EMBO/soda-nel huggingface dataset:
Iron overload inhibits late stage autophagic flux leading to insulin resistance (2019)
Figure 1

Figure 1. Validation of Iron overload (IO) model and insulin resistance induction after IO in L6 muscle cells

A Ferrozine-based intracellular iron concentration measurement in L6 cells after temporal analysis of iron treatment at 100 or 250 µM for multiple time points. *P < 0.05 (unpaired Student's t-test versus each time points with FeSO4 100 µM).

B Representative confocal microscope images of L6 cells stained with IP-1 (Iron Probe 1) or transfected with IRE-CFP (Iron Regulatory Element) reporter after iron treatment (FeSO4, 250 µM) for 24 h.

C Representative confocal images of L6 cells using iron sensitive fluorescent PGSK dye after iron treatment (250 µM, 24 h) with iron chelator DPD (500 µM).

D Relative gene expressions - ferritin heavy chain (FTH), ferritin light chain (FTL), ferroportin (SLC40A1, transferrin receptor 1 (tfr1, TFRC) - normalized to 18sr RNA expression after iron treatment (FeSO4, 250 µM) for 24 h. *P < 0.05 (unpaired Student's t-test versus Basal).

E Representative western blot images and quantification of ferritin (heavy chain) and tfr1 (indicated with arrowhead) over GAPDH after iron treatment (250 µM, 24 h). *P < 0.05 (unpaired Student's t-test versus Basal).

F Glucose uptake of L6 cells with insulin stimulation (10 nM or 100 nM, 20 min) after iron treatment (250 µM, 1 h or 24 h). *P < 0.05 (one-way ANOVA with multiple comparisons).

G Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) over GAPDH with insulin stimulation (10 nM or 100 nM, 5 min) after iron treatment (250 µM, 24 h). *P < 0.05 (one-way ANOVA with multiple comparisons). #P < 0.05 (unpaired Student's t-test versus Iron with insulin 100 nM).

Data information: All experiments were repeated five times. Data are expressed as means ± SEM. Scale bar = 20 µm.
Iron overload inhibits late stage autophagic flux leading to insulin resistance (2019)
Figure 2

Figure 2. Iron treatment transiently induced autophagy yet led to flux inhibition at 24 h iron treatment

A Representative confocal microscope image of L6 cells stably expressing tandem fluorescent-eGFP-mCherry LC3B treated with iron (FeSO4, 250 µM) for the indicated time points (4 h, 8 h, 24 h).

B Quantification of mean LC3B puncta per cell from (A). Experiments were repeated three times, and one representative experiment is presented here. *P < 0.05 (one-way ANOVA with Dunnett's posthoc test versus Basal)

C Representative western blot images and quantification of p62 to actin in L6 cells after iron treatment (FeSO4, 250 µM) at multiple time points. *P < 0.05 (multiple unpaired Student's t-test versus Basal).

D Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 4h iron treatment stimulated with chloroquine (CQ, 30 µM). *P < 0.05 (unpaired Student's t-test versus control, basal or iron without CQ)

E Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 24 h iron treatment stimulated with CQ 30 µM. *P < 0.05 (unpaired Student's t-test versus control, basal or iron without CQ).

F Representative confocal microscope z-stack image of mCherry-LC3B L6 cells after iron treatment (250 µM, 24 h).

G Quantification of LC3B puncta size from (F). Experiments were performed three times, and one representative experiment is presented here. *P < 0.05 (unpaired Student's t-test compared to Basal).

H Quantification of autophagosomes motility from live cell microscopy (Supplementary Movie 1) from mCherry-LC3B L6 cells under basal, iron (250 µM, 24 h) and starvation (amino acid free) condition. Experiments were performed three times, and one representative experiment is presented here. *P < 0.05 (unpaired Student's t-test compared to Iron 24h).

I Representative TEM images of autophagosome and autolysosomes in L6 cells after iron treatment (250 µM, 24 h).

J Representative confocal microscope images of eGFP-mCherry-LC3B L6 cells with Lysotracker DeepRed after iron treatment (250 µM, 24 h).

Data information: Data are expressed as means ± SEM. Western blot and confocal image analysis were performed three times. Scale bar (Confocal Microscope) = 10 µm. Scale bar (Electron Microscope) = 1 µm.
Iron overload inhibits late stage autophagic flux leading to insulin resistance (2019)
Figure 3

Figure 3. Iron treatment impaired mTOR restoration following autophagosomes degradation and enforced mTOR re-activation reversed autophagy defects and insulin resistance

A Representative western blot images of phospho-ULK1 (S757), phospho-S6Kp70 (T389), total S6K, GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points.

