EMBO/soda-nel huggingface dataset:
Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. (2012)
Figure 8

Membrane alterations induced by expression of individual HCV proteins.

Huh7.5 cells transfected with replicon RNAs (A, B) or Huh7-Lunet-T7 cells transfected with pTM-based expression constructs (C–H) specified in the left of each panel, respectively, were high pressure frozen 24 h after transfection, freeze substituted, embedded into epon resin and sections were analyzed by transmission EM. Representative images showing HCV-induced membrane alterations are shown. (I) Average diameter of specific vesicular structures, either DMVs or SMVs, detected in cells that had been transfected with constructs specified in the upper panels. Whiskers represent minimum and maximum values. (J) Number of vesicular structures detected in profiles of 10 transfected cells. Cells transfected with the pTM expression vector without HCV insert were used as reference. The number of vesicular structures per µm2 is given; whiskers represent minimum and maximum values. (K) Relative abundance of membranous structures. Note that only upon expression of the NS3-5A polyprotein and NS5A two different structures were observed. In all other cases, only one membranous structure was detected.
OMA1 reprograms metabolism under hypoxia to promote colorectal cancer development (2020)
Figure 1

Figure 1. OMA1 is upregulated in human colorectal cancer and is activated by hypoxia.

A. Relative levels of OMA1 mRNA in colorectal cancer tissues (n=105) compared with adjacent normal tissues (n=43) from colorectal cancer patients were shown (using the GEO dataset GSE21510). The data are presented as mean ± SEM, and statistical significance was determined by a Mann-Whitney test. ***p < 0.001.

B. Kaplan-Meier curves were constructed to analyze and compare between patients with high and low levels of OMA1 in colorectal cancer samples from the GEO dataset GSE17537. "Low" indicates patients with OMA1 mRNA levels less than the median. "High" indicates patients with OMA1 mRNA levels greater than the median. Statistical analysis was performed using log-rank tests, n = 55, p=0.0180.

C. The lysates of tumors (T) and adjacent normal (N) tissues from colorectal cancer patients were analyzed by Western blotting with antibodies against OPA1 or Tubulin. The "c, d and e" bands of OPA1 indicate cleaved OPA1 bands. Tubulin was used as a loading control.

D. Control and OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0 (normoxia), 24, or 48 hours. Cell lysates were then assessed by Western blotting with antibodies against OPA1, OMA1, or β-Actin. β-Actin was used as a loading control. Representative immunoblots were from n = 3 independent experiments.

E. OPA1-null mouse embryonic fibroblasts (MEFs) expressing control (empty vector) or OPA1 isoform 1 were cultured in normoxia or hypoxia (1% O2) for 24 hours. Cell lysates were analyzed by Western blotting with anti-OPA1 or anti-Tubulin antibody. Representative immunoblots were from n = 3 independent experiments.

F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were cultured in hypoxia (1% O2) for 0, 24, or 48 hours, and the cell lysates were assessed by western blot with antibodies against OMA1, and GAPDH. β-Actin was used as a loading control (representative data from three independent experiments). The asterisk indicates a nonspecific band.

G. The relative protein levels were evaluated by densitometry analysis using ImageJ software and were quantified for the ratio of OMA1-Flag/β-Actin or E324Q-Flag/β-Actin in hypoxia for 0, 24 or 48 hours (n = 3 independent experiments). The data are presented as mean ± SD.
OMA1 reprograms metabolism under hypoxia to promote colorectal cancer development (2020)
Figure 2

Figure 2. Loss of OMA1 inhibits the development of AOM/DSS-induced colorectal cancer in mice.

A. Schematic representation of the AOM/DSS procedure. To develop colitis-associated cancer (CAC), WT (n=11) and OMA1-/- (n=12) mice were injected intraperitoneally with AOM (10 mg/kg) on day 0. Then, three cycles of feeding water with 1.5% DSS treatment were administered. Mice were euthanized on day 105 and all intestinal tissues, tumors, and serum were collected. After mice were euthanized, intestines were removed and flushed with cold PBS. All experiments were repeated with three independent biological replicates.

B. Body weight changes of WT (n=11) and OMA1-/- (n=12) mice during AOM/DSS treatment. Data are presented as mean ± SEM. Statistical significance was assessed by a two-way ANOVA, **p < 0.01.

