Journal: Oncotarget

Article Title: Enhancement of the anti-tumor activity of FGFR1 inhibition in squamous cell lung cancer by targeting downstream signaling involved in glucose metabolism

doi: 10.18632/oncotarget.19279

Figure Lengend Snippet: (a) Total proteins were extracted from tissue samples obtained from LENTI-4 induced tumor xenografts from control and NVP-BEZ235, NVP-BGJ398 or NVP-BGJ398+NVP-BEZ235 treated BALB/c-Nude mice. Western Blotting was performed to evaluate src and FAK activation/expression. Results are representative of two independent experiments. (b) Total RNA was isolated from tissue samples obtained as in (a) . Human GLUT-1 mRNA levels were analyzed by RT-PCR. Amplifications were normalized to HPRT1 and PGK1. Fold changes were calculated by the ΔΔCT method and results were plotted as 2^-ΔΔCT ± SD. * p<0.05 vs C. (c) Immunofluorescence analysis of GLUT-1 protein expression in sections of tumors from controls (C) and NVP-BEZ235, NVP-BGJ398 or NVP-BGJ398+NVP-BEZ235 treated mice. Neoplastic cells are labelled by CD44 (red) and green fluorescence documents the expression of GLUT-1. In merged images, yellowish fluorescence corresponds to CD44pos tumor cells expressing GLUT-1 while bright red fluorescence indicates GLUT-1 negative cells that appear abundant in NVP-BGJ398+NVP-BEZ235 xenograft. Scattered clusters of GLUT-1pos/CD44neg cells are also present. Nuclei are stained by the blue fluorescence of DAPI. Scale bars: 100μm. (d) Bar graph of the quantitative analysis of the fractional area occupied by GLUT-1 positive cells; * p<0.05 vs C.

Article Snippet: Briefly, GLUT-1-stained sections were incubated with rat monoclonal anti-CD44 antibody (HCAM-IM7, Santa Cruz Biotechnology, Dallas, TX) followed by specific Cy3-conjugated secondary antibody (1:50 Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Control, Western Blot, Activation Assay, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Fluorescence, Staining

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

Journal: The Journal of Clinical Investigation

Article Title: A correctable immune niche for epithelial stem cell reprogramming and post-viral lung diseases

doi: 10.1172/JCI183092

Figure Lengend Snippet: ( A ) Protocol scheme for anti-CD44 mAb or control IgG treatment in mice with SeV infection compared with SeV-UV control. ( B ) Body weights for conditions in A . ( C ) Immunostaining for Krt5 with DAPI counterstaining in lung sections from conditions in A . Scale bar: 1 mm. ( D ) PAS and hematoxylin staining of lung sections for conditions in C . Scale bar: 1 mm. ( E ) Quantitation of staining in C and D . ( F ) Immunostaining for GPNMB plus CD11c and GPNMB plus F4/80 with DAPI counterstaining for conditions in A . Scale bar: 100 μm. ( G ) Quantitation of staining in F . Values represent mean ± SEM ( n = 5–8 mice per condition). * P < 0.05 by ANOVA and Tukey’s correction.

Article Snippet: Ab blockade was performed with goat anti-GPNMB Ab (R&D Systems, AF2330), rat anti-CD44 mAb (clone IM7; Bio X Cell, BE0039), or corresponding control IgG (R&D Systems, AB-108-C, and Bio X Cell, BE0130 and BE0094, respectively) administered by intraperitoneal injection.

Techniques: Control, Infection, Immunostaining, Staining, Quantitation Assay

Journal: The Journal of Clinical Investigation

Article Title: A correctable immune niche for epithelial stem cell reprogramming and post-viral lung diseases

doi: 10.1172/JCI183092

Figure Lengend Snippet: ( A ) Protocol scheme for cell isolation from mouse lungs at 21 days after SeV infection, then FACS for CD31 – CD45 – EpCAM + Aqp3 + epithelial cells and seeding into 3D organoid culture. ( B ) Levels of organoid formation from conditions in A and coculture with the indicated immune cell populations. ( C ) Levels of organoid formation in 3D cultures incubated with GPNMB (0–100 ng/mL) for conditions in A . ( D ) Photomicrographs of organoids for conditions in C without or with GPNMB (100 ng/mL). Scale bar: 80 μm. ( E ) Organoid formation for conditions in C but using incubation with GPNMB (10 ng/mL) with or without anti-CD44 mAb or control IgG. ( F ) Levels of Cxcl17 and Il33 mRNA for conditions in C . ( G ) Protocol scheme for human tracheobronchial epithelial cell isolation and 3D culture. ( H ) Photomicrographs of organoids for conditions in G without or with GPNMB (100 ng/mL). Scale bar: 80 μm. ( I ) Levels of organoid formation and CXCL17 and IL33 mRNA for conditions in G with GPNMB (0–100 ng/mL). For B – E and G , results are representative of non-disease control individuals ( n = 6–8 per condition). * P < 0.05 by ANOVA and Tukey’s correction.

