EMBO/soda-nel huggingface dataset:
miR-183 and miR-96 orchestrate both glucose and fat utilization in skeletal muscle (2021)
Figure 1

Figure 1. Loss of miR-183 and miR-96 enhances muscle oxidative phenotype.

A. Relative expression of miR-183 and miR-96 in GAS, TA and SOL muscles of WT and DKO mice (n=5).

B. SDH staining of GAS and SOL muscles in WT and DKO mice (n=5). Scale bars: 100 μm.

C. Representative photographs of GAS and SOL muscles dissected from WT and DKO mice (n=5). Scale bar: 1cm.

D. ATPase staining of GAS and SOL muscles of WT and DKO mice (left). Corresponding percentage of type I fibers were determined (right) (n=5). Scale bars: 100 μm.

E. Relative mRNA expression of MHC isoforms in GAS muscles of WT and DKO mice (n=3).

F. Oxygen consumption rate of WT and DKO mice in light and dark phases (n=5).

G. Relative mRNA expression of PGC1α and PLIN5 in GAS muscles of WT and DKO mice (n=3 for PGC1α analysis; n=5 for PLIN5 analysis).

H. Relative mRNA expression of PGC1α and PLIN5 in C2C12 cells transfected with miR-183 and miR-96 agomirs (Ago-183/96) or antagomirs (Ant-183/96) (n=3).

I. Western blot analysis of PLIN5 in C2C12 cells transfected with Ago-183/96 or Ant-183/96.

J, K. Mitochondrial respiration profile of C2C12 cells transfected with Ago-183/96 (J) or Ant-183/96 (K). Cellular oxygen consumption rate (OCR) was measured in real time and corresponding basal and maximal respiration were analyzed by overall OCR (n=3 or 4 per group).

Data information: Means ± SEM are shown for all panels except for left panels in panel J and K, in which means ± SD are shown. *P＜0.05 versus control; **P＜0.01 versus control; ***P＜0.001 versus control (Student's t-test). All experiments were performed at least three times, and representative data are shown.
miR-183 and miR-96 orchestrate both glucose and fat utilization in skeletal muscle (2021)
Figure 2

Figure 2. Loss of miR-183 and miR-96 alters glucose and lipid homeostasis and fuel metabolism in skeletal muscle.

A, B. Random (A) and fasting (B) blood glucose levels of WT and DKO mice (n=8 for DKO fasted group, n=10 for all other groups).

C. Glucose tolerance test (GTT) and area under the curve (AUC) data for GTT in WT and DKO mice (n=8 for DKO, n=10 for WT).

D. HE staining of the iWAT and eWAT of WT and DKO mice. Scale bars: 100 μm.

E. Analysis of fat mass of WT and DKO mice by NMR technique (n=7) (left) and tissue weight of iWAT, eWAT and iBAT (right) (n=4).

F. Serum levels of triglycerides (TAG) and non-esterified free fatty acids (NEFA) in WT and DKO mice (n=5).

G. Respiratory exchange ratio (RER) of WT and DKO mice in light and dark phases (n=5).

H. Relative mRNA expression of FoxO1 and PDK4 in GAS muscles of WT and DKO mice (n=5).

I. Western blot analysis of FoxO1, PDK4, p-PDHA1 and PDHA1 in GAS muscles of WT and DKO mice.

J. Western blot analysis of FoxO1, PDK4, p-PDHA1 and PDHA1 in C2C12 cells transfected with Ago-183/96 (left) or Ant-183/96 (right).

K. Relative mRNA expression of ATGL and HSL in GAS muscles of WT and DKO mice (n=5).

L. Western blot analysis of HSL and ATGL in GAS muscles of WT and DKO mice.

M. Western blot analysis of HSL and ATGL in C2C12 cells transfected with Ago-183/96 (left) or Ant-183/96 (right).

N-Q. LD area per cell was quantified at the indicated time points to measure LD turnover in C2C12 myocytes transfected with agomiR-183/96 (N) or antagomir-183/96 (P) stained with Nile red (n=5). Representative immunofluorescence images of lipid droplets in C2C12 cells transfected with agomiR-183/96 (O) or antagomir-183/96 (Q). Scale bars: 5 μm.

