Journal: The Journal of Clinical Investigation

Article Title: SIRT2 protects peripheral neurons from cisplatin-induced injury by enhancing nucleotide excision repair

doi: 10.1172/JCI123159

Figure Lengend Snippet: (A) Sirt2 KO in primary DRG neurons resulted in decreased cell survival after cisplatin treatment. Data were analyzed by 2-tailed Student’s t test (n = 3). (B and C) The viability of neuronally differentiated 50B11 (B) and PC12 (C) cells after various doses of cisplatin treatment was measured with trypan blue staining. Reexpression of WT-SIRT2, but not the enzymatically inactive mutant, HY-SIRT2, in Sirt2-KO 50B11 and PC12 cells increased cell resistance to cisplatin cytotoxicity. One-way ANOVA demonstrated a main effect for cell genotype (P < 0.001). *P < 0.05; **P < 0.01; ***P < 0.001 denote significance levels detected among various cell genotypes and at different time points by Tukey’s post hoc analysis. (D and E) The viability of control vector and Sirt2-KO LLC (D) and H1299 (E) cells after varying doses of cisplatin treatment, as measured by trypan blue staining. Sirt2 KO in LLC and H1299 cells shows no difference in cell viability following cisplatin treatment, as analyzed by 2-tailed Student’s t test.

Article Snippet: The mouse LLC cell line LL/2 (LLC1), human non–small cell lung cancer cell line H1299, and rat pheochromocytoma cell line PC12 were purchased from ATCC.

Techniques: Staining, Mutagenesis, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Zebrafish xenograft model for studying mechanism and treatment of non-small cell lung cancer brain metastasis

doi: 10.1186/s13046-021-02173-5

Figure Lengend Snippet: Establishment of zebrafish NSCLC BM xenograft models. ( A ) Schematic diagram of imaging and collecting zebrafish brain. The zebrafish brain was imaged from vertical view (blue arrow) and excised (red dotted box) to extract RNA. ( B ) The schematic diagram showed zebrafish embryos at 2 dpf. The red dots indicated cancer cells injected into the perivitelline space (PVs). ( C ) Images of brain at different developmental stages of Tg ( fli-1 : EGFP) zebrafish (2-6 dpf). The white short dashed line indicated the midbrain of the zebrafish, and the white long dashed line indicated the zebrafish’s eyes and hindbrain. ( D ) The expression of claudin-5 in the zebrafish brain at different developmental stages (2-7 dpf). ( E ) The expression of mfsd2aa and mfsd2ab in the zebrafish brain at different developmental stages (2-6 dpf). ( F ) Survival curves of 2 dpf zebrafish with different tumor-bearing amount in PVs. About 50-400 H1975 cells (red fluorescence) were injected into the PVs of 2 dpf zebrafish, and the number of deaths was counted till 8 dpi. ( G - I ) Three human NSCLC cell lines were involved: H1975, A549 and H1299. About 100 cells were injected into the PVs of zebrafish at different developmental stages, and the brain of zebrafish was imaged at 1 dpi. The white arrows indicated cancer cells in blood vessels of zebrafish brain. ( J - K ) Quantification of the BM cells number in zebrafish at 1 dpi. Significance was considered when P values were lower than 0.05. (ns) indicated statistical insignificance, (*) indicated statistical significance P < 0.05, (**) P < 0.01 and (***) P < 0.001. dpf: days post fertilization, dpi: days post injection

Article Snippet: Human NSCLC lung cancer cell lines, A549, H1975, H1299, originally from American Type Culture Collection, were cultured in accordance with standard requirements.

Techniques: Imaging, Injection, Expressing, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Zebrafish xenograft model for studying mechanism and treatment of non-small cell lung cancer brain metastasis

doi: 10.1186/s13046-021-02173-5

Figure Lengend Snippet: NSCLC cells could not proliferate in the midbrain of zebrafish at 4 dpi. ( A ) The schematic diagram showed zebrafish embryos at 2 dpf. The red dots indicated cancer cells injected into the midbrain of zebrafish. ( B ) Survival curves of zebrafish with different tumor-bearing amount at midbrain. About 50-400 H1975 cells (red fluorescence) were injected into the midbrain of 2 dpf Tg ( fli-1 : EGFP) zebrafish, and the number of deaths was counted till 8 dpi. ( C - G ) Five human cancer cell lines were involved: human breast cancer cell lines MDA-MB-231 (positive control) and MCF-7 (negative control), human NSCLC cancer lines H1975, A549 and H1299. About 100 cells were injected into the midbrain of zebrafish at 2 dpf, and the midbrain of zebrafish was imaged at 1 dpi and 4 dpi. (H-M) Quantification of cell proliferation in the midbrain of zebrafish at 1 dpi and 4 dpi. The fold change of cells in the midbrain was determined by dividing the measured values at 1 dpi and 4 dpi by the average measured values at 1 dpi. (ns) indicated statistical insignificance, (*) indicated statistical significance P < 0.05, (**) P < 0.01 and (***) P < 0.001

Article Snippet: Human NSCLC lung cancer cell lines, A549, H1975, H1299, originally from American Type Culture Collection, were cultured in accordance with standard requirements.

