Journal: Cell Death & Disease

Article Title: GW627368X inhibits proliferation and induces apoptosis in cervical cancer by interfering with EP4/EGFR interactive signaling

doi: 10.1038/cddis.2016.61

Figure Lengend Snippet: GW627368X induces apoptosis in cervical cancer cells HeLa, SiHa and ME 180 in time-dependent manner. Cells treated with 9, 10 and 10 μ M GW627368X for Hela, SiHa and ME 180, respectively, for indicated time periods, ( a ) stained with Rhodamine phalladoin and counter stained with DAPI and images captured at × 20 magnification. Nuclear fragmentation and disruption of actin cytoskeleton, characteristic of apoptotic cell death, was observed ( b ) TUNEL assay was performed and fluorescent images were captured. Time-dependent increase in TUNEL-positive green nuclei indicated induction of apoptosis, ( c and d ) Western blot analysis of apoptotic proteins, ( c ) Blots depicting expression profile of apoptotic and anti-apoptotic proteins in cervical cancer cells treated with GW627368X for indicated time periods compared with control treated with 0.1% DMSO, β -actin used as loading control ( d ) Quantification of protein expression by densitometry, normalized to β -actin, each representative of three different experiments each performed in triplicates, values presented as mean±S.D.; * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: For western blot and IHC rabbit monoclonal anti-COX-2, anti-apoptosis-inducing factor, anti-BAX, anti-Bcl-2, anti-vascular endothelial growth factor receptor, anti-p-vascular endothelial growth factor receptor, anti-EGFR, anti-p-EGFR, anti-Akt, anti-p-Akt, anti-MAPK, anti-p-MAPK, anti-Ki67, anti-Ras, anti-p-Ras, anti-Raf, anti-p-Raf, anti-GSK3 β , anti-p-GSK3 β , anti- β Catenin, anti-p- β Catenin, anti-CD31(Cell Signaling Technology, Beverly, MA, USA), mouse monoclonal anti- β -actin (Cell Signaling Technology) and goat monoclonal anti-EP4 (Imgenex, Bhuvaneswar, India), horseradish peroxidase conjugated goat anti-rabbit immunoglobulin G and goat anti-mouse immunoglobulin G (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Chemiluminescent peroxidase substrate, propidium iodide and MTT (Sigma Aldrich) ApopTag In Situ Apoptosis detection kit (Promega, Madison, WI, USA), Rhodamine phallaoidin (Invitrogen Corporation, CA, USA) Fetal bovine serum (FBS) (Gibco-BRL, Invitrogen Corporation, CA, USA), PGE2 immunoassay kit (Imgenex, India), Human VEGF quantikine ELISA kit (R&D systems, Minneapolis, MN, USA), and IHC detection system (Biogenex, Fremont, CA, USA) were purchased from corresponding companies.

Techniques: Staining, TUNEL Assay, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: GW627368X inhibits proliferation and induces apoptosis in cervical cancer by interfering with EP4/EGFR interactive signaling

doi: 10.1038/cddis.2016.61

Figure Lengend Snippet: GW627368X hinders tumor progression in vivo. Subcutaneous tumor-bearing nude mice were treated with GW627368X orally ranging from 0 to 10 mg/Kg body weight, thrice a week, for 4 weeks. ( a ) Representative picture showing subcutaneous tumor (ME 180)-bearing nude mice of different treatment groups. ( b ) Representative picture showing excised tumors from corresponding treatment groups of ME 180 xenografts. ( c ) Graph representing change in tumor volume (group average; ME 180 xenografts) in each test group measured every week. Data presented as mean±S.D. ( n =5), P =0.0125. ( d ) Representative picture showing tumors excised from different treatment groups of SiHa xenografts. ( e ) Graph representing change in tumor volume (group average; SiHa xenografts) in each test group measured every week. Data presented as mean±S.D. ( n =5), P =0.0002. Paraffin-embedded excised tumors from different treatment groups of ME 180 xenografts were sectioned, processed and ( f ) In situ TUNEL assay to assess induction of apoptosis within tumor and IHC specific for COX-2, Ki67(proliferation marker) and CD31(anigiogenesis marker) were performed ( g ) Photomicrographs representing IHC performed for specific signaling proteins, pMAPK, MAPK, pAkt, Akt, pEGFR and EGFR

