Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 1. Elevated expression of UNC13B in Wilms’ tumor cell lines. (A) Western blot analysis of UNC13B expression in WT‑CLS1, 17.94, G401, SK‑NEP‑1 and HK‑2 cells. Each lane was loaded with 20 µg protein and GAPDH was used as a reference. (B) Analysis of UNC13B transcription levels in different cell lines; n=5. (C) Changes in UNC13B transcription levels 48 h post‑shRNA‑mediated UNC13B knockdown in 17.94 cells compared with the scramble control; n=5. (D) UNC13B expression changes 48 h post‑shRNA‑mediated knockdown in 17.94 cells and (E) statistical analysis of the expression level changes. Each experiment was repeated 3 times, with GAPDH used as a reference. (F) Assessment of cell proliferation post‑knockdown using a Cell Counting Kit‑8 assay, measuring OD450 values at different time points; n=3. Cells were also transfected with non‑target scrambled shRNA as a control. ***P<0.001; ****P<0.0001. UNC13B, unc‑13 homolog B; sh, short hairpin; OD, optical density.

Article Snippet: The human Ewing sarcoma SK‐NEP‐1 (cat. no. HTB‐48) and human rhabdoid tumor G401 (cat. no. CRL‐1441) cell lines were purchased from the American Type Culture Collection (ATCC) and maintained in McCoy's 5A modified medium containing 15% FBS (HyClone, SH30088.03).

Techniques: Expressing, Wilms Tumor Assay, Western Blot, Knockdown, Control, CCK-8 Assay, Transfection, shRNA

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 2. UNC13B influences Wilms’ tumor sensitivity to chemotherapy drugs independent of the cell cycle. Evaluation of cell proliferation post‑shRNA‑medi ated UNC13B knockdown after treatment with varying concentrations of (A) vincristine, (B) actinomycin‑D and (C) doxorubicin for 48 h in 17.94 cells, and (D) vincristine, (E) actinomycin‑D and (F) doxorubicin in the G401 cell line, assessed using Cell Counting Kit‑8 assays. Changes in drug sensitivity were analyzed, with dashed lines representing fitted curves for half‑maximal inhibitory concentration calculated using GraphPad software, and the cell number ratio indicating the relative number of viable cells compared between initial cell number and different time points. (G) Cell cycle analysis of 1 µM doxorubicin treatment on control and shUNC13B knockdown cells, detected after 48 h post‑drug treatment. (H) Quantification of the G1, S and G2 phases of the scramble and shUNC13B groups. (I) Typical pseudocolor scatter plots of the apoptosis analysis of UNC13B‑knockdown 17.94 cells after 48 h treatment with 0.5 and 2 µM doxorubicin, and (J) statistical results. **P<0.01; ***P<0.001; ****P<0.0001. UNC13B, unc‑13 homolog B; sh, short hairpin.

Article Snippet: The human Ewing sarcoma SK‐NEP‐1 (cat. no. HTB‐48) and human rhabdoid tumor G401 (cat. no. CRL‐1441) cell lines were purchased from the American Type Culture Collection (ATCC) and maintained in McCoy's 5A modified medium containing 15% FBS (HyClone, SH30088.03).

Techniques: Wilms Tumor Assay, Knockdown, CCK-8 Assay, Concentration Assay, Software, Cell Cycle Assay, Control

Journal: Cancer Management and Research

Article Title: Long Non-Coding RNA XIST Promotes Wilms Tumor Progression Through the miR-194-5p/YAP Axis

doi: 10.2147/CMAR.S297842

Figure Lengend Snippet: miR-194-5p can bind to both XIST and YAP in WT tissues. XIST lncRNA regulates WT progression through the miR-194-5p/YAP axis. ( A ) XIST 3ʹ-UTR wild-type (XIST-wt) sequence containing the miR-194-5p binding site and sequence of the mutant (XIST-mut) miR-194-5p binding site. ( B and C ) Luciferase reporter gene assay (images and histograms) showed lower luciferase activity for miR-194-5p and XIST-wt than XIST-mut ( P < 0.05). TRAF6 was used as an internal control to verify the integrity of the luciferase gene reporter assay. ( D ) YAP 3ʹ-UTR wild-type (YAP-wt) sequence containing the miR-194-5p binding site and sequence of the mutant (YAP-mut) miR-194-5p binding site. ( E and F ) Luciferase reporter gene assay (images and histograms) showed lower luciferase activity for miR-194-5p and YAP-wt than YAP-mut ( P < 0.05). TRAF6 was used as an internal control to verify the integrity of the luciferase gene reporter assay. ( G ) XIST lncRNA expression and ( H ) miR-194-5p in WT G401 cells and normal renal epithelial HK2 cells. ( I ) RT-qPCR analysis of miR-194-5p after transfection of lentiviral XIST, NC, and sh-XIST in WT G401 cells. ( J ) Western blot analysis showed that YAP protein expression can be regulated by miR-194-5p and XIST. One-way ANOVA or two-tailed t -test was performed for comparisons between the two groups. * P < 0.05, *** P < 0.001.

Article Snippet: The packaged lentiviral vectors were then transfected into the human WT G401 rhabdoid cell line (ATCC, USA) according to the manufacturer’s protocol and were collected for analysis after 48 h. The miR-194-5p mimic and NC mimic plasmids (GenePharma, Shanghai, China) were transfected into the human WT G401 cell line using Lipofectamine TM RNAiMAX (Invitrogen, USA) according to the manufacturer’s protocol.

