EMBO/soda-nel huggingface dataset:
Bclaf1 regulates c-FLIP and protects cells from TNF induced apoptosis and tissue injury (2021)
Figure 7

Figure 7. Bclaf1 protects intestinal epithelia of mice from the TNF-induced damage.

A. Diagram showing the procedure used for siRNAs mediated knockdown of Bclaf1 in mice and mTNF treatment.

B-G. siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for Bclaf1 (B), cleaved caspase 3 (E), phospholated RIPK3 (F) and Hematoxylin-and-eosin staining (C) or western blotting analysis (G). Bclaf1 knockdown efficiency is detected by immunohistochemical analysis (B). Hematoxylin-and-eosin staining showing the morphological change (C). Villus height and crypt depth were measured (D). CC3+ and p-RIPK3+ positive cells in five fields per intestine were quantified. Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm in B, E and F; 100 μm in C.

H-K. HIEC-6 cells were transfected siCtrl or siBclaf1-1 and -2. Then the cells were treated with TNF for 24 hours were subjected to Annexin V/7AAD staining followed by flow cytometry analysis (H), or for protein extractions and western blotting analysis (I and J), or treated for indicated hours followed by total RNA extraction and RT-PCR analysis (K). Data are shown as mean ± SD. n=3 biological replicates. ****p<0.0001. One-way ANOVA test.

Expanded View Figure legends
MicroRNA-34/449 controls mitotic spindle orientation during mammalian cortex development (2016)
Figure 1

Figure 1. Screen for candidate miRNAs involved in cell division.

A. Live-cell imaging assay to detect mitotic perturbations. HeLa cells stably expressing a chromatin marker (H2B–mCherry; red) and a nuclear import substrate (IBB–eGFP; green) were imaged by automated live-cell microscopy and the duration of mitosis from prometaphase to anaphase onset was automatically determined for each dividing cell based on the time from nuclear envelope breakdown (mitotic entry) until nuclear reassembly (mitotic exit) (Schmitz et al., 2010).

B. miRNA mimic screen for miRNAs that regulate mitosis genes. miRNA-mimics from a library of all embryonic cortically expressed miRNAs were transfected individually into HeLa cells and prometaphase to anaphase onset duration was determined as in A. Individual points correspond to the mean z-score of the duration of prometaphase to anaphase onset determined in 2 independent experimental replicates for a given miRNA-mimic.

C. Cumulative histograms of mitotic progression for control cells and for cells transfected with miRNA mimics. Nuclear envelope breakdown is at t = 0 min. (n ≥ 71 in all conditions).

D. Confocal time lapse microscopy images of HeLa cells stably expressing H2B–mCherry (red) and a microtubule marker (α-Tub–eGFP; green) 48 h after transfection of miR-449a mimic, or non-targeting control siRNA. Scale bar, 10 μm.

E. Quantification of spindle rotation and duration from prometaphase to anaphase onset in time-lapse movies as in D. miR-449a mimic promotes spindle rotation. Duration from prometaphase to anaphase onset was defined as the time from nuclear envelope breakdown to anaphase onset. Spindle rotation during metaphase was measured as described in methods. Individual data points correspond to single cells (n ≥ 72 in all conditions). Normality was tested with Kolmogorov-Smirnov test. Variance between samples was tested using F test. Significance was tested by Welch’s t test: p-value = 2.598e-06 comparing spindle rotation of negative control versus miR-449a mimic-transfected cells (all data points are compared). p-value = 0.0005621 (for cells with prometaphase-anaphase onset duration lower than 60 min.
MicroRNA-34/449 controls mitotic spindle orientation during mammalian cortex development (2016)
Figure 2

Figure 2. miR-34/449 family is expressed in neural progenitors and is required for normal cortex development.

