EMBO/soda-nel huggingface dataset:
TFEB and TFE3 are Novel Components of the Integrated Stress Response (2015)
Figure 3

Figure 3. Contribution of Calcineurin to the activation of TFE3 in response to tunicamycin.

(A) ARPE-19 cells infected with either adenovirus expressing TFE3-WT-Myc or TFE3-S321A-Myc for 16h. Cells were fixed, permeabilized and stained with antibodies against Myc. Scale bar, 10 µm. Quantification of the percentage of ARPE-19 cells with nuclear TFE3-Myc (means ± SD of 3 independent experiments, students t-test, ****p < 0.0001; n>400 cells per condition).

(B) Immunoblots of protein lysates from ARPE-19 cells infected as indicated in (A). Data are representative of three independent experiments.

(C) Immunoblots of TFE3-Ser321 phosphorylation state in nuclear fractions of ARPE-19 cells incubated with DMSO (Ctrl.), Tunicamycin (Tun.), or Torin-1. Data are representative of three independent experiments.

(D) Immunoblots of protein of a GST pull-down from nuclear fractions of MEF cells treated with DMSO or Tunicamycin (5 µg/ml) (Tun.) for 16 h. Data are representative of three independent experiments.

(E) Immunoblots of protein lysates from ARPE-19 cells incubated in the presence of DMSO (Ctrl.), Tunicamycin (5 µg/ml) (Tun.), or starved in EBSS (Strv.) for 16 h. Data are representative of three independent experiments.

(F) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Tunicamycin + FK506 treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus Tunicamycin treated cells, ***p < 0.001; n>400 cells per condition).

(G) Quantification of the percentage of calcineurin depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Starvation (Strv.) treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment, ns= not significant, *p < 0.05, **p < 0.01, ***p < 0.001; n>400 cells per condition).

(H) Immunoblots of protein lysates from ARPE-19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB. Data are representative of three independent experiments.

(I) Relative Quantitative Real-Time PCR analysis of PPP3CA and PPP3CB mRNA transcript levels in ARPE-19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analsysis versus the siNon-Target (siNT) treated cells, ***p < 0.001).

(J) Quantification of the percentage of ATF4 depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin treatment for 16 h (mean ± SD of two independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment***p < 0.001; n>400 cells per condition).
TFEB and TFE3 are Novel Components of the Integrated Stress Response (2015)
Figure 4

Figure 4. TFEB- and TFE3-dependent up-regulation of lysosomal and autophagic genes upon Tunicamycin treatment.

(A) Relative Quantitative Real-Time PCR analysis of TFEB and TFE3 mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, student’s t-test versus siNon-Target (siNT) treated cells, ***p < 0.001).

(B) Relative Quantitative Real-Time PCR analysis of MCOLN1, ATP6V1C1, HEXA and UVRAG mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 upon incubation with DMSO (Control) or Tunicamycin (5 µg/ml) for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the same treatment condition in the siNon-Target (siNT) treated cells or versus the DMSO condition in siNon-Target (siNT) treated cells, *p < 0.05, **p < 0.01, ***p < 0.001).
TFEB and TFE3 are Novel Components of the Integrated Stress Response (2015)
Figure 5

Figure 5. Over-expression of TFEB or TFE3 enhances the ATF4-mediated stress response.

(A) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A, or TFE3 upon incubation with DMSO (Control), Tunicamycin (5 µg/ml) or starved in EBSS for 16 h. Data are representative of five independent experiments.

(B) Quantification of ATF4, CHOP and GADD34 protein levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, *p < 0.05, **p < 0.01, ***p < 0.001).

(C) Relative Quantitative Real-Time PCR analysis of ATF4, CHOP, GADD34 and MCOLN1 mRNA transcript levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the RNA fold change of indicated genes normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, **p < 0.01, ***p < 0.001).
TFEB and TFE3 are Novel Components of the Integrated Stress Response (2015)
Figure 6

Figure 6. TFEB and TFE3 enhance the integrated stress response by inducing transcriptional up-regulation of ATF4.

