(A) LAPTM5 mRNA levels in NB cell lines with or without MYCN amplification (amp.) and/or allelic loss of the LAPTM5 gene were determined by quantitative RT-PCR (qRT-PCR) and are indicated as bars. Levels of LAPTM5 mRNA were normalized with levels of GAPDH mRNA. GOTO and IMR32 cells were treated with 5-aza-2′-deoxycytidine (5-aza-dC, 10 µM for 5 days). (B) Frequencies of methylation at 17 CG sites within region-I around the transcriptional-start site (TSS) of the LAPTM5 gene were determined by bisulfite sequencing, as indicated in Supplementary Figure S1. An arrow indicates the TSS. (C) Methylation frequency and mRNA level of LAPTM5 in primary NB tumors at different stages. Levels of LAPTM5 mRNA measured by qRT-PCR are indicated as dots; whereas methylation frequencies at two CG sites (CG-I or –II) measured by Ms-SNuPE are indicated as dark gray and light bars, respectively. Levels of LAPTM5 mRNA were normalized with levels of GAPDH mRNA. n indicates the number of cases. Vertical lines, SD. (D) Representative results of bisulfite sequencing at CG sites around the TSS of LAPTM5 in primary NB tumors and ganglioneuromas (GNs). Methylation status is indicated as white (unmethylated) or black (methylated) circles. An arrow indicates the TSS. (E) Methylation frequency of LAPTM5 in primary NB tumors and GNs. The methylation frequencies at two CG site in 8 GNs were measured by Ms-SNuPE. The average frequency of methylation in primary NB tumors or GNs is indicated. n indicates the number of cases. Vertical lines, SD. (F) Representative images of immunostaining for LAPTM5 in tissue sections. Upper; representative images of hematopoietic cells, adrenal tissue within tumor sections, and GN. The enlarged image shows the adrenal medulla cells within adrenal tissue indicated by arrowheads. Lower; representative images of NB cells within a favorable tumor (stage-1) detected by mass-screening and an advanced-type tumor (stage-4a) with MYCN amplification.

(A) Western blot analysis of adenovirus-infected GOTO, IMR32, and SH-SY5Y cells. The cells were infected with the LacZ (Ad-LacZ) or LAPTM5 (Ad-LAPTM5) adenovirus at a MOI of 10 for GOTO and SH-SY5Y or 4 for IMR32. Two and four days after infection, whole-cell lysates was isolated, and analyzed by immunoblotting with an antibody to LAPTM5 or β-actin (internal control). Results shown are representative of two independent experiments. (B) Viability of adenovirus-infected GOTO, IMR32, and SH-SY5Y cells. GOTO (5×10⁴ cells/well), IMR32 (2×10⁴ cells/well), or SH-SY5Y (5×10⁴ cells/well) cells plated in 24-well plates were infected with Ad-LacZ or Ad-LAPTM5 at the indicated MOIs. Four days after infection, the percentage of surviving cells was determined by a colorimetric water-soluble tetrazolium salt (WST) assay. Vertical lines, SD for three experiments. No inf., no infection. (C) Frequency of dead cells among adenovirus-infected GOTO, IMR32, and SH-SY5Y cells. The cells were infected with Ad-LacZ or Ad-LAPTM5 under the same conditions as in (A) with or without treatment with the pan-caspase inhibitor zVAD-fmk at 100 µM in each cell line. Dead cells were counted 2 and 4 days after infection using the trypan blue exclusion method, and indicated as percentages. Vertical lines, SD for three experiments. (D) Western blot analysis of LAPTM5 in LAPTM5-infected GOTO cells treated with protein degradation inhibitors. Cells were infected with Ad-LacZ or Ad-LAPTM5 at a MOI of 10. One day after infection, cells were treated with Bafilomycin A1 (Baf.A1, lysosomal inhibitor), NH₄Cl (lysosomal inhibitor), ALLN (proteasome inhibitor), or MG-132 (proteasome inhibitor) at indicated concentration for 1 day. Whole-cell lysate was analyzed by immunoblotting with indicated antibodies. The results shown represent two independent experiments. (E) Frequency of dead cells among LAPTM5-infected GOTO cells treated with protein degradation inhibitors. Cells were infected and treated with Baf.A1 or ALLN, as indicated in (D). Dead cells were counted using the trypan blue exclusion method, and indicated as percentages. Vertical lines, SD for three experiments. *t-test; p<0.05. (F) Western blot analysis of LAPTM5 inhibition by siRNA transfection in LAPTM5-infected GOTO cells treated with protein degradation inhibitors. Cells were transfected with control- or LAPTM5-siRNA, and infected with Ad-LacZ or -LAPTM5 at a MOI of 10. The next day cells were treated with the inhibitor Baf.A1 or ALLN for 1 day. Whole-cell lysate was analyzed by immunoblotting with indicated antibodies. The results shown represent two independent experiments. (G) Effect of siRNA transfection on frequency of dead cells among LAPTM5-infected GOTO cells treated with protein degradation inhibitors. Cells were infected and treated with Baf.A1 or ALLN, as indicated in (F). Dead cells were counted using the trypan blue exclusion method, and indicated as percentages. Vertical lines, SD for three experiments. *t-test; p<0.05.

