EMBO/soda-nel huggingface dataset:
Systemically Targeted Cancer Immunotherapy and Gene Delivery using Transmorphic Particles. (2022)
Figure 6

Figure 6. Biodistribution and targeted systemic cancer immunotherapy with RGD4C.TPA.IL15IgK.

(A) Tumor-bearing BALB/c mice with established subcutaneous CT26.CL25 tumors were intravenously injected with a single dose, 5x1010 TU/mouse, of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Gene delivery was evaluated by quantifying the IL15IgK mRNA expression in tumors and healthy tissues at day 5 post TPA administration. Data are representative of one experiment, n = 3, and expressed as mean + SEM. Two-way ANOVA test was used for data analysis.

(B) Safety of dose escalation regimens assessed at day 5 following TPA delivery, by evaluating the serum levels of LDH from n = 2 mice/TPA dose of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Serums from untreated mice were also analysed. .

(C) Representative tumor-bearing mice showing bioluminescence imaging of luciferase on days 0 and 5 post administration with 5x1010 TU of RGD4C.TPA.IL15IgK (RGD4C) or non-targeted (NT) particles. The CT26.CL25 tumor cells stably express a luciferase Luc reporter gene. Untreated mice were also included as controls (CTRL).

(D) Changes in tumor viability in mice (n = 4) from day 0 to day 5 post TPA administration. Tumor viability was measured by bioluminescence activity. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Kruskal-Wallis test was used for data analysis.

(E) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from all experimental groups.

(F) Animal weights (n = 4) were monitored during therapy experiments ad expressed as mean + SEM.
Systemically Targeted Cancer Immunotherapy and Gene Delivery using Transmorphic Particles. (2022)
Figure 7

Figure 7. Tumor immune profiling after RGD4C.TPA.IL15IgK treatment and immunotherapy with RGD4C.TPA encoding IL12.

(A) The immune profile of CT26.CL25 tumors was investigated using RT-qPCR at day 5 after TPA.IL15IgK administration. A panel of immunological mRNA transcripts were selected for analysis and included CD8a and CD8b as markers for CD8+ T cell population; NKp46 for NK cell population. Markers for cytotoxic cell-killing by CD8+ T cells (GZMa, GZMb) and by NK cells (Prf1) were also analysed. Expression of TBX21 was evaluated as a marker for the Th1 response mediated by interferon gamma (IFN-γ), and FoxP3 for Treg cells. Data shown are representative of n = 5 mice and expressed as mean + SEM. Non-parametric Mann-Whitney test was used for GZMb and TBX2 data analysis, and independent t-test to analyse data of the other markers.

(B) TPA particles encoding IL12 were used to treat immunocompetent BALB/c mice bearing subcutaneous CT26.CL25 tumors labelled with a Luc reporter gene. Representative tumor-bearing mice at day 5 post-treatment with targeted RGD4C.TPA.IL12 (RGD4C) or non-targeted TPA.IL12 (NT).

(C) Analysis of total flux change in tumors, n = 4 mice, after IL12 immunotherapy. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Mann-Whitney test was used for data analysis.

(D) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from two experimental groups.

(E) Serial quantification of IL12 in media of B16.F10 cells treated with RGD4C.TPA.IL12 or controls. Data are representative of one experiment, n = 3 biological repeats, and shown as mean + SD Experiments were repeated three times. One-way ANOVA with Tukey's HSD test was used for data analysis.

(F) RGD4C.TPA.IL12 immunotherapy of subcutaneous B16.F10 tumors in C57BL/6 mice (n = 4). Data are shown as mean + SEM independent t-test was used for data analysis. Tumor volume changes are shown over time.

EXPENDED VIEW FIGURE LEGENDS
A Novel Form of JARID2 is Required for Differentiation in Lineage-Committed Cells (2018)
Figure 1

Figure 1: JARID2 exists as a LMW form (~80kDa) in many cell types.

A. A schematic diagram showing functional domains of JARID2: UIM, ubiquitin interaction motif; transcription repressive domain consisting of sub-domains 1-3; JmjN, JumonjiN; ARID, AT-rich DNA binding domain; GSGFP which is a SUZ12 binding domain; JmjC, JumonjiC; and ZF, zinc finger domain. Domains 1-3 constitute PRC2 associated domains. Domain 1 is required for PRC2 stimulation, domain 2 is needed for binding to EZH2 and domain 3 is needed for nucleosomal binding. The RNA binding domain has been indicated with dashed box.