Data information: Means ± SEM are shown for all panels. *P＜0.05 versus control; **P＜0.01 versus control; ***P＜0.001 versus control (Student's t-test). All experiments were performed at least three times, and representative data are shown.
miR-183 and miR-96 orchestrate both glucose and fat utilization in skeletal muscle (2021)
Figure 3

Figure 3. FoxO1, PDK4, and ATGL are target genes of miR-183 and miR-96 in skeletal muscle.

A, B. Sequence alignment of miR-183 and miR-96 and their target sites in the 3'UTR of FoxO1 (A) or PDK4 (B) from various species.

C. Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 agomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96 (right) (n=3).

D. Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96.

E. Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 antagomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96 (right) (n=3).

F. Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96.

G, H. Sequence alignment of miR-183 (G) and miR-96 (H) and their target sites in the 3'UTR of ATGL from various species.

I, J. Relative mRNA expression (I, left) and western blot analysis (J, top) of ATGL in C2C12 cells transfected with agomir for miR-183 (Ago-183) or miR-96 (Ago-96). Relative mRNA expression (I, right) and western blot analysis (J, bottom) of ATGL in C2C12 cells transfected with antagomir for miR-183 (Ant-183) or miR-96 (Ant-96). (n=5 per group for RT-PCR analysis)

K. Relative mRNA expression of ATGL and western blot analysis of FoxO1 and ATGL in C2C12 cells transfected with Ago-183/96 and FoxO1 as indicated (n=3).

Data information: Means ± SEM are shown for all panels. *P＜0.05 versus control; **P＜0.01 versus control; ***P＜0.001 versus control (Student's t-test). All experiments were performed at least three times, and representative data are shown.
miR-183 and miR-96 orchestrate both glucose and fat utilization in skeletal muscle (2021)
Figure 4

Figure 4. miR-183 and miR-96 modulate fat usage via FoxO1 and ATGL in muscle cells.

A. Relative mRNA expression of ATGL, PGC1α, PLIN5, and HSL in C2C12 cells transfected with ATGL (n=5).

B. Relative mRNA expression and western blot analysis of ATGL in C2C12 cells transfected with Ant-183/96 and siRNA of ATGL (siATGL) as indicated (n=3 per group for RT-PCR analysis).

C, D. Relative mRNA expression (C) and western blot analysis (D) of PGC1α, PLIN5, and HSL in C2C12 cells transfected with Ant-183/96 and siATGL as indicated (n=3 per group for RT-PCR analysis).

E, F. Relative mRNA expression of ATGL (E) and western blot analysis (F) of FoxO1 and ATGL in C2C12 cells transfected with Ago-183/96 and FoxO1 as indicated (n=5 per group for RT-PCR analysis).

G, H. Relative mRNA expression (G) and western blot analysis (H) of PGC1α, PLIN5, and HSL in C2C12 cells transfected with Ago-183/96 and FoxO1 as indicated (n=5 per group for RT-PCR analysis).

I. Relative mRNA expression of PGC1α, PLIN5, and HSL in C2C12 cells transfected with Ant-183/96 and siRNA of PPARδ (siPPARδ).

Data information: Means ± SEM are shown for all panels. *P＜0.05 versus control; **P＜0.01 versus control; ***P＜0.001 versus control (Student's t-test). The loading control was reused in Figure 4F and 4H. All experiments were performed at least three times, and representative data are shown.
miR-183 and miR-96 orchestrate both glucose and fat utilization in skeletal muscle (2021)
Figure 5

Figure 5. miR-183 and miR-96 levels are low in oxidative muscles and downregulated upon HFD feeding.

