EMBO/soda-nel huggingface dataset:
Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria (2010)
figf3

(a) Cells were transfected with control siRNA or with siRNA specific to IRGM (top) and DRP1 (bottom) for 48 h and protein samples from these cells were then analysed by immunoblotting with anti-IRGM and anti-DRP1 antibodies. Actin was used as a loading control. (b) HeLa cells were treated with siRNA specific to either IRGM or DRP1 as indicated and were then labelled with MTR and analysed by live-cell confocal microscopy. Scale bars, 5 μm. (c) Cells were treated with either control siRNA or with DRP1, IRGM, ATG7 or BECN1 (Beclin 1) siRNAs, labelled with MTR and the percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified. Definition of mitochondrial morphologies and quantification criteria are given in Supplementary Information, Figure S2. (d) Cells were transfected with either control siRNA or with siRNA specific to MFN1/MFN2, IRGM or IRGM and MFN1/MFN2, and then labelled with MTR and analysed for mitochondrial morphologies. Data are means ± s.e.m. (c, n = 3; d, n = 4). Asterisk indicates P 0.05, double asterisks indicate P 0.01 and dagger indicates P > 0.05 (t-test).
Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria (2010)
figf4

(a) U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP–LC3. Autophagy was induced by treatment with hIFN-γ. GFP–LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm. (b) LC3 puncta per cell were quantified from cells treated as in a. (c) LC3 puncta per cell quantified from cells treated as in a, except that autophagy was induced by starvation rather than treatment with hIFN- γ. (d) After transfection with either control siRNA or siRNA specific to MFN1/MFN2, endogenous LC3 puncta per cell were quantified. (e) U937 cells were transfected with the indicated siRNA and infected with BCG. Autophagy was induced by starvation and phagosome maturation was analysed using LysoTracker probe (LTR) as a reporter of acidification. (f) U937 cells were treated with the indicated siRNA and infected with virulent M. tuberculosis H37Rv. Autophagy was induced by starvation, or cells were kept in full medium, as indicated. Colony forming units were counted, and expressed as a percentage of the number of plated bacterial colonies from untreated cells, to assess bacterial survival. Data are means ± s.e.m. (n = 6; two independent transfections, 6 independent infections). Asterisk indicates P 0.05, and dagger indicates P > 0.05 (t-test) for the indicated data.
Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria (2010)
figf5

(a) Top: schematic representation of IRGM. The G1–G5 GTPase motifs and their sequences are shown. The change corresponding to the S47N mutation is indicated with an arrow. Bottom: splice variants of IRGM and Ras (p21). (b) HeLa cells were transfected with plasmids encoding fluorescent protein-tagged IRGMb, IRGMd WT (wild-type IRGMd) or IRGMS47N mutant (control cells; vector expressing GFP only), and after 48 h were labelled with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm. (c) Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP–IRGMd positive and one GFP–IRGMd negative, as indicated in the inset. (d) Quantification of GFP+ MTR+ cells from an experiment performed as in b. (e) Nitrocellulose filters spotted with the indicated lipids were incubated with GST or GST–IRGM. Binding was detected by immunoblotting. (f) Agarose beads (control beads) and cardiolipin-bound agarose beads (CL beads) were incubated with GST or GST–IRGMd. Proteins were eluted from the beads and immunoblotted. Two lanes on the left indicate immunoblotting of samples used to incubate beads. (g) Nitrocellulose filters spotted with the indicated lipids (defined in e) were incubated with wild-type IRGMd, IRGMdS47N, and the IRGM isoform IRGMb at the indicated concentrations. GST control is shown in Supplementary Information, Figure S1d. Membranes in e–g were probed with anti-GST antibody.
Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria (2010)
figf6

