EMBO/soda-nel huggingface dataset:
An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2 (2020)
Figure 1

Fig 1. Allelic imbalance of heterozygous FOXL2 transcripts in AGCT cells

A Bar graph and box-and-whisker plots are presented, which show the allelic proportions of WT FOXL2 mRNA and 402C>G FOXL2 mRNA in AGCT tissues from 46 patients analyzed by pyrosequencing. The box plot represents the minimum value, first quartile, median, third quartile and maximum value of a data set. X-axis indicates mRNAs of WT FOXL2 and 402C>G FOXL2. The whiskers extend to the most extreme data points not considered outliers, and the outliers are represented as dots. Comparisons between groups were performed using Student's t-test, and p values are presented.

B The relative abundances of WT and variant FOXL2 mRNA were analyzed in KGN and COV434 cells by pyrosequencing (left graph), allele-specific RT-PCR (middle graph) and real-time RT-PCR (right graph). gDNA was detected as a positive control. The relative abundances of the variant FOXL2 mRNA were normalized to that of WT mRNA (set to 1). FOXL2 mRNA levels detected by real-time RT-PCR were normalized to matching gDNA levels. The pyrosequencing data are presented from two independent experiments. The allele-specific semi-quantitative and real-time RT-PCR data are presented as the mean ± SEM from three independent experiments. The p values were analyzed by unpaired, two-tailed Student's t-test (***p < 0.001). n.d. not detected.

C RNA-decay rates of WT and 402C>G FOXL2 mRNAs in KGN cells were determined after treatment with 5 µg/mL ActD for the indicated times. The estimated half-lives of each transcript are presented. The data are presented as the mean ± SEM from three independent experiments.
An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2 (2020)
Figure 2

Fig 2. Selective downregulation of the variant FOXL2 mRNA allele by miR-1236

A, B Changes in WT and variant FOXL2 mRNA expression were assessed by RT-PCR (top) and real-time RT-PCR (bottom) (A) or by western blot analysis (B) after transfecting KGN cells with anti-miRNAs for 48 h.

C FOXL2 protein levels after transfection of control or miR-1236 were assessed in KGN and COV434 cells.

D The mRNA levels of WT and variant FOXL2 in control, miR-1236-/+, and miR-1236-/- KGN cells, with or without miR-1236 transfection, were determined by RT-PCR (top) or real-time RT-PCR (bottom).

E FOXL2 protein expression in control, miR-1236-/+, and miR-1236-/- KGN cells, and two independent miR-1236-/- (#1 and #2) COV434 cell lines were determined by western blotting.

Data information: Representative gel images are also shown. All quantified results (mean ± SEM) are from at least three independent experiments. Different letters (p < 0.0001; Student-Newman-Keuls test) or asterisks (*p < 0.05; Student's t-test) denote significant differences.
An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2 (2020)
Figure 3

Fig 3. Allele-specific downregulation of FOXL2 mRNA by miR-1236, which targets 402C>G

A Schematic representation of the luciferase-reporter constructs used to assay miR-1236 activity against a CDS target site in FOXL2 mRNA. The 231-bp human FOXL2 segments harboring either the C402 (WT) or the G402 (mutant) nucleotide were inserted in-frame into the CDS of the luciferase gene in the pGL3 control vector.

B Luciferase activity of the reporter constructs shown in (A) were measured in KGN cells after transfection with an miR-1236 mimic for 48 h. The black arrow indicates the position of 402C>G mutation site.

C A schematic diagram of the luciferase-reporter constructs generated by inserting the predicted miR-1236-target sequences of WT and 402C>G FOXL2 mRNAs in the 3′-UTR of luciferase.

D Luciferase activities were measured in KGN cells, using the reporter constructs shown in (C), after transfection with a control miRNA or an miR-1236 mimic for 48 h.

E, F miR-1236 mutants, in which the C that pairs with G402 of the FOXL2 mutant was substituted with either G (miR-1236-G) (E) or U (miR-1236-U) (F), were cotransfected into KGN cells with one of the reporter constructs described above. Luciferase activities were subsequently determined. Arrows indicate the mismatched sites.

