EMBO/soda-nel huggingface dataset:
MiR-182-3p targets TRF2 and impairs tumor growth of triple negative breast cancer (2022)
Figure 6

Figure 6. miR-182-3p impairs tumor growth by targeting TRF2 in PDTX-derived tumor cells (PDTCs) and patient-derived tumor xenografts (PDTXs)

A,B. PDTCs #1 and #2 underwent two rounds of transfection with miR-Control or miR-182-3p. Three days after the second transfection, miR-182-3p and TRF2 expression were analysed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control.

C,D. Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50μm. At least 85 3D cells were analysed for each experimental condition.

E. NSG mice implanted with breast PDTX (#2) were treated with LNPs-empty, LNPs-miR-Control or LNPs-miR-182-3p as indicated in the scheduling. Caliper measurement of tumors were taken at the indicated days. The mean of tumor volumes (n=5 per group) is shown.

F. miR-182-3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR.

G. Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in E. Scale bar: 50μm.

H. The histograms show: the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and Tunel. The analysis was performed on 3 mice per group, the points represent the number of field analysed for each condition.

Data information: For A-F and H, data are shown as mean ± SD. For A-F, P values are determined by unpaired two-tailed t test; for (H), P values are determined by Mann-Whitney test. For the experiments showed in A, B and C, D two or three biological replicates were performed, respectively.
MiR-182-3p targets TRF2 and impairs tumor growth of triple negative breast cancer (2022)
Figure 7

Figure 7. LNPs-miR-182-3p treatment does not cause toxicity or DNA damage in proliferative organs

A. Representative images of intestine sections from mice previously treated with LNPs-Empty or LNPs-miR-182-3p. H&E staining (scale bar: 200μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50μm).

B,C. Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX positive cells (%) (C) on intestine samples.

D. Representative H&E (scale bar: 200μm), TRF2 and γH2AX images of skin samples corresponding to LNPs-Empty or LNPs-miR-182-3p treated animals (scale bar: 50μm).

E,F. Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX positive cells (%) (F) on skin samples.

G. Representative H&E (scale bar: 200μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs-Empty or LNPs-miR-182-3p treated animals (scale bar: 50μm).

H,I. Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX positive cells (%) (I) on bone marrow samples.

Data information: For B,C,E,F,H,I, data are shown as mean ± SD. A Mann-Whitney test t test was used to calculate statistical significance. Four mice per group were analysed the points represent the number of field analysed for each condition.
Metabolic resistance to the inhibition of mitochondrial transcription revealed by CRISPR-Cas9 screen (2021)
Figure 1

Figure 1 - Whole-genome CRISPR-Cas9 genetic screen reveals mechanisms of resistance to IMT1.

A Dose-response viability curves in presence of increasing concentrations of IMT1 for one-week, viable cells were counted and normalized to DMSO-treated controls. Data are expressed as mean ± SD of n=3 independent experiments with four technical replicates. IC50 values were calculated with non-linear least square fit, RKO: 521.8 nM, MiaPaCa-2: 291.4 nM and HeLa: 29.9 nM.

B Mitochondrial transcript levels measured by qRT-PCR in RKO cells after 96 hours of 1 μM IMT1 treatment. Data are relative to DMSO-treated controls and are expressed as mean ± SD of n=4 independent experiments. Ordinary one-way ANOVA was used for comparisons to DMSO-treated controls (RNR1, RNR2, MT-COX2, MT-ATP6, MT-ND1: ***p<0,0001).

C Representative western blot images of OXPHOS protein levels of RKO cells at increasing concentrations of IMT1. ACTINB is showed as loading control.

D Volcano plot showing proteomic changes of RKO cells treated with 1 μM IMT1 for 96 hours. Data are plotted as average log2-fold change versus log10 of adjusted p-value of DMSO-treated controls; mitochondrial protein are highlighted in red.

E Schematic description of the experimental CRISPR-Cas9 approach: Cas-9 expressing-RKO cells were transduced with a genome-wide pooled knockout CRISPR library and passaged in the presence of either IMT1 or DMSO for guide selection. After cell collection and DNA isolation, the guides were sequenced and quantified in each population. Created with www.BioRender.com.