A. The expression levels of endogenous miR-34/449 family members were measured by RT-qPCR in ventricular zone samples derived by laser microdissection of mouse cortices at E14. The levels of the different miR-34/449 family members and miR-7a-1, a highly expressed miRNA relevant in cortical progenitor biology, were determined. All concentrations were normalized (norm.) using miR-7a-1 concentration (n = 8 cortices, 2 different litters). Error bars indicate standard error.

B, C. Expression analysis of miR-34/449 by in situ hybridization using locked nucleic acid (LNA) probes in wild type cortices at E14. Mature miR-449, miR-34b and miR-34c are preferentially expressed in the subventricular (SVZ) and ventricular (VZ) zones of the neocortex. Scale bar, 50 μm (A), 10 μm (B).

D, E. Brains of adult mice (P23), and quantification of brain weight. Dots indicate individual brains; red line indicates median. Mice lacking miR-449abc and miR-34bc (DKO) or miR-449abc, miR-34bc, and miR-34a (TKO) have significantly smaller brains compared to littermate controls (Het). Significance was tested by Pairwaise t-test with Bonferroni correction; Het (n = 6 brains, 2 different litters) vs. DKO (n = 4 brains, 2 different litters) p-value = 0.00215; Het (n = 6 brains, 2 different litters) vs. TKO (n = 4 brains, 2 different litters) p-value = 0.00054. Subfigure G was statistically tested as in E. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001.

F, G. Confocal images of coronal brain sections (P23) and quantification of cortex width from miR-34/449 DKO and TKO mice and littermate controls (Het). Sections were stained with DAPI. DKO or TKO mice have significantly thinner cortices compared to littermate controls (Het) in adult mice (P23). (n = 6 brains, 2 different litters) vs. DKO (n = 4 brains, 2 different litters) p-value = 0.00084, Het (n = 6 brains, 2 different liters) vs. TKO (n = 4 brains, 2 different litters) p-value = 0.00211. Scale bar, 500 μm.
MicroRNA-34/449 controls mitotic spindle orientation during mammalian cortex development (2016)
Figure 3

Figure 3. miR-34/449 family is required for timely cortical neurogenesis.

A. Confocal images of coronal sections from E16 brains of miR-34/449 KO mice and littermate controls (Het), stained with anti-Satb2-antibody to label neurons of layers II–IV (green) and DAPI to label all cell nuclei (blue). Scale bars: 50 μm.

B. Quantification of the number of Satb2-positive cells per 100 μm ventricular zone surface. Bars indicate mean ± SEM, as for all quantifications shown in this figure; n = 3 brains per genotype group, from two independent litters. Data was normalized (norm.) to the ventricular zone surface analyzed (100 µm) and relativized to the heterozygous control average value. Significance was tested by Welch’s t test: p-value = 0.01146 (Het vs. KO) (n = 4 cortices per genotype group, 2 independent litters). All subfigures were statistically tested as in B. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001.

C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:

C, D. E16 brains stained with anti-Ctip2 antibody to label V neurons (n = 4 brains per genotype group, 2 independent litters). Scale bars: 50 μm. Images in C were taken from same brain slices as shown in A. p-value = 0.03224 (Het vs. KO) (immuno co-staining of Satb2(green, A) and Ctip2(red, C)).

E, F. E14 brains stained with anti-Tbr1 antibody to label layer IV neurons p-value = 0.01392 (Het vs. KO) (n = 3 and 4 brains per genotype group from 2 independent litters). Scale bars: 50 μm.

G, H. E16 brains stained with anti-Tbr2 antibody to label intermediate progenitors. p-value = 0.01647 (Het vs. KO) (n = 7 brains for each genotype group, 5 independent litters). Scale bars: 50 μm.

I, J. E16 brains stained with anti-Pax6 antibody to label radial glia progenitors. p-value = 0.002768 (n = 5 brains per genotype group, 3 independent litters). Scale bars: 50 μm.

