Journal: JCI insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: Figure 3. SPINK1 secreted from cells induces cancer radioresistance in a paracrine manner. (A) HeLa tumor xenografts were stained with antibodies against a hypoxia marker, pimonidazole (green), or SPINK1 (red). Blue, counter staining with Hoechst 33342. The dotted line represents the outside edge of the pimonidazole-positive regions. Scale bar: 50 μm. (B–E) After 24 hours serum starvation, DU145 cells were precultured in the presence or absence of 100 ng/mL rSPINK1 for 24 hours, treated with the indicated dose of γ-ray irradiation, and subjected to the clonogenic survival assay (B), col- orimetric cell viability assay (C), and FACS analysis for the cell cycle status (D) and sub-G1 fraction (E). The cells were precultured and irradiated under the indicated oxygen conditions in C. (F and G) After transfection with either pcDNA4/SPINK1 (SPINK1) or its EV, the indicated cells were precultured under mild hypoxic conditions (O2 = 3%) for 48 hours, treated with the indicated dose of γ-ray irradiation under the same oxygen conditions as the

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Staining, Marker, Irradiation, Clonogenic Cell Survival Assay, Viability Assay, Transfection

Journal: JCI insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: Figure 4. SPINK1 accelerates tumor growth after radiotherapy. (A–C) DU145/EV and DU145/SPINK1 cells were cultured under the indicated oxygen conditions for 48 hours and subjected to qPCR (A) and the ELISA assay (B) or treated with the indicated dose of γ-ray irradiation for the clonogenic survival assay (C). (D and E) DU145/EV or SPINK1 xenografts were locally irradiated at a dose of 0 (solid lines) or 10 (dotted lines) Gy. When the volumes of the xenografts reached the same sizes as those on day 0, plasma SPINK1 levels were quantified by ELISA assays (D). Tumor growth was ana- lyzed after the treatment (E). Data are represented as mean ± SD (n = 3 in A and B, n = 6 in C, n = 5 in D, and n = 9–10 in E). Two-tailed Student’s t test. ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation, Clonogenic Cell Survival Assay, Clinical Proteomics, Two Tailed Test, Plasmid Preparation

Journal: JCI insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: Figure 5. SPINK1 decreases radiation-induced DNA damage and enhances radioresistance of cancer cells in a EGFR-dependent manner. (A–D) Four days after being transfected with either pcDNA4/SPINK1 or its EV, DU145/EGFP-53BP1-M (A and B) and DU145 (C and D) cells were irradiated with 0 or 4 Gy of γ-rays in the presence or absence of EGFR-I III (D), and the DNA DSBs detected as EGFP-53BP1 foci (A and B) or as γH2AX foci (C and D) were analyzed 2 hours (A and B) or 15 minutes (C and D) after the radiation. (A) Immunocytochemical analysis. Green, EGFP-53BP1 foci; blue, counter staining using Hoechst 33342. Scale bar: 10 μm. (B–D) The number of foci increased by 4 Gy γ-IR was calculated by subtracting the number of foci at 0 Gy from that at 4 Gy under each condition and represented as dot plots with mean ± SD. (E and F) DU145 cells were irradiated with 0 or 4 Gy of γ-ray in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 0.5 μM EGFR-I III (E), or with control IgG or 10 μg/mL cetuximab (F), and subjected to colori- metric cell viability assays. (G) The same experiment as in Figure 3B was conducted in the presence or absence of EGFR-I III. Data are represented as mean (n > 1000 in B–D) and mean ± SD (n = 3 in E and F, and n = 6 in G). Two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector. EV, empty vector; EGFR-I III, EGFR Inhibitor III; DSBs, double-strand breaks.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Transfection, Irradiation, Staining, Control, Two Tailed Test, Plasmid Preparation

Journal: JCI insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: Figure 6. SPINK1 decreases radiation-induced DNA damage and enhances radioresistance of cancer cells in a NRF2-dependent manner. (A and B) Four days after being transfected with either pcDNA4/SPINK1 (SPINK1) or its EV, DU145 cells were subjected to qPCR. (C–I) HeLa/scramble cells and HeLa/shSPINK1-1, HeLa/shSPINK1-2, and HeLa/shSPINK1-3 cells were cultured under severe hypoxic conditions (O2 < 0.1%) for 48 hours, irradiated with 0 (C, D, and I) or 4 (E–I) Gy of γ-rays and subjected to qPCR (C–H) or the DCFDA cellular ROS assay (I). Cells were irradiated in the presence or absence of the EGFR-I III (G and H). (J) DU145 cells were irradiated with γ-rays in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 2 μM ML385 and subjected to the colorimetric cell viability assay. Data are represented as mean ± SD (n = 3 in A–J). Two-tailed Student’s t test (A, B, and J). One-way ANOVA with Dunnett’s test (C–I). *P < 0.05, **P < 0.01, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; DCFDA, dichlorodihydrofluorescein diacetate; EGFR-I III, EGFR Inhibitor III.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Transfection, Cell Culture, Irradiation, ROS Assay, Viability Assay, Two Tailed Test, Plasmid Preparation

Journal: JCI insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: Figure 7. SPINK1 protein is expressed in severely hypoxic regions and secreted to the proximal regions of blood vessels. (A and B) Two pairs of serial sections of clinical human ccRCC tissues from 2 independent patients were stained with the indicated antibodies in A and B, respectively. Corresponding serial sections were stained with H&E. Scale bar: 50 μm. N, necrosis. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; ccRCC, clear cell renal cell carcinoma.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Staining, Plasmid Preparation

