cd44 im7 rat mab  (Cell Signaling Technology Inc)

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: Purinergic Signalling

Article Title: The P2X7 receptor antagonist JNJ-47965567 administered thrice weekly from disease onset does not alter progression of amyotrophic lateral sclerosis in SOD1 G93A mice

doi: 10.1007/s11302-020-09692-4

Figure Lengend Snippet: JNJ-47965567 does not alter proportions of T cell subsets in SOD1 G93A mice. Spleens and CNS draining and non-draining lymph nodes from end stage β-CD- and JNJ-47965567-treated SOD1 G93A mice were labelled with fluorochrome-conjugated mAb and the percentages of T cell subsets, as well as relative expression of CD39 (MFI), assessed by flow cytometry. Results are mean ± SEM

Article Snippet: Rat anti-murine CD16/CD32 (Mouse BD Fc Block), PerCP-Cy5.5-conjugated rat anti-murine CD4 (clone RM4-5), APC-Cy7-conjugated rat anti-murine CD8 (clone 53-6.7) and PE-Cy7-conjugated rat anti-murine CD44 (clone IM7) mAb were from BD Biosciences (San Diego, USA).

Techniques: Expressing, Flow Cytometry