Figure 1 ( A ) Human osteosarcoma U2OS GFP - LC3 cells were treated with a library of polyphenols and polyamines for 6 h And then GFP - LC3 dots were counted to measure autophagy activity . Top 20 hits were shown in the right frame . Human osteosarcoma U2OS cells ( B or neuroglioma H4 cells ( D stably expressing GFP - LC3 were treated with a library of chalcones ( 30 µM ) as indicated , or were left untreated for 6 h . The cells were then fixed and GFP - LC3 dots were counted as an indicator for autophagy . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . Representative images are shown in ( B , D ) . Scale bar equals 10 µm . ( C , E ) Acetylation : U2OS and H4 cells were treated as described above , followed by incubation with specific antibodies to block acetylated tubulin ; Thereafter immunofluorescence was conducted with antibodies against acetylated lysine residues and appropriate AlexaFluor conjugated secondary antibodies before the assessment of cytoplasmic fluorescence intensities . Representative images of acetylation are shown in ( C , E ) . Scale bar equals 10 µm . Human osteosarcoma U2OS cells F ) or neuroglioma H4 cells H ) stably expressing GFP - LC3 were treated with a library of chalcones ( 30 µM ) as indicated or rapamycin ( 10 µM ) , or were left untreated for 6 h . The cells were then fixed and GFP - LC3 dots were counted as an indicator for autophagy . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . Viability : U2OS and H4 cells were treated with 30 µM of the indicated chalcones for 24 h and then nuclei were stained with Hoechst 33342 and the number of cells harboring normal nuclei ( i . e . non pyknotic , " healthy cells " ) was determined . All chalcones were hierarchically clustered upon z - scoring the following phenotypes : autophagy ( number of GFP - LC3 dots ) , viability ( number of healthy cells ) and acetylation ( cytoplasmic fluorescence intensity of acetylated lysine residues ) in U2OS and H4 cells . Results are reported as a heatmap ( G , I ) . ( J , K ) U2OS cells were treated with 3 , 4 - DC in the presence or absence of dichloroacetate ( DCA ) , α - ketoisocaproic acid ( KIC ) , or L - leucine ( Leu ) . GFP - LC3 dots were quantified in ( J ) . Acetylation staining was performed as previously described , and acetylation intensity in the cytoplasm was measured in ( K ) . Data are means ± SD ( * * * = p < 0 . 001 versus DMSO / Ctr ; # # = p < 0 . 01 , # # # = p < 0 . 001 versus 3 , 4 - DC / Ctr ) .

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

Figure 1(A) Human osteosarcoma U2OS GFP-LC3 cells were treated with a library of polyphenols and polyamines for 6 h And then GFP-LC3 dots were counted to measure autophagy activity. Top 20 hits were shown in the right frame.Human osteosarcoma U2OS cells (B or neuroglioma H4 cells (D stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated , or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Representative images are shown in (B,D). Scale bar equals 10 µm. (C,E) Acetylation: U2OS and H4 cells were treated as described above, followed by incubation with specific antibodies to block acetylated tubulin; Thereafter immunofluorescence was conducted with antibodies against acetylated lysine residues and appropriate AlexaFluor conjugated secondary antibodies before the assessment of cytoplasmic fluorescence intensities. Representative images of acetylation are shown in (C,E). Scale bar equals 10 µm.Human osteosarcoma U2OS cells F) or neuroglioma H4 cells H) stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated or rapamycin (10 µM), or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Viability: U2OS and H4 cells were treated with 30 µM of the indicated chalcones for 24 h and then nuclei were stained with Hoechst 33342 and the number of cells harboring normal nuclei (i.e. non pyknotic, "healthy cells") was determined. All chalcones were hierarchically clustered upon z-scoring the following phenotypes: autophagy (number of GFP-LC3 dots), viability (number of healthy cells) and acetylation (cytoplasmic fluorescence intensity of acetylated lysine residues) in U2OS and H4 cells. Results are reported as a heatmap (G, I).(J, K) U2OS cells were treated with 3,4-DC in the presence or absence of dichloroacetate (DCA), α-ketoisocaproic acid (KIC), or L-leucine (Leu). GFP-LC3 dots were quantified in (J). Acetylation staining was performed as previously described, and acetylation intensity in the cytoplasm was measured in (K). Data are means ± SD (*** = p < 0.001 versus DMSO/Ctr; ##=p<0.01, ###=p<0.001 versus 3,4-DC/Ctr).


Figure 2Hepatoma HepG2 cells were treated with the indicated concentrations of 3 , 4 - DC for 8 h ( A , B ) GAPDH was measured as a loading control . Band intensities of p62 , GAPDH , LC3 - I and LC3 - II were measured and ratios of p62 or LC3 - II versus GAPDH ( LC3 - II / GAPDH , p62 / GAPDH ) and LC3 - II versus LC3 - I ( LC3 - II / LC3 - I ) were calculated in ( B Data are means ± SEM of at least three independent experiments ( LC3 - II / GAPDH : * = p < 0 . 05 , * * = p < 0 . 01 ; p62 / GAPDH : # = p < 0 . 05 , # # # = p < 0 . 001 ; LC3 - II / LC3 - I : $ = p < 0 . 05Hepatoma HepG2 cells were treated with 30 µM 3 , 4 - DC for the indicated time points ( C , D ) . Then , cells were processed to measure LC3 and p62 protein levels by SDS - PAGE and immunoblot GAPDH was measured as a loading control . Band intensities of p62 , GAPDH , LC3 - I and LC3 - II were measured and ratios of p62 or LC3 - II versus GAPDH ( LC3 - II / GAPDH , p62 / GAPDH ) and LC3 - II versus LC3 - I ( LC3 - II / LC3 - I ) were calculated in D ) . Data are means ± SEM of at least three independent experiments ( LC3 - II / GAPDH : * = p < 0 . 05 , * * = p < 0 . 01 ; p62 / GAPDH : # = p < 0 . 05 , # # # = p < 0 . 001 ; LC3 - II / LC3 - I : $ = p < 0 . 05 ) . ( E , F ) HepG2 were treated with 30 µM 3 , 4 - DC for 8 h in the presence or absence of chloroquine ( CQ , 50µM ) for 4 h . SDS - PAGE and immunoblot were performed Band intensities of LC3 - II and GAPDH were assessed and their ratio ( LC3 - II / GAPDH ) was calculated in ( F ) . Data are means ± SEM of at least three independent experiments ( * = p < 0 . 05 , * * = p < 0 . 01 versus untreated control ; # # = p < 0 . 01 versus CQ ) . ( G , H ) U2OS - GFP - LC3 cells were treated with 30 µM 3 , 4 - DC , 10 µM rapamycin , or left untreated for 16 h in the presence or absence of CQ for 4 h . GFP - LC3 dots were measured . Data are means ± SD ( * * * = p < 0 . 001 versus untreated ; # # # = p < 0 . 001 versus CQ ) . ( I , J ) U2OS cells stably expressing LC3 fused with tandem fluorescent GFP - RFP proteins ( GFP - RFP - LC3 ) were treated with the indicated raising doses of 3 , 4 - DC or 50 µM chloroquine ( CQ ) for 16 h . After fixation , GFP - LC3 and RFP - LC3 dots were measured by automated image acquisition and analysis . GFP and RFP double positive LC3 dots indicating autophagosomes ( GFP + ) , and GFP negative but RFP positive LC3 dots indicating autolysosomes ( GFP - ) were counted in ( J ) . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . Representative images are shown in I . Scale bar equals 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Hepatoma HepG2 cells were treated with the indicated concentrations of 3,4-DC for 8 h (A,B) GAPDH was measured as a loading control. Band intensities of p62, GAPDH, LC3-I and LC3-II were measured and ratios of p62 or LC3-II versus GAPDH (LC3-II/GAPDH, p62/GAPDH) and LC3-II versus LC3-I (LC3-II/LC3-I) were calculated in (B Data are means ± SEM of at least three independent experiments (LC3-II/GAPDH: * = p < 0.05, ** = p < 0.01; p62/GAPDH: # = p < 0.05, ### = p < 0.001; LC3-II/LC3-I: $=p<0.05Hepatoma HepG2 cells were treated with 30 µM 3,4-DC for the indicated time points (C,D). Then, cells were processed to measure LC3 and p62 protein levels by SDS-PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of p62, GAPDH, LC3-I and LC3-II were measured and ratios of p62 or LC3-II versus GAPDH (LC3-II/GAPDH, p62/GAPDH) and LC3-II versus LC3-I (LC3-II/LC3-I) were calculated in D). Data are means ± SEM of at least three independent experiments (LC3-II/GAPDH: * = p < 0.05, ** = p < 0.01; p62/GAPDH: # = p < 0.05, ### = p < 0.001; LC3-II/LC3-I: $=p<0.05).(E,F) HepG2 were treated with 30 µM 3,4-DC for 8 h in the presence or absence of chloroquine (CQ, 50µM) for 4 h. SDS-PAGE and immunoblot were performed Band intensities of LC3-II and GAPDH were assessed and their ratio (LC3-II/GAPDH) was calculated in (F). Data are means ± SEM of at least three independent experiments (* = p < 0.05, ** = p < 0.01 versus untreated control; ##=p<0.01 versus CQ).(G,H) U2OS-GFP-LC3 cells were treated with 30 µM 3,4-DC, 10 µM rapamycin, or left untreated for 16 h in the presence or absence of CQ for 4 h. GFP-LC3 dots were measured. Data are means ± SD (*** = p < 0.001 versus untreated; ###=p<0.001 versus CQ).(I,J) U2OS cells stably expressing LC3 fused with tandem fluorescent GFP-RFP proteins (GFP-RFP-LC3) were treated with the indicated raising doses of 3,4-DC or 50 µM chloroquine (CQ) for 16 h. After fixation, GFP-LC3 and RFP-LC3 dots were measured by automated image acquisition and analysis. GFP and RFP double positive LC3 dots indicating autophagosomes (GFP+), and GFP negative but RFP positive LC3 dots indicating autolysosomes (GFP-) were counted in (J). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001). Representative images are shown in I. Scale bar equals 10 µm.