C. Typical intestine images on day 105 after mice were euthanized.

D. Colorectal lengths (C) of WT (n=11 biological replicates) and OMA1-/- (n=12 biological replicates) mice were measured. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired student's t-test, *p < 0.05.

E. Representative images of colorectal tumors from WT (left) and OMA1-/- (right) mice.

F. The number of tumors in WT (n=11) and OMA1-/- (n=12) mice was measured. Each dot represents the tumor number of one individual mouse. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired student's t-test, ***p < 0.001.

G, H. The number of tumors of different sizes (diameter) in each mouse (G) and the distribution of different tumor sizes (H) were shown (WT n=11 biological replicates, and OMA1-/- n=12 biological replicates). Results are shown as mean ± SEM. p-values were calculated by unpaired student's t-test, **p < 0.01, ***p < 0.001.

I. Histological analysis of colorectal tumors of WT and OMA1-/- mice was shown by hematoxylin and eosin (H&E) staining.

J. Relative mRNA expression levels of pro-inflammatory genes (TNFα, IL-6, COX-2, CCL2) in colorectal homogenates of WT and OMA1_KO mice treated with AOM/DSS (n =4-5 per genotype). The data represent the mean ± SEM., n = 3 independent experiments, and statistical significance was determined by a two-tailed student's t-test. *p < 0.05, **p < 0.01.

K. The colorectal tissues of WT and OMA1-/- mice were stained with Ki67.
OMA1 reprograms metabolism under hypoxia to promote colorectal cancer development (2020)
Figure 3

Figure 3. OMA1-OPA1 axis promotes glycolysis under hypoxic conditions.

A, B. Control or OMA1 KO HCT116 cells expressing control (empty vector) or OMA1-Flag were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (A) and lactate production (B) were measured by using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD of three independent experiments, statistical significance was assessed by a two-way ANOVA, *P < 0.05. Cells were lysed and assessed by western blot with antibodies against OMA1 and GAPDH (A). The asterisk indicates a nonspecific band.

C. Control and OMA1 KO HCT116 cells were treated with DMSO or 1mM DMOG in fresh medium for 12 h. Subsequent injections of glucose, oligomycin (Oligo), and 2-deoxy-glucose (2-DG) were performed as indicated. The extracellular acidification rates (ECAR) were measured with a Seahorse Extracellular Flux Analyzer XF96. The data are presented as mean ± SD (n = 3 independent experiments).

D. Quantitative analysis of basal and maximal glycolytic capacity. Error bars are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-way ANOVA. N.S., not significant, ***p < 0.001.

E, F. Lactate production (E) and ATP production (F) were measured in control or OPA1 KO HCT116 cells upon DMSO or 2-DG treatment. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, **p < 0.01, ***p < 0.001.

G, H. Control or OPA1 KO HCT116 cells were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (G) and lactate production (H) were measured, and the values were normalized to the protein concentration. Statistical significance was assessed by a two-way ANOVA; error bars are presented as mean ± SD of three independent experiments; N.S., not significant, *p < 0.05, **p < 0.01.
OMA1 reprograms metabolism under hypoxia to promote colorectal cancer development (2020)
Figure 4

Figure 4. OMA1 stabilizes HIF-1α by increasing mitochondrial ROS generation under hypoxia

A. Control and OMA1 KO HCT116 cells were cultured in normoxia or in hypoxia (1% O2) for 24 or 48 hours. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, HK2, LDHA, PKM2, GPI, OMA1, or β-Actin. The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.

B. Control or OMA1 KO Hela cells were untreated (-) or CoCl2-treated (+) for 24 hours with 10 mM MG132 or vehicle added for the last 4 hours. Cell lysates were assessed by Western blotting with antibodies against HIF-1α or β-Actin. Relative protein levels were further evaluated by densitometry analysis. Error bars indicate the mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01.

C. Dihydroethidium (DHE) staining of optimum cutting temperature (OCT) covered tissues from the colons of WT and OMA1-/- mice; DAPI was used as counterstain. DHE was shown in red color, while DAPI in blue color (representative data from three independent experiments).

D. Quantification of DHE staining. Error bars are presented as mean ± SD (n=3 biological replicates), statistical significance was assessed by a two-way ANOVA, **p < 0.01, ***P< 0.001.