Journal: JCI insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: Figure 8. SPINK1 in plasma reflects the degree of hypoxia within tumor tissue in vivo. (A) A scatter plot for correlation analysis between mRNA levels of SPINK1 and CA9 in 36 HeLa tumor xenografts showed a good coefficient of determination, R2 = 0.9458. (B and C) After blood flow to the HeLa tumor xeno- grafts was decreased by ligaturing the leg for the indicated times, levels of SPINK1 mRNA (B) and SPINK1 protein (C) in the tumor tissues were quantified by qPCR and the ELISA assay, respectively. (D–I) After anemia treatment by phenylhydrazine administration, mRNA levels of EPO in the kidneys (D and E) and those of CA9 (E–H) and SPINK1 (F) in tumor tissues were quantified by qPCR. SPINK1 protein levels in tumors (G) and plasma (H and I) and the tumor volume (I) were measured by the ELISA assay and digital calipers, respectively. Scatter plots for correlation analysis between the 2 indicated factors (E–I). Data are represented as mean ± SD (B–D; n = 36 in A, n = 9–10 in B and C, n = 5 in D–I). Two-tailed Student’s t test (D). One-way ANOVA with Dunnett’s test (B and C). *P < 0.05, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; CA9, carbonic anhydrase 9; EPO, erythropoietin.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Clinical Proteomics, In Vivo, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Plasmid Preparation

Journal: Nature Communications

Article Title: EXO1 as a therapeutic target for Fanconi Anaemia, ZRSR2 and BRCA1-A complex deficient cancers

doi: 10.1038/s41467-025-63349-7

Figure Lengend Snippet: a Validation of EXO1 and FANCG synthetic lethal interaction in eHAP iCas9 cells by clonogenic assay (representative images, n = 3, well diameter 15.5 mm). b Quantification of clonogenic assay ( n = 3, mean with SD) in ( a ) with ordinary one-way ANOVA statistical analysis ( F = 158.8). c Validation of EXO1 and FANCG synthetic lethal interaction in eHAP iCas9 cells by CellTitre-Glo viability assay ( n = 3, mean with SD) with ordinary one-way ANOVA statistical analysis ( F = 132.5). d Apoptosis of EXO1 and FANCG double KO cells ( n = 3). e Quantification of cell cycle phases ( n = 4, based on gating shown in Supplementary Fig. , mean with SD). f Micronuclei increase in double KO cells. Paired t -test analyses were performed for each of the double KO to compare to ‘ EXO1 KO + sgNT’ ( n = 3, mean with SD, with each percentage calculated on the basis of >2181 nuclei; two-tailed P values). g Increase in phosphorylated RPA at serine 33 (RPA-pS33) foci in double KO cells. Dots in orange, blue and green represent the mean of a biological repeat, with the red line as their mean. Paired t -test analyses were performed on the means of biological repeat for each of the double KO cell lines in comparison to the ‘ EXO1 KO + sgNT’ cell line ( n = 3, mean with SD, with each percentage calculated on the basis of >754 nuclei; RPA-pS33 signal was measured within nuclei; two-tailed P values). h Immunoblotting of total cell extracts and chromatin for ‘WT + sgNT’, ‘WT + sg FANCG ’, ‘ EXO1 KO + sgNT’ and ‘ EXO1 KO + sg FANCG ’ eHAP iCas9 cells at day 5 post Cas9 expression induction. Samples were probed for phosphorylated levels of KAP1, p53, CHK2 and H2AX with controls, and EXO1, alpha-tubulin and histone H3 to control for sample identity and equal loading (representative experiment, n = 3). Source data for b – h are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-EXO1 (ab95068, Abcam, rabbit, 1:1000), anti-alpha-tubulin (clone B-5-1-2, T6074, Sigma, mouse, 1:10000), anti-vinculin (clone hVIN-1, ab11194, Abcam, mouse, 1:20000), anti-FANCG (clone F-8, sc-393382, Santa Cruz, mouse, 1:1000), anti-KAP1-pS824 (ab70369, Abcam, rabbit, 1:1000), anti-KAP1 (clone 20C1, ab22553, Abcam, mouse, 1:1000), anti-p53-pS15 (9284S, CST, rabbit, 1:1000), anti-p53 (clone 1C12, 2524S, CST, mouse, 1:1000), anti-CHK2-pT68 (2661S, CST, rabbit, 1:1000), anti-CHK2 (clone 7, 05-649, Millipore, mouse, 1:500), anti-H2AX-pS139 (gamma-H2AX, clone JBW301, 05-636, Millipore, mouse, 1:1000), anti-histone H3 (ab1791, Abcam, rabbit, 1:5000), anti-RPA-pS33 (A300-246A, Bethyl Laboratories, rabbit, 1:1000), anti-RPA (clone 9H8, ab2175, Abcam, mouse, 1:1000), anti-FANCD2 (clone EPR2302, ab108928, Abcam, rabbit, 1:1000), anti-RMI2 (ab122685, Abcam, rabbit, 1:500), anti-FAM175A/Abraxas1 (clone EPR6310(2), ab139191, Abcam, rabbit, 1:1000), anti-BRCC36 (A302-517A-M, Bethyl Laboratories, rabbit, 1:1000), anti-FANCC (clone 8F3, MABC524, Sigma, mouse, 1:1000).

Techniques: Biomarker Discovery, Clonogenic Assay, Viability Assay, Two Tailed Test, Comparison, Western Blot, Expressing, Control

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a Autophagy-related protein levels after treatment confirmed by western blot. b Schematic images of the autophagy initiation step involved in NRBF2. c Coimmunoprecipitation of NRBF2 and the VPS34 complex confirmed by western blot in GBM cell lines. d Autophagic flux analysis using the mCherry-EGFP reporter. A172 and U-87 MG cells transfected with mCherry-GFP-LC3B were treated with IR (3 Gy), siNRBF2 or both. The mean numbers of autophagosomes and autolysosomes are represented by yellow and red dots, respectively, in the merged images. The images were quantified using ImageJ ( n = 5). Scale bars, 10 µm. e IHC of LC3B in coronal sections from U-87 WT or NRBF2 knockout orthotopic xenograft mouse models treated with radiation (2 Gy × 5). Scale bars, 10 μm. ** p < 0.01, *** p < 0.001, **** p < 0.0001 using an unpaired t test.

