EMBO/soda-nel huggingface dataset:
Mitophagy bridges DNA sensing with metabolic adaption to expand lung cancer stem-like cells (2022)
Figure 1

Fig. 1 - Hypermitophagy drives Notch1 activity and CSC expansion

(A) mRNA expressions of PINK1 in CSCs and non-CSCs. Mean±SEM from 6 patients.

(B-C) Immunoblot analysis of PINK1 protein in non-CSCs and CSCs. Representative and mean±SEM from 9 patients.

(D-E) Immunoblot analysis of p-PINK1 protein in non-CSCs and CSCs. Representative and mean±SEM from 7 patients.

(F-G) Immunoblot analysis of Parkin protein in non-CSCs and CSCs. Representative and mean±SEM from 8 patients.

(H-I) PINK1 and Parkin protein levels in mitochondrial fractions and whole cell lyses of CSCs and non-CSCs. Representative and mean±SEM from 8 patients.

(J-K) Intracellular levels of p-Parkin protein in non-CSCs and CSCs were analyzed using flow cytometry. Representative histograms and mean±SEM from 6 patients.

(L-M) Immunoblot analysis of p-Parkin protein in non-CSCs and CSCs. Representative and mean±SEM from 11 patients.

(N-P) CSCs were treated with Mdivi-1 (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Scale bar 20 mm.

(Q) CSCs transfected with PINK1 shRNA or the control were analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments.

(R) CSCs were treated with DAPT (10 μM) and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments.

(S) CSCs were transfected with PINK1 shRNA or control, and analysed for activated Notch1. Mean±SEM from 6 patients.

(T) CSCs with or without transfection of Notch1 ICD were treated with Mdivi-1 (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.
Mitophagy bridges DNA sensing with metabolic adaption to expand lung cancer stem-like cells (2022)
Figure 2

Fig. 2 - Mitophagyhigh increases lysosomal mtDNA and TLR9 activation in CSCs

(A-B) Lysosomal DNA was identified with anti-LAMP1 and anti-DNA antibodies in non-CSCs and CSCs. Scale bar 5μm. Representative and mean±SEM with 50 cells from 5 patients.

(C) Lysosomal content of mtDNA in CSCs and non-CSCs was determined with qPCR. Mean±SEM from 6 patients.

(D) CSCs were treated with Mdivi-1 (50 μM) and analysed for lysosomal mtDNA. Mean±SEM from 6 patients.

(E) CSCs with or without transfection of PINK1 shRNA were analysed for lysosomal mtDNA. Mean±SEM from 6 patients.

(F-G) Protein expressions of TLR9 and MyD88 in CSCs and non-CSCs. Representative and mean±SEM from 6 patients.

(H) TLR9 proteins were immunoprecipitated from CSCs and non-CSCs and analyzed for the binding load of mtDNA. Mean±SEM from 6 patients.

(I) CSCs were transfected with mtDNA and analysed for expressions of TLR9 plus MyD88. Mean±SEM from 6 patients.

(J-K) CSCs treated with EtBr (50 ng/ml) were analysed for lysosomal mtDNA, TLR9, and MyD88. Mean±SEM from 6 patients.

(L) CSCs transfected with DNase II expression vector or the control were analyzed for lysosomal DNA plus MyD88 protein. Mean±SEM from 6 patients.

(M) CSCs transfected with DNase II shRNA or the control were analyzed for lysosomal DNA plus MyD88 protein. Mean±SEM from 6 patients.

Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.
Mitophagy bridges DNA sensing with metabolic adaption to expand lung cancer stem-like cells (2022)
Figure 3

Fig.3 - Lysosomal mtDNA-dependent TLR9 promotes CSC expansion

(A) CSCs were stimulated with CpG ODN (10 μg/ml) or control and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments.

(B-C) CSCs transfected with mtDNA were analyzed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments.

(D) CSCs were transfected with 2 different TLR9 shRNAs or the control, and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments.

(E-F) CSCs transfected with DNase II expression vector, DNase II shRNA, or the control was analyzed for sphere formation. Mean±SEM from 6 patients.

(G) CpG promoted tumor growth of PDX chimeras. Mean±SEM from 6 mice.

