EMBO/SourceData huggingface dataset:
Figure 4 (E) Heatmap of stemness association genes expression determined by mRNA sequencing comparing stiff and soft MCF-7 cells. n = 3. The unit for the color scale was z-score of log2 expression data shown.
Figure 5(A) The expression of BCL9L or β-catenin in stiff or soft 4T1, MCF-7, B16 or MP-1 cells was determined by western blot. (B) The expression of β-catenin and BCL9L from SGGFP, BCL9L-SGs-4T1, MCF-7, B16 or MP-1 cells were determined by western blot. Data information n = 3 independent experiments in (A and B).(E, F) The 100 soft SGGFP or BCL9L-SGs- 4T1, B16 or MP-1 cells were injected into the mammary fat pads (4T1) of BALB/c mice or tail vein (B16 and MP-1) of NSG mice. The tumor formation was recorded (E). Six to eight weeks later, the lung sections were H&E stained. The metastatic micronodules in the lung were counted and the metastasis index was calculated (F). Scale bar, 0.5 mm. n = 3 (for B16-F1) or 5 (for MP-1) mice with metastatic tumor. Data information: Bonferroni test The data represent mean ± SD.
Figure 6(A) The stiffness of primary breast (HBC, n = 3) and colon (HCC, n = 3) cancer cells screened from the outlet or backflow inlet was measured by AFM. n = 100 from each patient. Data information: Mann-Whitney test (A) The data represent mean ± SD.(C) The stiff and soft tumor cells were isolated from 3 patients with breast cancer and stained with anti-BCL9L antibody. The expression of BCL9L was observed under confocal microscope. Scale bar, 10 μ Data information: Paired Student's t test (C) The data represent mean ± SD.(E) The correlation between the expression of BCL9L from 18 melanoma patients and the grade of melanoma. Data information: Pearson's correlation test E) The data represent mean ± SD.(F) Overall survival compared to the BCL9L level in breast cancer (BRCA, n = 536) or melanoma (n = 376) patients. Data information: Log-rank survival analysis (F). The data represent mean ± SD.
Figure 1Ribosome occupancy map at the LINC00665 locus. MCF10A were either mock-treated or treated with TGF-β and Ribosome profiling on three biological replicates were shown.Relative levels of CIP2A-BP were determined by polysome profiling and qRT-PCR in MCF-10A and TNBC cells either mock treated or treated with TGF-β. Relative levels of LINC00665 were determined by qRT-PCR in MCF-10A and TNBC cells either mock treated or treated with TGF-β.Upper: putative ORFs in LINC00665. Lower: The ORFs were constructed into pcDNA3.1 vector and transfected to MCF-10A cells for 24h. The ORFs-His fusion proteins were determined by western blot with anti-His antibody.The indicated constructs were transfected into MCF-10A cells for 24h, then the GFP fluorescence was detected using fluorescence microscope (F) and fusion protein levels were determined by western blot with anti-GFP and anti-CIP2A-BP antibodies respectivelyDiagram of the His fusion constructs. The start codon ATG of the LINC00665 ORF is mutated to ATT. The indicated constructs were stably expressed in MDA-MB-231 cells, micropeptide CIP2A-BP was immuno-stained using anti-His (I) and anti-CIP2A-BP antibodies (K) respectively; CIP2A-BP-His fusion protein levels were determined by western blotting with anti-His and CIP2A-BP antibodiesMCF-10A cells incubated in the absence or presence of puromycin (200μM) were fractionated on sucrose gradients, and distribution of CIP2A-BP transcripts was quantified by qPCR.
Figure 2Micropeptide CIP2A-BP was immune-stained with anti-CIP2A-BP antibody in MCF-10A and MDA-MB-231 cells either mock treated or treated with TGF-β.Micropeptide CIP2A-BP levels were determined by western blot with anti-CIP2A-BP antibody in MCF-10A and TNBC cells either mock treated or treated with TGF-β.MCF-10A cells were first transfected with anti-LINC00665 translation-blocking antisense oligo, then micropeptide CIP2A-BP was detected.Micropeptide CIP2A-BP levels were detected by western blot with anti-CIP2A-BP antibody in the indicated non-metastatic TNBC tissues (non-metastasis) and metastatic TNBC tissues (metastasis). Micropeptide CIP2A-BP expression in MCF-10A cells was used as the positive control.Kaplan-Meier overall survival curves for TNBC patients with high or low micropeptide CIP2A-BP expression in Suzhou cohort (discovery set, n = 112) and Guangzhou cohort (validation set, n = 105).