(A) Effect of treatment with wortmannin or transfection with ATG5 siRNA in GOTO cells with a punctate distribution of LC3B. Cells (1×10⁵/well) were plated on coverslips in 24-well plates and the next day treated with wortmannin (Wort.; 200 nM) or DMSO (0.02%), or transfected with ATG5 siRNA (20 nM) or Luciferase siRNA (20 nM). Left; 4 days after the treatment or transfection, cells were treated with EBSS for 2 hours. Right; the plated cells were infected with Ad-LacZ or Ad-LAPTM5 at a MOI of 10 for 4 days with treatment with DMSO or wortmannin, or with transfection with Luciferase or ATG5 siRNA. Then, cells were fixed in 10% TCA, reacted with the LC3B antibody, and visualized with an FITC-conjugated secondary antibody. Upper; western blotting analysis of ATG5. Whole-cell lysate was analyzed by immunoblotting with an antibody to ATG5 or β-actin (internal control). Lower; frequency of cells showing a punctate distribution of LC3B among cells (at least 200 cells) was measured. *t-test, p<0.001 and **p<0.01. Vertical lines, SD for three experiments. (B) Effect of treatment with wortmannin or the knockdown of ATG5 during LAPTM5-induced cell death. GOTO cells (1×10⁴/well) were plated in 96-well plates and infected with Ad-LacZ or LAPTM5 at a MOI of 10 with treatment with DMSO or wortmannin (Wort.), or with transfection with Luciferase or ATG5 siRNA. Four days after infection, numbers of viable cells were assessed by WST assay. Vertical lines, SD for three experiments. (C) Western blot analysis of p62/SQSTM1 and ubiquitin in LAPTM5-infected GOTO cells. The same whole-cell lysates indicated in Figure 3A were analyzed by immunoblotting with indicated antibodies. The results shown represent two independent experiments. (D) Immunofluorescence microscopy for p62/SQSTM1 and ubiquitin in LAPTM5-infected GOTO cells. Upper; GOTO cells (1×10⁵/well) were plated on coverslips in 24-well plates, and infected with Ad-LAPTM5 at a MOI of 10. Four days after infection, the cells were fixed in 4% formaldehyde, reacted with p62/SQSTM1 and ubiquitin antibodies, and visualized with an FITC- or Alexa Fluor 594-conjugated secondary antibody. Arrowheads indicate the cells with p62/SQSTM1-ubiquitin positive inclusion-like body. DAPI was used for counterstaining. (E) Western blotting of ubiquitinated proteins. Four days after infection, GOTO cells were lysed in a 0.1% Triton-X solution. The Triton-insoluble pellet (P) was lysed in a 2% SDS buffer. The Triton-X soluble (S) or -insoluble (P) lysate was loaded on a 6% SDS-PAGE gel and analyzed by immunoblotting with an antibody to ubiquitin or α-tublin. For both soluble and insoluble samples, ubiquitinated proteins were found to be accumulated in Ad-LAPTM5-infected GOTO cells, compared with Ad-LacZ-infected GOTO cells. The results shown represent two independent experiments.

(A) Representative images of LysoTracker Rhodamine (LTR) staining in infected GOTO cells. GOTO cells (1×10⁵/well) were plated on coverslips in 24-well plates, and infected with Ad-LAPTM5 at a MOI of 10. Four days after infection, cells were stained with LTR (100 nM) for 30 min at 37°C, washed twice and fixed in 4% formaldehyde, and observed by fluorescence microscopy. (B) Acridine orange (AO) uptake analysis in infected GOTO cells. GOTO cells (2×10⁵/well) were plated in 6 well plates, and infected with Ad-LAPTM5 at a MOI of 10. Four days later, cells were stained with AO (5 µg/ml, 30 min). Detached cells were removed, and attached cells were collected by trypsinization and washed twice with PBS. Left-upper; scatter blots of FSC and SSC for Ad-LacZ or Ad-LAPTM5-infected cells. The main population of cells was gated and analyzed by FACS. Left-lower; the intensity of staining was measured by FACS using a channel of FL3; the range containing >99% of the cells without AO fluorescence was gated. Black line indicates Ad-LAPTM5-infected cells; red line, Ad-LacZ-infected cells. The frequency of cells with a reduction in AO staining was increased among Ad-LAPTM5-infected GOTO cells. Right; percentage of cells with a reduction in AO fluorescence. Vertical lines, SD for four separate experiments. *t-test, p<0.01. (C) Representative images of staining for cathepsin D (CTSD) in infected GOTO cells. GOTO cells (1×10⁵/well) were plated on coverslips in 24-well plates, and the next day infected with Ad-LacZ or Ad-LAPTM5 at a MOI of 10. Four days after infection, the cells were fixed in 10% TCA, reacted with a CTSD antibody, and visualized with an Alexa Fluo 594-conjugated secondary antibody. The percentage of cells that released CTSD (arrow) was measured. Vertical lines, SD for three separate experiments. *t-test, p<0.05. (D) Representative images of staining for CTSD, p62/SQSTM1, or ubiquitin in non-regressing or regressing areas within favorable NB tumors. Serial tumor sections were stained with each antibody. The area enclosed by the rectangle is enlarged at the right. Arrows indicate cells with ubiquitin-positive inclusion bodies.

