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: BioMed Research International

Article Title: Anti-Inflammatory and Protective Effects of Juncus effusus L. Water Extract on Oral Keratinocytes

doi: 10.1155/2022/9770899

Figure Lengend Snippet: Effects of the Juncus effusus water extract on chemokine production in Porphyromonas gingivalis lipopolysaccharide- (LPS-) stimulated oral keratinocytes. RT-7 cells were pretreated with the J. effusus water extract (20-fold dilution) for 3 min. After the removal of the medium, RT-7 cells were cultured with P. gingivalis LPS (1 μ g/mL) for 24 h. The detection of chemokines in cell culture supernatants was performed using the Human Chemokine Antibody Array 1. (a) The results shown are representative images of two independent experiments with similar results. (b) A densitometric analysis of various chemokine productions. Bars indicate the relative densitometric intensities after the values were normalized with both positive and negative controls and background. In particular, positive controls were used to normalize the values from different membranes being compared. Values represent the means ± standard deviations of two independent experiments. ∗ P < 0.05 and ∗∗ P < 0.01 show significant differences between the indicated groups.

Article Snippet: Chemokine detection in cell culture supernatants obtained from RT-7 cells treated with P. gingivalis LPS and/or the J. effusus L. water extract was performed using the Human Chemokine Antibody Array 1 (Ray Biotech, Inc., Norcross, GA, USA), according to the manufacturer's instructions.

Techniques: Cell Culture, Ab Array