Immunofluorescence analysis of total TDP - 43 and phosphorylated TDP - 43 ( p - TDP - 43 ) in sagittal brain sections by confocal microscopy . Representative images of one confocal layer in the midbrain . Zoom in on TDP - 43 aggregation in double knockout mouse brain sections of the midbrain . Scale bar indicates 25 µm . ( Animals 4 . 5 months of age ) .

Immunofluorescence analysis of total TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sagittal brain sections by confocal microscopy. Representative images of one confocal layer in the midbrain. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain. Scale bar indicates 25 µm. (Animals 4.5 months of age).


Western blot analysis of TDP - 43 and phosphorylated TDP - 43 ( p - TDP - 43 ) in sequential high salt ( HS ) / RIPA / Urea fractionations of total brain lysates from 4 . 5 - month - old mice with the indicated genotype . Analysis of C - terminal fragments of TDP - 43 ( CTF ) in RIPA fraction . p - TDP - 43 analyzed in Urea fraction . Quantification of ( C ) Protein expression was normalized to levels in wild type animals . Data represent the mean ± SD .

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Western blot analysis of TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sequential high salt (HS)/RIPA/Urea fractionations of total brain lysates from 4.5-month-old mice with the indicated genotype. Analysis of C-terminal fragments of TDP-43 (CTF) in RIPA fraction. p-TDP-43 analyzed in Urea fraction. Quantification of (C) Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.


, B . survival of Ssrp1fl / fl ; CreERT2 + / + mice of different ages ( weeks ) after the start of tamoxifen ( TMX ) treatment . B . Kaplan - Meier survival curves . Log - rank test p = 0 . 032 for the oldest male age group versus all other male groups ; p = 0 . 013 for all females versus all males ; for all other comparisons p > 0 . 05 . n = 6 - 10 mice .

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, B. survival of Ssrp1fl/fl; CreERT2+/+ mice of different ages (weeks) after the start of tamoxifen (TMX) treatment. B. Kaplan-Meier survival curves. Log-rank test p = 0.032 for the oldest male age group versus all other male groups; p = 0.013 for all females versus all males; for all other comparisons p > 0.05. n = 6-10 mice.


C . Normalized blood cell counts in Ssrp1fl / fl ; CreERT2 + / + mice treated with vehicle or tamoxifen on day 7 after start of treatment . Mean ± SD , * indicates p < 0 . 05 ( unpaired t - test ) . n = 3 mice .

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C. Normalized blood cell counts in Ssrp1fl/fl; CreERT2+/+ mice treated with vehicle or tamoxifen on day 7 after start of treatment. Mean ± SD, * indicates p < 0.05 (unpaired t-test). n = 3 mice.


A . H & E staining of mouse organ sections . Scale bars : spleen , colon 100 μM , bone marrow - 50 μM .

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A. H&E staining of mouse organ sections. Scale bars: spleen, colon 100 μM, bone marrow - 50 μM.


A , B . Effect of tamoxifen on replication in the spleen ( A ) and intestine ( B ) of Ssrp1fl / fl ; CreERT2 + / + mice . EdU was given to mice three days after the end of tamoxifen treatment ( 1 hour before tissue collection ) . Organ sections were stained for total DNA ( Hoechst ) , EdU , and SSRP1 . Scale bars - 100 μM . C . Quantitation of EdU staining . Mean number of EdU positive cells per field of view + / - SD . n > 5 fields of view per mouse . 3 mice were analyzed . D . Quantitation of cleaved caspase - 3 staining . Mean number of positive cells per fields of view + / - SD , n > 5 fields of view per mouse Two male and female mice were analyzed . Data information : P - values for C and D were calculated using the unpaired t - test .

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A, B. Effect of tamoxifen on replication in the spleen (A) and intestine (B) of Ssrp1fl/fl; CreERT2+/+ mice. EdU was given to mice three days after the end of tamoxifen treatment (1 hour before tissue collection). Organ sections were stained for total DNA (Hoechst), EdU, and SSRP1. Scale bars - 100 μM. C. Quantitation of EdU staining. Mean number of EdU positive cells per field of view +/- SD. n > 5 fields of view per mouse. 3 mice were analyzed. D. Quantitation of cleaved caspase-3 staining. Mean number of positive cells per fields of view +/- SD, n > 5 fields of view per mouse Two male and female mice were analyzed. Data information: P-values for C and D were calculated using the unpaired t-test.


D . Fold - change in the proportion of cells in each cluster shown in panel A in samples from tamoxifen - treated versus vehicle - treated mice . Cell types are identified by color as in panel B .

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D. Fold-change in the proportion of cells in each cluster shown in panel A in samples from tamoxifen-treated versus vehicle-treated mice. Cell types are identified by color as in panel B.


D . UMAP plots with color - coded intestinal stem cells identified by transcriptional signatures of intestinal stem cells or by expression of Lgr5 or Gkn3 genes .

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D. UMAP plots with color-coded intestinal stem cells identified by transcriptional signatures of intestinal stem cells or by expression of Lgr5 or Gkn3 genes.


A , B . Effect of 4 - OHT administration on colony formation by bone marrow cells of different genotypes . A . Photographs of colonies from bone marrow cells kept in culture for seven days in the presence or absence of 4 - OHT . B . Quantitation of bone marrow colony assay . Mean colony number per plate ± SD , n = 3 plates per mouse . P values < 0 . 05 are shown ( unpaired t - test ) . 2 mice of each genotype were analyzed . C , D . Growth of intestinal organoids in the presence or absence of 4 - OHT . C . Photographs of small intestine organoids on different days after plating . D . Number of organoids in the presence and absence of 4 - OHT . Mean fold change between number of organoids in control and 4 - OHT treated wells ± SD , n = 4 wells per mouse . P < 0 . 05 are shown ( unpaired t - test ) . Organoids were isolated from one mouse of each genotype in two independent experiments .

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A, B. Effect of 4-OHT administration on colony formation by bone marrow cells of different genotypes. A. Photographs of colonies from bone marrow cells kept in culture for seven days in the presence or absence of 4-OHT. B. Quantitation of bone marrow colony assay. Mean colony number per plate ± SD, n = 3 plates per mouse. P values < 0.05 are shown (unpaired t-test). 2 mice of each genotype were analyzed. C, D. Growth of intestinal organoids in the presence or absence of 4-OHT. C. Photographs of small intestine organoids on different days after plating. D. Number of organoids in the presence and absence of 4-OHT. Mean fold change between number of organoids in control and 4-OHT treated wells ± SD, n = 4 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from one mouse of each genotype in two independent experiments.


F , G . Effect of 4 - OHT administration on intestinal organoids of different genotypes kept in the presence ( undifferentiated ) or absence ( differentiated ) of R - spondin - 1 . F . The viability of organoids assessed by the resorufin assay . Mean ± SD , n = 3 wells per mouse . P < 0 . 05 are shown ( unpaired t - test ) . Organoids were isolated from two mice of each genotype . G . IF images of intestinal organoids from Ssrp1fl / fl ; CreERT2 + / + mice kept in the presence or absence of R - Spondin - 1 . The organoids were stained with antibodies to LGR5 and SSRP1 and counterstained with Hoechst ( DNA ) .

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F, G. Effect of 4-OHT administration on intestinal organoids of different genotypes kept in the presence (undifferentiated) or absence (differentiated) of R-spondin-1. F. The viability of organoids assessed by the resorufin assay. Mean ± SD, n = 3 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from two mice of each genotype. G. IF images of intestinal organoids from Ssrp1fl/fl; CreERT2+/+ mice kept in the presence or absence of R-Spondin-1. The organoids were stained with antibodies to LGR5 and SSRP1 and counterstained with Hoechst (DNA).


Effect of 4 - OHT administration on MSCs and their differentiated counterparts . H . Methylene blue viability assay for undifferentiated MSCs or MSCs differentiated into osteocytes or adipocytes for 21 days and then treated with vehicle or 4 - OHT . Mean ± SD , n = 3 plates per mouse . P < 0 . 05 are shown ( unpaired t - test ) . MSCs were isolated from two mice of each genotype .

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Effect of 4-OHT administration on MSCs and their differentiated counterparts. H. Methylene blue viability assay for undifferentiated MSCs or MSCs differentiated into osteocytes or adipocytes for 21 days and then treated with vehicle or 4-OHT. Mean ± SD, n = 3 plates per mouse. P < 0.05 are shown (unpaired t-test). MSCs were isolated from two mice of each genotype.


A . EU incorporation measured by flow cytometry . Histograms of EU fluorescent signals from two biological replicates for each condition ( except no EU ) . B . SSRP1 staining of the cells in A .

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A. EU incorporation measured by flow cytometry. Histograms of EU fluorescent signals from two biological replicates for each condition (except no EU). B. SSRP1 staining of the cells in A.


G . Box plots of mean read density ( y - axis ) at the gene body ( bins 21 - 100 out of 100 between TSS - TES ) for all genes and genes expressed at different levels . * * * - p < 1e - 10 ( paired t - test ) . n = 2 biological replicates . Central band is mean , box is interquartile range , whiskers are minimum and maximum .

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G. Box plots of mean read density (y-axis) at the gene body (bins 21-100 out of 100 between TSS - TES) for all genes and genes expressed at different levels. *** - p < 1e-10 (paired t-test). n = 2 biological replicates. Central band is mean, box is interquartile range, whiskers are minimum and maximum.