B. Immunoblot of whole cell lysates from multiple human lineage-committed cell-lines using a C-terminal anti-JARID2 antibody. The size of canonical form is 140kDa but an additional strong band was detected at around ~80kDa. The blot is divided according to cell-types, which are indicated in the figure.

C. qPCR measurements of JARID2 mRNA levels in different cell lines. The RNA levels were measured relative to 18S rRNA. Means and SEs are calculated over at least 2 independent experiments and 3 technical replicates. The data is represented as mean ± SE.

D. Immunoblot showing that the ~80kDa band disappears upon treatment of HEK293T and HaCaT cells with siRNAs against exon 3 and exon 15 of JARID2 RNA. Non-silencing control (NSC) was used as a transfection control. Densitometric measurements corresponding to this experiment are shown in Fig EV1A.
A Novel Form of JARID2 is Required for Differentiation in Lineage-Committed Cells (2018)
Figure 2

Figure 2: JARID2 LMW is a cleaved product of full length JARID2 isofrom-1.

A. A schematic showing three different isoforms of human JARID2, as predicted by ensembl annotations, is shown. The translation start site of each isoform is labelled using green arrow. Predicted sizes (140kDa, 120kDa and 106kDa) of proteins corresponding to three isoforms have been indicated.

B. CAGE-seq data showing the transcription start sites of JARID2 in different cell lines. CAGE peak is mainly observed at JARID2 isoform-1 in most cell types. JARID2 gene structure is shown at the top.

C. qPCR measurements of RNA levels (n=3) for the three isoforms of JARID2 in HaCaT cells. The RNA levels are plotted relative to 18S rRNA. Level of JARID2 isoform-1 is significantly higher than that of isoform-2 (***P<0.001) and isoform-3 (****P<0.0001). Data for three independent experiments is represented as mean ± SE. Multiple comparisons were performed and p-values were calculated using one- way ANOVA.

D. Agarose gel showing RT-PCR products amplified using primers in exon3 and exon15 (2.9kb) as well as RT-PCR product corresponding to JARID2 isoform-1 (3.7kb) amplified using the first (exon 1) and the last exon (exon18). Only one product at the right size is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2.

E. Ribosomal profiling data mapped on JARID2 isoform-1 and isoform-2. Main translation site corresponding to isoform-1 is highlighted using a dashed box.

F. CRISPR-Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in (E). Immunoblot revealed that ~80kDa form was removed from JARID2 KO lines. Densitometric measurements corresponding to the experiment are shown in Fig EV2D.

G. Immunoblot after a full length JARID2 isoform-1 was expressed from an exogenous vector in HaCaT cells showed an increase (2-3 fold) in ~80kDa level. Densitometric measurements are shown in Fig EV2E. An immunoblot with exogenous expression of 140kDa band in a control cell-line is shown in Fig EV2F.

H. Immunoprecipitation using anti-JARID2 antibody and followed by mass spectrometry identification of the ~80kDa band detected JARID2 peptides spanning only C-terminus of JARID2. The identified peptides are shown as red arrows on schematic of JARID2 sequence.
A Novel Form of JARID2 is Required for Differentiation in Lineage-Committed Cells (2018)
Figure 3

Figure 3. Effect of JARID2 knockout on differentiation markers.

A. The calcium-induced differentiation protocol used in this study. Up until day 0 cells were maintained in low calcium media and then induced to differentiate by growing them in high calcium medium for six days. Cells were harvested for immunoblot at day 0, day 1, day 3 and day 6 of differentiation.

B. Immunoblot showing increase in expression levels of differentiation markers Involucrin (IVL) and Transglutaminase-1 (TGase-1) as differentiation progressed indicating that keratinocyte differentiation protocol was successful. Protein levels on day 0 (D0), day 1 (D1), day 3 (D3) and day 6 (D6) of differentiation are shown.

C. Immunoblot for JARID2 during D0, D1, D3 and D6 of differentiation (as in B) are shown.

D. Effect of JARID2 removal on levels of differentiation markers Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as measured in qPCR experiment relative to 18S rRNA and the rescue using exogenous expression of ΔN-JARID2. The effect of exogenous expression of full-length JARID2 (FL-JARID2) in JARID2 knockout is also shown. Data from wildtype HaCaTs, two independent JARID2 knockout (KO1 and KO2) HaCaT lines as well as KO cells exogenously expressing empty vector control, ΔN-JARID2 and FL-JARID2 are shown. Data from three independent experiments (n=3) is represented as mean ± SE and multiple comparisons were performed using one-way ANOVA (**** P-value < 0.0001, *** P-value < 0.001, ** P-value < 0.01 and * P-value < 0.05).

