Journal: Oncotarget

Article Title: Melanoma cell therapy: Endothelial progenitor cells as shuttle of the MMP12 uPAR-degrading enzyme

doi:

Figure Lengend Snippet: Expression of CXCR4/SDF1 system in A375, MSC and ECFC cells evaluated by RT-PCR analysis in basal conditions (panel A), in A375 cells incubated with CM-MSC and with CM-ECFC (panel B). Panel C: SDF-1 expression after the knockdown of SDF-1 gene in MSC. Panel D: ECFC Matrigel invasion by adding in the lower compartment of Boyden chamber of: control media (picture 1); conditioned media from A375 (picture 2); CM-MSC (picture 3), and CM from cocolture A375/MSC (picture 5). Picture 4 and 6 show ECFC invasion upon addition in the lower compartment of CM from SDF1-silenced MSC or from A375/MSC SDF1-silenced cocolture, respectively; picture 7 and 8 show the invasion of ECFC in the presence of anti-CXCR4 antibody, in control condition (picure 7) and after CM-co-colture incubation (picture 8). Histogram on the right represent the quantification of Matrigel invasion performed by counting migrated cells as described in Figures . Asterisks (* P <0.05) indicate significant difference between the indicated experimental condition. Panel E: in vivo experiments. A375 melanoma cells were injected alone (control) or together with MSC in CD1-nude mice and the development of tumor was followed by measuring the tumor volume. Five mice were used for each experimental condition and the results are the mean of 3 different experiments performed in triplicate. Asterisks indicate significant difference (* P < 0.05) from control. Panel F: In vivo recruitment of ECFCs into tumor mass. CD1-nude mice were grafted subcutaneously with viable melanoma cells together with MSC and then ECFCs, radiolabelled with 111 In 8-oxyquinoline (oxine), were injected intravenously when the tumor reached about 150 mm 3 volume, in the absence or in the presence of anti-CXCR4 or irrelevant antibody. 24 hours after radiolabelled ECFCs administration, SPECT tomography was performed as described in material and method. SPECT images show a selective visualization of radioactive ECFCs in the tumor mass. Figure shows a representative experiment (n=3). On the left: SPECT images of ECFC engraftment in the tumor mass at 30 min and 24 h (upper panel) and ECFC engraftment in the presence of irrelevant IgG or anti-CXCR4 antibody (lower panel). On the right: relative histogram of uptake efficiency.

Article Snippet: In some experiments, conditioned medium (CM) was prepared with MSCs pretreated with small-interfering-RNA (siRNA) and negative control constructs obtained from Riboxx Life Sciences/Euroclone (IBONI siRNA-pool, targeting human SDF1). siRNA constructs were incorporated into RiboxxFECT transfection reagent according to the manufacturer's instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, In Vivo, Injection, Single Photon Emission Computed Tomography, Tomography

Journal: Oncotarget

Article Title: Melanoma cell therapy: Endothelial progenitor cells as shuttle of the MMP12 uPAR-degrading enzyme

doi:

Figure Lengend Snippet: ECFCs derived from cord blood, were transduced with lentiviral vectors encoding the matrix metalloproteinase 12 (MMP12). Subcutaneous tumors were developed by direct co-injection of A375 and MSCs. Six days after tumor cells implantation, genetically modified ECFCs labelled with 60 μCi of 111In 8-oxyquinoline (oxine) were injected intravenously into CD-1 nude (nu/nu). 24 hours later the scintigraphic imagine showed that radiolabeled ECFCs were localized only in the tumor mass. High expression of SDF1 by tumour cells and MSCs form a local gradient of the chemokine in the tumour region. CXCR4-expressing ECFCs are thus recruited to the tumour, where they release active MMP12 enzyme which cleaves uPAR impairing angiogenesis and tumor growth.

Article Snippet: In some experiments, conditioned medium (CM) was prepared with MSCs pretreated with small-interfering-RNA (siRNA) and negative control constructs obtained from Riboxx Life Sciences/Euroclone (IBONI siRNA-pool, targeting human SDF1). siRNA constructs were incorporated into RiboxxFECT transfection reagent according to the manufacturer's instructions.

