EMBO/soda-nel huggingface dataset:
CDK7 and MITF repress a transcription program involved in survival and drug tolerance in melanoma (2021)
Figure 6

Figure 6: Loss of SOX10 and MITF releases GATA6 expression

A. qRT-PCR analysis showing average TBP-normalized fold expression of SOX10, MITF and GATA6 in 501mel treated with either siCTL or siSOX10 for 48h.

B. Seurat UMAP of MM074 treated with siCTL or siSOX10 (24h, 48h and 72h post-treatment). The arrow indicated the trajectory from control to 72h post siSOX10 transfection.

C. UMAP of AUCell GATA6 regulon in MM074 shows that GATA6 regulon is up-regulated along the trajectory from siCTL to 72h post siSOX10 treatment (GSE116237) (Wouters et al., 2020). The arrow indicated the trajectory from siCTL to 72h post siSOX10 transfection. We considered cell with GATA6 regulon activity of AUCell > 0.15 as active (see Appendix Figure S5).

D-I. Graphs showing the average expression of the SOX10 (D), MITF (E), GATA6 (F), SERPINE1 (G), AMIGO2 (H) and ABCG2 (i) per individual melanoma cell measured by AUCell on MM074 at different time points post-transfection of siSOX10 (GSE116237) (Wouters et al., 2020) .

Data information: In (A), data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, *<0.05. In (D-I), data are presented as mean values + standard error of the mean (SEM) for six replicates (n=6).
CDK7 and MITF repress a transcription program involved in survival and drug tolerance in melanoma (2021)
Figure 7

Figure 7: MITF binds and represses the GATA6 locus

A. Gene track of 3HA-MITF signal occupancy showing a significant MITF binding peak (P1) in the GATA6 gene body in 501mel (GSE61967). Additional tracks indicate potential regulatory regions highlighted by ATAC-seq and H3K27ac, BRG1, SOX10 and H2A.Z deposition (GSE94488 and GSE61967). H3K27ac deposition is also shown in MM099 at the GATA6 locus. The scale bar indicates the size of the genomic region in kilobases (Kb). A magnification of the P1 region for MITF occupancy is shown in which the "intGATA6r" region is indicated in red and the two E-boxes in black.

B. ChIP experiment monitoring the fold enrichment (compare to control IgG) of MITF protein and H3K27ac mark at the "intGATA6r" region. Proteamine 1 (PRM1) and Tyrosinase (TYR) regulatory regions were used as negative and positive controlled, respectively (Laurette et al., 2015).

C. qRT-PCR analysis showing average TBP-normalized fold expression of MITF and GATA6 in 501mel treated with either siCTL or siMITF for 48h.

D. MM099MITF-SOX10-PAX3 expressing inducible MITF-SOX10-PAX3 genes was treated or not with Doxycycline (1μg/ml) for 24h and protein lysates were immuno-blotted for the indicated protein. The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software, and normalized relative to VINCULIN.

E. qRT-PCR analysis showing average TBP-normalized fold expression of GATA6, ABCG2, AMIGO2 or SERPINE1 in MM099MITF-SOX10-PAX3 treated or not with Doxycycline (1μg/ml) for 24h.

F. Left panel; Schematic representation of pCDNA-ieCMVenh-CMV-GFP (C1) or pCDNA-intGATA6r-CMV-GFP (C2) reporter vectors. The ieCMVenh sequence in C1 was replaced by the "intGATA6r" sequence to generate C2. Right panel; qRT-PCR analysis showing average TBP-normalized fold expression of GFP in MM099MITF-SOX10-PAX3 transfected with C1 or C2 vectors for 48h before treatment or not with Doxycycline (1μg/ml) for 24h.

Data information: In (B, C, E), data are presented as mean values + SD for three biological triplicates. The p-value (Student's t-test) is indicated, *<0.05, **<0.01; ***<0.001. In (F) data are presented as mean values + SD for three technical replicates (n=6). The p-value (Student's t-test) is indicated, ***<0.005, ns=non significant (>0.5).
CDK7 and MITF repress a transcription program involved in survival and drug tolerance in melanoma (2021)
Figure 8

Figure 8: GATA6 is expressed in MITF-low melanoma cells in vivo

A. Tumor sections were immuno-labelled (IHC) with anti-GATA6 (red) and anti-SOX10 (green) antibodies and images were captured by confocal microscopy at the indicated magnification. We analysed six tumor sections of metastases and observed significant GATA6 expression in only one of them. Scale bar 250 μM for 40x, 100μM for 100x.