(H) TLR9 inhibitor chloroquine abrogated the effect of CpG on tumor growth in PDX chimeras. Mean±SEM from 6 mice.

(I) CpG treatment increased the frequency of CD133-expressing CSCs in PDX chimeras. Mean±SEM from 6 mice.

(J) Chloroquine impeded the function of CpG in elevating CD133-expressing CSCs of PDX chimeras. Mean±SEM from 6 mice.

Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.
Mitophagy bridges DNA sensing with metabolic adaption to expand lung cancer stem-like cells (2022)
Figure 4

Fig.4 - TLR9 promotes CSC expansion through Notch1 hyperactivity

(A) CSCs transfected with TLR9 shRNA or control were analysed for protein expressions of activated Notch1 plus HES1. Mean±SEM from 6 patients.

(B) CSCs treated with or without EtBr (50 ng/ml) were analyzed for activated Notch1. Mean±SEM from 6 patients.

(C) CSCs transfected with DNase II expression vector or control were analyzed for activated Notch1 expression. Mean±SEM from 6 patients.

(D) CSCs with or without CpG treatment were analysed for interaction between TLR9 and Notch1. Representative and mean±SEM from 6 patients.

(E) CSCs were treated with CpG ODN (10 μg/ml) in the presence or absence of DAPT (10 μM), and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments.

(F) CSCs were transfected with 2 different Notch1 shRNAs or control, treated with CpG ODN (10 μg/ml), and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments.

(G-H) PDX chimeras treated by CpG with or without DAPT were analysed for tumor growth and CD133-expressing CSCs. Mean±SEM from 6 mice.

Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.
Mitophagy bridges DNA sensing with metabolic adaption to expand lung cancer stem-like cells (2022)
Figure 5

Fig. 5 - Lysosomal mtDNA-TLR9-Notch1 maintains mitochondrial metabolism for CSC expansion

(A) CSCs and non-CSCs were analyzed for mitochondrial metabolism using seahorse assay. Mean±SEM from 6 patients.

(B) CSCs were treated with 2-DG (10 mM) or Malonate (10 mM), and assayed for sphere formation plus tumorgenicity. Mean±SEM from 6 patients.

(C) CSCs treated with DAPT (10 μM) were analysed for mitochondrial OCR. Mean±SEM from 6 patients.

(D) CSCs transfected with Notch1 shRNA or control shRNA were analysed for mitochondrial OCR. Mean±SEM from 6 patients.

(E) CSCs treated with EtBr (50 ng/ml) were analysed for mitochondrial OCR. Mean±SEM from 6 patients.

(F) CSCs transfected with DNase II expression vector or control were analysed for mitochondrial OCR. Mean±SEM from 6 patients.

(G) CSCs treated with chloroquine (50 μM) were analyzed for mitochondrial OCR. Mean±SEM from 6 patients.

Data information: Paired t-test with Bonferroni method (A, C-G) and ANOVA test with Turkey's method (B). **p < 0.01, ***p < 0.001.
Mitophagy bridges DNA sensing with metabolic adaption to expand lung cancer stem-like cells (2022)
Figure 6

Fig. 6 - TLR9-Notch1 drives AMPKhigh to promote mitochondrial metabolism in CSCs

(A) CSCs stimulated with or without CpG ODN (10 μg/ml) were analyzed for expression of phosphor-AMPKα. Representative and mean±SEM from 6 patients.

(B) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of DAPT (10 μM), and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients.

(C) CSCs transfected with Notch1 shRNA or control were stimulated with CpG ODN (10 μg/ml) and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients.

(D) CSCs were treated with DAPT (10 μM) in the presence or absence of A769662 (10 μM), and analysed for mitochondrial OCR. Ctrl represents CSCs without any treatment. Mean±SEM from 6 patients.

(E) CSCs were stimulated with or without CpG ODN (10 μg/ml) and analyzed for mitochondrial OCR. Mean±SEM from 6 patients.

(F) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of Compound C (10 μM) and analysed for mitochondrial OCR. Mean±SEM from 6 patients.

(G) CSCs were transfected with 2 different AMPKα shRNA or the control, stimulated with CpG ODN (10 μg/ml), and analysed for mitochondrial OCR. Mean±SEM from 6 patients.