B Quantification of mTORC1 activity analyzed through phosphorylation of S6K T389 to total S6k and ULK1 S757 to GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points. *P < 0.05 (multiple unpaired Student's t-test versus Control at each time point).

C Representative epi-immunofluorescent images of L6 cells transfected with myc-RHEB Q46L and immuno-stained against LC3B and myc after iron treatment (FeSO4, 250 µM) for 24 h.

D Ferrozine-based colorimetric measurement of intracellular iron in wild-type (wt) L6 and RHEB-Q64L L6 cells after iron treatment (50 µM or 250 µM) for 24 h.

E Representative confocal images of wt L6 and RHEB-Q64L L6 cells pulsed with Lysotracker Deep Red and immuno-stained against LC3B after iron treatment (250 µM, 24 h).

F Quantification of LC3B-free lysosome numbers in (E). Experiments were repeated three times, and one representative experiment is presented here. *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)

G Representative TEM images of wt L6 and RHEB-Q64L cells after iron treatment (250 µM, 24 h).

H-I Representative western blot images and quantifications of phospho-IRS1 (Y612, indicated by arrowhead) and AKT (T308, indicated by arrowhead) to GAPDH in wt and RHEB-Q64L L6 cells stimulated with insulin (100 nM, 5 min) after iron treatment (250 µM, 24 h). *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)

Data information: All experiments were performed three times. Data are expressed as means ± SEM. Scale bar (confocal microscope) = 10 µm, (electron microscope) = 5 µm, and (epi-immunofluorescent microscope) = 25 and 5 µm
Iron overload inhibits late stage autophagic flux leading to insulin resistance (2019)
Figure 4

Figure 4. Restoration of lysosomal pools following iron withdrawal reversed insulin resistance

A Representative confocal microscope images of L6 cells pulsed with PGSK dye after iron treatment (250 µM, 24 h) followed by 24 h iron withdrawal.

B Ferrozine based colorimetric measurement of intracellular iron in L6 cells after iron treatment (250 µM, 24 h) followed by 24 h withdrawal. *P < 0.05, #P < 0.05 (multiple unpaired Student's t-test versus Basal or Iron).

C-D Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in L6 cells iron treatment (250 µM, 24 h) followed by 24 h withdrawal. *P < 0.05, #P < 0.05 (multiple unpaired Student's t-test versus Basal or Iron).

E Representative epi-immunofluorescent microscope images of L6 cells immuno-stained against LC3B and LAMP1 after iron treatment (250 µM, 24 h) followed by 3 h withdrawal.

F Quantification of autophagosome-free lysosomes in (E). Experiments were performed three times, and one representative experiment is presented here. Red line indicates median. *P < 0.05, #P < 0.05 (multiple unpaired Student's t-test versus Basal or Iron)

G Representative western blot images and quantification of ferritin to vinculin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h or 24 h. *P < 0.05, #P < 0.05 (multiple unpaired Student's t-test versus Basal or Iron)

Data information: All experiments were performed three times. Data are expressed as means ± SEM. Scale bar = (Confocal Microscope, Fig 4A) 10 µm and (Epi-Immunofluorescent Microscope) = 20, 10 and 5 µm
Iron overload inhibits late stage autophagic flux leading to insulin resistance (2019)
Figure 5

Figure 5. Molecular mechanisms underlying IO-induced mTOR activity suppression

A Representative western blot images and quantification of phospho-mTOR S2448, total mTOR and actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h. Experiments were performed three times. *P < 0.05, #P < 0.05 (multiple unpaired Student's t-test versus Basal or Iron).

B Representative western blot images of phospho-mTOR S2448, total mTOR phsophpo-TSC2 T1462, total TSC2, phospho-AKT S473, total AKT, actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without MK2206 (AKT inhibitor.)

C Representative western blot images of phospho-UVRAG S550 and total UVRAG in L6 cells transfected with FLAG-UVRAG and FLAG pulldown after iron treatment (250 µM, 4 h and 24 h) followed by withdrawal for 1 h and 3 h.

D Representative western blot images of endogenous phospho-UVRAG S550, total UVRAG expression in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without torin1 (200 nM).

E, F Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: lysosomal number (E) and speed (F) in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM). Experiments were performed three times, and all biological and technical replicates are plotted here. Red line indicates median. *P < 0.05, #P < 0.05, $P < 0.05 (one-way ANOVA with multiple comparisons).

