autophagy requires SCOC and WAC (2012)
Figure 1

Genome‐wide screen for starvation‐induced autophagy. (A) Cellomics images (top) of GFP–LC3‐HEK cells in full medium (FM) or after 2 h in starvation medium (ES) with leupeptin (EL). Scale bars=40 μm. Optimised automated analysis (bottom left) programme detecting hoechst‐labelled nuclei (purple line), cell outline (red line) and identifies GFP–LC3 spots (red spots) in FM and EL. Enlarged GFP–LC3 images in FM and EL (bottom right). Scale bars=10 μm. (B) SCPO, STIPO and STAPO after indicated siRNA treatment of GFP–LC3‐HEK cells incubated as in (A). Error bars represent s.e.m. Significance was determined using a two‐tailed paired t‐test compared with RISCfree (RF) in EL: SCPO siULK1, ***P=0.0002; siATG7, **P=0.0078; siNRBP2, **P=0.0088. STIPO siULK1, ***P0.0001; siATG7, **P=0.0060; siNRBP2, **P=0.0041. STAPO siULK1, ***P0.0001; siATG7, **P=0.0037; siNRBP2, **P=0.0028. The experiments were performed: RISCfree (n=5), siULK1 (n=5), siATG7 (n=3), siNRBP2 (n=3). (C) Overview of screening strategy. (D) Scatter plot of median SCPO Z‐scores from siRNA pools in the primary screen that met the minimum object count (OC) cutoff. Dashed lines represent approximate cutoff for 500 best increasers and decreasers. RP of known autophagy genes (ATG4C, ATG14, RB1CC1, ATG9A, ATG7) are given in Supplementary Table S2; AMBRA1 (FLJ20294), TBC1D25 (OATL1) and SH3GLB1 (BIF‐1) in Supplementary Table S1; LAMTOR2 has a RP=708.7 and RUBICON has a RP=1126.
Genome‐wide siRNA screen reveals amino acid starvation‐induced autophagy requires SCOC and WAC (2012)
Figure 2

In all, 51 hits including SCOC and WAC are validated by deconvolution and additional assays. (A) SCPO, STIPO and STAPO for the 51 genes validated by two out of four individual siRNA duplexes reproducing the GFP–LC3 spot phenotype, above a cutoff of greater or less than 20% of control, and passing additional criteria (Supplementary Table 3). Red boxes indicate an increase >1.2; pink boxes an increase between 1.1 and 1.2; white boxes are between 0.9 and 1.1; light blue boxes a decrease between 0.9 and 0.8; dark blue boxes a decrease to 0.8. Hits underlined were taken forward as three out of four4 hits, (§) not chosen because of restricted tissue expression, (*) are three out of four when alternate method of normalisation is used. (B) Table summarising results from further validation of 20 genes. mRNA levels scored as (+) or (−) indicate efficient or poor knockdown, respectively (see Supplementary Figures S1 and S2). Increasers (left) or decreasers (right) assayed for LC3 lipidation were scored (+) for yes, (−) for no, (+/−) subtle change and (nd) not done (Supplementary Figure S3). (C–E) STIPO results after incubation under different autophagy‐inducing conditions following (C) SCOC knockdown (D) SUPT5H knockdown and (E) WAC knockdown. siRNA‐treated cells were incubated for the indicated time in FL: full medium plus leupeptin; ES; EL: ES plus leupeptin; Torin: 250 nM Torin1 in FL; Resv: 128 μM Resveratrol in FL; LiCl: 10 mM LiCl in FM.
Genome‐wide siRNA screen reveals amino acid starvation‐induced autophagy requires SCOC and WAC (2012)
Figure 3

