Journal: Scientific Reports

Article Title: Critical role of SOX2–IGF2 signaling in aggressiveness of bladder cancer

doi: 10.1038/s41598-020-65006-z

Figure Lengend Snippet: SOX2 mediates growth of bladder cancer cells. ( a ) qPCR (upper left) and immunoblotting (lower left) analysis to assess SOX2 mRNA and protein expression, respectively, in T24 cells transduced with the lentiviral vector encoding SOX2 cDNA (SOX2) or empty control vector (Ctrl). Trypan blue cell exclusion analysis of T24 cells transduced with the lentiviral vector encoding SOX2 cDNA (SOX2) or empty control vector (Ctrl) for the indicated days. Results are the average of three replicates and expressed as the mean ± S.D. **P < 0.01 , ***P < 0.001 . ( b ) qPCR (upper left) and immunoblotting (lower left) analysis of SOX2 expression in 5637 cells transduced with the lentiviral vector encoding shRNA against SOX2 (shSOX2) or scrambled control vector (SC). Trypan blue cell exclusion analysis of 5637 cells transduced with the lentiviral vector encoding shSOX2 or scrambled control vector (SC) for the indicated days. Results are the average of three replicates and expressed as the mean ± S.D. The #1 and #2 indicate the two distinct shRNAs that target different regions within SOX2 . ***P < 0.001 . ( c ) Clonogenic analysis (left) to assess the SOX2 expression effect on the colony-forming ability in T24 cells transduced with the lentiviral vector encoding SOX2 cDNA (SOX2) or empty control vector (Ctrl). Clonogenic analysis (right) to assess the SOX2 knockdown effect on the colony-forming ability in 5637 cells transduced with the lentiviral vector encoding shSOX2 or scrambled control vector (SC). Colonies were subjected to crystal violet staining and quantified by ImageJ analysis. Results are the average of three replicates and expressed as the mean ± S.D. * P < 0.05, ** P < 0.01.

Article Snippet: T24 and 5637 cell lines were obtained in 2017 from BCRC (Bioresource Collection and Research Center, Taiwan).

Techniques: Western Blot, Expressing, Transduction, Plasmid Preparation, Control, shRNA, Knockdown, Staining

Journal: Scientific Reports

Article Title: Critical role of SOX2–IGF2 signaling in aggressiveness of bladder cancer

doi: 10.1038/s41598-020-65006-z

Figure Lengend Snippet: SOX2 induces IGF2/IGF1R signaling molecules in bladder cancer cells. ( a ) qPCR analysis to assess IGF2 expression in T24 cells transduced with the lentiviral vector encoding SOX2 cDNA (SOX2) or empty control vector (Ctrl). Results are the average of three replicates and expressed as the mean ± S.D. *** P < 0.001. ( b ) qPCR analysis to assess IGF2 expression in T24 versus 5637 cells (left) and in 5637 cells transduced with shSOX2 versus scrambled control (SC) vector (right). The #1 and #2 indicate the two distinct shRNAs that target different regions within SOX2 . *** P < 0.001. ( c ) RNA-seq analysis of SOX2 and IGF2 expression in ESC (HUES3) and differentiated fibroblast cells from GSE73211 database. ( d ) ChIP-qPCR analysis to assess H3K4me3 levels of IGF2 region in SOX2-expressing T24 cell (left) and SOX2-silenced 5637 cells (right). ** P < 0.01, *** P < 0.001. Primers targeting the H3K4me3 signal of IGF2 were designed at chr11: 2,158,962–2,159,038. ( e ) qPCR and immunoblotting analysis of IGFBP1 expression in SOX2-overexpressing T24 cells (left) and SOX2-silenced 5637 cells (right). Results are the average of three replicates and expressed as the mean ± S.D. * P < 0.05, *** P < 0.001.

Article Snippet: T24 and 5637 cell lines were obtained in 2017 from BCRC (Bioresource Collection and Research Center, Taiwan).

Techniques: Expressing, Transduction, Plasmid Preparation, Control, RNA Sequencing, ChIP-qPCR, Western Blot

