EMBO/SourceData huggingface dataset:
Figure 1(C) MRM mass spectrometry verification of the unique peptide (heavy) of SMIM26 in HEK293T cells. (D) PRM mass spectrometry verification of the unique peptide (heavy) of SMIM26 in HEK293T cells.(G) Immunofluorescence detected subcellular localization of SMIM26 (green) with mitochondria, F-actin, ER and Golgi. Nuclei were stained with DAPI (blue), and the colocalization between red (mitochondria, F-actin, ER, Golgi) and green (SMIM26) was measured. Scale bar, 10 μm. The colocalization of SMIM26 with mitochondria, F-actin, ER and Golgi in cells was measured by fluorescence intensity analysis with ImageJ software. The red line of statistical analysis centers on the co-location of SMIM26 and indicate cell organelle.(H, I) Mitochondria were purified and SMIM26 was enriched in mitochondria fractions (H), SMIM26 is a mitochondrial outer membrane protein as detected by proteinase K and Triton X-100 assayin HEK293T cells (I).(J) The degradation half-life of SMIM26 was detected in HEK293T. Three biological replicates. Data are shown as mean ± SEM.
Figure 2(A) Representative immunohistochemistry (IHC) images of SMIM26 protein level in multiple types of tumoral tissues (T) and adjacent non-tumorous tissues (N), showing that SMIM26 has significantly decreased expression in kidney cancer only. Mann-Whitney test, **P < 0.01; n = 5; Bar, SEM; Scale bar,100 μm.(B) Representative IHC images of SMIM26 protein levels in 368 ccRCC tissues (T) and 281 non-tumorous renal tissues (N). Mann-Whitney test, *** P < 0.001; Scale bar, 200 μm.(C) A Kaplan-Meier survival analysis of ccRCC patients with high or low expression levels of SMIM26 (P < 0.05, log-rank test).(E, The protein level (E) of SMIM26 were detected in five ccRCC cell lines by western blotting(G) The protein level of SMIM26 (up) and RNA level of LINC00493 (down) in ccRCC tissues (T) and their adjacent non-tumoral tissues (N) were detected by IHC staining and RNA in situ hybridization (ISH) staining. The right scatter plot shows no significant correlation between SMIM26 protein expression and LINC00493 RNA expression in ccRCC tissues (green dots, P=0.94) and adjacent non-tumorous renal tissues (purple dots, P=0.53). n = 72. Scale bar, 200 μm.
Figure 3(D) The expression of PABPC4 and SMIM26 in ccRCC tissues compared with adjacent non-tumor tissues was detected by western blotting.(F) RIP assay showing a strong binding of LINC00493 with PABPC4 in HEK293T and ACHN cells.(G) RNA levels of SMIM26 were determined by qRT-PCR in PABPC4 knockdown cells. Data are shown as mean ± SEM of three biological replicates. Unpaired two-tailed Student's t-test. n.s, nonsignificant. Bars, SEM.(I) Knocking down PABPC4 could not significantly alter the degradation half-life of SMIM26 protein. Data are shown as mean ± SEM of three biological replicates.(J) RNC of SMIM26 was detected in HEK293T and ACHN cells transfected with anti-PABPC4 siRNAs. Unpaired two-tailed Student's t-test, ***P < 0.001. Data are shown as mean ± SEM of three biological replicates.
Figure 4(D) Transwell assays were used to test the migration and invasion abilities of both ccRCC cells transfected with indicated constructs. Unpaired two-tailed Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. n.s, nonsignificant. Data are shown as mean ± SEM of three biological replicates. Scale bar, 600 μm.(E) Immunofluorescence staining of F-actin (red) in ACHN and 769-P cells transfected with indicated constructs. Nuclei were stained with DAPI (blue). The intensity of F-actin fluorescence was statistically plotted. Unpaired two-tailed Student's t-test, **P < 0.01, n.s, nonsignificant. Data are shown as mean ± SEM of three biological replicates. Scale bar, 20 μm.(F) Western blotting showed that mesenchymal marker of Vimentin and Fibronectin was decreased, and E-cadherin was increased in SMIM26-overexpressing cells.(J) NOD-SCID mice were transplanted with ACHN-luci cells with or without overexpression of SMIM26 via tail vein injection, respectively. Bioluminescent was visualized 5 weeks later by using an IVIS 200 Imaging System. Data are shown as mean ± SD of 5 mice per group. Unpaired two-tailed Student's t-test, ****P < 0.0001. n.s, nonsignificant.