F CRISPR guides enriched (positive hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value. mTORC1 and VHL pathway-related genes are reported in green and blue, respectively.
Metabolic resistance to the inhibition of mitochondrial transcription revealed by CRISPR-Cas9 screen (2021)
Figure 4

Figure 4 - Chronic dose-escalated IMT1 treatment confers resistance to RKO cells through an upregulation of mitochondrial function.

A Differences in sensitivity to increasing IMT1 doses between RKO cells and resistant RKO cells. The graph shows mean values ± SEM of n=3 independent experiments.

B Western blot analyses of OXPHOS protein steady state levels at increasing concentrations of IMT1 of RKO (left) and IMT1-resistant RKO cells (right). The samples were collected and analyzed after eight weeks of chronic IMT1 treatment of the resistant line; GAPDH is shown as loading control.

C 35S-labelling of newly synthesized mitochondrial-encoded proteins in RKO and IMT1-resistant RKO cells treated either with DMSO or with 1 μM IMT1 for four days. Left: pulse labelling, right: 24 hours chase in normal medium. Putative mitochondrial proteins are reported on the right-hand side of the gel, molecular weights on the left. Coomassie staining shows protein loading.

D Densiometric quantification ±SD of ND5, COX1, ND4 band from n=4 independent experiments. Statistical significance was calculated with paired t-test: RKO + IMT1 vs resistant + IMT1, pulse: *p=0,0139, chase: *p=0,0368.

E qRT-PCR relative quantification of mitochondrial transcript levels of RKO and IMT1-resistant RKO cells treated with DMSO or IMT1 for 96 hours. Data represent the mean values ± SEM of n=5 experiments. Statistical significance was calculated with one-way ANOVA test. RKO + IMT1 vs Resistant + IMT1: MT-ND1 **p=0,0092, MT-RNR1 *p=0,0112, MT-ATP6 **p= 0,0022.

F Northern blot analyses of mitochondrial transcripts of RKO cells treated with DMSO (RKO, "IMT1 -"), IMT1 (RKO "+ IMT1") and IMT1-resistant cells (Res, "IMT1 +"). n=3 independent experiments (Repl. 1, 2 and 3) were probed for COX2, ATP6, 16S and 12S mtRNA; the nuclear-encoded 18S RNA is shown as loading control.

G Relative mtDNA copy number measured in RKO and IMT1-resistant cells treated with IMT1 for 96 hours. Data are expressed as average from n=3 experiments ± SEM. Statistical significance was calculated with one-way ANOVA test. RKO + IMT1 vs Resistant + IMT1: MT-ND1 **p=0,0035, MT-ATP6 ***p=0,0002, MT-CYTB **p=0,0028.

H Cellular OCR measured by Seahorse extracellular flux analysis after sequential addition of different modulators of mitochondrial function in RKO and IMT1-resistant RKO cells treated with DMSO or IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. RKO DMSO vs RKO + IMT1: p=0,0008; RKO DMSO vs resistant DMSO: p=0,0343; RKO DMSO vs resistant + IMT1: p= 0,0092. Paired t-test of RKO + IMT1 vs resistant + IMT1: p= 0,135.
Metabolic resistance to the inhibition of mitochondrial transcription revealed by CRISPR-Cas9 screen (2021)
Figure 7

Figure 7 - Inhibition of mitochondrial translation and decrease in mtDNA copy number sensitize resistant cells to IMT1 treatment.

A Depleted CRISPR guides (negative hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value.

B Viability assessment of monolayer cultures of IMT1-resistant RKO cells treated for one-week with either DMSO, IMT1 (1 μM), CAP (1 μg/ml) alone or in combination with IMT1. Data are expressed as mean values ± SD of n=4 independent experiments each including four technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. IMT1 vs IMT1+ CAP: *p=0,0301.