(A-D) Relative Quantitative Real-Time PCR analysis of ATF4 (A), ASNS (B), CAT1 (C) and xCT (D) mRNA transcript levels in ATF4-depleted ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A upon incubation with DMSO (Control), Tunicamycin (5 µg/ml), or starved in EBSS for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected and siRNA Non-Target (siNT) treated cells, *p < 0.05, ***p < 0.001).

(E) ChIP-qPCR analysis of the ATF4 promoter and 2000bp upstream of the region of interest (ATF4-Control) from MEF cells that were untreated (control), starved for 2 h, or treated with Tunicamycin (0.1 µg/ml) for 16 h. Amplification regions are indicated by arrows. Chromatin DNA was immunoprecipitated with antibodies for anti-TFE3. Bar graphs show the amount of immunoprecipitated DNA detected by the real-time PCR assay. Values were normalized to the input and plotted as relative enrichment compared to untreated conditions (means ± SD of three independent experiments, students t-test, *p < 0.05, **p < 0.01).
TFEB and TFE3 are Novel Components of the Integrated Stress Response (2015)
Figure 7

Figure 7. TFEB and TFE3 regulate expression of critical regulators of the ER-stress response.

(A) Venn diagram showing overlapping genes in analyzed data sets.

(B-C) Schematic representations of TFE3 binding region in the ATF4 promoter in MEF cells treated with Tunicamycin (0.1 µg/ml) for 16 h (B) or starved in EBSS for 2 h (C). The distance upstream from the Transcription Start Site (TSS) of a potential E-box sequence is indicated in base pair (bp).

(D-E) E-box distance from transcription start site for targets categorized as lysosomal genes in MEF treated with Tunicamycin (0.1 µg/ml) for 16 h (D) or starved in EBSS for 2 h (E). Peaks called as TFE3 binding sites were analyzed for E-box elements using UCSC genome browser and distance from transcription start site assessed. Graphs represent the number of genes with E-box distance (in kbp) from the transcription start site.

(F) Relative Quantitative Real-Time PCR analysis of SUMF1, DERL1, ATF6, APEX1 and GPX1 mRNA transcript levels in ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, students t-test, **p < 0.01, ***p < 0.001).

(G) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A. Data are representative of three independent experiments.

(H) Quantification of IRE1α and APEX1 protein levels in ARPE-19 cells infected as indicated in (G) (mean ± SD of the fold increase of the indicated protein to actin ratio from three independent experiments, students t-test, ***p < 0.001).
TFEB and TFE3 are Novel Components of the Integrated Stress Response (2015)
Figure 8

Figure 8. ER stress response and susceptibility to apoptosis are regulated by TFEB and TFE3.

(A) Immunoblots of protein lysates from Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times. Data are representative of three independent experiments.

(B) Quantification of ATF4 protein levels in Null- or TFEB/3 knockout-MEF cells treated as indicated in (A) (mean ± SD of the ATF4 to actin protein ratio from three independent experiments, student’s t-test versus the corresponding time point in Null-MEF cells, ns= not significant, **p < 0.01).

(C) Cell viability was determined in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times (mean ± SD of the percentage of viable cells compared to control from three independent experiments, one-way ANOVA analysis versus the corresponding time point in Null-MEF cells ***p < 0.001).

(D) Chromatin condensation, an indication of cells undergoing apoptosis, was assessed in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Confocal microscopy images were obtained from fixed cells stained with Hoechst 33342. Scale bar, 20 µm. Data are representative of three independent experiments.

(E) Immunoblots of protein lysates from Null- or TFEB/3 knockout-MEF cells treated as described in (A). Data are representative of three independent experiments.

(F-H) Quantification of CHOP (F), PUMA (G) and NOXA (H) protein levels in Null- or TFEB/3 knockout-MEF cells treated as indicated in (E) (mean ± SD of the indicated protein to actin ratio from three independent experiments, student’s t-test analysis versus the corresponding time point in Null-MEF cells, ns= not significant, *p < 0.05, **p < 0.01).

(I) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A or TFE3. Data are representative of three independent experiments.

