EMBO/soda-nel huggingface dataset:
Long non-coding RNA ELDR enhances oral cancer growth by promoting ILF3-cyclin E1 signaling (2020)
Figure 3

Fig. 3: ELDR regulates EGFR signaling in OSCC.

A: Cal27 or JHU029 cells were transfected with control or ELDR siRNA, and EGFR mRNA expression was examined by qRT-PCR. 18S gene was used as internal control. n = 3 biological replicates and 3 technical repeates. Data are represented as the mean ± SD,

B: Cell lysates from control and ELDR depleted Cal27 or JHU029 cells using two different siRNAs to ELDR were subjected to Western blot analysis for the EGFR using specific antibodies. The membrane was reprobed with an antibody against Actin as an internal control. The same actin blot was used in Fig. 2D (for PCNA). The bottom panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,

C: Cal27 and JHU029 cells were transfected with siELDR, ELDR plasmid or both siELDR and ELDR plasmid. Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody. The membrane was reprobed with an antibody against Actin as internal control. The same actin blot was used in Fig. 2E (for PCNA). n = 2 biological replicates.

D: Cell lysates from control and ELDR overexpressed NOK were subjected to Western blot analysis for the EGFR using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. The right panel shows a quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,

E: Cell lysates from control and ELDR-depleted Cal27 and JHU029 cells were subjected to Western blot analysis for phsospho-STAT3 (pSTAT3 Y-705) and total STAT3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Right panel shows a quantitative representation of Western blot band intensities. n = 2 biological replicates. Data are represented as the mean ± SD,

Data information: Data were analyzed by Student's t test. Small bars indicate standard error (**, p<0.01; *** p<0.001).
Long non-coding RNA ELDR enhances oral cancer growth by promoting ILF3-cyclin E1 signaling (2020)
Figure 4

Fig. 4: ELDR interacts with ILF3 in OSCC.

A: Top hits of ELDR interacting proteins analyzed from the ELDR sense vs. antisense RNA pull-down assay followed by mass spectrometry in lysates from JHU022 and Cal27 cells.

B: ELDR interacting proteins identified by mass spectrometry classified by GENEONTOLOGY PANTHER classification system based on "Molecular Function".

C: Cell lysates from Cal27 and JHU022 cells were incubated with biotinylated ELDR sense and antisense RNA, pulled down and subjected to Western blot analysis for the ILF3 using specific antibodies. Bottom panel shows Western blot analysis of PUM-1 (an unrelated RNA binding protein).

D: Cal27 and JHU022 cell lysates were immunoprecipitated against control or ILF3 antibody and RNA was isolated from the precipitates. Relative expression of ELDR (left penal) and PCAT1 (right panel) was analyzed by qRT-PCR. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,

E: Cell lysates from control and ELDR depleted Cal27 and JHU029 cells were subjected to Western blot analysis for ILF3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Right panel shows the quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,

F: Control, ELDR overexpressed and ELDR-depleted Cal27 cells were probed with an antibody against ILF3 (red) and DAPI (blue). Representative confocal microscopic images show nuclear and cytoplasmic expression of ILF3. Arrows indicate cytoplasmic expression. Magnifications 60X, scale bar 20 μ.

Data information: Data were analyzed by Student's t test. Small bars indicate standard error (**, p<0.01; *** p<0.001).
Long non-coding RNA ELDR enhances oral cancer growth by promoting ILF3-cyclin E1 signaling (2020)
Figure 5

Fig. 5: ELDR- ILF3 interaction regulates Cyclin E1 signaling in OSCC.

A: Cal27 and JHU022 cell lysates were immunoprecipitated with control or ILF3 antibodies and RNA was isolated from the precipitates. Relative mRNA expression of EGFR was analyzed by qRT-PCR. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,

B: Cal27 and JHU029 cells were transfected with control or ILF3 siRNA and after 48hr cell lysates were subjected to Western blot analysis for EGFR and ILF3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. n = 3 biological replicates.

