Figure 3 ( A ) Representative immunofluorescence images showing different cell clusters in CCSC spheres . Single CCSCs with high - Notch - activity ( Notch - GFP ) were seeded to grow into spheres and immunofluorescence was performed to identify cell clusters using representative marker genes . Scale bar , 60 μm . ( B ) Pair - cell assay showing the generation of the five cell clusters by CCSC asymmetric division . Scale bar , 60 μm . ( C ) Edu incorporation assay showing the proliferative potential of cluster C1 ( CCNE2 + ) and cluster C2 ( CCNB2 + ) cells in CCSC spheres . Scale bar , 60 μm . ( D ) Pair - cell assay showing that cluster C1 ( CCNE2 + ) and cluster C2 ( CCNB2 + ) cells immediately enter the second division . Scale bar , 100 μm . ( E and F ) Ki67 staining showing the proliferative potential of cluster C1 ( CCNE2 + ) and cluster C2 ( CCNB2 + ) cells in xenografts ( E ) and clinical CRC tissues ( F ) . Scale bar , 60 μm . Quantification of the immunostaining for ( F ) shown on the right . Error bars represent SEM of five fields in each section . P - value was calculated based on two - tailed Student ' s t - test . ns ( p > 0 . 05 ) , no significant .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3(A) Representative immunofluorescence images showing different cell clusters in CCSC spheres. Single CCSCs with high-Notch-activity (Notch-GFP) were seeded to grow into spheres and immunofluorescence was performed to identify cell clusters using representative marker genes. Scale bar, 60 μm. (B) Pair-cell assay showing the generation of the five cell clusters by CCSC asymmetric division. Scale bar, 60 μm.(C) Edu incorporation assay showing the proliferative potential of cluster C1 (CCNE2+) and cluster C2 (CCNB2+) cells in CCSC spheres. Scale bar, 60 μm. (D) Pair-cell assay showing that cluster C1 (CCNE2+) and cluster C2 (CCNB2+) cells immediately enter the second division. Scale bar, 100 μm.(E and F) Ki67 staining showing the proliferative potential of cluster C1 (CCNE2+) and cluster C2 (CCNB2+) cells in xenografts (E) and clinical CRC tissues (F). Scale bar, 60 μm. Quantification of the immunostaining for (F) shown on the right. Error bars represent SEM of five fields in each section. P-value was calculated based on two-tailed Student's t-test. ns (p>0.05), no significant.


Figure 4 ( A and B ) RT - qPCR showing the enrichment of cluster C3 subpopulation ( CFAP54 + ) in CCSC1 ( A ) and CCSC2 ( B ) spheres treated with the chemotherapeutic agent 5FU or L - OHP . Error bars represent SD of three biological replicates . P - value was calculated based on two - tailed Student ' s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns , no significant . ( C ) Representative RNA - FISH images and quantification showing the level of cluster C3 subpopulation CFAP54 + cell ( red ) levels in L - OHP treated xenografts . Scale bar , 40 μm . Three biological replicates were analyzed . Data represent the mean ± SD . P - value was calculated based on two - tailed Student ' s t test . * , p < 0 . 05 . ( E ) Fluorescent images and quantification showing AP20187 - mediated ablation of cluster C4 ( PLAUR + ) cells from CCSC spheres carrying PLAUR - iCaspase9 - mCherry construct . Scale bar , 40 μm . Three biological replicates were analyzed . Data represent the mean ± SD . P - value was calculated based on two - tailed Student ' s t test . * * * , p < 0 . 001 . ( I ) Representative H & E images and quantification ( number of mice with invasive tumors ) showing the invasive potential of cluster C4 cells ( PLAUR + ) . CCSC sphere cells carrying PLAUR - iCaspase9 - mCherry construct were orthotopically injected into the mice . AP20187 was applied to ablate cluster C4 ( PLAUR + , red ) cells . Red arrows indicate invasive tumor . Scale bars , left , 100 μm ; right , 300 μm . ( K ) Left : Representative H & E images of liver tissue sections and quantification ( number of mice with liver metastasis ) showing the metastatic potential of cluster C4 cells ( PLAUR + ) . AP20187 was applied to ablate cluster C4 . Yellow arrows indicate liver metastasis . Scale bars , left , 2 mm ; right , 50 μm . Right : Quantification of liver metastasis in mice with intrasplenic injection . Error bars denote mean ± SEM of three mice per group . P - value was calculated based on Student ' s t - test . * * * , p < 0 . 001 .