H . Comparison of Hoechst 33342 binding to live control and 4 - OHT - treated MSCs from different genotypes . Data are presented as the median fluorescence intensity ± SD , n = 4 biological replicates . P < 0 . 05 is shown ( unpaired t - test ) .

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H. Comparison of Hoechst 33342 binding to live control and 4-OHT-treated MSCs from different genotypes. Data are presented as the median fluorescence intensity ± SD, n = 4 biological replicates. P < 0.05 is shown (unpaired t-test).


( F ) Representative images of GFP - positive HEK293 cells at day 7 post - transduction using 1x106 TU / cell of RGD4C . TPA . GFP or RGD4C . AAVP . GFP particles . Non - targeted ( NT ) particles were included as control . Data shown are representative of five independent experiments , performed each in triplicates . Scale bar , 100 μm .

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(F) Representative images of GFP-positive HEK293 cells at day 7 post-transduction using 1x106 TU/cell of RGD4C.TPA.GFP or RGD4C.AAVP.GFP particles. Non-targeted (NT) particles were included as control. Data shown are representative of five independent experiments, performed each in triplicates. Scale bar, 100 μm.


( A ) Quantification of gene expression in HEK293 cells after transduction with 0 . 5x106 , 1x106 , and 2x106 TU / cell of RGD4C . TPA , RGD4C . AAVP or non - targeted ( NT ) particles carrying Lucia . Luciferase activity was measured at days 1 to 6 post - transduction . Data shown are representative of three independent experiments ( n = 3 ) , and are expressed as mean + SEM . One - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(A) Quantification of gene expression in HEK293 cells after transduction with 0.5x106, 1x106, and 2x106 TU/cell of RGD4C.TPA, RGD4C.AAVP or non-targeted (NT) particles carrying Lucia. Luciferase activity was measured at days 1 to 6 post-transduction. Data shown are representative of three independent experiments (n = 3), and are expressed as mean + SEM. One-way ANOVA with Tukey's HSD test was used for data analysis.


( B ) Representative images of FITC - labelled TPA and AAVP particle diffusion in Matrigel at 5 and 30 minutes post - inoculation ( n = 3 ) . Scale bar , 100 μm . ( C ) Particle diffusion using a vertical trans - well system partitioned by Matrigel . Samples were collected from the wells below the Matrigel at days 1 to 7 , n = 3 , technical replicates . Data are expressed as mean + SEM , independent t - test was used for data analysis .

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(B) Representative images of FITC-labelled TPA and AAVP particle diffusion in Matrigel at 5 and 30 minutes post-inoculation (n = 3). Scale bar, 100 μm. (C) Particle diffusion using a vertical trans-well system partitioned by Matrigel. Samples were collected from the wells below the Matrigel at days 1 to 7, n = 3, technical replicates. Data are expressed as mean + SEM, independent t-test was used for data analysis.


( D ) Comparison of AAVP and TPA internalisation efficiency in HEK293 cells as evaluated by staining of the phage capsid proteins , then quantified by FACS at 2 and 4 hours post - transduction , 0 . 1X106TU / cell , and normalised to particle length . Data are expressed as mean + SEM of n = 3 independent experiments , independent t - test was used for data analysis .

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(D) Comparison of AAVP and TPA internalisation efficiency in HEK293 cells as evaluated by staining of the phage capsid proteins, then quantified by FACS at 2 and 4 hours post-transduction, 0.1X106TU/cell, and normalised to particle length. Data are expressed as mean + SEM of n = 3 independent experiments, independent t-test was used for data analysis.


( E ) Quantification of ITRs in the cytosol and nucleus of HEK293 cells 24 hours post - transduction with 0 . 5x106 TU / cell of RGD4C . TPA and RGD4C . AAVP , using qPCR with primers specific to AAV - 2 ITRs . Data are representative of n = 3 independent experiments and expressed as mean + SEM , independent t - test was used for data analysis .

(E) Quantification of ITRs in the cytosol and nucleus of HEK293 cells 24 hours post-transduction with 0.5x106 TU/cell of RGD4C.TPA and RGD4C.AAVP, using qPCR with primers specific to AAV-2 ITRs. Data are representative of n = 3 independent experiments and expressed as mean + SEM, independent t-test was used for data analysis.


( F ) Quantification of Lucia reporter gene expression following transduction of human primary SEBTA003 glioblastoma and G26 glioblastoma - derived neural stem cells with 1x106 TU / cell . Data are expressed as mean + SEM of n = 3 independent experiments . One - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(F) Quantification of Lucia reporter gene expression following transduction of human primary SEBTA003 glioblastoma and G26 glioblastoma-derived neural stem cells with 1x106 TU/cell. Data are expressed as mean + SEM of n = 3 independent experiments. One-way ANOVA with Tukey's HSD test was used for data analysis.


( G ) 3D primary GBM001 tumor spheres subjected to transduction by increasing doses of TPA and AAVP encoding Lucia . Experiments were repeated three times and performed in triplicates . Data are shown as mean + SEM and are representative of one experiment . Independent t - test was used for data analysis .

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(G) 3D primary GBM001 tumor spheres subjected to transduction by increasing doses of TPA and AAVP encoding Lucia. Experiments were repeated three times and performed in triplicates. Data are shown as mean + SEM and are representative of one experiment. Independent t-test was used for data analysis.


( B ) ELISA quantification of TNFα production in media collected on days 3 to 5 post - transduction of human primary GBM001 tumor spheres at 1x106 TU / cell of targeted ( RGD4C ) or non - targeted ( NT ) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences . Data are representative of one experiment and expressed as mean + SEM . Experiments were repeated twice in triplicates , one - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(B) ELISA quantification of TNFα production in media collected on days 3 to 5 post-transduction of human primary GBM001 tumor spheres at 1x106 TU/cell of targeted (RGD4C) or non-targeted (NT) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis.


( C ) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C . TPA . TNFαIL2 to NOD / SCID mice bearing subcutaneous human GBM001 . Tumors and healthy organs were harvested at day 18 post - transduction and analysed using RT - qPCR to quantify the TNFαIL2 transcripts . Data shown are expressed as mean + SEM , from n = 3 mice . Two - way ANOVA was used for data analysis .

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(C) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C.TPA.TNFαIL2 to NOD/SCID mice bearing subcutaneous human GBM001. Tumors and healthy organs were harvested at day 18 post-transduction and analysed using RT-qPCR to quantify the TNFαIL2 transcripts. Data shown are expressed as mean + SEM, from n = 3 mice. Two-way ANOVA was used for data analysis.


( D ) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene , at day 18 post targeted TNFαIL2 cytokine treatment . ( E ) Analysis of tumor growth in representative mice , from D , using the tumor bioluminescence data ( total flux ) and expressed as fold changes of tumor luminescence flux at different time points compared to day 0 .

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(D) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene, at day 18 post targeted TNFαIL2 cytokine treatment. (E) Analysis of tumor growth in representative mice, from D, using the tumor bioluminescence data (total flux) and expressed as fold changes of tumor luminescence flux at different time points compared to day 0.


( B ) Investigation of the TPA . IL15IgK in comparison to the native TPA . IL15 in B16 . F1 and CT26 . CL25 tumor cells . The TPA DNA constructs were transfected into tumor cells , then the bioactivity of secreted IL15 was assessed by evaluating the effects of media from transfected cells on the proliferation of mouse CTLL - 2 . Data are representative of one experiment and expressed as mean + SEM . Experiments were repeated twice and performed in triplicates , one - way ANOVA with Tukey ' s HSD test was used for data

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(B) Investigation of the TPA.IL15IgK in comparison to the native TPA.IL15 in B16.F1 and CT26.CL25 tumor cells. The TPA DNA constructs were transfected into tumor cells, then the bioactivity of secreted IL15 was assessed by evaluating the effects of media from transfected cells on the proliferation of mouse CTLL-2. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice and performed in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis


( C ) Targeted ( RGD4C ) and non - targeted ( NT ) TPA particles carrying IL15IgK were produced and used to transduce B16 . F1 or CT26 . CL25 cells at 0 . 5x106 or 1x106 TU / cell . Then secreted IL15 was quantified by ELISA in tumor cell media collected on day 5 post - transduction . RGD4C . TPA without IL15IgK ( mock ) was also included as control . Experiments were repeated twice and shown are the results from a representative experiment . Data are expressed as mean + SEM , one - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(C) Targeted (RGD4C) and non-targeted (NT) TPA particles carrying IL15IgK were produced and used to transduce B16.F1 or CT26.CL25 cells at 0.5x106 or 1x106 TU/cell. Then secreted IL15 was quantified by ELISA in tumor cell media collected on day 5 post-transduction. RGD4C.TPA without IL15IgK (mock) was also included as control. Experiments were repeated twice and shown are the results from a representative experiment. Data are expressed as mean + SEM, one-way ANOVA with Tukey's HSD test was used for data analysis.


( D ) Evaluation of CTLL - 2 proliferation in the presence of media collected from B16 . F1 or CT26 . CL25 tumor cells after transduction with TPA particles carrying IL15IgK . Cells treated with a recombinant IL2 cytokine were included as a positive control . Data are representative of one experiment , expressed as mean + SEM and shown as change in CTLL - 2 number relative to treatment initiation day . Experiments were repeated twice and in triplicates , one - way ANOVA with Tukey ' s HSD test was used to analyse the data .

