human pancreatic cancer cell line miapaca2  (ATCC)

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: Representative images from immunofluorescence analysis of MiaPaCa2 or BxPC3 tumors that were mock or vismodegib-treated (40 mg/kg). (A) Tumor sections were stained for the CAF's marker a-SMA (green) and the proliferation marker Ki67 (red). Magnified images from selected tumor areas indicate the presence of Ki67+/a-SMA (yellow) cells. (B) Quantification of Ki67+/a-SMA (yellow) tumor area fraction in MiaPaCa2 or BxPC3 control or vismodegib-treated tumors (n=8-10). Asterisks indicate statistically significant difference between compared groups (p<0.05). Scale bar: 25 μm.

Article Snippet: MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Immunofluorescence, Staining, Marker, Control

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: Representative images from immunofluorescence staining showing the effect of vismodegib (40 mg/kg) on (A) collagen (green) and (B) hyaluronan (HA, red), (C) CD31/Lectin and (D) CD31 levels compared to control-treated MiaPaCa2 or BxPC3 tumors. Quantification of collagen (E) area fractions were found to be significantly lower in vismodegib-treated compared to control-treated MiaPaCa2 tumors (p = 0.0001), whereas in BxPC3 tumors no significant changes were observed. Quantification of hyaluronan (F) area fractions were found to be significantly lower in both vismodegib-treated MiaPaCa2 and BxPC3 tumors (p = 0.02 and p = 3E-5, respectively), compared to control-treated tumors. For both vismodegib-treated pancreatic tumors the fraction of perfused vessels (G) increased compared to control tumors (p = 0.03, MiaPaCa2; p = 0.03, BxPC3). In contrast, quantification of CD31+ area (H) showed no significant change between the groups. Asterisks indicate statistically significant difference between compared groups (n = 8-10). Scale bar: 100 μm.

Article Snippet: MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Immunofluorescence, Staining, Control

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: (A) Schematic of the unconfined compression experiment. Desmoplastic tumors become stiff as they grow, exhibiting a higher elastic modulus and resisting stronger to compression. Desmoplasia reduces tumors hydraulic conductivity, resisting to fluid flow through their mass and thus, less fluid exits the tissue during compression. (B) Vismodegib-treated tumors in BxPC3 decreased the elastic modulus compared to control treatment (p = 0.002) whereas in MiaPaCa2 had no effect. Vismodegib-induced reduction in ECM content resulted in lower values of hydraulic conductivity of the tumor interstitial space (p = 0.005 for MiaPaCa2 and p = 0.04 for BxPC3) (C), which in turn caused alleviation of the interstitial fluid pressure (p = 4E-9 for MiaPaCa2 and p = 5E-5 for BxPC3) (D). Asterisks indicate statistically significant difference between compared groups (n = 8-10).

Article Snippet: MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Control

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: Tumor volume growth rates of (A) MiaPaCa2 and (B) BxPC3 pancreatic human tumors implanted in male NOD/SCID mice. Control treatment (MCT -0.5% methylcellulose, 0.2% Tween 80), vismodegib (40 mg/kg) or gemcitabine (50 mg/kg) alone had no effect on tumor growth in both pancreatic cancer cell lines. (A) Combination of vismodegib and gemcitabine significantly decreased tumor growth of MiaPaCa2 pancreatic tumors compared to gemcitabine monotherapy (p = 0.005 on day 60, n = 8-10). (B) In BxPC3 model combination of vismodegib and gemcitabine significantly delayed tumor growth compared to gemcitabine monotherapy (p = 7E-7 on day 26, n = 8-10). Asterisks indicate a statistically significant difference between compared groups (p < 0.05).

Article Snippet: MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Control

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: Quantification of doxorubicin (Dox) concentration in control and vismodegib-treated MiaPaCa2 pancreatic tumors as well as in kidney, liver and heart tissues. Doxorubicin (20 mg/kg) was injected intravenously to the animals 4 hours prior to sacrifice. Vismodegib increased the delivery of doxorubicin by 2-fold in vismodegib-treated compared to control-treated tumors (p < 0.05, n = 9). No significant differences were observed in drug delivery to normal tissues. Asterisk indicates statistically significant differences between compared groups (p < 0.05).