Localisation of SCOC with the Golgi, Atg9 and LC3. (A) Schematic of SCOC constructs. Numbers represent amino acids; CC is coiled‐coil domain. SCOC–myc was constructed with clone AF330205 (Van Valkenburgh et al, 2001). SCOC–GFP, GFP–SCOC and FLAG–SCOC were constructed with DNA corresponding to Uniprot isoform Q9UIL1‐3, Entrez gene isoform 5. (B) Endogenous SCOC shows colocalisation with Atg9 (red) and TGN46 (blue). HEK293 cells were fixed and permeabilised in saponin. Colocalisation indicated with arrows. (C) Endogenous SCOC (green top, red bottom) partially colocalises with endogenous LC3 (red, top) and GFP–LC3 (green, bottom) in HEK293 and GFP–LC3‐HEK cells, respectively, after 2 h amino‐acid starvation. Boxed region is at higher magnification in merge panel on right. Scale bars=10 μm.
Genome‐wide siRNA screen reveals amino acid starvation‐induced autophagy requires SCOC and WAC (2012)
Figure 4

SCOC is required for autophagy. (A) SCOC is required for autophagy in HEK293 cells. Anti‐ULK1, ‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siSCOC‐03 EL, *P=0.0419. (B) SCOC is required for p62 degradation. p62 levels were determined by western blot (Supplementary Figure S5) after incubation in FM, ES for 2 or 4 h, or ES with BafilomycinA1 (ES+BAF) for 2 and 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree ES 2 and 4 h, ***P>0.0001 and **0.0014, respectively. (C) SCOC–GFP and SCOC–myc levels are reduced by SCOC siRNA duplex‐03. Anti‐GFP, ‐Myc and ‐Actin blots of HEK293 cells after treatment with ULK1, RISCfree or SCOC siRNA and overexpression of GFP, SCOC–myc or SCOC–GFP. (D) Overexpression of SCOC–myc increases autophagy. Anti‐Actin, ‐LC3 and ‐Myc blots of HEK293 cells after transfection of pcDNA or SCOC–myc. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=5). SCOC–myc EL versus pcDNA EL, *P=0.0215. (E) Endogenous SCOC colocalises with endogenous FEZ1 in HEK293 cells. Arrows show partial colocalisation. Boxed region is at higher magnification in merge panel on bottom right. Scale bars=20 μm.
Genome‐wide siRNA screen reveals amino acid starvation‐induced autophagy requires SCOC and WAC (2012)
Figure 5

SCOC interacts with FEZ1. (A) Knockdown of FEZ1 enhances LC3 lipidation but does not affect p62 degradation. Anti‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells. Quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RISCfree ES versus siFEZ1‐01 ES, P=0.034. Quantification of p62/actin (Supplementary Figure S5) performed as in Figure 4B; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree 2 and 4 h P>0.0001 and P=0.0014, respectively. qRT–PCR showing FEZ1 mRNA levels after indicated knockdown in GFP–LC3‐HEK cells (error bars represent s.d.). (B) Overexpression of FEZ1 inhibits LC3 lipidation and p62 degradation. Anti‐Myc, ‐Actin and ‐LC3 blot after transfection with pcDNA or Myc–FEZ1 in HEK293 cells. Quantification of LC3II/actin; error bars represent s.e.m. (n=3); pcDNA ES versus Myc–FEZ1 ES, P=0.0318. Anti‐GFP, ‐Actin and ‐p62 blot after transfection with GFP or FEZ1–GFP and treatment with ES or ES plus Bafilomycin (EB) for 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); GFP FM versus GFP ES, P=0.0077; GFP FM versus GFP EB, P=0.0212; GFP ES versus FEZ1–GFP ES, P=0.048. (C) SCOC and FEZ1 interact. Left panel shows anti‐SCOC and ‐FEZ1 blots after co‐expression in HEK293 cells and co‐immunoprecipitation of SCOC–myc and FEZ1–GFP with anti‐GFP antibody. Input lysate (Inp), unbound supernatant (Unb). Right panel shows anti‐FEZ1 and ‐FLAG blots after co‐expression in HEK293 cells and co‐immunoprecipitation of FLAG–SCOC and Myc–FEZ1 with anti‐FLAG antibody. (D) FEZ1 interacts with SCOC in vitro. GST‐pulldown assays using in vitro translated 35S‐labelled Myc–FEZ1 (right) or 35S‐labelled Myc‐control (left) and GST or GST–SCOC (asterisk in bottom panel). Bound proteins were detected by autoradiography following SDS–PAGE (top), protein loading by coomassie blue (bottom). (E) The FEZ1–SCOC interaction requires the evolutionary conserved residues L254 and L260 in the coiled‐coil domain of FEZ1 as demonstrated in GST‐pulldown assays with in vitro translated 35S‐labelled WT or mutant Myc‐FEZ1 and GST or GST‐SCOC. Autoradiographs of 10% input and bound proteins (top panels) and Coomassie blue‐stained gels of bead‐bound GST and GST‐SCOC proteins used (bottom panels).
Genome‐wide siRNA screen reveals amino acid starvation‐induced autophagy requires SCOC and WAC (2012)
Figure 6