K. 3D reconstruction of a dividing radial glial progenitor at early anaphase. A coronal section of an E14 brain of a heterozygous control mouse was stained with anti-phospho-Vimentin antibody (red), anti-γ-tubulin antibody (green/yellow), phalloidin (magenta), and DAPI (blue) and imaged by 3D confocal microscopy. “s” indicates spindle axis, “α” indicates angle relative to ventricular surface plane, which was determined by a vector path as indicated by thin white lines located on the left side of the image. Yellow dots highlight the centrosomes of the spindle poles from the analyzed dividing cell. Scale bars: 5 μm.

L. Quantification of spindle orientation in radial glial cells as in K for E14 brains of miR-34/449 KO and littermate controls (Het). Each dot represents a single dividing cell; p-value = 3.166e-05 (Het vs. KO) (n = 131 vs. 107 cells, n = 4 brains per genotype group, 2 independent litters).
MicroRNA-34/449 controls mitotic spindle orientation during mammalian cortex development (2016)
Figure 4

Figure 4. JAM-A is expressed in radial glial cells and is a direct target of miR-34/449.

A. Quantification of mRNA expression by RT-qPCR of candidate miR-34/449 targets that might regulate spindle orientation in E14 cortex samples from miR-34/449 KO mice compared with littermate controls (Het). Expression was normlaized (norm.) to Phosphoglycerate kinase (PGK) mRNA levels. Significance was tested by Welch’s t test: p = 0.009756 (JAM-A (Het) vs. JAM-A (KO)), all the rest Het vs. KO comparisons p=n.s. (n = 4 cortices per genotype group, 2 independent litters). All subfigures were statistically tested as in A. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001. Red bar indicates median.

B. RT-qPCR quantification of JAM-A mRNA expression in HeLa cells 48 h after transfection of miR-449a mimic compared to cells transfected with non-targeting negative control siRNA. JAM-A mRNA Expression was normalized (norm.) to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. p = 0.0001218 (Neg. ctrol. vs. miR-449a mimic) (n = 9 measurements from 3 independent replicates).

C, D. mouse J110 cells were transfected with either miR-449a mimic or a negative control mimic. Whole cell extracts were subjected to immunoblot analysis to detect endogenous JAM-A and ACTIN. ACTIN was used to normalize JAM-A expression (norm.) as loading control. p = 0.04454 (Neg. ctrol. vs. miR-449a mimic) (n = 4 independent wells, 2 independent transfections).

E, F. HeLa cells were cotransfected with either miR-449a mimic or a negative control mimic, together with JAM-A-GFP fusion protein and GFP. Whole cell extracts were subjected to immunoblot analysis to detect GFP. GFP was used to normalize (norm.) by transfection efficiency as loading control. p = 0.001176 (JAM-A WT Neg. ctrol. vs. miR-449a mimic) (n = 9 independent wells, 4 independent transfections).

G, H. Confocal images of coronal sections from E14 brains of wild type mice stained with anti-JAM-A antibody (green), phalloidin to label actin (gray) and DAPI to label cell nuclei (blue). Scale bars: 10 μm (G), 5 μm (H).

I. Quantification of JAM-A protein expression in the ventricular zone of E14 brain coronal sections from miR-34/449 KO mice compared with littermate controls (Het) by immunofluorescence. ACTIN (phalloidin signal) was used to normalize (norm.) the expresion levels of JAM-A. p = 0.04842 (n = 5 (KO) or 7 (het) cortices per genotype group, 3 independent litters).
MicroRNA-34/449 controls mitotic spindle orientation during mammalian cortex development (2016)
Figure 5

Figure 5. JAM-A is required for normal spindle orientation and mediates miR-449-induced spindle defects.

A, D. Confocal time lapse images of metaphase HeLa cells stably expressing a centriole marker (Centrin2-GFP; green), the microtubule marker (α-tubulin–mRFP; red) and no extra protein (A) or mouse JAM-A (D), 48 h after transfection of miR-449a mimic, JAM-A siRNA, or non-targeting negative control siRNA. Scale bar, 10 μm.