E. Control or OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours were stained with mitoSOX, and then analyzed by confocal microscopy (representative data from three independent experiments).

F. The fluorescence intensity of mitoSOX was further analyzed by ImageJ software. Bars indicate the mean ± SD of three independent experiments by a two-way ANOVA, ***p < 0.001.

G-I. Control or OMA1_KO HCT116 cells were cultured in normoxia or hypoxia for 12 hours, and were treated with DMSO, NAC (10 mM) or Rotenone (1 μM) for 12 hours at the same time. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, and Tubulin (G). The glucose uptake (H) and lactate production (I) were measured using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-way ANOVA. N.S., not significant, *P < 0.05, ***P< 0.001.
OMA1 reprograms metabolism under hypoxia to promote colorectal cancer development (2020)
Figure 5

Figure 5. NDUFB5, NDUFB6, COX4L1 and NDUFA4 may be proteolytic substrates of OMA1.

A. 293T cells were transiently co-transfected with empty vector and NDUFB5-MYC, or OMA1-E324Q-Flag and NDUFB5-MYC. Cell lysates were used for co-immunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with anti-Flag or anti-MYC antibodies (n = 3 independent experiments).

B. 293T cells were transiently co-transfected with empty vector and NDUFB6-GFP, or OMA1-E324Q-Flag and NDUFB6-GFP. Cell lysates were used for co-immunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with antibodies against Flag or GFP (n = 3 independent experiments).

C. 293T cells were transiently co-transfected with empty vector and COX4L1-GFP, or OMA1-E324Q-Flag and COX4L1-GFP. Cell lysates were used for coimmunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with anti-Flag or anti-GFP antibodies (n = 3 independent experiments).

D. 293T cells were transiently co-transfected with empty vector and NDUFA4-GFP, or OMA1-E324Q-Flag and NDUFA4-GFP. Cell lysates were used for coimmunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with antibodies against Flag or GFP (n = 3 independent experiments).

E. HCT116 cells stably expressing control (empty vector) or OMA1-Flag, and HCT116 cells infected with control or shOMA1 (OMA1 knockdown) lentiviral particles and further cultured for 5 days, were then treated with DMSO or 20 μm CCCP for 4 hours. Cell lysates were analyzed by Western blotting with antibodies against NDUFA4, COX4L1, NDUFB5, DNUFB6, MT-CO2, OMA1 or Tubulin. The black arrowhead indicated the full-length COX4L1 or NDUFB6, and the red arrowhead pointed to the cleaved band. The asterisk indicates a nonspecific band.

F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were treated with CHX (100 µg/ml) plus CCCP (20 µM) for 0, 2, 4, 6 hours. The cell lysates were then assessed by western blot with antibodies against NDUFA4, COX4L1, NDUFB5, NDUFB6, OMA1 or HSPD1 (representative data from three independent experiments). The asterisk indicates a nonspecific band.

G. Control or OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0, 24, or 48 hours. Cell lysates were assessed by Western blotting with anti-NDUFB5, anti-NDUFB6, anti-COX4L1, anti-NDUFA4 antibodies, or anti-β-Actin. The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.
OMA1 reprograms metabolism under hypoxia to promote colorectal cancer development (2020)
Figure 6

Figure 6. OMA1 impairs the assembly of mitochondrial respiratory chain complexes under hypoxia

A. Mitochondria isolated from tumors (T) or adjacent normal (N) tissues of WT and OMA1-/- mice were subjected to blue native PAGE (BN-PAGE), and respiratory chain complexes were analyzed by Western blotting with anti-NDUFB8 (Complex I), anti-SDHA (Complex II), anti-COX4L1 (Complex IV), and anti-ATP5A1 (Complex V, ATP synthase) antibodies. SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.

B. Digitonin-solubilized mitochondria were isolated from control and OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours, and then analyzed by BN-PAGE and immunoblotting using the indicated antibodies against NDUFB8 (Complex I), SDHA (Complex II), NDUFA4 (Complex IV), and ATP5A1 (Complex V). SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, *p<0.05.

C. The lysates of adjacent normal tissues (N) or tumors (T) from WT and OMA1-/- mice were analyzed by Western blotting with antibodies against NDUFA4, COX4L1, NDUFB5, DNUFB6, UQCRC2, or Tubulin. Relative protein levels were further evaluated using ImageJ software. Error bars indicate the mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, ***p < 0.001.