G In vitro annealing kinetics of miR-1236 with 230 nt-long transcripts of WT or variant FOXL2. 32P-labeled miR-1236 (0.5 nM) was incubated with increasing concentrations of synthetic FOXL2 transcripts (0, 2.5, 12.5, 25, or 50 nM). FOXL2 mRNA-miR-1236 complexes were resolved on a 6% native gel and detected by autoradiography (left). The predicted Kds for the WT and 402C>G FOXL2 transcripts are presented in the right graph.

Data information: The data are expressed as the mean ± SEM from three independent experiments, performed in triplicate. The p values were analyzed by unpaired, two-tailed Student's t-test (***p < 0.001).
An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2 (2020)
Figure 4

Fig 4. Identification of AGO3 as the major miRISC regulator for variant FOXL2 mRNA degradation

A, B Changes in WT and variant FOXL2 mRNA-expression levels were assessed by real-time RT-PCR (A) or western blot analysis (B) after transfecting KGN cells with siRNAs against AGO mRNAs for 48 h. The data (mean ± SEM) are from three independent experiments, performed in triplicate.

C The mRNA levels of WT (left) and variant FOXL2 (right) were determined in KGN cells by real-time RT-PCR, after transfecting a control miRNA or miR-1236. The data (mean ± SEM) are from three independent experiments, performed in triplicate.

D The mRNA levels of WT and the variant FOXL2 in control (left) and miR-1236-/- KGN cells (right) after transfecting siRNAs against AGO mRNAs were determined by allele-specific real-time RT-PCR. The data (mean ± SEM) are from three independent experiments, performed in triplicate.

E 293T cells were transfected with an miR-1236 mimic (50 nM) for 24 h, followed by cotransfection with expression vectors encoding FLAG/HA-tagged variants of the indicated human AGOs and pGL3c-CDS-MT for 24 h. The empty p3XFLAG-CMV-10 vector was used as control. Co-immunoprecipitated mRNAs were reverse transcribed, and the cDNA products were used for allele-specific real-time PCR analysis of the FOXL2 variant and GAPDH mRNAs (top). The level of variant FOXL2 mRNA immunoprecipitated using FLAG-tagged AGO proteins was normalized using the level of GAPDH mRNA from the same lysates. The immunoprecipitated-AGO proteins were detected by western blotting (bottom). The data (mean ± SEM) are from three independent experiments.

F In vivo association of AGO3-mediated miRISC formation with FOXL2 mRNAs are shown. Following transfection of a control miRNA or the miR-1236-G mutant into KGN cells, AGO3-mediated RISC-associated RNAs were isolated by immunoprecipitation with an anti-AGO3 antibody. IgG was used as a control. The co-immunoprecipitated mRNAs were reverse transcribed using a FOXL2-430-R primer binding downstream of the 402C>G site. The cDNA products were used for FOXL2 allele-specific PCR analysis with the FOXL2-279F primer (Appendix Fig. S1), and a representative result (top left) is shown. Quantitative real-time RT-PCR results that examined FOXL2 mRNAs, normalized using the level of GAPDH mRNA (bottom left), are also presented. Western blot analysis of immunoprecipitated AGO3 and inputs are shown in the right panel. The data are presented as the mean ± SEM of two independent experiments.

G RNA-seq analysis was performed to determine AGO-expression levels (transcripts per million) from the individual tissues from 20 independent AGCT patients. X-axis represents mRNAs of AGO1 to 4. The box plot represents the minimum value, first quartile, median, third quartile and maximum value of a data set. The whiskers extend to the most extreme data points not considered outliers, and the outliers are represented as dots.

Data information: Different letters (p < 0.05; Student-Newman-Keuls test) or asterisks (***p < 0.001) denote statistically significant differences.
An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2 (2020)
Figure 6

Fig 6. FOXL2-mediated oncogenic effects of miR-1236 on cell death, cell cycle progression, and cell migration.

