EMBO/soda-nel huggingface dataset:
LncRNA-MIF, a c-Myc-activated long non-coding RNA, suppresses glycolysis by promoting Fbxw7-mediated c-Myc degradation (2016)
Figure 6

Figure 6. LncRNA-MIF inhibits the glycolysis via miR-586.

A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, acidification of the culture medium was evaluated by visually inspecting the color of the medium. Yellow medium indicates the presence of more lactate.

B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with the indicated antibodies.

C. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).

D. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).

E. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).

F. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).
LncRNA-MIF, a c-Myc-activated long non-coding RNA, suppresses glycolysis by promoting Fbxw7-mediated c-Myc degradation (2016)
Figure 8

Figure 8. LncRNA-MIF functions as a tumor suppressor to inhibit colony formation and cell proliferation.

A. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586mimics or NC mimics as indicated. Colonies were stained with crystal violet and counted after 14 days incubation.

B. HeLa cells expressing either MIF shRNA or control shRNA were transfected with miR-586 inhibitors or NC inhibitors as indicated. Colonies were stained with crystal violet and counted after 14 days incubation.

C. 2×106 HeLa cells expressing either lncRNA-MIF, MIF control RNA, MIF-shRNA or MIF control shRNA were individually injected subcutaneously into flanks of nude mice (n = 7 for each group). Representative photographs of xenograft tumors in situ were taken 3 weeks after injection.

D. Three weeks post graft, mice were euthanized, tumors were excised and weighed, and the mean tumor weight was plotted on the graph. Data shown are mean ± SD (n = 7; **P < 0.01, two-tailed t-test).

E. A schematic illustration of the proposed model depicting a role of c-Myc-induced lncRNA-MIF in regulating glycolysis via suppression of c-Myc.
Stress-induced host membrane remodeling protects from infection by 
non-motile bacterial pathogens (2018)
Figure 2

Figure 2. Host cellular stress inhibits Shigella binding to host cells

A and B. Representative images (A) and cfu quantification (B) of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated or not with arsenite. Ruffle formation induced by Shigella WT in panel (A) is indicated by white arrowheads.

C. Cfu quantification of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated with anisomycin or DMSO (control).

D and E. Representative images (D) and cfu quantification (E) of Shigella WT or ΔipaB mutant bound to HCT-8 cells pre-treated or not with arsenite.

Data information: Shigella infection was performed at MOI 10 for Shigella WT or at MOI 50 for the ΔipaB mutant strain; cells were incubated with the bacteria for 25 min. Results are shown as mean ± s.e.m. from 5 (C) or 6 (B, E) independent experiments; **p>0.01, ***p<0.001 (paired t-test). Scale bars, 25 µm.
Stress-induced host membrane remodeling protects from infection by 
non-motile bacterial pathogens (2018)
Figure 3

Figure 3. Inhibition of Shigella infection upon host cell stress is a consequence of ASM activation

A - F. Representative images (A, D), cfu (B, E) and qRT-PCR (C, F) quantification of intracellular Shigella in HeLa cells pre-treated with arsenite or anisomycin in the presence or not of amitriptyline, and corresponding controls.

G - I. Representative images (G), cfu (H) and qRT-PCR (I) quantification of intracellular Shigella in HeLa cells transfected with ASM siRNA or control siRNA and pre-treated or not with arsenite prior to infection.

J - M. 3D reconstruction of representative images of ASM (J, K) or ceramide (L, M) staining in HeLa cells treated with arsenite or anisomycin and corresponding controls. ASM, ceramide and Hoechst staining were surface converted by voxel distance.

Data information: Infection was performed with Shigella WT at MOI 10 and analyzed at 0.5 hpi. Results are shown as mean ± s.e.m. from 5 (H, I) or 7 (B-F) independent experiments; *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA). Scale bars, 50 µm (A, D, G) and 5 µm (J-M).
Stress-induced host membrane remodeling protects from infection by 
non-motile bacterial pathogens (2018)
Figure 4

Figure 4. MAPK p38 inhibition restores Shigella infection of host cells exposed to stress

A. Western blot analysis of p38 MAPK phosphorylation in HeLa cells treated with arsenite, TNF-α, H2O2 or anisomycin; β-actin was used as loading control.