(a) HeLa cells transfected with plasmids encoding GFP or GFP–IRGMd were stained with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm. (b–d) Quantification of mitochondrial morphologies in cells transfected with IRGMd, IRGMdS47N or IRGMb GFP fusions (control cells express GFP only). (e) Cells were transfected with the indicated siRNAs and vector encoding GFP–IRGMd, and labelled with MTR. The percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified. (f) Cells were transfected with the indicated siRNAs and vector encoding GFP–IRGMd (or vector encoding GFP only, as a control) and labelled with MTR. Percentages of GFP+ cells with MTR staining at 48 h were quantified. (g) Confocal microscopy images of HeLa cells transfected with vector encoding GFP–DRP1 (A) or GFP–IRGMd (B–G) and labelled with MTR (A–D) or antibodies against COX-IV (F). A, steady-state DRP1 distribution; B–D, translocation of IRGMd from the cytosol to mitochondria at indicated times post-transfection; E–G, co-localization of IRGMd and COX-IV (G is merged from E and F). Scale bars, 5 μm. (h) HeLa cells, co-transfected with GFP–IRGMd, and control, BECN1 or ATG7 siRNA, were labelled with MTR 48 h post-transfection, imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. (i) Cells from Atg7 wild-type (WT) or Atg7−/− MEFs transfected with vectors encoding GFP–IRGMd for 48 h were stained with MTR, imaged, and percentage of GFP+ cells that were also MTR+ cells was quantified. (j) Wild-type W2 (Bax/Bak+/+) or mutant D3 (Bax/Bak−/−) BMK cells were transfected with vectors encoding GFP–IRGMd and after 48 h were labelled with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. (k) HeLa cells were transfected with vectors encoding GFP–IRGMd, treated with z-VAD as indicated, and after 48 h stained with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. Data are means ± s.e.m. (n = 3). Dagger indicates P ≥ 0.05, asterisk indicates P 0.05 and double asterisks indicate P 0.01 (t-test) for the indicated data.
Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria (2010)
figf7

(a) Percentage of HeLa cells transfected with vectors encoding GFP or GFP–IRGMd that were rounded 48 h post-transfection. (b) HeLa cells transfected with plasmids encoding GFP or GFP–IRGMd were stained with 7-AAD after 48 h and percentages of GFP+ cells that were also 7-AAD+ were quantified. (c) HeLa cells transfected with vectors encoding GFP–IRGMd versus GFP–IRGMdS47N and YFP–IRGMb (vector encoding GFP only was transfected as a control) were stained with 7-AAD after 48 h, and percentages of GFP+ (for IRGMd and IRGMS47N) or YFP+ (for IRGMb) cells that were also 7-AAD+ were quantified. (d) HeLa cells were transfected with plamsids encoding GFP–IRGMd, GFP–IRGMDS47N or YFP–IRGMb (vector encoding GFP only was transfected as a control), and after 48 h were stained with propidium iodide (PI). Percentages of GFP+ or YFP+ cells that were also PI+ were quantified. (e) Images of HeLa cells transfected with vectors encoding GFP–IRGMd (bottom) were stained for active caspases 3 and 7 using FLICA dye. Staurosporine (STS; middle images) was used as a positive control. Cells were transfected with vectors encoding GFP only as a control (top). (f) HeLa cells were transfected with plasmids encoding GFP–IRGMd and were stained with antibodies against HMGB1 (absence of nuclear HMGB1 stain is a marker of HMGB1 release) or DAPI. Top: images indicate GFP–IRGMd (left), HMGB1 (middle) and DAPI (right) localization. Bottom: phase-contrast microscopy image of cells (left) and image merged from other panels (right). Scale bars, 5 μm. (g–i) Cells were transfected with plasmids encoding GFP or GFP–IRGMd, and control or DRP1 siRNA, as indicated. After 48 h, (g) cells were stained with 7-AAD and percentages of GFP+ cells that were also 7-AAD+ were quantified, (h) rounded cells were quantified, or (i) cells were stained with HMGB1 and HMGB1+ nuclei were quantified. Data are means ± s.e.m. (n = 3). Asterisk indicates P 0.05 and double asterisk indicate P 0.01 (t-test).
Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria (2010)
figf8

(a, b) HeLa cells were transfected with plasmids encoding GFP, CFP–IRGMa, GFP–IRGMc or GFP–IRGMd, and after 24 h (a) or 48 h (b), stained with MTR. Percentages of positively transfected cells that were also MTR+ were then quantified. (c, d) Cells were transfected as in a, b, and after 24 h (c) or 48 h (d) the percentages of positively transfected cells that were also rounded by morphology were quantified. (e, f) Cells were transfected as in a, b, and after 24 h (e) or 48 h (f), stained with 7AAD. Percentages of positively transfected cells that were also 7AAD+ were then quantified. (g, h) Cells were transfected as in a, b, and after 24 h (g) or 48 h (h), stained with antibodies against HMGB1. Cells positively transfected with the indicated plasmids were assessed for retention of HMGB1 in the nucleus. Data are means ± s.e.m. (n = 3, a–f; n = 4, g, h). Asterisk indicates P 0.05 and double asterisks indicate P 0.01 (t-test).
Regulation of hematogenous tumor metastasis by acid sphingomyelinase. (2015)
Figure 1