Article Snippet: Primary antibodies specific for LC3B were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA), and those specific for NRBF2, Ki67, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Transfection, Knock-Out

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a qRT‒PCR analysis of nrbf2 mRNA levels after irradiation and lidoflazine treatment in a GBM cell line. b Western blot analysis to evaluate autophagic flux after irradiation and treatment of lidoflazine in U-87 MG cells. c Autophagic flux analysis using the mCherry-EGFP reporter. U-87 MG and A172 cells transfected with mCherry-GFP-LC3B were treated with IR (3 Gy), lidoflazine (1 μM), or both for 24 h. The mean number of autophagosomes and autolysosomes are represented by yellow and red dots, respectively, in the merged images. The images were quantified using ImageJ ( n = 5). Scale bars, 10 µm. d Percentage of GBM cells stained positively for the Ki67 proliferation marker with different treatments. Differences among means were statistically significant, according to a t test. e Clonogenic assay after IR (3 Gy), lidoflazine (1 μM), or both in U-87 MG cells cultured in low glucose media containing 1 mM glucose. Representative images of single-cell clone proliferation stained with crystal violet. f IC 50 of lidoflazine with or without IR in U-87 MG cells was measured by CellTiter-Glo® Luminescent Cell Viability Assay. Cells were treated with increasing concentrations of lidoflazine and/or IR (3 Gy) for 48 h. g Clonogenic assay after IR (3 Gy), lidoflazine (1 μM), or both in ATG7 knockout U-87 MG cells cultured in low glucose media containing 1 mM glucose. Representative images of single-cell clone proliferation stained with crystal violet. h Transwell migration assay (upper) and invasion assay (lower) of U-87 MG cells cultured in low glucose media containing 1 mM glucose with treatment with lidoflazine (1 μM), IR (3 Gy), or both. i U-87 MG cells in a three-dimensional culture system were stained with α-tubulin (green) and DAPI (blue). Scale bars, 10 µm. * p < 0.05, *** p < 0.001, **** p < 0.0001 using an unpaired t test .

Article Snippet: Primary antibodies specific for LC3B were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA), and those specific for NRBF2, Ki67, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Irradiation, Western Blot, Transfection, Staining, Marker, Clonogenic Assay, Cell Culture, Cell Viability Assay, Knock-Out, Transwell Migration Assay, Invasion Assay

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a qRT‒PCR analysis of nrbf2 mRNA levels after IR (3 Gy) or additional treatment with lidoflazine (1 µM) in RR cells or untreated U-87 MG cells. b Western blot analysis of NRBF2 protein expression levels after IR (3 Gy), lidoflazine (1 µM), or both in RR cells or untreated U-87 MG cells. c Autophagic flux analysis using the mCherry-EGFP reporter. U-87 MG and RR cells transfected with mCherry-GFP-LC3B were treated with IR (3 Gy), lidoflazine (1 μM), or both for 24 h. The mean numbers of autophagosomes and autolysosomes are represented by yellow and red dots, respectively, in the merged images. The images were quantified using ImageJ ( n = 5). Scale bars, 10 µm. d Percentage of GBM cells stained positively for the Ki67 proliferation marker undergoing different treatments. e Cell reproductive death after treatment with ionizing radiation (3 Gy), lidoflazine (1 µM), or both was determined using crystal violet staining. f IC 50 of lidoflazine with or without IR in U-87 MG RR cells was measured by CellTiter-Glo® Luminescent Cell Viability Assay. Cells were treated with increasing concentrations of lidoflazine and/or IR (3 Gy) for 48 h. g Transwell migration and invasion assays were performed to analyze cell movement. Scale bars, 200 µm. h Control and RR cells in a three-dimensional culture system were stained with α-tubulin (green) and DAPI (blue). Scale bars, 100 µm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using an unpaired t test .

Article Snippet: Primary antibodies specific for LC3B were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA), and those specific for NRBF2, Ki67, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing, Transfection, Staining, Marker, Cell Viability Assay, Migration, Control

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a Scheme of the U87-Luc orthotopic xenograft mouse model and mouse treatment schedule. b Survival graph of the U87-Luc orthotopic xenograft mouse model with radiation (2 Gy x 5) and lidoflazine (1 mg/kg). Statistical analysis was performed with the log-rank (Mantel‒Cox) test. c Luminescence images (left) and relative luminescence units (right) of the indicated U87-Luc orthotopic xenografts after radiotherapy, lidoflazine treatment or not. d IHC of NRBF2 (upper), LC3B (middle), and Ki67 (lower) in coronal sections from U87-Luc orthotopic xenograft mouse control or treated with IR (2 Gy x 5) and lidofolfazine (1 mg/kg). Scale bars, 50 μm. e H&E staining of organ sections from mice treated with vehicle or lidofructose (1 mg/kg). Scale bars, 50 μm. f Schematic diagram illustrating how increased NRBF2 expression upon irradiation leads to GBM resistance.

Article Snippet: Primary antibodies specific for LC3B were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA), and those specific for NRBF2, Ki67, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, Staining, Expressing, Irradiation

Journal: Turkish Journal of Biology

Article Title: The protective impact of growth hormone against rotenone-induced apoptotic cell death via acting on endoplasmic reticulum stress and autophagy axis

doi: 10.55730/1300-0152.2639

Figure Lengend Snippet: Rotenone treatment reduced cell viability in SH-SY5Y wt cells more than in human autocrine GH-gained SH-SY5Y cells. A. GH secretion of SH-SY5Y following GH-pcDNA3.1 plasmid transfection was determined as 1.98 pg/mL by ELISA assay. B. MTT cell viability assay was performed in rotenone treated-SH-SY5Y and SK-N-AS cells with/without GH in a dose-dependent manner (0.1, 0.5, 1, 5, and 10 μM). * p < 0.05, **p < 0.01, ***p < 0.001. C. The effect of rotenone on MMP loss-induced cell death was determined by DiOC6, PI, and DAPI staining in both SH-SY5Y wt and GH+ cells. D. Cell survival assay was performed to show the effect of rotenone in a time-dependent manner in both SH-SY5Y and SK-N-AS wt (upper figure) and GH+ (lower figure) cells. E–F. The effect of 0.5 μM rotenone on anchorage-independent cell growth was examined by soft agar assay in SH-SY5Y (magnification: 40×) and SK-N-AS (magnification: 10×) cell lines. Scale bar: 200 μm. Average diameter of spheroids for both Figures E and F was analyzed by Olympus Image Analyzing program.