B-F (B, C and E) Representative images (B) and cfu quantification (C, E) of intracellular Shigella in HeLa cells pre-treated with arsenite or anysomycin in the absence or presence of the p38 inhibitor SB203580, and corresponding controls. (

D and F) Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre-treated or not with arsenite (D) or anisomycin (F) prior to infection.

G and H. 3D reconstruction of representative images of ceramide staining in HeLa cells treated with arsenite (G) or anisomycin (H), in the presence or absence of SB203580, and corresponding controls. Ceramide and Hoechst staining were surface converted by voxel distance.

Data information: Infection was performed with Shigella WT at MOI 10 and analyzed at 0.5 hpi. Results are shown as mean ± s.e.m. from 5 (E), 6 (D and F) or 7 (C) independent experiments; *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA). Scale bars, 50 µm (B) and 5 µm (G, H).
Stress-induced host membrane remodeling protects from infection by 
non-motile bacterial pathogens (2018)
Figure 5

Figure 5. Salmonella motility compensates for membrane remodeling in cells exposed to stress

A - C. Representative images (A), cfu (B) and qRT-PCR (C) quantification of intracellular Salmonella in HeLa cells pre-treated with arsenite or anisomycin and corresponding controls. Infection was performed with Salmonella WT and analyzed at 0.5 hpi.

D. Representative images of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT or Δ4 mutant strain for 10 min.

E and F. Violin plots showing the distribution of the number of bacteria bound per infected cell in HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT (E) or Δ4 mutant strain (F) for 10 min. Results are shown for 50 infected cells per condition and independent experiment (total 250 cells); white circles show the medians, box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; polygons extend to extreme values.

G and H. Representative images (G) and cfu quantification (H) of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella mutant strains ΔfliC, ΔfliC/pFliC or ΔflhC for 10 min.

I and J. Representative images (I) and cfu quantification (J) of Listeria bound to HeLa cells pre-treated or not with arsenite followed by incubation with the bacteria for 20 min.

K and L. Representative images (K) and cfu quantification (L) of Yersinia bound/internalized by HeLa cells pre-treated or not with arsenite followed by 70 min incubation with the bacteria.

Data information: Infection was performed at MOI 25 (A-C) or MOI 50 (D-H) for Salmonella, MOI 50 for Listeria (I, J) and Yersinia (K, L). Results are shown as mean ± s.e.m. from 5 (C, E, F, H), 6 (B - arsenite and L) or 7 (B - anisomycin and J) independent experiments; ***p<0.001 (paired t-test for B, C, J, L; Mann-Whitney U test for E, F; two-way ANOVA for H). Scale bars, 50 µm (A) and 25 µm (D, G, I, K).
Stress-induced host membrane remodeling protects from infection by 
non-motile bacterial pathogens (2018)
Figure 6

Figure 6. Re-infection by non-motile bacteria is inhibited by Shigella intracellular replication

A. Schematic representation of the experimental design for reinfection experiments. Cells were infected with Shigella-mCherry (primary infection) or mock-treated. At 3 hpi, cells were re-infected with Shigella or Salmonella expressing GFP (secondary infection). Secondary infection was analyzed at 0.5 hpi (i.e. 1 h after addition of bacteria to cells).

B - E. Representative images (B, D) and cfu quantification (C, E) of re-infection assays with Shigella WT (B, C) or Salmonella WT (D, E) of HeLa cells primarily infected with Shigella WT or ΔipaB/Inv mutant strain, or mock-treated. Secondary infection is shown in bottom images.

F and G. Representative images (F) and cfu quantification (G) of the re-infection assays with Salmonella ΔfliC, ΔfliC/pFliC or ΔflhC mutant strains in HeLa cells primarily infected with Shigella WT or mock-treated. Secondary infection is shown in bottom images.

H. Schematic representation of the experimental design for reinfections upon knockdown or inhibitor treatment.

I and J. Representative images (I) and cfu quantification (J) of the secondary infection with Shigella WT, in cells transfected with ASM siRNA or control siRNA.

