Journal: Molecular Cancer

Article Title: DCLK1 drives malignant progression and chemoresistance of bladder cancer by deubiquitinating HDAC6

doi: 10.1186/s12943-025-02560-y

Figure Lengend Snippet: DCLK1 knockdown suppresses stemness and enhances chemosensitivity in bladder cancer cells. A mRNA expression levels of DCLK1 in bladder cancer tissues compared with normal tissues. B qRT-PCR analysis of DCLK1 expression in BC cell lines and SV-HUC-1 cells. C qRT-PCR analysis confirming the knockdown efficiency of DCLK1. D Representative images of tumorsphere formation in DCLK1-depleted BC cells (scale bar = 100 μm). E Western blot analysis of stemness-associated markers (ALDH1, CD133, CD44, Nanog, and SOX2) in DCLK1-silenced BC cells. F The proportions of ALDH1⁺ and CD44⁺ cells were significantly higher in the control group than in the DCLK1-knockdown group under cancer stem cell-enriched conditions. G Cisplatin-induced apoptosis in BC cells analyzed by Annexin V/PI staining followed by flow cytometry. H Cisplatin-induced apoptosis in BC cells examined by the TUNEL assay (scale bar = 20 μm). Data are presented as mean ± SD. ** P < 0.01, *** P < 0.001

Article Snippet: DCLK1 (1:1000; Cell Signaling, 62257), HDAC6 (1:1000; Cell Signaling, 7558), USP10 (1:1000; Proteintech, 8501), E-cadherin (1:1000; Cell Signaling, 3195), Vimentin (1:1000; Cell Signaling, 5741), β-catenin (1:1000; Cell Signaling, 9562), Flag (1:1000; Sigma, F1804), HA (1:1000; Sigma, H6908), Myc-tag (1:1000; Proteintech, 16286-1-AP), α-tubulin (1:1000; Proteintech, 66031-1-Ig), ALDH1 (1:1000; Cell Signaling, 54135), CD133 (1:1000; Cell Signaling, 64326), CD44 (1:1000; Cell Signaling, 37259),SOX2 (1:1000; Cell Signaling, 23064),Nanog (1:2000; Cell Signaling, 4903), Notch1 (1:1000; Cell Signaling, 3608), c-Myc (1:1000; Cell Signaling, 13987), MAML1 (1:1000; Cell Signaling, 12166), HES1 (1:1000; Cell Signaling, 11988), HEY1 (1:1000; abcam, 154077), PD-L1 (1:1000; Cell Signaling, 13684), β-Actin (1:10000; Cell Signaling, 4967) and GAPDH (1:5000; Abcam, ab128915).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Control, Staining, Flow Cytometry, TUNEL Assay

Journal: Journal of Clinical Laboratory Analysis

Article Title: Correlation of integrin alpha 7 with clinicopathological characteristics and survival profiles, as well as its regulatory role in cell proliferation, apoptosis, and stemness in non‐small‐cell lung cancer

doi: 10.1002/jcla.22973

Figure Lengend Snippet: ITGA7 promoted cell proliferation, suppressed apoptosis, and enhanced stemness in A549 cells. A, ITGA7 mRNA expression after transfection. B, ITGA7 protein expression after transfection. C, Cell proliferation after transfection. D, E, Cell apoptosis after transfection. F, G, Proportion of CD133 + CD44 + cells after transfection. Comparison between two independent samples was determined by t test. P value < 0.05 was considered as significant. ITGA7, Integrin alpha 7

Article Snippet: Pre‐rinsed cells were, respectively, incubated with Alexa Fluor ® 647 conjugated CD133 Mouse mAb (Flow Specific) (1:50 dilution, CST) and FITC conjugated CD44 Rat mAb (1:100 dilution, CST) in incubation buffer at 37°C for an hour.