Data information: Paired t-test with Bonferroni method and ANOVA with Turkey method (D), **p < 0.01, ***p < 0.001, ****p < 0.0001.
Mitophagy bridges DNA sensing with metabolic adaption to expand lung cancer stem-like cells (2022)
Figure 7

Fig.7 - Notch1 induces metabolic stress and LKB1 phosphorylation for lysosomal AMPK activation in CSCs

(A) CSCs transfected with or without Notch1 ICD were analyzed for AMP/ATP ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients.

(B) CSCs treated with or without DAPT (10 μM) were analyzed for AMP/ATP ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients.

(C) CSCs treated with or without CpG ODN (10 μg/ml) were analysed for interactions between Notch1 and AMPK. Representative and mean±SEM from 6 patients.

(D) CSCs transfected with LKB1 shRNA（right）or the control (left) were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients.

(E) CSCs transfected with TAK1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients.

(F) CSCs transfected with LAMTOR1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients.

(G) CSCs transfected with AXIN1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients.

Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.
Mitophagy bridges DNA sensing with metabolic adaption to expand lung cancer stem-like cells (2022)
Figure 8

Fig. 8 - Targeting mitophagy and the TLR9-Notch1-AMPK pathway restricts CSC expansion in a NSCLC PDX model

(A) NSCLC PDX models treated with or without chemotherapy for the first 2 weeks were analyzed for tumor growth at the indicated time. Mean±SEM from 6 experiments.

(B) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with or without chemotherapy were determined after 4 weeks. Mean±SEM from 6 experiments.

(C-E) Flow cytometric analyses of protein expressions of TLR9, activated Notch1, and phosphor-AMPKα in cancer cells from NSCLC xenografts treated with or without chemotherapy after 4 weeks. Mean±SEM from 6 experiments.

(F) NSCLC PDX models were treated with chemotherapy alone or together with the indicated inhibitors, respectively, for the first 2 weeks. Tumor volume was determined at the indicated time. Mean±SEM from 6 experiments.

(G) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with chemotherapy alone or with the indicated inhibitors after 4 weeks. Mean±SEM from 6 experiments.

Data information: Paired t-test with Bonferroni (A-E) and ANOVA test with Turkey's (F-G) method. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Expanded View Figure legends
Stimulus-specific remodeling of the neuronal transcriptome through nuclear intron-retaining mRNAs (2022)
Figure 2

Figure 2. Nuclear intron-retaining transcripts are regulated by several forms of neuronal stimulation

(A) and (B) Efficiencies of bicuculline (A) and BDNF (B) stimulation were assessed by controlling the induction of ERK phosphorylation by immunostaining. Mouse primary neocortical cells (14 days in culture) were stimulated with bicuculline (20µM) to elevate the neuronal network excitation or with the brain-derived neurotrophic factor BDNF (50ng/mL) for 5min. Cells were stained with anti-phospho-ERK (green, bottom) and anti-MAP2 (white, top) antibodies and counterstained with Hoechst (blue, top). (four independent cultures)

(C) and (D) Volcano plots displaying the expression fold change (FC) of IR-transcripts and the corresponding z-score (absolute value) upon bicuculline (C) and BDNF (D) stimulation. DRB (50µM) was applied to mouse primary neocortical cells for 2hrs; 1hr before cell collection, bicuculline (20µM) or BDNF (50ng/mL) were applied or not (control). Every transcript retaining an intron (minPIR ≥ 20%) in unstimulated condition is plotted. IR-transcripts were considered down-regulated (light blue or orange) or up-regulated (dark blue or red) if the following applied: fold change of IR-transcript expression ≥ 20% and |z-score| ≥ 1.5. (three independent cultures)

(E) and (F) Pairwise comparison of the expression of regulated IR-transcripts in the nuclear and the cytosolic fractions of unstimulated mouse primary neocortical cells. Every transcript retaining an intron (minPIR ≥ 20%) in whole cell extract is plotted. Intron retaining transcripts down- or up-regulated upon bicuculline stimulation (E) are labeled in light blue and dark blue respectively and those down- or up-regulated upon BDNF stimulation (F) are labelled in orange and red respectively. (three independent cultures).
Stimulus-specific remodeling of the neuronal transcriptome through nuclear intron-retaining mRNAs (2022)
Figure 3