A-D KGN cells were transfected with 200 nM of scrambled control or FOXL2-specific siRNAs for 24 h. Then, KGN cells were further transfected with 20 nM of control miRNA, miR-1236, control anti-miRNA, or anti-miR-1236. The proportion of annexin-V-positive apoptotic cells (A and B) and the population at S phase (C and D) were analyzed by flow cytometry. The data are presented as the mean ± SEM of three independent experiments. Different letters denote statistically significant differences (p < 0.0001; Student-Newman-Keuls test). Efficient silencing of FOXL2 using specific siRNAs was confirmed by western blotting.

E, F KGN cells were transfected with control siRNA or siFOXL2 for 24 h. Then, KGN cells were further transfected with control miRNA or miR-1236 (E) or with control anti-miRNA or anti-miR-1236 (F) for 48 h, and Transwell-migration assays were performed. The migrated cells were imaged under a bright-field microscope (100 × magnification, scale bar = 100 µm). The results are from three independent experiments and represent fold-changes in the average number of cells/field (mean ± SEM). Different letters denote statistically significant differences (p < 0.01; Student-Newman-Keuls test).

G, H COV434 cells were transfected with miR-control or miR-1236 for 48 h, after which cell viabilities (G) and migration abilities (H) were measured. The data are presented as the mean ± SEM of three independent experiments. Immunoblots showing no change in FOXL2 protein are presented in the top panel, and images of migrated cells are presented in the right panel. The migrated cells were imaged under a bright-field microscope (100 × magnification, scale bar = 100 µm).

I-K The properties of miR-1236-/+ and miR-1236-/- KGN cells versus control KGN cells were assessed by measuring cell viability (I), cell proliferation (J), and cell migration (K). The data are presented as the mean ± SEM of three independent experiments. The migrated cells were imaged under a bright-field microscope (100 × magnification; top of Fig. 6K). Scale bar = 100 µm.

L No difference in the cell-migration activities of control and two independent miR-1236-/- (#1 and #2) COV434 cell lines. The migrated cells were imaged under a bright-field microscope (100× magnification, scale bar = 100 µm; top) and the results (bottom) represent fold-changes in the average number of cells/field. The data are presented as the mean ± SEM of three independent experiments.

Data information: Different letters denote statistically significant differences (p < 0.0001; Student-Newman-Keuls test).
An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2 (2020)
Figure 7

Fig 7. Inhibition of in vivo metastasis by miR-1236 KO and inverse expression between miR-1236 and the variant FOXL2 mRNA in tissues from AGCT patients

A-C The effect of miR-1236 KO on AGCT metastasis was assessed, using an in vivo xenograft mice model. (A) Representative images of tumor nodules (white arrows) formed in the intestines of nude mice xenografted with control or miR-1236-/- KGN cells are shown (left and right; scale bar = 5 mm). Hematoxylin and eosin staining confirmed the pathological characteristics of the metastasized GCT nodules (middle, black arrows; scale bar = 50 µm). The black dashed circles are metastasized nodules of xenografted KGN cells found in mouse intestines. (B) The number of tumor nodules formed in the intestines was counted in control (n = 8) and miR-1236-/- (n = 8) mice. (C) Allele-specific real-time RT-PCR analysis of the WT and 402C>G variant FOXL2 mRNAs was performed using RNA extracted from tumor nodules from control or miR-1236-/- mice. The p values were analysed by unpaired, two-tailed Student's t-test (**p < 0.01, ***p < 0.001).

D-F Box-and-whisker plots showing the relative expression of miR-1236 (D), variant FOXL2 mRNA (E), and WT FOXL2 mRNA (F), respectively, in 32 patients with non-metastasized AGCTs and 14 patients with metastasized AGCTs. X- axis indicates patient subgroups depending on whether they exhibit metastasized AGCTs (meta) or not (non-meta). The relative miR-1236 levels were measured using a TaqMan® microRNA RT-qPCR assay, with expression normalized to RNU6B. The levels of 402C>G or WT FOXL2 mRNA were determined by real-time RT-PCR, and the data were normalized to paired-gDNA levels. The relative levels of miR-1236 and FOXL2 mRNAs were quantified by setting the levels of AGCT #1 to 1. Real-time RT-PCR was performed in triplicate for each specimen. The box plot represents the minimum value, first quartile, median, third quartile and maximum value of a data set. The whiskers extend to the most extreme data points not considered outliers, and the outliers are represented as dots. Comparisons between groups were performed using Student's t-tests, and p values are presented.