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

Figure 4(A and B) RT-qPCR showing the enrichment of cluster C3 subpopulation (CFAP54+) in CCSC1 (A) and CCSC2 (B) spheres treated with the chemotherapeutic agent 5FU or L-OHP. Error bars represent SD of three biological replicates. P-value was calculated based on two-tailed Student's t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ns, no significant.(C) Representative RNA-FISH images and quantification showing the level of cluster C3 subpopulation CFAP54+ cell (red) levels in L-OHP treated xenografts. Scale bar, 40 μm. Three biological replicates were analyzed. Data represent the mean ± SD. P-value was calculated based on two-tailed Student's t test. *, p<0.05.(E) Fluorescent images and quantification showing AP20187-mediated ablation of cluster C4 (PLAUR+) cells from CCSC spheres carrying PLAUR-iCaspase9-mCherry construct. Scale bar, 40 μm. Three biological replicates were analyzed. Data represent the mean ± SD. P-value was calculated based on two-tailed Student's t test. ***, p<0.001.(I) Representative H&E images and quantification (number of mice with invasive tumors) showing the invasive potential of cluster C4 cells (PLAUR+). CCSC sphere cells carrying PLAUR-iCaspase9-mCherry construct were orthotopically injected into the mice. AP20187 was applied to ablate cluster C4 (PLAUR+, red) cells. Red arrows indicate invasive tumor. Scale bars, left, 100 μm; right, 300 μm.(K) Left: Representative H&E images of liver tissue sections and quantification (number of mice with liver metastasis) showing the metastatic potential of cluster C4 cells (PLAUR+). AP20187 was applied to ablate cluster C4. Yellow arrows indicate liver metastasis. Scale bars, left, 2 mm; right, 50 μm. Right: Quantification of liver metastasis in mice with intrasplenic injection. Error bars denote mean ± SEM of three mice per group. P-value was calculated based on Student's t-test. ***, p<0.001.


Figure 5 ( C ) RT - qPCR analysis of the signature genes of each cluster in CCSC sphere cells with control ( EV ) or ATF6 knockdown ( shATF6 - 1 and shATF6 - 2 ) . Error bars represent SD of three biological replicates . P - value was calculated based on Student ' s t - test . * , p < 0 . 05 ; * * , P < 0 . 01 . Diagram without asterisk indicates no statistical difference . ( D ) RT - qPCR analysis of signature genes of each cluster in control ( EV ) or ATF6 knockdown ( shATF6 - 1 and shATF6 - 2 ) under the treatment with L - OHP . Error bars represent SD of three biological replicates . P - value was calculated based on Student ' s t - test . * * * , p < 0 . 001 . Diagram without asterisk indicates no statistical difference . ( F ) Representative RNA - FISH of cluster C3 signature gene , CFAP54 ( up ) and immunohistochemistry of transcription factor ATF6 ( bottom ) showing the influence of ATF6 knockdown ( shATF6 - 1 and shATF6 - 2 ) on cluster C3 ( CFAP54 + , red ) population in xenografts . The mice were treated with vehicle or L - OHP after 14 days after CCSC sphere cells were implanted . Scale bars , upper , 40 μm ; lower , 80 μm . ( H ) RT - qPCR analysis of signature genes of each cell clusters in CCSC sphere cells with control ( EV ) or FOXQ1 knockdown ( shFOXQ1 - 1 and shFOXQ1 - 2 ) . Error bars denote the SD between triplicates . P - value was calculated based on Student ' s t test . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . ( I ) Representative H & E and immunofluorescence images showing the influence of FOXQ1 knockdown ( shFOXQ1 - 1 and shFOXQ1 - 2 ) on cluster C4 ( PLAUR + , red ) population and tumor invasion . CCSC sphere cells carrying control ( EV ) vector or FOXQ1 shRNAs were orthotopically injected into mice ( n = 5 ) . Scale bars , left , 300 μm ; right , 100 μm . Arrows indicate the location of tumor invasion .

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

Figure 5(C) RT-qPCR analysis of the signature genes of each cluster in CCSC sphere cells with control (EV) or ATF6 knockdown (shATF6-1 and shATF6-2). Error bars represent SD of three biological replicates. P-value was calculated based on Student's t-test. *, p<0.05; **, P<0.01. Diagram without asterisk indicates no statistical difference.(D) RT-qPCR analysis of signature genes of each cluster in control (EV) or ATF6 knockdown (shATF6-1 and shATF6-2) under the treatment with L-OHP. Error bars represent SD of three biological replicates. P-value was calculated based on Student's t-test. ***, p<0.001. Diagram without asterisk indicates no statistical difference.(F) Representative RNA-FISH of cluster C3 signature gene, CFAP54 (up) and immunohistochemistry of transcription factor ATF6 (bottom) showing the influence of ATF6 knockdown (shATF6-1 and shATF6-2) on cluster C3 (CFAP54+, red) population in xenografts. The mice were treated with vehicle or L-OHP after 14 days after CCSC sphere cells were implanted. Scale bars, upper, 40 μm; lower, 80 μm.(H) RT-qPCR analysis of signature genes of each cell clusters in CCSC sphere cells with control (EV) or FOXQ1 knockdown (shFOXQ1-1 and shFOXQ1-2). Error bars denote the SD between triplicates. P-value was calculated based on Student's t test. *, p<0.05; **, p<0.01; ***, p<0.001.(I) Representative H&E and immunofluorescence images showing the influence of FOXQ1 knockdown (shFOXQ1-1 and shFOXQ1-2) on cluster C4 (PLAUR+, red) population and tumor invasion. CCSC sphere cells carrying control (EV) vector or FOXQ1 shRNAs were orthotopically injected into mice (n=5). Scale bars, left, 300 μm; right, 100 μm. Arrows indicate the location of tumor invasion.