E. Levels of Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as above but measured on day 3 of differentiation.
A Novel Form of JARID2 is Required for Differentiation in Lineage-Committed Cells (2018)
Figure 4

Figure 4. Gene expression changes in JARID2-null cells.

A, B. Functional enrichment of (A) 2-fold down-regulated and (B) 2-fold up-regulated genes in JARID2-null cells as compared to WT on day 3 of differentiation. Functional categories are plotted on vertical axes and -log(p) values are plotted on horizontal axes.

C. A metagene plot showing enrichment of full-length JARID2 in human induced pluripotent cells at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plot is centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axis.

D. Metagene plots of average enrichment of EZH2 in ES cells and keratinocytes at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plots are centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axes.

E. Levels of H3K27me3 in WT, JARID2 KO lines and JARID2 KO lines expressing ΔN-JARID2. Histone H3 and Ponceau staining is used as a loading control. Densitometric measurements corresponding to this experiment are shown in Fig EV4A.

F. A bar-plot showing percentage of down- and up-regulated genes among genes which lose H3K27me3 vs which do not lose H3K27me3 modification during differentiation.
From network to phenotype: the dynamic wiring of an Arabidopsis transcriptional network induced by osmotic stress (2017)
Figure 3

Figure 3 - The regulatory connections of the osmotic stress-responsive GRN.

A The significant regulatory interactions identified by nCounter Nanostring at 1 h, 2 h, 4 h, 8 h and 24 h after induction of overexpression of a TF.

B The confirmed regulatory interactions between the 20 TFs part of the GRN, according to transient expression assays (n = 4 biological repeats). Green arrows represent activation and red arrows repression.

C Heatmap of the significant regulations upon induction of overexpression of the five members of the core network, the activators (green) ERF6, ERF59, ERF98 and the repressors (red) ERF8 and ERF9. Color code represents FDR-corrected p-values with thresholds at FDR=0.01, 0.05 and 0.1.

Data information: In A, data are extrapolated from estimated averages, n = 3 independent experiments, FDR-corrected p-values < 0.1 (mixed model analysis, user-defined Wald tests). The thickness of the arrows represents the FDR value. In B, data are presented as averages, n = 3 independent experiments. The intensity of the color of the arrows represents the strength of the regulation according to the TEA values and the thickness the FDR value of the nCounter Nanostring experiment. In C, data are represented as FDR-corrected p-values, n = 3 independent experiments.
From network to phenotype: the dynamic wiring of an Arabidopsis transcriptional network induced by osmotic stress (2017)
Figure 4

Figure 4 - Phenotypic analysis of loss-of-function (LOF) and gain-of-function (GOF) lines of the TFs under control conditions.

A At 22 days after stratification (DAS), leaf series of the LOF lines were made and the rosette area was calculated as the sum of the area of all individual leaves (n = 10 plants). The rosette area is presented relative to the corresponding wild type.

B Average area of the individual leaves of rap2.6L and myb51, two knock-out lines with a smaller average rosette area.

C Rosette area of two or three independent GOF lines per TF (calculated as the projected area or the sum of the leaves, n = 10 plants) germinated and grown on DEX-containing medium. Measurements were performed at 22 DAS relative to the control line. "Independent line 1" is the line with the highest overexpression level.

D A representative picture of the rosette of GOF lines with significant growth phenotypes in both independent lines at 22 DAS. Scale bar is 1 cm.

E, F Pavement cell number and area of the third leaf at 22 DAS of LOF (E) and GOF lines (F) that showed a significant rosette area phenotype.

Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey"s test). In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests). In E and F, data are presented as mean ± SEM, n = 3 independent experiments, . = P<0.1, * = P<0.05, ** = P<0.01, *** = P<0.001 (Tukey"s test).
From network to phenotype: the dynamic wiring of an Arabidopsis transcriptional network induced by osmotic stress (2017)
Figure 5

Figure 5 - Expression of four GA-biosynthesis and -degradation enzymes in wild-type plants upon mannitol treatment and in GOF lines showing an altered growth phenotype in both independent lines.

A The expression level of two GA-degradation and two GA-biosynthesis enzymes in expanding leaf tissue (third leaf at 15 DAS) of wild-type plants 20 min, 40 min, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h after mannitol treatment. The fold changes (FC) were calculated relative to control conditions.