D. A graphic model of mitochondrial stress sensor OMA1-coordinated glycolysis and mitochondrial oxidative phosphorylation in colorectal carcinogenesis and development. Hypoxia-induced OMA1 activation leads to the degradation of mitochondrial respiratory chain complexes (MRCC) components and OPA1, and results in cristae remodeling, all of which results in the inhibition of mitochondrial electron transport chain (ETC) and the generation of mitochondrial ROS. Elevated ROS can stabilize HIF1a, thereby promoting glycolysis. Thus, OMA1 promotes the Warburg effect by coordinating glycolysis and oxidative phosphorylation to facilitate colorectal cancer development.

Expanded View Figures
A new hERG allosteric modulator rescues genetic and drug-induced Long-QT Syndrome phenotypes in cardiomyocytes from isogenic pairs of patient induced pluripotent stem cells (2016)
Figure 1

Figure 1. The potent hERG allosteric modulator LUF7346 enhances IKr in HEK293 hERG cells.

A) Chemical structures of LUF7243, LUF7244, LUF7344, LUF7346 and LUF7389.

B) Percentage specific binding of [3H]dofetilide to the hERG channel after 6 minutes of dissociation induced by 10 µM dofetilide in the absence (control) or presence of 10 and 50 µM of LUF compounds. The specific binding of [3H]dofetilide in the absence of test compounds was set as Bcontrol, while the specific binding in their presence was set as B. * = p < 0.05 vs control; N: 3-4.

C) Representative traces of hERG activation (left), inactivation (middle), and deactivation (right) measured in HEK293 hERG cells under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). Insets: voltage clamp protocols.

D) Steady-state activation (empty symbols) and inactivation (filled symbols) curves for IKr under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). The corresponding Boltzmann’s fittings are superimposed to data points.* = p < 0.05 vs baseline. N: 11-14.

E) Plot of the time constants (τ) of deactivation derived from biexponential fittings under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). * = p < 0.05 vs respective baseline. N: 8.

Data information: B: Two-tailed unpaired t test. p < 0.0001 for all tested LUF coumpounds compared to the control group at both 10 and 50 µM. D: Repeated measures Two-ways ANOVA with Sidak’s multiple comparisons test. Adjusted p-values for inactivation: -100 mV: >0.9999; -90 mV: > 0.9999; -80 mV: 0.3922; -70 mV: 0.9636; -60 mV 0.0006; -50 mv < 0.0001; -40 mV: < 0.0001; -30 mV: < 0.0001; -20 mV: < 0.0001; -10 mV: <0.0001; -10 mV: 0.0011; 0 mV: 0.0111; 10 mV 0.0343; 20 mV: 0.4871; 30 mV: > 0.9999. Adjusted p-values for activation: -60 mv: > 0.9999; -50 mV: 0.9998; -40 mV: > 0.9999; -30 mV: 0.8854; -20 mV: 0.2129; -10 mV: 0.1188; 0 mV: 0.9944; 10 mV: >0.9999; 20 mV: 0.8340; 30 mV: 0.9841.

E: Paired Two-ways ANOVA with Sidak’s multiple comparisons test. Adjusted p-values for deactivation
A new hERG allosteric modulator rescues genetic and drug-induced Long-QT Syndrome phenotypes in cardiomyocytes from isogenic pairs of patient induced pluripotent stem cells (2016)
Figure 3

Figure 3. LUF7346 rescues genetic LQTS.

A) Representative MEA trace (left) showing the effect of increasing concentrations of LUF7346 on FP contour, measured in LQT2N996I-CMs. Average data (right) showing the effect of LUF7346 on QT interval duration relative to baseline in CMs derived from all the hiPSC lines used in this study. * = p<0.05 vs baseline. Colour in the heatmap defines the magnitude of QT shortening (blue) after treatment with increasing concentrations of LUF7346. N: 7-14.

B) Representative AP from LQT2corr-, LQT2N996I-, LQT1corr-, and LQT1R190Q-CMs paced at 1 Hz, under baseline conditions (black) and after application of increasing concentrations of LUF7346 (colour-code is shown).