Figure 3. Neuronal stimulation regulates intron-retaining transcripts through splicing and degradation processes

(A) and (B) Pairwise comparison of the expression fold change (FC) of intron-retaining isoforms (IR) and the spliced isoforms upon bicuculline (A) or BDNF (B) stimulation. In all conditions, DRB (50µM) was applied to mouse primary neocortical cells (14 days in culture) for 2hrs; 1hr before cell collection, bicuculline (20µM) or BDNF (50ng/mL) were applied or not (control). Every transcript containing a retained intron (minPIR>20) in control condition is plotted. IR-transcripts are considered regulated through splicing if the following applied: (1) IR-isoform expression fold change ≥ 20% and |z-score| ≥ 1.5, (2) spliced isoform expression fold change ≥ 20% and |z-score| ≥ 1.5, (3) expression of the IR-isoforms and the spliced isoforms evolved in opposite directions. IR-transcripts are considered regulated through degradation (IR-transcript specific decrease) if the following applied: (1) IR expression fold change ≥ 20% and |z-score| ≥ 1.5, (2) spliced expression fold change ≤ 5% and |z-score| ≤ 1. (three independent cultures)

(C) to (F) RT-PCR validations of regulated IR-transcripts through splicing upon bicuculline stimulation (C) and BDNF stimulation (D) and through degradation upon bicuculline stimulation (E) and BDNF stimulation (F). Expression of the IR-isoforms and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs (standard deviations) of PIR values are shown beneath each panel. In addition, fold changes in the expression of the IR-transcripts (red and orange) and spliced (dark grey) isoforms were assessed with real-time qPCR using three different primer sets, as represented in the top scheme. Means and SDs are displayed (three-four independent cultures); the p-values calculated with a one-tailed paired t-test are indicated (as numerical values when p>0.05, as * when p<0.05, as ** when p<0.01 and as *** when p<0.001). Note that the Gria3 spliced transcript (C) does not correspond to the canonical mRNAs and presumably arise from a first step of recursive splicing and thereby likely require splicing completion to generate fully mature Gria3 transcripts (Sibley et al, 2015).
Stimulus-specific remodeling of the neuronal transcriptome through nuclear intron-retaining mRNAs (2022)
Figure 5

Figure 5. Stimulus-specific regulation of sub-population of intron retentions

(A) Heatmap of the IR-transcript expression values in control, bicuculline-stimulated and BDNF-stimulated conditions. IR-transcripts regulated in at least one condition are displayed (fold change of intron-retaining transcript expression ≥ 20% and |z-score| ≥ 1.5).

(B) Pairwise comparison of the expression fold change (FC) of intron-retaining isoforms upon bicuculline and BDNF stimulation. Every transcript containing a retained intron (minPIR>20%) in control condition is plotted. IR-transcripts are considered regulated specifically upon bicuculline (light and dark blue) or BDNF (orange and red) stimulation if the following applied: (1) IR-transcript expression fold change ≥ 20% and |z-score| ≥ 1.5 upon bicuculline (or BDNF) stimulation (2) IR-transcript expression fold change ≤ 5% upon BDNF (or bicuculline) stimulation. IR-transcripts are considered commonly regulated upon bicuculline and BDNF stimulation (black) if the following applied: (1) IR-transcript expression fold change ≥ 20% and |z-score| ≥ 1.5 upon bicuculline stimulation (2) IR-transcript expression fold change ≥ 20% and |z-score| ≥ 1.5 upon BDNF stimulation (3) IR-transcripts evolved in the same direction upon bicuculline and BDNF stimulation. IR-transcripts are considered bi-directionally regulated upon bicuculline and BDNF stimulation (brown) if the following applied: (1) IR-transcript expression fold change ≥ 20% and |z-score| ≥ 1.5 upon bicuculline stimulation (2) IR-transcript expression fold change ≥ 20% and |z-score| ≥ 1.5 upon BDNF stimulation (3) IR-transcripts evolved in opposite directions upon bicuculline and BDNF stimulation.