Figure 6 ( G ) Representative immunohistochemistry images for the expression of PLAUR ( cluster C4 maker gene ) in primary CRC tumors and liver metastasis from CRC patients . Scale bar , 200 μm . ( H and I ) Representative immunohistochemistry images for ATF6 expression ( H ) and RNA - FISH for CFAP54 expression ( I ) in paired CRC tissues collected from CRC patients before chemotherapy or after chemotherapy . Scale bars , left , 200 μm ; right , 40 μm . Data represent the mean ± SEM . Three to five technical replicates were analyzed . P - value was calculated based on two - tailed Student ' s t test . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . ( J and K ) Kaplan - Meier survival curves for CRC patients ( n = 326 ) with different proportions of cluster C4 ( J ) and cluster C3 ( K ) . P value was calculated based on log - rank test .

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

Figure 6(G) Representative immunohistochemistry images for the expression of PLAUR (cluster C4 maker gene) in primary CRC tumors and liver metastasis from CRC patients. Scale bar, 200 μm.(H and I) Representative immunohistochemistry images for ATF6 expression (H) and RNA-FISH for CFAP54 expression (I) in paired CRC tissues collected from CRC patients before chemotherapy or after chemotherapy. Scale bars, left, 200 μm; right, 40 μm. Data represent the mean ± SEM. Three to five technical replicates were analyzed. P-value was calculated based on two-tailed Student's t test. *, p<0.05; **, p<0.01; ***, p<0.001.(J and K) Kaplan-Meier survival curves for CRC patients (n=326) with different proportions of cluster C4 (J) and cluster C3 (K). P value was calculated based on log-rank test.


Figure 1B . 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 . 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 . 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 ) .

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

Figure 1B. 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.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.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).


Figure 2B . 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 . 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 . 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 .

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


Figure 3A . 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 . 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 . G . Genomic position of a B4 SINE repeat and CACACA consensus motifs at the location of the RNAchIP peakH . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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. 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.G. Genomic position of a B4 SINE repeat and CACACA consensus motifs at the location of the RNAchIP peakH. 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.


Figure 4A . 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 ) . 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 .

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

Figure 4A. 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).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.


Figure 5C . 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 ) . 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 .

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

Figure 5C. 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).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.


Figure 6B . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6B. 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).


Figure 1Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Aβ , amyloid - β ; EPO , erythropoietin . Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Aβ , amyloid - β ; EPO , erythropoietin . Macrophages were detected by immunoblotting with the indicated antibodies ( n = 3 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPO , erythropoietin .

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

Figure 1Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. Aβ, amyloid-β; EPO, erythropoietin.Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. Aβ, amyloid-β; EPO, erythropoietin.Macrophages were detected by immunoblotting with the indicated antibodies (n = 3). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPO, erythropoietin.


Figure 3Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPOR - KO macrophages were PMs isolated from EPOR - MKO mice . Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPOR - KO macrophages were PMs isolated from EPOR - MKO mice . Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Macrophages were detected by immunoblotting with indicated antibodies ( n = 3 ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . EPOR - KO macrophages were PMs isolated from EPOR - MKO mice .

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

Figure 3Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPOR-KO macrophages were PMs isolated from EPOR-MKO mice.Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPOR-KO macrophages were PMs isolated from EPOR-MKO mice.Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Macrophages were detected by immunoblotting with indicated antibodies (n = 3). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPOR-KO macrophages were PMs isolated from EPOR-MKO mice.


Figure 4Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Epor + / + mice ( EPOR - C ) ( 8 w ) mice pretreated with rhEPO 10000 IU / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting ( A , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Epor + / + mice ( EPOR - C ) ( 8 w ) mice pretreated with ARA290 ( 0 . 03 mg / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting B , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA , ARA290Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Eporloxp / loxp mice ( EPOR - MKO ) ( 8 w ) mice pretreated with rhEPO 10000 IU / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Flag - Aβ42 was injected into the lateral ventricles of Lyz2 - Cre + / + / Eporloxp / loxp mice ( EPOR - MKO ) ( 8 w ) mice pretreated with ARA290 ( 0 . 03 mg / kg / d , i . p . ) for 2 days ( twice per day ) ( n = 3 ) . One day later , Flag - Aβ42 in spleens and brains was detected by immunoblotting E ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA , ARA290APP / PS1 - 21 mice ( 5 m ) treated with rhEPO ( 10000 IU / kg / d ) or ARA290 ( 0 . 03 mg / kg / d ) ( i . p . , once a day , for 14 days ) ( n = 10 ) , and Aβ plaques in cortex and hippocampus ( n = 5 ) were detected by IHC ( I ) . Data information : For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ARA , ARA290 ; IHC , Immunohistochemical staining . Proteins were extracted from spleens of 5 - or 11 - month - old APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Eporloxp / loxp mice ( APP / PS1 - 21 + EPOR - MKO ) or APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Epor + / + mice ( APP / PS1 - 21 + EPOR - C ) , and soluble Aβ levels were measured by ELISA ( n = 5 ) ( J Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . ELISA , 11 - month - old APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Eporloxp / loxp mice ( APP / PS1 - 21 + EPOR - MKO ) or APP / PS - 1 - 21 + / + / Lyz2 - Cre + / + / Epor + / + mice ( APP / PS1 - 21 + EPOR - C ) , Aβ plaques in the cortex and hippocampus of mice of 11 - month - old ( M ) ( n = 5 ) were detected by IHC . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IHC , Immunohistochemical staining . R Aβ plaques in the cortex and hippocampus ( n = 4 ) were detected by IHC . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IHC , Immunohistochemical staining .