Article Snippet: Secreted GH levels were determined in a filtered medium of 24 h serum-starved SH-SY5Y wt and GH+ cells by human GH ELISA Assay (Abcam, 190811) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Viability Assay, Staining, Clonogenic Cell Survival Assay, Soft Agar Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Towards a molecular understanding of the overlapping and distinct roles of UBQLN1 and UBQLN2 in lung cancer progression and metastasis

doi: 10.1016/j.neo.2021.11.010

Figure Lengend Snippet: Loss of UBQLN1 and UBQLN2 increases cell viability, clonogenic potential and combined loss of UBQLN1 and UBQLN2 exhibits additive effect on cell viability, clonogenic potential and induces EMT in A549 cells. Loss of UBQLN1 and UBQLN2 increases cell migration in A549 and HOP62 cells and combined loss of UBQLN1 and UBQLN2 exhibits additive effect on cell migration and cell invasion in A549 cells. (A) Cell viability assay in A549 cells. Cells (500,000) were seeded on 60mm plate and were transfected with either non targeting (si_NT) siRNA or positive control Kif11 (si_Kif11), two different siRNAs targeting UBQLN1 (si_U1 and si_U1-2), two different siRNAs targeting UBQLN2 (si_U2-5 and si_U2-6) and also combining two different siRNAs of UBQLN1 and UBQLN2 (si_U1+si_U2-5, si_U1-2+si_U2-6). After 24 hrs of transfection, cells were trypsinized and 1000 cells were reseeded in 96 well plates and cell viability was assessed for consecutive 4 days. Relative number of cells were compared by performing students’ t -test. * = significance compared to NT (* P < 0.05; ** P < 0.005). (B) Clonogenic assay in A549 cells. Cells were prepared as described in A. After 24 hrs of transfection, cells were trypsinized and 1000 cells were reseeded on 6 well plates in triplicates. Following the 10 days, cells were fixed and stained. (C) Quantification of relative number of colonies formed per 1000 number of cells seeded in B. Number of colonies formed were compared by performing students’ t-test. * = significance compared to NT (* P < 0.05; ** P < 0.005). (D) Western blot analysis of proteins involved in EMT. Cells were prepared as described in A. After 72 hrs of transfection, cells were harvested and analyzed for protein involved in EMT. * - UBQLN1; + - UBQLN2. (E) Cell migration assay/scratch assay in A549 and HOP62 cells. Representative bar graph showing percentage wound closure following loss of either UBQLN1, UBQLN2 or combined loss of UBQLN1 and UBQLN2. Cells were prepared as described in A. 24 hrs post siRNA transfections a pipette tip was used to scratch the dish to make a “wound”. Cells were examined after wound has been formed and successively for 24hr and 48hr post wound formation, photographed and quantified. Wound closure was analyzed using a Two-way ANOVA followed by a Tukey post-hoc test. * = significance compared to NT, $ = significance compared to U1, ^ = significance compared to U2. One symbol denotes a p -value < 0.05, two symbols denote a p -value < 0.005. (F) Boyden chamber cell migration and invasion assay in A549 cells. Cells were prepared as described in A and 24 h after siRNA transfection cells were seeded into Boyden chambers without or with matrigel. The lower chamber contained media with serum, whereas the upper chamber containing the cells was without serum. 48 h later cells on the underside of the membrane were fixed and stained. (G) Quantification of relative number of cells migrated or invaded through matrigel. Migration and invasion was analyzed using a one way ANOVA followed by a Tukey post-hoc test. * = significance compared to NT, $ = significance compared to U1, ^ = significance compared to U2. One symbol denotes a p -value < 0.05, two symbols denote a p -value < 0.005.

Article Snippet: Human lung adenocarcinoma cell line A549, neuroblastoma cell line SK-N-BE(2) and IMR90 fetal lung fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA).

Techniques: Migration, Viability Assay, Transfection, Positive Control, Clonogenic Assay, Staining, Western Blot, Cell Migration Assay, Wound Healing Assay, Transferring, Invasion Assay, Membrane

Journal: Neoplasia (New York, N.Y.)

Article Title: Towards a molecular understanding of the overlapping and distinct roles of UBQLN1 and UBQLN2 in lung cancer progression and metastasis

doi: 10.1016/j.neo.2021.11.010

Figure Lengend Snippet: RNAseq comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 and SK-N-BE(2) cells. (A) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 cells following RNAseq analysis. (B) Representative list of top 10 genes down-regulated specifically by siUBQLN1. (C) Representative list of top 10 genes down-regulated specifically by siUBQLN2. (D) Representative list of top 10 genes up-regulated specifically by siUBQLN1. (E) Representative list of top 10 genes up-regulated specifically by siUBQLN2. (F) Representative bar graph showing confirmation of genes that were significantly altered following loss of UBQLN1 in A549 and HOP62 cells. (G) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in SK-N-BE(2) cells following RNAseq analysis. (H) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 and SK-N-BE(2) cells following RNAseq analysis.

Article Snippet: Human lung adenocarcinoma cell line A549, neuroblastoma cell line SK-N-BE(2) and IMR90 fetal lung fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA).

Techniques: Knockdown

Journal: Neoplasia (New York, N.Y.)