Figure 3Migration and invasion of TNBC cells transfected with LINC00665 ORF and control lentiviruses for 48h. Right panel is the quantification of TNBC cells that migrated through the membrane without the Matrigel (upper) and invaded through Matrigel-coated membrane (lower).Migration and invasion of wild-type and CIP2A-BP knockout TNBC cells and CIP2A-BP re-expressed Hs578TKO and MDA-MB-231KO cells. Right panel is the quantification of cells that migrated through the membrane without the Matrigel (upper) and invaded through Matrigel-coated membrane (lower).Wound-healing assay on Hs578T and MDA-MB-231 cells transfected with LINC00665 ORF expressing and control lentiviruses. The wound edges were photographed at the indicated time points after wounding. Right panel is the quantification of the relative wound healing area.Wild-type and CIP2A-BP knockout TNBC cells and CIP2A-BP re-expressed Hs578TKO and MDA-MB-231KO were seeded on cell culture inserts for wound-healing assay. The wound edges were photographed at the indicated time points after wounding. Right panel is the quantification of the relative wound healing area.Macroscopic and histological analysis of the lungs of nude mice injected with the indicated Hs578T (G) and MDA-MB-231 cells (H) (1.0×105-106 cells/mouse) via tail vein; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.
Figure 4Immunoblotting analysis of expression of the indicated proteins in TNBC cells cultured with or without TGF-β.Chromatin immunoprecipitation showing Smad4 occupancy at the 4E-BP1 locus in TNBC cells. Co-precipitated DNA was analyzed for amplicons A-E by qPCR. Values represent the enrichment of bound protein fractions relative to input.Luciferase reporter assay was performed in Hs578T (F) and MDA-MB-231 (G) cells following co-transfection with wildtype or mutant 4E-BP1 promoter fragment and Smad4 over-expressed, or knocked down and respective controls for 36h and treatment with TGF-β for 24h. The reporter constructs were expressing the luciferase gene under 4E-BP1 promoter segment or 4E-BP1 promoter deleted 0 to +200 region.Regulation of 4E-BP1 expression and binding to eIF4E by TGF-β. TNBC cells were treated with TGF-β and equal amounts of proteins were subjected to immunoprecipitation and/or examined by immunoblotting analysis.Immunoblotting analysis of expression of micropeptide CIP2A-BP and β-actin in TNBC cells cultured with or without TGF-β.Smad4 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF-β. The indicated proteins were determined by Immunoblotting analysis.TNBC cells were pretreated with specific antagonist against Smad3 (SIS3, 3μM) for 1h and then cultured with or without TGF-β. The indicated proteins were determined by immunoblotting analysis.4E-BP1 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF-β. The indicated proteins were determined by immunoblotting analysis.
Figure 5LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation using anti-His antibody and immunoblot analysis with CIP2A antibody to verify that CIP2A bound to CIP2A-BP.LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation by CIP2A antibody and immunoblot analysis with CIP2A-BP antibodies to verify that CIP2A-BP bound to CIP2A.Endogenous CIP2A-BP and CIP2A were co-immunoprecipitated in MCF-10A cell line.LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were treated with 30 mg/mL RNase A for 30 min, the indicated complexes were co-immunoprecipitated by anti-His or anti-CIP2A antibody, and CIP2A or micropeptide CIP2A-BP were detected.Upper: A diagram of the domain structure of full length CIP2A protein. Red: RIP=Required for Interaction with PPP2R5C; Orange: RH=Required for Homodimerization; Blue, CC=Coiled coil. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A. Upper: A diagram of the domain structure of CIP2A protein fragment (1-245). Red: RIP=Required for Interaction with PPP2R5C. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A (amino acid 159-245).Co-immunoprecipitation assays showed that the interaction between CIP2A and B56γ in TNBC cells was affected by CIP2A-BP expression.