C Representative images and related plot of IMT1-resistant RKO spheroid areas in presence of the aforementioned compounds for two weeks (IMT1: 1 μM, CAP: 1 μg/ml); Scale bar: 1mm. Data show the mean values ± SD of n=4 experiments. Statistical significance was calculated with paired t-test: IMT1 vs IMT1+ CAP: *p=0,0242.

D Log2 fold changes in viable cell count in a panel of five IMT1-resistant cancer cell lines in presence of IMT1 + CAP (1 μg/ml). Data represent mean values of n=5 (resistant RKO cells) and n=3 (PANC1, Capan2, HCT-29 and Calu6) ±SD independent experiments. Paired t-test of IMT1 vs IMT1+CAP: resistant RKO cells **p=0,0014; PANC1, CAPAN2, HCT-29 and Calu-6: non-significant.

E Western blot analyses of TFAM protein steady state levels in IMT1-resistant RKO cells in presence siRNAs against controls (control #1, #2) and TFAM (TFAM #1, TFAM #2) and treated with DMSO or IMT1. Images are representative of n=3 independent experiments. ACTINB is showed as loading control.

F mtDNA levels in IMT1-treated resistant RKO cells in presence of siRNA against controls (control #1, #2) or TFAM (TFAM #1, TFAM #2). Data are expressed as relative to DMSO-treated resistant RKO cells treated with controls siRNAs (dotted line = 1) and show the average of n=2 independent experiments.

G Viable cells counts of IMT1-resistant RKO cells treated for one-week with either DMSO or IMT1 in presence of controls (control #1, #2) or TFAM (TFAM #1, TFAM #2) siRNAs. Data are expressed as mean values ± SD of n=5 independent experiments each including six technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Controls + IMT1 vs TFAM #1 + IMT1: p= 0,2148; Controls + IMT1 vs TFAM #2 + IMT1: *p=0,0415.

EV Figure legend
EGFR-mediated tyrosine phosphorylation of STING determines its trafficking route and cellular innate immunity functions (2020)
Figure 1

Figure 1: STING-mediated induction of a subset of genes needs EGFR kinase activity.

(A) Raw 264.7 cells were pre-treated with DMSO or the EGFR inhibitor gefitinib (10 µM) before cGAMP transfection; after 5h, IFNβ mRNA was measured by qRT-PCR (n=3). Bars are the mean ± SD of indicated (n) independent experiments. ***P < 0.001 when compared to gefitinib pretreated group by two-tailed Student's t-test.

(B) WT and EGFR knockdown Raw 264.7 cells were transfected with cGAMP; 4.5h post treatment, IFNβ mRNA was measured (n=3). Bars are the mean ± SD of indicated (n) independent experiments. ***P < 0.001 when compared to gefitinib treated by two-tailed Student's t-test.

(C) WT and EGFR-/- BMDMs were transfected with cGAMP; 5h post transfection, IFNα mRNA induction was measured (n=4). Bars are the mean ± SD of indicated (n) independent experiments. ***P < 0.001 when compared to EGFR-/- cells by two-tailed Student's t-test.

(D) WT and EGFR-/- HeLa cells were transfected with cGAMP; after 5h IFNβ mRNA was measured (n=3).

(E, F and G) MEF cells were pre-treated with gefitinib for 1h and then transfected with cGAMP; 4.5h post treatment, total RNA was harvested for analysis. (E) RNA-seq analysis (n=2). For RNA-seq analysis, poly T enriched cDNA library was prepared and 30 M single-end 50 bp reads were analyzed. The heat map for some of the genes is shown. (F) 259 mRNAs were induced by 2-fold or more upon cGAMP treatment; the induction of 86 of those genes was inhibited by gefitinib by 2-fold or more. (G) The RNA-seq analysis results were confirmed by qRT-PCR analysis of a gefitinib-sensitive and an insensitive mRNA (n=3). Bars are the mean ± SD of indicated (n) independent experiments. ***P < 0.001 when compared to gefitinib treated by two-tailed Student's t-test..