(C) to (F) RT-PCR validations of IR-transcripts commonly regulated (C), specifically regulated upon bicuculline stimulation (D), specifically regulated upon BDNF stimulation (E), and bi-directionally regulated (F). Expression of the IR-transcripts and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs of PIR values are shown beneath each panel. In addition, fold changes (FC) in the expression of the IR-transcripts (red and orange) and spliced isoforms (dark grey) were assessed with real-time qPCR. Means and SDs are displayed; the p-values calculated with a one-tailed paired t-test are indicated (as * when p<0.05, as ** when p<0.01, as *** when p<0.001 and as numerical values when p-value is close to the 0.05-significance threshold) (three-four independent cultures).
DGAT1 activity synchronises with mitophagy to protect cells from metabolic rewiring by iron depletion (2022)
Figure 4

Figure 4: DGAT1 activation drives lipid droplet biogenesis upon iron depletion.
a. Line diagram of temporal lipidomics workflow in human ARPE-19 cells.
b. Multivariate modelling of the lipidome in cells treated with DFP over time. Sparse PLS-DA (sPLS-DA) was performed using Metaboanalyst from LC-MS datasets from three independent biological experiments each with four technical replicates per time point in each experiment.
c. Lipidomic analysis. Hierarchical clustering heatmap analysis showing alterations in lipid species in ARPE19 cells treated with DFP for the length of time indicated. Each coloured cell on the map corresponds to a z-score value. The hierarchical clustering method of one minus Pearson correlation was applied (n=3, with 4 technical replicates per biological replicate).
d. DFP-induced Lipid droplet accumulation is dependent on DGAT1. Representative photomicrographs of ARPE19 cells treated with DFP (1mM) for 7 h followed by treatment with LD inhibitors (5 μM) as indicated. Cells were fixed after 24 h of DFP treatment. Neutral lipid droplets were stained by BODIPY493/503 and imaged by confocal microscopy after 24 h of DFP treatment ('i' denotes the presence of inhibitor e.g. DGAT1i = DGAT1-inhibition). Scale bar = 5 μm.
e. Quantitation of lipid droplets area per cell from experiments in 2d. Data are represented as mean ± SEM (n=3 with at least 80 cells analysed per condition in each biological replicate).
f. Acute inhibition of LD biosynthesis by DGAT1. Quantitation of mean LD numbers per ± SEM (n=2, with 3 technical replicates per biological experiment. Between 1000-2000 cells analysed per condition).

g. DGAT1 regulates triglyceride formation upon iron chelation. Hierarchical clustering heatmap analysis showing alterations in triglyceride lipid species in ARPE19 cells treated with DFP for 24 h and lipid droplet inhibitors (5 μM) for 17h as indicated (i denotes the presence of inhibitor e.g. DGAT1i = DGAT1-inhibition). Each coloured cell on the map corresponds to a z-score value. The hierarchical clustering method of one minus Pearson correlation was applied and statistical significance was determined by a T-test (n=3, with 5 technical replicates per biological replicate).
DGAT1 activity synchronises with mitophagy to protect cells from metabolic rewiring by iron depletion (2022)
Figure 5

Figure 5: LD biogenesis by iron depletion is autophagy-independent.
a. ARPE-19 ULK1 CRISPR KO cells with/without re-introduction of FLAG-ULK1 were treated with DFP for 24h as indicated. Cells were fixed and stained with BODIPY493/503 to visualise lipid droplets (green) or DAPI to visualise nuclei (blue). Scale bar, 10 μm.

b. Quantification from 5a, n=3 independent experiments, statical analysis by one-way ANOVA. *=P<0.05; **P=<0.01. Median represented by dashed line, quartiles by dotted lines.

c. Authentication of cell lines in 5a by immunoblot of protein lysates.

d. Representative flow cytometry assay of mito-QC ARPE-19 ULK1 CRISPR KO cells with/without re-introduction of FLAG-ULK1, treated with DFP for 24 h. A decrease/increase in GFP/mCherry expression was used quantified to measure the percent of cells undergoing mitophagy (indicated by number in each panel).

e. Quantitation of the flow data representing mean of n=2 ± SEM. Statistical analysis was performed with one-way ANOVA and a Tukey's multiple comparisons test. ****=P< 0.0001.