G Representative confocal images of time-lapse captures of lysosomes at 6 sec intervals for 30 sec. White and yellow arrows mark positions of two representative LAMP1 puncta over 30 seconds, and the traces of puncta were shown as lines in final panel.

H Schematic diagram of mTOR signaling regulation by IO.

Data information: Data are presented in Mean ± SEM. Scale bar = 10 µm.
Iron overload inhibits late stage autophagic flux leading to insulin resistance (2019)
Figure 6

Figure 6. Development of acute Iron Overload (IO) In Vivo model and validation of IO and insulin resistance in skeletal muscle

A Schematic diagram of iron-dextran injections experimental plan.

B Prussian Perl blue staining in liver and skeletal muscle after iron injections.

C-D Representative western blot images and quantification of ferritin and tfr1 (indicated by arrowhead) to tubulin in skeletal muscles 24 h after iron injections. *P < 0.05 (unpaired Student's t-test versus Control).

E Insulin tolerance test 24 h after iron injections.

F Glucose tolerance test (GTT) 24 h after iron injections.

G Quantification of area under curve in ITT (E). *P < 0.05 (unpaired Student's t-test versus Control).

H Quantification of area under curve for panel (F). *P < 0.05 (unpaired Student's t-test versus Control).

I-J Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in skeletal muscles 24 h after iron injection followed by i.p. insulin injection. *P < 0.05 (unpaired Student's t-test versus Control).

Data information: Results are presented in mean ± SEM. n = 6 males for ITT and GTT. n=3 males for western blot analysis. Scale bar = 50 µm.
Iron overload inhibits late stage autophagic flux leading to insulin resistance (2019)
Figure 7

Figure 7. Evidence of ALR defects in skeletal muscles after acute IO

A Representative confocal microscope images of skeletal muscle tissue sections immuno-stained against LC3B (Alexa 647) and LAMP1 (Alexa 555)

B Quantification of lysosomes (LAMP1 puncta) in skeletal muscles 24 h after iron injections. *P < 0.05 (unpaired Student's t-test versus Control).

C Representative TEM images of skeletal muscle 24 h after iron injections.

D Representative western blot images of phospho-UVRAG S550 and total UVRAG in skeletal muscle 24 h after iron injections.

E-F Representative western blot images and quantification of LC3-B and p62 to tubulin in skeletal muscles 24 h after iron injections. *P < 0.05 (unpaired Student's t-test versus Control).

Data information: Results are presented in mean ± SEM. n = 3 males. Scale bar (Confocal Microscope) = 20 µm. Scale bar (Electron Microscope) = 500 nm.
KRAS signaling in malignant pleural mesothelioma (2021)
Figure 1

Fig 1 - KRAS alterations in human MPM from published datasets and the cancer genome atlas (TCGA) pan-cancer MPM cohort.

A KRAS and TP53 mutation frequencies in MPM from the catalogue of somatic mutations in cancer (COSMIC) stratified by histologic subtype (n = 775 patients).

B Top 25 mutated genes from ten molecular studies of human MPM (n = 838 patients).

C-E KRAS and TP53 alterations in the cancer genome atlas (TCGA) pan-cancer MPM dataset (n = 86 patients). Shown are clinical and molecular data plot with alteration frequencies (C) and patients reclassified as KRAS- or TP53-altered (asterisks), copy number variation data summary (D), and segments of the KRAS and TP53 loci (E).

Data information: In (A), data are presented as cumulative percentages of patients tested mutant respective to patients tested for every gene. P, overall probability, two-way ANOVA. In (B), data are presented as cumulative numbers (n; numbers above bars) and percentages (%) of patients with KRAS (red bar), TP53 (blue bar), and other (grey bars) mutations. In (C), each column represents one patient and each row one clinical or molecular feature. Asterisks indicate KRAS and TP53 alterations not identified by the TCGA, but reclassified as altered in this study due to 12p gain, 17p loss, KRAS locus gain (z > 0.3), and/or TP53 locus loss (z < -0.3). In (D), data are presented as raw data points (circles), rotated kernel density distributions (violins), and patient numbers (n) between thresholds of normal (solid black line at z = 0), low amplification (dotted red line at z = 0.1), low loss (dotted blue line at z = -0.1), high amplification (solid red line at z = 0.3), and deep loss (solid blue line at z = -0.3). P, probability, paired Wilcoxon rank sum test. In (E), KRAS (red line) and TP53(blue line) loci segments of all 87 patients are shown. Each horizontal segment represents one patient. White and shades of red and blue indicate no change and magnitude of gain and loss, respectively.
KRAS signaling in malignant pleural mesothelioma (2021)
Figure 3

Fig 3 - KRAS and TP53 alterations in human MPM from Germany and human MPM cell lines from France.