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

Figure 4Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Epor+/+ mice (EPOR-C) (8 w) mice pretreated with rhEPO 10000 IU/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting (A, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Epor+/+ mice (EPOR-C) (8 w) mice pretreated with ARA290 (0.03 mg/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting B, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Eporloxp/loxp mice (EPOR-MKO) (8 w) mice pretreated with rhEPO 10000 IU/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Eporloxp/loxp mice (EPOR-MKO) (8 w) mice pretreated with ARA290 (0.03 mg/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting E). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290APP/PS1-21 mice (5 m) treated with rhEPO (10000 IU/kg/d) or ARA290 (0.03 mg/kg/d) (i.p., once a day, for 14 days) (n=10), and Aβ plaques in cortex and hippocampus (n = 5) were detected by IHC (I). Data information: For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290; IHC, Immunohistochemical staining.Proteins were extracted from spleens of 5- or 11-month-old APP/PS-1-21+/+/Lyz2-Cre+/+/Eporloxp/loxp mice (APP/PS1-21+EPOR-MKO) or APP/PS-1-21+/+/Lyz2-Cre+/+/Epor+/+ mice (APP/PS1-21+EPOR-C), and soluble Aβ levels were measured by ELISA (n = 5) (J Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ELISA, 11-month-old APP/PS-1-21+/+/Lyz2-Cre+/+/Eporloxp/loxp mice (APP/PS1-21+EPOR-MKO) or APP/PS-1-21+/+/Lyz2-Cre+/+/Epor+/+ mice (APP/PS1-21+EPOR-C), Aβ plaques in the cortex and hippocampus of mice of 11-month-old (M) (n = 5) were detected by IHC. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IHC, Immunohistochemical staining.R Aβ plaques in the cortex and hippocampus (n = 4) were detected by IHC. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IHC, Immunohistochemical staining.


Figure 5A , Cortex and hippocampus were applied for detection of inflammatory factor expression . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . B Cortex and hippocampus were applied for detection of inflammatory factor expression . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Both non - cognitive and cognitive behavioral impairments of APP / PS1 - 21 mice ( 11 m ) were analyzed by nest construction assay , social interaction assay , NORT , FCT ( n = 10 ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . NORT , novel object recognition test ; FCT , fear conditioning test .

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

Figure 5A, Cortex and hippocampus were applied for detection of inflammatory factor expression. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.B Cortex and hippocampus were applied for detection of inflammatory factor expression. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Both non-cognitive and cognitive behavioral impairments of APP/PS1-21 mice (11 m) were analyzed by nest construction assay, social interaction assay, NORT, FCT (n = 10). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. NORT, novel object recognition test; FCT, fear conditioning test.


Figure 6Splenic macrophages from APP / PS1 - 21 ( 5 - or matched WT mice were detected by immunoblotting with the indicated antibodies ( n = 3 ) ( A , Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Splenic macrophages from APP / PS1 - 21 11 - month - old ) or matched WT mice were detected by immunoblotting with the indicated antibodies ( n = 3 ) B ) . Data information : Unpaired t - tests were used for the comparison of two groups . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Old APP / PS1 - 21 mice ( 11 m ) and WT mice at the same age were treated with rhEPO ( 10000 IU / kg / d ) , ARA290 ( 0 . 03 mg / kg / d ) or vehicle ( i . p . , once a day , for 14 days ) . Splenic macrophages were isolated for detection by immunoblotting with the indicated antibodies ( n = 3 ) ( G ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . Old APP / PS1 - 21 mice ( 11 m ) and WT mice at the same age were treated with rhEPO ( 10000 IU / kg / d ) , ARA290 ( 0 . 03 mg / kg / d ) or vehicle ( i . p . , once a day , for 14 days ) . Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies ( n = 3 ) ( I ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IDE , insulin degrading enzyme ; NEP , neprilysin . PMs were treated with Aβ42 ( 0 , 5 , 7 . 5 or 10 μM ) for 12 hr , and cell lysates were detected by immunoblotting with the indicated antibodies ( n = 3 ) ( K ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . PMs were treated with Aβ42 ( 0 , 5 , 10 μM ) for 12 hr , and Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies ( n = 3 ) ( M ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IDE , insulin degrading enzyme ; NEP , neprilysin .

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

Figure 6Splenic macrophages from APP/PS1-21 (5- or matched WT mice were detected by immunoblotting with the indicated antibodies (n = 3) (A, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Splenic macrophages from APP/PS1-21 11-month-old) or matched WT mice were detected by immunoblotting with the indicated antibodies (n = 3) B). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Old APP/PS1-21 mice (11 m) and WT mice at the same age were treated with rhEPO (10000 IU/kg/d), ARA290 (0.03 mg/kg/d) or vehicle (i.p., once a day, for 14 days). Splenic macrophages were isolated for detection by immunoblotting with the indicated antibodies (n = 3) (G). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.Old APP/PS1-21 mice (11 m) and WT mice at the same age were treated with rhEPO (10000 IU/kg/d), ARA290 (0.03 mg/kg/d) or vehicle (i.p., once a day, for 14 days). Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies (n = 3) (I). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IDE, insulin degrading enzyme; NEP, neprilysin.PMs were treated with Aβ42 (0, 5, 7.5 or 10 μM) for 12 hr, and cell lysates were detected by immunoblotting with the indicated antibodies (n = 3) (K). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.PMs were treated with Aβ42 (0, 5, 10 μM) for 12 hr, and Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies (n = 3) (M). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IDE, insulin degrading enzyme; NEP, neprilysin.