B. Seurat cluster Heatmap was generated from published scRNA-seq performed on PDX tumour (n=674 cells) (GSE116237) (Rambow et al., 2018). The Heatmap shows 9 different clusters into which the cells can be divided according to the expression of different referenced genes (Z-score). The top 10 genes are indicated in the left for each cluster.

C. GO was used to analyze the genes characterizing each cluster identified above. The average P-value was retrieved for each cluster taking the 3 best GO per cluster, then z-score ((P-value of each biological process-average of P-value of each biological process)/standard deviation) was calculated. Clusters 2 and 3 were undefined (un).

D-E. Graphs showing the average expression of GATA6 (d) and MITF (e) (RPKM) for each phenotype cluster in T0 (drug naïve) (blue), phases 1-2 (MDR) (green and yellow) and phase 3 (drug-resistance) (red).

Data information: In (D-E), data are presented as mean values + SEM (n=6,574 cells from 5 PDX).

Expended View figure legends
Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing (2022)
Figure 1

Figure 1. Paraspeckle proteins interact with canonical BAF type SWI/SNF complex.

(a) Silver-stained gel image of the affinity-purified complexes of PSPC1, SFPQ and NONO. The respective proteins are marked along with AcTEV protease used for the elution of purified proteins.

(b) Venn diagram showing the overlap between the proteins co-purified in the mass spectrometry.

(c) GO-term analysis of common 685 interacting proteins using ShinyGO (http://bioinformatics.sdstate.edu/go/) identified by MudPIT in the affinity-purification of PSPC1, SFPQ and NONO.

(d,e) Heat map showing the enrichment (dNSAFavg*1000) of proteins normalized to the bait (PSPC1, SFPQ and NONO) focusing on (d) RNA processing and (e) SWI/SNF complex subunits.

(f) Western blotting of affinity purified complexes of PSPC1, SFPQ and NONO with SWI/SNF antibodies. cBAF, PBAF, ncBAF specific, as well as common subunits, are marked.
Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing (2022)
Figure 2

Figure 2. ARID1B SWI/SNF complex interacts with various paraspeckle components.

(a) Silver-stained gel image of the affinity-purified complexes of ARID1A and ARID1B. The bait and a few other SWI/SNF subunits are marked as per their molecular weight.

(b) GO-term analysis (Biological process) of the interacting proteins using ShinyGO identified by MudPIT in the affinity-purification of ARID1A and ARID1B.

(c-e) Heat maps showing the enriched proteins (dNSAFavg*1000) normalized to the bait identified in MudPIT analysis focusing on various (c) SWI/SNF subunits and (e) PSPs. (d) Graph showing the topmost processes picked up in the IPA (Ingenuity Pathway Analysis).

(f) Western blotting of affinity purified complexes of ARID1A and ARID1B with bonafide paraspeckle components. SMARCA4 was used as a positive control for purification.
Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing (2022)
Figure 3

Figure 3. SWI/SNF interacts with PSPs in a NEAT1-dependent manner.

(a) Halo purification was performed from nuclear extracts of scramble and various knockdown HEK293FRT cells expressing Halo-FLAG-tagged ARID1B. Input and eluted samples were resolved on a gel followed by western blotting with various antibodies. Western with SWI/SNF subunits- SMARCA4 and SMARCD1 were used as the control for purification, while beta-actin was used as a loading control.

(b-d) (b) Heat map of the dNSAF(*1000) values of PSPs and (d) various SWI/SNF subunits in control (C) and NEAT1 depleted (KD) cells for PSPC1, SFPQ and NONO purification from MudPIT analysis. (c) GO term analysis was performed for interactions lost post-depletion of NEAT1

(e, f) (e) RIP qPCR and (f) PCR analysis performed using an antibody against endogenous ARID1B. for monitoring NEAT1_2 levels. RIP with NONO and IgG was used as a positive and negative control respectively. GAPDH levels were also assessed in the assay. Data are expressed as a percent of Input. Error bars represent the standard error of three technical repeats (Two-tailed t-test ****P < 0.0001).
Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing (2022)
Figure 4

Figure 4. ARID1B subunit mediates the interaction of paraspeckles with SWI/SNF and numerous chromatin-associated proteins.