Figure 5Co-IP assays were performed to detect the interaction between SMIM26 and SLC25A11. Lysates from ACHN cells expressing SMIM26-flag were co-immunoprecipitated by anti-flag antibody (E) SMIM26 and SLC25A11 were detected by western blotting.(G) Co-localization of SMIM26 (green) with SLC25A11 (red) in ACHN was determined by immunofluorescence. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. The red line of statistical analysis centers on the co-location of SMIM26 and SLC25A11.(K-L) N-ter domain of SMIM26 fused with GFP-flag interacted with SLC25A11. The N-ter SMIM26-GFP-flag construct was transfected into HEK293T cells, co-immunoprecipitation was performed by using anti-flag antibody (K) and anti-SLC25A11 antibody (L).(O) Oxygen consumption rate profile was monitored in cells with indicated treatment by using a Seahorse XF24 analyzer. The metabolic inhibitors were injected at different time points, as indicated. Data are shown as mean ± SD of three biological replicates.(P) Transwell assays were used to test the migration and invasion abilities of both ccRCC cells with indicated treatment. Unpaired two-tailed Student's t-test, *P < 0.05, **P < 0.01. Data are shown as mean ± SEM of three biological replicates.
Figure 6(A-B) Immunoblotting validation of the p-AKT at Ser473 expression in ACHN and 769-P cells with overexpression (A) or knockdown (B) of SMIM26 compared with their corresponding control.(D) Co-IP assays were performed to detect the interaction between SMIM26 and AGK in ACHN and 769-P cells expressing SMIM26-flag. Lysates were co-immunoprecipitated by anti-flag antibody. (E) Co-IP assays were performed to detect the interaction between SMIM26 and AGK in ACHN and 769-P cells expressing SMIM26-flag. Lysates were co-immunoprecipitated by anti-AGK antibody.(G) Immunofluorescence of SMIM26 (purple), AGK (green) and mitochondrial (red) in ACHN cells with or without overexpression of SMIM26. Scale bar, 10 μm. Fluorescence intensity analysis of colocalization of AGK and mitochondria in cells treated with control or SMIM26. ImageJ software was used to analyze the fluorescence colocalization. The red line of statistical analysis centers on the co-location of AGK and mitochondria.The interaction of AGK and SLC25A11 was determined by immunoblotting in ACHN and 769-P cells with control or SMIM26 overexpressing (K)The interaction of AGK and SLC25A11 was determined by immunoblotting in ACHN and 769-P cells with control or SMIM26 knockdown (L).
Figure 1(B) Representative live-cell fluorescent images of HeLa cells co-transfected with EGFP-TECPR1 and LAMP1-mCherry and treated with 1 mM LLOMe for the indicated time. Scale bar = 10 µM.(D) Representative live-cell fluorescent images of HeLa cells co-transfected with EGFP-TECPR1 and LAMP1-mCherry and treated as indicated. Scale bars = 10 µM. (E) Quantification of the fold change in EGFP-TECPR1 lysosomal fluorescence from D. Grey points represent individual cells from three independent experiments. Red points represent the means of individual experiments (n > 25 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey's multiple comparisons tests. ** p = <0.005, **** p < 0.0001, ns = not significant.
Figure 2(B) Representative live-cell fluorescent images of HeLa cells co-transfected with the indicated EGFP-TECPR1 deletion mutant and LAMP1-mCherry before and after treatment with 1 mM LLOMe. Scale bars = 10 µM. To the right is the corresponding quantification of the fold change in EGFP-TECPR1 lysosomal fluorescence intensity after a 15-minute treatment with vehicle or 1 mM LLOMe. Grey points represent individual cells from three independent experiments.