Tumor metastasis is abrogated in Asm-deficient mice and restored by transplantation of wild-type platelets

A, B 1 × 105 B16F10 melanoma cells were intravenously injected into C57BL/6 wild-type (WT) or Asm-deficient (Asm−/−) mice, and the number of lung metastases was determined after 14 days. The photographs (A) show two representative results. The graph (B) shows the mean ± SD number of pulmonary metastases in 15 WT and 14 Asm-deficient animals.

C, D B16F10 melanoma grow as fast or slightly faster in Asm-deficient mice than in wild-type mice after subcutaneous injection at the flank (C) or transcutaneous direct intrapulmonary injection (D), excluding a general growth defect of the tumor in Asm-deficient mice. The size of tumors was determined 14 days after local injection at the flank or 10 days after injection into the lung.

E The number of lung metastases 25 days after intravenous injection does not differ from the number observed after 14 days in (B).

F 5 × 108 platelets isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice were injected intravenously into Asm-deficient or wild-type mice, respectively. After 120 min 1 × 105, B16F10 tumor cells were intravenously injected. The number of lung metastases was determined after 14 days. Displayed are the mean ± SD of the number of pulmonary metastases, n = 3 in WT platelets in WT mice, n = 5 in WT platelets in Asm−/− mice, and n = 4 in Asm−/− platelets in Asm−/− mice.

Data information: Shown is the mean ± SD, n = 5 (C–E). Statistical significance was determined by t-test for single comparisons or analysis of variance (ANOVA) followed by a Tukey's multiple comparisons test. P-values are given.
Regulation of hematogenous tumor metastasis by acid sphingomyelinase. (2015)
Figure 2

Co-incubation of B16F10 melanoma with platelets results in Asm secretion and activation and ceramide release to mediate metastasis

A Platelets were isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice by density-gradient centrifugation, and 1 × 107 platelets were incubated with 1 × 105 B16F10 cells. The samples were lysed, and Asm activity was determined. In unstimulated samples (time point of co-incubation 0 s), tumor cells and platelets were admixed after lysis.

B The formation of ceramide was determined by a diacylglycerol (DAG) kinase assay (upper panel) and mass spectrometry (lower panel). In unstimulated samples (time point of co-incubation 0 s), tumor cells and platelets were admixed after lysis.

C, D For determination of the secretion of Asm by platelets into the supernatants (C), tumor cells and wild-type (WT) or Asm-deficient (Asm−/−) platelets were co-incubated for 20 s, the samples were pelleted, the supernatants were removed and acidified, and the Asm activity was measured in the presence or absence of Zn2+. For measurement of surface Asm and ceramide, samples were co-incubated as indicated (C, D) and pelleted; the supernatants were discarded, incubated with anti-Asm antibodies, washed, and lysed; and Asm immunocomplexes were immobilized and subjected to immunocomplex enzyme assays. Surface ceramide was measured by incubation of intact cells with DAG kinase in the presence of [32P]γATP followed by extraction and measurement of [32P]-ceramide. Omission of one cell type indicated by “−” here and thereafter.

E Treatment of B16F10 tumor cells for 2 min with 1 U/ml ASM or 10 μM C16 ceramide restores in vivo metastasis in Asm-deficient mice. After treatment, the cells were injected intravenously into Asm-deficient (Asm−/−) mice. Controls were left untreated prior to injection. The number of metastases was determined 14 days after tumor cell injection.

Data information: Displayed is the mean ± SD of 4 (A–D) or 9 (E) experiments. Statistical significance was determined by analysis of variance (ANOVA) followed by a Tukey's multiple comparisons test. P-values are given.
Regulation of hematogenous tumor metastasis by acid sphingomyelinase. (2015)
Figure 3

Interaction of human or mouse melanoma cells with platelets results in Asm secretion and surface Asm activity independent of Asm expression in melanoma cells

Incubation of human melanoma (HM) cells with human platelets results in the release of Zn2+-dependent ASM into the supernatant, Zn2+-dependent activity of ASM on cell surfaces, and the formation of ceramide. The assay buffer contained 100 μM Zn2+.

