EMBO/SourceData huggingface dataset:
Figure 3Mitochondrial DNA (A) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationnuclear-encoded OXPHOS genes (B in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationrespirometry analysis (C in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationimmunoblots of mGPDH, phospho-Thr172 AMPK (p-AMPK), total AMPK (AMPK), phospho-Ser79-ACC (p-ACC), total ACC and PGC1α and corresponding quantifications represent mGPDH, p-AMPK, p-ACC and PGC1α protein level in C2C12 myocytes transfected with siRN for mGPDH 24 h after differentiationimmunoblots of mGPDH, phospho-Thr172 AMPK (p-AMPK), total AMPK (AMPK), phospho-Ser79-ACC (p-ACC), total ACC and PGC1α and corresponding quantifications represent mGPDH, p-AMPK, p-ACC and PGC1α protein levels (D-F) in C2C12 myocytes transfecte wit plasmid for mGPDH 24 h after differentiationImmunoblots of p-AMPK, p-ACC and PGC1α and corresponding quantifications represent p-AMPK, p-ACC and PGC1α protein levels (G in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiationmitochondrial DNA (H in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiationnuclear-encoded OXPHOS genes combined by NDUFS8, SDHb, Uqcrc1, COX5b and ATP5a1 (I) in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiationNAD+/NADH ratio (J in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationimmunoprecipitation analysis for PGC-1α acetyl-lysine (Ac-Lys) level (K) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiationImmunoblot of c-myc and myogenin (L in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h (L,M after differentiationcorresponding quantifications represent c-myc and myogenin protein levels (M) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h (L,Mrepresentative images of MyHC immunofluorescence (N), fusion index (O) and the distribution of nuclei per myotube (P) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC a 72 h (N-P) after differentiationImmunoblots of p-AMPK, p-ACC, PGC1α and myogenin in C2C12 myocytes transfected with mGPDH plasmid with the CaMKKβ inhibitor STO-609 at 24 h after differentiation. Quantifications represent p-AMPK, p-ACC, PGC1α and myogenin protein levelsImmunoblots of p-AMPK and p-ACC in C2C12 myocytes transfected with mGPDH plasmid with the Ca2+ chelator BAPTA-AM at 24 h after differentiation. Quantifications represent p-AMPK and p-ACC protein levels.
Figure 4Immunoblo of mGPD in GA muscles from obese patientIH of mGPD in GA muscles from obese patientImmunoblo of mGPDH and myogenin in GA muscles fro indicated micqRT-PCR of mGPDH in GA muscle of indicated mice at days 0 and 3 post CTX intramuscular injectionExperimental setup (upper of E), qRT-PCR of mGPDH, myogenin and myh3 (bottom of E in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injection), H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (F), distribution of the fiber CSA (G) in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injectionpercentage of myofibers with central nuclei (H) in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injectionExperimental setup (upper of I qRT-PCR of mGPDH, myogenin and myh3 (bottom of I) in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injectionH&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (J) in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injectiondistribution of the fibers CSA (K), percentage of myofibers with central nuclei (L in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injectionExperimental setup (uppe and muscle weight (bottom of M) in GA muscle from AAV-mGPDH-treated STZ-treated mice at da 14 (M) post CTX intramuscular injectionImmunoblots of mGPDH, p-AMPK, p-ACC, PGC1α and myogenin for the experiment described in E.
Figure 1(A) LncRNA CRNDE expression level was measured by qRT-PCR in cancerous tissues and adjacent normal tissues obtained from 35 cases of gastric cancer (GC) patients. Data information: Data are expressed as mean ± SD. Student's t-test **P<0.01.(C) LncRNA CRNDE expression level was measured by qRT-PCR in adjacent normal tissue-derived macrophages (NTMs) and tumor-associated macrophages (TAMs) obtained from 7 cases of GC patients. Data information: Data are expressed as mean ± SD. Student's t-test **P<0.01.
Figure 2(C) and (D) Left: representative electron micrograph of exosomes isolated from the medium of BMDMs, BMDM-polarized M2 macrophages, monocytes and monocyte-polarized M2 macrophages are depicted, showing the typical morphology of exosomes. Upper right: the protein levels of CD63 and CD81 were measured by Western blot. Lower left: Nanoparticle tracking analysis showed that most of the particles had a similar size of 50-150 nm in diameter with a peak at around 100 nm. Lower right: LncRNA CRNDE expression levels in different exosomes. Data information: Scale bar= 100 nm. The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test. **P<0.01 vs. BMDMs or monocytes or BMDMs-exo or monocytes-exo.