Figure 3(A,B) Representative live-cell fluorescent images of HeLa cells co-transfected with EGFP-TECPR1 and mCherry-Gal3 and treated with 1 mM LLOMe for the indicated time. Arrowheads in B indicate the appearance of TECPR1 (green arrow) and Gal3 (magenta arrow). Scale bars = 10 µM for A and 1 µM for B. (C) Quantification of TECPR1 and Gal3 recruitment to damaged lysosomes. Data are presented as mean ± SD from 5 independent experiments (each experiment represents a single cell with at least 3 individual lysosomes quantified).(D) Representative confocal image of a HeLa cell transfected with EGFP-TECPR1, treated with LLOMe for 5 minutes, and immunostained for CHMP2A and LAMP1. Scale bars = 10 µm for whole image and 1 µm for insets. Fluorescence intensity profiles of the indicated channels across the dotted lines are shown in the lower subpanel. (E) Representative confocal images of a HeLa cells transfected with EGFP-TECPR1 and TagBFP-TMEM192 (a lysosomal/late endosomal protein), treated with 1 mM LLOMe for the indicated time, and immunostained for ALIX and Gal3. Scale bars = 10 µm.
Figure 4(A) Western blot analysis of LC3 lipidation status in wild type (WT), FIP200 KO and ATG16L1 KO HeLa cells treated with the indicated concentrations of LLOMe for 30 minutes.(B) Quantification of LC3-II levels in HEK, HeLa and MEF WT and ATG16L1-deficient cell lines treated with and without 1 mM LLOMe for 30 minutes. Bars show mean ± SD from three biologically independent experiments represented as data points. Significance was determined from biological replicates using a Student's t tests. *p = 0.0177, ** p = 0.0082, *** p = 0.0001.(C) Western blot analysis of LC3 lipidation status in WT, ATG7 KO and ATG16L1 KO HeLa cells (top) and WT, ATG5-/- and ATG16L1Δ/Δ MEFs (bottom) treated with the indicated concentration of LLOMe for 30 minutes.(F) Western blot analysis of lyso-IP samples collected from HeLa ATG16KO cells treated with or without 1 mM LLOMe for 30 minutes. Bars show mean ± SD from three biologically independent experiments represented as data points.(G) Representative live-cell fluorescent images of HeLa cells co-transfected with TagBFP-TMEM192, EGFP-ATG5 and mCherry-TECPR1, before and after a 15-minute treatment with 1 mM LLOMe. Scale bars = 10 µm for whole images and 2 µm for zoom. To the right is the corresponding quantification of the fold change in ATG5/TECPR1 lysosomal fluorescence intensity after a 15-minute treatment with vehicle or 1 mM LLOMe. Grey points represent individual cells from three independent experiments. Green/magenta points represent the means of individual experiments (n > 25 cells per experiment).(H) Western blot analysis of LC3 lipidation status in HEK TECPR1/ATG16L1 KO cells treated with 1 mMLLOMe for 30 minutes. To the right is the corresponding quantification of LC3-II protein levels. Bars show mean ± SD from three or four biologically independent experiments which are represented as data points.
Figure 5(A) Representative live-cell images of HEK cells treated as indicated and stained with LysoTracker Red. Nuclei were stained with Hoechst 33342. Scale bars = 20 µm. (B) Quantification of LysoTracker Red puncta from A. Grey points represent individual cells from three independent experiments. Red points represent the means of individual experiments (n > 100 cells per experiment).