(a, b, d) Heat map showing the enrichment of (a) SWI/SNF subunits, (b) PSPs and (d) chromatin-associated proteins in MudPIT analysis. C is control and KD is ARID1B depleted cells.

(c) smFISH analysis was performed to detect NEAT1 signal in the nucleus. The scale bar corresponds to 10 µm. Graph showing the FISH signal quantification across the knockdowns in comparison to scramble control. The number of nuclei (n) used for quantification is mentioned. The error bars represent the standard error of three biological experiments (Two-tailed t-test **P< 0.0043, ns>0.05)
The novel lncRNA CALIC upregulates AXL to promote colon cancer metastasis (2019)
Figure 1

Figure 1. Identification of CALIC as a lincRNA upregulated in selected subpopulations having elevated metastatic activity.

A Schematic representation of the experimental method used to identify lincRNAs upregulated in metastatic subpopulations isolated from HCT116 colon cancer cells.

B Gene ontology (GO) analysis of upregulated genes in metastatic lesions.

C Cell migration assays were performed using Transwell migration chambers. HCT116 cells were transfected with siRNAs targeting 11 candidate lincRNAs and cell migration activities were evaluated by Transwell migration assays (n = 3). CALIC is the lincRNA targeted by si-LINC00920.

D Migration of HCT116 cells transfected with the indicated siRNAs or negative control. (n = 4)

E GO analysis of differentially expressed genes in CALIC knockdown HCT116 cells.

F Migration of colon cancer cells treated with an siRNA targeting CALIC. (n = 3)

G (Top) Schematic representation of the chromosomal location of the CALIC gene locus. Arrows indicate the orientation of transcription. (Bottom) ENCODE ChIP-seq data for H3K4Me3 is displayed in the UCSC browser illustrations. Gray boxes mark locations of exons (Ex1-2) of CALIC.

H Subcellular localization analysis of CALIC. RNAs were isolated from the nuclear and cytoplasmic fractions of HCT116 cells and quantified by qRT-PCR (n = 3). Nuclear controls: U6, MALAT1; Cytoplasmic control: GAPDH.

I CALIC expression levels in cancer tissues. N, normal mucosa; T, tumor.

J Expression of CALIC in primary skin tumors (P) and their metastases (M).

K Expression of CALIC in primary skin tumors of patients without (w/o) or with (w) metastases.

L qRT-PCR analysis of CALIC expression in early (I/II) and late (III/IV) stage colon cancers. [Normal (N) n = 8, stage I/II n = 14, stage III/IV n = 26]

M Kaplan-Meier curves for disease-free survival of colon cancer patients whose primary tumors expressed low (blue, n = 113) or high (red, n = 250) levels of CALIC.

Data information: Results are expressed as the mean ± SEM. * P < 0.05, **P < 0.01, *** P < 0.001 (Student's t-test).
The novel lncRNA CALIC upregulates AXL to promote colon cancer metastasis (2019)
Figure 2

Figure 2. CALIC interacts with hnRNP-L.

A Biotinylated full-length CALIC or antisense CALIC (negative control) generated in vitro were incubated with lysates from DLD-1 colon cancer cells and precipitated with streptavidin beads. Precipitated proteins were resolved by SDS-PAGE followed by silver staining. The protein band indicated by the arrow was excised and subjected to mass spectrometry.

B (Top) schematic of the full-length and mutants of CALIC used for the precipitation of hnRNP-L from HCT116 cell lysates. The mutated elements are indicated by the cross marks. Se, sense transcript; AS, antisense transcript. +, detectable binding activity; -, no detectable activity. (Bottom) precipitated hnRNP-L and biotin-labeled fragments of CALIC are shown.