Article Snippet: MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Concentration Assay, Control, Injection

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: Real-time PCR gene expression analysis and quantification of mouse (A) or human-specific (B) Gli1, Gli2, Gli3, Ptch1, Ptch2, Smo and Shh mRNA levels extracted from control-treated compared to vismodegib-treated MiaPaCa2 tumors, indicated that vismodegib suppressed Gli1 and Gli2 expression in mouse stromal cells but not in MiaPaCa2 human cancer cells. Relative expression for all genes in both groups was normalized based on the expression of beta-actin. Data represent the average of at least 3 independent experiments from 4 control and 4 vismodegib-treated tumors ± S.E. values and asterisks indicate statistically significant differences between compared groups (p < 0.05).

Article Snippet: MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Control, Expressing

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: Representative images from immunofluorescence analysis of MiaPaCa2 or BxPC3 tumors that were mock or vismodegib-treated (40 mg/kg). (A) Tumor sections were stained for the CAF's marker a-SMA (green) and the proliferation marker Ki67 (red). Magnified images from selected tumor areas indicate the presence of Ki67+/a-SMA (yellow) cells. (B) Quantification of Ki67+/a-SMA (yellow) tumor area fraction in MiaPaCa2 or BxPC3 control or vismodegib-treated tumors (n=8-10). Asterisks indicate statistically significant difference between compared groups (p<0.05). Scale bar: 25 μm.

Article Snippet: Cell culture MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Immunofluorescence, Staining, Marker, Control

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: Representative images from immunofluorescence staining showing the effect of vismodegib (40 mg/kg) on (A) collagen (green) and (B) hyaluronan (HA, red), (C) CD31/Lectin and (D) CD31 levels compared to control-treated MiaPaCa2 or BxPC3 tumors. Quantification of collagen (E) area fractions were found to be significantly lower in vismodegib-treated compared to control-treated MiaPaCa2 tumors (p = 0.0001), whereas in BxPC3 tumors no significant changes were observed. Quantification of hyaluronan (F) area fractions were found to be significantly lower in both vismodegib-treated MiaPaCa2 and BxPC3 tumors (p = 0.02 and p = 3E-5, respectively), compared to control-treated tumors. For both vismodegib-treated pancreatic tumors the fraction of perfused vessels (G) increased compared to control tumors (p = 0.03, MiaPaCa2; p = 0.03, BxPC3). In contrast, quantification of CD31+ area (H) showed no significant change between the groups. Asterisks indicate statistically significant difference between compared groups (n = 8-10). Scale bar: 100 μm.

Article Snippet: Cell culture MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Immunofluorescence, Staining, Control

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: (A) Schematic of the unconfined compression experiment. Desmoplastic tumors become stiff as they grow, exhibiting a higher elastic modulus and resisting stronger to compression. Desmoplasia reduces tumors hydraulic conductivity, resisting to fluid flow through their mass and thus, less fluid exits the tissue during compression. (B) Vismodegib-treated tumors in BxPC3 decreased the elastic modulus compared to control treatment (p = 0.002) whereas in MiaPaCa2 had no effect. Vismodegib-induced reduction in ECM content resulted in lower values of hydraulic conductivity of the tumor interstitial space (p = 0.005 for MiaPaCa2 and p = 0.04 for BxPC3) (C), which in turn caused alleviation of the interstitial fluid pressure (p = 4E-9 for MiaPaCa2 and p = 5E-5 for BxPC3) (D). Asterisks indicate statistically significant difference between compared groups (n = 8-10).

