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: Drug Delivery

Article Title: Machine learning-assisted design of immunomodulatory lipid nanoparticles for delivery of mRNA to repolarize hyperactivated microglia

doi: 10.1080/10717544.2025.2465909

Figure Lengend Snippet: Cellular characterization and behavior. (A) Left: Bar chart displaying the average size (left blue bars) and zeta potential (right red bars) of LNP formulations (mean ± SD for all 108 libraries) and HA-LNP (mean ± SD for all 108 libraries). Right: Percentage of encapsulation efficiency for two different libraries of LNP and HA-LNP, based on a sample size of n = 108 for each library. (B) Confocal microscopy images of BV2 cells across various activation states at 40X (upper row) and 63X (bottom row) magnification. Scale bars: 50 μm (upper row) and 10 μm (bottom row). The major characteristics of each morphology are indicated by yellow arrows in the bottom row. From left to right: the amoeboid morphology features a very large soma with few short processes, appearing rounded; the reactive morphology displays irregular cell bodies with a large soma and thickened processes; the homeostatic morphology has a small soma with thin, highly branched processes; and the rod morphology exhibits an enlarged cell body with processes that create a polar shape. (C) Left: Immunostaining of BV2 cells in different activation states. Fixed cells were stained with anti-CD44 (orange) and DAPI (blue) for nuclear visualization. CD44 served as a marker for pro-inflammatory activated cells. The immunostaining images were captured using a confocal microscope (40X, scale bar: 50 μm). Right: qPCR results for BV2 cells with or without activation. The left bar plot compares the expression of the pro-inflammatory gene CD86 between control BV2 cells (blue bars), LPS-activated BV2 cells (red bar), and IL4/IL13-activated BV2 cells (green bar). The right bar plot compares the expression of the anti-inflammatory gene CD206 between control BV2 cells (blue bar), IL4/IL13-activated BV2 cells (green bar), and LPS-activated BV2 cells (red bar). β-actin was used as the reference gene for normalization. All experiments were conducted in triplicate, and the data are presented as the mean ± SD of the three independent replicates. P-values are indicated as follows: ****<0.0001, ***<0.0002, **<0.0021, *<0.0332, ns = not significant.

Article Snippet: The primary antibody CD44 (clone IM7, RRID: AB_3662602, 39037) Rat mAb (1:400; Cell Signaling Technology, MA, USA) was applied to the BV2 cells.

Techniques: Zeta Potential Analyzer, Encapsulation, Confocal Microscopy, Activation Assay, Immunostaining, Staining, Marker, Microscopy, Expressing, Control

Journal: Drug Delivery

Article Title: Machine learning-assisted design of immunomodulatory lipid nanoparticles for delivery of mRNA to repolarize hyperactivated microglia

doi: 10.1080/10717544.2025.2465909

Figure Lengend Snippet: Validation of ML prediction on LPS-activated BV2 cells. (A) Fluorescence microscopy images of LPS-activated BV2 cells transfected with eGFP mRNA 24 h post-transfection. The top row shows bright-field images, while the bottom row displays fluorescent images captured using a fluorescence microscope (20X magnification, scale bar: 50 μm). LNP2 and HA-LNP2 are the same formulations used to validate the machine learning prediction. Lipofectamine MMAX was used as a control with the same mRNA dosage (final mRNA concentration of 2 μg/ml). (B) Normalized mean fluorescence intensity (MFI) after transfection with eGFP mRNA, quantified using ImageJ analysis, with lipofectamine MMax as a positive control. The sample sizes for both LNP and HA-LNP libraries are n = 108. (C) Blocking (competition) assay to evaluate the CD44 targeting capability of HA-LNP formulations. The MC3:DOPE:Chol:DMG-PEG2000 (40:20:38.5:1.5) formulation with an N/P ratio of 8 (final mRNA concentration of 2 μg/ml) was used to transfect LPS-activated BV2 cells under two conditions: Top row) BV2 cells treated with HA solution, bottom row) BV2 cells without blocking. Four hours post-transfection with Cy5-mRNA (red), cells were fixed, and the nucleus (DAPI) and F-actin (phalloidin, green) were stained to visualize the internalized mRNA (red). The immunostaining images were captured using a confocal microscope (63X magnification, scale bar: 10 μm). (D) Average bar plot illustrating the percentage of BV2 cell viability as determined by the MTT assay for various LNP groups (mean ± SD) and HA-LNP (mean ± SD), including controls: blank BV2 cells, naked mRNA, and commercial LipofectamineMMAX carrier encapsulated mRNA (mean ± SD). The mRNA cargo utilized was eGFP mRNA. (E) IL-10 ELISA assay results show cytokine secretion from LPS-activated BV2 cells at two time points post-transfection: 24 h (green bars) and 48 hours (blue bars). Data are presented for different formulations as carriers for IL-10 mRNA ( n = 3). (F) TNF-α ELISA assay results for TNF-α cytokine secretion from LPS-activated BV2 cells at the same time points: 24 h (red bars) and 48 h (orange bars) across different formulations ( n = 3). P-values are indicated as follows: **** <0.0001, *** <0.0002, ** <0.0021, * <0.0332, ns = not significant.

Article Snippet: The primary antibody CD44 (clone IM7, RRID: AB_3662602, 39037) Rat mAb (1:400; Cell Signaling Technology, MA, USA) was applied to the BV2 cells.

Techniques: Biomarker Discovery, Fluorescence, Microscopy, Transfection, Control, Concentration Assay, Positive Control, Blocking Assay, Competitive Binding Assay, Formulation, Staining, Immunostaining, MTT Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Super-enhancer profiling reveals ThPOK/ZBTB7B, a CD4 + cell lineage commitment factor, as a master regulator that restricts breast cancer cells to a luminal non-migratory phenotype

doi: 10.1101/2024.09.21.614267

Figure Lengend Snippet: a, b, d-f, h-i) Gene expression levels measured by qRT-PCR. Since three different shRNA against ThPOK were used, the circle for each gene is divided in thirds, one for each shRNA. The color of each third indicates the log 2 (fold-change) and the size the -log 10 (p-value), for a t -test relative to the control shRNA (shScr). Each gene was assessed in ≥3 biological replicates. Differential expression is shown in color scale as a log2 of the fold change, and p-values by the size of the circle as –log10. c) Morphological changes associated with ThPOK deficiency in Luminal and HER2+ cells (10X) magnification. Scale: each side of the square is 400µm. g) Flow cytometry analysis showing CD44 expression in Luminal and HER2+ scramble (Scr) and ThPOK-kd cells. j-k) Migration assays. j) Scratch assay showing increased migratory features of ThPOK-kd cells compared with control (scr) cells. Error bars represent the SD of ≥3 biological replicates. T47D cells p=4.07×10 −19 , MCF7 cells p=8.087×10 −8 . k) Boyden chamber assay. Error bars represent the SD of ≥3 biological replicates, p *<0.05, **<10 −4 .

Article Snippet: Cells were stained for 1 hour with an anti-CD44-PE antibody (Cell Signaling, cat# 88151) at a 1:300 dilution in FACS buffer.

Techniques: Gene Expression, Quantitative RT-PCR, shRNA, Control, Quantitative Proteomics, Flow Cytometry, Expressing, Migration, Wound Healing Assay, Boyden Chamber Assay