C, D RIP analysis was performed using control rabbit IgG or anti-hnRNP-L antibody. HPRT1 and the lncRNA MALAT1 were used as negative controls. Immunoblotting analysis of proteins precipitated by RIP assays using anti-hnRNP-L antibody are shown (D). (n = 3)

E ChIRP-immunoblotting analysis of hnRNP-L association with endogeneous CALIC in HCT116 cells.

F Migration of HCT116 cells transfected with the indicated siRNAs or negative control. (n = 4)

Data information: Results are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student's t-test).
The novel lncRNA CALIC upregulates AXL to promote colon cancer metastasis (2019)
Figure 3

Figure 3. CALIC imparts specificity to hnRNP-L-mediated gene expression.

A, B RNA-Seq analysis of hnRNP-L- and CALIC-regulated genes in HCT116 cells. Pie chart showing genes regulated by hnRNP-L or CALIC (A). Venn diagram showing the overlap of genes differentially expressed by hnRNP-L (blue) or CALIC (purple) knockdown (B, top). Expression levels of 59 genes regulated in common in both hnRNP-L and CALIC knockdown cells relative to control siRNA experiments (B, bottom). Two biological replicates (R1 and R2) were included for each siRNA treatment. The number of genes within each category is indicated.

C Scatterplots showing correlations between CALIC- and hnRNP-L-regulated genes in (B). Left, co-regulated 59 genes; Right, all differentially regulated genes (DEGs). The correlation was calculated using the data except for the gene indicated by the asterisk.

D Genes regulated in common and in the same direction by hnRNP-L and CALIC.

E Genes induced by CALIC are significantly enriched in those upregulated by hnRNP-L. GSEA comparing the genes repressed by CALIC and hnRNP-L knockdown. NES, normalized enrichment score.

F Heatmap showing the expression levels of genes positively regulated in common by both CALIC and hnRNP-L in colon and rectal cancers. Samples are ordered based on the expression levels of AXL.
The novel lncRNA CALIC upregulates AXL to promote colon cancer metastasis (2019)
Figure 4

Figure 4. Upregulation of AXL is essential for CALIC-mediated migration and metastasis.

A-D qRT-PCR (A, C) and immunoblotting (B, D) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALIC or hnRNP-L. (n = 3-4)

E qRT-PCR analyses of AXL in HCT116 cells infected with a lentivirus expressing wild-type CALIC (CALIC-full), CALIC-296-913 or CALIC-mut (see Fig 2B). (n = 3)

F HCT116 cells that had been treated with an siRNA targeting hnRNP-L or CALIC were transfected with an AXL reporter construct and subjected to luciferase assays. (n = 4)

G, H ChIP assays were performed using anti-hnRNP-L antibody or control mouse IgG. The promoter regions of GAPDH and Egr1 were used as negative and positive controls, respectively. Immunoblotting analysis of proteins precipitated by ChIP assays using anti-hnRNP-L antibody (H). (n = 4)

I ChIP analysis of hnRNP-L association with the AXL promoter region in CALIC knockdown HCT116 cells. (n = 4)

J ChIP analysis of hnRNP-L association with the AXL promoter region in HCT116 cells expressing lentiviral CALIC-full. (n = 3)

K ChIRP analysis of CALIC binding to the AXL promoter. Binding of CALIC to the promoter regions of the indicated genes was compared with that of LacZ. (n = 3)

L Migratory activity of AXL knockdown HCT116 cells. (n = 4)

M Lentiviral expression of AXL restores the migration activity of HCT116 cells transfected with an siRNA targeting CALIC. Immunoblotting and qRT-PCR analyses of HA-tagged AXL and CALIC expression, respectively (left). α-Tubulin was used as a loading control. (n = 3)

N Representative images of gross specimens (top) and H&E stained sections (bottom) of lung metastatic lesions in mice injected with CALIC or AXL knockdown HCT116 cells. Tumors were marked with yellow arrows. The dashed lines depict the boundary between normal and tumor tissues. (Right) Percentage of mice with lung metastases.

O Representative images of gross specimens of primary cecal tumors (top) and liver metastatic lesions (bottom) in mice orthotopically (cecal) injected with CALIC knockdown HCT116 cells. Tumors are marked with yellow arrows. (Right) Percentage of mice with primary tumors and liver metastases.