Figure 1A Representative fluorescent images of centrosomes in RPE1 WT, CEP250 KO and CROCC KO cells, without and with MT depolymerization using Nocodazole. PCNT was used to mark centrosomes and DNA was stained with DAPI. The boxes in the right-hand corner show enlargement of the centrosome signals of the cell in the main panel. White scale bars, 5 μm. Yellow scale bars, 1 μm. B Quantification of cells with separated centrosomes (centrosome distance > 2 μm) from RPE1 samples (A). N = 50 cells per experiment, n = 3 independent experiments. For WT with Nocodazole, N = 50 cells, n = 2 independent experiments. Bar and error represent mean and SD. Unpaired t-tests. n.s.: not significant p > 0.05, ** p < 0.01, *** p < 0.001.C Representative fluorescent images of centrosomes in HCT116 WT, CEP250 KO, and CROCC KO, without and with MT depolymerization using Nocodazole. γ-tubulin was used to mark centrosomes and DNA was stained with DAPI. The boxes in the right-hand corner show enlargements of the centrosome signals of the cell in the main panel. White scale bars, 10 μm. Yellow scale bars, 2 μm. D Quantification of cells with separated centrosomes (centrosome distance > 1 μm) from HCT116 samples (C). N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD. Unpaired t-tests. ** p < 0.01, *** p < 0.001, **** p < 0.0001.E Representative fluorescent images of RPE1 and HCT116 cells. Rootletin and CEP68 were stained, PCNT was used as centrosome marker and DNA was stained with DAPI. The boxes on top show the centrosome signals of the cell in the main panel. Scale bars in white: 5 μm. Scale bars in yellow: 1 μm. F Quantification of relative Rootletin and CEP68 intensity at the centrosome in RPE1 and HCT116 cells. Mean value of relative intensity at the centrosome from N = 50 in each replicate, n = 4 are shown. Bar and error represent mean and SD. Unpaired t-test. * p < 0.05, *** p < 0.001.G Abundance of Rootletin and CEP68 in RPE1 and HCT116 cells was examined by immunoblotting. Tubulin was used as loading control. Representative blot is shown. The original HCT116 protein sample was diluted 1:10. The relative abundance of Rootletin and CEP68 was normalized to the corresponding tubulin signal in each condition.
Figure 2A-H (A, C, E, G) Time-lapse images (4 min interval) of RPE1 WT, CEP250 KO, NIN KO and CROCC KO cells expressing γ-tubulin-mRuby2 (red) as marker for the centrosomes with and without Nocodazole treatment. Representative still images (top rows) and kymographs of centrosomes (bottom rows) are shown. The DNA was marked with SPY-DNA650 (grey). White scale bars, 5 μm. Yellow scale bars, 2 μm. (B, D, F, H) Intercentrosomal distances (μm) from (A), (C), (E) and (G) were plotted over time (n = 15 cells).
Figure 3A Indirect immunofluorescence analysis of Ninein at the centrosomes in RPE1 WT, CEP250 KO and CEP128 KO cells. CEP164 marks the mother centriole, γ-tubulin was used as centrosome marker. The model panel illustrates the position of the centrioles and centriole appendages. Scale bars: 1 μm. B STED super resolution image and model of Ninein at the centrosome in RPE1 WT, CEP250 KO and CEP128 KO cells. CEP164, C-Nap1 and GT335 were used to mark the mother centriole, the proximal end of centrioles, and the centriole wall, respectively. The model panel illustrates the position of the centrioles and appendages (colours as indicated). For WT and CEP250 KO a blue centriole model was used for better illustration of the different positions. Scale bars: 1 μm.E-H (E, G) Fluorescent images of centrosomes in RPE1 CEP250 KO and CEP128 KO cells with the indicated siRNA depletions, without and with MT depolymerization using Nocodazole. PCNT or γ-tubulin was used to mark centrosomes and DNA was stained with DAPI. The box in the right-hand corner shows the centrosome signals of the cell in the main panel. Scale bars in white: 5 μm. Scale bars in yellow: 1 μm. (F, H) Quantification of cells with separated centrosomes (centrosome distance > 2 μm) for RPE1 CEP250 KO (E) and CEP128 KO (G) samples. N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD. Unpaired t-test. n.s.: not significant p > 0.05, ** p < 0.01, *** p < 0.001.