(A) Serial tumor sections were stained with hematoxylin-eosin (HE) (a, b, e, and f) or with anti-LAPTM5 (c, d, g, and h). a–d; representative image of a type-I regressing area, with a loss of cells including differentiating NB cells. e–h; representative image of a type-II regressing area, with a large loss of undifferentiated NB cells. The images of b, d, f, and h are the areas enlarged by the rectangle in a, c, e, and g, respectively. The area surrounded with arrowheads in a and e indicates a typical regressing area. In d and h images, arrowheads and arrows indicate differentiating NB cells and degenerating NB cells, respectively. (B) Relationship of between LAPTM5-associated regression with patients and tumor characteristics. A comparison was made of the proportion of positive cases between mass-screened NBs and clinically detected NBs. The stage of each tumor was determined using the INSS (International Neuroblastoma Staging System). NA; cases with MYCN amplification. (C) Representative image of immunostaining of cleaved caspase-3. SH-SY5Y cells with or without treatment with Cisplatin (CDDP; 1.5 µg/ml) for 2 days or serial tumor sections were stained with an antibody to cleaved caspase-3 antibody.

(A) Representative transmission-electron micrographs of Ad-LacZ (a) or Ad-LAPTM5 (b and c) -infected GOTO cells 4 days after infection. The (c) image was enlarged from the area enclosed by the rectangle in (b), showing the accumulation of autophagic vacuoles (arrowheads). (B) Localization of GFP-LC3 or endogenous LC3B detected by immunofluorescence microscopy. GOTO cells stably expressing GFP-LC3 (1×10⁵/well) (upper panel) or parental GOTO cells (1×10⁵/well) (lower panel) were plated on coverslips in 24-well plates, and the next day infected with Ad-LacZ or Ad-LAPTM5 at a MOI of 10. Four days later, the GOTO cells stably expressing GFP-LC3 were fixed in 4% formaldehyde, and observed under a fluorescence microscope. For parental GOTO cells, the cells were fixed in 10% TCA 4 days after infection, reacted with the LC3B antibody, and visualized with an FITC-conjugated secondary antibody. Control cells starved of nutrients were treated with EBSS for 2 hours at 5 days of plating. The frequency of cells with a punctate distribution (dot pattern) of GFP-LC3 or LC3B among all GFP-positive cells or total cells (at least 200 cells), respectively, was measured. *t-test, p<0.005 and **p<0.05. Vertical lines, SD for three experiments. (C) Detection of LC3B-II by western blotting. As the time-courses indicated, whole-cell lysate from GOTO cells starved of nutrients or infected with Ad-LacZ and Ad-LAPTM5 at a MOI of 10 was analyzed by immunoblotting with an antibody to endogenous LC3B or β-actin (internal control). Arrows indicate form-I or -II of LC3B. Results shown are representative of two independent experiments.

(A) Representative image of the co-staining of Golgi-58K or LAMP2 (Green) and LAPTM5 (Red). GOTO cells (1×10⁵/well) were plated on coverslips in 24-well plates and the next day infected with Ad-LacZ or Ad-LAPTM5 at a MOI of 10. One or four days after infection, the cells were fixed in 10% TCA, reacted with Golgi-58K or LAMP2 and LAPTM5 antibodies, and visualized with FITC- or Alexa Fluor 594-conjugated secondary antibodies. DAPI was used for counterstaining. Arrowheads indicate the cells with a loss of Golgi apparatus structures. (B) Representative image of the co-staining of Golgi-58K or LAMP2 (Green) and LAPTM5 (Red). GOTO cells were infected with Ad-LacZ or Ad-LAPTM5 a MOI of 10, and next day treated with Bafilomycin A1 (Baf.A1) or ALLN for 1 day. Arrowheads indicate the cells with a loss of Golgi apparatus structures. Then immunofluorecent images were obtained as in (A).