Article Snippet: Cell culture MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Control

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: Tumor volume growth rates of (A) MiaPaCa2 and (B) BxPC3 pancreatic human tumors implanted in male NOD/SCID mice. Control treatment (MCT -0.5% methylcellulose, 0.2% Tween 80), vismodegib (40 mg/kg) or gemcitabine (50 mg/kg) alone had no effect on tumor growth in both pancreatic cancer cell lines. (A) Combination of vismodegib and gemcitabine significantly decreased tumor growth of MiaPaCa2 pancreatic tumors compared to gemcitabine monotherapy (p = 0.005 on day 60, n = 8-10). (B) In BxPC3 model combination of vismodegib and gemcitabine significantly delayed tumor growth compared to gemcitabine monotherapy (p = 7E-7 on day 26, n = 8-10). Asterisks indicate a statistically significant difference between compared groups (p < 0.05).

Article Snippet: Cell culture MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Control

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: Quantification of doxorubicin (Dox) concentration in control and vismodegib-treated MiaPaCa2 pancreatic tumors as well as in kidney, liver and heart tissues. Doxorubicin (20 mg/kg) was injected intravenously to the animals 4 hours prior to sacrifice. Vismodegib increased the delivery of doxorubicin by 2-fold in vismodegib-treated compared to control-treated tumors (p < 0.05, n = 9). No significant differences were observed in drug delivery to normal tissues. Asterisk indicates statistically significant differences between compared groups (p < 0.05).

Article Snippet: Cell culture MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Concentration Assay, Control, Injection

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

doi: 10.1016/j.jconrel.2017.06.022

Figure Lengend Snippet: Real-time PCR gene expression analysis and quantification of mouse (A) or human-specific (B) Gli1, Gli2, Gli3, Ptch1, Ptch2, Smo and Shh mRNA levels extracted from control-treated compared to vismodegib-treated MiaPaCa2 tumors, indicated that vismodegib suppressed Gli1 and Gli2 expression in mouse stromal cells but not in MiaPaCa2 human cancer cells. Relative expression for all genes in both groups was normalized based on the expression of beta-actin. Data represent the average of at least 3 independent experiments from 4 control and 4 vismodegib-treated tumors ± S.E. values and asterisks indicate statistically significant differences between compared groups (p < 0.05).

Article Snippet: Cell culture MiaPaCa2 human pancreatic cancer cell line and BxPC3 human primary pancreatic adenocarcinoma cell line were purchased from ATCC and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: REP1 Modulates Autophagy and Macropinocytosis to Enhance Cancer Cell Survival

doi: 10.3390/ijms18091866

Figure Lengend Snippet: Rab escort protein1 (REP1) depletion suppresses cell growth and survival. ( A ) MiaPaCa2 cells were transfected with control (CTL) and REP1 small interfering RNAs (siRNAs). After 48 h, immunoblotting was performed to analyze REP1 protein levels. Cells were treated with CTL and REP1 siRNAs and incubated for 24 h. Then, the cells were transfected with Flag-REP1 plasmid additionally and further incubated in the IncuCyte TM for monitoring cell proliferation. At the 72-h incubation time point, cell confluence levels were presented as a percentage using the IncuCyte TM analyzer. ( B , C ) MiaPaCa2 cells were transfected with control and REP1siRNAs, which replaced the following day with serum-, glucose-, or glutamine-free medium and then incubated for another 24 h.Cell morphology was observed by brightfield image. Scale bar: 50 μm ( B ). Cell death was assessed by using the Annexin V/propidium iodide (PI) assay ( C ). Error bars indicate mean +/− standard error for n = 3 independent experiments. ( D ) MiaPaCa2 cells were transfected with control (CTL) or REP1siRNAs, which replaced the following day with 1 µM rapamycin and then further incubated for monitoring cell confluence using IncuCyte TM . At 72 h time point, cell confluence levels were presented as percentage. Statistical significance was determined via t-test; * p < 0.05.

Article Snippet: The Hela cells, and Panc1, 8988T, and MiaPaCa2 human pancreatic cancer cell lines from the American Type Culture Collection (ATCC; Manassas, VA, USA) were kindly provided by Kyung-Tae Kim and Yun-Hee Kim (National Cancer Center, Goyang-si, Korea).