Figure 6The expression levels of the indicated proteins were detected by immunoblotting analysis in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective controls of TNBC cells.PP2A activity was measured using a protein phosphatase assay kit. PP2A activity assay was performed to evaluate PP2A activity in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control TNBC cells.Immunohistochemistry (IHC) staining of p-AKT from TNBC primary tumor (n = 20) with high or low micropeptide CIP2A-BP expression. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT.MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. Whole-cell lysates of these cells were subjected to immunoblot analysis with CIP2A, CIP2A-BP, PP2Ac, p-AKT, AKT and β-actin antibodies.CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. PP2A activity assay was performed to evaluate PP2A activity in the indicated cells.The indicated cells were pretreated with specific antagonist against AKT (MK-2206，3μM) for 1h. Then whole lysates of these cells were subjected to immunoblot analysis.
Figure 7Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.Immunohistochemistry (IHC) staining of p-AKT of mammary tumors from MMTV-PyMT;CIP2A-BP+/+ or MMTV-PyMT;CIP2A-BP-/- mice. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT .MMTV-PyMT mice were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad. Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.Representative IHC images of p-AKT of mammary tumors from MMTV-PyMT mice that were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad; Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT.Kaplan-Meier survival curves of nude mice transplanted with MDA-MB-231 cells and injected with CIP2A-BP or svCIP2A-BP (10 mice per group). The horizontal line indicates the time after the mice were injected with the cells via the tail vein.Macroscopic and histological analysis of the lungs of nude mice injected with MDA-MB-231 cells and CIP2A-BP or svCIP2A-BP via tail vein; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.
Figure 1(A) Live-cell nanoscopy of HeLa cells. COX8A-SNAP was labeled with SNAP-cell SiR and imaged with STED nanoscopy.(B) 3D architecture of cristae in HeLa cells visualized by FIB-SEM. The OM and the IBM are together shown as a transparent surface. Cristae of two mitochondria are depicted in light and dark blue. A twisted crista is shown in grey.(C) Live-cell recording as in (A). In addition, mitochondrial DNA was stained with PicoGreen and imaged by confocal laser scanning microscopy. Arrows mark sites where nucleoids but no lamellar cristae are present.(D) STED nanoscopy of mitochondria immunolabeled for Mic60 and dsDNA. Arrows mark sites where nucleoids are present.(E) Dual-color STED nanoscopy of cells labeled for COX8A-SNAP and immunolabeled for Mic60. Arrows mark Mic60 clusters in the absence of fully developed lamellar cristae.
Figure 2(A) Composition of MICOS in WT cells and MICOS mutants. Mic60 specific antibodies were used to pull-down Mic60 and interacting proteins from insolated mitochondria. ** Unspecific band, due to the cross reaction of the anti-Mic25 antibody with Mic19.(B) Oxygen consumption rate (OCR) of WT cells and MICOS mutants as analyzed by Seahorse Analyzer. Basal and maximal OCR are shown relative to the WT. Error bars: SEM, n=6.(C) Mitochondrial networks of WT and MICOS mutant cells. Cells were immunolabeled for TOM20 and visualized by confocal microscopy. The inset shows a magnification of the respective overview image. Scale bar: 10 µm. (D) Quantification of the mitochondrial networks as shown in (C). The evaluation was performed manually in a blinded approach based on pre-defined morphology criteria. Average and SD of 3 independent biological replicates are shown (>170 cells per sample and replicate).
Figure 3(A) Representative TEM recordings of WT cells and MICOS-KO mutants, as indicated.(B) Quantification of the overall cristae morphology on TEM recordings. Data information: n: number of mitochondrial sections(C) Quantification of detailed morphology of CMs in MICOS mutants (left). TEM recordings were assigned to 8 classes (right). Data information: n: number of mitochondrial sections(D) CJ frequency on TEM recordings. The number of CJs was manually determined and normalized to the length of the OM. Samples were compared to WT by a One-way ANOVA test. Error bars: SEM. Data information: n: number of CJs(E) Diameter of CJs. CJs were manually measured on TEM recordings. Red line: Average. Error bars: SD. Samples were compared to WT by a One-way ANOVA test. Data information: n: number of mitochondrial sections(F) Representative ET reconstructions of mitochondria from a WT cell (left), a Mic10-KO cell (center) and a Mic60-KO cell (right). The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.
