Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Workflow for comprehensive identification of transcriptionally active lncRNAs in the gastric mucosa of patients with GC. B Pie chart depicting gene loci harboring H3K4me3 peaks at transcription start sites (TSSs). These loci were categorized as protein-coding genes, lncRNA genes, or other genes. Peaks were categorized as patient-specific, healthy individual-specific, and common. C H3K4me3 peaks of representative genes in each category. D Summarized qRT-PCR results for 15 selected lncRNAs from eight GC cell lines. E qRT-PCR analysis of TCONS_00006264 (TM4SF1-AS1) and TM4SF1 in GC cell lines and normal stomach tissue. The locations of these two genes are shown at the top. ( n = 3). F Expression of TM4SF1-AS1 in primary GCs ( n = 374) and normal stomach tissues ( n = 32) from The Cancer Genome Atlas (TCGA) dataset. * P < 0.05.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Expressing

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A qRT-PCR analysis of TM4SF1-AS1 in the indicated GC cell lines transfected with siRNAs targeting TM4SF1-AS1 (si-1 and 2) or a control siRNA (si-Ctrl). ( n = 3). B Results of cell viability assays with GC cell lines transfected with the indicated siRNAs. ( n = 8). C qRT-PCR analysis of TM4SF1-AS1 in HSC-45 cells with inducible shRNAs targeting TM4SF1-AS1 (sh-1 and 2) or a control shRNA (sh-Ctrl). Cells were incubated for 8 days with or without doxycycline (Dox). ( n = 3). D Colony formation assays using HSC-45 cells with inducible shRNAs. Cells were incubated for 8 days with or without Dox. Summarized results are shown on the right; error bars represent SDs. ( n = 3). E Tumor growth in mice injected with HSC-45 cells inducibly expressing the indicated shRNAs. Mice were treated with or without Dox. Growth curves (left), resected tumors (middle), and tumor weights (right) are shown. F qRT-PCR analysis of TM4SF1-AS1 in the resected tumors in E . ( n = 4). G Tumor growth in mice injected with SNU638 cells stably expressing GFP or TM4SF1-AS1. Growth curves are shown on the left, and resected tumors are shown on the right. ( n = 4). H qRT-PCR analysis of TM4SF1-AS1 in the resected tumors in G . ( n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001, NS not significant.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Transfection, Control, shRNA, Incubation, Injection, Expressing, Stable Transfection

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A qRT-PCR analysis of TM4SF1-AS1 in the indicated cellular fractions from HSC-45 cells. U6 snRNA and ACTB served as endogenous controls. ( n = 3). B Association between TM4SF1-AS1 and Pur-α in the indicated subcellular fractions from HSC-45 cells. Pur-α was detected by western blotting in proteins pulled-down with BrU-labeled TM4SF1-AS1, antisense of TM4SF1-AS1 or beads. C Association between Pur-α and YB-1. Immunoprecipitated YB-1 or Pur-α from HSC-45 cell extracts were probed for co-precipitating proteins. D YB-1 was immunoprecipitated from HSC-45 cells with or without RNase A treatment, after which Pur-α was detected by western blotting. E YB-1 or Pur-α in the indicated subcellular fractions from HSC-45 cells were immunoprecipitated with or without RNase A treatment and then probed for co-precipitating proteins. F Results of RIP assays. Pur-α (upper) or YB-1 (lower) was immunoprecipitated from HSC-45 cells, after which co-precipitated TM4SF1-AS1 was detected with qRT-PCR. IgG served as a negative control. ( n = 3). G qRT-PCR analysis of Pur-α (upper) and YB-1 (lower) in HSC-45 cells transfected with the indicated siRNAs. ( n = 3). H Results of cell viability assays with HSC-45 cells transfected with the indicated siRNAs. ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Labeling, Immunoprecipitation, Negative Control, Transfection