K - M. Cfu quantification of the secondary infection with Shigella WT in HeLa cells following amitriptyline (K), transfection with MAPK p38 siRNA (L) or SB203580 treatment (M).

Data information: Infection was performed at MOI 10 for Shigella WT, MOI 350 for Shigella ΔipaB/Inv, MOI 25 for Salmonella WT (D, E, G), or MOI 50 for Salmonella WT, ΔfliC, ΔfliC/pFliC or ΔflhC mutant strains (F, G). Results are shown as mean ± s.e.m. from 5 independent experiments; *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA for C, E, J-M, two-way ANOVA for G). Scale bars, 50 µm.
Stress-induced host membrane remodeling protects from infection by 
non-motile bacterial pathogens (2018)
Figure 7

Figure 7. Intracellular Shigella replication induces remodeling of the host cell plasma membrane in vitro and in vivo

A and B. 3D reconstruction of representative images of ASM (A) or ceramide (B) staining in HeLa cells infected with Shigella WTor mock-treated, analyzed at 3 hpi. ASM, ceramide, Hoechst and Shigella staining were surface converted by voxel distance.

C and D. ASM enzymatic activity (C) and ceramide (D) measurement in HeLa cells infected with Shigella-WT or mock-treated, analyzed at 3 hpi. The ASM enzymatic activity was determined in the membrane fraction corresponding to 3.0×105 cells per condition. Ceramide is shown normalized to mock-treated cells.

E - H. Representative spinning-disk confocal images of colon tissue sections of non-infected (E and F) and Shigella infected (G and H) guinea pigs, collected at 4 hpi. E and G are montages of multiple image fields; dashed boxes in (E and G) are shown enlarged in (F and H), respectively.

I. Mean fluorescence intensity of ceramide signal in surface epithelial cells (outermost tissue layer) of non-infected or Shigella-infected guinea pig colon tissue sections.

J. Model depicting the effect of stress-induced host plasma membrane remodeling on infection by bacterial pathogens. Different stressors, including TNF-α stimulation and Shigella intracellular replication, induce the relocalization and activation of acid sphingomyelinase (ASM) to the host plasma membrane, leading to the conversion of sphingomyelin into ceramide. As a consequence of membrane remodeling, the binding of non-motile bacteria, such as Shigella, to host cells is inhibited. Motile bacteria, such as Salmonella, are able to scan the host membrane for the remaining permissive binding sites, and thus circumvent this defense mechanism.

Data information: Shigella infection was performed at MOI 100 (A, B). Results are shown as mean ± s.e.m. from 9 (C) or 6 (D) independent experiments; whiskers in the box-plot (I) correspond to min/max from 10 (non-infected) or 13 (Shigella) tissue sections. *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA for C, D; unpaired t-test for I). Scale bars, 5 µm (A, B), 50 µm (E, G) and 12.5 µm (F, H).

EXPANDED VIEW FIGURE LEGENDS
Optogenetic control of mitochondrial protonmotive force to impact cellular stress resistance (2020)
Figure 1

Figure 1 - Light-activated proton pump mitochondria-ON (mtON) is expressed in mitochondria.

A Schematic depicting the targeting strategy to localize mtON to the mitochondrial inner membrane (IM). The electron transport chain (ETC) complexes generate the endogenous mitochondrial PMF by proton pumping, represented by the + and - across the IM. Mitochondrial ATP synthase utilizes the PMF to convert ADP to ATP. The N-terminal mitochondria target sequence from the IMMT1 protein shown in red; GFP shown in green. In response to light, mtON pumps protons from the mitochondrial matrix to the intermembrane space (IMS).

B Confocal images demonstrate overlap of GFP tagged mtON with MitoTrackerTM Red CMXRos stained C. elegans hypodermal mitochondria. Scale bar 10 µm.

C Immunoblot comparing the cytosolic supernatant and the mitochondria-enriched pellet of isolation fractions. GFP-tagged mtON migrates at the predicted molecular weight of 82 kDa accounting for the mitochondria-target sequence, the proton pump, and GFP. mtON is observed only in the mitochondrial fraction compared to marker proteins HSP60 (mitochondria) and actin (cytosol). All blots are from the same lanes on one membrane.
Optogenetic control of mitochondrial protonmotive force to impact cellular stress resistance (2020)
Figure 2

Figure 2 - mtON activation increases the PMF.