Figure 3(B) BMDM-polarized M2 macrophages were transfected with Cy3-labelled CRNDE and exosomes were isolated from the medium (Cy3-CRNDE-EXO). Then, MFC cells were incubated with Cy3-CRNDE-EXO. Left: Confocal microscopy was used to detect the Cy3-labelled CRNDE in MFC cells. The nuclei were stained with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. MFC cells incubated with the control EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test.(D) Monocyte-polarized M2 macrophages were transfected with Cy3-labelled CRNDE and exosomes were isolated from the medium (Cy3-CRNDE-EXO). Then, SGC7901 cells were incubated with Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. SGC7901 cells incubated with the control EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test.(E) SGC7901 cells were incubated with chlorpromazine (CPZ, 50 μM), genistein (GEN, 200 μM), 5-(n-ethyl-n-isopropyl)-amiloride (EIPA, 100 μM), or methyl-β-cyclodextrin (MβCD, 10 mM), following by the incubation of Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA.(F) SGC7901 cells were incubated with 0, 25, 50, 100 μM CPZ, following by the incubation of Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA.
Figure 4Exosomes were isolated from the media of BMDMs (BMDMs-exo), BMDM-polarized M2 macrophages (M2-exo), BMDM-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and BMDM-polarized M2 macrophages transfected with the negative control of si-CRNDE (M2-si-control-exo). (A) LncRNA CRNDE expression in indicated exosomes was detected by qRT-PCR. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. : one-way ANOVA. **P<0.01.Exosomes were isolated from the media of BMDMs (BMDMs-exo), BMDM-polarized M2 macrophages (M2-exo), BMDM-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and BMDM-polarized M2 macrophages transfected with the negative control of si-CRNDE (M2-si-control-exo). Then, MFC cells were treated with indicated exosomes and cisplatin (CDDP; 1μg/ml) for 48 h. (C) Representative images of colony formation assay performed on MFC cells.Exosomes were isolated from the medium of monocytes (monocytes-exo), monocyte-polarized M2 macrophages (M2-exo), monocyte-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and monocyte-polarized M2 macrophages transfected with si-control (M2-si-control-exo). (E) LncRNA CRNDE expression were measured. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.Exosomes were isolated from the medium of monocytes (monocytes-exo), monocyte-polarized M2 macrophages (M2-exo), monocyte-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and monocyte-polarized M2 macrophages transfected with si-control (M2-si-control-exo). Then, SGC7901 cells were treated with indicated exosomes and CDDP (1μg/ml). (G) cell proliferation were measured.
Figure 53×105 MFC cells were subcutaneously injected into nude mice. Ten days after injection, BMDMs-exo, M2-exo, M2-si-control-exo, or M2-si-CRNDE-exo (10 µg) was injected into the center of the homograft tumors of mice (n=7 each group), followed by intraperitoneal injection of CDDP (10 mg/kg). Six days after CDDP treatment, all mice were sacrificed and the homograft tumors were collected. (A) Growth curves of MFC subcutaneous homograft tumors (The homograft tumor sizes were recorded from the day of CDDP treatment). *P<0.05, **P<0.01 vs. BMDMs-exo; #P<0.05, ##P<0.01 vs. M2-si-control-exo. Data information: Data are expressed as mean ± SD. one-way ANOVA.3×105 MFC cells were subcutaneously injected into nude mice. Ten days after injection, BMDMs-exo, M2-exo, M2-si-control-exo, or M2-si-CRNDE-exo (10 µg) was injected into the center of the homograft tumors of mice (n=7 each group), followed by intraperitoneal injection of CDDP (10 mg/kg). Six days after CDDP treatment, all mice were sacrificed and the homograft tumors were collected. (D) LncRNA CRNDE expression in homograft tumors of mice. **P<0.01. Data information: Data are expressed as mean ± SD. : one-way ANOVA.
Figure 6(C) SGC7901 cells were transfected with pcDNA-CRNDE or its negative control (Mock) before MG132 treatment (10 µM, 6 h). The Co-immunoprecipitation (Co-IP) assay was performed to examine the combination of PTEN and NEDD4-1.(E) The expressions of PTEN and NEDD4-1 were measured in SGC7901 cells transfected with pcDNA-CRNDE or Mock at four time-points after cycloheximide (CHX, 100 μg/mL) treatment. GAPDH was served as an internal control. Left: Representative bands. Right: Quantitative results. The results shown are from three biological replicates and are expressed as mean ± SD. Student's t-test. *P<0.05, **P<0.01 vs. Mock.G) The ubiquitylation of PTEN was measured in 293T cells transfected with indicated vectors.