Techniques: Injection, Fluorescence, Positive Control, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Zebrafish xenograft model for studying mechanism and treatment of non-small cell lung cancer brain metastasis

doi: 10.1186/s13046-021-02173-5

Figure Lengend Snippet: Zebrafish NSCLC BM xenograft models discriminated the BM potentials of different cell lines. ( A ) Transwell culture system was used to assess cell invasion ability in vitro , and five cancer cell lines were involved: MDA-MB-231, MCF-7, H1975, A549 and H1299. Invaded cells were stained with crystal violet (0.5 %) and imaged after 24 h incubation. ( B ) Quantification and comparison of invaded cells in vitro . Colonies were quantified using Image Pro Plus. ( C ) Five cancer cell lines were involved: MDA-MB-231, MCF-7, H1975, A549 and H1299. About 100 cells (red fluorescence) were injected into the PVs of Tg ( fli-1 : EGFP) zebrafish at 2 dpf, and the brain of zebrafish was imaged at 4 dpi. The white arrows indicated cancer cells in blood vessels of zebrafish brain. ( D ) Quantification of BM rate at 4 dpi. The number of BM cells in the same zebrafish at 4 dpi was divided by the number of BM cells at 1 dpi. The ratio of these two was named as BM potential. If the BM potential was greater than 1, it was considered that the zebrafish had brain metastasis. ( E - I ) Quantification of BM cells at 1 dpi and 4 dpi. The two dots connected by a straight line represented the number of BM cells of the same zebrafish at 1 dpi and 4 dpi. ( J ) Quantification and comparison of cell BM potential. The BM potential was determined by dividing the number of BM cells in the same zebrafish at 4 dpi by the number of BM cells at 1 dpi. (ns) indicated statistical insignificance, (*) indicated statistical significance P < 0.05, (**) P < 0.01 and (***) P < 0.001

Article Snippet: Human NSCLC lung cancer cell lines, A549, H1975, H1299, originally from American Type Culture Collection, were cultured in accordance with standard requirements.

Techniques: In Vitro, Staining, Incubation, Comparison, Fluorescence, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Zebrafish xenograft model for studying mechanism and treatment of non-small cell lung cancer brain metastasis

doi: 10.1186/s13046-021-02173-5

Figure Lengend Snippet: Zebrafish NSCLC BM xenograft models simultaneously discriminated the BM potentials of different cell lines. ( A ) The schematic diagram showed zebrafish embryos at 2 dpf. The red dots and green dots indicated cancer cells labeled with two different dyes were co-injected into the PVs of zebrafish. ( B ) About 50 H1975 cells (red fluorescence) and 50 H1975 cells (green fluorescence) were co-injected into the PVs of wild zebrafish at 2 dpf, and the brain of zebrafish was imaged at 4 dpi. The white long dashed line indicated the brain of zebrafish. ( C ) About 50 H1975 cells (red fluorescence) and 50 A549 cells (green fluorescence) were co-injected into the PVs of wild zebrafish at 2 dpf, and the brain of zebrafish was imaged at 4 dpi. ( D - G ) Quantification of BM cells in co-injection at 1 dpi and 4 dpi. The two dots connected by a straight line represented the number of BM cells of the same zebrafish at 1 dpi and 4 dpi. ( H and I ) The percentage of cells with two different dyes in the brain of zebrafish. The percentage of the cell line (red/green fluorescence) at 1 dpi was determined by dividing the number of BM cells (red/green fluorescence) by the total number of BM cells at 1 dpi. (ns) indicated statistical insignificance, (*) indicated statistical significance P < 0.05, (**) P < 0.01 and (***) P < 0.001

Article Snippet: Human NSCLC lung cancer cell lines, A549, H1975, H1299, originally from American Type Culture Collection, were cultured in accordance with standard requirements.