C: Cal27 and JHU029 cell lysates were transfected with either siELDR or siILF3 and relative mRNA expression of Cyclin E1 was analyzed by qRT-PCR. 18S was used as internal control. n = 3 biological replicates. Data are represented as the mean ± SD,

D: Control or ELDR depleted Cal27 and JHU029 cell lysates using two different siRNAs to ELDR were subjected to Western blot analysis for Cyclin E1 using specific antibody. The membrane was reprobed with an antibody against Actin as an internal control. The same actin blot was used in Fig. 2D (for PCNA) and 3B (for EGFR) fir siRNA(ii). Bottom panels show quantitation. n = 4 biological replicates. Data are represented as the mean ± SD,

E: Control or ELDR overexpressed NOK lysates were subjected to Western blot analysis for Cyclin E1 using specific antibody. The membrane was reprobed with an antibody against Actin as internal control. Bottom panel shows quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,

F: Control or ILF3depleted Cal27 and JHU029 cell lysates were subjected to Western blot analysis for Cyclin E1 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Bottom panels show quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,

G: Cal27 and JHU029 cells were transfected with either siELDR or siILF3 and after 48hr cell lysates were subjected to Western blot analysis for phospho-RB and total RB using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Bottom panels show quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,

H: Control or ELDR knockdown synchronized Cal27 and JHU029 cells were harvested, fixed and stained with propidium iodide. DNA content was analyzed by flow cytometry. Results are represented cell population in G1, S, and G2/M phases of the cell cycle. Right panels show percentage of cell population in different phases of cell cycle. n = 4 biological replicates. Data are represented as the mean ± SD,

I: Schematic representation of ELDR-ILF3 mediated regulation of Cyclin E1 signaling. The arrow indicates activation.

Data information: Data were analyzed by Student's t test. Small bars indicate standard error (*, p<0.05; **, p<0.01; *** p<0.001).
Long non-coding RNA ELDR enhances oral cancer growth by promoting ILF3-cyclin E1 signaling (2020)
Figure 6

Fig. 6: ELDR inhibits miR-7 expression to regulate EGFR.

A: Cal27 and JHU029 cells were transfected with control or ELDR siRNA and relative expression of miR-7 was analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates. Data are represented as the mean ± SD,

B: Cal27 and JHU029 cells were transfected with control or ELDR plasmid and relative expression of miR-7 was analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates. Data are represented as the mean ± SD,

C: Cal27 and JHU029 cells were transfected with control or mimic miR-7 and relative expression of ELDR was analyzed by qRT-PCR. 18S rRNA was used as an internal control. n = 2 biological replicates; n= 3 technical replicates. Data are represented as the mean ± SD,

D: Cal27 and JHU029 cells were transfected with miR-7 mimic, ELDR plasmid or both miR-7 and ELDR plasmid. Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody. The membrane was reprobed with an antibody against Actin as an internal control. Representative bands were cropped from same gel. Right panels show quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,

E: Relative expression of miR-7 in Cal27 and JHU029 cells compared to NOK cell line analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,

F: Schematic representation of ELDR and miR-7 mediated regulation of EGFR signaling. Sharp arrow indicates activation and blunt arrow indicates inhibition.

Data information: Data were analyzed by Student's t test. Small bars indicate standard error (*, p<0.05; **, p<0.01; *** p<0.001).
Long non-coding RNA ELDR enhances oral cancer growth by promoting ILF3-cyclin E1 signaling (2020)
Figure 7

Fig. 7: Therapeutic potential of ELDR in a OSCC xenograft model. JHU029 cells (1X106) cells were injected subcutaneously into the flank of nude mice. Tumor bearing mice were randomly divided into two groups, and10µg of siELDR or control oligoes were injected intratumorally at an interval of 4 days until the end of experiment.

A: Body weight was measured in control and siELDR treated mice. n=5 animal per group

B: Representative images of tumors in control and treatment groups.

C: Tumors were measured using a slide caliper and tumor volumes were calculated. Arrow indicates starting point siELDR treatment. n=5 animal per group and tumor volume presented as the mean ± SD

D: ELDR knockdown was examined by analyzing expression of ELDR in treated tumors compared to the control tumors. 18S rRNA gene was used as internal control. n=5 animal per group and 3 technical replicates from each tumor.

E: Control or siELDR treated tumor lysates were subjected to Western blot analysis for EGFR and Cyclin E1expression using specific antibodies and representative bands are shown. The blot was reprobed with an antibody against Actin for normalization. Right panels show quantitation. n= 3 biological replicates from each tumor

F: OSCC-PDX tissue was implanted subcutaneously into the flank of NSG mice. When the tumor volume reached >70 mm3, mice were divided into two groups. 10µg of siELDR or control oligoes were injected intratumorally as described above. Body weight was measured in control and siELDR treated mice. n=5 animal per group

G: Representative images of OSCC-PDX tumors in control and treatment groups.