Journal: Scientific Reports

Article Title: Critical role of SOX2–IGF2 signaling in aggressiveness of bladder cancer

doi: 10.1038/s41598-020-65006-z

Figure Lengend Snippet: SOX2-induced IGF2/IGF1R signaling is essential for bladder cancer cells. ( a ) qPCR (upper) and immunoblotting (lower) analysis of 5637 cells transduced with the lentiviral vector encoding shRNA against IGF2 (shIGF2) or scrambled control vector (SC). The #1 and #2 indicate the two distinct shRNAs that target different regions within IGF2 . ***P < 0.001. ( b ) Clonogenic assay of 5637 cells transduced with shIGF2 or scrambled control vector (SC). Colonies were subjected to crystal violet staining (top) and quantified by ImageJ analysis. Results are the average of three replicates and expressed as the mean ± S.D. ** P < 0.01. ( c ) qPCR (upper) and immunoblotting (lower) analysis of 5637 cells transduced with the lentiviral vector encoding shRNA against IGF1R (shIGF1R) or scrambled control vector (SC). The #1 and #2 indicate the two distinct shRNAs that target different regions within IGF1R . ***P < 0.001. ( d ) Clonogenic assay of 5637 cells transduced with shIGF1R or scrambled control vector (SC). Colonies were subjected to crystal violet staining (top) and quantified by ImageJ analysis. Results are the average of three replicates and expressed as the mean ± S.D. ** P < 0.01. ( e ) Immunoblotting analysis to assess the expression of phosphorylated AKT at Ser473 and total AKT expression in SOX2 -expressing T24 cells under low-serum (1% FBS) condition in the presence or absence of linsitinib (5 μM) for 48 hr. ( f ) 3D colony-forming analysis to assess linsitinib effect on colony formation of SOX2 -expressing T24. The cells were grown under the low-attached condition in low-serum medium (1% FBS) in the presence or absence of linsitinib (5 μM) for 7 days. Photos (left) are representative images of the colony and colony sizes were quantified (right). Results are the average of three independent experiments and expressed as the mean ± SD. *** P < 0.001.

Article Snippet: T24 and 5637 cell lines were obtained in 2017 from BCRC (Bioresource Collection and Research Center, Taiwan).

Techniques: Western Blot, Transduction, Plasmid Preparation, shRNA, Control, Clonogenic Assay, Staining, Expressing

Journal: iScience

Article Title: KNSTRN knockdown impairs autophagy flux to inhibit bladder cancer progression

doi: 10.1016/j.isci.2026.114734

Figure Lengend Snippet: Lysosomal reactivation restores autophagy damage caused by KNSTRN knockdown (A and B) T24 and 5637 cells pretreated with clioquinol (ClioQ, T24, 10 μM; 5637, 20 μM) for 1 h and subsequently transfected with si-NC and si-KNSTRN for 48 h. And subjected to western blot analysis with antibodies against KNSTRN and P62. Representative western blot images and the relative quantification of protein expression are shown. (C) 5637 cell transfected with mCherry-GFP-LC3 were treated with si-NC and KNSTRN-silenced for 48 h in the absence or presence of clioquinol (ClioQ, 20 μM). The number of LC3 puncta (yellow for autophagosomes, red for autolysosomes) was quantified. And images were captured using a fluorescence microscope. Scale bars, 20 μm. (D) Lysosomal acidity was analyzed by LysoTracker red (scale bar: 20 μm). Representative fluorescent images of the cells are shown. The fluorescence intensity is calculated using ImageJ software. Significance is determined by one-way ANOVA. Data are presented as the mean ± SD ( n = 3). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: 5637 cell line was obtained from Servicebio (Wuhan, China).

Techniques: Knockdown, Transfection, Western Blot, Quantitative Proteomics, Expressing, Fluorescence, Microscopy, Software

Journal: iScience

Article Title: KNSTRN knockdown impairs autophagy flux to inhibit bladder cancer progression

doi: 10.1016/j.isci.2026.114734

Figure Lengend Snippet: KNSTRN knockdown-induced ROS is responsible for impairing lysosomal activity and autophagy (A) Reactive oxygen species (ROS) levels were detected through inverted fluorescence microscope in T24 and 5637 cells transfected with si-NC and si-KNSTRN. (B) Lysosomal acidity was analyzed by LysoTracker red (scale bar: 20 μm). Representative fluorescent images of the cells are shown. The fluorescence intensity is calculated using ImageJ software. (C) T24 and 5637 cells pretreated with N-acetylcysteine (NAC, T24, 2 mM; 5637, 5 mM) for 1 h and subsequently transfected with si-NC and si-KNSTRN for 48 h. And subjected to western blot analysis with antibodies against KNSTRN and P62. Representative western blot images and the relative quantification of protein expression are shown. (D) 5637 cell transfected with mCherry-GFP-LC3 were treated with si-NC and KNSTRN-silenced for 48 h in the absence or presence of N-acetylcysteine(NAC;5 mM). The number of LC3 puncta (yellow for autophagosomes, red for autolysosomes) was quantified. Scale bars, 20 μm. Significance is determined by unpaired t test (two groups) and one-way ANOVA (more than two groups). Data are presented as the mean ± SD ( n = 3). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: 5637 cell line was obtained from Servicebio (Wuhan, China).

Techniques: Knockdown, Activity Assay, Fluorescence, Microscopy, Transfection, Software, Western Blot, Quantitative Proteomics, Expressing