Article Title: Towards a molecular understanding of the overlapping and distinct roles of UBQLN1 and UBQLN2 in lung cancer progression and metastasis

doi: 10.1016/j.neo.2021.11.010

Figure Lengend Snippet: RNAseq comparisons of overlapping genes differentially expressed upon UBQLN1 knockdown in differentiated iPSC-WNT and mESC-EGFR cells. (A) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 knockdown in unmanipulated iPSC and differentiated iPSC-WNT epithelial and iPSC-EGF neuronal cells following RNAseq analysis. (B) Representative list of overlapping 17 genes that were significantly altered following UBQLN1 knockdown in unmanipulated iPSC, differentiated iPSC-WNT epithelial and iPSC-EGF neuronal cells following RNAseq analysis. (* = found in A549 UBQLN1/UBQLN2 knockdown common signature). (C) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 knockdown in unmanipulated mESC, differentiated mESC-WNT epithelial and mESC-EGF neuronal cells following RNAseq analysis. (D) Representative list of overlapping 21 genes that were significantly altered following UBQLN1 knockdown in unmanipulated mESC, differentiated mESC-WNT epithelial and mESC-EGF neuronal cells following RNAseq analysis.

Article Snippet: Human lung adenocarcinoma cell line A549, neuroblastoma cell line SK-N-BE(2) and IMR90 fetal lung fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA).

Techniques: Knockdown

Journal: Nature Communications

Article Title: EXO1 as a therapeutic target for Fanconi Anaemia, ZRSR2 and BRCA1-A complex deficient cancers

doi: 10.1038/s41467-025-63349-7

Figure Lengend Snippet: a Validation of EXO1 and FANCG synthetic lethal interaction in eHAP iCas9 cells by clonogenic assay (representative images, n = 3, well diameter 15.5 mm). b Quantification of clonogenic assay ( n = 3, mean with SD) in ( a ) with ordinary one-way ANOVA statistical analysis ( F = 158.8). c Validation of EXO1 and FANCG synthetic lethal interaction in eHAP iCas9 cells by CellTitre-Glo viability assay ( n = 3, mean with SD) with ordinary one-way ANOVA statistical analysis ( F = 132.5). d Apoptosis of EXO1 and FANCG double KO cells ( n = 3). e Quantification of cell cycle phases ( n = 4, based on gating shown in Supplementary Fig. , mean with SD). f Micronuclei increase in double KO cells. Paired t -test analyses were performed for each of the double KO to compare to ‘ EXO1 KO + sgNT’ ( n = 3, mean with SD, with each percentage calculated on the basis of >2181 nuclei; two-tailed P values). g Increase in phosphorylated RPA at serine 33 (RPA-pS33) foci in double KO cells. Dots in orange, blue and green represent the mean of a biological repeat, with the red line as their mean. Paired t -test analyses were performed on the means of biological repeat for each of the double KO cell lines in comparison to the ‘ EXO1 KO + sgNT’ cell line ( n = 3, mean with SD, with each percentage calculated on the basis of >754 nuclei; RPA-pS33 signal was measured within nuclei; two-tailed P values). h Immunoblotting of total cell extracts and chromatin for ‘WT + sgNT’, ‘WT + sg FANCG ’, ‘ EXO1 KO + sgNT’ and ‘ EXO1 KO + sg FANCG ’ eHAP iCas9 cells at day 5 post Cas9 expression induction. Samples were probed for phosphorylated levels of KAP1, p53, CHK2 and H2AX with controls, and EXO1, alpha-tubulin and histone H3 to control for sample identity and equal loading (representative experiment, n = 3). Source data for b – h are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-EXO1 (ab95068, Abcam, rabbit, 1:1000), anti-alpha-tubulin (clone B-5-1-2, T6074, Sigma, mouse, 1:10000), anti-vinculin (clone hVIN-1, ab11194, Abcam, mouse, 1:20000), anti-FANCG (clone F-8, sc-393382, Santa Cruz, mouse, 1:1000), anti-KAP1-pS824 (ab70369, Abcam, rabbit, 1:1000), anti-KAP1 (clone 20C1, ab22553, Abcam, mouse, 1:1000), anti-p53-pS15 (9284S, CST, rabbit, 1:1000), anti-p53 (clone 1C12, 2524S, CST, mouse, 1:1000), anti-CHK2-pT68 (2661S, CST, rabbit, 1:1000), anti-CHK2 (clone 7, 05-649, Millipore, mouse, 1:500), anti-H2AX-pS139 (gamma-H2AX, clone JBW301, 05-636, Millipore, mouse, 1:1000), anti-histone H3 (ab1791, Abcam, rabbit, 1:5000), anti-RPA-pS33 (A300-246A, Bethyl Laboratories, rabbit, 1:1000), anti-RPA (clone 9H8, ab2175, Abcam, mouse, 1:1000), anti-FANCD2 (clone EPR2302, ab108928, Abcam, rabbit, 1:1000), anti-RMI2 (ab122685, Abcam, rabbit, 1:500), anti-FAM175A/Abraxas1 (clone EPR6310(2), ab139191, Abcam, rabbit, 1:1000), anti-BRCC36 (A302-517A-M, Bethyl Laboratories, rabbit, 1:1000), anti-FANCC (clone 8F3, MABC524, Sigma, mouse, 1:1000).

Techniques: Biomarker Discovery, Clonogenic Assay, Viability Assay, Two Tailed Test, Comparison, Western Blot, Expressing, Control

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a Scheme of the U-87 Luc orthotopic xenograft mouse model and mouse irradiation schedule. b Microarray experiments were performed using cDNA samples obtained from orthotopic xenograft GBM tumor sections treated or not treated with 2Gy × 5 radiation, and microarray results were deposited in the Gene Expression Omnibus database (GEO: GSE117126). c Hematoxylin and eosin (H&E) staining (left panel) and immunohistochemistry (IHC) of Ki67 and NRBF2 (middle panel and right panel) of coronal sections from U-87 MG orthotopic xenograft mouse control or treated with IR (2Gy × 5). Scale bars, 10 μm. d Real-time qRT‒PCR analysis of the nrbf2 mRNA levels in the GBM orthotopic xenograft mouse model. e Real-time qRT‒PCR analysis of the nrbf2 mRNA levels in GBM cell lines. f Western blot analysis of control and IR-treated GBM cells shows IR-induced expression of NRBF2. g The gene expression profiles were collected from The Cancer Genome Atlas (TCGA) database available through Gliovis (PMID: 28031383). mRNA expression of nrbf2 was measured in 10 nontumor tissues and 528 GBM tissues. h Nrbf2 gene expression profiles were collected from The Cancer Genome Atlas (TCGA, Pan-Cancer Atlas) database ( https://www.cbioportal.org/ ). mRNA expression of nrbf2 was measured in nine different cancer types, including GBM. ** p < 0.01, **** p < 0.0001 using an unpaired t test.