Article Snippet: Ab blockade was performed with goat anti-GPNMB Ab (R&D Systems, AF2330), rat anti-CD44 mAb (clone IM7; Bio X Cell, BE0039), or corresponding control IgG (R&D Systems, AB-108-C, and Bio X Cell, BE0130 and BE0094, respectively) administered by intraperitoneal injection.

Techniques: Cell Isolation, Infection, Incubation, Control

Journal: eLife

Article Title: Delineating the transcriptional landscape and clonal diversity of virus-specific CD4 + T cells during chronic viral infection

doi: 10.7554/eLife.80079

Figure Lengend Snippet:

Article Snippet: Antibody , BUV395 rat monoclonal anti-mouse CD44 (IM7) , BD Biosciences , Cat #740215 RRID: AB_2739963 , 1:500.

Techniques: Virus, Multiplex Assay, Software

Journal: Cell reports

Article Title: Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages

doi: 10.1016/j.celrep.2022.111804

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat monoclonal anti-mouse CD44 - 162Dy , Fluidigm , Cat#3162030B; RRID:AB_2814898.

Techniques: Purification, Recombinant, Activation Assay, SYBR Green Assay, Reverse Transcription, Detection Assay, Enzyme-linked Immunosorbent Assay, Selection, Software

Journal:

Article Title: Murine Model of Interstitial Cytomegalovirus Pneumonia in Syngeneic Bone Marrow Transplantation: Persistence of Protective Pulmonary CD8-T-Cell Infiltrates after Clearance of Acute Infection

doi:

Figure Lengend Snippet: Phenotypes of phase 1 and phase 2 pulmonary infiltrate T cells. Pulmonary infiltrate cells were isolated at 4 weeks (phase 1) and at 10 weeks (early in phase 2) after BMT and mCMV infection. (A and B) Three-color cytofluorometric analysis was performed for the marker combinations FITC (FL-1)-CD8, PE (FL-2)-TCRα/β, and PE-Cy5 (FL-3)-CD4. A gate was set on lymphocytes, and the analysis was restricted to ca. 20,000 α/β T cells by a second gate set on positive FL-2. (C and D) Three-color cytofluorometric analysis was performed for the marker combinations FITC (FL-1)-CD44, PE (FL-2)-CD8, and RED613 (FL-3)-CD62L. A gate was set on lymphocytes, and the analysis was restricted to ca. 10,000 CD8 T cells by a second gate set on positive FL-2. FL-3 (ordinate) versus FL-1 (abscissa) log fluorescence intensities are shown for gated cells as contour plots in a 70% log-density mode (threshold, 2%; smoothing factor, 5). Percentages of relevant T-cell subsets and the ratios of CD8 to CD4 T cells (CD8/CD4) are indicated.

Article Snippet: Pulmonary infiltrate cells retrieved from the Ficoll interphase were labeled with FITC-conjugated MAb anti-CD44 (clone IM7, rat IgG2b; catalog no. 01224D; PharMingen) and PE-conjugated MAb anti-CD8a (see above).

Techniques: Isolation, Infection, Marker, Fluorescence

Journal:

Article Title: Murine Model of Interstitial Cytomegalovirus Pneumonia in Syngeneic Bone Marrow Transplantation: Persistence of Protective Pulmonary CD8-T-Cell Infiltrates after Clearance of Acute Infection

doi:

Figure Lengend Snippet: Production of IFN-γ after polyclonal CD3ɛ signaling. (A) BMT and mCMV infection. (B) BMT with no infection. Pulmonary infiltrate cells (phase 1, 4 weeks; phase 2, 16 weeks) were stimulated for 5 h with MAb anti-CD3ɛ in the presence of brefeldin A. Control groups were treated accordingly except for polyclonal CD3ɛ stimulation. Three-color cytofluorometric analysis was performed for the marker combination FITC (FL-1)-CD62L, PE (FL-2)-IFN-γ, and PE-Cy5 (FL-3)-CD8. A gate was set on lymphocytes, and the analysis was restricted to ca. 25,000 CD8 T cells by a second gate set on positive FL-3. FL-1 (ordinate) versus FL-2 (abscissa) log fluorescence intensities are shown for gated cells as dot plots, with 10,000 dots displayed. Percentages are indicated for CD62Lhi CD8 T cells (upper left quadrants) and for CD62Llo CD8 T cells with intracellular accumulation of IFN-γ (lower right quadrants). The isotype controls (PE-conjugated rat IgG1) are shown for cells stimulated with anti-CD3ɛ.

Article Snippet: Pulmonary infiltrate cells retrieved from the Ficoll interphase were labeled with FITC-conjugated MAb anti-CD44 (clone IM7, rat IgG2b; catalog no. 01224D; PharMingen) and PE-conjugated MAb anti-CD8a (see above).

Techniques: Infection, Marker, Fluorescence