(J) Quantification of PUMA protein levels in ARPE-19 cells infected as indicated in (I) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the adenovirus-Null infected cells, *p < 0.05, ***p < 0.001).
Bacteria elicit a phage tolerance response subsequent to infection of their neighbors (2021)
Figure 2

Figure 2. SigX is required for plaque constriction

(A) The indicated strains were infected with SPP1 (10-6 PFU/ml), spread over MB plates, and plaque diameter was monitored after 20 hrs of incubation. Shown is average plaque diameter and SD for each strain (n ≥ 54).

(B-C) Plaque formation dynamic of SPP1 (B) and Phi29 (C) was monitored by automated scanning Levin-Reisman et al., 2010() on a lawn of infected (10-6 PFU/ml) PY79 (WT) or ET19 (∆sigX) cells spread over an MB agar plate. Shown are average values and SD from kinetic of random plaques for each strain (n ≥ 7).

(D) PY79 (WT) and ET19 (∆sigX) cells were infected with SPP1 at either high (phages:bacteria 1:1) or low (1:20) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, and the average values and SD of 8 technical repeats.

(E) BDR2637 (Pveg-mCherry) (WT, purple) and ET191 (PrrnE-gfp, ∆sigX) (∆sigX, cyan) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from mCherry (purple) and GFP (cyan) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm. Corresponds to Movie EV4.

(F) ET27 (PsigX-gfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluorescence from GFP (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.
Bacteria elicit a phage tolerance response subsequent to infection of their neighbors (2021)
Figure 3

Figure 3. SigX is activated in non-infected cells upon infection of their neighbors

(A) Phage sensitive PY79 (WT) cells were mixed with phage resistant BS12 (ΔyueB, PsigX-gfp) cells and the mixture was infected with SPP1, as illustrated. Infection was conducted at 2:1 (phages:bacteria) MOI and fluorescence intensity from PsigX-gfp (AU) was followed at 2.5 min intervals. Uninfected mixed population served as a control and its fluorescence was subtracted from the overall GFP signal of the infected culture. Shown is a representative experiment out of 3 biological repeats, and the average values and SD of 8 technical repeats.

(B) Phage sensitive PY79 (WT) cells were infected with SPP1 at 2:1 (phages:bacteria) MOI, and placed in the outer ring of a transwell, as illustrated. Phage resistant BS12 (ΔyueB, PsigX-gfp) were placed in the inner ring. Fluorescence intensity from PsigX-gfp (AU) of the inner compartment was followed at 2.5 min intervals. Uninfected population served as a control, and its fluorescence was subtracted from the overall GFP signal of the infected culture. Shown is a representative experiment out of 3 biological repeats, and the average values and SD of 8 technical repeats.

(C) ET26 (PsigX-sigX-gfp) cells were visualized by fluorescence microscopy. Shown are signal from SigX-GFP (cyan) (left panel), and an overlay image of phase contrast (grey) and signal from SigX-GFP (cyan) (right panel). Scale bar 1 μm.

(D) BDR2637 (Pveg-mCherry) (WT, purple) and ET261 (∆yueB, PsigX-sigX-gfp) (cyan) cells were mixed, infected with SPP1 at 5:1 (phages:bacteria) MOI, placed on an agarose pad and followed by time lapse fluorescence microscopy. Shown are overlay images from mCherry (purple), SigX-GFP (cyan), and phase contrast (grey), captured at the indicated time points post infection. Scale bar 1 μm.
Bacteria elicit a phage tolerance response subsequent to infection of their neighbors (2021)
Figure 4

Figure 4. SigX expression confers phage tolerance

(A) PY79 (WT) and ET28 (PIPTG-sigX) cells were infected with SPP1 or Phi29 (t=60 min) at 1:20 (phages:bacteria) MOI, and OD600nm was followed at 2 min intervals. IPTG was added 30 min before infection (t=30 min). Shown is a representative experiment out of 3 biological repeats, and the average values and SD of 4 technical repeats.