Article Snippet: Primary antibodies specific for LC3B were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA), and those specific for NRBF2, Ki67, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Irradiation, Microarray, Gene Expression, Staining, Immunohistochemistry, Control, Western Blot, Expressing

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a Real-time qRT‒PCR analysis of nrbf2 mRNA levels in U-87 MG and A172 cells after nrbf2 knockdown. b NRBF2 protein levels after NRBF2 siRNA transfection confirmed by western blot. c , d Representative images of the transwell migration assay ( c ) and invasion assay ( d ) of GBM cell lines cultured in low glucose media containing 1 mM glucose with NRBF2 or IR (3 Gy) treatment siRNA or both. Scale bars, 200 µm. e Clonogenic assay after NRBF2 knockdown, IR (3 Gy) or both in GBM cells cultured in low glucose media containing 1 mM glucose. Representative images of single-cell clone proliferation stained with crystal violet. f DepMap ( https://depmap.org/portal/ ) analysis of the expression of NRBF2 in CNS, glioma, medulloblastoma, and meningioma. g Survival graph of U-87 Luc WT or NRBF2 knockout orthotopic xenograft mouse model with radiation (2Gy × 5). Statistical analysis was performed with the log-rank (Mantel‒Cox) test. h Luminescence images (left) and relative luminescence units (right) of the indicated U87-Luc orthotopic xenografts.

Article Snippet: Primary antibodies specific for LC3B were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA), and those specific for NRBF2, Ki67, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Knockdown, Transfection, Western Blot, Transwell Migration Assay, Invasion Assay, Cell Culture, Clonogenic Assay, Staining, Expressing, Knock-Out

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a Autophagy-related protein levels after treatment confirmed by western blot. b Schematic images of the autophagy initiation step involved in NRBF2. c Coimmunoprecipitation of NRBF2 and the VPS34 complex confirmed by western blot in GBM cell lines. d Autophagic flux analysis using the mCherry-EGFP reporter. A172 and U-87 MG cells transfected with mCherry-GFP-LC3B were treated with IR (3 Gy), siNRBF2 or both. The mean numbers of autophagosomes and autolysosomes are represented by yellow and red dots, respectively, in the merged images. The images were quantified using ImageJ ( n = 5). Scale bars, 10 µm. e IHC of LC3B in coronal sections from U-87 WT or NRBF2 knockout orthotopic xenograft mouse models treated with radiation (2 Gy × 5). Scale bars, 10 μm. ** p < 0.01, *** p < 0.001, **** p < 0.0001 using an unpaired t test.

Article Snippet: Primary antibodies specific for LC3B were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA), and those specific for NRBF2, Ki67, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Transfection, Knock-Out

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured by a Seahorse XFp Analyzer in U-87 MG and A172 cells with or without IR (3 Gy), IR-treated U-87 MG cells with or without NRBF2 knockout, and IR-treated A172 cells with or without NRBF2 knockdown. b OCR and ECAR were measured in IR-treated patient-derived GBM cells with or without NRBF2 knockdown. c – g Each bar graph shows the metabolite levels in WT and NRBF2 knockout U-87 MG cells. c ATP levels were determined in WT and NRBF2 knockout U-87 MG cells after irradiation (3 Gy). d TCA cycle metabolites such as citrate, succinate, fumarate, and malate were detected. e Metabolites of glycolysis-related pathways were determined by measuring the NADP + /NADPH ratio, glycerol-3-phosphate, and serine levels. f WT and NRBF2 knockout U-87 MG cells were incubated with [1- 14 C] oleic acid, and 14 CO 2 production from the complete b-oxidation of [1- 14 C] oleic acid was analyzed. g After incubation with [9,10- 3 H] oleic acid (pulse) followed by incubation in DMEM (chase), media equivalent to 500 μg of cell protein were analyzed for radioactivity in the form of 3 H 2 O. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using an unpaired t test.

Article Snippet: Primary antibodies specific for LC3B were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA), and those specific for NRBF2, Ki67, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Knock-Out, Knockdown, Derivative Assay, Irradiation, Incubation, Radioactivity

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a qRT‒PCR analysis of nrbf2 mRNA levels after irradiation and lidoflazine treatment in a GBM cell line. b Western blot analysis to evaluate autophagic flux after irradiation and treatment of lidoflazine in U-87 MG cells. c Autophagic flux analysis using the mCherry-EGFP reporter. U-87 MG and A172 cells transfected with mCherry-GFP-LC3B were treated with IR (3 Gy), lidoflazine (1 μM), or both for 24 h. The mean number of autophagosomes and autolysosomes are represented by yellow and red dots, respectively, in the merged images. The images were quantified using ImageJ ( n = 5). Scale bars, 10 µm. d Percentage of GBM cells stained positively for the Ki67 proliferation marker with different treatments. Differences among means were statistically significant, according to a t test. e Clonogenic assay after IR (3 Gy), lidoflazine (1 μM), or both in U-87 MG cells cultured in low glucose media containing 1 mM glucose. Representative images of single-cell clone proliferation stained with crystal violet. f IC 50 of lidoflazine with or without IR in U-87 MG cells was measured by CellTiter-Glo® Luminescent Cell Viability Assay. Cells were treated with increasing concentrations of lidoflazine and/or IR (3 Gy) for 48 h. g Clonogenic assay after IR (3 Gy), lidoflazine (1 μM), or both in ATG7 knockout U-87 MG cells cultured in low glucose media containing 1 mM glucose. Representative images of single-cell clone proliferation stained with crystal violet. h Transwell migration assay (upper) and invasion assay (lower) of U-87 MG cells cultured in low glucose media containing 1 mM glucose with treatment with lidoflazine (1 μM), IR (3 Gy), or both. i U-87 MG cells in a three-dimensional culture system were stained with α-tubulin (green) and DAPI (blue). Scale bars, 10 µm. * p < 0.05, *** p < 0.001, **** p < 0.0001 using an unpaired t test .