Figure 3A , B ) HepG2 cells were treated for the indicated time with 30 µM 3 , 4 - DC ( A ) or indicated dose of 3 , 4 - DC for 8 h ( B ) . Thereafter , cells were collected and SDS - PAGE and immunoblots were performed as described before . Total p70S6K , phosphorylated p70S6K at T389 ( P - p70S6K ) , Atg14 , Lamp1 , LC3 , p62 , and GAPDH protein levels were measured with specific antibodies . Samples for immunoblots in A and B were run in parallel instances , then cut into horizontal stripes and probed separately . Blots probed for P - p70S6K were stripped and reprobed to detect p70 independently from its phosphorylation status . The same applies for LAMP1 and GAPDH . ( C , D ) H4 - GFP - LC3 cells were treated with 30 µM 3 , 4 - DC in the presence or absence of CHX or AMD or with CQ for 16 h as controls , as indicated . GFP - LC3 dots were quantified in ( D ) . Data are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 versus untreated control ; # = p < 0 . 05 versus untreated with CQ ) . ( E ) RNA expression levels 16 h after exposure to 3 , 4 - DC of the indicated genes in HepG2 cells were measured by RT - PCR and normalized to the expression of the housekeeping gene ( GAPDH ) . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . , G ) U2OS GFP - LC3 cells were enucleated to obtain cytoplasts , which were further treated with 3 , 4 - DC and torin1 . GFP - LC3 dots in cytoplasts were counted as shown in ( G ) . Data are means ± SD ( * * = p < 0 . 01 ) . Representative images with cytoplast marked by a white square are shown in ( F ) . Scale bar equals 10 µm .

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

Figure 3A, B) HepG2 cells were treated for the indicated time with 30 µM 3,4-DC (A) or indicated dose of 3,4-DC for 8 h (B). Thereafter, cells were collected and SDS-PAGE and immunoblots were performed as described before. Total p70S6K, phosphorylated p70S6K at T389 (P-p70S6K), Atg14, Lamp1, LC3, p62, and GAPDH protein levels were measured with specific antibodies. Samples for immunoblots in A and B were run in parallel instances, then cut into horizontal stripes and probed separately. Blots probed for P-p70S6K were stripped and reprobed to detect p70 independently from its phosphorylation status. The same applies for LAMP1 and GAPDH.(C, D) H4-GFP-LC3 cells were treated with 30 µM 3,4-DC in the presence or absence of CHX or AMD or with CQ for 16 h as controls, as indicated. GFP-LC3 dots were quantified in (D). Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus untreated control; # = p < 0.05 versus untreated with CQ).(E) RNA expression levels 16 h after exposure to 3,4-DC of the indicated genes in HepG2 cells were measured by RT-PCR and normalized to the expression of the housekeeping gene (GAPDH). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001).,G) U2OS GFP-LC3 cells were enucleated to obtain cytoplasts, which were further treated with 3,4-DC and torin1. GFP-LC3 dots in cytoplasts were counted as shown in (G). Data are means ± SD (** = p < 0.01). Representative images with cytoplast marked by a white square are shown in (F). Scale bar equals 10 µm.


Figure 4U2OS cells stably expressing GFP - TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h . GFP intensities in nuclei and cytoplasm were measured and the ratio of GFP intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei ( A ) . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . U2OS cells stably expressing GFP - TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h . Representative images are shown in ( B ) . Scale bar equals 10 µm . ( C , D ) U2OS cells were treated with 30 µM 3 , 4 - DC for 6 h , and then endogenous TFEB translocation was assessed by immunostaining ( C ) . TFEB intensities in nuclei and cytoplasm were measured and the ratio of TFEB intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei ( D ) . Data are means ± SD ( * * * = p < 0 . 001 ) . Representative images are shown in ( C ) . Scale bar equals 10 µm . ( E , F ) U2OS cells were treated with 30 µM 3 , 4 - DC for 6 h or were left untreated . Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 by SDS page . MEF wild type and TSC2 knockout ( TSC2 KO ) cells were treated with 3 , 4 - DC for 6 h . The cells were fixed and immunofluorescence was performed to detect endogenous TFEB . The ratio of TFEB intensities between nuclei and cytoplasm was calculated in ( H ) . Data are means ± SD ( * * * = p < 0 . 001 versus Ctr ; # # # = p < 0 . 001 versus WT / 3 , 4 - DC ) . Representative images are shown in ( G ) . Scale bar equals 10 µm . MEF wild type and TSC2 knockout ( TSC2 KO ) cells were treated with 3 , 4 - DC for 6 h . ( I ) After the treatment , nuclei were isolated and western blot was performed to detect nuclear and cytosol TFEB protein levels . ( J - L ) MEF wild type and TSC2 KO cells were collected and lysed for western blot after the treatment with 3 , 4 - DC . Antibodies against LC3 , p62 , TSC2 , or GAPDH were administrated to detect protein levels , and phosphorylation of mTOR substrate p70 S6K at Threonine 389 ( T389 ) was measured with the phosphorylation specific antibody ( P - p70 ( T389 ) ) and anti - p70 antibody . The bands intensities of LC3 - II , p62 , and GAPDH were measured with Image J , and the ratios of LC3 - II / GAPDH and p62 / GAPDH were calculated in ( K ) and ( L ) . Data are means ± SEM of at least three independent experiments ( * * = p < 0 . 01 , * * * = p < 0 . 001 vs Ctr ; # = p < 0 . 05 , # # = p < 0 . 01 vs WT / 3 , 4 - DC ) ( M , N ) U2OS cells treated with 3 , 4 - DC or Torin for 6 h were collected and processed for western blot . TFEB phosphorylation at Serine 211 was checked with the phosphorylation specific antibody ( P - TFEB ( S211 ) ) and total TFEB protein level was also detected with anti - TFEB . The bands intensities of P - TFEB and total TFEB were measured with Image J , and their ratio was calculated in ( N ) . Data are means ± SEM of at least three independent experiments ( * = p < 0 . 05 vs Ctr ) .

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

Figure 4U2OS cells stably expressing GFP-TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h. GFP intensities in nuclei and cytoplasm were measured and the ratio of GFP intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (A). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001).U2OS cells stably expressing GFP-TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h. Representative images are shown in (B). Scale bar equals 10 µm.(C, D) U2OS cells were treated with 30 µM 3,4-DC for 6 h, and then endogenous TFEB translocation was assessed by immunostaining (C). TFEB intensities in nuclei and cytoplasm were measured and the ratio of TFEB intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (D). Data are means ± SD (*** = p < 0.001). Representative images are shown in (C). Scale bar equals 10 µm.(E, F) U2OS cells were treated with 30 µM 3,4-DC for 6 h or were left untreated. Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 by SDS page.MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. The cells were fixed and immunofluorescence was performed to detect endogenous TFEB. The ratio of TFEB intensities between nuclei and cytoplasm was calculated in (H). Data are means ± SD (*** = p < 0.001 versus Ctr; ###=p<0.001 versus WT/3,4-DC). Representative images are shown in (G). Scale bar equals 10 µm.MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. (I) After the treatment, nuclei were isolated and western blot was performed to detect nuclear and cytosol TFEB protein levels.(J-L) MEF wild type and TSC2 KO cells were collected and lysed for western blot after the treatment with 3,4-DC. Antibodies against LC3, p62, TSC2, or GAPDH were administrated to detect protein levels, and phosphorylation of mTOR substrate p70 S6K at Threonine 389 (T389) was measured with the phosphorylation specific antibody (P-p70(T389)) and anti-p70 antibody. The bands intensities of LC3-II, p62, and GAPDH were measured with Image J, and the ratios of LC3-II/GAPDH and p62/GAPDH were calculated in (K) and (L). Data are means ± SEM of at least three independent experiments (** = p < 0.01, *** = p < 0.001 vs Ctr; #=p<0.05, ##=p<0.01 vs WT/3,4-DC)(M,N) U2OS cells treated with 3,4-DC or Torin for 6 h were collected and processed for western blot. TFEB phosphorylation at Serine 211 was checked with the phosphorylation specific antibody (P-TFEB(S211)) and total TFEB protein level was also detected with anti-TFEB. The bands intensities of P-TFEB and total TFEB were measured with Image J, and their ratio was calculated in (N). Data are means ± SEM of at least three independent experiments (* = p < 0.05 vs Ctr).