B, E. Quantification of spindle rotation for cells as shown in A, D and G. Each dot represents a single cell. Red bar indicates median. Significance was tested by Pairwaise t-test with Bonferroni correction. p-value = 2e-11 (Neg. control versus miR449 mimic), p-value = 0.01928 (Neg. control versus JAM-A siRNA), p-value = 0.17492 (Neg. control versus miR449 mimic, under JAM-A overexpression). * indicates p ≤ 0.05; ** p ≤ 0.01; p*** p ≤ 0.001 for all panels.

C, F. Cumulative histograms of mitotic duration from nuclear envelope breakdown until anaphase onset, measured for cells as shown in A and D; n ≥ 20 cells for all conditions, 2 independent replicates.
Identification of a dynamic gene regulatory network required for pluripotency factor-induced reprogramming of mouse fibroblasts and hepatocytes (2020)
Figure 1

Figure 1. Identification of nine TRs required for the generation of iPSCs from cells of different origins.

(A) Venn diagram depicting the total number of genes upregulated in MEFs (1504) or mHEPs (532) undergoing reprogramming and the 454 common genes upregulated in both MEFs and mHeps at day 6 of reprogramming. Of these, 30 genes, listed in the adjacent table, encode for Transcriptional Regulators (TRs). The top diagram indicates the experimental outline of the DNA Microarrays expression studies performed in MEFs and mHEPs undergoing reprogramming.

(B) Bar graph summarizing the efficiency of single RNAi knockdowns for 28 out of the 30 TRs, and their effects in the reprogramming efficiency in comparison to control cells expressing scramble shRNA. The efficiency of each TR's knockdown (KD) does not correlate with its effect in reprogramming efficiency. The knockdown of 9 (Class III) out of the 28 TRs decreased the efficiency of the reprogramming in a statistically significant manner (unpaired two-tailed Student's t-test, p-value <0.05). Data are shown as mean ± SEM of at least two independent experiments. Class I corresponds to one TR inhibiting reprogramming, whereas Class II corresponds to 18 TRs having a weak or no effect in repogramming. Shown are the effects of 28 out of 30 TRs, because we did not succeed in knocking down Cphx and Phox2a.

(C) Left: Shown is a line graph depicting normalized mRNA expression levels of the 9 TRs at the indicated time points during the reprogramming of MEFs. The data were plotted after normalization with the endogenous Gapdh using the "ΔCt method". The grey vertical dashed line depicts day 6 in which the expression levels of nearly all TRs peaks. Notice that the y-axis is broken to accommodate the large spread of expression levels. Right: Shown are western blots using antibodies specific for Irf6, Tead4, Taf1c, Rcan1 and Tfap4. Whole cell extracts were prepared from MEFs at day 6 of reprogramming and run side-by-side with extracts from control samples (day 0). Gapdh, mTOR and b-Tubulin were used as loading controls.

(D) Same as in (B) except MEFs were co-transduced with lentiviruses expressing all possible pairwise combinations of shRNAs for the nine TRs. Left: Heat map depicting the effects of paired KD combinations in the Reprogramming Efficiency (RE%). Shown are the average REs values from at least two independent experiments. The REs of the double KDs were evaluated against the RE of the corresponding single KDs. Right: Alkaline Phosphatase (AP) stained cultures of terminal reprogrammed MEFs upon representative single and double KDs of our shRNA-based screens.
Identification of a dynamic gene regulatory network required for pluripotency factor-induced reprogramming of mouse fibroblasts and hepatocytes (2020)
Figure 3

Figure 3. The expression of the nine TRs is regulated by direct binding of the OSKM reprogramming factors to putative regulatory regions.