Techniques: Expressing, Transfection, Comparison

Journal: International Journal of Oncology

Article Title: Mechanism of SMYD2 promoting stemness maintenance of bladder cancer stem cells by regulating PYCR1 expression and PINK1/Parkin mitophagy pathway

doi: 10.3892/ijo.2025.5747

Figure Lengend Snippet: PYCR1 sustains bladder CSC stemness. (A) RT-qPCR and (B) western blot were used to assess PYCR1 mRNA and protein expression in CD44 + CD133 + and CD44 − CD133 − cells. Detection of PYCR1 (C) mRNA and (D) protein expression using western blot and RT-qPCR. (E) Detection of CD44 + CD133 + cells by flow cytometry. (F) Measurement of in vitro colony formation ability. (G) CSC sphere-forming assay was performed to examine tumor stemness. (H) RT-qPCR and (I) western blotting were performed to assess expression of stemness marker proteins Nanog and Sox2 mRNA and protein. * P<0.05, ** P<0.01, *** P<0.001. PYCR1, pyrroline-5-carboxylate reductase 1; CSC, cancer stem cell; RT-q, Reverse transcription quantitative; si, small-interfering; NC, negative control; oe, overexpression.

Article Snippet: Cells were cultured in the presence of antibodies labeled with CSC markers, such as CD44 (IM7) Rat mAb (Alexa Fluor ® 555 Conjugate; cat. no. #95235) and CD133 (A8N6N) Mouse mAb (Alexa Fluor ® 647 Conjugate; cat. no. #53276; both 1:50; both Cell Signaling Technology, Inc.) on ice without light for 30 min, followed by PBS rinsing three times.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry, In Vitro, Marker, Reverse Transcription, Negative Control, Over Expression

Journal: International Journal of Oncology

Article Title: Mechanism of SMYD2 promoting stemness maintenance of bladder cancer stem cells by regulating PYCR1 expression and PINK1/Parkin mitophagy pathway

doi: 10.3892/ijo.2025.5747

Figure Lengend Snippet: SMYD2 promotes stemness retention in bladder CSCs. (A) Analysis of SMYD2 expression in normal bladder tissue and bladder cancer using Gene Expression Profiling Interactive Analysis database. SMYD2 (B) mRNA and (C) protein expression using RT-qPCR and western blotting. (D) Flow cytometry to determine CD44 + CD133 + cell levels. (E) In vitro colony formation capability. (F) CSC sphere-forming assay to examine tumor stemness. (G) RT-qPCR and (H) western blotting were used to evaluate the expression of Nanog and Sox2. * P<0.05, ** P<0.01, *** P<0.001. SMYD2, SET and MYND domain-containing protein 2; CSC, cancer stem cell; RT-qPCR, reverse transcription quantitative polymerase chain reaction; si, small-interfering; NC, negative control; oe, overexpression; ns, not significant; BLCA, bladder urothelial carcinoma.

Article Snippet: Cells were cultured in the presence of antibodies labeled with CSC markers, such as CD44 (IM7) Rat mAb (Alexa Fluor ® 555 Conjugate; cat. no. #95235) and CD133 (A8N6N) Mouse mAb (Alexa Fluor ® 647 Conjugate; cat. no. #53276; both 1:50; both Cell Signaling Technology, Inc.) on ice without light for 30 min, followed by PBS rinsing three times.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Western Blot, Flow Cytometry, In Vitro, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Over Expression

Journal: International Journal of Oncology

Article Title: Mechanism of SMYD2 promoting stemness maintenance of bladder cancer stem cells by regulating PYCR1 expression and PINK1/Parkin mitophagy pathway

doi: 10.3892/ijo.2025.5747

Figure Lengend Snippet: SMYD2 regulates PYCR1 expression to potentiate bladder SCS stemness. PYCR1 (A) mRNA and (B) protein expression by reverse transcription-quantitative PCR and western blot. (C) Assessment of CD44 + CD133 + cell level by flow cytometry. (D) In vitro colony formation ability. (E) Tumor stemness testing utilizing CSC sphere-forming assay. Examination of Sox2 and Nanog (F) mRNA and (G) protein expression by RT-qPCR and western blot. * P<0.05, ** P<0.01, *** P<0.001. SMYD2, SET and MYND domain-containing protein 2; PYRC1, pyrroline-5-carboxylate reductase 1; si, small-interfering; NC, negative control; oe, overexpression; CSC, cancer stem cell.