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(D) Evaluation of CTLL-2 proliferation in the presence of media collected from B16.F1 or CT26.CL25 tumor cells after transduction with TPA particles carrying IL15IgK. Cells treated with a recombinant IL2 cytokine were included as a positive control. Data are representative of one experiment, expressed as mean + SEM and shown as change in CTLL-2 number relative to treatment initiation day. Experiments were repeated twice and in triplicates, one-way ANOVA with Tukey's HSD test was used to analyse the data.


( A ) Tumor - bearing BALB / c mice with established subcutaneous CT26 . CL25 tumors were intravenously injected with a single dose , 5x1010 TU / mouse , of targeted ( RGD4C ) or non - targeted ( NT ) TPA . IL15IgK . Gene delivery was evaluated by quantifying the IL15IgK mRNA expression in tumors and healthy tissues at day 5 post TPA administration . Data are representative of one experiment , n = 3 , and expressed as mean + SEM . Two - way ANOVA test was used for data analysis .

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(A) Tumor-bearing BALB/c mice with established subcutaneous CT26.CL25 tumors were intravenously injected with a single dose, 5x1010 TU/mouse, of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Gene delivery was evaluated by quantifying the IL15IgK mRNA expression in tumors and healthy tissues at day 5 post TPA administration. Data are representative of one experiment, n = 3, and expressed as mean + SEM. Two-way ANOVA test was used for data analysis.


( B ) Safety of dose escalation regimens assessed at day 5 following TPA delivery , by evaluating the serum levels of LDH from n = 2 mice / TPA dose of targeted ( RGD4C ) or non - targeted ( NT ) TPA . IL15IgK . Serums from untreated mice were also analysed .

(B) Safety of dose escalation regimens assessed at day 5 following TPA delivery, by evaluating the serum levels of LDH from n = 2 mice/TPA dose of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Serums from untreated mice were also analysed.


( C ) Representative tumor - bearing mice showing bioluminescence imaging of luciferase on days 0 and 5 post administration with 5x1010 TU of RGD4C . TPA . IL15IgK ( RGD4C ) or non - targeted ( NT ) particles . The CT26 . CL25 tumor cells stably express a luciferase Luc reporter gene . Untreated mice were also included as controls ( CTRL ) .

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(C) Representative tumor-bearing mice showing bioluminescence imaging of luciferase on days 0 and 5 post administration with 5x1010 TU of RGD4C.TPA.IL15IgK (RGD4C) or non-targeted (NT) particles. The CT26.CL25 tumor cells stably express a luciferase Luc reporter gene. Untreated mice were also included as controls (CTRL).


( D ) Changes in tumor viability in mice ( n = 4 ) from day 0 to day 5 post TPA administration . Tumor viability was measured by bioluminescence activity . The central band represents the median of the data . The boxes upper and lower lines represent quartile 3 and quartile 1 of the data , respectively . The whiskers represent the maximum and minimum outliers of the data . Non - parametric Kruskal - Wallis test was used for data analysis .

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(D) Changes in tumor viability in mice (n = 4) from day 0 to day 5 post TPA administration. Tumor viability was measured by bioluminescence activity. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Kruskal-Wallis test was used for data analysis.


( E ) Kaplan Meier curves showing survival benefit for tumor - bearing mice ( n = 4 ) from all experimental groups .

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(E) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from all experimental groups.


( F ) Animal weights ( n = 4 ) were monitored during therapy experiments ad expressed as mean + SEM .

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(F) Animal weights (n = 4) were monitored during therapy experiments ad expressed as mean + SEM.


( A ) The immune profile of CT26 . CL25 tumors was investigated using RT - qPCR at day 5 after TPA . IL15IgK administration . A panel of immunological mRNA transcripts were selected for analysis and included CD8a and CD8b as markers for CD8 + T cell population ; NKp46 for NK cell population . Markers for cytotoxic cell - killing by CD8 + T cells ( GZMa , GZMb ) and by NK cells ( Prf1 ) were also analysed . Expression of TBX21 was evaluated as a marker for the Th1 response mediated by interferon gamma ( IFN - γ ) , and FoxP3 for Treg cells . Data shown are representative of n = 5 mice and expressed as mean + SEM . Non - parametric Mann - Whitney test was used for GZMb and TBX2 data analysis , and independent t - test to analyse data of the other markers .

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(A) The immune profile of CT26.CL25 tumors was investigated using RT-qPCR at day 5 after TPA.IL15IgK administration. A panel of immunological mRNA transcripts were selected for analysis and included CD8a and CD8b as markers for CD8+ T cell population; NKp46 for NK cell population. Markers for cytotoxic cell-killing by CD8+ T cells (GZMa, GZMb) and by NK cells (Prf1) were also analysed. Expression of TBX21 was evaluated as a marker for the Th1 response mediated by interferon gamma (IFN-γ), and FoxP3 for Treg cells. Data shown are representative of n = 5 mice and expressed as mean + SEM. Non-parametric Mann-Whitney test was used for GZMb and TBX2 data analysis, and independent t-test to analyse data of the other markers.


( B ) TPA particles encoding IL12 were used to treat immunocompetent BALB / c mice bearing subcutaneous CT26 . CL25 tumors labelled with a Luc reporter gene . Representative tumor - bearing mice at day 5 post - treatment with targeted RGD4C . TPA . IL12 ( RGD4C ) or non - targeted TPA . IL12 ( NT ) .

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(B) TPA particles encoding IL12 were used to treat immunocompetent BALB/c mice bearing subcutaneous CT26.CL25 tumors labelled with a Luc reporter gene. Representative tumor-bearing mice at day 5 post-treatment with targeted RGD4C.TPA.IL12 (RGD4C) or non-targeted TPA.IL12 (NT).


( C ) Analysis of total flux change in tumors , n = 4 mice , after IL12 immunotherapy . The central band represents the median of the data . The boxes upper and lower lines represent quartile 3 and quartile 1 of the data , respectively . The whiskers represent the maximum and minimum outliers of the data . Non - parametric Mann - Whitney test was used for data analysis .

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(C) Analysis of total flux change in tumors, n = 4 mice, after IL12 immunotherapy. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Mann-Whitney test was used for data analysis.


( D ) Kaplan Meier curves showing survival benefit for tumor - bearing mice ( n = 4 ) from two experimental groups .

(D) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from two experimental groups.


( E ) Serial quantification of IL12 in media of B16 . F10 cells treated with RGD4C . TPA . IL12 or controls . Data are representative of one experiment , n = 3 biological repeats , and shown as mean + SD Experiments were repeated three times . One - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(E) Serial quantification of IL12 in media of B16.F10 cells treated with RGD4C.TPA.IL12 or controls. Data are representative of one experiment, n = 3 biological repeats, and shown as mean + SD Experiments were repeated three times. One-way ANOVA with Tukey's HSD test was used for data analysis.


( F ) RGD4C . TPA . IL12 immunotherapy of subcutaneous B16 . F10 tumors in C57BL / 6 mice ( n = 4 ) . Data are shown as mean + SEM independent t - test was used for data analysis . Tumor volume changes are shown over time .

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(F) RGD4C.TPA.IL12 immunotherapy of subcutaneous B16.F10 tumors in C57BL/6 mice (n = 4). Data are shown as mean + SEM independent t-test was used for data analysis. Tumor volume changes are shown over time.


B . Genome views of RNA read density profiles on representative loci enriched ( left ) or low ( right ) in FLAG - HP1γ RNAchIP ( IP ) relative to input RNA ( inp . ) in overlaid replicate samples from HP1γ cells stimulated or not with PMA . Orange arrow , position of the locus analyzed in D .

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B. Genome views of RNA read density profiles on representative loci enriched (left) or low (right) in FLAG-HP1γ RNAchIP (IP) relative to input RNA (inp.) in overlaid replicate samples from HP1γ cells stimulated or not with PMA. Orange arrow, position of the locus analyzed in D.


C . Genome - wide scatter plot of IP and input normalized ( norm . ) RNA read counts per gene for the mean of the triplicates ; n = 21 , 754 ; r , Pearson ' s correlation coefficient between IP and input .

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C. Genome-wide scatter plot of IP and input normalized (norm.) RNA read counts per gene for the mean of the triplicates; n=21,754; r, Pearson's correlation coefficient between IP and input.


D . Relative quantities of RNA in the RNAchIP samples , detected by RT - qPCR with primers aligning on the intron1 of the Fosl1 gene as depicted in B ( orange arrow ) . n = 3 independent experiments . Data information : Histograms represent mean and s . d . Dots represent individual data points . P - values indicate significantly higher difference between IP in HP1γ and in KO cells ( * * * P < 0 . 001 ; two - tailed Student ' s t - test ) .

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D. Relative quantities of RNA in the RNAchIP samples, detected by RT-qPCR with primers aligning on the intron1 of the Fosl1 gene as depicted in B (orange arrow). n=3 independent experiments. Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test).


E . Heat maps of RNA IP and input signal centered on the summit of RNAchIP peaks of IP versus input detected by MACS2 analysis ( 10 , 013 and 2609 peaks ) for a representative replicate ( replicate 1 ) . Black arrows represent peak summit .

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E. Heat maps of RNA IP and input signal centered on the summit of RNAchIP peaks of IP versus input detected by MACS2 analysis (10,013 and 2609 peaks) for a representative replicate (replicate 1). Black arrows represent peak summit.