Article Snippet: For western blot and IHC rabbit monoclonal anti-COX-2, anti-apoptosis-inducing factor, anti-BAX, anti-Bcl-2, anti-vascular endothelial growth factor receptor, anti-p-vascular endothelial growth factor receptor, anti-EGFR, anti-p-EGFR, anti-Akt, anti-p-Akt, anti-MAPK, anti-p-MAPK, anti-Ki67, anti-Ras, anti-p-Ras, anti-Raf, anti-p-Raf, anti-GSK3 β , anti-p-GSK3 β , anti- β Catenin, anti-p- β Catenin, anti-CD31(Cell Signaling Technology, Beverly, MA, USA), mouse monoclonal anti- β -actin (Cell Signaling Technology) and goat monoclonal anti-EP4 (Imgenex, Bhuvaneswar, India), horseradish peroxidase conjugated goat anti-rabbit immunoglobulin G and goat anti-mouse immunoglobulin G (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Chemiluminescent peroxidase substrate, propidium iodide and MTT (Sigma Aldrich) ApopTag In Situ Apoptosis detection kit (Promega, Madison, WI, USA), Rhodamine phallaoidin (Invitrogen Corporation, CA, USA) Fetal bovine serum (FBS) (Gibco-BRL, Invitrogen Corporation, CA, USA), PGE2 immunoassay kit (Imgenex, India), Human VEGF quantikine ELISA kit (R&D systems, Minneapolis, MN, USA), and IHC detection system (Biogenex, Fremont, CA, USA) were purchased from corresponding companies.

Techniques: In Vivo, In Situ, TUNEL Assay, Marker

Journal: Oncogene

Article Title: Involvement of small ArfGAP1 (SMAP1), a novel Arf6-specific GTPase-activating protein, in microsatellite instability oncogenesis.

doi: 10.1038/onc.2013.211

Figure Lengend Snippet: Figure 1. SMAP1 mutations in human MSI CRCs. (a) SMAP1 mutation frequency according to tumor stage. Stage II: in situ tumors (n ¼ 40); stage III: tumors with lymph node metastasis (n ¼ 32); stage IV: tumors with distant synchronous or metachronous metastasis (n ¼ 21). (b) SMAP1 expression pattern by immunohistochemistry in normal colic mucosa, in MSI CRCs harboring a heterogeneous pattern, and in MSI CRC harboring a homogeneous pattern. Upper images: 20; lower images: 100. *Po0.05.

Article Snippet: Immunohistochemical analyses Blocks of formalin-fixed paraffin-embedded CRC tissue comprising an area of normal colonic mucosa adjacent to the tumor were selected, as described.22 Antigen retrieval was performed on 4-mm sections in citrate buffer (pH 6.0) using a microwave technique (15 min at 95 1C), sections were then incubated for 2 h at room temperature with a rabbit antibody against SMAP1 (1/75 dilution, HPA030574, Atlas Antibodies, Stockholm, Sweden) and revealed using the Biogenex Detection kit (Biogenex, Fremont, CA, USA).

Techniques: Mutagenesis, In Situ, Expressing, Immunohistochemistry

Journal: Oncogene

Article Title: Involvement of small ArfGAP1 (SMAP1), a novel Arf6-specific GTPase-activating protein, in microsatellite instability oncogenesis.

doi: 10.1038/onc.2013.211

Figure Lengend Snippet: Figure 2. Characterization of SMAP1 and SMAP2 mutations arising in tumor-derived cell lines, and SMAP1 transcript and protein expression according to tumor SMAP1 mutations. (a) SMAP1 gene sequencing in cell lines derived from MSS or MSI tumors of various tissue origins. (b) Distribution of MSI tumor-derived cell lines from various tissue origins according to SMAP1 or SMAP2 mutation status, wild-type (no mutation), monoallelic or biallelic mutations. (c) Relative SMAP1 transcript expression in MSS and MSI tumor-derived cell lines. Hatched box: MSS cell lines derived from noncolorectal tumors; black box: MSS CRC-derived cell lines; closely punctuated box: MSI CRC-derived cell lines with wild-type SMAP1 allele only; largely punctuated box: MSI CRC-derived cell lines harboring a monoallelic SMAP1 mutation; white box: MSI CRC-derived cell lines harboring biallelic SMAP1 mutations. (d) Relationship between SMAP1 transcript level in tumor-derived cell lines according to SMAP1 mutation status using the Mann–Whitney test. wt: wild-type; mono: monoallelic mutation; bi: biallelic mutations. (e) Representation of wild-type and mutant SMAP1 proteins. The 1 bp deletion leads to the loss of both the clathrin box (CB) and the clathrin assembly lymphoid myeloid leukemia (CALM) binding domain (CALM BD). The epitope recognized by the antibody used in f and g is located within the ArfGAP domain (101–115 aa, amino acid). (f) SMAP1 expression was analyzed in HeLa cells (SMAP1wt) (left lane), in HeLa cells transfected with the expression vector of the HA-tagged truncated SMAP1 form corresponding to the 1 bp deletion mutation (SMAP1-A9-HA) (middle lane) and in the HCT116 cell line carrying a homozygous 1 bp deletion SMAP1 mutation (SMAP1-A9) (right lane). The membrane was revealed with an antibody directed against the SMAP1 peptide 101–115 aa (Sigma); the 55 and 21 kDa bands correspond to the endogeneous wild-type SMAP1 protein and the SMAP1-A9 truncated protein, respectively. Loading controls were obtained using an horseradish peroxidase (HRP)-conjugated anti-GAPDH antibody. (g) HCT116 cells were transfected with pcDNA3.1–SMAP1wt-HA (left), pcDNA3.1–SMAP1-A9-HA (middle) or pcDNA3.1 control vector (right lane) and treated during 8 h with MG132 (5 mM), a proteasome inhibitor or vehicle. The p53 protein used as a control for proteasome inhibition was detected with the DO-1 monoclonal mouse antibody. *Po0.05.