Techniques: Transfection, Control, Western Blot, Incubation, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: REP1 Modulates Autophagy and Macropinocytosis to Enhance Cancer Cell Survival

doi: 10.3390/ijms18091866

Figure Lengend Snippet: REP1 knockdown down-regulates mTORC1 signaling pathway. ( A ) Cells were treated with CTL orREP1siRNAs for 48 h. Then, cells harvested and the lysates were immunoblotted with antibodies against REP1, p70S6K, pS6, total 70S6K, S6 ribosomal (S6) protein, and beta actin; ( B ) MiaPaCa2 cells were with CTL or REP1siRN as for 24 h. Then, Flag-REP1 plasmid were transfected to the cells for overexpression, which were harvested and immunoblotted with antibodies against p70S6Kand a total 70S6K. These results are representative of three independent experiments; ( C ) cells were treated with CTL orREP1siRNAs for 48 h. After 2 h incubation in the presence or absence of amino acids, cells were stained with antibodies against mTOR and LAMP2, and analyzed by fluorescence microscopy to monitor mTOR localization. Co-localization coefficient was quantified by co-localization function of image-based software (ZEN) provided by the microscope system. Error bars indicate mean +/− standard error for n = 3 independent experiments. Statistical significance was determined via a Student’s t -test;* p < 0.05. Scale bar: 20 μm.

Article Snippet: The Hela cells, and Panc1, 8988T, and MiaPaCa2 human pancreatic cancer cell lines from the American Type Culture Collection (ATCC; Manassas, VA, USA) were kindly provided by Kyung-Tae Kim and Yun-Hee Kim (National Cancer Center, Goyang-si, Korea).

Techniques: Knockdown, Plasmid Preparation, Transfection, Over Expression, Incubation, Staining, Fluorescence, Microscopy, Software

Journal: International Journal of Molecular Sciences

Article Title: REP1 Modulates Autophagy and Macropinocytosis to Enhance Cancer Cell Survival

doi: 10.3390/ijms18091866

Figure Lengend Snippet: REP1 depletion suppresses starvation-induced autophagy. ( A ) Hela and MiaPaCa2 cells stably expressing GFP-LC3 with a retroviral vector system were treated with CTL or REP1 siRNA and incubated for 48 h. Then, after 2 h of incubation in amino acid-deprived media, cells were harvested and immunoblotted for LC3, P62, and GFP. These results are representative of more than three independent experiments. GFP-LC3 puncta were analyzed by live imaging using fluorescence microcopy in ( B ) Hela and ( C ) MiaPaCa2 cells. Scale bar: 20 μm. Quantification data for GFP-LC3 puncta area are expressed as a percentage of the DAPI area within the cell. Hela and MiaPaCa2 cells stably expressing mCherry-GFP-LC3 with a retroviral vector system were treated with CTL or REP1 siRNA and incubated for 48 h. Then, after 2 h of incubation in Hank’s balanced saline solution (HBSS) media, mCherry puncta from mCherry-GFP-LC3 were imaged by fluorescence microcopy in ( D ) Hela and ( E ) MiaPaCa2 cells. Scale bar: 20 μm. Quantification data for mCherry puncta area derived from mCherry GFP-LC3 are expressed as a percentage of the DAPI area within the cell. Images shown are representative of at least three independent experiments. Statistical significance was determined via t -test; * p < 0.05.

Article Snippet: The Hela cells, and Panc1, 8988T, and MiaPaCa2 human pancreatic cancer cell lines from the American Type Culture Collection (ATCC; Manassas, VA, USA) were kindly provided by Kyung-Tae Kim and Yun-Hee Kim (National Cancer Center, Goyang-si, Korea).