Addition of human or mouse recombinant ASM to human melanoma or B16F10 cells, respectively, results in binding of ASM to the tumor cell surfaces as determined by flow cytometry. Cytochalasin B was added to control for internalization of added ASM.

Suppression of Asm in B16F10 tumor cells using siRNA technology reduces Asm activity in B16F10 cells (right panel), but does not alter release or surface activity of the Asm after co-incubation with wild-type platelets (left panels).

Data information: In (A) and (C) are shown the mean ± SD, n = 4. In (B) representative data from four independent experiments each are displayed. Statistical significance was determined using ANOVA followed by a Tukey's multiple comparisons test. P-values are indicated (Ctrl: control).
Regulation of hematogenous tumor metastasis by acid sphingomyelinase. (2015)
Figure 4

The primary interaction of tumor cells and platelets is not affected by Asm deficiency, but Asm expression in platelets is required for tumor cell adhesion

Platelets were isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice by density-gradient centrifugation, and 1 × 107 platelets were incubated with 1 × 105 B16F10 cells or left untreated. Co-incubation of washed platelets with B16F10 melanoma cells resulted in upregulation of GPIIbIIIa and CD62P on both wild-type and Asm-deficient platelets in FACS analyses. Platelets were gated in FCS/SSC.

Aggregation properties of platelets after co-incubation with collagen or ADP are independent of Asm as determined by aggregometry measurements.

Incubation of tumor cells with platelets resulted in marked change of platelet shape and degranulation as determined by FACS analysis using forward versus side scatter analysis indicating global activation of platelets.

4 × 104 B16F10 tumor cells were incubated with 2 × 107 wild-type (WT), Asm-deficient (Asm−/−) platelets (Plts) or a 45:55 of WT:Asm−/− platelet mixture in the presence or absence of 50 ng/ml PGE1. Controls were stimulated with Mn2+. Tumor cells were then incubated for 60 s on fibronectin-coated cover slips, washed, and fixed, and adhesion of the tumor cells was determined. The graph displays the mean ± SD of tumor cells adhering to fibronectin-coated cover slips, n = 7 for WT-platelets, n = 9 for Asm-deficient-platelets, n = 4 for 45:55 WT:Asm−/− platelets, and all others n = 3. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. P-values are indicated.

Data information: Displayed are representative results of each four independent experiments (A–C).
Regulation of hematogenous tumor metastasis by acid sphingomyelinase. (2015)
Figure 5

Acid sphingomyelinase induces clustering and activation of α5 and β1 integrins in ceramide-enriched membrane domains resulting in tumor cell adhesion and metastasis

A–C 1 × 105 B16F10 cells were incubated with (A) 1 U/ml purified acid sphingomyelinase (ASM) or (B) 5 × 107 wild-type (WT), Asm-deficient (Asm−/−) platelets (Plts), or a mixture of 45:55 wild-type:Asm-deficient platelets. Cells were fixed with 2% paraformaldehyde for 15 min and stained with fluorescein isothiocyanate (FITC)-coupled anti-α5 integrin, Cy3-labeled anti-β1 integrin, and Cy5-labeled anti-ceramide antibodies. Samples were analyzed by confocal microscopy. Shown are representative examples from four independent experiments (A, B) or the quantitative analysis of cells positive for ceramide/β1 integrin clusters from at least 100 cells/sample (C). Given is the mean ± SD, n = 4, ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.

D ASM-induced B16F10 tumor cell adhesion to fibronectin-coated cover slips is abrogated by the inhibition of integrins with RGD peptides. Shown is the mean ± SD of the number of cells adherent to the cover slip. Statistical significance was determined by ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.

E Intravenous injection of CFSE-labeled B16F10 cells into wild-type mice results in formation of ceramide-enriched domains that contain β1 integrin clusters on tumor cells in vivo, while injection of tumor cells into Asm-deficient mice does not result in ceramide and integrin clustering. Please note that the cell in the lower panel still binds a platelet. Shown are representative results from four independent experiments each.
Regulation of hematogenous tumor metastasis by acid sphingomyelinase. (2015)
Figure 6

Acid sphingomyelinase induces clustering and activation of β1 integrins in ceramide-enriched membrane domains in human melanoma cells