Figure 5U2OS - GFP - LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB ( siTFEB - # 1 , siTFEB - # 2 , siTFEB - # 3 ) or scrambled siRNA ( siCtr ) were treated with 3 , 4 - DC as indicated for 16 h . TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS - PAGE and immunoblotU2OS - GFP - LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB ( siTFEB - # 1 , siTFEB - # 2 , siTFEB - # 3 ) or scrambled siRNA ( siCtr ) were treated with 3 , 4 - DC as indicated for 16 h . GFP - LC3 dots were quantified as indicator of autophagy Data are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 versus siCtr or WT cells treated with 3 , 4 - DC ) . Representative images are shown Scale bar equals 10 µm . U2OS wild type ( wt ) and TFEB knockout ( TFEB KO ) GFP - LC3 cells were treated with 3 , 4 - DC as indicated for 16 h . TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS - PAGE and immunoblotU2OS wild type ( wt ) and TFEB knockout ( TFEB KO ) GFP - LC3 cells were treated with 3 , 4 - DC as indicated for 16 h . GFP - LC3 dots were quantified as indicator of autophagy ata are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 versus siCtr or WT cells treated with 3 , 4 - DC ) . Representative images are shown cale bar equals 10 µm . U2OS wild type ( WT ) , TFEB or TFE3 knockout , or double knockout ( TF DKO ) cells were treated with 30 µM 3 , 4 - DC for 16 h . Following , cells were collected and SDS - PAGE and immunoblots were performed as described before . TFEB , TFE3 , LC3 , p62 , and GAPDH protein levels were measured with specific antibodies ( G ) . U2OS wild type ( WT ) , TFEB or TFE3 knockout , or double knockout ( TF DKO ) cells were treated with 30 µM 3 , 4 - DC for 16 h . GFP - LC3 dots were quantified as indicator of autophagy ( H ) . Data are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 versus Ctr , respectively ) . ( I ) U2OS WT , knockout for TFEB ( TFEB - / - ) or TFE3 ( TFE3 - / - ) , double knockout ( DKO ) were treated with 3 , 4 - DC for 24 h and stained with LysoTracker Red for 30 min . Thereafter , the red ( positive ) dots were measured . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . ( J ) Relative mRNA expression levels of key autophagy genes such as Lamp1 , Lc3b and p62 were detected in controls and 3 , 4 - DC treated U2OS cells . Data is depicted as a heatmap showing means of at least three independent experiments ( * * * = p < 0 . 001 versus WT / Ctr and # = p < 0 . 05 , # # = p < 0 . 01 , as compared to WT treated with 3 , 4 - DC ) .

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

Figure 5U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblotU2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown Scale bar equals 10 µm.U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblotU2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy ata are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown cale bar equals 10 µm.U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. Following, cells were collected and SDS-PAGE and immunoblots were performed as described before. TFEB, TFE3, LC3, p62, and GAPDH protein levels were measured with specific antibodies (G).U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. GFP-LC3 dots were quantified as indicator of autophagy (H). Data are means ± SD (* = p < 0.05, ** = p < 0.01, *** = p < 0.001 versus Ctr, respectively).(I) U2OS WT, knockout for TFEB (TFEB-/-) or TFE3 (TFE3-/-), double knockout (DKO) were treated with 3,4-DC for 24 h and stained with LysoTracker Red for 30 min. Thereafter, the red (positive) dots were measured. Data are means ± SD (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).(J) Relative mRNA expression levels of key autophagy genes such as Lamp1, Lc3b and p62 were detected in controls and 3,4-DC treated U2OS cells. Data is depicted as a heatmap showing means of at least three independent experiments (*** = p < 0.001 versus WT/Ctr and # = p < 0.05, ## = p < 0.01, as compared to WT treated with 3,4-DC).


Figure 6 ( A - D ) Female C57 / BL6 animals were treated with 3 , 4 - DC for 24 h . Following , liver and heart tissue was excised and cells were subjected to subcellular fractionation . Thereafter proteins were separated by SDS PAGE and immunoblots were performed to detect the nuclear translocation of TFEB and TFE3 ( A , C ) . GAPDH and H3 were used as controls for cytosolic and nuclear fractions , respectively . Band intensities of nuclear TFEB , TFE3 and H3 were measured and ratios of nuclear TFs versus H3 ( TFsnuc / H3 ) were calculated in ( B , D ) . Data are means ± SEM of at least three mice ( * * * = p < 0 . 001 versus Ctr ) GFP - LC3 expressing mice were i . p . injected with 3 , 4 - DC for 24 h . Leupeptin ( Leu ) was used to test autophagic flux in vivo and GFP - LC3 dots were measured in liver tissue . Data are means ± SEM of at least three mice ( * = p < 0 . 05 versus ctr without Leu ; # = p < 0 . 05 versus ctr with Leu ) . C57 / BL6 mice were i . p . injected with 3 , 4 - DC or were starved for 24 hours . Following liver tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control . Band intensities of GAPDH and LC3 - II were measured and ratios LC3 - II versus GAPDH ( LC3 - II / GAPDH ) were calculated Data are means ± SEM of at least three mice ( * = p < 0 . 05 , * * = p < 0 . 01 ) . GFP - LC3 expressing mice were i . p . injected with 3 , 4 - DC for 24 h . Leupeptin ( Leu ) was used to test autophagic flux in vivo and GFP - LC3 dots were measured in heart ( I , J ) tissue . Data are means ± SEM of at least three mice ( * = p < 0 . 05 versus ctr without Leu ; # = p < 0 . 05 versus ctr with Leu ) . C57 / BL6 mice were i . p . injected with 3 , 4 - DC or were starved for 24 hours . Following heart tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control . Band intensities of GAPDH and LC3 - II were measured and ratios LC3 - II versus GAPDH ( LC3 - II / GAPDH ) were calculated Data are means ± SEM of at least three mice ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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

Figure 6(A-D) Female C57/BL6 animals were treated with 3,4-DC for 24 h. Following, liver and heart tissue was excised and cells were subjected to subcellular fractionation. Thereafter proteins were separated by SDS PAGE and immunoblots were performed to detect the nuclear translocation of TFEB and TFE3 (A, C). GAPDH and H3 were used as controls for cytosolic and nuclear fractions, respectively. Band intensities of nuclear TFEB, TFE3 and H3 were measured and ratios of nuclear TFs versus H3 (TFsnuc/H3) were calculated in (B, D). Data are means ± SEM of at least three mice (*** = p < 0.001 versus Ctr)GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in liver tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu).C57/BL6 mice were i.p. injected with 3,4-DC or were starved for 24 hours. Following liver tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of GAPDH and LC3-II were measured and ratios LC3-II versus GAPDH (LC3-II/GAPDH) were calculated Data are means ± SEM of at least three mice (* = p < 0.05, ** = p < 0.01).GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in heart (I, J) tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu).C57/BL6 mice were i.p. injected with 3,4-DC or were starved for 24 hours. Following heart tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of GAPDH and LC3-II were measured and ratios LC3-II versus GAPDH (LC3-II/GAPDH) were calculated Data are means ± SEM of at least three mice (* = p < 0.05, ** = p < 0.01).


Figure 7 ( A ) Twelve weeks - old wild - type and cardiac specific Atg7 knockout ( Atg7cKO ) mice were injected with corn oil ( vehicle control , Ctr ) or 3 , 4 - DC 24 h before surgery and subjected to 3 h prolonged ischemia . Representative images of left ventricular ( LV ) myocardial sections after alcian blue and triphenyltetrazolium chloride ( TTC ) staining are depicted . Scale bar equals 1 mm . ( B , C ) The size of the infarction area ( indicated by pale color ) per area at risk ( AAR ) ( B ) and the global AAR per LV ( C ) were measured ( mean value ± SEM , * * p < 0 . 01 , # p < 0 . 05 vs WT / Ctr ) .

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

Figure 7(A) Twelve weeks-old wild-type and cardiac specific Atg7 knockout (Atg7cKO) mice were injected with corn oil (vehicle control, Ctr) or 3,4-DC 24 h before surgery and subjected to 3 h prolonged ischemia. Representative images of left ventricular (LV) myocardial sections after alcian blue and triphenyltetrazolium chloride (TTC) staining are depicted. Scale bar equals 1 mm.(B,C) The size of the infarction area (indicated by pale color) per area at risk (AAR) (B) and the global AAR per LV (C) were measured (mean value ±SEM, **p<0.01, #p<0.05 vs WT/Ctr).