F . Percentage of RNAchIP peaks overlapping with indicated chromatin features in MEF samples from ENCODE database ( green bars ) . Overlaps was evaluated by comparison with the list of merged peaks whose genomic location was randomized among genes with one hundred permutations ( random , gray bars ) . Data information : Histograms represent mean and s . d . Dots represent individual data points . P - values indicate significantly higher difference between IP in HP1γ and in KO cells ( * * * P < 0 . 001 ; two - tailed Student ' s t - test ) .

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F. Percentage of RNAchIP peaks overlapping with indicated chromatin features in MEF samples from ENCODE database (green bars). Overlaps was evaluated by comparison with the list of merged peaks whose genomic location was randomized among genes with one hundred permutations (random, gray bars). Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test).


B . RNAchIP peaks are enriched in CACACA and GAGAGA motifs . Number of peaks containing at least one exact hexameric motif as indicated , compared to the average number of motifs in peaks whose genomic location was randomized among genes with ten permutations ( random , light bars , represent mean and s . d . ) .

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B. RNAchIP peaks are enriched in CACACA and GAGAGA motifs. Number of peaks containing at least one exact hexameric motif as indicated, compared to the average number of motifs in peaks whose genomic location was randomized among genes with ten permutations (random, light bars, represent mean and s.d.).


C , D . Distribution profiles of average RNAchIP signal in both IP ( warm colors ) and input ( blue colors ) over ± 2kb centered on intronic ( C ) and exonic ( D ) CACACA hexameric motifs oriented in the same orientation ( sense ) or in opposite orientation ( antisense ) relative to the overlapping annotated transcript . The antisense CACACA motifs were obtained by querying the transcript sequences with the TGTGTG motif .

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C, D. Distribution profiles of average RNAchIP signal in both IP (warm colors) and input (blue colors) over ±2kb centered on intronic (C) and exonic (D) CACACA hexameric motifs oriented in the same orientation (sense) or in opposite orientation (antisense) relative to the overlapping annotated transcript. The antisense CACACA motifs were obtained by querying the transcript sequences with the TGTGTG motif.


E . Left , representative example of a locus on the Ppp3ca gene surrounding an RNAchIP merged peak , showing the RNAchIP read density as in Fig 1B . Red and orange arrowheads correspond to oriented CACACA and GAGAGA motifs , respectively . Right , sequence of the RNA surrounding a CACACA motif highlighted in red , as well as a second a neighboring imperfect motif . The sequence was used to design a CACACA - containing RNA oligonucleotide probe ( CACACA ) , compared to a control probe ( no - CA ) devoid of any related motif .

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E. Left, representative example of a locus on the Ppp3ca gene surrounding an RNAchIP merged peak, showing the RNAchIP read density as in Fig 1B. Red and orange arrowheads correspond to oriented CACACA and GAGAGA motifs, respectively. Right, sequence of the RNA surrounding a CACACA motif highlighted in red, as well as a second a neighboring imperfect motif. The sequence was used to design a CACACA-containing RNA oligonucleotide probe (CACACA), compared to a control probe (no-CA) devoid of any related motif.


F , Gel mobility shift assays of bacterially expressed , purified HP1γ - Hinge domain , fused to GST proteins , and tested for its direct interaction with the Cy3 - labeled RNA oligonucleotide probes The probes in the gels , either free ( white arrowheads ) or as shifted protein / RNA complexes ( red arrowheads ) were detected by their Cy3 fluorescence . Representative of 3 independent experiments .

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F, Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled RNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.


G , Gel mobility shift assays of bacterially expressed , purified HP1γ - Hinge domain , fused to GST proteins , and tested for its direct interaction with the Cy3 - labeled dsDNA oligonucleotide probes The probes in the gels , either free ( white arrowheads ) or as shifted protein / RNA complexes ( red arrowheads ) were detected by their Cy3 fluorescence . Representative of 3 independent experiments .

G, Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled dsDNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.


H . Gel mobility shift assays of bacterially expressed , purified HP1γ - Hinge domain , fused to GST proteins , and tested for its direct interaction with the Cy3 - labeled RNA oligonucleotide probes The probes in the gels , either free ( white arrowheads ) or as shifted protein / RNA complexes ( red arrowheads ) were detected by their Cy3 fluorescence . Representative of 3 independent experiments .

H. Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled RNA oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.


A . Fold enrichment values on all repeat masker classes and on Refseq genes , on the basis of all uniquely aligned reads , shown as mean and s . d . , n = 6 biological replicates . Dashed line represents an IP / Input ratio of 1 , taken as a reference .

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A. Fold enrichment values on all repeat masker classes and on Refseq genes, on the basis of all uniquely aligned reads, shown as mean and s.d., n=6 biological replicates. Dashed line represents an IP/Input ratio of 1, taken as a reference.


C . Profiles of average RNAchIP signal as in Fig 2B over ± 2kb centered on intragenic LINE , LTR , or SINE repeats annotated in Repeat masker database ( n = 34 , 000 ) . All repeats are oriented in the same orientation ( sense ) relative to the overlapping annotated transcript , unless otherwise specified ( antisense , shuffled ) , and as depicted by an oriented white box overlapping the transcript in 5 ' to 3 ' orientation ( black line with arrowhead ) . D . Profiles of average RNAchIP signal , as in ( C ) centered on the 5 major families of intragenic SINE repeats .

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C. Profiles of average RNAchIP signal as in Fig 2B over ±2kb centered on intragenic LINE, LTR, or SINE repeats annotated in Repeat masker database (n=34,000). All repeats are oriented in the same orientation (sense) relative to the overlapping annotated transcript, unless otherwise specified (antisense, shuffled), and as depicted by an oriented white box overlapping the transcript in 5' to 3' orientation (black line with arrowhead). D. Profiles of average RNAchIP signal, as in (C) centered on the 5 major families of intragenic SINE repeats.


E . Proportions of the families of CACACA - containing SINE repeats within each family , as a percentage of total intragenic sense SINEs .

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E. Proportions of the families of CACACA-containing SINE repeats within each family, as a percentage of total intragenic sense SINEs.


F . Profiles of average RNAchIP signal , as in ( D ) centered on the subset of intragenic B4 SINEs containing a consensus CACACA motif .

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F. Profiles of average RNAchIP signal, as in (D) centered on the subset of intragenic B4 SINEs containing a consensus CACACA motif.


G . Genomic position of a B4 SINE repeat and CACACA consensus motifs at the location of the RNAchIP

G. Genomic position of a B4 SINE repeat and CACACA consensus motifs at the location of the RNAchIP peak


H . North - western blot assay showing direct association between the indicated bacterially expressed , purified GST - fusion proteins and in vitro transcribed , biotinylated RNA probes based on the sequence of the B4 SINE depicted in ( G ) . Top panels , binding of a 256nt probe corresponding to the CACACA - containing B4 SINE sequence , was compared to an identical B4 SINE deleted of its CACACA by truncation of its 3 ' portion ( B4 SINE - Δ3 ' ) . RNA probes hybridized on the membranes were detected by their Cy3 fluorescence . Total GST - fusion protein loading was visualized by Ponceau S staining . Representative of two independent experiments .

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H. North-western blot assay showing direct association between the indicated bacterially expressed, purified GST-fusion proteins and in vitro transcribed, biotinylated RNA probes based on the sequence of the B4 SINE depicted in (G). Top panels, binding of a 256nt probe corresponding to the CACACA-containing B4 SINE sequence, was compared to an identical B4 SINE deleted of its CACACA by truncation of its 3' portion (B4 SINE-Δ3'). RNA probes hybridized on the membranes were detected by their Cy3 fluorescence. Total GST-fusion protein loading was visualized by Ponceau S staining. Representative of two independent experiments.


A . Box plot showing the distribution of fold enrichment values per genes sorted by exon number , n = 66 , 247 , 389 , and 574 genes with 1 , 2 , 3 , and 4 exons , respectively . Dashed line represents an IP / Input ratio of 1 , taken as a reference . B . Reads from input and IP counted in unstimulated and PMA stimulated triplicates give significant enrichment values of 1 . 4 and 0 . 7 on introns and exons , respectively , shown as mean and s . d . , n = 3 biological replicates . Dots represent individual data points . C . Box plot of reads belonging to spliced transcripts versus all reads in input or RNAchIP samples pooled between + and - PMA , calculated as a ratio between split reads and total reads , n = 3 biological replicates . Dots represent individual data points . Data information : All box boundaries represent 25th and 75th percentiles ; center line represents median ; whiskers indicate ± 1 . 5 × IQR ; points are values of outliers . All P - values are * P < 0 . 01 , * * P < 0 . 005 , * * * P < 0 . 001 ; two - tailed Student ' s t - test .

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A. Box plot showing the distribution of fold enrichment values per genes sorted by exon number, n= 66, 247, 389, and 574 genes with 1, 2, 3, and 4 exons, respectively. Dashed line represents an IP/Input ratio of 1, taken as a reference. B. Reads from input and IP counted in unstimulated and PMA stimulated triplicates give significant enrichment values of 1.4 and 0.7 on introns and exons, respectively, shown as mean and s.d., n=3 biological replicates. Dots represent individual data points. C. Box plot of reads belonging to spliced transcripts versus all reads in input or RNAchIP samples pooled between + and -PMA, calculated as a ratio between split reads and total reads, n=3 biological replicates. Dots represent individual data points. Data information: All box boundaries represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. All P-values are * P < 0.01, ** P < 0.005, ***P < 0.001; two-tailed Student's t-test.