Figure 4(A Nanoscopy of mitochondria of WT and Mic10-KO cells, as indicated. (A) Mitochondria were immunolabeled for Mic60 and ATPB and visualized by 2D STED nanoscopy.(B) Mitochondria were immunolabeled for TOM20 and ATPB and recorded with 3D STED nanoscopy. Image data were deconvolved. Depicted are cross-sections of a mitochondrion (side view).(C) 3D MINFLUX nanoscopy. Mic10-KO cells were immunolabeled for Mic60 using a directly labeled antibody. Colors encode depth information.(D) 3D SIM of living WT and Mic10-KO cells. Cells were labeled with Mitotracker Green. Images show maximum intensity projections.(E) FIB-SEM of Mic10-KO cells. Shown is a representative reconstructed mitochondrion. Dashed box: Orthoslice view on the smaller mitochondrion. The OM and IBM are displayed together in clear grey, the CM in is shown in blue.(F) 2D STED nanoscopy of mitochondria. Mic19-KO cells were transfected with a plasmid for Mic10-FLAG expression under the control of a tetracycline-inducible promoter. Left panel: Mitochondria from noninduced cell. Right: Mitochondria from induced cell expressing Mic10-FLAG.
Figure 5(A) Cells were immunolabeled for Mic60 and DNA and visualized by confocal fluorescence microscopy before and after induction of Mic60 expression for 48 h.(B) Protein levels of MICOS proteins after Mic60 induction. Cell lysates were analyzed by western blotting at the indicated time points.(C) Recovery of mitochondrial networks upon Mic60 re-expression. Cells were induced with doxycycline hyclate for 72 h, immunolabeled for TOM20 and visualized by confocal microscopy.(D-G) TEM of Mic60-TO cells before and after induction. (D) TEM recordings of Mic60-TO cells at the indicated time points. (E) Quantification of the cristae morphology. (F) CJ frequency. The number of CJs on TEM recordings was normalized to the length of the OM. At least 90 mitochondrial sections were analyzed for each sample. (G) Quantification of the cristae morphology of Mic60-TO cells. Only mitochondria exhibiting at least one CJ were analyzed. Data information: n: Number of mitochondrial sections. Scale bars: 20 µm (A), 10 µm (C), 500 nm (D).
Figure 6(A,B) Live-cell nanoscopy of Mic10-TO cells. Cells expressing COX8A-SNAP were labeled with SNAP-cell SiR and recorded with STED nanoscopy. (A) Cristae architecture in noninduced cells. (B) Cristae architecture after Mic10 re-expression (24 h induction with doxycycline). STED image data were deconvolved.(C) STED nanoscopy of fixed Mic10-TO cells. Cells were induced for 24 h and immunolabeled for Mic60 and Mic10-FLAG. Upper row: Cell with weak Mic10 expression level. Lower row: Cell with strong Mic10 expression level.(D-G) TEM of Mic10-TO cells. (D) Cells induced for the indicated period of time were recorded with TEM. Arrow indicates an intermediately shaped crista. (E) Quantification of the cristae morphology. n: Number of mitochondrial sections. (F) CJ frequency in Mic10-TO cells. Number of CJs was normalized to the length of the OM. The same sections were analyzed as in (E). (G) Diameter of CJs estimated on TEM recordings. Data information: n: Number of analyzed mitochondrial sections (E) or CJs (G)..
Figure 7(A) FIB-SEM of Mic10-TO cells. Cells were induced for 0 h, 16 h and 24 h. The representative reconstructions show mitochondria from noninduced, rescued and intermediate cells, as indicated. The OM is shown together with the IBM in clear grey, the CM in blue.(B) ET of Mic10-TO cells. Mic10 expression was induced for 0 h and 16 h. Shown are reconstructions of representative noninduced and almost completely rescued mitochondria. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.
Figure 8Knockdown (KD) of OPA1 in HeLa cells. WT and Mic60-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 5 d. (A) Cell lysates were analyzed by western blotting. (B) TEM recordings from OPA1 depleted WT and Mic60-KO cells. Arrows point to a septum.(C,D) Quantification of the cristae morphology of OPA1 deficient WT (C) and Mic60-KO (D) cells. The number of CJs was normalized to the length of the OM (C). The number of septated mitochondria in Mic60-KO cells was estimated (D).(E,F) ET of mitochondria from OPA1-KD cells. WT cells were transfected with a scrambled control (E) or siRNA pools against OPA1 (F) for 48 h. Reconstructions of representative mitochondria are displayed. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.
