Additional file 3 of Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors

Additional file 3: Figure S3. Expression of extracellular signal-regulated kinase (ERK) upon treatments. Increasing concentrations of RTKi were used for the treatment, and levels of phospho-mTOR, phospho-PI3K, phospho-AKT and phospho-ERK (pERK) and total ERK were evaluated by western blot. GAPDH served as a loading control protein. D = DMSO; the numbers indicate the concentrations used in μM

Additional file 4 of Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors

Additional file 4: Figure S4. Autophagy activation in SH-SY5Y cell line upon treatment with RTKi. Autophagy activation upon treatment with Afatinib and Sorafenib was validated using immunofluorescence in order to visualize a creation of autophagosomes; 60X magnification. DMSO treatment was used as control. Green—autophagy vacuoles; Blue—Hoechst, nuclear staining

Additional file 1 of Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors

Additional file 1: Figure S1. Caspase-3 and PI measurement upon treatments. A) Apoptosis activation was evaluated by measuring the intensity of PARP cleavage (89 kDa proteolytic PARP fragment), and by evaluating a decrease in the pro-Caspase-3 (35 kDa protein) level. Active Caspase-3 forms are observed as cleaved proteins with molecular weight below 20 kDa. GAPDH served as a loading control protein. D = DMSO; Af—Afatinib (8 μM); Sor—Sorafenib (14 μM); TP—TP-0903 (0.15 μM). B) Percentage of PI positive cells with respect to total cell number was presented as mean ± SD out of triplicates. D—DMSO; Af—Afatinib; Sor—Sorafenib; TP—TP-0903. p value is marked as **p < 0.01; n.s.—non-significant. C) Changes in the proliferation rate upon treatment with the three RTKi were determined by means of PCNA expression. The numbers indicate relative expression (densitometry) obtained after normalization to GAPDH protein level, which was used a loading control, and with respect to DMSO control (DMSO = 1)

Additional file 2 of Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors

Additional file 2: Figure S2. Colony formation assay. Effects on the capacity of SH-SY5Y cells to form colonies upon treatment with RTKi alone or in combined treatment with CQ or SP1 was measured. Upper image is the representative out of triplicate experiment. Lower graph represents a colony number calculated as a percentage of control (DMSO; 100%), and presented as the mean ± SD out of triplicate measurement. p value is marked as **p < 0.01; n.s.—non significant (Dunnett’s test). CQ—Chloroquine; SP1—Spautin-1; Af—Afatinib

MOESM1 of Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors

Additional file 1: Figure S1. Caspase-3 and PI measurement upon treatments. A) Apoptosis activation was evaluated by measuring the intensity of PARP cleavage (89 kDa proteolytic PARP fragment), and by evaluating a decrease in the pro-Caspase-3 (35 kDa protein) level. Active Caspase-3 forms are observed as cleaved proteins with molecular weight below 20 kDa. GAPDH served as a loading control protein. D = DMSO; Af—Afatinib (8 μM); Sor—Sorafenib (14 μM); TP—TP-0903 (0.15 μM). B) Percentage of PI positive cells with respect to total cell number was presented as mean ± SD out of triplicates. D—DMSO; Af—Afatinib; Sor—Sorafenib; TP—TP-0903. p value is marked as **p < 0.01; n.s.—non-significant. C) Changes in the proliferation rate upon treatment with the three RTKi were determined by means of PCNA expression. The numbers indicate relative expression (densitometry) obtained after normalization to GAPDH protein level, which was used a loading control, and with respect to DMSO control (DMSO = 1)

The c.-456_-453delCCTT does not alter the steady-state levels of <i>CDKN1B</i> allelic mRNA nor the promoter usage pattern.

<p>(A) The wild type and mutated alleles were present at similar level in blood-derived RNA. The amount of both alleles are expressed as differences between Cq values obtained for mRNA minus Cq for genomic DNA for removing the intrinsic variation between the two qPCR assays. (B) A single <i>CDKN1B</i> promoter is used in lymphocytes from both deletion carrier (P) and a normal control (NC). M, molecular marker; -, negative control. (C) Both wild type and mutated alleles are transcribed in pancreatic lesion.</p

Comparison of the <i>CDKN1B</i> gene among species.

<p>(A) Schematic representation of the <i>CDKN1B</i> gene in different species. The uORF has been reported as grey box while <i>CDKN1B</i> as a yellow one. Similarities at nucleotide level with human sequences have been reported within boxes. Intercistronic length is also shown. * In chicken <i>CDKN1B</i> 5′UTR a second uORF has been described. (B) uORF amino acid sequences in seven species. In all but one, conservation of the uORF encoded peptide is even higher than p27^KIP1. The considered nucleotide and protein sequences are the following: Human, NM_004064.3, NP_04055.1; Mouse, NM_009875.4, NP_034005.2; Dog, HE804769, CCH35981; Opossum, HE804772, CCH35984; Chicken, NM_204256, NP_989587; Sloth, HE804770, CCH35982; Megabat, HE804771, CCH35983. Nucleotide and protein sequences alignments among species have been performed by LALIGN and PRALINE, respectively.</p

Constructs used in transfection experiments.

<p>(A) Either the wild type, the c.-456_-453delCCTT or the c.-469C>T containing 5′UTRs were cloned upstream the firefly luciferase gene. (B) The wild type/c.-456_-453delCCTT 5′UTR were subcloned upstream the <i>CDKN1B</i> gene. (C) The introduction of the c.-428A>T substitution in the two former plasmids reported in panel A restores uORF length and intercistonic distance. (D) Constructs reported in panel B were mutated introducing a c.-74insC leading to the translation of a chimeric protein in the double mutant.</p

Expression of p27^KIP1 and Ki-67 in pancreatic tumors and control tissue.

<p>(A–C) Immunohistochemical staining with anti-p27^KIP1 antibody. (D) Staining with anti-Ki67 antibody. (A) staining of a control human tonsil. Cells in the lymphatic nodule show the expected strong nuclear positivity for p27^KIP1, while only few of the highly proliferating germinal center (GC) cells express p27^KIP1. (B) Sporadic endocrine pancreatic tumor of an individual with no known germline mutations. The tumor cells show intermediate levels of nuclear immunoreactivity for p27^KIP1. (C) Endocrine pancreatic tumor of the patient having the c.-456_-453delCCTT mutation. Tumor cells show low expression of p27^KIP1 in the nucleus but also expression in the cytoplasm. Endothelial cells (arrows) show the typical strong nuclear positivity and serve as internal control to check for the adequacy of the staining. (D) Same tissue as in C stained for the proliferation marker Ki-67. Positive nuclei are indicated with arrowheads. The proliferation rate was estimated to be ca. 1%. Bar, 20 µM. Original magnification: A–D, 200×.</p

Polysome profiling of lymphoblastoid cells from the -456_-453delCCTT mutation carrier.

<p>(A) Representative absorbance profile for RNA separated by velocity sedimentation through a 15–50% sucrose gradient. The positions of the 40S, 60S, 80S, and polysomal peaks are indicated. (B) The levels of <i>CDKN1B</i> mRNA (wt and c.-456_-453del) in each gradient fraction were measured by quantitative real-time PCR and plotted as a percentage of the total <i>CDKN1B</i> mRNA levels (wt or c.-456_-453del, respectively) in that sample. Data represent three independent experiments, with mean ± SD reported. (C) The levels of both alleles in each fraction were expressed as relative quantities calculated from differences in Cq values between mRNA and genomic DNA for removing the intrinsic variation between the two qPCR assays. Data represent the mean of three independent experiments ± SD.</p