(A-I) Shown are ChIP-seq big wig files in the IGV browser depicting the binding of Oct4 (Red), Sox2 (Blue), Klf4 (Green) and c-Myc (Black) to putative regulatory regions in the nine TRs genes in MEFs undergoing reprogramming (18 hrs, D3 and D5) and in control MEFs, mESCs and miPSCs. All peaks have been normalized against input DNA. The relative sizes of the represented genomic loci and the corresponding TSSs are also indicated. The side color bar depicts the expression levels of each TR at the indicated time points.
Identification of a dynamic gene regulatory network required for pluripotency factor-induced reprogramming of mouse fibroblasts and hepatocytes (2020)
Figure 4

Figure 4. Identification of a transcription gene regulatory network essential for the acquisition of pluripotency.

(A) Schematic representation of the experimental data sets and the integration procedure used to reconstruct the 9TR GRN. DEGs: Differentially Expressed Genes; TRs: Transcriptional Regulators; KDs: Knockdowns; RE: Reprogramming Efficiency; GRN: Gene Regulatory Network

(B) The sequential assembly of a novel gene regulatory network required for reprogramming. Each connection (line) represents the regulatory interaction between the indicated nine TRs during the reprogramming course. The red connections indicate functional synergy between the TRs. The light grey circles shown at 18 hrs timepoint represent the TR genes that have not yet been expressed. The O/S/K/M labeled beads placed on the gene circles denote the direct O/S/K/M binding at putative gene regulatory regions as deciphered from our ChIP-seq binding profiles. O: Oct4, S: Sox2, K: Klf4 and M: c-Myc.

(C) Heatmap summarizing the effects of each of the nine TRs single knockdowns on the expression of the other TRs on day 6; The white color denotes no change in expression levels.

(D) Scatter plots depicting the extent of co-expression of either the broadly- or early-iPSCs-specifically expressed TRs in individual cells obtained from the early-iPSCs colonies or from the rest of the population (left and right panel respectively). Each dot depicts the percentage of cells co-expressing a unique combination of two or three TRs. Data are shown as mean ± SEM of at least two independent experiments.

(E) Heatmap summarizing the most striking effects of double nine TRs KDs on the expression of the other TRs and on the expression of the Epcam and Lin28a pluripotency markers; the white color indicates no change in expression levels. The genes examined have been ranked in the heatmap according to their order of upregulation during reprogramming; Lin28a is the latest upregulated gene.
Identification of a dynamic gene regulatory network required for pluripotency factor-induced reprogramming of mouse fibroblasts and hepatocytes (2020)
Figure 5

Figure 5. Validation of the 9TR GRN

(A) The effect of overexpression of the indicated TRs (Nanog, Irf6, Tfap4 and Irf6+Tfap4) on the expression of the other TRs of the network was determined by RT-qPCR analyses. Dark blue color denotes TRs upregulated more than 2 fold. Reduction by more than 2 fold in the expression is depicted by the absence of the corresponding TR. The light red and blue colored circles indicate downregulation or upregulation in the range of 1.3-2 fold, respectively, while grey circles indicate no change of gene expression. O: Oct4, S: Sox2, K: Klf4 and M: c-Myc. The light grey-green and red boxes depict the upstream and downstream layers of the 9TR GRN. The first panel on the left denotes the native 9TR GRN as shown in figure 4B (Phase III).

(B) Shown is a bar graph depicting the effect of overexpression of the indicated TRs in reprogramming efficiency. Data are shown as mean ± SEM of at least two independent experiments. All values are normalized to "empty vector". The y axis labeled as % reprogramming efficiency refers to the AP+ colonies scored as iPSCs.

(C) Shown is a bar graph depicting the effects of rescue experiments in reprogramming efficiency. MEFs undergoing reprogramming were knocked down using shRNAs for the endogenous Nanog or Irf6 expression and were co-infected with a vector expressing an shRNA-resistant human homologue of Nanog or Irf6 (hNanog OE, hIrf6 OE). Data are shown as mean ± SEM of two independent experiments.
Extracellular Vesicles deliver Mycobacterium RNA to promote host immunity and bacterial killing (2018)
Figure 1

Figure 1. EVs Released by M.tb-infected Macrophages Stimulate RIG-I/MAVS-dependent Type I Interferon Expression in Host Cells.