Figure 9WT and Mic10-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 72 h. (A,B) STED nanoscopy of OPA1 depleted WT (A) and Mic10-KO (B) cells. Cells were immunolabeled for Mic60 and ATPB.(C) TEM of OPA1 depleted Mic10-KO cells. Left: Representative TEM recordings showing CJs marked by arrows in a control cell and a similar view for an OPA1-KD cell. The CMs are colored in green and the OM and IBM together in magenta. Right: Quantification of mitochondria containing CJs or septa junctions. Sections that contained CJ-like structures were analyzed. Of these, the number of sections exhibiting septa or cristae structures (other) was quantified.(D Depletion of ATP5ME in HeLa cells. Cells were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d. TEM recording (left) and quantification of the number of cristae per mitochondrial section (right).(E) Cells depleted of ATP5ME were immunolabeled for Mic60 and recorded with STED. The arrows point to crossing points of the opposite Mic60 distribution bands.
figf1(a) Top: immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of changes in total LRRK2 (n = 6 independent experiments). SH-SY5Y cells were untreated or incubated with 5 μM lactacystin (Lact), 10 mM 3MA or 20 mM NH4Cl and 100 μM leupeptin (NL) for 9.5 h.(b) Top: representative immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of LRRK2 relative to that in untreated samples (n = 3 or 4 independent experiments). Brain slices from mouse cortex, midbrain or cerebellum were incubated without additions or with NL or 3MA for 2 h at 37 °C.(c,d) Homogenate (Hom) and lysosomal fractions (Lys) isolated from SH-SY5Y cells (c) or the indicated mouse brain regions (d), immunoblotted for the indicated proteins.(e) HEK293 cells expressing GFP-tagged LRRK2 and transduced with lentivirus control (Ctr) or carrying shRNA against LAMP-2A (L2A(−)), incubated or not with NL for 12 h and immunoblotted for the indicated proteins.(f) Immunoblots for LRRK2 and LAMP-2A of homogenates and lysosome-enriched fractions isolated from control fibroblasts (Ctr) or fibroblasts stably interfered for LAMP-2A (L2A(−)), maintained in the presence or absence of serum. Actin and LAMP-1 (L-1) are shown as loading controls of homogenate and lysosomes, respectively. All values are mean + s.e.m.; *P 0.05, unpaired t-test. Full-length blots and gels in Supplementary Figure 12.
figf2(b) Binding, association and uptake calculated from quantification of LRRK2 in immunoblots of lysosomes from starved rat livers, untreated or pretreated with protease inhibitors (PI) and incubated with LRRK2. Inset: representative immunoblot. LAMP-2A is shown as lysosomal loading control. Input (Inp), one-fifth of LRRK2 added. n = 7 independent experiments. Uptake: difference between LRRK2 associated with lysosomes with and without PI.(c) Effect of increasing concentrations of LRRK2 on the degradation of a radiolabeled pool of cytosolic proteins by intact lysosomes. Values are percentage of the degradation without LRRK2 (n = 5 or 6 independent experiments).(d) Association of GAPDH to rat liver lysosomes in the presence of increasing concentrations of LRRK2. Values are percentage of the GAPDH associated with lysosomes in the absence of LRRK2 (n = 4-6 independent experiments in duplicate). Inset: representative immunoblot. Inp: 1 μg of GAPDH(e,f) Association of LRRK2 and RNase A (e) or LRRK2 and GAPDH (f) with PI-treated rat liver lysosomes incubated with increasing concentrations of GAPDH (e) or RNase A (f) or ovalbumin (Ov). Left: representative immunoblots. Right: protein association expressed as a percentage of the value when incubated with lysosomes alone (n = 5 or 6 independent experiments). Inp: RNase A (0.4 μg), LRRK2 (0.04 μg) or GAPDH (1 μg).(g,h) Association of LRRK2 to lysosomes incubated with GAPDH (g) or RNase A (h) at 4 °C (n = 3 independent experiments).(i,j) Immunoblots of brain lysosomes incubated with LRRK2 and increasing concentrations of GAPDH (i) or RNase A (j). All values are mean + s.e.m.; differences with lysosomes incubated with the protein alone were significant at *P 0.05, unpaired t-test. Full-length blots and gels in Supplementary Figure 12.