(H) MEF cells were pretreated with gefitinib for 1h and followed with cGAMP or Poly (I:C) transfection; 4.5h post treatment, total RNA was harvested and IFNα mRNA induction was measured (n=3). Bars are the mean ± SD of indicated (n) independent experiments. ***P < 0.001 when compared to gefitinib pretreated group by two-tailed Student's t-test, NS means no significance.
EGFR-mediated tyrosine phosphorylation of STING determines its trafficking route and cellular innate immunity functions (2020)
Figure 2

Figure 2: STING-mediated IFN induction and attenuation of HSV1 replication and pathogenesis require EGFR.

(A) MEF cells were pre-treated with gefitinib or DMSO for 1 hour, and then infected with HSV1 at m.o.i. of 1. 4 hours post infection (h.p.i.), IFNβ mRNA induction was monitored by qRT-PCR (n=3). Uninfected cells were used as negative control (U). Bars are the mean ± SD of indicated (n) independent experiments. ***P < 0.001 when compared to gefitinib pretreated group by two-tailed Student's t-test.

(B) DNA was extracted from gefitinib-treated or untreated infected MEFs 0 and 16 h.p.i. and viral DNA was measured by qPCR (n=3). Bars are the mean ± SD of indicated (n) independent experiments. **P < 0.01 when compared to DMSO group by two-tailed Student's t-test.

(C) WT and EGFR KO HeLa cells were infected with HSV1, RNA was extracted at 4 h.p.i. and IFNα mRNA induction was measured (n=3). Bars are the mean ± SD of indicated (n) independent experiments. **P < 0.01 when compared to EGFR-KO group by two-tailed Student's t-test.

(D) Survival of uninfected mice treated with gefitinib and HSV1-infected mice treated or untreated with gefitinib.

(E) Survival of WT and EGFRfl+/-LysMCre+/+ mice infected with HSV1.

(F) The infection was as in "E"; HSV1 titers in the brains of the live mice at day 9 were determined. Bars are the mean ± SD of indicated (n) independent experiments. ***P < 0.001 when compared to WT by two-tailed Student's t-test.
EGFR-mediated tyrosine phosphorylation of STING determines its trafficking route and cellular innate immunity functions (2020)
Figure 3

Figure 3: EGFR kinase activity is required for IRF3 activation.

(A) EGFR was not required for ligand-induced STING dimerization: MEFs were transfected with HA-tagged STING and treated with gefitinib or DMSO before cGAMP transfection, 4h later STING was immunoprecipitated with anti-HA antibodies and analyzed by Western blot.

(B) EGFR was required for the activation of IRF3, but not TBK1: MEFs were pretreated with gefitinib for 1h, then treated with cGAMP for 2h; cell lysate were subjected to Western blot analysis using the indicated antibodies.

(C) Nuclear translocation of IRF3, but not NFκB p65, required EGFR: MEFs were pre-treated with gefitinib (+) or DMSO (-) for 1 hour before transfecting cGAMP. Cells were harvested and nuclei were isolated at the indicated time points; total cell extracts (T) and nuclear extracts (N) were analyzed by Western blot.

(D) IRF3, but not TBK1, binding to STING required EGFR: MEFs were treated as in B, cell lysates were immunoprecipitated with anti-STING antibody and analyzed by Western blot.

(E) STING-IRF3 interaction in human cells required EGFR: WT and EGFR-/- (KO) HeLa cells were transfected with cGAMP, 2h later cells were lysed, immunoprecipitated with anti-STING antibody and analyzed by Western blot.

(F) STING and IRF3 co-localized in the endosomes of stimulated cells: STING-/- MEF cells were transfected with STING-HA for 24 hours then treated with cGAMP for 1h. The cells were then stained with the HA (purple), CD63 (red) and IRF3 (green) antibodies. Imaging data were analyzed by the Fuji software to reveal co-localization as white dots. Scale bars represent 10 µ m.

(G) EGFR was not required for ligand-induced phosphorylation of STING Ser366: EGFR-/- HeLa cells or gefitinib-treated and untreated WT HeLa cells were treated with cGAMP for the indicated time and cell lysates were analyzed by Western blot.