G, H The estimated regression line superimposed on the scatter plot of miR-1236 levels with 402C>G FOXL2 mRNA (G) or WT FOXL2 mRNA (H) in AGCT samples (n = 46) is shown, along with correlation coefficient (r) and p values.

I The proposed model for FOXL2 haploinsufficiency induced by the 402C>G mutation during AGCT development.
Class IA PI3Ks regulate subcellular and functional dynamics of IDO1 (2020)
Figure 1

Figure 1. Class I PI3K p110 activity is required for IDO1 signaling and immunosuppressive effects in pDCs.

A Expression of transcripts coding for PI3K subunit genes in pDCs (n =12 biological replicates). In the boxplot, the middle, the bottom, and the top of the box represent the median, the 1st quantile and the 3rd quantile, respectively; the height of the box is given by the difference between the 3rd and the 1st quantiles (IQR, interquartile range); the whiskers length is 1.5 times the interquartile range. Data points beyond the whiskers represent values more extreme than 1.5 times the interquartile range above or below the 3rd or the 1st quantile, respectively.

B Selectivity of PI3K inhibitors used in this study.

C Kyn levels in culture supernatants of pDCs incubated for 24 h with medium alone (Ctrl) or IFN-γ after 1−h pretreatment with PI3K inhibitors.

D Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs stimulated for 18 h with TGF-β, in the presence or absence (Ctrl, control) of catalytic PI3K inhibitors, normalized to the expression of Gapdh and presented relative to results in untreated cells (dotted line, 1-fold).

E In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs conditioned in vitro with TGF-β (or medium) for 24 h in the presence or absence (Ctrl) of catalytic inhibitors as in C. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).

F Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs transfected with p110α-, β-, δ-specific, or negative control (nc) siRNA and stimulated for 18 h with TGF-β, normalized to the expression of Gapdh and presented relative to results in unstimulated cells (dotted line, 1-fold).

G In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs transfected with siRNA as in (F) and conditioned in vitro with TGF-β (or medium) for 24 h. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).

Data information: In C-G, one representative experiment of three is shown. Data represent the mean ± SD (n=3 technical replicates). Statistical analysis was performed using two-tailed unpaired Student's t-test (C) or ANOVA followed by post-hoc Bonferroni's test (D and F) or paired Student's t test (E and G). *P < 0.05; **P < 0.01; ***P < 0.001.
Class IA PI3Ks regulate subcellular and functional dynamics of IDO1 (2020)
Figure 2

Figure 2. IDO1 contains a YENM motif that binds the PI3K p85 subunit.

A Alignment of the amino acid sequences of IDOs restricted to the stretch of amino acids containing the YxxM motif (where x is any amino acid). Residues are highlighted based on increasing conservation level of symbols in the multiple alignment (light gray ≥ 44.4%; dark grey ≥ 66.6%; black ≥ 88.8%). IDOs, homologues of IDO1. Proto-IDOs, homologues of IDO2. Accession number for each specific gene is indicated.

B Cartoon model of human IDO1 (gray) and the SH2 domain of PI3K p85 (orange) complex as resulting from docking study. Residues of the YENM motif are colored in yellow and shown in sticks. Residues of phosphotyrosine binding site in p85 are colored in cyan and shown in sticks. Heme and l-tryptophan are shown in sticks and colored in red and green, respectively.

C Pull-down (PD) assay of lysates from P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated IDO1 peptides (pYENM, pITIM1, and pITIM2), analyzed by sequential immunoblotting with anti−p85, −SHP-1, and -SHP-2 antibodies.

D Immunoblotting with anti-IDO1 and -p85 antibodies of IDO1 immunoprecipitates obtained from P1.HTR cells transfected with IDO1.WT or IDO1.Y149F. Mock-transfected P1.HTR cells were used as a control.

E Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from lysates of P1.HTR transfectants as in D.

F Kyn release over 24 h by P1.HTR transfectants as in D.

Data information: One of two (C and D) and three (E and F) representative experiments is shown. Data represent the mean ± SD (n = 3 technical replicates). Statistical analysis was performed using ANOVA followed by post-hoc Bonferroni's test. **P < 0.01.
Class IA PI3Ks regulate subcellular and functional dynamics of IDO1 (2020)
Figure 3

Figure 3. IDO1 colocalizes with EE in tumor transfectants.

A Confocal immunofluorescence microscopy images of P1.HTR cells either untransfected (Untr) or transfected with IDO1.WT (WT) or vector alone (Mock) and stained with antibodies recognizing IDO1 (green). Nuclei were stained with DAPI (blue). Scale bar, 20 µm.

B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT, co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (as indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.

C Immunoblotting analysis of EE purified from P1.IDO1.WT cells. Mock-transfected cells as in A were used as control.

D Distribution of the IDO1.WT protein expressed by P1.HTR transfected cells in isopycnic sucrose gradients. Fractions were immunoblotted using antibodies specific for IDO1 and intracellular organelles or structures as indicated.

Data information: In all panels, one of three representative experiments is shown.
Class IA PI3Ks regulate subcellular and functional dynamics of IDO1 (2020)
Figure 4

Figure 4. The YENM Motif is required for IDO1 EE localization in tumor transfectants

A Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT or IDO1.Y149F and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.

B Pearson's coefficient of IDO1 colocalization with EEA1 in P1.HTR cells transfected as in A, with each point representing colocalization within an image stack. Data represent mean ± SD (n > 24 cells from three independent experiments). Statistical analysis was performed using two-tailed unpaired Student's t-test. ***P < 0.001.

C Distribution of IDO1.WT and the IDO1.Y149F mutants in homogenates of P1.HTR cells along the isopycnic sucrose gradient as in Fig 3D. Fractions were immunoblotted using antibodies specific for IDO1.

D The distribution of IDO1.WT and IDO1.Y149F proteins along the isopycnic sucrose gradient is shown in term of densitometric analysis in each fraction (mean ± SD of three experiments).
Class IA PI3Ks regulate subcellular and functional dynamics of IDO1 (2020)
Figure 5

Figure 5. Class IA PI3K p110 activity is required for IDO1 signaling and EE localization in tumor transfectants

A Pull-down assay of lysates from untransfected P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated (pYENM) IDO1 peptides, analyzed by immunoblotting with anti-p85, -p110α, -p110β, -p110γ, and -p110δ antibodies. Whole cell lysates (WCL) were used as control of lysate amount.

B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT incubated with class IA PI3K p110 isoform inhibitors (indicated) or medium alone (Ctrl) for 24 h and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.

C Pearson's coefficient of IDO1 colocalization with EEA1 in P1.HTR cells transfected and treated as in (B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from two independent experiments).

D Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from P1.HTR cells transfected with IDO1.WT cells and treated with PI3K catalytic inhibitors (indicated) or medium alone (ctrl). Data represent the mean ± SD (n = 3 technical replicates).

Data information: One of two (A) and three (B and D) representative experiments is shown. Statistical analysis was performed using ANOVA followed by post-hoc Bonferroni's test.

***P < 0.001.
Class IA PI3Ks regulate subcellular and functional dynamics of IDO1 (2020)
Figure 6

Figure 6. The signaling but not catalytic form of IDO1 colocalizes with EE in pDCs

A Confocal immunofluorescence microscopy images of freshly purified pDCs co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 5 µm.

B Confocal immunofluorescence microscopy images of pDCs incubated for 24 h with TGF-β or IFN-γ and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). Scale bar, 5 µm.

C Pearson's coefficient of IDO1 colocalization with EEA1 in pDCs either untreated or stimulated with TGF-β or IFN-γ (as in B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from three independent experiments).

Data information: In A and B, one representative experiment of three is shown. Statistical analysis was performed using ANOVA followed by post-hoc Bonferroni's test. *P < 0.05; **P < 0.01.