D , E . Average distribution profiles of the depicted hexameric motifs ( D ) , or SINE and LINE repeats ( E ) on RefSeq genes , computed over exons ( black box ) and on ± 0 . 7kb of surrounding introns ( left panels ) , or over entire gene bodies including introns ( pre - mRNA ; middle panels ) , or as metagenes over exons only ( Metagene mRNA ; right panels ) .

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D, E. Average distribution profiles of the depicted hexameric motifs (D), or SINE and LINE repeats (E) on RefSeq genes, computed over exons (black box) and on ±0.7kb of surrounding introns (left panels), or over entire gene bodies including introns (pre-mRNA; middle panels), or as metagenes over exons only (Metagene mRNA; right panels).


F . Box plot showing the distribution of fold enrichment values on series of introns of comparable size ( 4kb in average , ranging from 3 to 5kb ) sorted by number of sense CACACA motifs per intron ( 2 + , two or more motifs ) , n = 5782 , 899 , 960 , and 3234 introns for all introns , 0 , 1 , and 2 + CACACA categories , respectively . R2 , Linear regression coefficient between 0 , 1 , and 2 + . Dashed line represents an IP / Input ratio of 1 , taken as a reference . Data information : All box boundaries in Fig 4 represent 25th and 75th percentiles ; center line represents median ; whiskers indicate ± 1 . 5 × IQR ; points are values of outliers . All P - values are * P < 0 . 01 , * * P < 0 . 005 , * * * P < 0 . 001 ; two - tailed Student ' s t - test .

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F. Box plot showing the distribution of fold enrichment values on series of introns of comparable size (4kb in average, ranging from 3 to 5kb) sorted by number of sense CACACA motifs per intron (2+, two or more motifs), n= 5782, 899, 960, and 3234 introns for all introns, 0, 1, and 2+ CACACA categories, respectively. R2, Linear regression coefficient between 0, 1, and 2+. Dashed line represents an IP/Input ratio of 1, taken as a reference. Data information: All box boundaries in Fig 4 represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. All P-values are * P < 0.01, ** P < 0.005, ***P < 0.001; two-tailed Student's t-test.


G , H . Average read profiles on two groups of RefSeq genes , in the size - range 4 to 15kb , containing the highest ( G ) or lowest ( H ) CACACA motif density .

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G, H. Average read profiles on two groups of RefSeq genes, in the size-range 4 to 15kb, containing the highest (G) or lowest (H) CACACA motif density.


C . Box plot depicting the length of exon junctions found differentially regulated by MAJIQ between HP1γ ( green ) and KO ( orange ) in three categories as in Fig EV5A , all classified differential HP1γ / KO junctions , annotated alternative exon junctions , non - annotated de novo cryptic junctions detected in KO only ; n , differential events . Box boundaries represent 25th and 75th percentiles ; center line represents median ; whiskers indicate ± 1 . 5 × IQR ; points are values of outliers . Data information : All graphes represent mean and s . d . ( * * P < 0 . 005 , * * * P < 0 . 001 ; two - tailed Student ' s t - test ) .

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C. Box plot depicting the length of exon junctions found differentially regulated by MAJIQ between HP1γ (green) and KO (orange) in three categories as in Fig EV5A, all classified differential HP1γ/KO junctions, annotated alternative exon junctions, non-annotated de novo cryptic junctions detected in KO only; n, differential events. Box boundaries represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; two-tailed Student's t-test).


D . RNAchIP read density per kb on the indicated introns for input and IP in samples from both untreated and PMA stimulated cells , n = 6 biological replicates , or 3 - times randomized intervals , n = 18 samples . Dots represent individual data points . HP1γ / KO introns are all 272 intronic intervals between exon junctions differentially detected in HP1γ and KO . Random introns are a matching library of random intronic intervals . E . RNAchIP read density per kb on the genes corresponding to the introns tested in ( D ) , and analyzed as in ( D ) , n = 6 biological replicates , or 3 - times randomized intervals , n = 18 samples . Data information : All graphes represent mean and s . d . ( * * P < 0 . 005 , * * * P < 0 . 001 ; two - tailed Student ' s t - test ) .

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D. RNAchIP read density per kb on the indicated introns for input and IP in samples from both untreated and PMA stimulated cells, n=6 biological replicates, or 3-times randomized intervals, n=18 samples. Dots represent individual data points. HP1γ/KO introns are all 272 intronic intervals between exon junctions differentially detected in HP1γ and KO. Random introns are a matching library of random intronic intervals. E. RNAchIP read density per kb on the genes corresponding to the introns tested in (D), and analyzed as in (D), n=6 biological replicates, or 3-times randomized intervals, n=18 samples. Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; two-tailed Student's t-test).


F . Reads from input and IP counted on the indicated features in combined unstimulated and PMA stimulated triplicate samples , n = 6 biological replicates . Dots represent individual data points . Data information : All graphes represent mean and s . d . ( * * P < 0 . 005 , * * * P < 0 . 001 ; one - tailed Student ' s t - test ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Reads from input and IP counted on the indicated features in combined unstimulated and PMA stimulated triplicate samples, n=6 biological replicates. Dots represent individual data points. Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; one-tailed Student's t-test).


G . Average distribution profiles of sense SINE repeats on indicated exons and on ± 0 . 7kb of surrounding introns .

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G. Average distribution profiles of sense SINE repeats on indicated exons and on ±0.7kb of surrounding introns.


B . RT - PCR analysis of the RNAs produced in the splicing assay with the Prl3 wt or Prl3 CAmut constructs as depicted in A transfected into HP1γ or KO cell lines . The top 210bp and bottom 120bp amplicons correspond to the included form ( beta globin with Prl exon , more included in lane1 , blue arrow , compared to lane 2 ) , and the skipped form ( beta globin exons only ) of RNA , respectively . Orange arrowheads , position of the HBGEX - F and HBGEX - R primers used in PCR analysis . C . Histogram showing the loss of inclusion of the Prl3 exon upon depletion of HP1γ ( in KO ) relative to inclusion in the presence of HP1γ with the Prl3 wt ( red ) or Prl3 CA mut ( grey ) reporters . Based on the quantitation of 4 experiments as the one depicted in B . Values represent the fold change relative to HP1γ as a reference ( dashed line ) . Graph represents mean and s . d . ( * * * P < 0 . 001 ; two - tailed Student ' s t - test ; n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RT-PCR analysis of the RNAs produced in the splicing assay with the Prl3 wt or Prl3 CAmut constructs as depicted in A transfected into HP1γ or KO cell lines. The top 210bp and bottom 120bp amplicons correspond to the included form (beta globin with Prl exon, more included in lane1, blue arrow, compared to lane 2), and the skipped form (beta globin exons only) of RNA, respectively. Orange arrowheads, position of the HBGEX-F and HBGEX-R primers used in PCR analysis. C. Histogram showing the loss of inclusion of the Prl3 exon upon depletion of HP1γ (in KO) relative to inclusion in the presence of HP1γ with the Prl3 wt (red) or Prl3 CA mut (grey) reporters. Based on the quantitation of 4 experiments as the one depicted in B. Values represent the fold change relative to HP1γ as a reference (dashed line). Graph represents mean and s.d. (*** P < 0.001; two-tailed Student's t-test; n=4).


( a ) Electron micrographs of the IM ( black arrowheads ) associated with the ER ( black arrows ) from Atg4BC74A - expressing NIH 3T3 cells . The IM appears to expand between the two ER cisternae and forms an autophagosome . The white arrowhead indicates vesicular clusters visible in the vicinity of the IM . The white arrow indicates the autophagosome .

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

(a) Electron micrographs of the IM (black arrowheads) associated with the ER (black arrows) from Atg4BC74A-expressing NIH 3T3 cells. The IM appears to expand between the two ER cisternae and forms an autophagosome. The white arrowhead indicates vesicular clusters visible in the vicinity of the IM. The white arrow indicates the autophagosome.


( b ) The ratio of ER - associated autophagic structures ( ER - IM complexes ) to total autophagic structures . Data are expressed as the mean ± s . d . of triplicates from representative experiments .

O O O O O O O O O O O O O O O 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) The ratio of ER-associated autophagic structures (ER-IM complexes) to total autophagic structures. Data are expressed as the mean ± s.d. of triplicates from representative experiments.


( c ) Localization of GFP - DFCP1 with Atg16L1 in both control and Atg4BC74A - expressing cells . Insets show enlarged images of boxed regions .

(c) Localization of GFP-DFCP1 with Atg16L1 in both control and Atg4BC74A-expressing cells. Insets show enlarged images of boxed regions.


( d - g ) Gold - enhanced immunogold electron microscopy revealed that GFP - DFCP1 localized to ER - IM complexes in both control ( d , e ) and Atg4BC74A - expressing cells ( f ) . Immunogold particles are indicated by small arrows . No specific labelling was observed on ER - IM complexes in cells expressing GFP ( g ) . Large arrows and arrowheads indicate the ER and the IM , respectively . Scale bars , 200 nm in a and d - g , an d 10 μm in c .

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(d-g) Gold-enhanced immunogold electron microscopy revealed that GFP-DFCP1 localized to ER-IM complexes in both control (d, e) and Atg4BC74A-expressing cells (f). Immunogold particles are indicated by small arrows. No specific labelling was observed on ER-IM complexes in cells expressing GFP (g). Large arrows and arrowheads indicate the ER and the IM, respectively. Scale bars, 200 nm in a and d-g, an d 10 μm in c.