A Representative TMRE fluorescence traces in arbitrary units (a.u.) before and after Δψm dissipation with FCCP. Dashed lines indicate where FCCP was added. +/- ATR traces were performed in the absence of succinate. mtON activation was continuous throughout the traces. Light green trace is from mitochondria with ATR, gray traces are without.

B Quantification of change in fluorescence (ΔF) normalized to the maximum change in fluorescence given by succinate respiration (ΔFmax) for each mitochondrial preparation. Data are from the maximum light dose in Fig. EV 3A. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0469, +ATR succinate vs. +ATR +light p = 0.9978, n = 6 mitochondrial isolations. Data show mean ± SEM.

C Representative BCECF-AM 490/440 nm ratio trace. Ratio of 545 nm fluorescence intensity at either 440 or 490 nm excitation. Dashed lines are where light or FCCP treatment occurred. Light green trace is from mitochondria with ATR, gray traces are without.

D Quantification of change in BCECF-AM fluorescence ratio normalized to maximum change given by succinate matrix alkalization. Two-way ANOVA with Sidak's test for multiple comparisons, -ATR succinate vs. -ATR, +light *p = 0.0212, +ATR succinate vs. +ATR light p = 0.999, +ATR succinate vs. +ATR,-light p = 0.0237, +ATR, +light vs, +ATR, -light p = 0.0474, n = 3 mitochondrial isolations. Some individual points overlap. Data show mean ± SEM.

E ATP levels normalized to total ATP synthesis given by succinate respiration. Data from Fig. EV 3B. Succinate data shown for comparison after normalization. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0011, +ATR succinate vs +ATR light p = 0.5680, n = 3,7,3,7, biological replicates for each bar from left to right. Some individual points overlap. Data show mean + SEM.

F O2 required to consume 50 nmoles of ADP after mtON activation. Data are the maximum illumination from Fig. EV 4D using time-matched dark conditions, One-way ANOVA *p = 0.013 (-ATR, -light vs. +ATR, +light p = 0.010. -ATR, +light vs. +ATR, +light p = 0.013.) -ATR, -light n = 5, rest n = 6 mitochondrial preparations. Data show mean + SEM.
Optogenetic control of mitochondrial protonmotive force to impact cellular stress resistance (2020)
Figure 3

Figure 3 - mtON reverses mitochondrial dysfunction.

A Day 1 adult animals expressing mtON were exposed to 50 µM rotenone (ETC complex I inhibitor) for 5 hours and survival was scored. Illumination was continuous throughout toxin exposure (see methods). ATR alone was protective (-ATR, -light vs. +ATR, -light p = 0.016.) The effect of mtON activation was greater than the ATR alone effect, one-way ANOVA with Tukey's post hoc test *p = 0.01. (-ATR, -light vs +ATR, +light p = 0.0002. -ATR, +light vs. +ATR, +light p = 0.0009. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM.

B mtON-expressing animals were exposed to 50 µM antimycin A (complex III inhibitor) and survival was scored 18 hours later. One-way ANOVA with Tukey's post hoc test *p = 0.02 (-ATR, -light vs +ATR, +light p = 0.03, -ATR, +light vs. +ATR, +light p = 0.01. n = 5 plates each condition with at least 15 animals per plate). Data show mean ± SEM.

C mtON-expressing animals were exposed to 0.25 M azide (complex IV inhibitor) for 1 hour and scored for survival 1 hour after recovering, one-way ANOVA with Tukey's post hoc test *p = 0.0002. (-ATR, -light vs +ATR, +light p < 0.0001, -ATR, +light vs. +ATR, +light p <0.0001. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM.

D mtON-expressing animals were exposed to 31 µM oligomycin A (ATP synthase inhibitor) and survival was scored 18 hours later. No significant differences were found by one-way ANOVA (n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM.