Article Snippet: Cells were cultured in the presence of antibodies labeled with CSC markers, such as CD44 (IM7) Rat mAb (Alexa Fluor ® 555 Conjugate; cat. no. #95235) and CD133 (A8N6N) Mouse mAb (Alexa Fluor ® 647 Conjugate; cat. no. #53276; both 1:50; both Cell Signaling Technology, Inc.) on ice without light for 30 min, followed by PBS rinsing three times.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, In Vitro, Quantitative RT-PCR, Negative Control, Over Expression

Journal: International Journal of Oncology

Article Title: Mechanism of SMYD2 promoting stemness maintenance of bladder cancer stem cells by regulating PYCR1 expression and PINK1/Parkin mitophagy pathway

doi: 10.3892/ijo.2025.5747

Figure Lengend Snippet: PYCR1 promotes bladder CSC stemness sustenance via the PINK1/Parkin pathway. (A) Immunofluorescence detection of mitochondrial (MTG; green) and autophagy marker (LC3B; red) expression. (B) Assessment of the protein levels of LC3B II/I, p62, PINK1 and Parkin by western blot. (C) Measurement of CD44 + CD133 + cell level by flow cytometry. (D) Colony formation assay. (E) CSC sphere-forming assay to estimate tumor stemness. Expression of stemness marker proteins Nanog and Sox2 (F) mRNA and (G) protein was assessed by reverse transcription-quantitative PCR and western blot. * P<0.05, ** P<0.01, *** P<0.001. SMYD2, SET and MYND domain-containing protein 2; PYRC1, pyrroline-5-carboxylate reductase 1; si, small-interfering; NC, negative control; oe, overexpression; PINK1, PTEN-induced putative kinase 1; MTG, Mito-Tracker Green; CSC, cancer stem cell.

Article Snippet: Cells were cultured in the presence of antibodies labeled with CSC markers, such as CD44 (IM7) Rat mAb (Alexa Fluor ® 555 Conjugate; cat. no. #95235) and CD133 (A8N6N) Mouse mAb (Alexa Fluor ® 647 Conjugate; cat. no. #53276; both 1:50; both Cell Signaling Technology, Inc.) on ice without light for 30 min, followed by PBS rinsing three times.

Techniques: Immunofluorescence, Marker, Expressing, Western Blot, Flow Cytometry, Colony Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Over Expression

Journal: International Journal of Oncology

Article Title: Mechanism of SMYD2 promoting stemness maintenance of bladder cancer stem cells by regulating PYCR1 expression and PINK1/Parkin mitophagy pathway

doi: 10.3892/ijo.2025.5747

Figure Lengend Snippet: SMYD2 increases the maintenance of bladder cancer stem cell stemness by upregulating PYCR1 to stimulate the PINK1/Parkin pathway. Tumor (A) size, (B) volume and (C) weight. (D) Hematoxylin-eosin staining to detect pathological changes in tumor tissue. (E) Western blot to measure SMYD2, PYCR1, PINK1 and Parkin levels in nude mouse tissue homogenate. (F) Immunohistochemistry was performed to detect the number of SMYD2-, H3K4me3-, CD44- and CD133-positive cells in tumor tissues. (G) Protein expression of autophagy markers LC3B II/I and p62 was determined by western blotting. (H) Expression of stemness marker proteins Nanog and Sox2 mRNA was measured by reverse transcription-quantitative PCR. (I) Western blot assay to detect protein levels of Nanog and Sox2 in tissue homogenates of nude mice. * P<0.05, ** P<0.01, *** P<0.001. SMYD2, SET and MYND domain-containing protein 2; PYCR1, pyrroline-5-carboxylate reductase 1; PINK1, PTEN-induced putative kinase 1; H3K4me3, histone H3 lysine 4 trimethylation; BC, bladder cancer; Lv, lentiviral; oe, overexpression; NC, negative control; si, small-interfering.

Article Snippet: Cells were cultured in the presence of antibodies labeled with CSC markers, such as CD44 (IM7) Rat mAb (Alexa Fluor ® 555 Conjugate; cat. no. #95235) and CD133 (A8N6N) Mouse mAb (Alexa Fluor ® 647 Conjugate; cat. no. #53276; both 1:50; both Cell Signaling Technology, Inc.) on ice without light for 30 min, followed by PBS rinsing three times.

Techniques: Staining, Western Blot, Immunohistochemistry, Expressing, Marker, Reverse Transcription, Real-time Polymerase Chain Reaction, Over Expression, Negative Control