Atg4BC74A - expressing cells ( for Atg16L1 ) or control cells ( for PDI , COP II and β - COP ) were labelled with specific antibodies against the indicated markers . The left and right anti - Atg16L1 and anti - PDI images are from two different ER - IM complexes . Arrows , arrowheads and open arrowheads indicate the ER , IMs and VTCs , respectively . Small arrows in the image from the anti - COP II experiment indicate immunogold particles . Scale bars , 200 nm .

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

Atg4BC74A-expressing cells (for Atg16L1) or control cells (for PDI, COP II and β-COP) were labelled with specific antibodies against the indicated markers. The left and right anti-Atg16L1 and anti-PDI images are from two different ER-IM complexes. Arrows, arrowheads and open arrowheads indicate the ER, IMs and VTCs, respectively. Small arrows in the image from the anti-COP II experiment indicate immunogold particles. Scale bars, 200 nm.


( e ) Images show 1 . 5 - nm tomographic slices of the boxed region in c , and depict the IM ( black arrow ) , whose edge ( red arrowhead ) is connected to the associated ER ( black arrowhead ) . Numbers indicate the depth of the tomographic slices . Scale bars , 200 nm in a , 100 nm in b and c , and 50 nm in d and e .

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(e) Images show 1.5-nm tomographic slices of the boxed region in c, and depict the IM (black arrow), whose edge (red arrowhead) is connected to the associated ER (black arrowhead). Numbers indicate the depth of the tomographic slices. Scale bars, 200 nm in a, 100 nm in b and c, and 50 nm in d and e.


A 2 - nm tomographic slice of each ER - IM complex , reconstructed from a 200 - nm thick section , and their 3D models are depicted in the left panels . Red arrows indicate sites at which an edge of the IM is connected to the ER . Boxed regions are enlarged in the right panels . 3D models of the boxed regions , shown in a different orientation , depict the connection between the IM and ER ( red arrows ) . Tomographic slices of the boxed regions show the edge of the IM ( red arrowheads ) is connected to the ER . Numbers indicate the depth of the tomographic slices . Scale bars , 100 nm .

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

A 2-nm tomographic slice of each ER-IM complex, reconstructed from a 200-nm thick section, and their 3D models are depicted in the left panels. Red arrows indicate sites at which an edge of the IM is connected to the ER. Boxed regions are enlarged in the right panels. 3D models of the boxed regions, shown in a different orientation, depict the connection between the IM and ER (red arrows). Tomographic slices of the boxed regions show the edge of the IM (red arrowheads) is connected to the ER. Numbers indicate the depth of the tomographic slices. Scale bars, 100 nm.


A , B Intracellular NAD levels and NAD / NADH ratio in DC and age - matched healthy control fibroblasts . All values are presented as mean ± SD of four and eight replicates in A and B , respectively . Student ' s t test was performed on DC cells vs controls .

O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B Intracellular NAD levels and NAD/NADH ratio in DC and age-matched healthy control fibroblasts. All values are presented as mean ± SD of four and eight replicates in A and B, respectively. Student's t test was performed on DC cells vs controls.


Immunoblots of the expression of NAD synthesis and consuming proteins , and their activities in DC and control cells . Protein levels are normalized to GAPDH .

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Immunoblots of the expression of NAD synthesis and consuming proteins, and their activities in DC and control cells. Protein levels are normalized to GAPDH.


E Immunoblots of the expression of NAD synthesis and consuming proteins Quantification values are presented as mean ± SD of four controls versus five DC samples as shown in D . Student ' s t test was performed on DC cells vs controls . Con : control fibroblasts .

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E Immunoblots of the expression of NAD synthesis and consuming proteins Quantification values are presented as mean ± SD of four controls versus five DC samples as shown in D. Student's t test was performed on DC cells vs controls. Con: control fibroblasts.


F Effects of NR and NAM supplementation ( 24 hours ) on the NAD levels in DC fibroblasts . All values are presented as mean ± SD of four replicates . One - Way ANOVA was performed on DC cells in indicated conditions .

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F Effects of NR and NAM supplementation (24 hours) on the NAD levels in DC fibroblasts. All values are presented as mean ± SD of four replicates. One-Way ANOVA was performed on DC cells in indicated conditions.


Quantitative RT - PCR and immunoblots of the expression of CD38 and SARM1 in DC and age - matched health control fibroblasts . All values are presented as mean ± SD of three replicates in quantitative RT - PCR . Protein levels are normalized to GAPDH , and mean ( ± SD ) quantification values of 4 controls vs 5 DC samples are shown . Student ' s t test was performed on DC cells vs controls . Irrelevant intervening lanes have been removed for clarity ( full blots are available online as Source Data ) .

Quantitative RT-PCR and immunoblots of the expression of CD38 and SARM1 in DC and age-matched health control fibroblasts. All values are presented as mean ± SD of three replicates in quantitative RT-PCR. Protein levels are normalized to GAPDH, and mean (± SD) quantification values of 4 controls vs 5 DC samples are shown. Student's t test was performed on DC cells vs controls. Irrelevant intervening lanes have been removed for clarity (full blots are available online as Source Data).


Immunoblots and quantification of indicated proteins in DC fibroblasts treated with DMSO and with 2 μM ATM inhibitor , KU - 55933 for 48 hours . Mean ( ± SD ) quantification values of three DC lines with and without ATMi treatment are shown . P values on the basis of Student ' s t test .

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Immunoblots and quantification of indicated proteins in DC fibroblasts treated with DMSO and with 2 μM ATM inhibitor, KU-55933 for 48 hours. Mean (± SD) quantification values of three DC lines with and without ATMi treatment are shown. P values on the basis of Student's t test.


The efficacy of CD38 shRNA ( sh - 1 and sh - 2 ) was verified by quantitative RT - PCR in DC fibroblasts and by RT - PCR and immunoblots in A549 cells . The relative CD38 mRNA values in the CD38 knockdown cells were normalized to the scrambled shRNA . All values are presented as mean ± SD of three replicates . Student ' s t test was performed on DC and A549 cells in indicated conditions .

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The efficacy of CD38 shRNA (sh-1 and sh-2) was verified by quantitative RT-PCR in DC fibroblasts and by RT-PCR and immunoblots in A549 cells. The relative CD38 mRNA values in the CD38 knockdown cells were normalized to the scrambled shRNA. All values are presented as mean ± SD of three replicates. Student's t test was performed on DC and A549 cells in indicated conditions.


Intracellular NAD levels in the scramble and CD38 knockdown DC fibroblasts . The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib ( 400nM ) and the SIRT1 inhibitor EX 527 ( 1 μM ) for 24 hours . Data are representative four replicates . All values are presented as mean ± SD of four replicates . One - way ANOVA was performed on DC cells in indicated conditions .

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Intracellular NAD levels in the scramble and CD38 knockdown DC fibroblasts. The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib (400nM) and the SIRT1 inhibitor EX 527 (1 μM) for 24 hours. Data are representative four replicates. All values are presented as mean ± SD of four replicates. One-way ANOVA was performed on DC cells in indicated conditions.


immunoblots of the expression of indicated proteins in the scramble and CD38 knockdown DC fibroblasts . The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib ( 400nM ) and the SIRT1 inhibitor EX 527 ( 1 μM ) for 24 hours . Data are representative four replicates . All values are presented as mean ± SD of four replicates . One - way ANOVA was performed on DC cells in indicated conditions .

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immunoblots of the expression of indicated proteins in the scramble and CD38 knockdown DC fibroblasts. The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib (400nM) and the SIRT1 inhibitor EX 527 (1 μM) for 24 hours. Data are representative four replicates. All values are presented as mean ± SD of four replicates. One-way ANOVA was performed on DC cells in indicated conditions.


Intracellular NAD levels in brain tissues from 6 - month - old G1 and G3 Tert - / - female mice .

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Intracellular NAD levels in brain tissues from 6-month- old G1 and G3 Tert-/- female mice.


immunoblots of the expression of NAD consuming related proteins and their activities in brain tissues from 6 - month - old G1 and G3 Tert - / - female mice . The CD38 antibody was validated using the CD38 - / - mouse brain tissues .

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immunoblots of the expression of NAD consuming related proteins and their activities in brain tissues from 6-month- old G1 and G3 Tert-/- female mice. The CD38 antibody was validated using the CD38-/- mouse brain tissues.


C Quantification of immunoblots from the indicated proteins in B . Data information : All values are presented as mean ± SD of G1 Tert - / - mouse brain tissues versus G3 Tert - / - mouse brain tissues . n = 4 in each group . P values on the basis of Student ' s t test .

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C Quantification of immunoblots from the indicated proteins in B. Data information: All values are presented as mean ± SD of G1 Tert-/- mouse brain tissues versus G3 Tert-/- mouse brain tissues. n=4 in each group. P values on the basis of Student's t test.


D NADase activity in G1 and G3 Tert - / - mouse brain lysate was determined at various time points during the reaction . NADase activity was not detectable in the extracts treated with the CD38 inhibitor , 78c , at a concentration of 50nM .

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D NADase activity in G1 and G3 Tert-/- mouse brain lysate was determined at various time points during the reaction. NADase activity was not detectable in the extracts treated with the CD38 inhibitor, 78c, at a concentration of 50nM.


A Representative immunoblots of the expression of PGC1 - 1α in DC cells treated with vehicle or 3 mM NR . Quantification of the indicated proteins is from three immunoblots .