SCOC regulates FEZ1's interaction with the ULK1 complex. (A) FEZ1 interacts with both the kinase domain (1–278) and the proline–serine rich spacer of ULK1 (279–828). GFP‐ or Myc‐tagged ULK1 fragments were in vitro translated with 35S‐methionine and subjected to GST‐pulldown assays using GST and GST–FEZ1 purified from E. coli (bottom). Bound proteins were detected by autoradiography following SDS–PAGE (top). (B) ULK1 interacts with FEZ1. ULK1 was immunoprecipitated from HEK293 lysates with anti‐ULK1 and bound ULK1 and FEZ1 were detected with anti‐ULK1 and anti‐FEZ1 antibodies. Input lysate (Inp). (C) The FEZ1–GFP and Myc–ULK1 interaction is unaffected by amino‐acid starvation. Anti‐Myc and ‐GFP blots after HEK293 cells were co‐transfected with FEZ1–GFP and Myc–ULK1, incubated in FM or ES for 2 h, harvested and the complex was immunoprecipitated using anti‐GFP antibody. (D) BN–PAGE reveals SCOC and FEZ1 are in a complex. FEZ1–GFP, FLAG–SCOC and Myc–ULK1 wild type or kinase‐inactive ULK1K46I (ULK1KI) were co‐expressed in HEK293 cells. Anti‐GFP, ‐FLAG and ‐Myc western blots are shown for the BN–PAGE gel (top) or the SDS–PAGE gel (bottom). In GFP and FLAG western blots, two different exposures of the same BN–PAGE blot are shown spliced together. (E) siRNA depletion of ULK1 does not affect the interaction of FEZ1 with SCOC. RF or ULK1 siRNA‐treated cells were co‐transfected with FEZ1–GFP and FLAG–SCOC, then incubated and immunoprecipitated as in (C). Western blot of lysates for ULK1, GFP and FLAG, and immunoprecipitates with GFP and FLAG antibodies. (F) siRNA depletion of SCOC reduces FEZ1–ULK1 complex. HEK293 cells treated with either SCOC or ULK1 siRNAs were transfected with FEZ1–GFP. FEZ1–GFP was detected with anti‐GFP in both BN–PAGE and SDS–PAGE. Endogenous ULK1 was detected with anti‐ULK1 antibody. Arrowheads indicate a FEZ1–GFP and endogenous ULK1 complex at 200 kDa and a larger MW complex >1236 kDa also detected in (D) in the triple transfection. The 200 KDa complex is sensitive to loss of SCOC and ULK1. (G) SCOC can reduce the interaction of ULK1 with MBP–FEZ1. Immobilised MBP–FEZ1 was incubated with in vitro 35S‐methionine‐labelled Myc–ULK1. Addition of increasing amounts of in vitro translated 35S‐methionine SCOC reduced the amount of Myc–ULK1 bound. Top and bottom panels are as described in (A).
Genome‐wide siRNA screen reveals amino acid starvation‐induced autophagy requires SCOC and WAC (2012)
Figure 7

