Journal: Nature Communications

Article Title: RNA splicing is a key mediator of tumour cell plasticity and a therapeutic vulnerability in colorectal cancer

doi: 10.1038/s41467-022-30489-z

Figure Lengend Snippet: a Representative images of BrdU-stained mouse small intestines with the indicated genotypes, 5-days after tamoxifen induction. Letters ‘ c ’ and ‘ v ’ indicate demarcated crypt and villus compartments respectively and the red line highlights the proliferative zone. Scale bar 250 µm. b Quantification of proliferative cells in the crypts and villi of the indicated genotypes, n = 4 (WT) vs 3 (Srsf1 fl/+ ) vs 5 (Apc fl/fl ) vs 4 (Apc; fl/fl Srsf1 fl/+ ) biologically independent mice. c Representative images of clonogenicity assays using villi-derived intestinal organoids of indicated genotypes. Scale bar 500 µm. d Quantification of villi-derived organoid clonogenicity, n = 10 vs 7 independent experiments. e qPCR gene expression of Srsf1 in organoids normalised to β-actin, n = 3 vs 4 independent experiments. f qPCR quantification of upregulated late transit-amplifying (differentiated) cell markers in Apc ; fl/fl Srsf1 fl/+ mouse intestinal tissue, relative to Apc fl/fl intestinal tissue, using Gapdh loading control, n = 3 vs 3 biologically independent mice. g Prox1 stem cell marker qPCR quantification in mouse small intestines of indicated genotypes, normalised to Gapdh , n = 3 vs 3 biologically independent mice. h qPCR-derived ratio of Kras splicing isoforms in the indicated genotypes, n = 7 (WT) vs 3 (Srsf1 fl/+ ) vs 8 (Apc fl/fl ) vs 4 (Apc; fl/fl Srsf1 fl/+ ) biologically independent mice. i Images of organoids of the indicated genotype, treated with control or Srsf1 shRNA. AKP ( Apc; ∆/∆ Kras; G12D/+ Trp53 ∆/∆ ), KPN ( Kras; G12D/+ Trp53 ; fl/fl Rosa26 N1icd/+ ). Scale bar 500 µm. j Relative clonogenicity and k size of surviving organoids following control or Srsf1 shRNA treatment, n = 3 vs 3 independent experiments. Data in bar charts are represented as mean and error bars are SD with data analysed with two-tailed, unpaired t-tests, p values are indicated in figure panels. All biological replicates are shown as individual value plots. See also Fig. .

Article Snippet: The following antibodies were used: BrdU (BD Biosciences, 347580, pH6, 1/500), PROX1 (R&D systems, AF2727, pH6, 1/100 for human tissue array, 1/200 for mouse sections), SLC13A2 (Atlas antibodies, HPA014963, pH8, 1/100), SRSF1 (Invitrogen/Thermo Fisher Scientific, 32-4600, pH8, 1/10,000), Ki67 (Abcam, ab15580, pH6, 1/2000), Active Caspase-3 (R&D systems, AF835, pH6, 1/800).

Techniques: Staining, Derivative Assay, Gene Expression, Control, Marker, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: RNA splicing is a key mediator of tumour cell plasticity and a therapeutic vulnerability in colorectal cancer

doi: 10.1038/s41467-022-30489-z

Figure Lengend Snippet: a Representative histological images of mouse colons and tumours stained with hematoxylin and eosin (H&E). Scale bars are 1 mm (2.5x), 500 µm (5x) and 250 µm (10x). Tumours were isolated when mice showed clinical signs of intestinal tumourigenesis. b Quantification of the presence or absence of invasive tumours in mice from the indicated genotypes, with statistical difference calculated using a two-sided Chi-square test, n = 23 vs 34 biologically independent mice. c Box and whisker plot showing the number of invasive tumours as a percentage of the total number of tumours for each mouse for each genotype. The box extends from 25th to 75th centiles, the centre line is median and whiskers extend to minima and maxima, n = 23 vs 34 biologically independent mice. d Representative histological images of p53 fl/fl and p53; fl/fl Srsf1 fl/+ mouse intestines and tumours stained for PROX1 (stem cell marker) and SLC13A2 (differentiation marker). Scale bars are 2.5 mm (1.25x), 500 µm (5x) and 50 µm (40x). e Histoscore and staining strength quantification for PROX1, n = 5 vs 6 biologically independent mice and f SLC13A2, n = 7 vs 5 biologically independent mice. g Schematic depiction of the experimental strategy used to investigate the invasive potential of tumour-derived intestinal organoids. h Representative images of calcein-stained organoids once they had invaded through Matrigel and through the porous membrane at the bottom of the assay well. Scale bar 500 µm. i Quantification of the invasive potential of p53 fl/fl tumour-derived organoids following control or Srsf1 shRNA manipulation, n = 9 vs 9 independent experiments. Data in bar charts are represented as mean and error bars are SD with data analysed with two-tailed, unpaired t -tests, p values are indicated in figure panels. All biological replicates are shown as individual value plots and n > 3. See also Fig. .