Techniques: Derivative Assay, Transduction, Injection, Genetically Modified, Expressing

Journal: Oncotarget

Article Title: Melanoma cell therapy: Endothelial progenitor cells as shuttle of the MMP12 uPAR-degrading enzyme

doi:

Figure Lengend Snippet:

Article Snippet: In some experiments, conditioned medium (CM) was prepared with MSCs pretreated with small-interfering-RNA (siRNA) and negative control constructs obtained from Riboxx Life Sciences/Euroclone (IBONI siRNA-pool, targeting human SDF1). siRNA constructs were incorporated into RiboxxFECT transfection reagent according to the manufacturer's instructions.

Techniques:

Journal: iScience

Article Title: Sox9 regulates melanocytic fate decision of adult hair follicle stem cells

doi: 10.1016/j.isci.2023.106919

Figure Lengend Snippet: Recombination in adult Nestin-CreER T2 transgenic mice targets Sox9-expressing hair follicle stem cells (A) Schematic drawing of an adult hair follicle (HF) showing the localisation of Nestin-expressing cells (Nestin + ) (green) and melanocytic cells in various differentiation stages; ORS = outer root sheath. (B) Schematic drawing depicting the applied tamoxifen protocol to track Nestin + cells and their progeny in vivo ; Nestin-CreER T2 ; GFP mice (Ctrl mice) were intraperitoneally injected with tamoxifen every 12 h for 5 consecutive days starting at P56 and were killed 7 days post-injection (dpi). (C) Representative immunohistochemical staining for GFP (green) and Sox9 (red) in tail skin of tamoxifen-treated Ctrl mice 7 dpi. Yellow arrows point toward recombined GFP + /Sox9 + cells in the bulge; dashed line marks the hair follicle; E = Epidermis, HF = Hair follicle, SG = Sebaceous gland, IF = Interfollicular area, B = Bulge, ORS = Outer root sheath, Bu = Bulb; DAPI is depicted in blue (scale bar = 50 μm). (D) Immunohistochemical staining of GFP (green), Sox9 (red) and Nestin (white) in the hair follicle of Ctrl mice; Magnifications show signals in the bulge (D′); Yellow arrows point toward Sox9 + /Nestin + /GFP + cells; dashed line marks the hair follicle; HF = Hair follicle, SG = Sebaceous gland, B = Bulge; DAPI is depicted in blue (scale bar = 20 μm). (E) The graphs show the quantification of Sox9 + /GFP + over total GFP + cells per HF in tamoxifen-treated Ctrl mice 7 dpi; n = 4 mice, 4–10 HF per mice. (F) Quantification of Sox9 + /Nestin + /GFP + over total Nestin + /GFP + cells per HF in tamoxifen-treated Ctrl mice 7 dpi; n = 4 mice, 4–10 HF per mice. Data are presented as mean ± SEM.

Article Snippet: On the following day, we transfected the cells with siRNA pool against human Sox9 (siTools Biotech GmbH, Planegg, Germany) or a control siPool (siCTR), respectively, using the LipofectamineTM RNAiMAX transfection reagent (Life Technologies, Darmstadt, Germany).

Techniques: Transgenic Assay, Expressing, In Vivo, Injection, Immunohistochemical staining, Staining

Journal: iScience

Article Title: Sox9 regulates melanocytic fate decision of adult hair follicle stem cells

doi: 10.1016/j.isci.2023.106919

Figure Lengend Snippet: Expression of Sox9 in ORS melanocytic cells (A) Quantification of Sox9 + /DCT + /GFP + cells over total DCT + /GFP + cells per HF in Ctrl mice; 7 dpi; n = 4 mice, 4–10 HF per mice; data are presented as mean ± SEM. (B) Immunohistochemical staining of GFP (green), Sox9 (red) and DCT (white) in tamoxifen-treated Ctrl mice 7 dpi. Magnifications show signals in the ORS (B′) and hair bulb (B''); Yellow arrows point toward Sox9 + /DCT + /GFP + (upper panels, ORS) and yellow arrowheads point toward Sox9 neg /DCT + /GFP + cells (lower panels, Bulb); dashed line marks the hair follicle; E = Epidermis, HF = Hair follicle, B = Bulge, ORS = Outer root sheath, Bu = Bulb; DAPI is depicted in blue (scale bar = 20 μm).