P A schematic model showing the mechanism of AXL regulation by CALIC and hnRNP-L. CALIC interacts with hnRNP-L to induce AXL expression and thereby facilitates metastasis.

Data information: Results are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student's t-test).

Expanded View Figure Legends
Dual control of dopamine in Drosophila myeloid-like progenitor cell proliferation and regulation of lymph gland growth (2022)
Figure 1

Fig 1: Dopamine expression during lymph gland growth.

(A-D) Immunostaining with dopamine antibody at late 3rd instar (96h AEL) in control background (domeMESO-GFP/+). The inset shows DomeGFP+ cells (B') and the corresponding dopamine levels (C') in them.

(E-L) Similar expression analysis of dopamine at early 3rd (72h AEL, E-H) and early 2nd instar (48h AEL, I-L).

(D, H and L) The spectral mode of iDopamine representation with blue pixels indicating low and red pixels indicating high intensity.

(M) Quantifications of dopamine levels in DomeGFP+ progenitors (domeMESO-Gal4, UAS-GFP/+, n= 14 at 48h, n=13 at 72h and n=16 at 96h AEL).

Data information: DNA is stained with Hoechst in blue, intracellular Dopamine (iDopamine) in red, domeMESO expression in green (representative of progenitors in the medullary zone, MZ). The iDopamine channel has been converted to spectral mode in panels D, H and L. All image panels show a 20µm scale bar. The inset represents a 2X zoom of region of interest (ROIs) marked in white dashed square. AEL indicates After Egg Laying. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line for segregation between Dome+ and Dome- cells. The quantification in M shows median with whiskers indicating the maximum and minimum values,. box indicates the lower and upper quartiles. The statistical analysis applied is One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.
Dual control of dopamine in Drosophila myeloid-like progenitor cell proliferation and regulation of lymph gland growth (2022)
Figure 3

Fig 3: Dopamine sensing in the progenitors via dopamine receptor Dop2R and dopamine transporter DAT contributes to lymph gland size regulation.

(A-C') Expression profile of dopamine activation based receptor sensor (DA1m) in the progenitors using domeMESO-Gal4 driver in a temporal manner- 48h, (A-A'), 72h, (B-B') and 96h AEL, (C-C').

(D) Quantification of the number DA1m+ cells, %DA1m positive cells (domeMESO-Gal4; DA1m, n=10 at 96h, n=16 at 72h and n=10 at 48h AEL).

(E-G') Temporal expression analysis of dopamine transporter, DAT.

(H) A line plot representing the trend of DAT expression at 96h AEL. The distance (x) axis indicates 0µm as a line beginning from the core region to the periphery.

(I-K) The loss of progenitor Dop2R and DAT function using domeMESO-Gal4, UAS-GFP analysed at wandering 3rd instar, 120h AEL.

(L) Quantifications of lymph gland size upon loss of Dop2R and DAT in the progenitors (domeMESO-Gal4, UAS-GFP/+, n= 46, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 44, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 37).

(M) Quantifications of the proportions of %Dome (green bar), %negative (blue bar), %overlap (Dome and P1, yellow bar) and %P1 (red bars) upon loss of Dop2R and DAT in the progenitors. For Dome positive area (domeMESO-Gal4, UAS-GFP/+, n= 28, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23), negative area (domeMESO-Gal4, UAS-GFP/+, n= 28, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23), overlap area (domeMESO-Gal4, UAS-GFP/+, n= 26, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 25) and P1 area (domeMESO-Gal4, UAS-GFP/+, n= 27, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23).

(N) Quantification of the number of PPO positive crystal cells upon dopamine sensing perturbation (domeMESO-Gal4, UAS-GFP/+, n= 29, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 26, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 25).

Data information: DNA is stained with Hoechst in blue, Dopamine transporter (DAT) in red, lifeAct-RFP to mark progenitors in red, Dopamine receptor-based reporter (DA1m) in green, domeMESO (representative of progenitors) expression in green, P1 (representative of mature plasmatocytes) in red. All image panels show a 20µm scale bar. AEL indicates After Egg Laying. The quantification in L and N represents mean with standard deviation, M represents mean, D represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in L and N is One-way ANOVA with Tukey's multiple comparisons test while that applied in D and M is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.