B The expression of GA2-OX6 and GA20-OX1 in expanding leaf tissue (third leaf - 15 DAS), 8 h after transfer to DEX-containing medium to induce overexpression. The FC was calculated relative to control conditions.

C The effect of ERF6, ERF9, ERF98 and WRKY15 on the GA2-OX6 promoter determined with transient expression assays. The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats).

Data information: In A, data are presented as mean ± SEM, n = 4 independent experiments, *= FDR<0.05 (unpaired two-sided Student"s t-test) In B, data are presented as mean ± SEM, n = 3 independent experiments, * = FDR < 0.1 (mixed model analysis, user-defined Wald tests). In C, data are presented as mean ± SEM, n = 3 independent experiments.
From network to phenotype: the dynamic wiring of an Arabidopsis transcriptional network induced by osmotic stress (2017)
Figure 6

Figure 6 - Phenotypic analysis under mild osmotic stress of loss-of-function (LOF) and gain-of-function (GOF) lines of every TF.

A At 22 DAS, leaf series were made of the LOF lines (n = 10 plants) grown under control conditions or mild osmotic stress conditions, and the reduction under mild osmotic stress was calculated. The rosette area reduction is presented relative to the corresponding wild type.

B Leaf series of stz and erf11 mutants grown on control and mild osmotic stress conditions.

C The rosette area of two or three independent GOF lines (n = 10 plants) germinated on DEX-containing control or mild osmotic stress medium was measured at 22 DAS and the reduction by mild osmotic stress was calculated relative to the control line. "Independent line 1" is the line with the highest overexpression level.

Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey"s test). In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests).
From network to phenotype: the dynamic wiring of an Arabidopsis transcriptional network induced by osmotic stress (2017)
Figure 7

Figure 7 - Five different effects of two TFs on a common target gene.

A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes ERF11 (A), ERF5 (B), MYB51 (C), RAP2.6L (D) and ERF6 (E). The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation.

Data information: Data are presented as mean ± SEM, n = 3 independent experiments.
The AhR-SRC axis as a therapeutic vulnerability in BRAFi-resistant melanoma. (2022)
Figure 1

Figure 1. Loss of AhR reduces the invasive phenotype of BRAFi-resistant melanoma cells.

A. Vem sensitivity was established in BRAFi-resistant SK28 cells before and after knockout out of AhR by CRISPR/Cas9, by cell density measurements for four days after treatment (every 2 days), with an increasing concentration of BRAFi (vemurafenib, dabrafenib, or encorafenib). The IC50 (M) was calculated using GraphPad (PRISM9.0®).

B-C. Wound healing assays were performed using IBIDI® chambers to evaluate the role of the AhR on cell migration. Images of the wound were captured using an Axio Vert.A1 inverted microscope (Carl Zeiss®) at 5x magnification. The histogram represents the mean ± s.d. Wound closure was determined by measuring the distance between the edges of the wound at time 0 and 15 h (n = 3 independent technical experiments for each cell lines or conditions) and compared using unpaired t-tests with the Sidak-Bonferroni method. B. Results obtained with BRAFi-sensitive or resistant SK28 cells KO for the AhR in the absence of treatment. (n = 3 independent technical experiments for each cell lines). C. Results obtained for the migration assay (0 to 15 h) for SK28 R cells KO or not for the AhR after treatment or not with 10 μM CH-223191. (n = 3 independent technical experiments for each cell lines or conditions, mean ± s.d.). Statistical analysis using unpaired t-tests with the Sidak-Bonferroni method has been performed between the mean of the 3 independent experiments.

D. Three-dimensional spheroid growth of BRAFi-sensitive or resistant SK28 cells KO before or after knockout of AhR by CRISPR/Cas9 in the absence of treatment. Images were captured four days after implantation of the spheroids into collagen gel. (n = 4 independent technical experiments, mean ± s.d.).

E. Three-dimensional spheroid growth of BRAFi-resistant SK28 cells KO before or after knockout of AhR by CRISPR/Cas9 after daily treatment with the specific AhR inhibitor CH-223191 (5 μM) for one week or in the absence of treatment. Images were captured four days after implantation of the spheroids into collagen gel. (n = 3 independent technical experiments, mean ± s.d.). Statistical analysis using unpaired t-tests method has been performed with the Sidak-Bonferroni method, (p<0.01 ##, **; p<0.001 ###,***).
The AhR-SRC axis as a therapeutic vulnerability in BRAFi-resistant melanoma. (2022)
Figure 2

Figure 2. The activated form of AhR rescues the invasive and BRAFi-resistant phenotype of melanoma cell lines

A. AhR Protein levels in 501Mel CTR cells and those transduced with sgRNA targeting AhR (#1, #2) were analyzed by western blotting.