(B) ET29 (Pveg-mCherry, PIPTG-sigX) (purple) cells were grown in the presence of IPTG and mixed with PY79 (WT) cells. The mixture was infected with SPP1-lysin-yfp 5:1 (phages:bacteria) MOI, placed on an IPTG-containing agarose pad, and followed by time lapse fluorescence microscopy. Shown are overlay images of phase contrast (grey), signal from mCherry labeled cells (purple), and signal from Lysin-SPP1-YFP (cyan), captured at the indicated time points post infection (upper panels). Corresponding signal from Lysin-SPP1-YFP (cyan) is shown separately (lower panels). Arrows highlight the delayed infection of ET29 cells. Scale bar 1 μm.

(C) Quantification of the experiment described in (B). Shown is the percentage of phage infected PY79 (WT) and ET29 (Pveg-mCherry, PIPTG-sigX) cells at the indicated time points, scored by the Lysin-SPP1-YFP signal, with average values and SD (n ≥ 200 cells for each time point). Of note, the majority of WT cells were lysed at t=75 min post infection.

(D) PY79 (WT) and ET28 (PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1 (1:1 MOI) for 10 min. Next, phage adsorption was monitored before and after cell dilution (x100 fold). Percentage of phage adsorption was calculated as follows: (P0-P1)x100/P0, where P0 is the initial phage input in the lysate (PFU/ml), and P1 is the titer of free phages (PFU/ml) 10 min after infection. Shown are average values and SD of a representative experiment out of 3 independent experiments.

(E) OF83 (Ppen-lacIΔ11-cfp) (WT) and ET40 (Ppen-lacIΔ11-cfp, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1-delX110lacO64 at 5:1 (phages:bacteria) MOI. The formation of LacI-CFP foci on phage DNA was monitored 10 min post infection. Non-infected OF83 cells were used for comparison. Shown are overlay images of phase contrast (grey) and signal from LacI-CFP (cyan). Scale bar 1 µm.

(F) Quantification of the experiment described in (E). Shown is the percentage of LacI-CFP foci 10 min post infection of OF83 and ET40 cells by SPP1, with average values and SD (n ≥ 850 cells for each population).
Bacteria elicit a phage tolerance response subsequent to infection of their neighbors (2021)
Figure 5

Figure 5. The dlt operon mediates the SigX-induced phage tolerance response

(A) ET28 (PIPTG-sigX) and ET42 (∆dltA, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1 at 1:20 (phages:bacteria) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, with the average values and SD of 8 technical repeats.

(B) PY79 (WT) and ET42 (ΔdltA, PIPTG-sigX) cells, grown in the presence of IPTG, were infected with SPP1 (1:1 MOI) for 10 min. Next, phage adsorption was monitored before and after cell dilution (x100 fold). Percentage of phage adsorption was calculated as follows: (P0-P1) x100/P0, where P0 is the initial phage input in the lysate (PFU/ml), and P1 is the titer of free phages (PFU/ml) 10 min after infection. Shown are average values and SD of 5 biological repeats.

(C) PY79 (WT), ET28 (PIPTG-sigX), and ET42 (ΔdltA, PIPTG-sigX), grown in the presence of IPTG, were transduced with SPP1-pBT163 lysate, and the number of transductants was monitored by plating the cells on selective plates. Transduction unit (TRU) was calculated as the number of transductant colonies /total colony forming unit (CFU). Shown are average values and SD of 3 biological repeats.

(D) PY79 (WT) and ET41 (ΔdltA) cells were infected with SPP1 (10-6 PFU/ml), spred over an MB agar plate, and plaque diameter was monitored after 20 hrs of incubation. Shown is plaque diameter distributions for each strain (n ≥ 40).

(E) Plaque formation dynamic of SPP1 was monitored by automated scanning Levin-Reisman et al., 2010() on a lawn of infected (10-6 PFU/ml) PY79 (WT) or ET41 (ΔdltA) cells grown on an MB agar plate. Shown are average values and SD from kinetics random plaques for each strain (n ≥ 10).

(F) AR16 (PrrnE-gfp) (WT, cyan) and ET411 (Pveg-mCherry, ∆dltA) (∆dltA, purple) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from GFP (cyan) and mCherry (purple) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 150 µm.