Figure 8A . Induction of autophagy in murine MCA205 fibrosarcomas . Cells were treated with 3 , 4 DC alone or in combination with chloroquine , and were harvested 6 hours later for immunoblot detection of LC3 lipidation . ( C - E ) . Cytofluorometric characterization of tumor infiltrating lymphocytes ( TIL ) , in particular FOXP3 + regulatory T cells ( Treg ) , CD8 + cytotoxic T lymphocytes and the ratio of CD8 + T cells over Treg . Growth kinetic of MCA205 fibrosarcomas that were wild type and were evolving in immunocompetent C57Bl / 6 mice treated as indicatedGrowth kinetic of MCA205 fibrosarcomas that were either wild type or Atg5KD ( H - I ) and were evolving in immunocompetent C57Bl / 6 mice or immunodeficient nu / nu mice ( G ) , treated as indicated ( J - L ) Immunocompetent C57Bl / 6 mice were subcutaneously inoculated with TFEB / TFE3 double knockdown MCA205 cells or its scramble control cells ( K ) . When tumors became palpable , mice were treated as indicated Tumor growth curves from mice subjected to 3 , 4 - DC administration alone or in combination with MTX are shown ( J , L ) . Asterisks indicate significant effect of MTX with respect to untreated controls ( mean value ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) , while hash symbols refer to the comparison of the effects of MTX plus 3 , 4 - DC to MTX alone ( # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 ) ( n = 6 - 14 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O 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 8A. Induction of autophagy in murine MCA205 fibrosarcomas. Cells were treated with 3,4 DC alone or in combination with chloroquine, and were harvested 6 hours later for immunoblot detection of LC3 lipidation.(C-E). Cytofluorometric characterization of tumor infiltrating lymphocytes (TIL), in particular FOXP3+ regulatory T cells (Treg), CD8+ cytotoxic T lymphocytes and the ratio of CD8+ T cells over Treg.Growth kinetic of MCA205 fibrosarcomas that were wild type and were evolving in immunocompetent C57Bl/6 mice treated as indicatedGrowth kinetic of MCA205 fibrosarcomas that were either wild type or Atg5KD (H-I) and were evolving in immunocompetent C57Bl/6 mice or immunodeficient nu/nu mice (G), treated as indicated(J-L) Immunocompetent C57Bl/6 mice were subcutaneously inoculated with TFEB/TFE3 double knockdown MCA205 cells or its scramble control cells (K). When tumors became palpable, mice were treated as indicated Tumor growth curves from mice subjected to 3,4-DC administration alone or in combination with MTX are shown (J,L). Asterisks indicate significant effect of MTX with respect to untreated controls (mean value ±SEM, *p<0.05, **p<0.01, ***p<0.001), while hash symbols refer to the comparison of the effects of MTX plus 3,4-DC to MTX alone (#p<0.05, ##p<0.01, ###p<0.001)(n=6-14).


Figure 1 ( B ) Violinplots showing the number of detected mRNAs and lincRNAs across distinct cell types by 10x Genomics . Total 7 , 935 cells were collected from 2 replicates of 6 mice .

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Figure 1(B) Violinplots showing the number of detected mRNAs and lincRNAs across distinct cell types by 10x Genomics. Total 7,935 cells were collected from 2 replicates of 6 mice.


Figure 2 ( C ) Boxplot showing the number of detected lincRNAs across distinct neuron types by two scRNA - seq methods . Total 1 , 818 neurons from 10x Genomics were collected from 2 replicates of 6 mice . And 197 neurons from Smart - seq2 ( analyzed in B , C and D ) were collected from lumbar 5 DRG from five adult male mice as described in previous study .

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Figure 2(C) Boxplot showing the number of detected lincRNAs across distinct neuron types by two scRNA-seq methods. Total 1,818 neurons from 10x Genomics were collected from 2 replicates of 6 mice. And 197 neurons from Smart-seq2 (analyzed in B, C and D) were collected from lumbar 5 DRG from five adult male mice as described in previous study.


Figure 3 ( E ) Representative images ( Left ) of smFISH in DRG section , tSNE plot of 10x Genomics ( Middle ) , and quantitative result ( Right ) showed that C530044C16Rik was co - localized with Gal - Cre ; Ai9 tdTomato neurons ( n = 7 ) , but not with Sst - Cre neurons ( n = 6 ) . ( F ) Representative smFISH images ( Left ) , tSNE plot of 10x Genomics ( Middle ) , and quantitative result ( Right ) showed that CLAP was co - localized with Sst - Cre ; Ai9 tdTomato neurons ( n = 6 ) , but not with Gal - Cre neurons ( n = 29 ) . ( G ) Representative smFISH images ( Left ) , tSNE plot of 10x Genomics ( Middle ) , and quantitative result ( Right ) showed that Gm11549 was co - localized with Slc17a8 - Cre ; Ai9 tdTomato neurons ( n = 5 ) , but not with Mrgprd - Cre neurons ( n = 6 ) . ( H ) Representative smFISH images ( Left ) , tSNE plot of 10x Genomics ( Middle ) , and quantitative result ( Right ) showed that 2310002F09Rik was co - localized with Slc17a8 - Cre ; Ai9 tdTomato neurons ( n = 6 ) , but not with Slc17a8 - Cre neurons ( n = 6 ) . ( I - K ) Representative images ( Right ) of double smFISH showed that 2310002F09Rik was rarely co - localized with C530044C16Rik ( I ) , CLAP ( J ) and Gm11549 ( K ) . Scale bar , 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3(E) Representative images (Left) of smFISH in DRG section, tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that C530044C16Rik was co-localized with Gal-Cre; Ai9 tdTomato neurons (n = 7), but not with Sst-Cre neurons (n = 6). (F) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that CLAP was co-localized with Sst-Cre; Ai9 tdTomato neurons (n = 6), but not with Gal-Cre neurons (n = 29). (G) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that Gm11549 was co-localized with Slc17a8-Cre; Ai9 tdTomato neurons (n = 5), but not with Mrgprd-Cre neurons (n = 6). (H) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that 2310002F09Rik was co-localized with Slc17a8-Cre; Ai9 tdTomato neurons (n = 6), but not with Slc17a8-Cre neurons (n = 6).(I-K) Representative images (Right) of double smFISH showed that 2310002F09Rik was rarely co-localized with C530044C16Rik (I), CLAP (J) and Gm11549 (K). Scale bar, 10 µm.


Figure 4 ( D ) Representative smFISH images in DRG sections and quantitative results showed that 2310002F09Rik and Gm14273 were more enriched in the nucleus , whereas CLAP and Gm11549 were mainly distributed in the cytoplasm . DAPI served as a nuclear marker . Scale bar , 10 μm .

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Figure 4(D) Representative smFISH images in DRG sections and quantitative results showed that 2310002F09Rik and Gm14273 were more enriched in the nucleus, whereas CLAP and Gm11549 were mainly distributed in the cytoplasm. DAPI served as a nuclear marker. Scale bar, 10 μm.


Figure 5 ( B ) Sequence conservation analysis of lincRNA CLAP and upstream mRNA Pirt in 9 different species including mouse , human , chimpanzee , rhesus monkey , rat , cow , dog , chicken and zebrafish , which were visualized with the VISTA browser . CDS , coding sequence . UTR , untranslated region . ( D ) qPCR showed that CLAP was highly expressed in the DRG and other visceral organs such as small intestine , stomach and lung . The results are presented as mean ± SEM ( n = 3 ) . ( E ) qPCR showed that the expression of CLAP was gradually increased from E13 . 5 to adult DRGs . The results are presented as mean ± SEM ( n = 3 ) . ( F ) Representative bright - field and fluorescent images showing the Sst - cre ; Ai9 neurons for calcium imaging following siCLAP transfection . Scale bar , 10 μm . ( G ) qPCR showed that the expression of CLAP was decreased by siRNA treatment in cultured Sst - Cre ; Ai9 DRG neurons . The results are presented as mean ± SEM ( n = 3 ) . * * P < 0 . 01 versus siNC . The percentage of Sst - Cre ; Ai9 neurons evoked by histamine ( I ) was decreased by knockdown of CLAP , whereas the percentage of these neurons affected by 5 - HT ( J ) , chloroquine ( K ) , capsaicin ( L ) were not changed . The results are presented as mean ± SEM ( n = 4 ) . * * P < 0 . 01 versus siNC .

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

Figure 5(B) Sequence conservation analysis of lincRNA CLAP and upstream mRNA Pirt in 9 different species including mouse, human, chimpanzee, rhesus monkey, rat, cow, dog, chicken and zebrafish, which were visualized with the VISTA browser. CDS, coding sequence. UTR, untranslated region.(D) qPCR showed that CLAP was highly expressed in the DRG and other visceral organs such as small intestine, stomach and lung. The results are presented as mean ± SEM (n = 3). (E) qPCR showed that the expression of CLAP was gradually increased from E13.5 to adult DRGs. The results are presented as mean ± SEM (n = 3).(F) Representative bright-field and fluorescent images showing the Sst-cre; Ai9 neurons for calcium imaging following siCLAP transfection. Scale bar, 10 μm.(G) qPCR showed that the expression of CLAP was decreased by siRNA treatment in cultured Sst-Cre; Ai9 DRG neurons. The results are presented as mean ± SEM (n = 3). ** P < 0.01 versus siNC.The percentage of Sst-Cre; Ai9 neurons evoked by histamine (I) was decreased by knockdown of CLAP, whereas the percentage of these neurons affected by 5-HT (J), chloroquine (K), capsaicin (L) were not changed. The results are presented as mean ± SEM (n = 4). ** P < 0.01 versus siNC.


Figure 6 ( B ) qPCR showed that the CLAP level was significantly decreased by siCLAP in the DRG following intrathecal injection . The results are presented as mean ± SEM ( siNC = 12 , siCLAP = 15 ) . * * * P < 0 . 001 versus siNC . ( C - E ) Knockdown of CLAP reduced itch scratching evoked by histamine ( C ) , but not 5 - HT ( D ) and chloroquine ( E ) . The results are presented as mean ± SEM ( For histamine and 5 - HT , siNC = 12 ， siCLAP = 15 ; for chloroquine , siNC = 5 ， siCLAP = 8 ) . * * P < 0 . 01 versus siNC .

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Figure 6(B) qPCR showed that the CLAP level was significantly decreased by siCLAP in the DRG following intrathecal injection. The results are presented as mean ± SEM (siNC = 12, siCLAP = 15). *** P < 0.001 versus siNC.(C-E) Knockdown of CLAP reduced itch scratching evoked by histamine (C), but not 5-HT (D) and chloroquine (E). The results are presented as mean ± SEM (For histamine and 5-HT, siNC = 12，siCLAP = 15; for chloroquine, siNC = 5，siCLAP = 8 ). ** P < 0.01 versus siNC.