A-D Pleural fluid cell pellets and supernatants from 45 patients (called ASK #) with pleural effusion from Munich, Germany (Klotz et al, 2019a and 2019b) were subjected to digital droplet polymerase chain reaction (ddPCR) for the detection of mutant (MUT) copies of KRAS codon 12/13 (KRASG12/13) and KRAS codon 61 (KRASQ61), as well as copies of TP53 and TERT. Diagnoses were benign pleural effusion (n = 5), lung adenocarcinoma (LUAD; n = 16), MPM (n = 12), as well as other extrathoracic cancers (n = 12). The assays were designed for detection of down to 1:20000 copies using EKVX (KRASWTTP53G610T), A549 (KRASG12STP53WT), NCI-H460 (KRASQ61HTP53WT), NCI-H3122 (KRASWTTP53E285V), and NCI-H3255 (KRASWTTP53G560-1A) human LUAD cells as controls. Shown are individual patient (KRAS plot) and individual sample (TP53 plot) allelic frequencies with color code and limits of normal TP53 allelic frequency as vertical dashed lines in the TP53 plot (A), representative gated dotplots of codon 12/13 KRAS ddPCR (B) and TP53/TERT (C), and results summary table (D). Any number of KRAS-mutant droplets detected in any sample (KRAS plot in A) and any patient that failed to achieve normal TP53 ploidy by any sample (TP53 plot in A) was deemed altered.

E-G Results summary (E), representative KRAS CNA segments (F), and data summary of individual cell line CNA z-score (G) from Affymetrix CytoScanHD Arrays of 33 primary MPM cell lines (called MESO #) from Nantes, France (GEO dataset GSE134349). Red lines denote the KRAS locus on chromosome 12p12.1.

H Data summary of mutant allelic frequency of KRAS compared with NF2 and BAP1 in all mutated samples from (A-G).

Data information: In (A), data are presented as data summary of the highest mutant copy percentage detected per individual sample (KRAS plot) or of all individual samples assessed (TP53 plot). In (D), data are presented as number of patients (n). P, probability, hypergeometric test for enrichment of KRAS mutations in MPM versus other tumors. In (E), data are presented as individual cell lines (columns), genes (rows), legend, and number of patients (n in table). P, probability, hypergeometric test for enrichment of KRAS mutations in TP53-mutant MPM. In (G), data are presented as raw data points (circles), rotated kernel density distribution (violins), and cell line numbers (n) outside thresholds of amplification (dotted red line at 2.3) and loss (solid blue line at 1.7). P, probability, paired Wilcoxon rank sum test. In (H), data are presented as raw data points (circles), rotated kernel density distributions (violins), and medians (lines). P, overall probability, one-way ANOVA. * and **: P < 0.05 and P < 0.01, respectively, compared with KRAS, Tukey's post-test.
KRAS signaling in malignant pleural mesothelioma (2021)
Figure 4

Fig 4 - KRAS and TP53 alterations in MPM patients from France and Turkey.

A, B Pleural fluid cell pellets and supernatants from 10 patients (called CRCINA #) with pleural effusion from Nantes, France (Gueugnon et al, 2011; Smeele et al, 2018) and pleural tumor samples from 17 patients (called TR#) with MPM from Istanbul, Turkey, were subjected to digital droplet polymerase chain reaction (ddPCR) for the detection of mutant (MUT) copies of KRAS codon 12/13 (KRASG12/13) and KRAS codon 61 (KRASQ61), as well as copies of TP53 and TERT. Diagnoses were lung adenocarcinoma (LUAD; n = 4) and MPM (n = 23). The assays were designed for detection of down to 1:20000 copies using EKVX (KRASWTTP53G610T), A549 (KRASG12STP53WT), NCI-H460 (KRASQ61HTP53WT), NCI-H3122 (KRASWTTP53E285V), and NCI-H3255 (KRASWTTP53G560-1A) human LUAD cells as controls. Shown are individual patient (KRAS plot) and individual sample (TP53 plot) allelic frequencies with color code and limits of normal TP53 allelic frequency as vertical dashed lines in the TP53 plot (A) and results summary table (B). Any number of KRAS-mutant droplets detected in any sample (KRAS plot in A) and any patient that failed to achieve normal TP53 ploidy by any sample (TP53 plot in A) was deemed altered.