SCOC interacts with UVRAG and the interaction is reduced in starvation. (A) SCOC interacts with UVRAG. HEK293 cells were transfected with FLAG–SCOC and Myc–UVRAG, lysed and incubated with anti‐FLAG (left) or anti‐Myc (right) antibodies. Co‐immunoprecipitation of Myc–UVRAG (left) and FLAG–SCOC (right) was confirmed using anti‐Myc and anti‐FLAG antibodies for western blotting. Asterisk in (A–C) and (D) indicates non‐specific bands. (B) Interaction of SCOC with UVRAG is unaffected by FEZ1–GFP. As in (A), co‐immunoprecipitation was preformed in HEK293 cells transfected with FLAG–SCOC, Myc–UVRAG and FEZ1–GFP. Myc–UVRAG was detected as in (A), FEZ1–GFP was detected with anti‐GFP antibody. (C) The SCOC and UVRAG interaction is reduced by starvation, and this reduction is inhibited by FEZ1–GFP. HEK293 cells were transfected as in (B) and 24 h later either treated with FM or ES for 2 h. FLAG–SCOC or Myc–UVRAG was immunoprecipitated with anti‐FLAG or anti‐Myc antibodies and the bound proteins were detected with anti‐Myc, ‐GFP or ‐FLAG antibodies by western blotting. Quantification of the amount of FLAG–SCOC co‐immunoprecipitated with Myc–UVRAG in FM or ES, without overexpressed FEZ1–GFP (left) or with overexpressed FEZ1–GFP (right). Significance was determined using a two‐tailed paired t‐test (n=3). (D) FEZ1 interacts directly with endogenous UVRAG and is required for SCOC interaction. HEK293 cells were transfected with FEZ1–GFP, GFP or FLAG–SCOC alone or in combination. Top panel input, bottom panel immunoprecipitation with anti‐GFP or anti‐FLAG. Western blot for endogenous UVRAG, GFP–FEZ1 GFP or FLAG–SCOC as indicated. Arrowhead: endogenous UVRAG. (E) UVRAG can interact with both FEZ1 and SCOC in vitro. GST, GST–FEZ1 or GST–SCOC were immobilised and incubated with 35S‐labelled Myc–UVRAG as indicated. Arrowheads indicate GST–FEZ1 (left) and GST–SCOC (right). (F) Endogenous UVRAG partially colocalises with endogenous FEZ1 in HEK293 cells. Arrows show colocalisation. Scale bar=10 μm.
Genome‐wide siRNA screen reveals amino acid starvation‐induced autophagy requires SCOC and WAC (2012)
Figure 8

WAC is required for autophagy and negatively regulates the UPS and degradation of mutant Htt. (A) Depletion of endogenous WAC. Anti‐WAC and ‐Actin blot and WAC mRNA levels by qRT–PCR after treatment with RF or individual WAC siRNAs (W‐01 to W‐04) in HEK293 cells. qRT–PCR error bars represent standard deviation of triplicates (n=2). (B) WAC is required for autophagy. Anti‐ULK1, ‐WAC, ‐Actin and ‐LC3 blots after siRNA treatment in HEK293 cells and incubation in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siWAC‐03 EL, **P=0.0079. (C) p62 degradation requires WAC. Anti‐p62 and ‐Actin blot after siRNA treatment of GFP–LC3‐HEK cells in FM or ES for 2 h in duplicate. Bars represent p62/actin levels normalised to RF FM; quantification of p62/actin; error bars represent s.e.m. (n=3): RF FM versus RF ES, **P=0.0057; RF FM versus siWAC‐03 FM, **P=0.0025; RF ES versus siWAC‐03 ES, *P=0.0386. (D) WAC depletion accelerates degradation of a UPS reporter. Anti‐p62, ‐GFP, ‐Actin, ‐LC3, ‐WAC and ‐Atg16 blots after siRNA treatment in UbG76V–YFP MelJuSo cells and incubation conditions FM, ES or FM with 10 μM MG132 (MG) for 2 h. Quantification of normalised average GFP intensity in FM of RISCfree compared with WAC‐03 siRNA treatment (n=3) and compared with ATG16 siRNA treatment (n=2). Error bars represent s.e.m.: RF FM versus siWAC‐03 FM, **P=0.0082. (E) Soluble HttQ25–CFP degradation is increased after WAC depletion. Anti‐WAC, ‐Actin and ‐GFP blots after indicated siRNA treatment (siWAC duplexes ‐02, ‐03, ‐04) of HttPolyQ‐mutant HeLa cell lines and incubation conditions DMSO or DOX (Doxycyline) for 3 days. The insoluble fraction was obtained by pelleting the HttQ103 lysates (see Materials and methods). Quantification of normalised average HttQ25–CFP intensity in DMSO of RISCfree compared with WAC siRNA treatment; error bar represents s.e.m. (n=3): HttQ25–CFP/actin RF DMSO versus siWAC DMSO, *P=0.0350; soluble HttQ103–CFP/actin RF DMSO versus siWAC DMSO, *P=0.0380.
L-arginine homeostasis governs adult neural stem cell activation by modulating energy metabolism in vivo (2023)
Figure 1