Figure 7 ( D ) The expression changes of C2 - specific and itch - related genes upon CLAP knockdown by RNA - seq . Each dot represents an RNA - seq sample , and results are presented as mean ± SEM ( n = 3 ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 versus siNC . ( F ) qPCR showed that downregulated CLAP and C2 - specific genes including Sst , Plcb3 and Cysltr2 by siCLAP was fully rescued by overexpression of CLAPR in L4 / 5 DRGs of mice . The results are presented as mean ± SEM ( siNC , n = 10 ; siCLAP , n = 6 ; siCLAP + CLAPR , n = 12 ) . * P < 0 . 01 versus siNC and # P < 0 . 05 , # # P < 0 . 01 versus the indicated treatment . ( I ) qPCR showed that downregulated Plcb3 by siCLAP was fully rescued by knockdown of MSI2 in L4 / 5 DRGs of mice . The results are presented as mean ± SEM ( siNC , n = 8 ; siCLAP , n = 6 ; siCLAP + siMSI2 , n = 5 ) . * P < 0 . 05 , * * * P < 0 . 001 versus siNC and # # P < 0 . 01 versus the indicated treatment .

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

Figure 7(D) The expression changes of C2-specific and itch-related genes upon CLAP knockdown by RNA-seq. Each dot represents an RNA-seq sample, and results are presented as mean ± SEM (n = 3). * P < 0.05, ** P < 0.01 and *** P < 0.001 versus siNC.(F) qPCR showed that downregulated CLAP and C2-specific genes including Sst, Plcb3 and Cysltr2 by siCLAP was fully rescued by overexpression of CLAPR in L4/5 DRGs of mice. The results are presented as mean ± SEM (siNC, n = 10; siCLAP, n = 6; siCLAP + CLAPR, n = 12). * P < 0.01 versus siNC and # P < 0.05, ## P < 0.01 versus the indicated treatment.(I) qPCR showed that downregulated Plcb3 by siCLAP was fully rescued by knockdown of MSI2 in L4/5 DRGs of mice. The results are presented as mean ± SEM (siNC, n = 8; siCLAP, n = 6; siCLAP + siMSI2, n = 5). * P < 0.05, *** P < 0.001 versus siNC and ## P < 0.01 versus the indicated treatment.


Figure 1 ( A ) Daily 10 μg 4 - AP enhanced recovery of sciatic nerve motor function as compared with treatment with vehicle at 3 days post injury ( dpi ) through 8 dpi . ( * : Day3 ( D3 ) , p = 0 . 0131 ; D5 , p = 0 . 0475 ; D8 , p = 0 . 0472 ; n = 6 ; ANOVA with post - hoc comparisons using two - tailed unpaired t - test ) ( B ) Daily 10 μg 4 - AP administration also enhanced recovery of nerve conduction velocity ( NCV ) as observed beginning at 21 days post - injury , eventually restoring NCV to near - normal values while NCV in vehicle - treated mice remained less than half that of uninjured animals ( * : D21 , p = 0 . 0454 ; D28 , p = 0 . 0487 ; D35 , p = 0 . 0475 ; n = 5 ; ANOVA with post - hoc comparisons using two - tailed unpaired t - test ) . ( C , D ) Analysis of withdrawal latency in response to thermal ( C ) or mechanical ( D ) stimuli revealed that 4 - AP treatment did not worsen these diagnostics of neuropathic pain syndromes . In the case of response to thermal hyperalgesis , 4 - AP treated mice showed a significantly more rapid return to baseline levels . ( * : ( C ) D3 , D5 , D8 : p < 0 . 001 saline vs . baseline ; D3 p = 0 . 006 ; 4 - AP vs . baseline ; ( D ) D3 , p = 0 . 014 ; D5 , p = 0 . 008 ; saline versus baseline ; D3 , p = 0 . 006 4 - AP vs . baseline ; D8 : p = 0 . 0472 ; n = 10 ; ANOVA with post - hoc comparisons using two - tailed unpaired t - test . ) In this and all other figures data is mean + SEM and shows a representative experiment from 2 - 3 repetitions .

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

Figure 1(A) Daily 10 μg 4-AP enhanced recovery of sciatic nerve motor function as compared with treatment with vehicle at 3 days post injury (dpi) through 8 dpi. (*: Day3 (D3), p= 0.0131; D5, p = 0.0475; D8, p =0.0472; n=6; ANOVA with post-hoc comparisons using two-tailed unpaired t-test)(B) Daily 10 μg 4-AP administration also enhanced recovery of nerve conduction velocity (NCV) as observed beginning at 21 days post-injury, eventually restoring NCV to near-normal values while NCV in vehicle-treated mice remained less than half that of uninjured animals (*: D21, p= 0.0454; D28, p = 0.0487; D35, p =0.0475; n=5; ANOVA with post-hoc comparisons using two-tailed unpaired t-test).(C, D) Analysis of withdrawal latency in response to thermal (C) or mechanical (D) stimuli revealed that 4-AP treatment did not worsen these diagnostics of neuropathic pain syndromes. In the case of response to thermal hyperalgesis, 4-AP treated mice showed a significantly more rapid return to baseline levels. (*: (C) D3, D5, D8 : p<0.001 saline vs. baseline; D3 p= 0.006; 4-AP vs. baseline; (D) D3, p= 0.014; D5, p = 0.008; saline versus baseline; D3, p = 0.006 4-AP vs. baseline; D8: p =0.0472; n=10 ; ANOVA with post-hoc comparisons using two-tailed unpaired t-test.) In this and all other figures data is mean + SEM and shows a representative experiment from 2-3 repetitions.


Figure 2 ( A ) Local 4 - AP treated crushed sciatic nerve ( black : vehicle PLGA films ; red : ( 4 - AP ) - PLGA films ) regained partial walking ability as early as 3 days post - injury compared to vehicle treated group . ( * : D5 , p = 0 . 0004 ; D8 , p = 0 . 0012 ; D11 , p = 0 . 003 ; D14 , p = 0 . 0089 ; n = 6 ; ANOVA , with post - hoc comparisons using two - tailed unpaired t - test ) . ( B ) Local 4 - AP treated crushed sciatic nerve ( black : vehicle PLGA films ; red : ( 4 - AP ) - PLGA films ) showed faster improvement in NCV restoration compared with vehicle - treated mice , beginning at 21 days post - injury .

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Figure 2(A) Local 4-AP treated crushed sciatic nerve (black: vehicle PLGA films; red: (4-AP)-PLGA films) regained partial walking ability as early as 3 days post-injury compared to vehicle treated group. (*: D5, p= 0.0004; D8, p = 0.0012; D11, p =0.003; D14, p=0.0089; n=6; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test).(B) Local 4-AP treated crushed sciatic nerve (black: vehicle PLGA films; red: (4-AP)-PLGA films) showed faster improvement in NCV restoration compared with vehicle-treated mice, beginning at 21 days post-injury.


Figure 3 ( A ) Electron microscopy images of healthy , crushed , and 4 - AP treated crushed sciatic nerve at 21 days post - injury . Scale bar at 3000x = 5m ; scale bar at 8000x = 2m ) ( B ) Comparison of the axonal area of randomly chosen individual axons of vehicle treated and 4 - AP treated mice ( n = 4 for each experimental group ; 40 axons analyzed per mouse ) . 4 - AP treated sciatic nerve showed statistically greater axonal area compared to the vehicle - treated group ( p < 0 . 05 ; ANOVA ; restricted - maximum - likelihood ) . Moreover , 4 - AP treated mice had a greater proportion of axons with areas greater than the mean value for uninjured mice ( shown in the green line ) , with inset figure displaying all axons with values above this mean . This experiment represents a single group of mice of one of three replicates on NCV recovery .

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Figure 3(A) Electron microscopy images of healthy, crushed, and 4-AP treated crushed sciatic nerve at 21 days post-injury. Scale bar at 3000x = 5m; scale bar at 8000x = 2m) (B) Comparison of the axonal area of randomly chosen individual axons of vehicle treated and 4-AP treated mice (n=4 for each experimental group; 40 axons analyzed per mouse). 4-AP treated sciatic nerve showed statistically greater axonal area compared to the vehicle-treated group (p<0.05; ANOVA; restricted-maximum-likelihood). Moreover, 4-AP treated mice had a greater proportion of axons with areas greater than the mean value for uninjured mice (shown in the green line), with inset figure displaying all axons with values above this mean. This experiment represents a single group of mice of one of three replicates on NCV recovery.