Figure 4A Representative fluorescent images of MT asters nucleated from the centrosome (γ-tubulin) after in vivo MT regrowth assay. The enlargements on the right show the centrosome signal of the cell in the main panel. Scale bars in white: 10 μm; scale bars in yellow: 1 μm.B Quantification of the average number (left) and length (right) of MTs re-nucleated from the centrosomes in (A). N = 50 cells per experiment, n ≥ 5 independent experiments. Bar and error represent mean and SD. Unpaired t-test. n.s.: not significant p > 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001.C Representative fluorescent images of RPE1 CEP250 KO and CEP250 KO expressing C-Nap1-HA-mNeonGreen (CEP250 KO + CEP250). The indicated antibodies were used for indirect fluorescent analysis. Scale bars in white: 5 μm; scale bars in yellow: 1 μm. D Quantification of centrosome separation from (C). N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD Unpaired t-test. n.s.: not significant p > 0.05, **** p < 0.0001. E Quantification of the average number (left) and length (right) of MTs re-nucleated from the centrosomes in (A) from RPE1 WT and CEP250 KO cells with PLK4 OE. N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD Unpaired t-test. ** p < 0.01.
Figure 5B Fluorescent images of centrosomes in Dox-inducible Flag-PLK4 RPE1 WT, CEP250 KO, NIN KO and CROCC KO cells, with and without Dox induction. Flag-PLK4 was stained with Flag antibodies, PCNT was used as centrosome marker and DNA was stained with DAPI. The boxes on top show enlargement of the centrosome signals of the cell in the main panel. Scale bars in white: 5 μm; scale bars in yellow: 1 μm.D Fluorescent images of centrosomes in Dox-inducible Flag-PLK4 RPE1 WT, CEP250 KO, NIN KO and CROCC KO cells upon 48 h Dox induction, without and with MT depolymerization using Nocodazole. DNA was stained with DAPI. Scale bars: 5 μm. E Quantification of the diameter of amplified centrosomes from (D) as outlined in (A). A representative dataset from six replicates (three replicates per experiment) is shown. N = 50 cells. Dot represents the diameter of amplified centrosomes in each cell with amplified centrosomes. The red lines represent the median. Unpaired t-test. n.s.: not significant p > 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001.G The mean of I50 values from (F). N ≥ 22 cells per replicate, n=3 biological replicates. Bar and error represent mean and SD. Unpaired t-test. n.s.: not significant p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 6A Representative images of live cell imaging analysis of RPE1 WT, CEP250 KO, WT PLK4 OE and CEP250 KO PLK4 OE cells expressing LaminB1-mNeonGreen (magenta) and γ-tubulin-mRuby2 (green) as markers for the NE and centrosomes, respectively. Yellow arrows indicate first NE fold. White arrows indicate first NE discontinuation. The inlet boxes show enlargement of first NE discontinuation. Scale bars: 5 μm.C Quantification of NEBD preparation time. WT: n = 54 cells from 6 independent experiments, CEP250 KO: n = 56 cells from 6 independent experiments, WT PLK4 OE: n = 48 cells from 7 independent experiments, CEP250 KO PLK4 OE: n = 47 cells from 8 independent experiments. D Quantification of NEBD duration. WT: n = 54 cells from 6 independent experiments, CEP250 KO: n = 56 cells from 6 independent experiments, WT PLK4 OE: n = 48 cells from 7 independent experiments, CEP250 KO PLK4 OE: n = 47 cells from 8 independent experiments.H Representative images from live cell imaging analysis of WT, CEP250 KO, WT PLK4 OE and CEP250 KO PLK4 OE cells expressing γ-tubulin-mRuby2 (red) as a marker for the centrosomes. The DNA was marked by SPY-DNA650 dye (cyan). Gray arrows highlight chromosome segregation errors. Scale bars: 5 μm.I Fluorescent images of RPE1 WT, CEP250 KO, WT PLK4 OE and CEP250 KO PLK4 OE cells. BUB1 and MAD1 were stained to analyse the signal distribution or accumulation/persistence. CENP-C was used as centromere marker, α-tubulin to visualize the mitotic spindle and DNA was stained with DAPI. Scale bars: 5 μm.