B. BRAFi sensitivity was established in 501Mel cells or with endogenous overexpression of the constitutive form of the AhR (sgRNA, CRISPRi), by measuring cell density for four days after treatment (every 2 days), with an increasing concentration of BRAFi (vemurafenib). The IC50 (M) was calculated using GraphPad (PRISM9.0®). Statistical analysis (2way ANOVA) has been performed between the mean of 4 independent experiments (SK28R KO vs SK28R) at different concentrations; p<0.001 ***.

C. Three-dimensional spheroid growth of 501Mel cells. Images were captured seven days after spheroid implantation (n = 3 independent technical experiments, mean ± s.d.). Each histogram represents the mean ± s.d. Comparisons with control were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.001 ***.

D. AhR Protein levels in SK28R WT, KO, and KO CA-AhR cells were analyzed by western blotting.

E. BRAFi sensitivity was established in SK28R cells in the absence of AhR (SK28R KO (CRISPR-Cas9) or after overexpression of the constitutive form of AhR (CA-AhR), by measuring the cell density for four days after treatment (every 2 days), with increasing concentrations of BRAFi (vemurafenib). The IC50 (M) was calculated using GraphPad (PRISM9.0®). Statistical analysis (2way ANOVA) has been performed between the mean of 4 independent experiments (SK28R KO or SK28R KO CA-AhR vs SK28R) at different concentrations; p<0.01 **, p<0.001 ***.

F. Three-dimensional spheroid growth of SK28 cells KO or not for AhR by CRISPR/Cas9 and rescued by the constitutive active form of AhR (KO CA-AhR). Images were captured four days after spheroid implantation (n = 3 independent technical experiments, mean ± s.d.). Comparisons were performed using unpaired t-tests with the Sidak-Bonferroni method.

G. Expression heatmap for various gene signatures (established by the median of expression for specific genes) (invasive vs proliferative, alpha, beta, resistant, melanocytic, transitory, neural crest-like, and undifferentiated, see Appendix Table S1) in SK28 BRAFi-sensitive or resistant cell lines KO or not for AhR by CRISPR/Cas9 and rescue with the constitute active form of AhR (KO CA-AhR) (GSE166617). Genes and clusters with similar expression profiles across the cohort are placed close to each other in the grid. The scale corresponds to the Z scores.
The AhR-SRC axis as a therapeutic vulnerability in BRAFi-resistant melanoma. (2022)
Figure 4

Figure 4. Canonical activation of AhR induces the activation of SRC/FAK associated with an increased activation of the focal adhesion pathway in BRAFi-resistant and dedifferentiated melanoma cell lines.

A. At the basal level, AhR is located at the membrane layer in a protein complex that includes HSP90, p23, XAP, and SRC. Immunoprecipitation of SRC was performed with specific antibodies and SRC/AhR interaction analyzed by western blotting.

B. Protein levels of the AhR, p-SRC (Y416), SRC, p-FAK (Y576/577), and FAK in the four different melanoma cell lines (n=3). The level of BRAFi resistance corresponds to our measure of IC50 (Vemurafenib) for the different cell lines 501Mel (0,23uM), SKMel28 (0,29uM), M229 (0,89uM) and M238 (2,16uM). These cell lines correspond to the sensitive parental cells.

C. Protein levels of the AhR, p-SRC (Y416), SRC, p-FAK (Y576/577), and FAK in the SK28R cell line KO or not for AhR by CRISPR/Cas9 or after rescue with the activated-form of the AhR (CA-AhR).

D. Protein levels of AhR, p-SRC (Y416), and SRC, in the SK28R cell line after treatment with different AhR ligands for 24h (5 μM BaP, 5 μM indirubin, 5 μM ITE, 10 nM TCDD, and 5 μM FICZ).

E. Protein levels of AhR, p-SRC (Y416), and SRC, in the SK28R cell line after 24 h of treatment with increasing doses of ITE.

F. Expression heatmap for SRC, FAK, focal-adhesion, and integrin signatures in four different BRAFi-sensitive and resistant melanoma cell lines.

G. Expression heatmap for SRC, FAK, focal-adhesion, and Integrin signatures in the SK28R cell line KO or not for AhR by CRISPR/Cas9 or after rescue with the activated-form of the AhR (CA-AhR). The scale corresponds to the Z scores.