(H) Kinetics of STING and IRF3 co-localization in the endosomes of stimulated cells: STING-/- MEF cells were transfected with STING-HA for 24 hours and treated with cGAMP for the indicated time. The cells were then stained with the HA (purple), CD63 (red) and IRF3 (green) antibodies and DAPI. Imaging data were analyzed by the Fuji software to reveal co-localization as white dots. Scale bars represent 10 µ m.
EGFR-mediated tyrosine phosphorylation of STING determines its trafficking route and cellular innate immunity functions (2020)
Figure 4

Figure 4: Upon cGAMP-stimulation, EGFR binds STING and mediates its Tyr-phosphorylation.

(A) STING was Tyr-phosphorylated in cGAMP-treated cells. Raw 264.7 cells were tranfected with cGAMP; after 60 min, cell lysates were immuno-precipitated with anti-pTyr antibodies and then subjected to anti-STING Western blot.

(B) STING Tyr-phosphorylation was inhibited by gefitinib. MEF cells were pre-treated with gefitinib before cGAMP-stimulation for 60 min. Cell lysates were immuno-precipitated with anti-pTyr antibody and then analyzed by anti-STING Western blot. NTC: no treatment control.

(C) STING Tyr-phosphorylation required EGFR. Raw 264.7 cells expressing EGFR shRNA (KD) or non-target shRNA (CO) were transfected with cGAMP for 2 hours; cell lysates were analyzed by Western blot before or after immunoprecipitation with anti-pTyr antibodies.

(D) cGAMP stimulation triggered EGFR-binding to STING. Raw 264.7 cells were transfected with cGAMP for 1h; cell lysates were immunoprecipitated with anti-STING antibody and analyzed by Western blot.

(E) STING co-localized with EGFR after cGAMP stimulation. STING-/- MEF cells were transfected with STING-GFP (Green) for 24hours and stimulated with cGAMP for 1h, cells were fixed and labeled with EGFR (Red) and Calnexin (Purple) antibodies. Co-localization was analyzed by Fuji and co-localized dots are shown in white. Scale bars represent 10 µ m.

(F) STING-EGFR interaction was transient. Raw 264.7 cell lysates were prepared at indicated time points after cGAMP transfection, immunoprecipitated with anti-EGFR antibodies and subjected to Western blot.

(G) Gefitinib did not block, but prolonged STING-EGFR interaction. Raw 264.7 cells were pre-treated with gefitinib or DMSO for 1 hour before cGAMP stimulation. Cell lysates were prepared at indicated time points, immuno-precipitated with anti-STING antibody and subjected to Western blot.

(H) Poly I:C did not activate EGFR. STING-/- MEF cells were transfected with cGAMP or Poly (I:C); after 3h, p-EGFR were detected by Western blot.

(I) Kinetics of EGFR activation. Raw 264.7 cells were treated with cGAMP for the indicated time. Cell lysates were analyzed for p-EGFR via Western blot.

(J) STING-bound EGFR was phosphorylated. Raw 264.7 cells were stimulated for 1h, cell lysates were immunoprecipitated with anti-STING antibody and subjected to Western blot.

(K) Kinetics of phosphorylation. Raw 264.7 cells were treated with cGAMP for the indicated time. Cell lysates were subjected to Western blot using the indicated antibodies.

(L) Poly I:C did not trigger Tyr or Ser phosphorylation of STING. HeLa cells were transfected with cGAMP or Poly (I:C); after 3h, cell lysates were analyzed by Western blot using the indicated antibodies.
EGFR-mediated tyrosine phosphorylation of STING determines its trafficking route and cellular innate immunity functions (2020)
Figure 5

Figure 5: In the absence of EGFR, cGAMP-stimulation causes STING to translocate to autophagosomes but not to late endosomes.

(A) In the absence of EGFR, after cGAMP stimulation, STING translocated out of the ER. STING-/- MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h; cells were fixed and labeled with Calreticulin (ER marker, Red) antibody. Co-localization (Col) is indicated by white dots. Scale bars represent 10 µ m.