Figure 7E Aβ plaques in the cortex and hippocampus ( n = 5 ) were detected by IHC . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . G Expression of MAP - 2 or the apoptotic neurons of hippocampus were detected by IF staining and then statistically quantified ( n = 5 ) . Data information : vehicles were PBS . For multiple groups , one - way ANOVA was used . Results are presented as mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ; ns denotes no statistical significance . IF , Immunofluorescent ; MAP - 2 , microtubule - associated protein 2 ; NeuN , neuronal nuclei ; TUNEL , Terminal dUTP Nick End Labeling .

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

Figure 7E Aβ plaques in the cortex and hippocampus (n = 5) were detected by IHC. Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.G Expression of MAP-2 or the apoptotic neurons of hippocampus were detected by IF staining and then statistically quantified (n = 5). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IF, Immunofluorescent; MAP-2, microtubule-associated protein 2; NeuN, neuronal nuclei; TUNEL, Terminal dUTP Nick End Labeling.


Figure 4B . Pull - down assay of Sec62 domain with different biotin - labeled Sec63 C - terminal peptides . Sec63 - C2P denotes di - phosphorylated Sec63 - C2 . C . ITC - thermogram obtained from ITC after titration of Sec62 domain with increasing amounts of Sec63 - C2P . F . Pull - down assay of the Sec62 domain and a mutant in the interaction interface ( Sec62R51E ) with biotin - labeled Sec63 - C2P .

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Figure 4B. Pull-down assay of Sec62 domain with different biotin-labeled Sec63 C-terminal peptides. Sec63-C2P denotes di-phosphorylated Sec63-C2.C. ITC-thermogram obtained from ITC after titration of Sec62 domain with increasing amounts of Sec63-C2P.F. Pull-down assay of the Sec62 domain and a mutant in the interaction interface (Sec62R51E) with biotin-labeled Sec63-C2P.


figf1 ( a ) Representative photographs of Bax / Bak − / − MEFs treated with etoposide . Bax / Bak − / − MEFs were treated with 20 μM etoposide in the presence of PI ( 2 μM ) for the indicated times , and were analysed under a phase - contrast and fluorescence ( PI ) microscope . ( b , c ) Reduced viability of Bax / Bak − / − MEFs after exposure to etoposide . WT ( circles ) and Bax / Bak − / − ( squares ) MEFs were treated with 20 μM etoposide in the presence ( closed symbols ) or absence ( open symbols ) of 100 μM zVAD - fmk . Then cell viability was measured by the PI staining ( b ) and CTB assay ( c ) , and expressed as a percentage of the initial value without etoposide . Data are shown as mean ± s . d . ( n = 4 ) . ( d ) Reduced viability of etoposide - treated Bax / Bak − / − MEFs as assessed by proliferation assay . Bax / Bak − / − MEFs were either not treated ( 0 h ) or were treated with 20 μM etoposide for 12 and 24 h , then all cells were recovered and 5 , 000 cells were reseeded . Viable cell numbers were measured on the indicated days by the CTB assay . Results were normalized by adjusting the day 0 value . n = 4 . e ) Reduced viability of Bax / Bak − / − MEFs after exposure to etoposide and staurosporine , as assessed by clonogenicity assay . MEFs were treated with etoposide ( 20 μM ) or staurosporine ( 1 μM ) at the indicated times , collected , and 2 , 000 cells were seeded in the normal medium . After 1 week , colonies were counted . n = 4f - i ) Induction of autophagy in Bax / Bak − / − MEFs by etoposide . ( f ) Electron micrograph ( × 14 , 800 ) of Bax / Bak − / − MEF treated with etoposide ( 20 μM ) for 18 h . Representative features of Bax / Bak − / − MEFs that have received no treatment ( NT ; that is , are healthy ) are also shown ( NT : × 14 , 000 ) . ( g ) In magnified photographs , a large number of autolysosomes / autophagosomes ( left : × 26 , 000 ) were observed , but the mitochondria were unaffected ( right : × 17 , 000 ) . ( h , i ) Punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs treated with etoposide . GFP - LC3 - transfected cells were incubated with and without etoposide ( 20 μM ) for 24 h ( h ) or the indicated times ( i ) , and then examined by fluorescent microscopy . ( h ) Representative photographs of healthy ( NT ) and etoposide - treated Bax / Bak − / − MEFs are shown . ( i ) The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - positive cells . Data are shown as mean ± s . d . ( n = 4 ) .