Article Snippet: On the following day, we transfected the cells with siRNA pool against human Sox9 (siTools Biotech GmbH, Planegg, Germany) or a control siPool (siCTR), respectively, using the LipofectamineTM RNAiMAX transfection reagent (Life Technologies, Darmstadt, Germany).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: iScience

Article Title: Sox9 regulates melanocytic fate decision of adult hair follicle stem cells

doi: 10.1016/j.isci.2023.106919

Figure Lengend Snippet: Increased expression of Sox9 in immature/dedifferentiated melanocytic cells (A) Immunohistochemical staining of GFP (green), Sox9 (red) and DCT (white) in the epidermis of tamoxifen-treated Ctrl mice 7 dpi; n = 3 mice, 2–5 20-μm-thick sections of 1 cm long mice tail skin per mice. Yellow arrowheads point toward Sox9 neg /DCT + cells; dashed line marks the epidermis; E = Epidermis; DAPI is depicted in blue (scale bar = 25 μm). (B and C) cDNA microarray (n = 3; Table S2 , for details, please see Link-Paulus et al. ) and (C) qRT-PCR (n = 3; normalized to β-Actin) analyzing Sox9 expression in melanoblast-related-cells (MBrc) compared to its expression in normal human epidermal melanocytes (NHEM). (D) Representative Western Blot image, lanes were cut out of a blot image including other, irrelevant lanes (n = 3; normalized to β-Actin); (B-D) students two-sided t- test was applied to determine statistical significance (∗p< 0.05). Data are presented as mean ± SEM.

Article Snippet: On the following day, we transfected the cells with siRNA pool against human Sox9 (siTools Biotech GmbH, Planegg, Germany) or a control siPool (siCTR), respectively, using the LipofectamineTM RNAiMAX transfection reagent (Life Technologies, Darmstadt, Germany).

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: Sox9 regulates melanocytic fate decision of adult hair follicle stem cells

doi: 10.1016/j.isci.2023.106919

Figure Lengend Snippet: Sox9 ablation leads to a decrease in the number of DCT + (/Nestin + )/GFP + cells in the HF (A) Schematic demonstration of the applied tamoxifen protocol to specifically ablate Sox9 in Nestin + cells in HF of adult mice in vivo ; Either Nestin-CreER T2 ; GFP mice (Ctrl) or Nestin-CreER T2 ; GFP with floxed alleles of Sox9 (Sox9KO) were intraperitoneally injected with tamoxifen every 12 h for 5 consecutive days starting at P56 and sacrificed 7 dpi. (B) Quantification of Sox9 + /GFP + cells over total GFP + cells per HF in Sox9KO mice and Ctrl mice 7 dpi confirming successful knock out of Sox9; n = 4 mice per group; 4–10 HF per mice; data are presented as mean ± SEM. (C) Quantification of the mean cell number of GFP + or Nestin + /GFP + cells per HF in Sox9KO mice and Ctrl mice 7 dpi; n = 4 mice per group; 12 HF per mice; data are presented as mean ± SEM. (D) Immunohistochemical staining for GFP (green), Nestin (red) and DCT (white) in Sox9KO mice and Ctrl mice 7 dpi; dashed line marks the hair follicle; E = Epidermis, IF = Interfollicular area, B = Bulge, ORS = Outer root sheath, Bu = Bulb; Magnifications show signals in the bulge (upper panels), the ORS (middle panels) and the hair bulb (lower panels); DAPI is depicted in blue (scale bar = 20 μm); yellow arrows point toward DCT + /Nestin + /GFP + cells (upper, middle panels) and DCT + /Nestin neg /GFP + cells (middle, lower panels); yellow arrowheads point toward DCT neg /Nestin neg /GFP + cells (upper panels) and DCT + /Nestin neg /GFP neg cells (middle panels (Sox9KO), lower panels). (E) Quantification of the mean cell number of DCT + /GFP + cells in the bulge/ORS and bulb in Sox9KO mice and Ctrl mice 7 dpi; n = 4 mice per group; 5–12 HF per mice; data are presented as mean ± SEM. (F) Quantification of the mean cell number of DCT + /Nestin + /GFP + cells in the bulge in Sox9KO mice and Ctrl mice 7 dpi; n = 4 mice per group; 5–12 HF per mice; data are presented as mean ± SEM. (G) Quantification of the mean cell number of DCT + cells per HF in Sox9KO mice and Ctrl mice 7 dpi; n = 4 mice per group; 12–15 HF per mice; data are represented as mean ± SEM. (B, E, F, G) students two-sided t -test was applied to determine statistical significance (∗: p < 0.05, ∗∗: p < 0.01).