Techniques: Sequencing, Binding Assay, Mutagenesis, Luciferase, Reporter Gene Assay, Activity Assay, Control, Reporter Assay, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Two Tailed Test

Journal: Cancer Management and Research

Article Title: Long Non-Coding RNA XIST Promotes Wilms Tumor Progression Through the miR-194-5p/YAP Axis

doi: 10.2147/CMAR.S297842

Figure Lengend Snippet: XIST promotes the proliferation, migration, and invasion of G401 cells, and inhibits apoptosis in vitro. ( A and B ) Stable XIST overexpression (lentiviral XIST and negative control, NC) and XIST knockdown (lentiviral sh-RNA and negative control, sh-NC) were successfully established in WT G401 cells. ( C ) CCK-8 cell viability assay profiles showed that XIST overexpression enhanced G401 cell proliferation, while ( D ) XIST knockdown decreased cell proliferation. ( E ) Flow cytometry showed that XIST overexpression decreased apoptosis while XIST knockdown promoted apoptosis. ( F ) Scratch assay (100×magnification) ( G ) and transwell assay showed that XIST overexpression promoted G401 cell migration and invasion in vitro (100×magnification). Three independent replicates were performed. One-way ANOVA or two-tailed t -test was performed for comparisons between the two groups. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The packaged lentiviral vectors were then transfected into the human WT G401 rhabdoid cell line (ATCC, USA) according to the manufacturer’s protocol and were collected for analysis after 48 h. The miR-194-5p mimic and NC mimic plasmids (GenePharma, Shanghai, China) were transfected into the human WT G401 cell line using Lipofectamine TM RNAiMAX (Invitrogen, USA) according to the manufacturer’s protocol.

Techniques: Migration, In Vitro, Over Expression, Negative Control, Knockdown, CCK-8 Assay, Viability Assay, Flow Cytometry, Wound Healing Assay, Transwell Assay, Two Tailed Test

Journal: Cancer Management and Research

Article Title: Long Non-Coding RNA XIST Promotes Wilms Tumor Progression Through the miR-194-5p/YAP Axis

doi: 10.2147/CMAR.S297842

Figure Lengend Snippet: miR-194-5p can bind to both XIST and YAP in WT tissues. XIST lncRNA regulates WT progression through the miR-194-5p/YAP axis. ( A ) XIST 3ʹ-UTR wild-type (XIST-wt) sequence containing the miR-194-5p binding site and sequence of the mutant (XIST-mut) miR-194-5p binding site. ( B and C ) Luciferase reporter gene assay (images and histograms) showed lower luciferase activity for miR-194-5p and XIST-wt than XIST-mut ( P < 0.05). TRAF6 was used as an internal control to verify the integrity of the luciferase gene reporter assay. ( D ) YAP 3ʹ-UTR wild-type (YAP-wt) sequence containing the miR-194-5p binding site and sequence of the mutant (YAP-mut) miR-194-5p binding site. ( E and F ) Luciferase reporter gene assay (images and histograms) showed lower luciferase activity for miR-194-5p and YAP-wt than YAP-mut ( P < 0.05). TRAF6 was used as an internal control to verify the integrity of the luciferase gene reporter assay. ( G ) XIST lncRNA expression and ( H ) miR-194-5p in WT G401 cells and normal renal epithelial HK2 cells. ( I ) RT-qPCR analysis of miR-194-5p after transfection of lentiviral XIST, NC, and sh-XIST in WT G401 cells. ( J ) Western blot analysis showed that YAP protein expression can be regulated by miR-194-5p and XIST. One-way ANOVA or two-tailed t -test was performed for comparisons between the two groups. * P < 0.05, *** P < 0.001.

Article Snippet: Human WT G401 and normal renal tubular epithelial HK-2 cell lines (ATCC, USA) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, USA) at 37°C, in a 5% CO 2 humidified incubator.

Techniques: Sequencing, Binding Assay, Mutagenesis, Luciferase, Reporter Gene Assay, Activity Assay, Control, Reporter Assay, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Two Tailed Test

Journal: Cancer Management and Research

Article Title: Long Non-Coding RNA XIST Promotes Wilms Tumor Progression Through the miR-194-5p/YAP Axis

doi: 10.2147/CMAR.S297842

Figure Lengend Snippet: XIST promotes the proliferation, migration, and invasion of G401 cells, and inhibits apoptosis in vitro. ( A and B ) Stable XIST overexpression (lentiviral XIST and negative control, NC) and XIST knockdown (lentiviral sh-RNA and negative control, sh-NC) were successfully established in WT G401 cells. ( C ) CCK-8 cell viability assay profiles showed that XIST overexpression enhanced G401 cell proliferation, while ( D ) XIST knockdown decreased cell proliferation. ( E ) Flow cytometry showed that XIST overexpression decreased apoptosis while XIST knockdown promoted apoptosis. ( F ) Scratch assay (100×magnification) ( G ) and transwell assay showed that XIST overexpression promoted G401 cell migration and invasion in vitro (100×magnification). Three independent replicates were performed. One-way ANOVA or two-tailed t -test was performed for comparisons between the two groups. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human WT G401 and normal renal tubular epithelial HK-2 cell lines (ATCC, USA) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, USA) at 37°C, in a 5% CO 2 humidified incubator.

Techniques: Migration, In Vitro, Over Expression, Negative Control, Knockdown, CCK-8 Assay, Viability Assay, Flow Cytometry, Wound Healing Assay, Transwell Assay, Two Tailed Test

Journal: Cancer cell

Article Title: ATRX in-frame fusion neuroblastoma is sensitive to EZH2 inhibition via modulation of neuronal gene signatures

doi: 10.1016/j.ccell.2019.09.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: G401 , ATCC , CRL-1441.

Techniques: Virus, Microarray, Recombinant, Magnetic Beads, Bicinchoninic Acid Protein Assay, Cell Culture, DNA Library Preparation, Extraction, Expressing, Sequencing, Software