E mtON-expressing animals in a complex I mutant background (gas-1) were scored for locomotion speed in the presence of food by counting body bends per minute. mtON activation rescued the decreased locomotion of the gas-1 mutant background, one-way ANOVA with Tukey's post hoc test, -ATR, +light vs. +ATR, +light *p = 0.0145, +ATR, +light vs. wildtype (WT) *p = 0.0008 (-ATR, -light vs +ATR, +light p = 0.0272, +ATR, -light vs. +ATR, +light p = 0.0332, all conditions vs WT, p < 0.001, n = 30, 30,30,41,41 animals each bar from left to right). Data show mean ± standard deviation.
Optogenetic control of mitochondrial protonmotive force to impact cellular stress resistance (2020)
Figure 4

Figure 4 - mtON affects whole-animal energy sensing.

A Top: Immunoblot assessing the effect of mtON activation on AMPK phosphorylation status. Top bands (~62 kDa) are phosphorylated AMPK signal, and bottom bands (~43 kDa) are actin signal. Image is from the same membrane cut to separately probe for phosphorylated AMPK (pAMPK) and actin. Bottom: Densitometry analysis showing decreased pAMPK to actin ratio, as there is no known antibody directed against total AMPK in C. elegans. Phosphorylation increases in the absence of food, but low phosphorylation is preserved when mtON is activated, two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0203, n = 3,3,3,4,4 biological replicates each bar from left to right. Data show mean + standard deviation.

B Locomotion was assessed by counting body bends per minute. Wild type animals were compared to aak-2(ok524) mutant animals. 2-sample, 2-tailed unpaired t-test *p < 0.0001, wild type n = 35, aak-2 n = 39 animals across at least 3 days. Data show mean ± standard deviation.

C Locomotion in response to mtON activation. Illumination was continuous throughout body bends measurement (see methods). For AAK-2 activation, animals were exposed to 1 mM AICAR for 4 hours before body bends measurement. One-way ANOVA with Tukey's post hoc test, -ATR, +light vs +ATR, +light *p < 0.0001, +ATR, +light vs. AICAR *p < 0.0001, +ATR,+light vs AICAR +light *p < 0.0001. n = 36, 39, 37, 46, 36, 36 animals each bar from left to right. Data show mean ± standard deviation.
Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1 (2014)
Figure 1

Ca2+ oscillations in immature DCs migrating in micro‐channels

A Left, kymograph from sequential epifluorescence (20×) images showing cytosolic Ca2+ oscillations in speed‐fluctuating immature DC after slow motility phases (upper and lower panel) and a change in direction (lower panel). DCs were loaded with the Ca2+ dye Oregon Green BAPTA 1‐AM, introduced in micro‐channels, and imaged every 10 s. Right, sequential epifluorescence (20×) images (1 image/20 s is shown). Instantaneous velocities and intracellular calcium oscillations were measured as described in Materials and Methods. Scale bar: 10 μm.
B, C Mean cell velocity of immature DCs (n > 125 cells from two independent experiments) (B) or CD8+ T lymphocytes (n > 40 cells from one experiment) (C) migrating in micro‐channels with or without BAPTA. Boxes illustrate 10–90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal–Wallis test.
D Mean intracellular calcium concentration in speed‐fluctuating (ΔV/V0 > 0.3; ΔV/V0 relative speed variation divided by the global median velocity) and non‐speed‐fluctuating (ΔV/V0 0.3) immature DCs (n = 187 cells from more than three independent experiments). P‐values were calculated using a Mann–Whitney test.
E, F Velocity fluctuations (ΔV/V0) (E) or mean Ca2+ activities (F) displayed by DCs migrating in micro‐channels in the presence of 100 ng/ml CCL3. P‐values were calculated using a Mann–Whitney test, with respect to non‐treated DCs (NT).
G Mean Ca2+ activities during phases of fast (> 50% of max velocity) and slow ( 50% of max velocity) motility in speed‐fluctuating DCs (Supplementary Fig S2F) (n = 64 cells, from three independent experiments). Activities were normalized to the mean Ca2+ activity shown by each cell during the entire movie. The P‐value was calculated using a Mann–Whitney test.