(A) Quantitative RT-PCR analysis of M.tb RNA in EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) BMMs. ND, not detected. Quantitative RT-PCR analysis for IFN-β mRNA (B) or IFN-β protein (C) levels in wild type BMMs 5 and 24 hr post-treatment with EVs respectively (1 μg/ml equals approximately 3 x 108 vesicles/ml). (D) IFN-β mRNA level was quantified in wild type BMMs treated with EVs at a concentration of 10μg/ml for the times indicated. Quantitative RT-PCR analysis for IFN-β mRNA (E) or IFN-β protein (F) levels in either WT, MAVS- and/or RIG-I-knockdown BMMs treated for 4 hr (E) or 24 hr (F) with 10µg/ml EVs from uninfected or M.tb-infected macrophages. (Control) negative control siRNA. (G) and (H) Similar to above except using WT and Mavs -/- BMMs. (I) Schematic of transwell assay for measuring IFN-β induction. BMMs were uninfected or infected with WT M.tb at a MOI of 5. Twenty-four hr post-infection, cells were transferred into the top chamber and co-cultured with naïve BMMs (Bottom chamber). The IFN-β mRNA levels in BMMs (bottom) were quantified by qRT-PCR after 24 hr. (J) Top chamber: WT BMMs; Bottom chamber: WT or Mavs -/- BMMs. (K) Western blot analysis for phospho-TBK1 (Ser172) in WT and MAVS-knockdown BMMs treated for 4 hr with EVs from uninfected or M.tb-infected macrophages. β-actin served as a loading control. (L) Western blot analysis of IRF3 under the conditions described above for TBK1. Histone H3 (H3) and β-actin were used as loading controls for nuclear fraction and whole-cell lysate (WCL) respectively. Densitometry of the western blots for (K) and (L) are shown. (M) Fluorescence microscopy analysis for nuclear translocation of IRF3 in wild type BMMs untreated (Mock) or treated with EVs from uninfected or M.tb-infected macrophages. Scale bar, 20 μM. (N) IRF3 nuclear translocation was analyzed in RIG-I- or MAVS-knockdown BMMs. Scale bar, 20 μM. Data shown in (A) - (H), and (J) are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).
Extracellular Vesicles deliver Mycobacterium RNA to promote host immunity and bacterial killing (2018)
Figure 2

Figure 2. EVs-induced Type I Production in Macrophages Requires the M.tb SecA2 Secretion System.

(A) Nanosight analysis for EVs isolated from BMMs infected with WT, ∆secA2 or secA2-complemented (∆secA2-C) M.tb CDC 1551 strains. (B) Similar to (A), but EVs from cells infected with WT, ∆esxA or esxA-complemented (∆esxA-C) M.tb Erdman strains. (C) Yield of purified EVs from BMMs infected with various M.tb CDC 1551 strains based on Nanosight analysis. (D) Similar to (C) but EVs from cells infected with WT, ∆esxA or esxA-complemented (∆esxA-C) M.tb Erdman strains. qRT-PCR analysis for M.tb RNA in EVs from BMMs infected with various M.tb CDC 1551 strains (E) or Erdman strains (F). ND, not detected. qRT-PCR analysis for IFN-β mRNA (G) or IFN-β protein (H) levels in WT BMMs treated with EVs for 4 hr and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb CDC 1551 strains. (I) ELISA analysis for IFN-β secreted by BMMs treated with EVs plus liposomes containing M.tb RNA. qRT-PCR analysis for IFN-β mRNA (J) or protein (K) levels in WT BMMs treated with EVs for 4 and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb Erdman strains. Data shown in (C), (D) and (E - K) are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).