A. Relative expression of miR-183 and miR-96 mRNAs in GAS and SOL muscles of C57 BL/6 mice (n=3).

B. Relative mRNA expression of ATGL, HSL, and PLIN5 in GAS and SOL muscles of C57 BL/6 mice (n=3).

C. Western blot analysis of ATGL, HSL, and PLIN5 in GAS and SOL muscles of C57 BL/6 mice.

D. Relative expression of miR-183 and miR-96 mRNAs in GAS muscles of C57 BL/6 mice fed with HFD for 2 months (n=5).

E. Relative mRNA expression of ATGL, HSL, and PLIN5 in GAS muscles of C57 BL/6 mice fed with HFD for 2 months (n=5).

F. Western blot analysis of ATGL, HSL, and PLIN5 in GAS muscles of C57 BL/6 mice fed with HFD for 2 months.

G. The role of skeletal muscle miR-183 and miR-96 in fuel metabolism. The effects of miR-183 and miR-96 deficiency (genetic ablation) and downregulation (HFD feeding), and low levels of miR-183 and miR-96 (more oxidative myofibers) on gene expression, enzyme or transcription factor activity, and metabolic pathways involved in fuel usage are shown by up/down arrows or cross in red.

Data information: Means ± SEM are shown for all panels. *P＜0.05 versus control; **P＜0.01 versus control; ***P＜0.001 versus control (Student's t-test). All experiments were performed at least three times, and representative data are shown.
miR-183 and miR-96 orchestrate both glucose and fat utilization in skeletal muscle (2021)
Figure 6

Figure 6. Loss of miR-183 and miR-96 protects mice from diet-induced obesity and glucose intolerance.

A. Representative photographs of WT and DKO mice after HFD feeding for 2 months. Scale bar: 1cm.

B, C. Body weight, body weight gain after HFD feeding (B) and tissue weight of iWAT and eWAT (C) of WT and DKO mice fed with HFD for 2 months (n=10-11).

D. Concentration of serum triglyceride (TAG) (left) and non-esterified fatty acid (NEFA) (right) in WT and DKO mice after HFD feeding for 2 months (n=5).

E. Relative mRNA expression of FoxO1, PDK4, and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months (n=5).

F. Western blot analysis of FoxO1, PDK4, p-PDHA1, PDHA1 and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months.

G, H. Relative mRNA expression of PGC1α, PLIN5 (G) and HSL (H) in GAS muscles of WT and DKO mice fed with HFD for 2 months (n=5).

I. Fasting blood glucose levels (left) and random blood glucose levels (right) in WT and DKO mice after HFD feeding (n=10-11).

J. Glucose tolerance test (GTT) and area under the curve (AUC) data for GTT in WT and DKO mice after HFD feeding (n=10-11).

Data information: Means ± SEM are shown for all panels. *P＜0.05 versus control; **P＜0.01 versus control; ***P＜0.001 versus control (Student's t-test). All experiments were performed at least three times, and representative data are shown.
Identification of lectin receptors for conserved SARS-CoV-2 glycosylation sites (2021)
Figure 2

Figure 2. Identification of lectins that bind to Spike and RBD of SARS-CoV-2.

(A,B) ELISA screen of the lectin-Fc library against full-length trimeric SARS-CoV-2 Spike (A) or monomeric RBD (B). Results are shown as mean OD values of 2 replicates normalized against a BSA control and ranked by value. Lectin-Fc fusion proteins with a normalized OD > 0.5 in either (A) or (B) are indicated in both panels. See Dataset EV2 for primary ELISA data.

(C) Lectin-Fc and human ACE2-mIgG1 Fc-fusion protein (hACE2) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike by ELISA. hACE2-mIgG1 was used as control for complete denaturation of Spike protein. Results are shown as mean OD values ± SD normalized to the BSA control (technical replicates, N=3).

(D) Lectin-Fc binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized to BSA controls (technical replicates, N=3).

(E,F) Surface plasmon resonance (SPR) analysis with immobilized full-length trimeric Spike, probed with various concentrations of Clec4g-Fc (E) and CD209c-Fc (F). See Table 1 for kinetics values.

