human mouse cd44 apc  (Multi Sciences (Lianke) Biotech Co Ltd)

Journal: bioRxiv

Article Title: Immunogenicity and protective efficacy of an intranasal neuraminidase-based influenza virus vaccine adjuvanted with bacterial cell membrane-derived adjuvants

doi: 10.1101/2025.02.26.640278

Figure Lengend Snippet: a. Representative flow cytometry plots showing the percentage of IL-2- and TNFα-producing CD4 + EM T-lymphocytes in mouse lungs and spleens 10 days after the booster immunization (gated on live CD3 + CD19 ‒ CD4 + CD62L ‒ CD44 + cells). Cells were incubated with overlapping peptides, covering the whole sequence of N1 NA-protein for 6h in the presence of Brefeldin A and co-stimulatory anti-CD28 antibodies. b. Percentage of different cytokine-producing cell populations within the total CD4 + EM T-cell subset after background subtraction. Average and SE values are shown. Statistical analyses were performed using one-way ANOVA with Tukey posthoc test. P-values for the groups with statistically significant differences are shown on the plots.

Article Snippet: For the analysis of T- and B-cell populations in lungs, spleen, and NALTs, cells were stained with the antibody cocktail, including the following antibodies: CD38-AF488 (0.125 μl, BioLegend), PD-1-PE/Dazzle (0.5 μl, BioLegend), CD273-RB744 (0.25 μl, BD Biosciences), CD138-PE (0.25 μl, BioLegend), CD103-PE-Cy5.5 (0.5 μl, BioLegend), CD44-PE/Cy7 (0.25 μl, Tonbo Biosciences), GL7-APC (0.25 μl, BioLegend), CD69-AF700 (1 μl, BioLegend), CD62L-APC/Cy7 (0.125 μl, BioLegend), CD185-BV421 (0.5 μl, BioLegend), CD80-BV605 (1 μl, BioLegend), IgD-BV650 (0.25 μl, BioLegend), CD3-BV711 (0.5 μl, BioLegend), CD8-BV785 (0.25 μl, BioLegend), CD4-BUV496 (0.125 μl, BD Biosciences), IgM-BUV737 (1 μl, BD Biosciences), CD19-BUV805 (0.125 μl, BD Biosciences).

Techniques: Flow Cytometry, Incubation, Sequencing

Journal: bioRxiv

Article Title: Immunogenicity and protective efficacy of an intranasal neuraminidase-based influenza virus vaccine adjuvanted with bacterial cell membrane-derived adjuvants

doi: 10.1101/2025.02.26.640278

Figure Lengend Snippet: a. Body weight dynamics and survival after the heterologous challenge with 5 × LD 50 of A/bald eagle/Florida/W22-134-OP/2022 (H5N1 reassortant with A/Puerto Rico/8/1934 vaccine backbone) and A/New Caledonia/20/1999 (H1N1) (n = 8). b. Viral titers in lungs and nasal turbinates on day 4 after the challenge (n = 4). c. Representative flow cytometry plots showing the percentage of IL-2- and TNFα-producing CD4 + EM T-lymphocytes (gated on live CD3 + CD19 ‒ CD4 + CD62L ‒ CD44 + cells) in mouse lungs and spleens 5 days after the challenge with 0.1 × LD 50 of A/bald eagle/Florida/W22-134-OP/2022 (H5N1). Cells were incubated with overlapping peptides, covering the whole sequence of N1 NA-protein for 6h in the presence of Brefeldin A and co-stimulatory anti-CD28 antibodies. d. Percentage of different cytokine-producing cell populations within the total CD4 + EM T-cell subset after background subtraction. Average and SE values are shown. Statistical analyses were performed using one-way ANOVA with Tukey posthoc test. P-values for the groups with statistically significant differences are shown on the plots.