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

figf1(a) Representative photographs of Bax/Bak−/− MEFs treated with etoposide. Bax/Bak−/− MEFs were treated with 20 μM etoposide in the presence of PI (2 μM) for the indicated times, and were analysed under a phase-contrast and fluorescence (PI) microscope.(b, c) Reduced viability of Bax/Bak−/− MEFs after exposure to etoposide. WT (circles) and Bax/Bak−/− (squares) MEFs were treated with 20 μM etoposide in the presence (closed symbols) or absence (open symbols) of 100 μM zVAD-fmk. Then cell viability was measured by the PI staining (b) and CTB assay (c), and expressed as a percentage of the initial value without etoposide. Data are shown as mean ± s.d. (n = 4).(d) Reduced viability of etoposide-treated Bax/Bak−/− MEFs as assessed by proliferation assay. Bax/Bak−/− MEFs were either not treated (0 h) or were treated with 20 μM etoposide for 12 and 24 h, then all cells were recovered and 5,000 cells were reseeded. Viable cell numbers were measured on the indicated days by the CTB assay. Results were normalized by adjusting the day 0 value. n = 4.e) Reduced viability of Bax/Bak−/− MEFs after exposure to etoposide and staurosporine, as assessed by clonogenicity assay. MEFs were treated with etoposide (20 μM) or staurosporine (1 μM) at the indicated times, collected, and 2,000 cells were seeded in the normal medium. After 1 week, colonies were counted. n = 4f-i) Induction of autophagy in Bax/Bak−/− MEFs by etoposide. (f) Electron micrograph (×14,800) of Bax/Bak−/− MEF treated with etoposide (20 μM) for 18 h. Representative features of Bax/Bak−/− MEFs that have received no treatment (NT; that is, are healthy) are also shown (NT: ×14,000).(g) In magnified photographs, a large number of autolysosomes/autophagosomes (left: ×26,000) were observed, but the mitochondria were unaffected (right: ×17,000).(h, i) Punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs treated with etoposide. GFP-LC3-transfected cells were incubated with and without etoposide (20 μM) for 24 h (h) or the indicated times (i), and then examined by fluorescent microscopy. (h) Representative photographs of healthy (NT) and etoposide-treated Bax/Bak−/− MEFs are shown. (i) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-positive cells. Data are shown as mean ± s.d. (n = 4).


figf2 ( a , b ) Bax / Bak − / − MEFs were treated with 20 μM etoposide in the absence or presence of 10 mM 3 - MA for 24 h , and then were examined by phase - contrast microscopy ( a ) and electron microscopy ( × 8 , 400 ) ( b ) . Autolysosomes / autophagosomes were hardly observed . ( c , d ) Reduction of punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs by 3 - MA . Bax / Bak − / − MEFs that were transfected with GFP - LC3 were incubated without ( NT ) or with 20 μM etoposide in the presence or absence of 10 mM 3 - MA for 24 h , and then were examined by confocal fluorescent microscopy . ( c ) Representative photograph of etoposide - treated Bax / Bak − / − MEFs in the presence of 3 - MA . ( d ) The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . Data are shown as mean ± s . d . ( n = 4 ) . ( c , d ) Reduction of punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs by 3 - MA . Bax / Bak − / − MEFs that were transfected with GFP - LC3 were incubated without ( NT ) or with 20 μM etoposide in the presence or absence of 10 mM 3 - MA for 24 h , and then were examined by confocal fluorescent microscopy . ( c ) Representative photograph of etoposide - treated Bax / Bak − / − MEFs in the presence of 3 - MA . ( d ) The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . Data are shown as mean ± s . d . ( n = 4 ) . ( e - g ) Inhibition of etoposide - and staurosporine - induced cell death by 3 - MA . Bax / Bak − / − MEFs were treated with 20 μM etoposide ( e - g ) or 1 μM staurosporine ( g ) in the absence ( open symbols ) or presence ( closed symbols ) of 10 mM 3 - MA for the indicated time . Cell viability was measured by PI staining ( e ) , CTB assay ( f ) and clonogenicity assay ( g ) ( as in Fig . 1e ) . Upper panels in g : viable cells were visualized by staining with calcein - AM . n = 4 .

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

figf2(a, b) Bax/Bak−/− MEFs were treated with 20 μM etoposide in the absence or presence of 10 mM 3-MA for 24 h, and then were examined by phase-contrast microscopy (a) and electron microscopy (×8,400) (b). Autolysosomes/autophagosomes were hardly observed. (c, d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by 3-MA. Bax/Bak−/− MEFs that were transfected with GFP-LC3 were incubated without (NT) or with 20 μM etoposide in the presence or absence of 10 mM 3-MA for 24 h, and then were examined by confocal fluorescent microscopy. (c) Representative photograph of etoposide-treated Bax/Bak−/− MEFs in the presence of 3-MA. (d) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).(c, d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by 3-MA. Bax/Bak−/− MEFs that were transfected with GFP-LC3 were incubated without (NT) or with 20 μM etoposide in the presence or absence of 10 mM 3-MA for 24 h, and then were examined by confocal fluorescent microscopy. (c) Representative photograph of etoposide-treated Bax/Bak−/− MEFs in the presence of 3-MA. (d) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).(e-g) Inhibition of etoposide- and staurosporine-induced cell death by 3-MA. Bax/Bak−/− MEFs were treated with 20 μM etoposide (e-g) or 1 μM staurosporine (g) in the absence (open symbols) or presence (closed symbols) of 10 mM 3-MA for the indicated time. Cell viability was measured by PI staining (e), CTB assay (f) and clonogenicity assay (g) (as in Fig. 1e). Upper panels in g: viable cells were visualized by staining with calcein-AM. n = 4.