Data information: (C) t-test with Holm-Sidak correction for multiple comparisons. (D) One-way ANOVA with Tukey's multiple comparisons; **P<0.01; ***P<0.001; ns: not significant.
Identification of lectin receptors for conserved SARS-CoV-2 glycosylation sites (2021)
Figure 3

Figure 3. Single molecule, real time imaging of lectin-Spike binding.

(A) Schematic overview of single molecule force spectroscopy (SMFS) experiments using full-length trimeric Spike coupled to an atomic force microscopy (AFM) cantilever tip and surface coated murine Clec4g-Fc or CD209c-Fc. Arrow indicates pulling of cantilever.

(B) Representative force traces showing sequential bond ruptures in the SMFS experiments. Measured forces are shown in pico-Newtons (pN).

(C) Experimental probability density function (PDF) of unbinding forces (in pN) determined by SMFS (black line, measured data). The three distinct maxima fitted by a multi-Gaussian function reveal rupture of a single bond (blue dotted line), or simultaneous rupture of 2 (red dotted line) and 3 (green dotted line) bonds, respectively.

(D) SMFS-determined binding probability for the binding of trimeric Spike to Clec4g and CD209c. Data are shown as mean binding probability ± SD of single, double, triple or quadruple bonds (technical replicates, N=4).

(E) High speed AFM of single trimeric Spike visualizing the real-time interaction dynamics with lectins. Top panel shows 5 frames of trimeric Spike alone imaged on mica. Middle and bottom panels show 5 sequential frames of trimeric Spike/Clec4g and trimeric Spike/CD209c complexes, acquired at a rate of 153.6 and 303 ms/frame, respectively. Association and dissociation events between lectin and Spike are indicated by white and red arrows, respectively. The blue dotted ellipses display the core of the complexes showing low conformational mobility. Color schemes indicate height of the molecules in nanometers (nm). Volumes of single trimeric Spike, trimeric Spike/Clec4g and trimeric Spike/CD209c complexes are indicated, as well as numbers of lectins bound to trimeric Spike, averaged over the experimental recording period.
Identification of lectin receptors for conserved SARS-CoV-2 glycosylation sites (2021)
Figure 4

Figure 4. Characterization of human lectin-Spike interactions.

(A) ELISA analyses of human lectin-hIgG1 Fc-fusion protein (hCLEC4g, hCD209, hCD299) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike. A human ACE2-hIgG1-Fc fusion protein (hACE2) was used to control for the complete denaturation of Spike. Results are shown as mean OD values ± SD normalized to a BSA control (technical replicates, N=3).

(B) hCLEC4g, hCD209, and hCD299 binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized against the BSA control (technical replicates, N=3).

(C, D) SPR analysis with immobilized full-length trimeric Spike, probed with various concentrations of hCLEC4G (C) and hCD209 (D). See Table 1 for kinetics values.

(E) High speed AFM of single trimeric Spike visualizing the real-time interaction dynamics with lectins. Top and bottom panels show 5 sequential frames of trimeric Spike/hCLEC4G and trimeric Spike/hCD209 complexes, acquired at a rate of 303 ms/frame. Association and dissociation events between hCLEC4G or hCD209 and Spike are indicated by white and red arrows, respectively. The blue dotted ellipses display the core of the complexes showing low conformational mobility. Color schemes indicate height of the molecules in nanometers (nm). Volumes of single trimeric Spike and trimeric Spike/Clec4g and trimeric Spike/CD209c complexes are indicated, as well as numbers of lectins bound to trimeric Spike, averaged over the experimental recording period.

Data information: (A) t-test with Holm-Sidak correction for multiple comparisons. (B) One-way ANOVA with Tukey's multiple comparisons; *P<0.05; ***P<0.001; ns: not significant.
Identification of lectin receptors for conserved SARS-CoV-2 glycosylation sites (2021)
Figure 6

Figure 6. Functional determination of lectin-Spike binding in SARS-CoV-2 infections.