Article Snippet: The following antibodies were used: BrdU (BD Biosciences, 347580, pH6, 1/500), PROX1 (R&D systems, AF2727, pH6, 1/100 for human tissue array, 1/200 for mouse sections), SLC13A2 (Atlas antibodies, HPA014963, pH8, 1/100), SRSF1 (Invitrogen/Thermo Fisher Scientific, 32-4600, pH8, 1/10,000), Ki67 (Abcam, ab15580, pH6, 1/2000), Active Caspase-3 (R&D systems, AF835, pH6, 1/800).

Techniques: Staining, Isolation, Whisker Assay, Marker, Derivative Assay, Membrane, Control, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: RNA splicing is a key mediator of tumour cell plasticity and a therapeutic vulnerability in colorectal cancer

doi: 10.1038/s41467-022-30489-z

Figure Lengend Snippet: a A sample of cores taken from a human colon cancer tissue microarray (TMA), with the same respective core stained for SRSF1 or PROX1 on different sections. Scale bar 500 µm. b Linear regression analysis showing the correlation of SRSF1 and PROX1 staining (based on histoscore) on the TMA shown in a , with each datapoint representing a core taken from a patient. c Relationship between SRSF1 immunohistochemistry staining and tumour stage in human patients, using TMA CO2081b, n = 54 vs 56 biologically independent tumour cores. d Representative images of patient-derived organoids (PDOs) treated with control or Srsf1 shRNAs. MD175 (polyp), MD20853 (CRC), MD20910 (CRC), MD19648 (FAP rectum Tumour), MD20043 (rectal carcinoma), C-002 (liver metastasis). Scale bar 1000 µm. e Number of surviving organoid clones after shRNA treatment and f size of indicated PDOs, n = 3 vs 3 independent experiments. g Viability of PDO MD20043 after Srsf1 shRNA treatment and h mRNA expression (qPCR) of indicated genes, n = 3 vs 3 independent experiments. i Model outlining the role of SRSF1 in modulating tumour cell plasticity and invasion in colorectal cancer. Data in bar charts are represented as mean and error bars are SD with data analysed with two-tailed, unpaired t- tests, p values are indicated in figure panels. All biological replicates are shown as individual value plots and n > 3. See also Figure .

Article Snippet: The following antibodies were used: BrdU (BD Biosciences, 347580, pH6, 1/500), PROX1 (R&D systems, AF2727, pH6, 1/100 for human tissue array, 1/200 for mouse sections), SLC13A2 (Atlas antibodies, HPA014963, pH8, 1/100), SRSF1 (Invitrogen/Thermo Fisher Scientific, 32-4600, pH8, 1/10,000), Ki67 (Abcam, ab15580, pH6, 1/2000), Active Caspase-3 (R&D systems, AF835, pH6, 1/800).

Techniques: Microarray, Staining, Immunohistochemistry, Derivative Assay, Control, Clone Assay, shRNA, Expressing, Two Tailed Test

Journal: Arthritis Research & Therapy

Article Title: TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

doi: 10.1186/ar2038

Figure Lengend Snippet: TWEAK/Fn14 interaction induces RANTES production by MC3T3-E1 cells. (a) MC3T3-E1 cells were cultured in the presence or absence of 100 ng/ml TWEAK for 48 hours. The culture supernatants were then collected and subjected to a cytokine protein array. The table indicates the corresponding cytokines on the protein array membrane. (b) MC3T3-E1 cells were stimulated with the indicated doses of TWEAK for 48 hours in the presence or absence of the indicated doses (ng/ml) of mouse Fn14-Fc chimera or control mouse immunoglobulin G (mIgG). The culture supernatants were then collected, and the RANTES concentrations were measured by enzyme-linked immunosorbent assay. (c) MC3T3-E1 cells were stimulated with the indicated doses of TWEAK for 96 hours. Viable cell number was then measured by WST assay. The data were expressed as OD units. Values represent the mean ± standard deviation. * p < 0.05 compared with corresponding control. Similar results were obtained in at least three independent experiments. Fn14, fibroblast growth factor-inducible 14; G-CSF, granulocyte-cell-stimulating factor; GM-CSF, granulocyte macrophage-colony-stimulating factor; IFN-γ, interferon-γ; IL, interleukin; IP-10, inositol phosphate-10; M-CSF, macrophage-colony-stimulating factor; MIG, monokine induced by IFN-γ; MIP-1α, viral macrophage inflammatory protein-1α; OD, optical density; RANTES, regulated upon activation, healthy T cell expressed and secreted; TNF-α, tumour necrosis factor-α; TWEAK, tumour necrosis factor-like weak inducer of apoptosis; VEGF, vascular endothelial growth factor; WST, water-soluble tetrazolium.

Article Snippet: The amounts of several cytokines in the culture supernatants were then determined by using a cytokine protein array (TranSignal Mouse Cytokine Antibody Array 1.0; Panomics, Inc., Fremont, CA, USA) according to the manufacturer's instructions.

Techniques: Cell Culture, Protein Array, Membrane, Control, Enzyme-linked Immunosorbent Assay, WST Assay, Standard Deviation, Activation Assay