H: PDX tumors were measured using a slide caliper and tumor volumes were calculated. n=5 animal per group and tumor volume presented as the mean ± SD

I: ELDR knockdown was examined by analyzing expression of ELDR in treated OSCC-PDX tumors compared to the control tumors. 18S gene was used as internal control. n=5 animal per group and 3 technical replicates presented as the mean ± SD

Data information: Data were analyzed by Student's t test. Small bars indicate standard error (*, p<0.05; **, p<0.01; *** p<0.001).

Expanded View Figure Legends
Mitochondrial F1FO ATP synthase determines the local proton motive force at cristae rims (2021)
Figure 1

Figure 1 - Characterization of cell lines used: metabolic profile, mitochondrial membrane potential and morphology.

A) Metabolic profiles of HeLa cells in dependence on IF1 levels under glycolytic and OXPHOS conditions generated by an automatic flux analyzer measuring oxygen consumption rates and extracellular acidification rates. n=3 independent experiments (error bars = SD).

B) Mitochondrial membrane potential (∆ψm) under the influence of overexpressed IF1-H49K and the absence of IF1 in IF1-KO cells. To estimate ∆ψm, HeLa cells were stained with TMRE (7 nM). In addition, HeLa cells were stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM) for normalization to the mitochondrial area. Calculated TMRE/MTG ratios from n=4 independent experiments are displayed for each cell. Statistics: one-way ANOVA with post hoc Scheffé test, *, p≤0.05, **, p≤0.01, ***, p≤0.001.

C) MiNA analysis of HeLa cells stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM) to visualize and determine the mitochondrial area (= mitochondrial footprint). Left: HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Co-localization shown in yellow. Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green).

D) Left: HeLa IF1-KO cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green).

E) Left: HeLa IF1-H49K cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). Co-localization shown in yellow. Scale bars = 10 µm.

F) Mitochondrial footprint in µm2 determined via MiNa analysis of HeLa cells. Cells were stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM).

G) Mean branch length in µm determined via MiNA analysis.

H) Number of networks determined via MiNA analysis.

I) Number of individuals determined via MiNA analysis.

Data information: Statistics: one-way ANOVA with post hoc Scheffé test, *, p≤0.05, **, p≤0.01, ***, p≤0.001. Parameters from n=4 independent experiments are displayed for each cell. (F-I). Single dots indicate data from single cells (B, F-I). The error bars denote SD, the boxes represent the 25th to 75th percentiles. The vertical lines in the boxes represent the median values, whereas the square symbols in the boxes denote the respective mean values. The minimum and maximum values are denoted by x. Whiskers: include outliners, coefficient 1.5.
Mitochondrial F1FO ATP synthase determines the local proton motive force at cristae rims (2021)
Figure 2

Figure 2 - Assembly of OXPHOS complexes.

A) BN-PAGE and subsequent immuno-blotting of mitochondrial extracts from indicated HeLa cell lines showing the assembly of F1FO ATP synthase and binding of IF1. Monomers (M), dimers (D) and oligomers (O) were detected. Left panel: Coomassie-stained gel. Right panels: Immunoblotting and detection of ATP synthase and IF1. WT: wild type HeLa cells; IF*: cells expressing IF1-H49K. Stars indicate oligomeric IF1 forms. I: complex I; III: complex III.

B) Relative levels of ATP synthase monomers, dimers and oligomers. Semi-quantification of immunoblot shown in (A).

C) Assembly of F1FO ATP synthase SU e-sEcGFP (Sue#) into monomeric, dimeric and oligomeric ATP synthase (CV). Proteins were separated by BN-PAGE and immunoblotted as indicated. Left panel: detection of subunit β (α-ATP5B); right panel: detection of sEcGFP with α−GFP; O: oligomeric ATP synthase; D: dimeric (blue **); M: monomeric F1F0 ATP synthase (blue*).