Article Snippet: Immunohistochemical analyses Blocks of formalin-fixed paraffin-embedded CRC tissue comprising an area of normal colonic mucosa adjacent to the tumor were selected, as described.22 Antigen retrieval was performed on 4-mm sections in citrate buffer (pH 6.0) using a microwave technique (15 min at 95 1C), sections were then incubated for 2 h at room temperature with a rabbit antibody against SMAP1 (1/75 dilution, HPA030574, Atlas Antibodies, Stockholm, Sweden) and revealed using the Biogenex Detection kit (Biogenex, Fremont, CA, USA).

Techniques: Derivative Assay, Expressing, Sequencing, Mutagenesis, MANN-WHITNEY, Binding Assay, Transfection, Plasmid Preparation, Membrane, Control, Inhibition

Journal: Oncogene

Article Title: Involvement of small ArfGAP1 (SMAP1), a novel Arf6-specific GTPase-activating protein, in microsatellite instability oncogenesis.

doi: 10.1038/onc.2013.211

Figure Lengend Snippet: Figure 3. Effect of SMAP1 expression on tumor growth. (a) Western blot analysis of SMAP1 expression in HeLa (SMAP1wt), parental HCT116 (SMAP1-null) and three clones derived from HCT116 cells by stably transfecting SMAP1; immunofluorescence detection of SMAP1 is shown for HCT116 cells and a representative HCT116–SMAP1 clone. (b) After cell injection in nude mice, tumor volume was monitored twice a week to follow tumor growth according to SMAP1 expression. Results represent the mean tumor volumes (mm3±s.e.m.) from day 11 to day 29 postinjection. (c) Pictures of tumors after the mice were killed. Four tumors per group are represented. ***Po0.001; ****Po0.0001.

Article Snippet: Immunohistochemical analyses Blocks of formalin-fixed paraffin-embedded CRC tissue comprising an area of normal colonic mucosa adjacent to the tumor were selected, as described.22 Antigen retrieval was performed on 4-mm sections in citrate buffer (pH 6.0) using a microwave technique (15 min at 95 1C), sections were then incubated for 2 h at room temperature with a rabbit antibody against SMAP1 (1/75 dilution, HPA030574, Atlas Antibodies, Stockholm, Sweden) and revealed using the Biogenex Detection kit (Biogenex, Fremont, CA, USA).

Techniques: Expressing, Western Blot, Clone Assay, Derivative Assay, Stable Transfection, Injection

Journal: Molecular Cancer

Article Title: LncRNA PVT1 triggers Cyto-protective autophagy and promotes pancreatic ductal adenocarcinoma development via the miR-20a-5p/ULK1 Axis

doi: 10.1186/s12943-018-0845-6

Figure Lengend Snippet: Knockdown of PVT1 prohibited PDA growth in vivo. a Down-regulation of PVT1 in HPAF-II cells inhibited tumor growth after 4 weeks in vivo ( n = 5 per group). Upper: negative control group. Lower: knockdown groups formed in nude mice subcutaneously injected with PVT1-silencing HPAF-II cells; b , c Growth curves ( b ) and tumor weight ( c ) of xenograft tumors after subcutaneous injection with PVT1-silencing HPAF-II cells. The tumor volumes were investigated every week after injection with PVT1-silencing HPAF-II cells ( n = 5). Diminished expression of PVT1 markedly attenuated the tumor growth in PDA; d , e Representative images ( d ) and photon influx ( e ) of xenograft tumors after subcutaneous injection with PVT1-silencing HPAF-II cells by IVIS ( n = 5 per group). f Representative images of HE staining, TUNEL, immunohistochemical staining of Ki67 and ULK1; g-i The quantitation of Ki67 ( g ), TUNEL ( h ) and ULK1 ( i ) was calculated. Immunohistochemical staining of Ki67 and ULK1 showed that decreased expression of PVT1 led to suppression of Ki67 proliferation index and ULK1 expression, while TUNEL positive cells were increased. The data were represented as the mean ± S.D. ( n = 5). * P < 0.05, ** P < 0.01 and *** P < 0.001 by Student’s t-test

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using primary antibodies against Ki67 (#9449, Cell Signaling Technology, Danvers, MA, USA) and ULK1 (ab128859, Abcam, Cambridge, MA, USA), as well as horseradish peroxidase-conjugated IgG, and proteins were visualized in situ with 3, 3-diaminobenzidine kit (BioGenex, Fremont, CA, USA).