Techniques: Stable Transfection, Expressing, Retroviral, Plasmid Preparation, Incubation, Imaging, Fluorescence, Saline, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: REP1 Modulates Autophagy and Macropinocytosis to Enhance Cancer Cell Survival

doi: 10.3390/ijms18091866

Figure Lengend Snippet: REP1 depletion changes lysosomal function and intracellular localization. ( A ) MiaPaCa2 cells were treated with CTL or REP1 siRNA and incubated for 48 h. Then the cells were treated with DQ-BSA at a final concentration 10 μg/mL for 30 min. Intracellular fluorescent signals were analyzed by fluorescence microcopy and presented as quantification values based on images; ( B ) Hela and MiaPaCa2 cells stably transfected with GFP-LC3 were treated with CTL or REP1 siRNA and incubated for 24 h. Then the cells were further incubated in the media with pH 7.4 or 6.8 overnight and cells were treated with a lysotracker for 30min. Intracellular fluorescent signals were analyzed by fluorescence microcopy and co-localization levels were presented as co-localization coefficient based on image quantification. Error bars indicate mean +/− standard error for n = 3 independent experiments. Statistical significance was determined via a Student’s t -test; * p < 0.05.

Article Snippet: The Hela cells, and Panc1, 8988T, and MiaPaCa2 human pancreatic cancer cell lines from the American Type Culture Collection (ATCC; Manassas, VA, USA) were kindly provided by Kyung-Tae Kim and Yun-Hee Kim (National Cancer Center, Goyang-si, Korea).

Techniques: Incubation, Concentration Assay, Fluorescence, Stable Transfection, Transfection

Journal: International Journal of Molecular Sciences

Article Title: REP1 Modulates Autophagy and Macropinocytosis to Enhance Cancer Cell Survival

doi: 10.3390/ijms18091866

Figure Lengend Snippet: REP1localization is affected by nutrient status and mTOR activity. ( A ) Hela and ( B ) MiaPaCa2 cells were plated and incubated for 24 h, which were further incubated in treatment with Torin2 at 20 nM or replaced with amino acid-deplete media for 4 h. Then the cells were stained with a lysotracker and intracellular fluorescent signals were analyzed by fluorescence microcopy. Co-localization levels were presented as a co-localization coefficient based on image quantification, described in materials and methods. Error bars indicate mean +/− standard error for n = 3 independent experiments. Statistical significance was determined via Student’s t -test; * p < 0.05.

Article Snippet: The Hela cells, and Panc1, 8988T, and MiaPaCa2 human pancreatic cancer cell lines from the American Type Culture Collection (ATCC; Manassas, VA, USA) were kindly provided by Kyung-Tae Kim and Yun-Hee Kim (National Cancer Center, Goyang-si, Korea).

Techniques: Activity Assay, Incubation, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: REP1 Modulates Autophagy and Macropinocytosis to Enhance Cancer Cell Survival

doi: 10.3390/ijms18091866

Figure Lengend Snippet: REP1 depletion induces macro pinocytosis ( A ) MiaPaCa2 cells were treated with CTL or REP1 siRNA for 24 h and replaced with serum deplete media to incubate further for another 12 h. Then the cells were treated with FITC-dextran at final concentration of 10 µg/mL for 30min and analyzed for cellular uptake of FITC-dextran by fluorescence microscopy. The level of macropinocytic uptake was quantified by image-based determination of the total macro pinocytic vesicle area compared with DAPI-stained area within cell. Data are expressed as a percentage of the DAPI-stained area within the cell area Statistical significance was determined via t -test; * p < 0.05; ( B ) MiaPaCa2 cells were transfected with CTL or REP1 siRNA for 24 h and replaced with either serum complete- or deplete-media for further incubation. Concomitantly with media replacement, either EIPA or Chloroquine (CQ) was treated respectively at final concentration of 15 μM in either REP1 or CTL siRNA. After 48 h of further treatment, cell death assay was performed using Annexin V/PI staining to show the cell apoptotic rate. Error bars indicate mean +/− standard error for n = 3 independent experiments.

Article Snippet: The Hela cells, and Panc1, 8988T, and MiaPaCa2 human pancreatic cancer cell lines from the American Type Culture Collection (ATCC; Manassas, VA, USA) were kindly provided by Kyung-Tae Kim and Yun-Hee Kim (National Cancer Center, Goyang-si, Korea).

Techniques: Concentration Assay, Fluorescence, Microscopy, Staining, Transfection, Incubation