Figure 4 ( A , B ) Sustained local 4 - AP administration was associated with increased myelin area and close - to - normal Garea - ratio compared to untreated group , as determined by analysis of 40 randomly chosen myelinated axons from sections of 4 nerves for each group . ( * * : p < 0 . 0001 ; ANOVA , with post - hoc comparisons using two - tailed unpaired t - test ) . 4 - AP treated mice had a greater proportion of axons for which the associated myelin area was greater than the mean value for uninjured mice ( shown in the green line ) , with inset figure displaying all axons with values above this mean . ( C , D ) 4 - AP treated nerves also showed increases in the levels of P0 protein as detected by western blot analysis ( * * : p < 0 . 01 ; ANOVA , with post - hoc comparisons using two - tailed unpaired t - test ) . ( C , D ) 4 - AP treated nerves also showed increases in the levels of P0 protein as detected by western blot analysis ( * * : p < 0 . 01 ; ANOVA , with post - hoc comparisons using two - tailed unpaired t - test ) . ( E ) Increases in P0 protein over time also were observed by immunofluorescence analysis at different time points , with P0 protein expression increasing to a greater extent in nerves of mice treated with 4 - AP . Scale bar = 200 m . ( F , G ) 4 - AP treatment increased the number of myelinated axons ( * * : p = 0 . 002 , n = 4 ; ANOVA , with post - hoc comparisons using two - tailed unpaired t - test ) . Even though the 4 - AP treated group also exhibited a greater number of total axons , this difference was not statistically significant . All myelinated and total axons were counted in 5 randomly chosen grids from each of 4 nerves for each group . This experiment represents a single group of mice of one of three replicates on NCV recovery .

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

Figure 4(A, B) Sustained local 4-AP administration was associated with increased myelin area and close-to-normal Garea-ratio compared to untreated group, as determined by analysis of 40 randomly chosen myelinated axons from sections of 4 nerves for each group. (**: p<0.0001; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test). 4-AP treated mice had a greater proportion of axons for which the associated myelin area was greater than the mean value for uninjured mice (shown in the green line), with inset figure displaying all axons with values above this mean.(C, D) 4-AP treated nerves also showed increases in the levels of P0 protein as detected by western blot analysis (**:p<0.01; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test).(C, D) 4-AP treated nerves also showed increases in the levels of P0 protein as detected by western blot analysis (**:p<0.01; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test).(E) Increases in P0 protein over time also were observed by immunofluorescence analysis at different time points, with P0 protein expression increasing to a greater extent in nerves of mice treated with 4-AP. Scale bar = 200 m.(F, G) 4-AP treatment increased the number of myelinated axons (**: p=0.002, n=4; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test). Even though the 4-AP treated group also exhibited a greater number of total axons, this difference was not statistically significant. All myelinated and total axons were counted in 5 randomly chosen grids from each of 4 nerves for each group. This experiment represents a single group of mice of one of three replicates on NCV recovery.


Figure 5 ( A ) At 1 day post - injury , a single dose of 10 μg 4 - AP significantly improved walking function as determined by SFI analysis ( * : p = 0 . 001 , n = 5 ) . ( B ) In contrast , even higher doses of 4 - AP administration ( 50 μg , ip ) had no effect on SFI in mice with transected nerves ( n = 10 ) . ( C ) In contrast with effects of 4 - AP , treatment with neostigmine did not cause improvements in SFI . ( p = 0 . 0024 for 4 - AP vs . saline , n = 8 , 1 - way ANOVA with Tukey ' s multiple comparison test ) .

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Figure 5(A) At 1 day post-injury, a single dose of 10 μg 4-AP significantly improved walking function as determined by SFI analysis (*: p=0.001, n=5). (B) In contrast, even higher doses of 4-AP administration (50 μg, ip) had no effect on SFI in mice with transected nerves (n=10). (C) In contrast with effects of 4-AP, treatment with neostigmine did not cause improvements in SFI. (p = 0.0024 for 4-AP vs. saline, n=8, 1-way ANOVA with Tukey's multiple comparison test).


Figure 1 ( A ) HEK293T ‐ Fpn cells were incubated with FAC ( 10 μM Fe ) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A . Cells were then incubated with and without 100 μM chloroquine or 10 μM leupeptin for 10 h and harvested . The ferritin content was determined by ELISA . Induction of Fpn resulted in decreased ferritin levels and this was not prevented by treatment with chloroquine or leupeptin . The data are presented as the standard deviation from three different experiments . ( B ) Cells treated as in ( A ) were incubated in the presence of 1 μg / ml EGF for 2 h . Cells were fixed and processed for immunofluorescence using mouse anti ‐ EGF receptor and Alexa 594 conjugated goat anti ‐ mouse IgG . Chloroquine and leupeptin effectively inhibit degradation of EGF receptor .

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

Figure 1(A) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then incubated with and without 100 μM chloroquine or 10 μM leupeptin for 10 h and harvested. The ferritin content was determined by ELISA. Induction of Fpn resulted in decreased ferritin levels and this was not prevented by treatment with chloroquine or leupeptin. The data are presented as the standard deviation from three different experiments.(B) Cells treated as in (A) were incubated in the presence of 1 μg/ml EGF for 2 h. Cells were fixed and processed for immunofluorescence using mouse anti‐EGF receptor and Alexa 594 conjugated goat anti‐mouse IgG. Chloroquine and leupeptin effectively inhibit degradation of EGF receptor.


Figure 2 ( A ) HEK293T ‐ Fpn cells were incubated with FAC ( 10 μM Fe ) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A . Cells were then treated with or without 10 μM MG132 or 10 μM lactacystin in the presence of Ponasterone A for 10 h and harvested . The ferritin content was determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate . ( B ) Samples treated as in ( A ) were immunoprecipitated using anti ‐ ferritin antibodies and the immunoprecipitate examined for the presence of ferritin or ubiquitin by Western blot analysis . The arrows indicate the migration of H and L chains . ( C ) Cells were incubated with 1 . 0 × 10 − 7 M Tf ( 59Fe ) 2 for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A . Cells were then treated with or without 10 μM MG132 in the presence of Ponasterone A for 10 h and harvested and the amount of 59Fe in immunoprecipitatedferritin was determined . ( D ) Cells were treated as described in ( A ) and cell extracts were applied to size exclusion chromatography and the ferritin content , in selected fractions , was determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate . ( E ) Cells were treated as described in ( C ) , ferritin was then immunoprecipitated , eluted using 100 mM glycine , pH 2 . 5 and measured by ELISA . The amount of ferritin ‐ associated 59Fe was measured and the specific activity of ferritin determined . Error bars represent the standard deviation from three different experiments in duplicate . ( F ) Samples were treated as in ( D ) , and ferritin was immunoprecipitated and analyzed by Western blot analysis using an antibody to ubiquitin .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O 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) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then treated with or without 10 μM MG132 or 10 μM lactacystin in the presence of Ponasterone A for 10 h and harvested. The ferritin content was determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(B) Samples treated as in (A) were immunoprecipitated using anti‐ferritin antibodies and the immunoprecipitate examined for the presence of ferritin or ubiquitin by Western blot analysis. The arrows indicate the migration of H and L chains.(C) Cells were incubated with 1.0 × 10−7 M Tf(59Fe)2 for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then treated with or without 10 μM MG132 in the presence of Ponasterone A for 10 h and harvested and the amount of 59Fe in immunoprecipitatedferritin was determined.(D) Cells were treated as described in (A) and cell extracts were applied to size exclusion chromatography and the ferritin content, in selected fractions, was determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(E) Cells were treated as described in (C), ferritin was then immunoprecipitated, eluted using 100 mM glycine, pH 2.5 and measured by ELISA. The amount of ferritin‐associated 59Fe was measured and the specific activity of ferritin determined. Error bars represent the standard deviation from three different experiments in duplicate.(F) Samples were treated as in (D), and ferritin was immunoprecipitated and analyzed by Western blot analysis using an antibody to ubiquitin.


Figure 3 ( A ) FM3A ( black ) and ts85 ( grey ) cells were transiently transfected with a plasmid containing CMV ‐ regulated Fpn ‐ GFP and incubated in the presence of FAC ( 10 μM Fe ) and 0 . 5 μM hepcidin for 24 h . Hepcidin was either maintained ( + ) or removed ( − ) to allow Fpn ‐ GFP localization at the plasma membrane . Cells were maintained at the permissive temperature ( 33 ° C ) or moved to the restrictive temperature ( 39 ° C ) and incubated for 6 h . Cells were harvested and ferritin content determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate . ( B ) FM3A and ts85 cells were treated as in ( A ) but FAC was replaced with 1 . 0 × 10 − 7 M Tf ( 59Fe ) 2 . Ferritin was immunoprecipitated and the specific activity of 59Fe ‐ ferritin determined . Error bars represent the standard deviation from three different experiments in duplicate . ( C ) ts85 cells were treated as in ( B ) and cell extracts applied to size exclusion chromatography . Ferritin content in selected fractions was measured by ELISA . Black bars represent assembled ferritin ( > 400 kDa ) , gray bars represent monomeric ( 50 kDa ) . ( D ) FM3A and ts85 cells were treated as in ( A ) but cells that had been incubated at the restrictive temperature ( 39 ° C ) were then returned to the permissive temperature ( 33 ° C ) and incubated in the presence of cycloheximide ( 75 μg / ml ) and 10 μM FAC for 1 h . Cells were then harvested and ferritin levels determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FM3A (black) and ts85 (grey) cells were transiently transfected with a plasmid containing CMV‐regulated Fpn‐GFP and incubated in the presence of FAC (10 μM Fe) and 0.5 μM hepcidin for 24 h. Hepcidin was either maintained (+) or removed (−) to allow Fpn‐GFP localization at the plasma membrane. Cells were maintained at the permissive temperature (33°C) or moved to the restrictive temperature (39°C) and incubated for 6 h. Cells were harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(B) FM3A and ts85 cells were treated as in (A) but FAC was replaced with 1.0 × 10−7 M Tf(59Fe)2. Ferritin was immunoprecipitated and the specific activity of 59Fe‐ferritin determined. Error bars represent the standard deviation from three different experiments in duplicate.(C) ts85 cells were treated as in (B) and cell extracts applied to size exclusion chromatography. Ferritin content in selected fractions was measured by ELISA. Black bars represent assembled ferritin (>400 kDa), gray bars represent monomeric (50 kDa).(D) FM3A and ts85 cells were treated as in (A) but cells that had been incubated at the restrictive temperature (39°C) were then returned to the permissive temperature (33°C) and incubated in the presence of cycloheximide (75 μg/ml) and 10 μM FAC for 1 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.