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A Representative immunoblots of the expression of PGC1-1α in DC cells treated with vehicle or 3 mM NR. Quantification of the indicated proteins is from three immunoblots.


B - D Cellular and mitochondrial ROS in DC and age - matched healthy control cells supplemented with vehicle and NR were measured by flow cytometry from three replicates .

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B-D Cellular and mitochondrial ROS in DC and age-matched healthy control cells supplemented with vehicle and NR were measured by flow cytometry from three replicates.


Cellular and mitochondrial ROS in DC and age - matched healthy control cells in the scramble and CD38 knockdown DC cells ( D ) were measured by flow cytometry from three replicates .

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Cellular and mitochondrial ROS in DC and age-matched healthy control cells in the scramble and CD38 knockdown DC cells (D) were measured by flow cytometry from three replicates.


E Representative images captured by transmission electron microscopy of DC1 cells treated with vehicle or NR . White arrows : mitochondria . Yellow arrow : autophagosome - like structures with engulfed mitochondria . Enlarged image within white frame is shown . F - H Quantifications of percentage of damaged mitochondria per cell ( F ) , mitochondrial length ( G ) , and mitochondrial diameter ( H ) . A minimum of 200 mitochondria counted per group .

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E Representative images captured by transmission electron microscopy of DC1 cells treated with vehicle or NR. White arrows: mitochondria. Yellow arrow: autophagosome-like structures with engulfed mitochondria. Enlarged image within white frame is shown. F-H Quantifications of percentage of damaged mitochondria per cell (F), mitochondrial length (G), and mitochondrial diameter (H). A minimum of 200 mitochondria counted per group.


Representative images of mitophagy showing co - localization between the mitophagy dye ( red ) and the lysosome dye ( green ) in DC and control fibroblasts treated with vehicle or 3 mM NR . White arrows : mitophagy . B The mean value of fluorescence intensity / cells in each image was scored . At least 15 images ( ~ 200 cells ) were counted per group . The relative values in each group was normalized to Con1 .

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Representative images of mitophagy showing co-localization between the mitophagy dye (red) and the lysosome dye (green) in DC and control fibroblasts treated with vehicle or 3 mM NR. White arrows: mitophagy. B The mean value of fluorescence intensity/cells in each image was scored. At least 15 images (~ 200 cells) were counted per group. The relative values in each group was normalized to Con1.


C Representative immunoblots of the expression of PINK1 and PARKIN in DC cells treated with vehicle or 3 mM NR . Quantification of immunoblots from the indicated proteins is from three replicates .

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C Representative immunoblots of the expression of PINK1 and PARKIN in DC cells treated with vehicle or 3 mM NR. Quantification of immunoblots from the indicated proteins is from three replicates.


A PCR amplification efficiency at the telomere in the mock - and FPG - treated DC and age - matched healthy control fibroblasts supplemented with vehicle or 3 mM NR . All values are presented as mean ± SD of three replicates . The relative telomere PCR amplification in each sample was normalized to the 36B4 reference gene .

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A PCR amplification efficiency at the telomere in the mock- and FPG-treated DC and age-matched healthy control fibroblasts supplemented with vehicle or 3 mM NR. All values are presented as mean ± SD of three replicates. The relative telomere PCR amplification in each sample was normalized to the 36B4 reference gene.


B Telomere restriction fragment analysis in DC and control fibroblasts treated with vehicle or 3 mM NR . Genomic DNA was isolated from the indicated cell lines at 0 d or 14 d , with or without 3 mM NR treatment . At left , DNA molecular mass markers , in kilobase pairs ( Kb ) .

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B Telomere restriction fragment analysis in DC and control fibroblasts treated with vehicle or 3 mM NR. Genomic DNA was isolated from the indicated cell lines at 0 d or 14 d, with or without 3 mM NR treatment. At left, DNA molecular mass markers, in kilobase pairs (Kb).


C , D Representative images of γH2AX ( green ) , telomere immuno - FISH ( red ) in DC cells treated with vehicle or 3 mM NR . White arrows : co - localization of γH2AX foci and telomeric DNA ( or TIF ) . Enlarged views of co - localizing foci are shown at the right panel and in Figure EV1 . Percentage of DC and control cells with indicated TIFs per nuclei . ~ 100 cells per condition were scored .

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C, D Representative images of γH2AX (green), telomere immuno-FISH (red) in DC cells treated with vehicle or 3 mM NR. White arrows: co-localization of γH2AX foci and telomeric DNA (or TIF). Enlarged views of co-localizing foci are shown at the right panel and in Figure EV1. Percentage of DC and control cells with indicated TIFs per nuclei. ~ 100 cells per condition were scored.


A Cumulative population doubling analysis of the proliferation of representative scramble and CD38 knockdown DC fibroblasts or DC fibroblasts treated with vehicle or 3 mM NR . Each data point is represented as mean ± SD of three replicates .

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A Cumulative population doubling analysis of the proliferation of representative scramble and CD38 knockdown DC fibroblasts or DC fibroblasts treated with vehicle or 3 mM NR. Each data point is represented as mean ± SD of three replicates.


B , C Representative images of BrdU ( red ) and DAPI ( blue ) staining of DC and age - matched healthy control fibroblasts treated with vehicle or NR . Percentage of BrdU - positive cells per condition . Each dot represents the percentage of cells with BrdU staining per image . ~ 400 cells were counted per condition . All values are presented as mean ± SD .

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B, C Representative images of BrdU (red) and DAPI (blue) staining of DC and age-matched healthy control fibroblasts treated with vehicle or NR. Percentage of BrdU -positive cells per condition. Each dot represents the percentage of cells with BrdU staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD.


D , E Representative images of SPiDER - β - gal ( green ) and DAPI ( blue ) staining of DC and age - matched healthy control cells treated with vehicle or 3 mM NR . Percentage of SA - β - gal - positive cells per condition . Each dot represents the percentage of cells with SA - β - gal staining per image . ~ 400 cells were counted per condition . All values are presented as mean ± SD .

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D, E Representative images of SPiDER-β-gal (green) and DAPI (blue) staining of DC and age-matched healthy control cells treated with vehicle or 3 mM NR. Percentage of SA-β-gal-positive cells per condition. Each dot represents the percentage of cells with SA-β-gal staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD.


F The levels of IL - 6 , IL - 8 , and MCP - 1 in both supernatant and cell lysate of indicated DC fibroblasts treated with vehicle or 3 mM NR . All values are represented as mean ± SD of three replicates .

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F The levels of IL-6, IL-8, and MCP-1 in both supernatant and cell lysate of indicated DC fibroblasts treated with vehicle or 3 mM NR. All values are represented as mean ± SD of three replicates.


G , H Representative immunoblots of p21 and p16 in DC fibroblasts treated with vehicle or 3 mM NR . Quantification of the indicated proteins are presented as mean ± SD from three immunoblots .

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G, H Representative immunoblots of p21 and p16 in DC fibroblasts treated with vehicle or 3 mM NR. Quantification of the indicated proteins are presented as mean ± SD from three immunoblots.


Immunofluorescence staining of LN229 , U87and SNB19 tumor cells with primary antibodies against αv , β3 or β5 integrin subunits . Scale bars , 20μm for LN229 and SNB19 , and 50μm for U87 .

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Immunofluorescence staining of LN229, U87and SNB19 tumor cells with primary antibodies against αv, β3 or β5 integrin subunits. Scale bars, 20μm for LN229 and SNB19, and 50μm for U87.


Targeted gene delivery into LN229 , U87 and SNB19 cells by RGD4C / AAVP - Luc carrying the Luc reporter gene . Cells were seeded in 48 well plates before transduction . Non - targeted / AAVP - Luc was used as negative control for targeted transduction . Results represent the average Relative Luminescence Units ( RLU ) / 1μg protein . Data shown are representative of two experiments , n = 3 .

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Targeted gene delivery into LN229, U87 and SNB19 cells by RGD4C/AAVP-Luc carrying the Luc reporter gene. Cells were seeded in 48 well plates before transduction. Non-targeted/AAVP-Luc was used as negative control for targeted transduction. Results represent the average Relative Luminescence Units (RLU)/1μg protein. Data shown are representative of two experiments, n=3.


Western blot analysis of the endogenous Grp78 protein expression levels in LN229 , U87 and SNB19 cells following treatment with increasing concentrations of TMZ . Grp78 level was normalized to GAPDH . Data shown in right panel are representative of two experiments , n = 3 .

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Western blot analysis of the endogenous Grp78 protein expression levels in LN229, U87 and SNB19 cells following treatment with increasing concentrations of TMZ. Grp78 level was normalized to GAPDH. Data shown in right panel are representative of two experiments, n=3.


Induction of Grp78 promoter activity by TMZ in RGD4C / AAVP - Grp78 - Luc cells . LN229 , U87 and SNB19 cells stably transduced with RGD4C / AAVP - Grp78 - Luc or RGD4C / AAVP - CMV - Luc were grown in the presence of TMZ for the indicated times . Results represent the average RLU / 1μg protein . Data shown are representative of two experiments , n = 3 . Two - way ANOVA with Bonferroni correction ( GraphPad Prism 6 ) was used for data analysis .

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Induction of Grp78 promoter activity by TMZ in RGD4C/AAVP-Grp78-Luc cells. LN229, U87 and SNB19 cells stably transduced with RGD4C/AAVP-Grp78-Luc or RGD4C/AAVP-CMV-Luc were grown in the presence of TMZ for the indicated times. Results represent the average RLU/1μg protein. Data shown are representative of two experiments, n=3. Two-way ANOVA with Bonferroni correction (GraphPad Prism 6) was used for data analysis.