Article Snippet: For the analysis of T- and B-cell populations in lungs, spleen, and NALTs, cells were stained with the antibody cocktail, including the following antibodies: CD38-AF488 (0.125 μl, BioLegend), PD-1-PE/Dazzle (0.5 μl, BioLegend), CD273-RB744 (0.25 μl, BD Biosciences), CD138-PE (0.25 μl, BioLegend), CD103-PE-Cy5.5 (0.5 μl, BioLegend), CD44-PE/Cy7 (0.25 μl, Tonbo Biosciences), GL7-APC (0.25 μl, BioLegend), CD69-AF700 (1 μl, BioLegend), CD62L-APC/Cy7 (0.125 μl, BioLegend), CD185-BV421 (0.5 μl, BioLegend), CD80-BV605 (1 μl, BioLegend), IgD-BV650 (0.25 μl, BioLegend), CD3-BV711 (0.5 μl, BioLegend), CD8-BV785 (0.25 μl, BioLegend), CD4-BUV496 (0.125 μl, BD Biosciences), IgM-BUV737 (1 μl, BD Biosciences), CD19-BUV805 (0.125 μl, BD Biosciences).

Techniques: Flow Cytometry, Incubation, Sequencing

Journal: Nature Communications

Article Title: Aggresome formation promotes ASK1/JNK signaling activation and stemness maintenance in ovarian cancer

doi: 10.1038/s41467-024-45698-x

Figure Lengend Snippet: a A multiomic integration approach was developed to screen the key factor controlling self-renewal and maintenance of OCSC. SKOV3 floating spheres and the derived differentiated cells were subjected to RNA-seq analysis, which is sorted by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$-{\log }_{10}\,{P\; {{{{\rm{Value}}}}}}$$\end{document} − log 10 P Value . The obtained differentially expressed genes were further screened based on the gene dataset of susceptibility loci for ovarian cancer and their negatively correlation with tumor prognosis. SDC Sphere Differentiated Cells. FS Floating Sphere. b Kaplan–Meier survival curves revealed a negative correlation between OTUD1 expression level and progression-free survival (PFS) in individuals with serous ovarian cancer ( https://kmplot.com/analysis/ ). n = 535 patients with serous ovarian cancer. c The protein level of OTUD1 were measured in SKOV3 floating spheres and the derived differentiated cells. SDC Sphere Differentiated Cells. FS Floating Sphere. d Immunohistochemistry analysis of OTUD1 protein abundance in ovarian serous cancer tissue microarray; representative images of indicated pathological grade are shown (Scale bar, 20 μm). LGSOC samples, n = 4; HGSOC samples, n = 54. HGSOC, High grade serous ovarian carcinoma. LGSOC Low grade serous ovarian carcinoma. Representative images ( e ) and statistical analysis ( f ) demonstrating the effects of OTUD1 knockout on floating sphere-forming capacity in SKOV3 and OVCAR8 cell lines ( n = 3). Scale bar, 50 μm. g Flow cytometry analysis of CD44 + and CD133 + CSCs in SKOV3 OTUD1 depleted cell line. Numbers in the charts indicate the percentages of corresponding subpopulations ( n = 3). Tumor images ( h ) and volumes ( i ) in mice injected with negative control and OTUD1 knockout SKOV3 cells ( n = 6 mice per group). Tumor sizes were monitored and the protein level of xenografts were analyzed by western blot ( j ). P values are calculated using two-tailed unpaired Student’s t test ( d ), one-way ANOVA ( f , g ) and two-way ANOVA ( i ). n.s. not significant. Representative of n = 3 independent experiments ( c , e , g ). Source data are provided as a Source Data file.

Article Snippet: Goat anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab15007, 1:200), Goat anti-Rabbit IgG H&L (Alexa Fluor 555) (ab150078, 1:200), APC Mouse Anti-Human CD44(G44-26) (559942, 1:50), and FITC Mouse Anti-Human CD133(W6B3C1) (567029, 1:50) were purchased from BD Pharmingen.