Figure 7Normal, si-control-transfected, or si-PTEN-transfected SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. (A) The protein levels of PTEN, phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), p-glycoprotein (p-gp), B-cell lymphoma 2 (Bcl-2) were measured. GAPDH was served as an internal control.Normal, si-control-transfected, or si-PTEN-transfected SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. The cell apoptosis in SGC7901 cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.SGC7901 cells were divided into four groups: M2-si-control-exo, M2-si-CRNDE-exo, M2-si-CRNDE-exo +PTEN inhibitor (SF1670; 50 μmol/l), M2-si-control-exo+PTEN inhibitor. All cells were treated with CDDP. The cell survival rate were measured. Data information: The results shown are from seven biological replicates. Data are expressed as mean ± SD. : one-way ANOVA. **P<0.01.SGC7901 cells were divided into four groups: M2-si-control-exo, M2-si-CRNDE-exo, M2-si-CRNDE-exo +PTEN inhibitor (SF1670; 50 μmol/l), M2-si-control-exo+PTEN inhibitor. All cells were treated with CDDP. The cell apoptosis were measured. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.
Figure 1(b) Kinematics of the embryo surface. A kymograph of the embryo surface (φ axis) from a Tg (β-actin:m-GFP) embryo shows the continuous displacement of the EVL / yolk margin, which accelerates after 50% epiboly, while the yolk cell membrane does not show any shift (see Movie EV1). (c) Theoretical kymograph from 50 % to 70 % epiboly superimposed, between the red lines, on the experimental kymograph(b). Membrane signal intensity is color-coded (blue to red). White arrowheads point to flow lines (black).(d) AV velocities, normalized to EVL / yolk margin velocity, were measured by PIV of time-lapse recordings of the surface of lectin-soaked embryos. The plots correspond to three different epiboly stages (30 %, blue; 60 %, red; and 90 %, green). Blue and red arrows point to the width of the E-YSL at 30 and 60 % epiboly respectively (from Appendix Figure S1).(e) The yolk cell membrane is highly convoluted (white brackets) ahead of the EVL leading cells and condenses over time. Tg (β-actin:m-GFP) embryo at 30 % (left) and 60 % (right) epiboly. Scale bar 25 μm. Inset shows a high magnification of the wrinkled area (arrowhead). Scale bar 10 μm.(f) Actin accumulates within wrinkles at the E-YSL. The yolk cell membrane of LifeAct-GFP injected embryos (actin - green) was stained with lectin-TRITC (red) (top). Orthogonal projection of the wrinkled area (bottom). Wrinkles are outlined with a white dotted line. Scale bar 2 μm.
Figure 4(a) Yolk microrheology. Mean squared displacement (MSD) of thermal fluctuations of fluorescent nanoparticles embedded in the yolk of zebrafishembryos. Solid and dashed black lines are Mean ± Standard Error computed from 99 particles. Red line is the fit of the Stokes-Einstein equation. MSD of nanoparticles embedded in the yolk exhibits a proportional dependence on time lag, Δt, consistent with a Newtonian liquid behavior with a viscosity of 129 mPa·s. A linear time dependence of MSD with similar values of yolk viscosity has been recently reported in Drosophila (He et al., 2014). Interestingly, the viscosity of the zebrafish yolk is two orders of magnitude lower than that of C. elegansembryos (Daniels et al., 2006) and of the cytoplasm of several cell types (Wirtz, 2009).(b) Viscoelasticity of the yolk cortex. Effective complex modulus (g*) measured by indenting the surface of zebrafishembryos with AFM. Red symbols are the elastic modulus (g') and yellow symbols are the viscous modulus (g''), respectively. Standard Errors are displayed. In all the measured frequencies cortex rheology is dominated by a solid-like behavior with the viscous modulus 5 times lower that the elastic modulus. No significant frequency dependence was found in the elastic (ANOVA, p = 0.123) and viscous modulus (ANOVA, p = 0.719).(c) Flow lines from snapshots of the PIV measurements (at 40, 60 and 80 % epiboly) of a two-photon video time-lapse (from Movie EV4) of a Tg (β-actin:m-GFP) embryo (top). Fitted flow lines using HR ar shown at the bottom. The relative mean square errors (RMSE) of the fitting are displayed as a percentage at the bottom left of each panel. Scale bar 100 μm.(d) Mechanical power maps over time obtained by HR of the same embryos as in (c) (from Movie EV5). The RMSE of the power is shown as a percentage at the bottom left of each panel. The mechanical work to produce doming (40 % epiboly) arises mainly at the blastoderm (left); at 60 % epiboly (middle) DCs undergo ingression (red) while the E-YSL resists constriction (blue). At 80 % epiboly (right), the active work leading to the active movement of the EVL / yolk margin mainly maps at the E-YSL (red) while the blastoderm undergoes elastic resistance (blue). White arrows point to the main observed events. Scale bar 100 μm.(e) AV (red) and CC (green) stresses and their differences (blue) normalized values in arbitrary units from the regression analysis (Movie EV6) were plotted as a function of the φ angle (0-1) along the cortex from animal to vegetal. The equator - dotted yellow line -, yolk surface - purple shadow - and the RMSE of the mechanical pressure as a percentage for each time point are displayed (40 %; 60 %; 80 % epiboly). The CC stress steeps up from animal to vegetal and the AV shows a more complex profile. Their difference changes senses around the EVL / yolk margin.