(G) ET43 (dltA-yfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluoresce from DltA-YFP signal (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.
Syncytia formation by SARS-CoV-2 infected cells. (2020)
Figure 2

Figure 2. Impact of TMPRSS2 and IFITMs on syncytia formation by U2OS-ACE2 infected cells.

Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h.

A. TMPRSS2 increases fusion and cell mortality. Right panel: Areas of GFP+ cells and PI+ cells

B. IFITM1, but not IFITM2 and 3, inhibits SARS-CoV-2-induced syncytia formation. Right panels: Area of GFP+ cells

C. The fusion of IFITM1+ cells with U2OS-ACE2 syncytia is drastically reduced. Right panel: Area of IFITM+ GFP+ cells

D. IFITM1 decreases the number of infected cells. Right panel: Fraction of cells positive for S, normalized to control cells (transduced with pQCXIP-empty vector ).

Data information: Left panels: one representative experiment is shown. Scale bars: 100 µm. Right panels: Data are mean±sd of 3-9 independent experiments. Statistical analysis: B-C: One-Way ANOVA, D: Two-Way ANOVA. ns: non-significant, ∗p < 0. 05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Syncytia formation by SARS-CoV-2 infected cells. (2020)
Figure 3

Figure 3: Impact of TMPRSS2 and IFITMs on the kinetics of fusion by S-expressing 293T cells.

A. 293T-GFP1-10 and -GFP11 cells (1:1 ratio) were co-transfected with S, ACE2, TMPRSS2, IFITM or control plasmids. Cell fusion was quantified by measuring the GFP+ area by high-content imaging after 18h. (B and C) or analyzed over time by video microscopy (D and E).

B. Representative images of cell-cell fusion. Scale bar: 100 µm.

C. Quantification of GFP+ areas. Results are mean±sd from 5 independent experiments.

D. TMPRSS2 accelerates fusion. Cells were monitored by video microscopy and the GFP area was quantified over time. Left panel: one representative experiment. Results are mean±sd from 3 fields per condition. Right panel: Mean±sd from 7 independent experiments (at 12h post transfection).

E. Impact of TMPRSS2 and IFITMs on the kinetics of fusion by S-expressing 293T cells. One representative out of three independent experiment is shown.

Data information: Statistical analysis: B, C: One-Way ANOVA, ns: non-significant, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. D: Wilcoxon matched-pairs signed rank test, ∗∗p < 0.01.
Syncytia formation by SARS-CoV-2 infected cells. (2020)
Figure 4

Figure 4: Effect of TMPRSS2 and IFITM on S- or ACE2-expressing cells.

A. S-expressing cells (Donor cells), ACE2-expressing cells (Acceptor cells) were co-transfected with TMPRSS2, IFITM or control plasmids. Cell fusion was quantified by measuring the GFP+ area after 18h. The indicated combinations were tested. The impact of IFITMs was measured in absence of TMPRSS2 (left panel), in presence of TMPRSS2 in donor (middle panel) or acceptor cells (right panel). Data are mean±sd of 4 independent experiments. Statistical analysis: One-Way ANOVA, ns: non-significant, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

B-D. Impact of TMPRSS2 on IFITMs, ACE2 and S levels. B. TMPRSS2 does not decrease IFITM amounts measured by flow cytometry. 293T cells were transfected with the indicated IFITM plasmids, with or without TMPRSS2, and analyzed 18h later. C. Impact of TMPRSS2 on S and ACE2, measured by Western Blot. 293T cells were transfected with or without IFITM1, TMPRSS2, S or ACE2 plasmids, and analyzed 18h later. D. TMPRSS2 decreases S surface levels, measured by flow cytometry. 293T cells were transfected with S plasmid, with or without TMPRSS2, and analyzed 18h later. Murine polyclonal anti-S antibodies, one serum from a convalescent COVID-19 patient (sera n°111), and two monoclonal antibodies (anti-S1 and anti-S2) were tested. Data are representative of three independent experiments.

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