Fig 1: Mitochondrial trafficking is modulated by SNPH.(a) LN229 cells transfected with control siRNA (Ctrl) or SNPH-directed siRNA were stained with antibodies against Tom20, β-tubulin and DAPI and analysed by confocal microscopy. Top, 3D rendering of mitochondria localization by fluorescence microscopy. Bottom, 3D drawings represent the cells above, and show mitochondrial repositioning to the cortical cytoskeleton. Scale bar, 10 μm. (b,c) Cells were labelled with MitoTracker (b) or Tom20 (c) and analysed for mitochondrial recruitment to the cortical cytoskeleton. Symbols correspond to individual cells. Data are represented as mean±s.e.m. **P=0.003; ***P<0.0001 by Student's t test. (d) PC3 cells transduced with the indicated shRNA were transfected with vector or shRNA-insensitive SNPH cDNA and analysed for mitochondrial trafficking to the cortical cytoskeleton. Symbols correspond to individual cells. ***P<0.0001 by one-way ANOVA and Bonferroni's post test. (e,f) Cells transfected with control or SNPH-directed siRNA and expressing mitochondria-targeted RFP were imaged by time lapse microscopy, and individual mitochondria were tracked through the stack to calculate trafficking parameters (e), and the speed of individual mitochondria per condition (f). Data are represented as mean±s.e.m. (n indicated on the individual bars). ***P<0.0001 by Student's t test. (g,h) The displacement of mitochondria relative to the initial location was calculated in 2D plots (g) and the distance travelled by individual mitochondria was quantified (h). Each tracing on g represents individual mitochondria and are colour coded according to Euclidean distance displacement. Each dot on h correspond to individual mitochondria (n indicated in the individual panels). ***P<0.0001 by Student's t test.
Fig 2: SNPH regulation of tumour cell motility.(a) Schematic diagram of the genome-wide shRNA screening to identify novel mitochondrial regulators of tumour cell invasion modulated by Gamitrinib. (b,c) Bioinformatics analysis of mitochondrial regulators of tumour cell invasion modulated by Gamitrinib. (d,e) PC3 cells transfected with control siRNA (Ctrl) or SNPH-directed siRNA (top) or vector or SNPH cDNA (bottom) were analysed for invasion across Matrigel-coated inserts. Representative micrographs of DAPI-stained cells (d) and quantification of invaded cells (e). Data are represented as mean±s.e.m. (n=3). ***P=0.0006–<0.0001 by Student's t test. Scale bar, 200 μm. (f) LN229 cells labelled with Talin-RFP and transfected with control (Ctrl) or SNPH-directed siRNA were analysed for FA complex dynamics by time lapse microscopy. Representative merged frames at 0 h (magenta) and 2 h (cyan) are shown. Scale bar, 5 μm. (g) FA complex dynamics in LN229 cells transfected with control siRNA or SNPH-directed siRNA (n=380). (h) LN229 cells were transfected with the indicated siRNAs and analysed for 2D chemotaxis by time-lapse video microscopy. Colour-identified cutoff velocities of cell motility are indicated. Each tracing corresponds to an individual cell. Sample sizes were Ctrl (n=60), SNPH (n=89). (i) shRNA-transduced PC3 cells were reconstituted with vector or SNPH cDNA and analysed for cell motility in a 2D chemotaxis chamber with quantification of speed of cell migration. Data are represented as mean±s.e.m. Sample sizes were: pLKO,Vector (n=46), SNPHsh,Vector (n=48), SNPHsh,SNPHcDNA (n=46). *P=0.0209; ***P=0.0007 by one-way ANOVA and Bonferroni's post-test.
Fig 3: SNPH regulation of mitochondrial dynamics.(a) LN229 cells transfected with control siRNA (Ctrl) or SNPH-directed siRNA were labelled with Mito-RFP and mitochondrial fission and fusion events were quantified in individual frames of time lapse microscopy. (b) Combined histograms of mitochondrial size and velocity data. Large mitochondria are slow moving (speed <0.05 μm s−1) compared with smaller mitochondria (speed up to 0.25 μm s−1). (c) PC3 cells stably expressing pLKO or SNPH-targeting shRNA #0 were transfected with siRNA against MFN1 or MFN2 and analysed for Matrigel invasion. Data are expressed as mean±s.e.m. (n=3). ***P<0.001 by one-way ANOVA and Bonferroni's post test. (d,e) PC3 cells stably expressing pLKO or SNPH-targeting shRNA were transfected with cDNAs for SOD2 or PRX-III and analysed by Western blotting (d), with quantification of bands by densitometry and normalization to β-actin levels (e). Data are expressed as mean±s.e.m. (n=3).*P<0.05 by one-way ANOVA and Bonferroni's post test. (f) Schematic model for mitochondrial trafficking and immobilization in neurons. The implicated molecules in the SNPH network are indicated. (g) PC3 cells stably expressing pLKO or SNPH-directed shRNA were transfected with siRNA against KIF5B, Miro1 or Miro2, and analysed for Matrigel invasion. Data are expressed as mean±s.e.m. (n=3). ***P<0.001 by one-way ANOVA and Bonferroni's post test. ns, not significant. (h) PC3 cells were transfected with pGFP, full length (FL) SNPH or SNPH deletion mutants lacking the microtubule-binding domain (ΔMBD), kinesin-binding domain (ΔKBD) or LC8-binding domain (ΔLBD) and analysed for Matrigel invasion. Data are represented as mean±s.e.m. (n=3). ***P<0.0001 by Student's t test. (i) LN229 cells transfected with the indicated siRNAs were treated with the small molecule PI3K inhibitor, PX-866 (5 μM, 48 h) to induce mitochondrial repositioning to the cortical cytoskeleton, and analysed by fluorescence microscopy. Magnification, × 60. None, untreated.
Fig 4: SNPH controls tumour progression and metastasis.(a) Bioinformatics analysis of SNPH or Miro2 differential expression in cancer versus normal tissues in public databases (Oncomine, TCGA, Prognoscan). , upregulation; , downregulation; C/N, cancer/normal; HR, hazard ratio. (b) IHC analysis of SNPH expression in breast carcinoma versus normal breast epithelium. Bulk, main lesion; IF, invasive front. Scale bar, 100 μm. (c) SNPH-positive cells in normal breast, CIS, bulk breast carcinoma or invasive front (IF). Each point corresponds to individual patients. ***P<0.0001 by unpaired t test with Welch's correction. (d) SNPH-positive cells in molecular subtypes of breast cancer. Lum, luminal. HER2, HER2-enriched. Each point corresponds to individual patients. **P=0.006–0.007; *P=0.01 by unpaired t test with Welch's correction. (e) SNPH-positive cells in breast cancer with no lymph node metastasis, ⩽10% of metastatic lymph nodes (<10%) or >10% metastatic lymph nodes (>10%). Data are represented as mean±s.e.m. **P=0.007 by Mann Whitney test. (f) Histologic analysis of liver metastases after intrasplenic injection of PC3 cells stably expressing vector or SNPH cDNA. Scale bar, 500 μm. (g) Number of liver metastatic foci per field in animals from f. Data are represented as mean±s.e.m. (n=15). ***P<0.0001 by Student's t test.
Supplier Page from MilliporeSigma for Anti-SNPH antibody produced in rabbit