Article Snippet: Primary antibodies specific for LC3B were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA), and those specific for NRBF2, Ki67, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Irradiation, Western Blot, Transfection, Staining, Marker, Clonogenic Assay, Cell Culture, Cell Viability Assay, Knock-Out, Transwell Migration Assay, Invasion Assay

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a qRT‒PCR analysis of nrbf2 mRNA levels after IR (3 Gy) or additional treatment with lidoflazine (1 µM) in RR cells or untreated U-87 MG cells. b Western blot analysis of NRBF2 protein expression levels after IR (3 Gy), lidoflazine (1 µM), or both in RR cells or untreated U-87 MG cells. c Autophagic flux analysis using the mCherry-EGFP reporter. U-87 MG and RR cells transfected with mCherry-GFP-LC3B were treated with IR (3 Gy), lidoflazine (1 μM), or both for 24 h. The mean numbers of autophagosomes and autolysosomes are represented by yellow and red dots, respectively, in the merged images. The images were quantified using ImageJ ( n = 5). Scale bars, 10 µm. d Percentage of GBM cells stained positively for the Ki67 proliferation marker undergoing different treatments. e Cell reproductive death after treatment with ionizing radiation (3 Gy), lidoflazine (1 µM), or both was determined using crystal violet staining. f IC 50 of lidoflazine with or without IR in U-87 MG RR cells was measured by CellTiter-Glo® Luminescent Cell Viability Assay. Cells were treated with increasing concentrations of lidoflazine and/or IR (3 Gy) for 48 h. g Transwell migration and invasion assays were performed to analyze cell movement. Scale bars, 200 µm. h Control and RR cells in a three-dimensional culture system were stained with α-tubulin (green) and DAPI (blue). Scale bars, 100 µm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using an unpaired t test .

Article Snippet: Primary antibodies specific for LC3B were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA), and those specific for NRBF2, Ki67, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing, Transfection, Staining, Marker, Cell Viability Assay, Migration, Control

Journal: Experimental & Molecular Medicine

Article Title: NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

doi: 10.1038/s12276-022-00873-2

Figure Lengend Snippet: a Scheme of the U87-Luc orthotopic xenograft mouse model and mouse treatment schedule. b Survival graph of the U87-Luc orthotopic xenograft mouse model with radiation (2 Gy x 5) and lidoflazine (1 mg/kg). Statistical analysis was performed with the log-rank (Mantel‒Cox) test. c Luminescence images (left) and relative luminescence units (right) of the indicated U87-Luc orthotopic xenografts after radiotherapy, lidoflazine treatment or not. d IHC of NRBF2 (upper), LC3B (middle), and Ki67 (lower) in coronal sections from U87-Luc orthotopic xenograft mouse control or treated with IR (2 Gy x 5) and lidofolfazine (1 mg/kg). Scale bars, 50 μm. e H&E staining of organ sections from mice treated with vehicle or lidofructose (1 mg/kg). Scale bars, 50 μm. f Schematic diagram illustrating how increased NRBF2 expression upon irradiation leads to GBM resistance.

Article Snippet: Primary antibodies specific for LC3B were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA), and those specific for NRBF2, Ki67, GAPDH, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, Staining, Expressing, Irradiation

Journal: Cancers

Article Title: UBQLN Family Members Regulate MYC in Lung Adenocarcinoma Cells

doi: 10.3390/cancers15133389

Figure Lengend Snippet: Loss of UBQLN1 and UBQLN2 increases cell viability, cell proliferation, cell cycle progression, and clonogenic potential in lung adenocarcinoma cells. ( A ) Cell viability assay in H2030 and H2009 cells. Cells were transfected as indicated. After 24 h of transfection, cells were trypsinized and 1000 cells were reseeded in 96-well plates and cell viability was assessed for 5 consecutive days. ( Bi , ii ) AnnexinV and 7AAD staining in A549 cells. Cells were prepared as described in methodology followed by incubation with AnnexinV antibody for one hour. After subsequent washing, cells were incubated with 7AAD and analyzed for apoptosis by flow cytometry analysis. ( Ci , ii ) Cell proliferation assay in A549 cells. Cells were prepared as described in methodology followed by incubation with anti-BrdU antibody for one hour. After subsequent washing, cells were incubated with 7AAD and analyzed by flow cytometry analysis. ( D ) Western blot analysis of cell cycle progression markers in A549, HOP62, and H23 cells. ( Ei , ii ) Clonogenic assay in H2030, H2009, and A549 cells (* p < 0.05, ** p < 0.01). The original western blots is .

Article Snippet: Human lung adenocarcinoma cell lines A549, HOP62, H23, and H2009 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA).

Techniques: Viability Assay, Transfection, Staining, Incubation, Flow Cytometry, Proliferation Assay, Western Blot, Clonogenic Assay

Journal: Cancers

Article Title: UBQLN Family Members Regulate MYC in Lung Adenocarcinoma Cells

doi: 10.3390/cancers15133389

Figure Lengend Snippet: Loss of UBQLN1 and/or UBQLN2 stabilizes MYC. ( A ) Western blot analysis of MYC in A549, HOP62, H23, and H2009 cells. ( B ) Western blot analysis of MYC in A549 cells. Cells were prepared as described in methodology, followed by treatment with either vehicle alone or with cycloheximide for indicated time points. After 72 h of transfection, cells were harvested and analyzed for MYC expression. ( C ) Western blot analysis of MYC in A549 and HOP62 cells. Cells were prepared as described in methodology, except cells were also transfected with combination of siRNA targeting UBQLN1 along with siRNA targeting UBQLN2 (si_U1+si_U2-5). ( D ) Western blot analysis of MYC in PC9 and H358 cells. The original western blots is .