Figure 4 ( A ) Cells were then incubated at the restrictive temperature ( 39 ° C ) for 6 h in the presence or absence of 100 μM DFO , with or without 100 μM chloroquine or 10 μM MG132 . Cells were then harvested and ferritin content determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate . ( B ) Cells treated as in ( A ) were incubated in the presence or absence of 5 mM 3 ‐ methyladenine .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were then incubated at the restrictive temperature (39°C) for 6 h in the presence or absence of 100 μM DFO, with or without 100 μM chloroquine or 10 μM MG132. Cells were then harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.(B) Cells treated as in (A) were incubated in the presence or absence of 5 mM 3‐methyladenine.


Figure 5 ( A ) Δccc1 / pGAL ‐ H + L cells were transformed with a plasmid containing a methionine regulated CCC1 ( pMET3CCC1 ) . Cells were grown in medium with galactose for 20 h , washed and incubated in galactose ( • ) , glucose with 10 × methionine ( ▵ ) , glucose ( ○ ) or glucose without methionine ( ▾ ) for 10 h . Cells were then harvested and ferritin levels determined by ELISA . Error bars represent the standard deviation from three different experiments in duplicate . The absence of methionine leads to expression of the vacuolar iron transporter Ccc1p . ( B ) erg6 ‐ 2 and erg6 ‐ 2 pGAL ‐ H + L strains were grown in medium with galactose and 250 μM FeSO4 for 24 h . Cells were then washed and incubated in galactose or glucose in the absence or presence of 50 μM MG132 for 7 h . ( C ) Cells were then harvested , ferritin levels determined by ELISA and immunoprecipitated using anti ‐ ferritin antibodies and the immunoprecipitate examined for the presence of ubiquitin by Western analysis . Cells with the erg6 ‐ 2 allele permit the entry of MG132 ( Lee and Goldberg , 1996 ) .

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

Figure 5(A) Δccc1/pGAL‐H+L cells were transformed with a plasmid containing a methionine regulated CCC1 (pMET3CCC1). Cells were grown in medium with galactose for 20 h, washed and incubated in galactose (•), glucose with 10 × methionine (▵), glucose (○) or glucose without methionine (▾) for 10 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate. The absence of methionine leads to expression of the vacuolar iron transporter Ccc1p.(B) erg6‐2 and erg6‐2 pGAL‐H+L strains were grown in medium with galactose and 250 μM FeSO4 for 24 h. Cells were then washed and incubated in galactose or glucose in the absence or presence of 50 μM MG132 for 7 h.(C) Cells were then harvested, ferritin levels determined by ELISA and immunoprecipitated using anti‐ferritin antibodies and the immunoprecipitate examined for the presence of ubiquitin by Western analysis. Cells with the erg6‐2 allele permit the entry of MG132 ( Lee and Goldberg, 1996).


Figure 2B ) Western blot of the NOTCH3 EGF1 - 5 WT and R133C purified proteins . The eluate fractions were visualized by western blot using an α - myc antibody . C ) Western blot of NOTCH3 EGF1 - 5 WT and R133C aggregated proteins . The incubated fractions of NOTCH3 EGF1 - 5 WT and R133C were visualized on a western blot using an α - myc antibody . The purified proteins and the aggregates were visualized after 1 - 5 days incubation by western blot using α - myc antibody under non - reducing conditions .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2B) Western blot of the NOTCH3 EGF1-5 WT and R133C purified proteins. The eluate fractions were visualized by western blot using an α-myc antibody.C) Western blot of NOTCH3 EGF1-5 WT and R133C aggregated proteins. The incubated fractions of NOTCH3 EGF1-5 WT and R133C were visualized on a western blot using an α-myc antibody. The purified proteins and the aggregates were visualized after 1-5 days incubation by western blot using α-myc antibody under non-reducing conditions.


Figure 4A ) Representative images of TgN3R182C150 , sham - and NOTCH3 EGF1 - 5 - immunized mice at 7 months of age and TgN3R182C150 at 18 months of age . Representative images show brain arteries of TgN3R182C150 ( 7 and 18 months ) , sham and NOTCH3 EGF1 - 5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) and an α - SMA antibody ( green ) . Scale bar = 20µm . B ) Quantification of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF1 - 5 - immunized ( n = 10 ) , sham ( n = 8 ) and non - vaccinated ( n = 6 ) TgN3R182C150 mice at 7 months of age . NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C150 mice at 18 months ( n = 3 ) of age versus NOTCH3 EGF1 - 5 - immunized ( n = 10 ) , sham ( n = 8 ) and non - vaccinated ( n = 6 ) TgN3R182C150 mice at 7 months of age . Statistical significance was assessed using a Brown - Forsythe and Welch ANOVA tests followed by Dunnett ' s T3 multiple comparisons . ( NOTCH3 ECD deposits ( % of vessel area ) : 7 m . o . vs . Sham , ns P = 0 . 9424 ; 7 m . o . vs . Vaccinated , ns P = 0 . 9809 ; 7 m . o . vs . 18 m . o . * * P = 0 . 0046 ; Sham vs . Vaccinated , ns P = 0 . 9999 ; Sham vs . 18 m . o . * * P = 0 . 0032 ; Vaccinated vs . 18 m . o . * * P = 0 . 0032 ) . NOTCH3 ECD deposits ( number / 1000 um2 ) : 7 m . o . vs . Sham , ns P = 0 . 7877 ; 7 m . o . vs . Vaccinated , ns P = 0 . 8358 ; 7 m . o . vs . 18 m . o . * * P = 0 . 0069 ; Sham vs . Vaccinated , ns P = 0 . 9995 ; Sham vs . 18 m . o . * * P = 0 . 0044 ; Vaccinated vs . 18 m . o . * P = 0 . 0104 ) . NOTCH3 ECD deposits size : 7 m . o . vs . Sham , ns P = 0 . 1169 ; 7 m . o . vs . Vaccinated , ns P > 0 . 9999 ; 7 m . o . vs . 18 m . o . * * * P = 0 . 0009 ; Sham vs . Vaccinated , ns P = 0 . 0577 ; Sham vs . 18 m . o . * * P = 0 . 007 ; Vaccinated vs . 18 m . o . * * * * P < 0 . 0001 , ns = non - significant ) . Error bars indicate standard error of the mean ( SEM ) .

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

Figure 4A) Representative images of TgN3R182C150, sham- and NOTCH3 EGF1-5-immunized mice at 7 months of age and TgN3R182C150 at 18 months of age. Representative images show brain arteries of TgN3R182C150 (7 and 18 months), sham and NOTCH3 EGF1-5-immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an α-SMA antibody (green). Scale bar =20µm. B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF1-5 -immunized (n=10), sham (n=8) and non-vaccinated (n=6) TgN3R182C150 mice at 7 months of age. NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C150 mice at 18 months (n=3) of age versus NOTCH3 EGF1-5 -immunized (n=10), sham (n=8) and non-vaccinated (n=6) TgN3R182C150 mice at 7 months of age. Statistical significance was assessed using a Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons. (NOTCH3 ECD deposits (% of vessel area): 7 m.o. vs. Sham, ns P=0.9424; 7 m.o. vs. Vaccinated, ns P=0.9809; 7 m.o. vs. 18 m.o.**P=0.0046; Sham vs. Vaccinated, ns P=0.9999; Sham vs. 18 m.o. **P=0.0032; Vaccinated vs. 18 m.o. **P=0.0032). NOTCH3 ECD deposits (number/1000 um2): 7 m.o. vs. Sham, ns P=0.7877; 7 m.o. vs. Vaccinated, ns P=0.8358; 7 m.o. vs. 18 m.o.**P=0.0069; Sham vs. Vaccinated, ns P=0.9995; Sham vs. 18 m.o. **P=0.0044; Vaccinated vs. 18 m.o. *P=0.0104). NOTCH3 ECD deposits size: 7 m.o. vs. Sham, ns P=0.1169; 7 m.o. vs. Vaccinated, ns P>0.9999; 7 m.o. vs. 18 m.o.***P=0.0009; Sham vs. Vaccinated, ns P=0.0577; Sham vs. 18 m.o. **P=0.007; Vaccinated vs. 18 m.o. ****P<0.0001, ns=non-significant). Error bars indicate standard error of the mean (SEM).