Article Snippet: On the following day, we transfected the cells with siRNA pool against human Sox9 (siTools Biotech GmbH, Planegg, Germany) or a control siPool (siCTR), respectively, using the LipofectamineTM RNAiMAX transfection reagent (Life Technologies, Darmstadt, Germany).

Techniques: In Vivo, Injection, Knock-Out, Immunohistochemical staining, Staining

Journal: iScience

Article Title: Sox9 regulates melanocytic fate decision of adult hair follicle stem cells

doi: 10.1016/j.isci.2023.106919

Figure Lengend Snippet: Sox9 ablation leads to a reduced number of proliferating melanocytic cells accompanied by a hyperproliferation of stem cells (A) Immunohistochemical staining of GFP (green), Ki67 (red) and Nestin (white) in Ctrl mice and Sox9KO mice 7 dpi; magnifications show signals in the bulge in Ctrl mice (A′) and Sox9KO mice (A''); dashed line marks the hair follicle; B = Bulge; DAPI is depicted in blue (scale bar = 20 μm). Yellow arrows point toward triple positive cells (Ki67 + /Nestin + /GFP + ). (B) Quantification of the mean number of Ki67 + /GFP + cells in Sox9KO and Ctrl mice 7 dpi; n = 4 mice per group; 6–12 HF per mice; data are presented as mean ± SEM. (C) Quantification of the mean number of Ki67 + /Nestin + /GFP + cells in the bulge of Sox9KO and Ctrl mice 7 dpi; n = 4 mice per group; 4–12 HF per mice; data are presented as mean ± SEM. (D) Quantification of the mean number of Ki67 + /DCT + /GFP + cells per HF in Sox9KO and Ctrl mice 7 dpi; n = 4 mice per group; 6–12 HF per mice; data are presented as mean ± SEM. (E) Immunohistochemical staining of GFP (green), Ki67 (red) and DCT (white) cells in Ctrl mice and Sox9KO mice 7 dpi; magnifications show signals in the bulb of Ctrl mice (E′) and SoxKO mice (E''); dashed line marks the hair follicle; Bu = Bulb; DAPI is depicted in blue (scale bar = 20 μm); Yellow arrows point toward Ki67 + /DCT + /GFP + cells; yellow arrowheads point toward Ki67 neg /DCT + /GFP neg cells (C) – (D) students two-sided t -test was applied to determine statistical significance (∗: p < 0.05, ∗∗: p < 0.01).

Article Snippet: On the following day, we transfected the cells with siRNA pool against human Sox9 (siTools Biotech GmbH, Planegg, Germany) or a control siPool (siCTR), respectively, using the LipofectamineTM RNAiMAX transfection reagent (Life Technologies, Darmstadt, Germany).

Techniques: Immunohistochemical staining, Staining

Journal: iScience

Article Title: Sox9 regulates melanocytic fate decision of adult hair follicle stem cells