Techniques: Knockdown, In Vivo, Negative Control, Injection, Expressing, Staining, TUNEL Assay, Immunohistochemical staining, Quantitation Assay

Journal: BMC Microbiology

Article Title: HIV-1 expression induces cyclin D 1 expression and pRb phosphorylation in infected podocytes: cell-cycle mechanisms contributing to the proliferative phenotype in HIV-associated nephropathy

doi: 10.1186/1471-2180-2-26

Figure Lengend Snippet: In situ immunodetection of phosphorylated serine 780 on pRb in Tg26 kidneys. Phospho-pRb (Ser780) is not detected in the nuclei of epithelium of normal kidneys ( A ), but is readily detected in podocytes (short arrows) and tubular epithelial cells (long arrows) in hyperplastic nephrons of diseased transgenic kidneys at cortical ( B) , juxta-medullary ( C ), and medullary ( D ) depths. The prominent staining in many, but not all nuclei, is characteristic of asynchronously proliferating cells. (magnification = 40×)

Article Snippet: Four micron full coronal kidney sections from each animal were adhered to lysine-coated slides and used for immunodetection of phospho-Rb (Ser780) as follows: sections were deparaffinized and hydrated by placing in xylene for 8 minutes, followed by 3 minutes each of 100%, 96%, 80%, 70%, 50%, 30% ethanol, ending with 10 minutes in water; sections then underwent antigen retrieval (Glyca Reagent, Biogenex) followed by immunodetection (Super Sensitive Detection Kit, Biogenex) of phospho-Rb (Ser780) after a 12 hour incubation with a 1:1000 dilution of rabbit anti-phospho-Rb (Ser780) antibody (Cell Signaling) or control rabbit anti-sera (Biogenex) at 4°C.

Techniques: In Situ, Immunodetection, Transgenic Assay, Staining

Journal: BMC Microbiology

Article Title: HIV-1 expression induces cyclin D 1 expression and pRb phosphorylation in infected podocytes: cell-cycle mechanisms contributing to the proliferative phenotype in HIV-associated nephropathy

doi: 10.1186/1471-2180-2-26

Figure Lengend Snippet: Expression of cyclin D 1 and phospho-pRb (Ser780) in HIV-1-infected podocytes. ( A ) Northern and western blot detection of cyclin D 1 expression in wild-type, control-infected, and HIV-1-infected podocytes during subconfluent (SC) growth at 33°C and during 14 days of differentiation of confluent podocytes at 37°C shows that neither the cyclin D 1 transcript nor the elevated cyclin D 1 protein in HIV-1-infected podocytes (full-length, singly-spliced, and multiply spliced HIV-1 transcripts) are significantly down-regulated by podocyte cell-cell contact or by podocyte differentiation (synaptopodin transcript) when compared to wild-type or control-infected (one transcript from LTR sequences) podocytes. ( B ) Immunodetection of phospho-pRb (Ser780) on several phosphorylated forms (arrows) of pRb in the asynchronously proliferating, HIV-1-infected podocytes that is absent in the quiescent, control-infected podocytes after 14 days of differentiation at 37°C. ( C ) Abundant foci formation in confluent HIV-1-infected podocytes but not in confluent control-infected podocytes due to the ongoing proliferation of HIV-1-infected podocytes after cell-cell contact. Blots are representative of two independent experiments.

Article Snippet: Four micron full coronal kidney sections from each animal were adhered to lysine-coated slides and used for immunodetection of phospho-Rb (Ser780) as follows: sections were deparaffinized and hydrated by placing in xylene for 8 minutes, followed by 3 minutes each of 100%, 96%, 80%, 70%, 50%, 30% ethanol, ending with 10 minutes in water; sections then underwent antigen retrieval (Glyca Reagent, Biogenex) followed by immunodetection (Super Sensitive Detection Kit, Biogenex) of phospho-Rb (Ser780) after a 12 hour incubation with a 1:1000 dilution of rabbit anti-phospho-Rb (Ser780) antibody (Cell Signaling) or control rabbit anti-sera (Biogenex) at 4°C.

Techniques: Expressing, Infection, Northern Blot, Western Blot, Control, Immunodetection