Techniques: Labeling, Injection, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Zebrafish xenograft model for studying mechanism and treatment of non-small cell lung cancer brain metastasis

doi: 10.1186/s13046-021-02173-5

Figure Lengend Snippet: Expression of microRNA-330-3p (miR-330-3p) affected the NSCLC BM potential in zebrafish xenograft models. ( A and B ) Quantification of the miR-330-3p expression in vitro . The appropriate transfection concentration was determined between 1 nM and 50 nM, according to the instructions. ( C and D ) The expression of miR-330-3p in H1975 cell line was knocked down by anti-miR-330-3p inhibitor and the expression of miR-330-3p in A549 cell line was overexpressed by over-miR-330-3p mimics. Transwell culture system was used to evaluate the effectiveness of transfection in vitro . Invaded cells were stained with crystal violet (0.5 %) and imaged after 24 h incubation. ( E and F ) Quantification of invaded cells in vitro . Colonies were quantified using Image Pro Plus. ( G and H ) H1975, H1975 with under-expressed miR-330-3p, A549 and A549 with over-expressed miR-330-3p were involved. About 100 cells (red fluorescence) were injected into the PVs of Tg ( fli-1 : EGFP) zebrafish at 2 dpf to evaluate the effectiveness of transfection in vivo , and the brain of zebrafish was imaged at 4 dpi. The white arrows indicated cancer cells in blood vessels of zebrafish brain. ( I - L ) Quantification of BM cells at 1 dpi and 4 dpi. The two dots connected by a straight line represented the number of BM cells of the same zebrafish at 1 dpi and 4 dpi. ( M and N ) Quantification and comparison of the BM potentials of the transfected cell lines and the original cell lines. (ns) indicated statistical insignificance, (*) indicated statistical significance P < 0.05, (**) P < 0.01 and (***) P < 0.001

Article Snippet: Human NSCLC lung cancer cell lines, A549, H1975, H1299, originally from American Type Culture Collection, were cultured in accordance with standard requirements.

Techniques: Expressing, In Vitro, Transfection, Concentration Assay, Staining, Incubation, Fluorescence, Injection, In Vivo, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Zebrafish xenograft model for studying mechanism and treatment of non-small cell lung cancer brain metastasis

doi: 10.1186/s13046-021-02173-5

Figure Lengend Snippet: Zebrafish NSCLC BM xenograft models discriminated different chemosensitivities within 4 days. ( A and B ) NSCLC cell lines H1975 and A549 were involved. Transwell culture system was used to evaluate the effectiveness of osimertinib (1 µM) and gefitinib (2 µM) against cell invasion in vitro . Invaded cells were stained with crystal violet (0.5 %) and imaged after 24 h incubation. ( C and D ) Quantification of invaded cells in vitro. Colonies were quantified using Image Pro Plus. ( E and F ) NSCLC cell lines H1975 and A549 were involved. Tg ( fli-1 : EGFP) zebrafish embryos were injected about 100 cells (red fluorescence) into the PVs at 2 dpf and administered with osimertinib (1 µM) and gefitinib (13 µM) by intracardiac injection. ( G and H ) Quantification of cell brain metastasis at 4 dpi. Fold change of BM cell number was determined by dividing the number of BM cells in the same zebrafish at 4 dpi by the number of BM cells at 1 dpi. (ns) indicated statistical insignificance, (*) indicated statistical significance P < 0.05, (**) P < 0.01 and (***) P < 0.001

Article Snippet: Human NSCLC lung cancer cell lines, A549, H1975, H1299, originally from American Type Culture Collection, were cultured in accordance with standard requirements.

Techniques: In Vitro, Staining, Incubation, Injection, Fluorescence

Journal: Glia

Article Title: Endoplasmic Reticulum Stress Amplifies Cytokine Responses in Astrocytes via a PERK / eIF2α / JAK1 Signaling Axis

doi: 10.1002/glia.70067

Figure Lengend Snippet: JAK1 and PERK are required to augment TNF‐α induced gene expression in human glioma cells. (A) 1321N1 cells stably expressing Cas9 were transfected with non‐targeting (NT) or JAK1 guide RNAs (gRNA) to establish non‐clonal cell lines. These cells were then treated with IFN‐γ (10 ng/mL) for 30 min followed by immunoblotting. (B) NT and JAK1 gRNA (#2) cells were treated with thaps (1 μM), TNF‐α (10 ng/mL), or both for 4 h followed by qPCR. (C) 1321N1 cells stably expressing Cas9 were transfected with NT or PERK gRNA to establish non‐clonal cell lines. These cells were then treated with thaps (1 μM) for the indicated times followed by immunoblotting. (D) NT and PERK gRNA cells were treated with thaps (1 μM), TNF‐α (10 ng/mL), or both for 4 h followed by qPCR. N = 4, data are means ± standard deviation.

Article Snippet: 1321N1 human astrocytoma cell line stably expressing Cas9 nuclease was purchased from GeneCopoeia.

Techniques: Gene Expression, Stable Transfection, Expressing, Transfection, Western Blot, Standard Deviation