doi: 10.1016/j.isci.2023.106919

Figure Lengend Snippet: Sox9 ablation leads to an increase of GFP + /GFAP + cells in the interfollicular area (A) Quantification of DCT neg /Nestin + /GFP + and DCT + /Nestin + /GFP + cells in Nestin + /GFP + cells in the bulge of Sox9KO mice and Ctrl mice 7 dpi; n = 4 mice per group; 5–12 HF per mice; data are presented as mean ± SEM. (B) Quantification of the mean cell number of DCT neg /Nestin + /GFP + cells in the bulge in Sox9KO mice and Ctrl mice 7 dpi; n = 4 mice per group; 5–11 HF per mice; data are presented as mean ± SEM. (C) Schematic demonstration of the experimental mouse model used to specifically ablate Sox9 in Nestin + stem cells in the hair follicle of adult mice in vivo ; adult Nestin-CreER T2 ;GFP mice (Ctrl mice) with floxed alleles of Sox9 (Sox9KO mice) were intraperitoneally injected with Tamoxifen every 12 h for 5 consecutive days starting at P56 and killed 28 dpi. (D) Immunohistochemical staining of GFP (green) in the IF of tail skin of Sox9KO mice and Ctrl mice 28 dpi; schematic drawing demonstrates the localisation of the region of interest; dashed line marks the hair follicle; E = Epidermis, IF = Interfollicular area, B = Bulge, SG = Sebaceous gland; yellow arrows point toward GFP + cells; DAPI is depicted in blue (scale bar = 50 μm). (E) Quantification of the mean cell number of GFP + cells per IF in Sox9KO mice and Ctrl mice 28 dpi; n = 4 mice per group; 4 IF per mice; data are presented as mean ± SEM. (F) Immunohistochemical staining of GFP (green) and GFAP (red) in the IF area of tail skin of Sox9KO mice and Ctrl mice 28 dpi; E = Epidermis, IF = Interfollicular area; yellow arrows point toward GFAP + /GFP + cells; yellow arrowheads point toward GFAP neg /GFP + cells; Magnification shows GFAP + /GFP + cells (F′); DAPI is depicted in blue (scale bar = 50 μm). (G) Quantification of the mean cell number of GFAP + /GFP + cells per IF in Sox9KO mice and Ctrl mice 28 dpi; n = 4 mice per group; 4 IF per mice; data are presented as mean ± SEM. (A, B, E, H) students two-sided t-test was applied to determine statistical significance (∗: p < 0.05, ∗∗: p < 0.01).

Article Snippet: On the following day, we transfected the cells with siRNA pool against human Sox9 (siTools Biotech GmbH, Planegg, Germany) or a control siPool (siCTR), respectively, using the LipofectamineTM RNAiMAX transfection reagent (Life Technologies, Darmstadt, Germany).

Techniques: In Vivo, Injection, Immunohistochemical staining, Staining

Journal: iScience

Article Title: Sox9 regulates melanocytic fate decision of adult hair follicle stem cells

doi: 10.1016/j.isci.2023.106919

Figure Lengend Snippet:

Article Snippet: On the following day, we transfected the cells with siRNA pool against human Sox9 (siTools Biotech GmbH, Planegg, Germany) or a control siPool (siCTR), respectively, using the LipofectamineTM RNAiMAX transfection reagent (Life Technologies, Darmstadt, Germany).

Techniques: Reverse Transcription, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Western Blot, Sequencing

Journal: bioRxiv

Article Title: Androgen receptor-negative prostate cancer is vulnerable to SWI/SNF-targeting degrader molecules

doi: 10.1101/2024.03.24.586276

Figure Lengend Snippet: a, ATAC-seq differential accessibility plot from WCM1078 organoids treated for 24h with 1µM A947 or A858. b, RT-PCR measurement of TCF7L2 mRNA in WCM1078 after treatment with indicated drugs. The housekeeping gene is GAPDH. c, Immunoblot of indicated proteins 72h after transfection with indicated siRNA. GAPDH and Ponceau S serve as loading control. d, Growth data measured by live-cell imaging (Incucyte S3) upon transfection of indicated siRNA. Data are presented as mean values +/− SEM and analyzed using two-way ANOVA (ns= not significant, *p < 0.05). e, Proliferation was measured with CellTiter Glo 2.0 upon transfection of the indicated siRNA. Data are presented as mean values +/− SEM and analyzed using Students t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). f The TOPFlash TCF/LEF reporter assay was measured after 48h upon transfection and 24h upon treatment with indicated drugs in HEK293T cells. FOPFlash serves as a negative control. Data are presented as mean values +/− SEM and analyzed using Students t-test (ns= not significant, *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: After overnight incubation, cells were transfected with siTools TCF7L2 siRNA pool using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) to the proportions of 2 μL of 20nM siRNA per well.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Live Cell Imaging, Reporter Assay, Negative Control