Figure 7A Fluorescent image of mitotic spindles in Dox-inducible Flag-PLK4 RPE1 WT, CEP250 KO, NIN KO and CROCC KO cells upon 48 h Dox induction (PLK4 OE). CW069 was used to partially inhibit HSET. DMSO is solvent control. White scale bars: 5 μm. B Quantification of mitotic cells with multipolar spindle from (A). N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD. Unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. C Quantification of multipolar spindle/chromosome mis-segregation of Dox-inducible Flag-PLK4 RPE1 WT and CEP250 KO cells upon 48 h Dox induction expressing γ-tubulin-mScarlet-I (red) as markers for the centrosomes with or without CW069 treatment. The DNA was marked with SPY-DNA650 (grey). WT PLK4 OE: nDMSO = 28 cells, nCW069 = 40 cells, CEP250 KO PLK4 OE: nDMSO = 26 cells, nCW069 = 31 cells; from 2 independent live cell experiments. Bar represents mean. Unpaired t-test. * p < 0.05.D Time-lapse analysis of RPE1 WT PLK4 OE and CEP250 KO PLK4 OE cells expressing γ-tubulin-mScarlet-I (red) as markers for the centrosomes followed by 30 h siHSET. The DNA was marked with SPY-DNA650 (grey). (D) shows representative still images of live cell imaging analysis. Scale bars: 10 μm. E Quantification of multipolar spindle/chromosome mis-segregation from (D) in RPE1 WT PLK4 OE and CEP250 KO PLK4 OE with siControl and siHSET for 30 h. WT PLK4 OE: nsiControl = 59 cells, nsiHSET = 59 cells, CEP250 KO PLK4 OE: nsiControl = 45 cells, nsiHSET = 32 cells; from 3 independent experiments. Bar and error represent mean and SD. Unpaired t-test. * p < 0.05, ** p < 0.01.
Figure 1B. Significant increase of survival in Ppt1-/- animals transplanted with HSCPs or BM isolated from Ppt1+/+ mice; Log-rank: HSPCs/BM+/+ tx vs BM-/- tx p<0.001; BM-/- tx vs Ppt1-/- UT p < 0.001.D. Representative fluorescence microscope photomicrographs showing the distribution of donor-derived cells (transduced with D.NGFR reporter gene, red) and autofluorescent storage material (AF, green) in the brain of a Ppt1-/- mouse euthanized at 240 days of age, after transplantation with HSPCs isolated from Ppt1+/+ mice. Insets in D show high magnification representative confocal scanning photomicrographs of DNGFR+AF+ cells in different regions of the CNS. Arrows show DNGFR+ cells engulfed with autofluorescent (AF) storage material. Scale bar (main figure) = 1mm; scale bar (insets) = 25μm.G. Representative brightfield photomicrographs of Nissl-stained brain sections from wild type Ppt1+/+ mice and untreated or transplanted Ppt1-/- mice. Black dashed box highlights a portion of the cortex, shown in the high magnification insets. Yellow lines represent the segments applied to measure cortical thickness (see Materials and Methods for details on the quantification). Scale bar (main figure) = 1mm; scale bar (insets) = 25μm. H. Histograms showing the quantification of cortical thickness. SM = somatomotor; SS = somatosensory; V = visual cortex. ** = p < 0.01; Kruskal Wallis followed by Dunn's post-hoc test (for SM) or ANOVA followed by Tukey's post-hoc test (for SS and V). D
Figure 2C. Significant increase of the survival in Ppt1-/- mice receiving the administration of hPPT1-LV transduced HSPCs.E, F. Significant prevention of pathological symptoms (DSS higher than 4 and limbs stiffness) in Ppt1-/- mice transplanted with hPPT1-LV transduced HSPCs.
Figure 3E. Representative fluorescence microscope photomicrographs showing autofluorescent storage material (AF, green) in different brain regions of Ppt1-/- mice transplanted with hPPT1-LV transduced HSPCs at 400-420 days of age, when the study was terminated. Mock transplanted and untreated Ppt1 -/- mice at about 250 days, i.e. humane end point, are shown as reference; 450 days-old untreated wild type Ppt1+/+ are shown as control. Neurotrace fluorescent Nissl staining (NT, red) is used to highlight neurons in different brain regions. Arrows highlight the surviving Purkinje cells in the cerebellum of IV and ICV+IV transplanted mice; arrowheads highlight the high number of AF+ cells in the Purkinje cell layer of ICV transplanted or Ppt1-/- untreated (UT) mice. Scale bar = 100μm.