Article Snippet: Human lung adenocarcinoma cell lines A549, HOP62, H23, and H2009 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Sigma, St Louis, MO, USA).

Techniques: Western Blot, Transfection, Expressing

Journal: PLoS ONE

Article Title: Combination of Gefitinib and DNA Methylation Inhibitor Decitabine Exerts Synergistic Anti-Cancer Activity in Colon Cancer Cells

doi: 10.1371/journal.pone.0097719

Figure Lengend Snippet: (A) and (B) SW1116 and LOVO cells were cultured in control conditions (DMSO) or in the presence of the indicated concentrations of decitabine (DAC) and gefitinib (GEF), alone or in combination, for 48 h, and then assessed for viability by MTT assay. Results are means of duplicate assessments from one out of three independent experiments. (C) and (D) SW1116 cells and LOVO cells were plated, treated, and processed as in A and B. The dose–response curve of each drug was determined and combination index (CI) values for DAC/GEF concentration ratios (2.5∶1 in SW1116 cells, 2∶1 in LOVO cells) were calculated according to the Chou–Talalay's method at the 48 h time point, with the biological response being expressed as the fraction of affected cells. Rectangle symbol and diamond symbol designate the CI value for each fraction affected (effect). CI<1, CI = 1, CI>1 indicate synergistic, additive and antagonistic effects, respectively. The effect ranges from 0 (no inhibition) to 1 (complete inhibition). The data are representative of three independent experiments. (E) Influence of SW1116 cells and LOVO cells on the number of colony-forming cells, as evaluated by clonogenic assay. For colony-forming assay, the clonogenic assay was done as described in . Columns, mean of three determinations; bars, SD. Results shown are representative of three independent experiments. **, P <0.01, ***, P <0.001, compared with DAC-treated cells. ##, P <0.01, ###, P <0.001, compared with GEF-treated cells.

Article Snippet: For clonogenic cell survival experiments, the attached cells from the same 10 cm culture dish that were trypsinized with 1 mL trypsin–EDTA (Gibco; Life Technologies, Carlsbad, CA) and inactivated with media containing 10% FBS.

Techniques: Cell Culture, Control, MTT Assay, Concentration Assay, Inhibition, Clonogenic Assay

Journal: International Journal of Oncology

Article Title: Development of single nanometer-sized ultrafine oxygen bubbles to overcome the hypoxia-induced resistance to radiation therapy via the suppression of hypoxia-inducible factor-1α

doi: 10.3892/ijo.2018.4248

Figure Lengend Snippet: Oxygen nanobubble water suppresses HIF-1α accumulation in hypoxic cancer cells. (A) HIF-1α and HSC70 protein expression in the EBC-1 lung cancer cell line and MDA-MB-231 breast cancer cell line was evaluated by western blot analysis after 6 and 24 h of exposure ot hypoxia. Oxygen nanobubble medium clearly suppressed HIF-1α induction under hypoxic conditions. (B) Hypoxia-induced radiation resistance was validated in both the EBC-1 and MDA-MB-231 cells. DW, normal medium; O 2 , oxygen nanobubble medium.

Article Snippet: The EBC-1 human lung cancer cell line was purchased from the RIKEN Cell Resource Center of Biomedical Research (Tsukuba, Japan), and the MDA-MB-231 human breast cancer cell line and BEAS-2S non-cancerous human bronchial cell line were from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Western Blot

Journal: International Journal of Oncology

Article Title: Development of single nanometer-sized ultrafine oxygen bubbles to overcome the hypoxia-induced resistance to radiation therapy via the suppression of hypoxia-inducible factor-1α

doi: 10.3892/ijo.2018.4248

Figure Lengend Snippet: Oxygen nanobubble medium reverses hypoxia-induced radiation resistance in EBC-1 lung cancer and MDA-MB-231 breast cancer cells. (A) Cell viability assay showed that oxygen nanobubble medium suppressed hypoxia-induced radiation resistance in EBC-1 cells. A similar effect was observed with the MDA-MB-231 cells, although this was not significant; however, a similar tendency was validated. (B) Clonogenic assay revealed that oxygen nanobubble medium suppressed the hypoxia-induced resistance of EBC-1 and MDA-MB-231 cells to radiation. DW, normal medium; O 2 , oxygen nanobubble medium.

Article Snippet: The EBC-1 human lung cancer cell line was purchased from the RIKEN Cell Resource Center of Biomedical Research (Tsukuba, Japan), and the MDA-MB-231 human breast cancer cell line and BEAS-2S non-cancerous human bronchial cell line were from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Viability Assay, Clonogenic Assay

Journal: International Journal of Oncology

Article Title: Development of single nanometer-sized ultrafine oxygen bubbles to overcome the hypoxia-induced resistance to radiation therapy via the suppression of hypoxia-inducible factor-1α

doi: 10.3892/ijo.2018.4248

Figure Lengend Snippet: Oxygen nanobubble medium treatment under normoxic conditions is non-toxic to cancer and non-cancer cell lines. (A) CCK8 assay revealed that oxygen nanobubble medium did not reduce the viability of EBC-1 lung cancer, MDA-MB-231 breast cancer, or non-cancerous BEAS-2B bronchial cells compared to normal medium. (B) Clonogenic assay revealed that oxygen nanobubble medium did not affect the radiation sensitivity of EBC-1, MDA-MB-231, or BEAS-2B cells compared to normal medium.

Article Snippet: The EBC-1 human lung cancer cell line was purchased from the RIKEN Cell Resource Center of Biomedical Research (Tsukuba, Japan), and the MDA-MB-231 human breast cancer cell line and BEAS-2S non-cancerous human bronchial cell line were from the American Type Culture Collection (Manassas, VA, USA).

Techniques: CCK-8 Assay, Clonogenic Assay