Journal: Oncotarget

Article Title: Reciprocal expression of INSM1 and YAP1 defines subgroups in small cell lung cancer

doi: 10.18632/oncotarget.20572

Figure Lengend Snippet: A. Survival analysis based upon INSM1 IHC scores from the second TMA. Restricted to those patients with OS ≥ 3 mo. The median INSM1 expression score was 15 (range: 0 - 240) and the mean was 51 (STD: 70.9). B. Correlation of INSM1 IHC score with chemo-response ( N = 28) in the second TMA cohort. Response was graded as complete response (CR), partial response (PR), progressive disease (PD) or stable disease (SD) using RECIST criteria. Mean IHC values are shown by horizontal bars. C. IC50 values for irinotecan inhibition of cell growth listed for individual cell lines (left) as well as for cell line subgroups (right). Individual cell line IC50 values represent mean ± SEM of 1-2 independent experiments and are shown as either red (Group I) or blue (Group II) bars depending on their subgroup assignment. Cells are arranged on x-axis, left to right, in identical order to the clustering diagram in Figure . *Cell line not tested. Boxplots represent mean ± SEM of individual IC50 values for a Group with p values showing significance between Groups. D. Western blot of cell lysates shown as in Figure but probed for BCL2 and BCLxL. E. Same as in panel C except for ABT199. F. Growth curves for SW1271 and H841 cells after knockdown by 500 nM YAP1 siRNA (KD) or non-targeting siRNA (NT) compared to untreated (CON) or mock-transfected cells. Bottom shows western blot validating YAP1 knockdown after 4 days of treatment.

Article Snippet: Knockdown of RB1 was accomplished with RB1 siRNA, sc-29468 (Santa Cruz Biotechnology).

Techniques: Expressing, Inhibition, Western Blot, Knockdown, Transfection

Journal: Oncotarget

Article Title: Reciprocal expression of INSM1 and YAP1 defines subgroups in small cell lung cancer

doi: 10.18632/oncotarget.20572

Figure Lengend Snippet: A. Western blots of protein lysates shown as in Figure . Targeted protein listed on right. YAP1 blot is reproduced from Figure for easy comparison. RB1 exon mutation status obtained from COSMIC shown in parentheses after cell line name. B. Western blot results of transient knockdown of RB1 or YAP1 in SW1271 and H841. Cells were treated with gene-specific siRNA (Rx) or non-targeting siRNA (NT) and results compared to untreated (Con) or mock-transfected (Mock) cells. Protein lysates were prepared after the indicated numbers of days. IC50 values for CDK4/6 inhibitors LY2835219 C. and PD0332991 D. on individual cell lines. Individual cell line IC50 values are represented as in 4C. Bars with no error bars have IC50 values > 2 µM. E. RB1 IHC on consecutive sections of same four tumor cores as shown in Figure , each shown at two magnifications (shown to right). Slides were counterstained to show histology. F. Plots of RB1 versus YAP1 and INSM1 IHC scores using data from . Size of circles proportional to number of cores represented. Blue circles represent cores with a high YAP1 score. G. Survival analysis based upon RB1 mutation status in the genomic cohort ( N = 64). A binary (+/-) scoring system was used for mutation status and did not consider the type of mutation or the number of RB1 mutations per tumor. H. Correlation of RB1 mutation status with initial chemo-response in genomic cohort of panel G ( N = 61). Response was graded as chemo-sensitive (CS) or chemo-refractory (CR) based upon the standard RECIST criteria for SCLC.

Article Snippet: Knockdown of RB1 was accomplished with RB1 siRNA, sc-29468 (Santa Cruz Biotechnology).

Techniques: Western Blot, Comparison, Mutagenesis, Knockdown, Transfection

Journal: BMC Cell Biology

Article Title: Evidence for a mitochondrial localization of the retinoblastoma protein

doi: 10.1186/1471-2121-10-50

Figure Lengend Snippet: Rb protein is localized in mitochondria . A . Equal protein amounts (40 μg) of total extract (T), nuclear (N) and mitochondrial (M) fractions of human (HT1080 and HF) and rat (PC12 and FR3T3) cells cultured either untreated or etoposide-treated (16 h), were loaded onto gel and then immunoblotted with: anti-Rb (G3-245) antibody, the mitochondrial (COX II or cytochrome c), the cytosolic (Tubulin) or the nuclear marker antibodies (PCNA or Lamin A). HyperPh or hypoPh represents the phosphorylated state of Rb. ( Lower panel ) The quantification (Image J software) of the Rb and the PCNA protein levels (or Lamin A for FR3T3) in nuclear and mitochondrial fractions illustrated as mitochondrial/nuclear ratio (untreated cells). B . Effect of Rb siRNA on the presence of Rb in the mitochondria. FR3T3 cells were incubated for 48 h with Rb siRNA (siRb), control siRNAs (siCtrl) or non-transfected (NT). The total cell extract and mitochondrial fractions (20 μg) were loaded onto gel and subjected to immunoblotting with anti-Rb (G3-245) antibody. Quantifications were performed with respect to Enolase (for the total extract) and COX II (for the mitochondrial extract). Student's tests were performed (** P < 0.01).

Article Snippet: Then, 10 nM Rb siRNA (Rn_Rb1_1_HP siRNA, Qiagen) or negative control (non-silencing, Qiagen) and HiPerFect Transfection Reagent (Qiagen) were mixed in culture medium and then added to the cells, according to the manufacturer's recommendations (HiPerFect Transfection Reagent Handbook, Qiagen).

Techniques: Cell Culture, Marker, Software, Incubation, Control, Transfection, Western Blot