Journal: bioRxiv

Article Title: Androgen receptor-negative prostate cancer is vulnerable to SWI/SNF-targeting degrader molecules

doi: 10.1101/2024.03.24.586276

Figure Lengend Snippet: a, ATAC-seq read-density tornado plots from WCM1078 organoids treated with 1µM A858 or 1µM A947 for 24h (n = 4 biological replicates). Venn diagram indicating lost and gained regions. b, Motifs enriched in depleted peaks from WCM1078 treated for 24h with 1µM A947 identified using HOMER on ATAC-seq. c, Growth data measured by live-cell imaging (Incucyte S3) upon transfection of indicated siRNA or plasmid (EV or rescue). Immunoblot of indicated proteins at 72h after transfection. GAPDH serves as loading control. Data are presented as mean values +/− SEM and analyzed using two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). EV: empty vector. d, Spheroid formation measured by live-cell imaging (Incucyte SX5) upon transfection of indicated siRNA. Data are presented as mean values +/− SEM and analyzed using two-way Anova (*p < 0.05, **p < 0.01, ***p < 0.001). e, Immunohistochemistry (IHC) and staining intensity of TCF7L2 on indicated organoids upon treatment with 1µM A858 or 1µM A947 for 24h. Violin plot from TCF7L2 staining intensity analyzed using two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Scale: 100µm (MSK-PCa16), 50µm (WCM1078). f, Immunoblot of indicated proteins at indicated time upon treatment with 1µM A947. GAPDH serves as loading control. g, TOPFlash TCF/LEF reporter assay measured after 48h upon transfection and 24h upon treatment with indicated drugs in MSK-PCa16. FOPFlash serves as a negative control. Data are presented as mean values +/− SEM and analyzed using Students t-test (ns= not significant, *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: After overnight incubation, cells were transfected with siTools TCF7L2 siRNA pool using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) to the proportions of 2 μL of 20nM siRNA per well.

Techniques: Live Cell Imaging, Transfection, Plasmid Preparation, Western Blot, Immunohistochemistry, Staining, Reporter Assay, Negative Control

Journal: bioRxiv

Article Title: Androgen receptor-negative prostate cancer is vulnerable to SWI/SNF-targeting degrader molecules

doi: 10.1101/2024.03.24.586276

Figure Lengend Snippet: a, ChIP-seq read-density tornado plots from WCM1078 organoids treated with 1µM A858 or 1µM A947 for 24h (n = 2 biological replicates). Venn diagram indicating lost and gained regions. b, Venn diagram indicating lost and gained regions from ChIP-seq data. c, Motifs enriched in depleted peaks from WCM1078 treated for 24h with 1µM A947 identified using HOMER from TCF7L2 ChIP-seq. d, GSEA of TCF7L2 ChIP-seq identified depleted peaks upon A947-treatment. e, Immunoblot of indicated proteins at an indicated time upon treatment with 1µM A947. GAPDH serves as loading control. f, Dose-response curves with indicated drugs after measurement of proliferation with Celltiter Glo 2.0 after 7-day treatment (n=2 independent biological experiments).

Article Snippet: After overnight incubation, cells were transfected with siTools TCF7L2 siRNA pool using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) to the proportions of 2 μL of 20nM siRNA per well.

Techniques: ChIP-sequencing, Western Blot

Journal: bioRxiv

Article Title: Androgen receptor-negative prostate cancer is vulnerable to SWI/SNF-targeting degrader molecules

doi: 10.1101/2024.03.24.586276

Figure Lengend Snippet: a, Immunoblot of indicated proteins at indicated time upon transfection with siTCF7L2 after 72h. Vinculin serves as a loading control. b, Correlation plot comparing ATAC-seq and PRO-cap from A947-treated WCM1078. c, Correlation plot comparing TCF7L2 ChIP-seq and PRO-cap from A947-treated WCM1078. d, Density tornado plot from overlap of TCF7L2 ChIP-seq and PRO-cap. e, Immunoblot of indicated proteins at the indicated time upon transfection with siJUN after 72h. Actin serves as loading control. f, PLA with indicated antibodies after treatment with 1µM A858 or A947 for 24h. DAPI was used for nuclear staining. Scale bar: 10 µm. g, Immunoblot of indicated proteins after co-immunoprecipitation with β-Catenin antibody from MSK-PCa16 nuclear extracts after 24h treatment with 1µM control A858 or A947. Rabbit IgG serves as the isotype control, and Histone 3 (H3) serves as the control for nuclear extract.

Article Snippet: After overnight incubation, cells were transfected with siTools TCF7L2 siRNA pool using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) to the proportions of 2 μL of 20nM siRNA per well.

Techniques: Western Blot, Transfection, ChIP-sequencing, Staining, Immunoprecipitation