Techniques: Derivative Assay, RNA Sequencing Assay, Expressing, Immunohistochemistry, Microarray, Knock-Out, Flow Cytometry, Injection, Negative Control, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Aggresome formation promotes ASK1/JNK signaling activation and stemness maintenance in ovarian cancer

doi: 10.1038/s41467-024-45698-x

Figure Lengend Snippet: 1 × 10 4 , 1 × 10 5 or 1 × 10 6 SKOV3 cells were subcutaneously injected into BALB/c nude female mice ( n = 6 mice per group). After 25, 32, or 54 days, the mice were sacrificed and the tumor tissues were collected as shown in a . The table shows the number of harvested tumors in each group ( b ). Tumor growth curve of indicated xenografts are created based on tumor volume of indicated time ( c ). EV empty vector. d Effect of WT OTUD1 or Δ105–164 mutant on CD44 + or CD133 + cells proportions were evaluated by flow cytometry analysis. EV empty vector. e Representative images of the effects of depletion of OTUD1 IDR region (105–164 aa) on sphere-formation in SKOV3 cells. Scale bar, 50 μm. IDR intrinsically disordered protein region. EV empty vector. f Representative images of the effect of WT (OTUD1 WT ) or IDR depleted mutant OTUD1 (OTUD1 Δ105–164 ) on anchor-independent growth capacity in SKOV3 cells. Scale bar, 400 μm. EV empty vector. g qPCR was employed to confirm the expression of stemness-associated genes (ie, CD133, CD44, CD24, OCT4, SOX2, NOTCH1 ) in SKOV3 OTUD1 WT or OTUD1 Δ105-164 expressing cells ( n = 3). EV empty vector. h Immunofluorescence analysis was performed to examine the correlation of OTUD1 WT or OTUD1 Δ105–164 expression and ASK1 protein level or distribution in vivo. Scale bar, 15 μm. i Statistical analysis showing the OTUD1-based aggresome average diameter of OTUD1 (WT) or mutant OTUD1 proteins (Δ105–164, M1) in SKOV3 xenografted tumor samples ( n = 25 cells examined over 2 xenografted tumor samples). EV, empty vector. P values are calculated using two-way ANOVA ( c ), one-way ANOVA ( g ), two-tailed unpaired Student’s t test ( h ). Representative of n = 3 independent experiments ( d – f ). Source data are provided as a Source Data file.

Article Snippet: Goat anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab15007, 1:200), Goat anti-Rabbit IgG H&L (Alexa Fluor 555) (ab150078, 1:200), APC Mouse Anti-Human CD44(G44-26) (559942, 1:50), and FITC Mouse Anti-Human CD133(W6B3C1) (567029, 1:50) were purchased from BD Pharmingen.

Techniques: Injection, Plasmid Preparation, Mutagenesis, Flow Cytometry, Expressing, Immunofluorescence, In Vivo, Two Tailed Test

Journal: Nature Communications

Article Title: Aggresome formation promotes ASK1/JNK signaling activation and stemness maintenance in ovarian cancer

doi: 10.1038/s41467-024-45698-x

Figure Lengend Snippet: a Representative images demonstrate the effects of IN-8 (2 μg/mL), selonsertib (SE, 1 μM), or ibrutinib (2.5 μg/mL) treatment on SKOV3 sphere-formation with or without ectopic OTUD1, respectively. EV empty vector. Scale bar, 50 μm. b Statistical analysis reveals the effect of inhibitors (IN8, Selonsertib, and Ibrutinib) on number of floating spheres of indicated SKOV3 cells in Fig. 6a ( n = 3). EV empty vector, SE selonsertib. c The soft agar assay showing anchorage-independent growth of OTUD1 cells in SKOV3 treated with or without IN-8 (2 μg/mL), Selonsertib (SE, 1 μM), or Ibrutinib (2.5 μg/mL). Scale bar, 400 μm. d Statistical analysis illustrates the effect of inhibitors (IN8, Selonsertib, and Ibrutinib) on number of colonies in indicated SKOV3 cells in Fig. 6c ( n = 3). EV empty vector, SE selonsertib. e The qPCR assay was utilized to confirm the expression of stemness-associated genes (i.e., CD44, OCT4 , and SOX2 ) in SKOV3 cells expressing OTUD1 with indicated treatment ( n = 3). EV empty vector, SE selonsertib. f Flow cytometry analysis of CD44 + and CD133 + CSCs in SKOV3 OTUD1 WT cell with indicated treatment. Numbers in the charts indicate the percentages of corresponding subpopulations. EV empty vector. g Statistical analysis showing the effect of those three inhibitors (IN8, Selonsertib, and Ibrutinib) on the percentages of CSCs in Fig. 6f ( n = 3). EV empty vector. h The chemotherapy sensitivity assay by using SKOV3 cells expressing OTUD1 with or without IN-8 (2 μg/mL), Ibrutinib (2.5 μg/mL), or/and DPP (1 μg/mL) treatment. DPP Cisplatin, EV empty vector. i Statistical analysis displays the effect of those three inhibitors (IN8, Selonsertib, and Ibrutinib) on the clonal formation in Fig. 6h ( n = 3). EV empty vector. P values are calculated using unpaired Student’s t test ( b , d , e , g ), one-way ANOVA ( i ). Representative of n = 3 independent experiments ( a , c , f , h ). Source data are provided as a Source Data file.