Figure 1. Arg-II deficiency leads to increased adult hippocampal neurogenesis at young adult age.

(A) Experimental scheme for assessing cell proliferation by a 2-hour BrdU pulse chase in P60 mice.

(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.

(F) Experimental scheme for assessing Nestin-GFP+ RGLs in P60 mice.

(G) Representative images of GFAP+Nestin-GFP+Mcm2+ RGLs in the DG. Scale bar = 50 μm.

(H) Quantification of GFAP+Nestin-GFP+Mcm2+ RGLs (WT vs. KO, Student's t-test, p = 0.98) in the DG of WT and KO mice. WT, n = 4 mice; KO, n = 4 mice.

(I) Percentage of GFAP+Nestin-GFP+Mcm2+ activated RGLs among total GFAP+Nestin-GFP+ RGLs (WT vs. KO, Student's t-test, p < 0.001) in the DG of WT and KO mice. WT, n = 4 mice; KO, n = 4 mice.

(J) Experimental scheme for assessing adult-born neurons by a 30-day BrdU pulse chase in P60 mice.

(K) Representative images of NeuN+BrdU+ adult-born neurons in the DG. Scale bar = 100 μm.

(L) Quantification of NeuN+BrdU+ adult-born neurons (WT vs. KO, Student's t-test, p = 0.0087) in the DG of WT and KO mice at P90. WT, n = 5 mice; KO, n = 5 mice.

Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.
L-arginine homeostasis governs adult neural stem cell activation by modulating energy metabolism in vivo (2023)
Figure 2

Figure 2. Arg-II deficiency leads to RGL overactivation.

(A) Experimental scheme for assessing RGLs and their progeny after a 30-day post-tamoxifen induction in P60 mice.

(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.

(D) Experimental scheme for assessing the effect of Arg-II deficiency on activated RGLs return to quiescence by a BrdU labeling-retention assay.

(E) Representative images of BrdU-retaining RGLs (tdT+BrdU+) in the DG of WT and cKO mice. Scale bar = 20 μm.

(F) Quantification of BrdU-retaining RGLs (BrdU+tdT+GFAP+, WT vs. cKO, Student's t-test, p = 0.73) in the DG of WT and cKO mice. WT, n = 3 mice; cKO, n = 3 mice.

(G) Experimental scheme for assessing the effect of Arg-II deficiency on the maintenance of quiescent RGLs by a BrdU labeling.

(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.

(J) Experimental scheme for assessing cell cycle exit of RGLs.

(K) Representative images of tdT+BrdU+ RGLs co-stained with Ki67 in the DG. Scale bar = 50 μm.

(L) The proportion of activated RGLs that exited cell cycle (tdT+BrdU+Ki67-, WT vs. KO, Student's t-test, p < 0.001) or reenter cell cycle (tdT+BrdU+Ki67+, WT vs. KO, Student's t-test, p < 0.001) among total BrdU labeled tdT+ RGLs in the DG of WT and KO mice. WT, n = 4 mice; KO, n = 3 mice.

Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01