Human melanoma cells were stimulated for 10 min with 1 U/ml purified ASM or 10 μM C16 ceramide (C16-Cer) in the presence or absence of 500 ng ceramidase (CDase) or 1 μg/ml neutralizing anti-ceramide antibodies (anti-Cer), and lysed and active β1 integrin was immunoprecipitated using the HUTS-4 antibody. Octylglucopyranoside (OGP, final concentration 0.01%) served to resuspend C16 ceramide. Internalization was excluded by addition of cytochalasin B (CTB) as indicated. Samples were separated by SDS-PAGE and blotted with an activation-independent anti-β1 Integrin antibody to detect the amount of immunoprecipitated, that is active β1 integrin. Control immunoprecipitates (control Ipt) were performed with an irrelevant isotype control antibody. Activation of β1 integrin in human melanoma cells by incubation with Asm or C16 ceramide was confirmed by FACS analysis upon staining with FITC-labeled HUTS-4 antibodies.

Incubation of human melanoma with human platelets or ASM for 5 min triggers co-clustering of ceramide and activated β1 integrin on the surface of the tumor cells. Cells were stained with FITC-coupled anti-active β1 integrin (HUTS-4) antibodies and Cy3-coupled anti-ceramide antibodies. Shown are representative stainings from each four independent experiments.

Source data are available online for this figure.
Regulation of hematogenous tumor metastasis by acid sphingomyelinase. (2015)
Figure 7

Asm-mediated β1 integrin activation mediates adhesion of tumor cells in the lung in vivo

B16F10 tumor cells or B16F10 transfected with siRNA targeting Asm expression or with scrambled control siRNA were labeled with [3H]thymidine, washed, and treated (+) for 10 min with 1 U/ml ASM, 10 μM C16 ceramide, 10 μM S1P, 10 μM PDMP, 20 μM sphingosine kinase inhibitor SKI II, and 10 μM myriocin, or left untreated (−). The cells were then injected intravenously into wild-type or Asm-deficient mice as indicated. If indicated, 10 μg of arginine-glycine-aspartic acid (RGD) peptide or 10 μg/ml neutralizing anti-β1 integrin antibodies were added to B16F10 tumor cells together with ASM for 15 min prior injection. Mice were sacrificed 30 min after tumor cell injection, the lungs carefully flushed via the right heart, and the radioactivity in the lung as a measurement for adherent tumor cells was determined. Shown is the mean ± SD of the counts per minute (cpm) in the lung lysates from three independent experiments. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey's test for multiple comparison to determine P-values.
Regulation of hematogenous tumor metastasis by acid sphingomyelinase. (2015)
Figure 8

Targeting the Asm system prevents melanoma metastasis

A Amitriptyline (2 mg/kg, Ami) was intraperitoneally injected into C57BL/6 mice for five times every 12 h. Sixty minutes after the last injection, 1 × 105 B16F10 tumor cells were intravenously injected. Control experiments confirmed that amitriptyline inhibited Asm activity in the blood by approximately 85%. Asm heterozygous mice were injected with 1 × 105 B16F10 tumor cells. The number of lung metastases was determined after 14 days. Shown is the mean ± SD from six mice each, ANOVA followed by Tukey's test for multiple comparison. P-values are indicated.

B, C MT/ret cells (105) suspended in 200 μl Matrigel/PBS (1:1) were injected s.c. in the left and right flanks of 8-week-old Asm-deficient mice or wild-type mice. Mice were sacrificed at day 20 after injection, and spleens were inspected for the presence of metastases. The representative photographs (B) show the results from one of four experiments, and the quantitative analysis is displayed in (C).

D Platelets were isolated from wild-type or Asm-deficient mice by density-gradient centrifugation, and 1 × 107 platelets were incubated with 1 × 105 MT/ret melanoma cells. The samples were lysed, and Asm activity in cell lysates was determined. In unstimulated samples (time point 0 of co-incubation), tumor cell and platelet lysates were admixed after lysis.

E To measure secretion of Asm by platelets, tumor cells and platelets were co-incubated for 30 s, the samples were pelleted, the supernatants were acidified, and the Asm activity was measured. All Asm activity measurements were performed in the presence of 100 μM Zn2+.

F Local tumor growth in wild-type or Asm-deficient mice at the flank was measured at day 20.

Data information: Displayed is the mean ± SD of each three or four experiments. Statistical significance was determined by t-test (C, E, F) or analysis of variance followed by Tukey's multiple comparisons test (D). P-values are indicated.