(A, B) Binding activity of the full-length trimeric Spike coupled to an AFM cantilever tip to the surface of Vero E6 cells in the presence of (A) hCLEC4G and hCD209 or (B) mouse Clec4g or murine CD209c, probed at the indicated concentrations in SMFS experiments. A definite decrease in binding was observed at concentrations of 37 - 75 nM for Clec4g and 33 - 71 nM for hCLEC4G. Data are normalized to the untreated control and shown as mean ± SD (biological replicates, N=4).

(C) Infectivity of mouse Clec4g, CD209c, Asgr1 and hCLEC4G pre-treated SARS-CoV-2 virus in Vero E6 cells. Data represent two to three independent experiments for Clec4g, CD209c and hCLEC4G lectins (N=5-9) and, one experiment for Asgr1 (N=2-4).

(D) Infectivity of mouse Clec4g, CD209c, Asgr1 and hCLEC4G pre-treated SARS-CoV-2 virus in human airway epithelial Calu3 cells measured in triplicates and compared to non-treated SARS-CoV-2 infected Calu3 cells measured in quadruplicates (technical replicates).

Data information: (C) and (D) Viral RNA was measured with qRT-PCR 15 hours after infection with 0.02 MOI (103 PFUs) of SARS-CoV-2. Data are presented as fold changes of viral loads over mock (only SARS-CoV-2 was added) treated controls (mean log10 values ± SD). All no virus controls were negative for SARS-CoV-2. (A-C) ANOVA with Dunett's multiple comparisons test with the mock-treated group; *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001.

ka,1

[M-1s-1]

kd,1

[s-1]

Kd,1

[M]

koff

xβ

[nm]

Table 1. Values computed for surface plasmon resonance (SPR) and atomic force microscopy (AFM).

SPR. Kinetic association (ka,1), kinetic disssociation (kd,1), and equlibrium dissociation (Kd,1) constants of the first binding step fitted from the bivalent analyte model, assuming two-step binding and dissociation of the lectins to adjacent immobilized Spike trimer binding sites under spontaneous thermodynamic energy barriers; no reasonable fit was obtained with the simple 1:1 binding model. AFM. Kinetic off-rate constants (koff) and lengths of dissociation paths (xβ) of single lectin bonds, originating from force-induced unbinding in single molecule force spectroscopy (SMFS) experiments and computed using Evans's (Bell, 1978; Evans and Ritchie, 1997) model, assuming a sharp single dissociation energy barrier.

Expanded View
A Salmonella protein causes macrophage cell death by inducing autophagy. (2003)
Figure 1.

An S. typhimurium strain devoid of its SPI-1 TTSS effector proteins is able to induce programmed cell death in macrophages. BMDPM from wild-type (A) or caspase-1−/− (B) mice (Kuida et al., 1995) were infected with wild-type S. typhimurium or mutant derivative either lacking all TTSS effector proteins (effectorless) or defective for TTSS secretion by virtue a having a mutation on an essential component of this system (invG). 6 h after infection, cytotoxicity was evaluated by lactate dehydrogenase release or by terminal deoxynucleatide transferase (TUNEL) staining. Values for the lactate dehydrogenase release represent the average of at least three independent measurements. Equivalent results were obtained in several repetitions of these experiments.
A Salmonella protein causes macrophage cell death by inducing autophagy. (2003)
Figure 2.

Transient expression of SipB in BMDPM from caspase-1−/−mice results in cell death. Plasmids expressing either SipB, SipC, or SipD fused to YFP were introduced into BMDPM from caspase-1−/− mice as indicated in the Materials and methods section. Macrophages were then fixed, stained with DAPI (to visualize chromatin), and observed under a fluorescence microscope. Cells expressing SipC or SipD do not show any signs of cytotoxicity. In contrast, cells expressing SipB show clear signs of cytotoxicity and chromatin condensation. Arrows indicate the nucleus of transfected cells.
A Salmonella protein causes macrophage cell death by inducing autophagy. (2003)
Figure 3.