D) Assembly of sEcGFP-tagged subunits CoxVIIIa-sEcGFP (CIV#) into complex IV, and assembly of subunits γ-sEcGFP (SUγ#) and SU e-sEcGFP (Sue#) into ATP synthase. Proteins were separated by BN-PAGE and immunoblotted. Lanes #1-#10 were cut into three pieces for further evaluation. Lanes 1-2: complex IV assembly in WT and CoxVIIIa-sEcGFP-expressing cells, detection of CIV with anti-Cox1. Lanes 3-4: complex V assembly in WT and SUγ-sEcGFP-expressing cells, detection with anti-SU γ. Lanes 8-10: assembly of CIV# into CIV, SU γ-sEcGFP (SUγ#) into CV, and SU e-sEcGFP (SUe#) into CV. At lane #6 in the Coomassie-stained gel, the positions of molecular weight markers (M) are shown. SC: respiratory supercomplexes; CIV2: complex IV dimers; CIV: complex IV monomer; s1: F1-c8 subcomplexes; s2: subcomplexes of CV containing subunits b, e and g He et al., 2018(). Blue **: dimeric F1F0 ATP synthase; blue*: monomeric F1F0 ATP synthase. Red asterisks: respiratory supercomplexes
Mitochondrial F1FO ATP synthase determines the local proton motive force at cristae rims (2021)
Figure 3

Figure 3 - Single particle diffusion analysis of CV-SU g reveals restrictive localization and diffusion of the oligomeric ATP synthase holo-complex compared to ATP synthase monomers and subcomplexes.

A,A' Trajectory maps of ATP synthase labeled at SU γ (A) and SU g (A') in single mitochondria. Insets: Detailed views of the diffusion of CV-SU γ (A) and CV-SU g (A'). Scale bars: 1 μm overview and 0.5 μm insets.

B,B' Generation of transformation matrices for linearization of mitochondria to analyze the directionality of the movement. The number of individual trajectories is shown in brackets.

C,C' Analysis of the step directions with respect to the longitudinal axis (±180° and 0°) and the orthogonal axis (from -90° and +90°) represented on a graduated disk. For the determination of directionality of CV-SU γ and CV-SU g, 5 successive steps were integrated. The number of analyzed steps is indicated in D and D'.

D,D' Heat maps showing the step magnitude in dependence on the step direction.

E Diffusion coefficients (-log D) for CV labeled at SU γ and SU g in comparison. Left: probability density function (PDF) and cumulative density functions (CDF) for D from all trajectories. Right: box and whiskers plot. Mean diffusion coefficients D (open squares) calculated from all individual trajectories shown in A and A', respectively.

F Step size diagram (PDF and CDF) for steps of SU γ and SU g in comparison.

Data information: Statistics: Mann Whitney test (E, F). Diffusion coefficients of trajectories shown as single dots. The error bars denote SD, the boxes represent the 25th to 75th percentiles. The vertical lines in the boxes represent the median values, whereas the square symbols in the boxes denote the respective mean values. The minimum and maximum values are denoted by x.
Mitochondrial F1FO ATP synthase determines the local proton motive force at cristae rims (2021)
Figure 5

Figure 5 - sEcGFP as a pH sensor.

A) Fluorescence emission spectrum of mt-sEcGFP in HeLa cells in situ at different pH values (λex.=405 nm, λem.=415-705 nm; λ-emission scan of transfected cells with 10 nm steps using a cLSM microscope). Before and after the isosbestic point (indicated), the pH dependence of the emission is reversed, allowing for a readout of pH-dependent fluorescence emission in the two indicated channels and ratiometric imaging (λ511/λ464). The λ range for the first channel is larger than the λ range for the second channel, allowing a better match of the signal intensities of the two different channels.

B) Scheme showing the positions of the pH sensor fused to subunit of different OXPHOS complexes and MPP in the matrix. CIV: Cox8a (CoxVIIIa)-sEcGFP; CV SU e-sEcGFP, CV SU γ-sEcGFP. Matrix: MPP-sEcGFP. CI: complex I, CII: complex II, CIII: complex III, CIV: complex VI, CV2: dimeric complex V. ICS: intracristal space, CM: crista membrane.

C) Images showing matrix pH equilibration with extracellular pH. Shown are ratiometric fluorescence images of MPP-sEcGFP before and after addition of the uncouplers and inhibitors FCCP, nigericin and oligomycin (plus 0.002 % w/v Triton X-100 where indicated). False color images (λ511/λ464) indicate ratio values.

D) Ratiometric imaging of mitochondrial pH after equilibration with extracellular pH values pH=8, pH=7.4 and pH=6.5. Scale bars = 10 µm.