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Heatmap showing expression of genes suppressed by TM4SF1-AS1 knockdown in HSC-45 cells. B GO analysis using the genes in A . C qRT-PCR analysis of representative IRDS genes in HSC-45 cells transfected with the indicated siRNAs. ( n = 3). D GSEA analysis using the microarray data. Gene sets with an FDR < 0.01 are shown on the left. Enrichment plots of indicated gene sets are shown on the right. E Western blot analysis of total and phosphorylated STAT1 in HSC-45 cells transfected with the indicated siRNAs. (F, G) qRT-PCR analysis of interferon genes in HSC-45 cells transfected with the indicated siRNAs ( F ) or in SNU638 cells stably transfected with the indicated genes ( G ). ( n = 3). H Western blot analysis of total and phosphorylated STAT1 in SNU638, SNU638-GFP, and SNU638-TM4SF1-AS1 cells. I qRT-PCR analysis of IRDS genes in SNU638-GFP and SNU638-TM4SF1-AS1 cells. ( n = 3). J Western blot analysis of STAT1 and Pur-α in SNU638-TM4SF1-AS1 cells transfected with the indicated siRNAs targeting Pur-α. K qRT-PCR analysis of IRDS genes in SNU638-TM4SF1-AS1 cells transfected with the indicated siRNAs. ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Transfection, Microarray, Western Blot, Stable Transfection

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Workflow of ChIRP coupled with mass spectrometry (ChIRP-MS) or RNA-seq (ChIRP-RNA-seq) to identify molecules associated with TM4SF1-AS1. B qRT-PCR confirming enrichment of TM4SF1-AS1 in ChIRP products derived from SNU638-TM4SF1-AS1 cells. ( n = 3). C Protein-protein interaction (PPI) network among the proteins identified by ChIRP-MS. Functional categories of the proteins are indicated by node colors. D RIP-qPCR assays validating the ChIRP-MS results. The indicated proteins in HSC-45 cells were immunoprecipitated, and co-precipitated TM4SF1-AS1 was detected by qRT-PCR. IgG served as a negative control. ( n = 3). E Immunofluorescence images showing staining of the SG marker G3PB2 in SNU638, SNU638-GFP, and SNU638-TM4SF1-AS1 cells treated with or without sodium arsenite (SA). Summarized results are shown on the right ( n = 5). Scale bars = 10 μm. F Immunofluorescence indicating G3BP2 (green), TIA1 (white), and inducible MS2-tagged TM4SF1-AS1 (red) in SNU638 cells. Cells were transfected with a MS2 coat protein (MCP)-RFP plasmid and incubated for 8 days with or without Dox. Magnified views of the respective markers are shown on the right. Scale bars = 10 μm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Mass Spectrometry, RNA Sequencing Assay, Quantitative RT-PCR, Derivative Assay, Functional Assay, Immunoprecipitation, Negative Control, Immunofluorescence, Staining, Marker, Transfection, Plasmid Preparation, Incubation