Figure 5(a) Correlation between the differential tension (cyan) represented by the recoil velocities differences (m/s) between CC and AV laser cuts and their difference (blue) calculated by regression analysis at the E-YSL during epiboly (%). The difference values in normalized relative units were fitted applying a constant adjustment ratio at all time points.(b) AV tension (averaged recoil velocity in m/s) in the yolk cortex at different distances from the EVL (0, 10, 20, 40 and 60 m) and at different epiboly stages (50 %, blue; 60-70 %, red; 80 % epiboly, green). Significance changes are highlighted and Standard Errors are displayed.(c) Probing mechanical parameters of living embryos with AFM. Force-indentation curves recorded at cantilever velocity of 10 m/s (blue) fitted with a "liquid balloon" model (red). Inset: AFM force-indentation description.(d) Averaged values of mean surface tension (pN / μm) on the yolk obtained with AFM at different epiboly stages (left), and the significant difference of tension at 60-70 % epiboly (p = 0.0245) between the E-YSL and the vegetal pole (right). Standard Errors are displayed.
Figure 6(a) Kymograph showing the deformation of the cortex along a segment perpendicular to a laser cut (red line time point) in the E-YSL of a Myosin-GFP transgenic embryo [Tg (β-actin:myl 12.1-e-GFP)]. After a rapid opening, an active contraction displaces the cut centroid vegetalward indicating that the EVL is more easily stretched (elastic) than the vegetal yolk cellmembrane/cortex. When the gap is closed, the cut center returns to its initial position and the elastic equilibrium is restored. Scale bar 10 μm.(d) Dynamic simulation of AV and CC stresses normalized values in arbitrary units (from Movie EV9 - 40 %; 65 %; 80 % epiboly) represented as in Figure 1a. The AV and CC stresses are equal at the poles, as the epiboly simulation progresses, the CC stress steeps up from animal to vegetal and the difference between CC and AV stresses change sense at the EVL / yolk margin.
Figure 7(a) Speed of the EVL advancing front at different epiboly stages (φ angle) for two distinct simulated conditions: 1) a difference of elastic tension between the EVL and the yolk associated to a two-phase reduction of the contractile E-YSL width (from 20º to 5º) as epiboly advances (from 50º to 155º) (wild type - blue). The contractile E-YSL width decreases at a slower rate until 80º and shrinks afterwards at a rate 2.25 times faster, and 2) Same assumptions as in 1 but with no shrinkage of the E-YSL (constant 20º) (contractileless - red).(b) Experimental values for the EVL advancing front speeds at different epiboly stages (φ angle - top) and at different times (bottom) for wild type controls (blue) and Msn1yolk morphants with reduced E-YSL contractility (red). Experimental profiles closely approach to those inferred in the simulations (compare to (a)).(c) Timeline showing medial sections and power isolines for wild type (top) and Msn1yolk morphants (bottom) along epiboly. Mechanical parameters were inferred by HR from experimental velocity fields (Movie EV10). The RMSE of power is shown as a percentage at each time point. DCs in the Msn1yolk morphants undergo ingression on schedule at 50 % epiboly (yellow arrow). Importantly, as in wild type controls, the active work in morphants is detected at the E-YSL (red arrow). This power supply fades away as epiboly slows down and eventually fails. Scale bar 100 μm.(d) Tensional profiles normalized values in arbitrary units inferred by HR for wild type at 80 % epiboly and its temporal equivalent (9 hours after egg laying) for Msn1yolk morphants. The characteristic profiles of longitudinal and latitudinal stresses and the animal to vegetal stress gradient that are built up during epiboly are never properly developed in morphants.
Figure 1RNA-Seq analysis of: different time points of primary CD4+ T-cells infected in vitro with HIV-1 or mock infected Expression levels were normalized as Log2 fold change in infected vs matched mock-infected controls For (A) data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in (A) depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots illustrate the pathway enrichment analysis of genes up-regulated in infected vs matched mock-infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed genes (DEGs) in the pathway; grey dots are not statistically significantRNA-Seq analysis of PBMCs of macaques infected with SIVmac239 before and after suppression of viremia with ART Expression levels were normalized as Log2 fold change or as enrichment score in ART-treated vs ART-naïve animals (B). (B) data were analyzed by Gene Set Enrichment Analysis (GSEA, number of animals =8). Symbols in the GSEA enrichment plots represent the position of the gene set members in the transcriptome ranked by differential expression between ART-naïve and ART-treated time points. Red color indicates leading edge genes. All analyses were conducted on the genes of the pathway GO cellular response to oxidative stress (184 genes; GO:0034599).