"CausalXtract: a flexible pipeline to extract causal effects from live-cell time-lapse imaging data" datasets

Datasets from the article: CausalXtract: a flexible pipeline to extract causal effects from live-cell time-lapse imaging data by Franck Simon, Maria Colomba Comes, Tiziana Tocci, Louise Dupuis, Vincent Cabeli, Nikita Lagrange, Arianna Mencattini, Maria Carla Parrini, Eugenio Martinelli, Hervé Isambert. The original videos are uploaded as: "20161230.zip", "20170105.rar", "Video_2017_0517.zip". Details for each experiment can be found in: "Experiments' details.zip". The ROIs (ROI: Region of Interest) are uploaded as .tif files in: "EXTRACTED ROIs.zip". The MATLAB data is uploaded in "MATLAB_DATA.rar" and includes: the cancer cells' trajectories (subfolder: "TUMOR TRAJECTORIES"), the immune cells' trajectories (subfolder: "IMMUNE TRAJECTORIES"), the ROIs videos as .mat files for the detection of cells (subfolder: "ROI MAT"), the ROIs further cropped for the extraction of shape descriptors (folder: "ROI_TU MAT"). The ROIs videos .mat files included in the last two subfolders are stopped after their apoptosis has been detected

RalGPS2 Is Essential for Survival and Cell Cycle Progression of Lung Cancer Cells Independently of Its Established Substrates Ral GTPases

<div><p>The human genome contains six genes coding for proteins validated <i>in vitro</i> as specific activators of the small GTPases “Ras-related protein Ral-A” and “Ras-related protein Ral-B”, generically named Ral-guanine nucleotide exchange factors (RalGEF). Ral proteins are important contributors to Ras oncogenic signaling, and <i>RAS</i> oncogenes are important in human Non-Small Cell Lung Carcinoma (NSCLC). Therefore in this work, RalGEF contribution to oncogenic and non-oncogenic features of human NSCLC cell lines, as anchorage-dependent and independent growth, cell survival, and proliferation, was investigated. Among all human RalGEF, silencing of <i>RGL1</i> and <i>RALGPS1</i> had no detectable effect. However, silencing of either <i>RGL2</i>, <i>RGL3</i>, <i>RALGDS</i> or, to a larger extent, <i>RALGPS2</i> inhibited cell population growth in anchorage dependent and independent conditions (up to 90 and 80%, respectively). <i>RALGPS2</i> silencing also caused an increase in the number of apoptotic cells, up to 45% of the cell population in transformed bronchial BZR cells. In H1299 and A549, two NSCLC cell lines, <i>RALGPS2</i> silencing caused an arrest of cells in the G0/G1-phase of cell cycle. Furthermore, it was associated with the modulation of important cell cycle regulators: the E3 Ubiquitin Protein Ligase S-phase kinase-associated protein 2 (Skp2) was strongly down-regulated (both at mRNA and protein levels), and its targets, the cell cycle inhibitors p27 and p21, were up-regulated. These molecular effects were not mimicked by silencing <i>RALA</i>, <i>RALB</i>, or both. However, <i>RALB</i> silencing caused a modest inhibition of cell cycle progression, which in H1299 cells was associated with Cyclin D1 regulation. In conclusion, <i>RALGPS2</i> is implicated in the control of cell cycle progression and survival in the <i>in vitro</i> growth of NSCLC cell lines. This function is largely independent of Ral GTPases and associated with modulation of Skp2, p27 and p21 cell cycle regulators.</p></div

<i>RALGPS2</i> silencing induces pronounced anchorage-dependent and anchorage-independent cell population growth inhibition.

<p>(A—F) Cell population growth in adherence was measured using the resazurin reduction assay and is expressed as percentage of untreated (non-transfected) cells (mean ± SEM,). It was evaluated from 72 to 120 h after cell transfection, depending on the optimized condition for each individual cell line. Each panel depicts the effect of silencing one RalGEF with two or three independent siRNA oligonucleotides: (A) <i>RGL1</i>; (B) <i>RGL2</i>; (C) <i>RGL3</i>; (D) <i>RALGDS</i>; (E) <i>RALGPS1</i>; (F) <i>RALGPS2</i>. The effect of each siRNA was statistically compared with the effect obtained with siNT by Two-way ANOVA and Bonferroni posttests (*<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001; <i>n</i> = 2 to 4 mean values from independent experiments, each done in quadruplicate wells). (G) Anchorage-independent growth of H1299 cells was evaluated over 14 days and is expressed as mean relative colony number with respect to siNT-treated cells. Data were collected from independent experiments where siPLK1 (positive control) originated none or very few colonies. (H) Illustrative images of H1299 colonies in agarose at 14 days of growth after siNT, siPLK1, siRalGPS2 #231, and siRalGPS2 pool, as indicated. Statistical comparison was done by One-way ANOVA with Dunnett’s posttest. (*<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001; <i>n</i> = 2 (siRGL1 and siRALGPS1), 3 (siRalGPS1 and siRalGPS2) or 4 (siRGL2, siRGL3 and siRalGDS) independent experiments, each performed in triplicate wells.</p

Ral-GTP levels in H1299 cells after RalGPS2 depletion.

<p>H1299 “starved” cells were obtained by overnight (~19 h) culture without serum before lysis at 48 h post-transfection. (A) Pull-down was performed as independent duplicates from the same lysates at 0.4 mg protein/pull-down/lane (0.5 mg/ml). The input corresponds to 25-fold less protein (16 μg/lane). 20 μg GST-Sec5-RBD and 40 μl beads were used per pull-down. (B) Quantification of immunoblots (IB) Ral-GTP/total Ral ratios from “starved” cells and expressed relatively to siNT (mean ± SD, <i>n</i> = 2 or, in the case of siRalGPS2_ft10, 3 independent pull-downs). (C) IB signal quantification from the experiment with cells “not starved” (mean ± SD, <i>n</i> = 2 independent pull-downs). No significant differences were found among means at a significance level of 0.05 (Two-way ANOVA with Bonferroni posttests).</p

Taqman qPCR probe sets.

<p>Taqman qPCR probe sets.</p

RalGPS2 depletion increases p21 and p27 protein expression.

<p>(A and B) Representative Western blots and quantification (mean ± SEM) of up to 4 independent experiments of the expression of Cyclin D1 and Cyclin D3, and (C and D) of the Cyclin dependent kinase inhibitors p21 and p27. (E and F) RalA and RalB protein downregulation efficiency is shown together with Cyclin E expression and its respective quantification data (mean ± SEM) corresponding to 1 and 2 independent experiments in H1299 and A459, respectively. Data were collected 72 h after siRNA transfection. Light-grey bars—siRalA, medium-grey bars—siRalB, and dark-grey bars—siRalGPS2. Statistics: the difference from the reference (theoretical value 1) was evaluated by One sample t test, two-tailed, *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01. The vertical white lines in the Western blots indicate positions were gel images were cut in order to juxtapose non-adjacent lanes coming from the same gel.</p

Histology and Ras mutation type of the cell lines used in this work.

<p>Histology and Ras mutation type of the cell lines used in this work.</p