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Immunofluorescence indicating RACK1 (green), G3BP1 (white), and inducible MS2-tagged TM4SF1-AS1 (red) in SNU638 cells. The cells were transfected with a MS2 coat protein (MCP)-RFP plasmid and incubated for 8 days with or without Dox. Magnified views of the respective markers are shown on the right. Scale bars = 10 μm. B Immunofluorescence images of G3PB2 in HSC-45 cells incubated for 8 days with or without Dox and expressing the indicated inducible shRNAs. Representative images are shown on the left. Summarized results are on the right. ( n = 5). C , D Apoptosis ( C ) and cell cycle ( D ) assays in HSC-45 cells incubated with or without Dox for 8 days and expressing the indicated inducible shRNAs. Representative results are shown on the left. Summarized results are shown on the right. ( n = 3). E , F Western blot analysis of PARP, caspase-3 ( E ) and p38 ( F ) in HSC-45 cells incubated with or without Dox for 8 days and expressing the indicated inducible shRNAs. Cleaved PARP and cleaved caspase-3 are indicated by arrows ( E ). Phosphorylated p38 is indicated by an arrow ( F ). ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Incubation, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A , B Immunofluorescence images showing Pur-α (A) or YB-1 (B), TIA1 and inducible MS2-tagged TM4SF1-AS1 in SNU638 cells incubated for 8 days with or without Dox. Scale bars = 10 μm. C Immunofluorescent staining of G3PB2 in SNU638-TM4SF1-AS1 cells transfected with the indicated siRNAs. Representative results are shown on the left. Summarized results are shown on the right. ( n = 5). Scale bars = 10 μm. D Localization of GFP-tagged Pur-α or YB-1, G3BP1 and TIA1 immunofluorescence in SNU638 cells. Cells were transfected with vectors encoding GFP (upper), GFP-tagged Pur-α (middle) or GFP-tagged YB1 (bottom). Magnified views of the respective markers are shown on the right. Scale bars = 10 μm. E Cell cycle analysis of HSC-45 cells expressing the indicated siRNAs. ( n = 3). F , G Western blot analysis of PARP and caspase-3 ( F ) and total and phosphorylated p38 ( G ) in HSC-45 cells transfected with the indicated siRNAs. Cleaved PARP, cleaved caspase-3 and phosphorylated p38 are indicated by arrows. ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Immunofluorescence, Incubation, Staining, Transfection, Cell Cycle Assay, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: In the early steps of gastric tumorigenesis, TM4SF1-AS1 is transcriptionally activated. TM4SF1-AS1 sequesters RACK1 within SGs, which leads to suppression of stress-responsive MAPK signaling and inhibition of apoptosis (created with BioRender.com).

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Inhibition

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry

Journal: Stem Cells International

Article Title: Dot1l Regulates the Spontaneous Bone Regeneration of Periosteum-Derived Stem Cells by Regulating Chac1 Expression

doi: 10.1155/sci/1508850

Figure Lengend Snippet: Mandibular defect repair and periosteal PDSCs isolation: (A) animal experimental design, (B) surgical image of a rat model of jaw defect with periosteum preserved, (C) X-ray images of the defect area in rats at day 7 and 8 weeks later, (D) morphologic photographs of periosteum-derived stem cells in sham and model rats, (E) the proportions of CD44+, CD90+, CD31+, and CD45+ cells in PDSCs isolated from the jaws of sham-operated rats were analyzed using flow cytometry, and (F) the proportion of CD44+, CD90+, CD31+, and CD45+ cells in PDSCs isolated from jaws of model rats were analyzed using flow cytometry.

Article Snippet: For the identification of PDSCs cells, the cells were washed and incubated with CD44 antibody (15675-1-AP, Proteintech), CD90 antibody (66766-1-Ig, Proteintech), CD3 antibody (17617-1-AP, Proteintech), and CD45 antibody (20103-1-AP, Proteintech) to label PDSCs cells (CD44+CD90+CD3-CD45-).

Techniques: Isolation, Derivative Assay, Flow Cytometry

Journal: Stem Cells International

Article Title: Dot1l Regulates the Spontaneous Bone Regeneration of Periosteum-Derived Stem Cells by Regulating Chac1 Expression

doi: 10.1155/sci/1508850

Figure Lengend Snippet: Expression pattern of Dot1l during mandibular defect healing, and Dot1l is important for the osteogenesis of PDSCs. (A) The volcano plot showed the differential genes in PDSCs isolated from the sham operation group and the periosteum-preserved jaw defect model group. (B) GO functional enrichment analysis showing the top 30 significantly enriched biological functions and roles. (C) The mRNA expression level of differential genes was detected using RT-qPCR assay, data were analyzed by using one-way ANOVA, with the Tukey's post hoc test. ⁣ ∗∗∗∗ p < 0.001. (D) The protein expression level of Dot1l was detected using western blotting assay. (E) The proportion of CD44, CD90, CD31, and CD45 positive expression cells in PDSCs from 4-week-old SD rats was analyzed by flow cytometry. (F) ARS staining was used to detect calcium deposition in PDSCs cells, ALP staining was used to detect ALP activity of PDSCs cells, oil red O staining was used to detect the lipogenic ability of PDSCs cells. (G) Detection of the Dot1l mRNA in PDSCs treated with Dot1l ctrl/overexpression via RT-qPCR assay, data were analyzed by t -test analysis, ⁣ ∗∗∗ p < 0.001. (H) Detection of the Dot1l mRNA in PDSCs treated with Dot1l ctrl/overexpression via western blot assay. (I) Detection of the mRNA expressions of osteogenic differentiation related genes in PDSCs treated with Dot1l ctrl/overexpression via RT-qPCR assay, data were analyzed by using one-way ANOVA, with the Tukey's post hoc test. ⁣ ∗∗∗∗ p < 0.001. (J) ALP staining was used to detect ALP activity of PDSCs cells treated with Dot1l ctrl/overexpression. (K) ARS staining was used to detect calcium deposition of PDSCs cells treated with Dot1l ctrl/overexpression.