C) Effect of LUF7346 on APD90, APD70, and APD50, measured in LQT2corr- (blue), LQT2N996I- (green), LQT1corr- (purple), LQT1R190Q- (dark blue) paced at 1 Hz; * = p<0.05 vs baseline. The colour of the asterisk indicates comparisons and respective statistical significance. ° = p<0.05 vs the genetically-matched corrected control. # = p<0.05 LQT1corr vs LQT2corr. N: 12-15.

D) Effect of LUF7346 on APA and Ediast, measured in LQT2corr- (blue), LQT2N996I- (green), LQT1corr- (purple), and LQT1R190Q-CMs (dark blue) paced at 1 Hz. N: 12-15.

Data information: A: Repeated measurements ANOVA with Sidak’s multiple testing correction. Adjusted p-values: LQT1R594Q: Baseline vs LUF7346 5µM, 10µM, 20µM, 30µM: <0.0001. JLNSR594Q: Baseline vs LUF7346 10µM: 0.0051. Baseline vs LUF7346 20µM, 30 µM: < 0.0001. WT: Baseline vs LUF7346 5µM: 0.0329. Baseline vs LUF7346 10µM, 20µM, 30µM: 0.0001. LQT2corr: Baseline vs LUF7346 10µM: 0.0205. Baseline vs LUF7346 20µM, 30µM: 0.0001. LQT2N996I: Baseline vs LUF7346 10µM: 0.0004. Baseline vs LUF7346 20µM, 30µM: <0.0001. LQT1R190Q: Baseline vs LUF7346 5µM: 0.0042. Baseline vs LUF7346 10µM, 20µM, 30µM: 0.0001. LQT1corr: Baseline vs LUF7346 10µM: 0.0006. Baseline vs LUF7346 20µM, 30µM: <0.0001.

C: Paired Two-ways ANOVA with Holm-Sidak’s multiple comparisons test. APD90: Tyr LQT2corr vs Tyr LQT2N996I: 0.0013. Tyr LQT1corr vs Tyr LQT1R190Q: <0.0001. Tyr LQT2corr vs Tyr LQT1R190Q: <0.0001. Tyr LQT2corr vs Tyr LQT1corr: 0.0278. Tyr LQT2N996I vs Tyr LQT1R190Q: 0.0008. Tyr LQT2N996I vs LQT1corr: 0.31071. Tyr LQT1R190Q vs Tyr LQT1corr: <0.0001. LQT2corr: Tyr vs LUF7346 3µM: 0.0433. Tyr vs LUF7346 5µM: <0.0001. LQT2N996I: Tyr vs LUF7346 3µM, 5µM: <0.0001. APD70: Tyr LQT2corr vs Tyr LQT2N996I: 0.0478. Tyr vs LUF7346 5µM: 0.0018. LQT2N996I: Tyr vs LUF7346 3µM, 5µM: <0.0001. APD50: Tyr vs LUF7346 5µM: 0.0023. LQT2N996I: Tyr vs LUF7346 3µM, 5µM: <0.0001. LQT1corr: Tyr vs LUF7346 3µM: 0.0006; Tyr vs LUF7346 5µM: <0.0001. LQT1R190Q: Tyr vs LUF7346 3µM, 5µM: <0.0001.

D: Repeated measures Two-ways ANOVA with Holm-Sidak’s multiple testing correction. All comparisons were not significant.
Genetic screen of yeast environmental stress response dynamics uncovers distinct regulatory phases (2019)
Figure 1

Figure 1: A parametric view of dynamic transcriptional response

Hsp12-GFP accumulation (Y axis) vs. time (X axis) in response to different levels of KCl stress. Points reflect the measured values, and lines correspond to best model fit.

Similar to (B), in different genetic backgrounds at 0.4M KCl.

A scheme of parametric decomposition. (i) Start from protein abundance measurements in different timepoints following stress. (ii) Fit RNA parameters based on a simplified model for transient transcriptional response (Weiner et al, 2012) (iii) Compare different conditions/perturbations in parameter space.

Representation of the experiments shown in (B,C) in parameter space. Connected line shows the progression in the response to increasing KCl levels (See B).

The inferred tonparameter (Y axis) vs. KCl concentration (X axis) of the KCl gradient experiment (See B).

The inferred total open time - toff− ton(Y axis) vs. the inferred production rate (X axis) of the KCl gradient experiment (See B).