figf3 ( a , b ) Bax / Bak − / − MEFs were treated with the indicated siRNAs ( 10 μg ) for 24 h and then incubated with 20 μM etoposide for the indicated times . Expression of APG5 - APG12 complex ( a ) , Beclin 1 ( b ) and VDAC ( loading control ) was analysed by Western blot analysis . ( c ) Inhibition of etoposide - induced autophagy by silencing of Beclin 1 . Bax / Bak − / − MEFs with silencing of Beclin 1 were treated with 20 μM etoposide at 18 h and were analysed by electron microscopy ( × 8 , 500 ) . d ) Reduction of punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs by silencing of Beclin 1 and APG5 . Bax / Bak − / − MEFs that were transfected with GFP - LC3 together with the indicated siRNAs , were incubated without ( NT ) or with 20 μM etoposide for 24 h , and then examined by confocal fluorescent microscopy . The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . Data are shown as mean ± s . d . ( n = 4 ) . ( e , f ) Inhibition of etoposide - and staurosporine - induced cell death by silencing of Beclin 1 and APG5 . Bax / Bak − / − MEFs that were treated with the indicated siRNAs were incubated with 20 μM etoposide ( e , f ) or 1 μM staurosporine ( f ) for the indicated time . Cell viability was measured by CTB assay ( e ) and clonogenicity assay ( f ) . n = 4 .

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

figf3(a, b) Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times. Expression of APG5-APG12 complex (a), Beclin 1 (b) and VDAC (loading control) was analysed by Western blot analysis.(c) Inhibition of etoposide-induced autophagy by silencing of Beclin 1. Bax/Bak−/− MEFs with silencing of Beclin 1 were treated with 20 μM etoposide at 18 h and were analysed by electron microscopy (×8,500).d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were transfected with GFP-LC3 together with the indicated siRNAs, were incubated without (NT) or with 20 μM etoposide for 24 h, and then examined by confocal fluorescent microscopy. The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).(e, f) Inhibition of etoposide- and staurosporine-induced cell death by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were treated with the indicated siRNAs were incubated with 20 μM etoposide (e, f) or 1 μM staurosporine (f) for the indicated time. Cell viability was measured by CTB assay (e) and clonogenicity assay (f). n = 4.


figf4 ( a , b ) APG5 - dependent punctate GFP - LC3 fluorescence in Bcl - 2 - transfected MEFs after etoposide treatment . APG5 + / + and APG5 − / − MEFs that were transfected with both GFP - LC3 and the indicated plasmids ( Bcl - 2 or vector ) were incubated without or with 20 μM etoposide for 24 h , then examined by confocal fluorescent microscopy ( a ) . The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells ( b ) . ( c ) Accumulation of Beclin 1 and APG5 - APG12 complex in Bcl - 2 - transfected MEFs after etoposide treatment . WT MEFs were transfected with the indicated plasmids , and were incubated without ( NT ) or with 20 μM etoposide for 24 h . ( d , e ) Occurrence of etoposide - induced 3 - MA - inhibitable non - apoptotic death in Bcl - xL - overexpressing APG5 + / + MEFs , but not APG5 − / − MEFs . APG5 + / + ( open columns ) and APG5 − / − ( closed columns ) MEFs were transfected with the indicated plasmids ( 2 μg ) . After 24 h , transfected cells were incubated with 20 μM of etoposide in the presence or absence of 100 μM of zVAD or 10 mM of 3 - MA for 24 h , and cell viability was measured by the CTB assay ( d ) and apoptotic nuclear morphology ( e ) . Data are shown as mean ± s . d . ( n = 4 ) . ( f ) Overexpression of Bcl - xL does not sensitize Beclin 1 - silenced MEFs to etoposide - induced 3 - MA - inhibitable non - apoptotic death . WT MEFs with silencing of control GFP ( open columns ) or Beclin 1 ( closed columns ) were transfected with the indicated plasmids ( 1 μg ) . After 24 h , transfected cells were incubated with 20 μM of etoposide in the presence or absence of 10 mM of 3 - MA for 24 h , and cell viability was measured by the CTB assay . n = 3 .

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

figf4(a, b) APG5-dependent punctate GFP-LC3 fluorescence in Bcl-2-transfected MEFs after etoposide treatment. APG5+/+ and APG5−/− MEFs that were transfected with both GFP-LC3 and the indicated plasmids (Bcl-2 or vector) were incubated without or with 20 μM etoposide for 24 h, then examined by confocal fluorescent microscopy (a). The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells (b).(c) Accumulation of Beclin 1 and APG5-APG12 complex in Bcl-2-transfected MEFs after etoposide treatment. WT MEFs were transfected with the indicated plasmids, and were incubated without (NT) or with 20 μM etoposide for 24 h.(d, e) Occurrence of etoposide-induced 3-MA-inhibitable non-apoptotic death in Bcl-xL-overexpressing APG5+/+ MEFs, but not APG5−/− MEFs. APG5+/+ (open columns) and APG5−/− (closed columns) MEFs were transfected with the indicated plasmids (2 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 100 μM of zVAD or 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay (d) and apoptotic nuclear morphology (e). Data are shown as mean ± s.d. (n = 4).(f) Overexpression of Bcl-xL does not sensitize Beclin 1-silenced MEFs to etoposide-induced 3-MA-inhibitable non-apoptotic death. WT MEFs with silencing of control GFP (open columns) or Beclin 1 (closed columns) were transfected with the indicated plasmids (1 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay. n = 3.