Transient expression of SipB in COS-2 cells. (A) A plasmid expressing YFP-tagged SipB was transfected into COS-2 cells, and its localization was examined under a confocal microscope. (B) SipB colocalized with mitochondrial markers in transfected cells. COS-2 cells were transfected with a plasmid encoding either SipB-CFP (top) or SipB-YFP (bottom). Transfected cells were stained with MitoTracker® Red (Molecular Probes, Inc.) to visualize mitochondria and were examined by confocal microscopy. The SipB-induced structures were readily stained with mitochondrial markers (top). Mitochondria were also seen contained within the SipB-induced structures (bottom). (C) SipB-induced structures were labeled with the ER marker Sec-61. COS-2 cells were cotransfected with plasmids encoding SipB-YFP and Sec61-CFP, and transfected cells were examined by confocal microscopy.
A Salmonella protein causes macrophage cell death by inducing autophagy. (2003)
Figure 4.

Immuno-EM of SipB-transfected COS-2 cells. COS-2 cells were transfected with a plasmid expressing SipB-YFP, and 24 h after transfection, cells were fixed and prepared for immuno-EM using an antibody specific for GFP and a gold-labeled anti–rabbit antibody. SipB was seen localized to structures made up of closely apposed membranes that were often in close proximity to mitochondria (A). Mitochondria were often seen apparently fusing to the SipB-induced membranous structure (B, see arrows in inset on top; C, inset to the right). Mitochondria were often seen contained with the SipB-induced membrane structures (D). Bars, 250 nm.
A Salmonella protein causes macrophage cell death by inducing autophagy. (2003)
Figure 5.

The nonfusogenic SipB428–593 mutant localizes to the mitochondria, but does not form multivesicular structures and interferes with the activity of the wild-type protein. (A) COS-2 cells were transfected with a plasmid expressing YFP-tagged SipB428–593, stained with an antibody to the mitochondrial protein CoxIV, subunit VIc (Molecular Probes, Inc.), and examined by confocal microscopy. SipB428–593-YFP localized to mitochondria and did not induce the formation of multivesicular structures seen after expression of the wild-type protein. (B) COS-2 cells were cotransfected with plasmids expressing either SipB428–593-YFP or SipB-CFP, and transfected cells were examined under a confocal microscope. Expression of SipB428–593-YFP prevented wild-type SipB-CFP from forming multivesicular structures (notice that vesicular structures are only apparent in the absence, but not in the presence, of SipB428–593-YFP; right).
A Salmonella protein causes macrophage cell death by inducing autophagy. (2003)
Figure 6.

The S. typhimurium SPI-1 TTSS–secreted protein SipB localizes to the mitochondria of infected macrophages. (A) Bone marrow–derived macrophages were infected with either wild-type S. typhimurium or the TTSS translocation-defective sipD mutant strain with a multiplicity of infection of 50. 3 h after infection, macrophages were collected and processed for immuno-EM using gold-labeled antibodies. SipB could be readily detected in macrophages infected with wild-type (A and B), but not in macrophages infected with the sipD mutant strain (C). Bars, 100 nm. (B) Alternatively, mitochondria were isolated from the infected macrophages and the presence of SipB or the resident mitochondrial protein CoxIV, subunit IV, and were examined by Western immunoblot analysis using specific antibodies.
A Salmonella protein causes macrophage cell death by inducing autophagy. (2003)
Figure 7.

S. typhimurium-infected macrophages exhibited signs of autophagocytosis. Caspase-1−/− BMDPM were infected with wild-type S. typhimurium or it isogenic translocation-defective sipD mutant. 6 h after infection, macrophages were either stained with MDC to label autophagosomes (I) or processed for EM (II) as described in the legend to Fig. 3. Macrophages infected with wild-type S. typhimurium (but not those infected with the sipD mutant) exhibited a large number of vesicular structures that stained with MDC (I). Under the electron microscope (II), macrophages infected with wild-type Salmonella exhibited multivesicular structures resembling autophagosomes (A–D) Often, electron-dense material, presumably damaged organelles, were readily observed within the multivesicular structures (B–D). In contrast, macrophages infected with type III secretion-defective sipD mutant did not exhibit the large number of multivesicular structures observed in wild-type–infected cells. Bars (A and E) 500 nm; (B, C, D and F) 250 nm.