E) Calibration curves for the different sensor-fusion proteins. Ratiometric response of sEcGFP to the local pH. Extracellular buffers with different pH values used: MES (2-(N-morpholino)ethanesulfonic acid, 20 mM, pH 5.6-6.1), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 20 mM, pH 6.1-7.6) or HEPPSO (4-(2-hydroxyethyl)-piperazine-1-(2-hydroxy)-propanesulfonic acid, 20 mM, pH 7.1-8.0) in 125 mM KCl, 20 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgSO4 plus FCCP (10 µM), nigericin (1 µM) and oligomycin (5 µg/mL) Each data point represents n=2 independent experiments and mean values ± s.d. of at least 15 cells. NFO: nigericin + FCCP + oligomycin; NFOD: nigericin + FCCP + oligomycin + digitonin; NFOT: nigericin + FCCP + oligomycin + Triton X-100.

F) One-way ANOVA comparison of pH values at SU γ of CV at the matrix site after treating cells with the cocktail (FCCP, nigericin and oligomycin) for pH equilibration. Box (75%) and whisker plots, median (horizontal line in box), mean (box in box). ***, p<0.001; **, p<0.01; *, p<0.5, n.s.: non-significant (N=3 technical replicates, ≥10 cells analyzed). NFO: nigericin + FCCP + oligomycin; NFOD: nigericin + FCCP + oligomycin + digitonin (0.006 % w/v); NFOT: nigericin + FCCP + oligomycin + Triton X-100 (0.002 % w/v).

G) Local pH values at CoxVIIIa, SU e, SU γ and in the matrix bulk in cells supplied with high glucose (High Glc, 25 mM) before and after the addition of FCCP, nigericin and oligomycin, demonstrating successful pH equilibration with extracellular pH 7.3. Three technical replicates from same clones. Bars = mean, error bars ± SD. Statistics: one-way ANOVA with post hoc Scheffé test.
Mitochondrial F1FO ATP synthase determines the local proton motive force at cristae rims (2021)
Figure 6

Figure 6 - Local pH values in glycolytic and oxidative mitochondria.

A) Ratiometric imaging of pH in living HeLa cells at different OXPHOS complexes and in the matrix. WT matrix: MPP-sEcGFP; WT SU γ: SU γ fused to sEcGFP; WT SU e: SU e fused to sEcGFP; WT CoxVIIIa: subunit CoxVIIIa fused to sEcGFP (all C-terminally). HGlc: high glucose; Gal: galactose. Ratio-images for IF1-H49K OE cells are displayed in Figure EV4. Scale bars: 10 µm.

B) Mitochondrial pH values at SU e, SU γ and in the matrix bulk in hyper-glycolytic cells (high glucose: HGlc, 25 mM).

C) pH at SU e, SU γ and in the matrix in respiring cells (Gal, 10 mM).

D) pH at CoxVIIIa at complex IV in the ICS before and after stimulation of the respiratory chain.

Data information: Box (75%) and whiskers plots. Outlier included. The error bars denote SD, the boxes represent the 25th to 75th percentiles. The vertical lines in the boxes represent the median values, whereas the square symbols in the boxes denote the respective mean values. The minimum and maximum values are denoted by x. Statistics: one-way ANOVA with post hoc Scheffé test: ***, p≤0.001; **, p≤0.01; *, p≤0.05. Three technical replicates from same clones (B-D).
Mitochondrial F1FO ATP synthase determines the local proton motive force at cristae rims (2021)
Figure 7

Figure 7 - Inhibitor effects on the local pH value at SU e in the intracristal space

A) Mitochondrial pH at CV SU e measured in HeLa WT in glycolytic (HGlc) and respiring (Gal) conditions. Where indicated, 5 µg/mL oligomycin was added to inhibit CV activity.

B) Mitochondrial pH at CV SU e in IF1-KO cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin.

C) Mitochondrial pH at CV SU e in IF1-OE cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin.

D) Fluorescence image showing the localization of SU e-sEcGFP in WT cells. Scale bar: 10 µm.

E) pH calibration curve for testing the effect of antimycin A on local pH at SU e.

F) Effect of inhibiting the respiratory chain at complex III (CIII) with antimycin A (AA) on the local pH at SU e in WT cells (glycolytic conditions).

G) Local pH at SU e in IF1-KO cells after inhibiting CIII.

H) Local pH at SU e in IF1-OE cells after inhibiting CIII.

Data information: Box (75%) and whiskers plots. Whiskers: outlier included. The error bars denote SD, the boxes represent the 25th to 75th percentiles. The vertical lines in the boxes represent the median values, whereas the square symbols in the boxes denote the respective mean values. The minimum and maximum values are denoted by x. Statistics: one-way ANOVA with post hoc Scheffé test: ***, p≤0.001; n.s.: non-significant. Technical replicates from same clones: 3 (A-C,), 2 (F-H).