figf5 ( a ) Enhancement of etoposide - induced non - apoptotic death by overexpression of human Bcl - 2 and Bcl - xL in Bax / Bak − / − MEFs in a Beclin 1 - dependent manner . Bax / Bak − / − MEFs with silencing of control Bax or Beclin 1 were transfected with the indicated plasmids ( 1 μg ) . After 24 h , the transfected cells were incubated with 20 μM etoposide for 24 h , and cell viability was assessed by the CTB assay . Data are shown as mean ± s . d . ( n = 3 ) . ( b ) Bax / Bak − / − MEFs were transiently transfected with 10 μg of siRNA for Bcl - 2 , Bcl - x , or Bax ( negative control ) for 24 h . Expression levels of Bcl - 2 , Bcl - xL and Lamin B ( loading control ) were analysed by Western blot analysis . ( c - e ) Inhibition of etoposide - induced autophagy in Bax / Bak − / − cells treated with siRNA for Bcl - x . ( c ) Bax / Bak − / − MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide for 24 h , then examined by phase - contrast microscopy . ( d ) Bax / Bak − / − MEFs that were transfected with siRNA for Bcl - x and then treated with etoposide for 18 h were analysed by electron microscopy ( × 8 , 500 ) ( e ) Reduction of punctate GFP - LC3 fluorescence in etoposide - treated Bax / Bak − / − MEFs by silencing of Bcl - x . Bax / Bak − / − MEFs that were transfected with both GFP - LC3 and the indicated siRNAs were incubated without ( NT ) or with etoposide for 24 h , and then were examined by confocal fluorescent microscopy . The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . n = 4 . f ) Inhibition of etoposide - induced cell death by silencing of Bcl - x . Bax / Bak − / − MEFs were transfected with siRNA ( circles , siRNA for Bax ; squares , siRNA for Bcl - x ) were treated with 20 μM etoposide in the absence ( open symbols ) or presence ( closed symbols ) of 10 mM 3 - MA , and cell viability was assessed by the CTB assay at the indicated times . n = 4 . ( g ) Clonogenicity assay of Bax / Bak − / − MEFs after exposure to etoposide and staurosporine . Bax / Bak − / − MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide or 1 μM staurosporine at 24 h . After collection , 2 , 000 cells were seeded in the normal medium . After one week , colonies were counted . h , i ) Inhibition of APG5 - APG12 induction by silencing of Bcl - x or Beclin 1 . Bax / Bak − / − MEFs were treated with the indicated siRNAs ( 10 μg ) for 24 h and then incubated with 20 μM etoposide for the indicated times . Expression of APG5 - APG12 complex , Beclin 1 and GAPDH / VDAC ( loading control ) was analysed by Western blot analysis .

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

figf5(a) Enhancement of etoposide-induced non-apoptotic death by overexpression of human Bcl-2 and Bcl-xL in Bax/Bak−/− MEFs in a Beclin 1-dependent manner. Bax/Bak−/− MEFs with silencing of control Bax or Beclin 1 were transfected with the indicated plasmids (1 μg). After 24 h, the transfected cells were incubated with 20 μM etoposide for 24 h, and cell viability was assessed by the CTB assay. Data are shown as mean ± s.d. (n = 3).(b) Bax/Bak−/− MEFs were transiently transfected with 10 μg of siRNA for Bcl-2, Bcl-x, or Bax (negative control) for 24 h. Expression levels of Bcl-2, Bcl-xL and Lamin B (loading control) were analysed by Western blot analysis.(c-e) Inhibition of etoposide-induced autophagy in Bax/Bak−/− cells treated with siRNA for Bcl-x. (c) Bax/Bak−/− MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide for 24 h, then examined by phase-contrast microscopy.(d) Bax/Bak−/− MEFs that were transfected with siRNA for Bcl-x and then treated with etoposide for 18 h were analysed by electron microscopy (×8,500)(e) Reduction of punctate GFP-LC3 fluorescence in etoposide-treated Bax/Bak−/− MEFs by silencing of Bcl-x. Bax/Bak−/− MEFs that were transfected with both GFP-LC3 and the indicated siRNAs were incubated without (NT) or with etoposide for 24 h, and then were examined by confocal fluorescent microscopy. The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. n = 4.f) Inhibition of etoposide-induced cell death by silencing of Bcl-x. Bax/Bak−/− MEFs were transfected with siRNA (circles, siRNA for Bax; squares, siRNA for Bcl-x) were treated with 20 μM etoposide in the absence (open symbols) or presence (closed symbols) of 10 mM 3-MA, and cell viability was assessed by the CTB assay at the indicated times. n = 4.(g) Clonogenicity assay of Bax/Bak−/− MEFs after exposure to etoposide and staurosporine. Bax/Bak−/− MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide or 1 μM staurosporine at 24 h. After collection, 2,000 cells were seeded in the normal medium. After one week, colonies were counted.h, i) Inhibition of APG5-APG12 induction by silencing of Bcl-x or Beclin 1. Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times. Expression of APG5-APG12 complex, Beclin 1 and GAPDH/VDAC (loading control) was analysed by Western blot analysis.