Figure 5A ) Representative images of TgN3R182C150 , sham - and NOTCH3 EGF1 - 5 - immunized mice at 3 , 7 and 18 months of age . Representative images show brain arteries and capillaries of TgN3R182C150 , sham and NOTCH3 EGF1 - 5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) and an anti - perlecan antibody ( green ) . Scale bar = 20µm . B ) Quantification of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 - ECD stained area and average size per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non - vaccinated 3 months old TgN3R182C150 ( n = 3 ) and 7 months old TgN3R182C150 ( n = 6 ) mice and 18 months old TgN3R182C150 ( n = 3 ) . Quantification of NOTCH3 - ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 - ECD stained area and average size per vessel revealed a significant decrease in NOTCH3 - ECD deposition in brain arteries and capillaries between NOTCH3 EGF1 - 5 - immunized ( n = 11 ) , sham ( n = 8 ) and non - vaccinated TgN3R182C150 ( n = 6 ) mice . Statistical significance was assessed using a Brown - Forsythe and Welch ANOVA tests followed by Dunnett ' s T3 multiple comparisons . ( NOTCH3 ECD deposits ( % of vessel area ) : 3 m . o . vs . 18 m . o . * * P = 0 . 0029 ; 3 m . o . vs . 7 m . o . * * * P = 0 . 0004 ; 7 m . o . vs . Sham , ns P = 0 . 7326 ; 7 m . o . vs . Vaccinated , * * P = 0 . 003 ; Sham vs . Vaccinated , * * * * P < 0 . 0001 ) . NOTCH3 ECD deposits ( number / 1000 um2 ) : 3 m . o . vs . 18 m . o . * * P = 0 . 0038 ; 3 m . o . vs . 7 m . o . * * * P = 0 . 0002 ; 7 m . o . vs . Sham , ns P = 0 . 9913 ; 7 m . o . vs . Vaccinated , * P = 0 . 021 ; Sham vs . Vaccinated , * * * P = 0 . 001 ) . NOTCH3 ECD deposits size : 3 m . o . vs . 18 m . o . * P = 0 . 0215 ; 3 m . o . vs . 7 m . o . * * P = 0 . 0029 ; 7 m . o . vs . Sham , ns P = 0 . 3622 ; 7 m . o . vs . Vaccinated , * P = 0 . 0428 ; Sham vs . Vaccinated , * * P = 0 . 0038 , ns = non - significant ) . Dotted lines indicate quartiles and dashed thicker lines are the median .

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

Figure 5A) Representative images of TgN3R182C150, sham- and NOTCH3 EGF1-5-immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C150, sham and NOTCH3 EGF1-5-immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an anti-perlecan antibody (green). Scale bar =20µm. B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3-ECD stained area and average size per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non-vaccinated 3 months old TgN3R182C150 (n=3) and 7 months old TgN3R182C150 (n=6) mice and 18 months old TgN3R182C150 (n=3). Quantification of NOTCH3-ECD deposits (numbers per 1,000 μm2) and NOTCH3-ECD stained area and average size per vessel revealed a significant decrease in NOTCH3-ECD deposition in brain arteries and capillaries between NOTCH3 EGF1-5 -immunized (n=11), sham (n=8) and non-vaccinated TgN3R182C150 (n=6) mice. Statistical significance was assessed using a Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons. (NOTCH3 ECD deposits (% of vessel area): 3 m.o. vs. 18 m.o. **P=0.0029; 3 m.o. vs. 7 m.o. ***P=0.0004; 7 m.o. vs. Sham, ns P=0.7326; 7 m.o. vs. Vaccinated, **P=0.003; Sham vs. Vaccinated, ****P<0.0001). NOTCH3 ECD deposits (number/1000 um2): 3 m.o. vs. 18 m.o. **P=0.0038; 3 m.o. vs. 7 m.o. ***P=0.0002; 7 m.o. vs. Sham, ns P=0.9913; 7 m.o. vs. Vaccinated, *P=0.021; Sham vs. Vaccinated, ***P=0.001). NOTCH3 ECD deposits size: 3 m.o. vs. 18 m.o. *P=0.0215; 3 m.o. vs. 7 m.o. **P=0.0029; 7 m.o. vs. Sham, ns P=0.3622; 7 m.o. vs. Vaccinated, *P=0.0428; Sham vs. Vaccinated, **P=0.0038, ns= non-significant). Dotted lines indicate quartiles and dashed thicker lines are the median.


Figure 6A ) NOTCH3 ECD was detected in the whole blood serum of the non - treated TgN3R182C150 mice at three months ( n = 9 ) of age and further increased at seven months ( n = 6 ) of age . Serum from Notch3 - / - and C57Bl6 / J WT mice were included as negative controls . Statistical analysis was performed using unpaired student t test with Welch ' s correction . ( 3 m . o . vs 7 m . o . * * * P = 0 . 0005 ) . Error bars indicate standard error of the mean ( SEM ) . B ) NOTCH3 ECD in the TgN3R182C150 mice was significantly reduced in the vaccinated ( n = 10 ) TgN3R182C150 mice vs sham ( n = 9 ) . Statistical analysis was performed using unpaired student t test with Welch ' s correction . ( Sham vs Vaccinated * P = 0 . 0196 ) . Error bars indicate standard error of the mean ( SEM ) .

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

Figure 6A) NOTCH3 ECD was detected in the whole blood serum of the non-treated TgN3R182C150 mice at three months (n=9) of age and further increased at seven months (n=6) of age. Serum from Notch3 -/- and C57Bl6/J WT mice were included as negative controls. Statistical analysis was performed using unpaired student t test with Welch's correction. (3 m.o. vs 7 m.o. ***P=0.0005). Error bars indicate standard error of the mean (SEM).B) NOTCH3 ECD in the TgN3R182C150 mice was significantly reduced in the vaccinated (n=10) TgN3R182C150 mice vs sham (n=9). Statistical analysis was performed using unpaired student t test with Welch's correction. (Sham vs Vaccinated *P=0.0196). Error bars indicate standard error of the mean (SEM).


Figure 7A ) Representative images show microglia stained with anti - CD68 antibody ( red ) and Iba1 antibody ( green ) . Scale bar = 20µm . B ) Quantification of CD68 - stained area revealed a significant increase in the % of microglia and microglia area between N3 EGF1 - 5 - immunized ( n = 6 ) , sham ( n = 4 ) and non - vaccinated TgN3R182C150 ( n = 5 ) mice . Statistical significance was assessed using an ordinary one - way ANOVA followed by Tukey ' s multiple comparisons test . ( % microglia with CD68 staining : 7 m . o . vs . Sham ns P = 0 . 7393 ; Sham vs . Vaccinated * * P = 0 . 0087 . CD68 staining ( % of microglia area ) : 7 m . o . vs . Sham ns P = 0 . 9131 ; Sham vs . Vaccinated * P = 0 . 0157 , ns = non - significant ) . Error bars indicate standard error of the mean ( SEM ) . C ) Representative images of TgN3R182C150 , sham - and NOTCH3 EGF1 - 5 - immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) and an antibody against microglia ( Iba1 , green ) . Scale bar = 20µm . D ) Quantification of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF1 - 5 - immunized ( n = 11 ) , sham ( n = 8 ) and non - vaccinated TgN3R182C150 ( n = 6 ) mice at 7 months of age . Statistical significance was assessed using an ordinary one - way ANOVA followed by Tukey ' s multiple comparisons test . ( % microglia with NOTCH3 ECD deposits : 7 m . o . vs . Sham ns P = 0 . 7997 ; Sham vs . Vaccinated ns P = 0 . 0526 . NOTCH3 ECD deposits ( % of microglia area ) : 7 m . o . vs . Sham ns P = 0 . 5362 ; Sham vs . Vaccinated ns P = 0 . 8059 . NOTCH3 ECD deposits ( number / 1000 um2 ) : 7 m . o . vs . Sham ns P = 0 . 8392 ; Sham vs . Vaccinated ns P = 0 . 5777 . NOTCH3 ECD deposits size : 7 m . o . vs . Sham ns P = 0 . 6664 ; Sham vs . Vaccinated ns P = 0 . 8787 , ns = non - significant ) . Error bars indicate standard error of the mean ( SEM ) .

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

Figure 7A) Representative images show microglia stained with anti-CD68 antibody (red) and Iba1 antibody (green). Scale bar=20µm. B) Quantification of CD68-stained area revealed a significant increase in the % of microglia and microglia area between N3 EGF1-5-immunized (n=6), sham (n=4) and non-vaccinated TgN3R182C150 (n=5) mice. Statistical significance was assessed using an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (% microglia with CD68 staining: 7 m.o. vs. Sham ns P=0.7393; Sham vs. Vaccinated **P=0.0087. CD68 staining (% of microglia area): 7 m.o. vs. Sham ns P=0.9131; Sham vs. Vaccinated *P=0.0157, ns=non-significant). Error bars indicate standard error of the mean (SEM).C) Representative images of TgN3R182C150, sham- and NOTCH3 EGF1-5-immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an antibody against microglia (Iba1, green). Scale bar=20µm. D) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF1-5-immunized (n=11), sham (n=8) and non-vaccinated TgN3R182C150 (n=6) mice at 7 months of age. Statistical significance was assessed using an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (% microglia with NOTCH3 ECD deposits: 7 m.o. vs. Sham ns P=0.7997; Sham vs. Vaccinated ns P=0.0526. NOTCH3 ECD deposits (% of microglia area): 7 m.o. vs. Sham ns P=0.5362; Sham vs. Vaccinated ns P=0.8059. NOTCH3 ECD deposits (number/1000 um2): 7 m.o. vs. Sham ns P=0.8392; Sham vs. Vaccinated ns P=0.5777. NOTCH3 ECD deposits size: 7 m.o. vs. Sham ns P=0.6664; Sham vs. Vaccinated ns P=0.8787, ns=non-significant). Error bars indicate standard error of the mean (SEM).