(B) STING was present in the ERGIC of stimulated cells in the presence or the absence of gefitinib. With the same treatment as in A, the cells were labeled with P58 (ERGIC marker, Red) antibody. Scale bars represent 10 µ m.

(C) STING translocated to the autophagosomes in the absence of EGFR. With the same treatment in A, the cells were labeled with LC3 (Autophagosome marker, Red) antibody. Scale bars represent 10 µ m.

(D) No STING was detected in the autophagosomes even after 3h after stimulation if EGFR was not inhibited. The procedures were as in C. Scale bars represent 10 µ m.

(E) In gefitinib-treated cells, STING was detected in the autophagosomes as early as 0.5h. The procedures were as in C. Scale bars represent 10 µ m.

(F) STING translocation to late endosomes required EGFR activity. STING-/- MEF cells were transfected with STING-GFP (Green) for 24hours, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time periods; cells were fixed and labeled with CD63 (late endosome marker, Red) antibody. Scale bars represent 10 µ m.
EGFR-mediated tyrosine phosphorylation of STING determines its trafficking route and cellular innate immunity functions (2020)
Figure 6

Figure 6: EGFR phosphorylates Tyr245 of STING.

(A) EGFR phosphorylates STING in vitro. Purified STING-GFP was incubated with increasing amounts of EGFR for in vitro kinase assays; the products were analyzed on phos-tag gel by Western blot using GFP antibody (upper panel). The substrate was analyzed, before the assay incubations, by the same method (lower panel).

(B) Y245F STING mutant is not a substrate of EGFR. WT and the indicated mutants of STING-GFP were purified and used as substrates for EGFR and analyzed as in A.

(C) LC-MS/MS analysis of STING identifies phosphorylated Y245. STING-GFP was purified from cGAMP-stimulated cells, subjected to protease digestion and LC-MS/MS analysis. Phosphopeptides corresponding to 239VYSNSIpYELLENGQR253 containing pY245 were identified. The MS/MS spectra for the phosphorylated peptide VYSNSIpYELLENGQR is shown. This doubly charged peptide has an observed m/z of 932.9266 Da and is within -0.96 ppm of the expected mass. The mass difference between the y8 and y9 ions is consistent with phosphorylation at Y245.

(D) Evolutionary conservation of the STING Y245.

(E) Gefitinib pretreatment inhibits phosphorylation of Y245. STING-GFP-293XL cells were pretreated with gefitinib and then treated with cGAMP. LC-MS/MS analysis was done, as above, for detecting pY245 (n=3). Bars are the mean ± SD of indicated (n) independent experiments. ***P < 0.001 when compared to gefitinib pretreated group by two-tailed Student's t-test.

(F) EGFR is required for STING Tyr phosphorylation. STING-GFP was expressed in 293XL cells in which EGFR expression had been knocked down. STING phosphorylation was analyzed by LC-MS/MS as described above (n=3). Bars are the mean ± SD of indicated (n) independent experiments. ***P < 0.001 when compared to EGFR KD group by two-tailed Student's t-test.

(G) Y245F STING mutant cannot trigger IFNβ mRNA induction. RNA was isolated from cells, expressing WT or Y245F STING, 3h after stimulation and subjected to qRT-PCR analysis (n=3). Bars are the mean ± SD of indicated (n) independent experiments. ***P < 0.001 when compared to WT by two-tailed Student's t-test.

(H) Y245F STING is phosphorylated on S366 after stimulation of cells. Phosphorylation of S366 was detected in cells expressing the WT or the mutant STING after stimulation for the indicated time.

(I) TBK1, but not IRF3, can bind to Y245F STING and be phosphorylated in cGAMP-stimulated cells. Lysates from cells expressing the WT or the mutant STING were analyzed by Western blot after (upper panel) or before (lower panel) GFP pull down.

(J) Y245F mutant induced more LC3 lipidation after 1h cGAMP treatment of 293XL cells expressing WT or mutant STING.

(K) Inhibition of EGFR by gefitinib induced more LC3II after 1.5h cGAMP treatment of L929 cells.n