Data information: In (A), data are presented as data summary of the highest mutant copy percentage detected per individual sample (KRAS plot) or of all individual samples assessed (TP53 plot). In (B), data are presented as number of patients (n). P, probability, χ2 test.
KRAS signaling in malignant pleural mesothelioma (2021)
Figure 5

Fig 5 - Human-like malignant pleural mesotheliomas and effusions of mice with pleural mesothelial-targeted oncogenic KRASG12D and/or Trp53 deletion.

Wild-type (Wt), KRASG12D, and Trp53f/f mice (all C57BL/6) were intercrossed and all possible offspring genotypes received 5 x 108 PFU intrapleural Ad-Cre [n is given in survival table in (C)].

A Representative photographs of the thorax before (top) and after (bottom) chest opening (t, tumors; l, lungs; cw, chest wall; h, heart; dashed lines, effusion; ppt, parietal pleural tumors).

B Kaplan-Meier survival plot.

C Survival table.

D Data summary of pleural effusion volume and nucleated cells [n is given in table in (C)].

E Incidence of pleural tumors and effusions.

F Representative May-Gruenwald-Giemsa-stained pleural fluid cytocentrifugal specimen from a KRASG12D;Trp53f/f mouse showing macrophages (MΦ, black arrow), lymphocytes (LΦ, purple arrow), and neutrophils (NΦ, green arrow) and summary of cellular and biochemical features of effusions of KRASG12D;Trp53f/f mice (n = 10).

G Gross macroscopic and microscopic images of visceral and parietal tumors stained with hematoxylin and eosin or PCNA [n is given in table in (E)].

Data information: In (B) and (C), data are presented as Kaplan-Meier survival estimates (lines), censored observations (line marks) 95% confidence interval (shaded areas) and number of mice at risk. P, overall probability, log-rank test. ** and ***: P < 0.01 and P < 0.001, respectively, for the comparisons indicated, log-rank test. In (D), data are presented as raw data points (circles), rotated kernel density distribution (violins), and medians (lines). P, overall probability, one-way ANOVA. ****: P < 0.0001, for comparison with all other groups, Bonferroni post-tests. In (E), data are presented as number of mice (n). P, probability for comparison with the top-three groups, Fischer's exact test. In (F), data are presented as mean ± 95% confidence interval. Wt, wild-type; KRASG12D, Lox-STOP-Lox.KRASG12D; Trp53f/f, conditional Trp53-deleted; Ad, adenovirus type 5; PFU, plaque-forming units; Cre, CRE recombinase gene; PCNA, proliferating cell nuclear antigen; LDH, lactate dehydrogenase; ANOVA, analysis of variance; VEGF, vascular endothelial growth factor.
KRAS signaling in malignant pleural mesothelioma (2021)
Figure 6

Fig 6 - Molecular phenotyping of murine mesothelioma.

Wild-type (Wt), KRASG12D, and Trp53f/f mice were intercrossed, and all possible offspring genotypes received 5 x 108 PFU intrapleural or intratracheal Ad-Cre and were sacrificed when moribund. In parallel, C57BL/6 mice received ten consecutive weekly intraperitoneal injections of 1 g/Kg urethane and were sacrificed after six months. Data summary (heatmap) and representative images of immunoreactivity of tissue sections of pleural and pulmonary tissues and tumors from these mice for different markers of human malignant pleural mesothelioma (MPM) and lung adenocarcinoma (LUAD). n = 10 mice/group were analyzed for each marker. Brown color indicates immunoreactivity and blue color nuclear hematoxylin counterstaining. Note the ubiquitous strong expression of Wilms' tumor 1 (WT1), patchy moderate expression of vimentin (VIM), ubiquitous moderate expression of mesothelin (MSLN), ubiquitous strong expression of calretinin (CALB2), podoplanin (PDPN), and osteopontin (SPP1), patchy moderate expression of cytokeratin 5/6 (CK5/6), and the absence of expression of surfactant protein C (SFTPC) in murine KRAS-driven mesotheliomas. Note also the ubiquitous strong expression of WT1, the patchy moderate expression of VIM, the ubiquitous low-level expression of MSLN, the ubiquitous strong expression of CALB2 and SPP1, the ubiquitous low-level expression of PDPN, the variable moderate expression of CK5/6, and the ubiquitous moderate expression of SFTPC in murine KRASG12D-driven and urethane-induced LUAD.