Article Snippet: For the identification of PDSCs cells, the cells were washed and incubated with CD44 antibody (15675-1-AP, Proteintech), CD90 antibody (66766-1-Ig, Proteintech), CD3 antibody (17617-1-AP, Proteintech), and CD45 antibody (20103-1-AP, Proteintech) to label PDSCs cells (CD44+CD90+CD3-CD45-).

Techniques: Expressing, Isolation, Functional Assay, Quantitative RT-PCR, Western Blot, Flow Cytometry, Staining, Activity Assay, Over Expression

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 4 ch5E6 treatment leads to a substantial decrease in the growth of organoids derived from PC patients or pancreatic and lung cancer genetically engineered mouse models. a PC patient and b KPC mouse organoid staining with ch5E6 indicating MUC16 expression (green) and specific binding of mAb compared to no binding with isotype control mAb huIgG1. The binding of ch5E6 to the ductal cells (yellow) was illustrated by colocalization with CK-19 (red) staining in human and mouse pancreatic tumor organoids. Scale bar 10 µm.The representative images for ch5E6 treated. c Human PC patient. d KPC and (e) KPA mouse-derived organoids obtained by real-time kinetics using Incucyte live cell imaging system. The data were quantitated for organoid counts using essence incucyte software, plotted as a graph of change in organoid counts or area over time (3–5 days) for both ch5E6 and huIgG1 treatments, and is shown in parallel. Error bars indicate SEM. *P < 0.05; **P < 0.01.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Derivative Assay, Staining, Expressing, Binding Assay, Control, Live Cell Imaging, Software

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 6 Inhibition of EMT by ch5E6 is validated in PC and NSCLC cell line-derived xenografts. a Immunofluorescence analysis showing a decrease in pFAK(Y397) and N-cadherin expression in xenograft tumors of SW1990 cells treated with ch5E6 compared to isotype control mAb huIgG1 group (n = 6–8 fields/tissue: three animals). The data was plotted for changes in fluorescence intensity using GraphPad Prism 9 and is shown in parallel. Nuclei were stained with DAPI. b Immunofluorescence analysis of ch5E6 treated SW1573 cell line-derived xenografts showing a reduction in pFAK(Y397) and N-cadherin levels compared to isotype control mAb huIgG1 group (n = 6–8 fields/tissue: 3 animals). Scale bar, 10 µm; magnified images, 2 µm. No significant changes in the intensity of MUC16 were seen in the ch5E6 treated versus isotype control tumors derived from both cancers. c, d Immunoblot analysis of ch5E6 treated PDAC and NSCLC tumor lysates showing a substantial decrease in phosphorylated levels of FAK(Y397), p70S6K(T389) and N-cadherin as compared to huIgG1 treatment. Error bars indicate SEM. Scale bar, 400 μm; magnified images, 100 μm; *P < 0.05; **P < 0.01.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Inhibition, Derivative Assay, Expressing, Control, Staining, Western Blot