TMZ induction of phospho - eIF2α and ATF6 - p90 expression . Human glioblastoma cells transduced with RGD4C / AAVP - Grp78 were analyzed by Western blot following treatment with TMZ ( 100 μM for LN229 and SNB19 , 60 μM for U87 ) . GAPDH was used as a control .

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TMZ induction of phospho-eIF2α and ATF6-p90 expression. Human glioblastoma cells transduced with RGD4C/AAVP-Grp78 were analyzed by Western blot following treatment with TMZ (100 μM for LN229 and SNB19, 60 μM for U87). GAPDH was used as a control.


RT - PCR analysis of constitutive expression and splicing of XBP1 in glioblastoma cells transduced with RGD4C / AAVP - Grp78 , in the presence of TMZ . Sizes of the PCR products were 289 bp for unspliced XBP1 and 263 bp for spliced XBP1 , the lower size band is not specific . Time points were selected among those tested in the Western blot in ( A ) , subsequently the image containing 0 , 1 and 2 hours was juxtaposed to images of 6 , 12 and 24 hours . A white line has been added between the gel pieces that have been juxtaposed .

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RT-PCR analysis of constitutive expression and splicing of XBP1 in glioblastoma cells transduced with RGD4C/AAVP-Grp78, in the presence of TMZ. Sizes of the PCR products were 289 bp for unspliced XBP1 and 263 bp for spliced XBP1, the lower size band is not specific. Time points were selected among those tested in the Western blot in (A), subsequently the image containing 0, 1 and 2 hours was juxtaposed to images of 6, 12 and 24 hours. A white line has been added between the gel pieces that have been juxtaposed.


Tumor cell killing in vitro by the HSVtk / GCV approach . Glioblastoma cells stably transduced with RGD4C / AAVP - Grp78 - HSVtk or RGD4C / AAVP - CMV - HSVtk were treated with either GCV ( 10 μM ) or TMZ ( 100 μM for LN229 and SNB19 , 60 μM for U87 ) or combination of both GCV and TMZ . Cells were stained with MitoSOX and analyzed by FACS at day 4 post - treatment . Data shown are representative of three experiments , n = 3 . Two - way ANOVA with Tukey ' s multiple comparison test ( GraphPad Prism 6 ) was used for data analysis .

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Tumor cell killing in vitro by the HSVtk/GCV approach. Glioblastoma cells stably transduced with RGD4C/AAVP-Grp78-HSVtk or RGD4C/AAVP-CMV-HSVtk were treated with either GCV (10 μM) or TMZ (100 μM for LN229 and SNB19, 60 μM for U87) or combination of both GCV and TMZ. Cells were stained with MitoSOX and analyzed by FACS at day 4 post-treatment. Data shown are representative of three experiments, n=3. Two-way ANOVA with Tukey's multiple comparison test (GraphPad Prism 6) was used for data analysis.


Immuofluorescent staining for αv integrin expression in brain sections including intracranial tumor ( T ) and the surrounding healthy brain ( B ) . Scale bar , 100μm .

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Immuofluorescent staining for αv integrin expression in brain sections including intracranial tumor (T) and the surrounding healthy brain (B). Scale bar, 100μm.


Co - staining of CD31 ( red ) and phage ( green ) in brain sections comprising tumor and healthy brain . Scale bar , 100μm . A high - magnification view from the low - magnification insert of the tumor section is shown . Scale bar , 25 μm .

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Co-staining of CD31 (red) and phage (green) in brain sections comprising tumor and healthy brain. Scale bar, 100μm. A high-magnification view from the low-magnification insert of the tumor section is shown. Scale bar, 25 μm.


Quantification of the relative homing ability of RGD4C / AAVP - Grp78 - HSVtk to brain tumors , brain tissue and pancreas after intravenous administration . The data were normalized both to non - targeted vector and to tissue weight then expressed as relative TU . Data shown are representative of two experiments , n = 5 . One - way ANOVA with Tukey ' s multiple comparison test ( GraphPad Prism 6 ) was used for data analysis .

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Quantification of the relative homing ability of RGD4C/AAVP-Grp78-HSVtk to brain tumors, brain tissue and pancreas after intravenous administration. The data were normalized both to non-targeted vector and to tissue weight then expressed as relative TU. Data shown are representative of two experiments, n=5. One-way ANOVA with Tukey's multiple comparison test (GraphPad Prism 6) was used for data analysis.


In vivo BLI of Luc expression for evaluation of tumor viability and tumor size of representative tumor - bearing mice from all experimental groups before initiation of therapy ( day 9 post intracranial cell implantation ) and at the end of treatments ( day 27 post intracranial cell implantation ) .

In vivo BLI of Luc expression for evaluation of tumor viability and tumor size of representative tumor-bearing mice from all experimental groups before initiation of therapy (day 9 post intracranial cell implantation) and at the end of treatments (day 27 post intracranial cell implantation).


Individual tumor Luc intensities before ( filled circles ) and after therapy ( open circles ) , on days 9 and 27 post cell implantation , respectively . Data shown are representative of two experiments , n = 5 .

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Individual tumor Luc intensities before (filled circles) and after therapy (open circles), on days 9 and 27 post cell implantation, respectively. Data shown are representative of two experiments, n=5.


Average tumor Luc activity showing glioblastoma progression in each experimental group . Pre - treatment day 9 was set at 100 % . Data shown are representative of two experiments , n = 5 .

Average tumor Luc activity showing glioblastoma progression in each experimental group. Pre-treatment day 9 was set at 100%. Data shown are representative of two experiments, n=5.


Survival benefit for tumor - bearing mice from all treatment groups . Data shown are representative of two experiments , n = 5 . Statistical significance was determined by Kaplan - Meier method ( Kaplan - Meier survival fractions ) . Log - rank ( Mantel - Cox ) test was used .

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Survival benefit for tumor-bearing mice from all treatment groups. Data shown are representative of two experiments, n=5. Statistical significance was determined by Kaplan-Meier method (Kaplan-Meier survival fractions). Log-rank (Mantel-Cox) test was used.


Hematoxylin and eosin staining ( H & E ) and immunostaining for the proliferation marker protein Ki67 of representative tumor sections from all experimental groups , Scale bars , 200 μm for H & E and 100 μm for Ki67 . T , tumor ; B , brain . The low - magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase - 3 immunostainings .

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Hematoxylin and eosin staining (H&E) and immunostaining for the proliferation marker protein Ki67 of representative tumor sections from all experimental groups, Scale bars, 200 μm for H&E and 100 μm for Ki67. T, tumor; B, brain. The low-magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase-3 immunostainings.


Blood vessel staining with CD31 ( red ) . DAPI staining of the sections is shown in blue . Scale bar , 200 μm . T , tumor ; B , brain . The low - magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase - 3 immunostainings .

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Blood vessel staining with CD31 (red). DAPI staining of the sections is shown in blue. Scale bar, 200 μm. T, tumor; B, brain. The low-magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase-3 immunostainings.


Tumor immunostaining for apoptosis using an anti - caspase - 3 antibody . Scale bar , 100 μm .

Tumor immunostaining for apoptosis using an anti- caspase-3 antibody. Scale bar, 100 μm.


Fluorescence - activated cell sorting ( FACS ) analysis with antibodies against Sox - 2 , Nestin and CD133 stem cell markers .

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Fluorescence-activated cell sorting (FACS) analysis with antibodies against Sox-2, Nestin and CD133 stem cell markers.


Immunofluorescent staining for αv , β3 or β5 integrins in primary HSJD - GBM - 001 and G26 GBM cells .

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Immunofluorescent staining for αv, β3 or β5 integrins in primary HSJD-GBM-001 and G26 GBM cells.


Targeted transduction of HSJD - GBM - 001 and G26 cells , over a time course , by RGD4C / AAVP - Luc . Non - targeted / AAVP - Luc was used as negative control . Results represent RLU / 1μg protein of triplicate wells . Data shown are representative of two experiments , n = 3 . Scale bar , 100 μm .

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Targeted transduction of HSJD-GBM-001 and G26 cells, over a time course, by RGD4C/AAVP-Luc. Non-targeted/AAVP-Luc was used as negative control. Results represent RLU/1μg protein of triplicate wells. Data shown are representative of two experiments, n=3. Scale bar, 100 μm.


Immunofluorescent staining for αv , β3 or β5 integrins in human normal astrocytes , chondrocytes , skin and lung fibroblasts .

Immunofluorescent staining for αv, β3 or β5 integrins in human normal astrocytes, chondrocytes, skin and lung fibroblasts.


Treatment of chondrocytes , astrocytes , lung and skin fibroblasts with RGD4C / AAVP - Luc vector , alone or in the presence of TMZ . Non - targeted / AAVP - Luc was used as negative control . Results represent RLU / 1μg protein . Data shown are representative of two experiments , n = 3 . Scale bars , 160 μm for chondrocytes and 80 μm for all other cells .

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Treatment of chondrocytes, astrocytes, lung and skin fibroblasts with RGD4C/AAVP-Luc vector, alone or in the presence of TMZ. Non-targeted/AAVP-Luc was used as negative control. Results represent RLU/1μg protein. Data shown are representative of two experiments, n=3. Scale bars, 160 μm for chondrocytes and 80 μm for all other cells.