Article Snippet: Goat anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab15007, 1:200), Goat anti-Rabbit IgG H&L (Alexa Fluor 555) (ab150078, 1:200), APC Mouse Anti-Human CD44(G44-26) (559942, 1:50), and FITC Mouse Anti-Human CD133(W6B3C1) (567029, 1:50) were purchased from BD Pharmingen.

Techniques: Plasmid Preparation, Soft Agar Assay, Expressing, Flow Cytometry, Sensitive Assay

Journal: Cancers

Article Title: MACC1 Regulates LGR5 to Promote Cancer Stem Cell Properties in Colorectal Cancer

doi: 10.3390/cancers16030604

Figure Lengend Snippet: CD44-enriched CSCs from the PDOs expressed higher levels of MACC1 and LGR5. ( a ) mRNA expression analyses of a total of eight PDO samples, performed directly after FACS. ( b ) Summarized expression of single qRT-PCR values. (** = p < 0.01, *** = p < 0.001).

Article Snippet: CD44-APC Mouse Anti-Human CD44 antibody (Clone G44-26, BD PharmingenTM, Heidelberg, Germany) and APC Mouse IgG2b κ Isotype Control (Clone 27-35, BD PharmingenTM) were added to a final dilution of 1:10 to PBS-washed cells.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancers

Article Title: MACC1 Regulates LGR5 to Promote Cancer Stem Cell Properties in Colorectal Cancer

doi: 10.3390/cancers16030604

Figure Lengend Snippet: CD44-high/CD166+ stem cell-enriched populations display higher tumorigenicity in vivo, as illustrated by stem cell populations of two PDOs. ( a ) Experimental design. ( b ) qRT-PCR of CD44, CD166, LGR5, MACC1 and S100A4 in CD44-low/CD166+ and CD44-high/CD166+ stem cell-enriched populations. ( c ) Tumor volumes as measured in the respective mouse models. (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Article Snippet: CD44-APC Mouse Anti-Human CD44 antibody (Clone G44-26, BD PharmingenTM, Heidelberg, Germany) and APC Mouse IgG2b κ Isotype Control (Clone 27-35, BD PharmingenTM) were added to a final dilution of 1:10 to PBS-washed cells.

Techniques: In Vivo, Quantitative RT-PCR

Journal: Cancers

Article Title: MACC1 Regulates LGR5 to Promote Cancer Stem Cell Properties in Colorectal Cancer

doi: 10.3390/cancers16030604

Figure Lengend Snippet: Expression analyses of MACC1 and LGR5 in CD44-low and CD44-high cell populations derived from CRC-PDX. ( a ) Experimental design. ( b ) Analyzed PDOs. ( c ) Expression of CD44, MACC1 and LGR5 in CD44-sorted cell population of PDOs. (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Article Snippet: CD44-APC Mouse Anti-Human CD44 antibody (Clone G44-26, BD PharmingenTM, Heidelberg, Germany) and APC Mouse IgG2b κ Isotype Control (Clone 27-35, BD PharmingenTM) were added to a final dilution of 1:10 to PBS-washed cells.

Techniques: Expressing, Derivative Assay