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 7 MUC16 and N-cadherin are clinically correlated in patient tumors. a, b Representative images and quantitative illustration of immunohistochemical analyses demonstrate a strong positive correlation between MUC16 and N-cadherin (R = 0.84) in both primary PC tumors (n = 10) and liver metastasis (R = 0.99) samples (n = 8). Scale bar, 400 µm; magnified images, 100 µm. *P < 0.05; **P < 0.01. c Representative images of immunofluorescence analysis showing coexpression of MUC16 (green) and N-cadherin (red) in primary PDAC tumors compared to no MUC16 and N-cadherin in normal pancreatic sections. Scale bar, 20 µm; magnified images, 5 µm. d Schematic diagram representing ch5E6 induced downregulation of MUC16 mediated EMT resulting in its anti-tumor potential in PC and NSCLC. Overall, anti- MUC16 chimeric mAb5E6 (ch5E6) binds to the cell surface-tethered domain of MUC16, interferes with oncogenic pFAK/p70S6K/N-cadherin signaling associated with MUC16-mediated EMT, and reduces tumor burden in both PC and NSCLC models.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Immunohistochemical staining

Journal: bioRxiv

Article Title: Epstein–Barr Virus BALF0/1 Subverts the Caveolin and ERAD Pathways to Target B-cell Receptor Complexes for Degradation

doi: 10.1101/2024.01.04.574276

Figure Lengend Snippet: BALF0/1 mediated BCR degradation requires the Ig heavy chain cytoplasmic tail (A) Immunoblot analysis of WCL from Cas9+ P3HR-1 cells expressing the indicated CD79A targeting sgRNA, 4-HT induced into lytic cycle for 24 h. (B) Immunoblot analysis of WCL from Cas9+ P3HR-1 cells expressing the indicated CD79B sgRNA, 4-HT induced into lytic cycle for 24 h. (C) Immunoblot analysis of WCL from Cas9+ P3HR-1 cells expressing the indicated CD79A and CD79B sgRNA, 4-HT induced into lytic cycle for 24 h. (D) Mean ±SEM percentage of cells with PM IgM signals as in Figure S6 of P3HR-1 cells expressing the indicated control or dual CD79A/CD79B sgRNA, uninduced or 4-HT induced, as indicated, using data from 12 randomly selected panels of 240 cells from n=3 replicates, analyzed using the ImageJ ComDet plugin. (E) Immunofluorescence analysis of CD79A, CD79B, IgM and calnexin in P3HR-1 cells expressing control sgRNA and 4-HT induced for lytic replication in the presence of bortezomib for 24h. (F) Fluorescence intensity line scanning for calnexin (magenta) and IgM (green) signals from the white rectangle region (E). (G) Immunofluorescence analysis of IgM, calnexin, CD79A and CD79B in Cas9+ P3HR-1 cells expressing CD79A and CD79B sgRNA. Cells were 4-HT induced for lytic replication and treated with bortezomib for 24 h. (H) Fluorescence intensity line scanning for calnexin (magenta) and IgM (green) signals from the white rectangle region in (G). (I) Mean ±SEM percentage of cells with overlapping calnexin and IgM signals as in panel E, G and Figure S6 of P3HR-1 cells expressing the indicated control or dual CD79A/CD79B sgRNA, uninduced or 4-HT induced in the absence or presence of bortezomib as indicated, using data from 12 randomly selected panels of 240 cells from n=3 replicates, analyzed using the ImageJ ComDet plugin. (J) Schematic model of the BCR complex immunoglobulin heavy (IgH) chain, light chain and the CD79A and CD79B signaling chains. The C’ IgM cytoplasmic tail residues are shown. (K) Immunoblot analysis of WCL from 293T cells transiently expressing wildtype (WT) or cytoplasmic tail deleted (ΔKVK) EGFP-tagged IgM heavy chain alone or together with either BXLF1 or BALF0/1. (L) Immunoblot analysis of WCL from 293T transiently expressing the IgA heavy chain, alone or together with BALF0/1 or BXLF1. Statistical analysis was performed with Student’s t-test unless otherwise specified. ns p > 0.05. White bars indicate scale. See also Figure S 6. Blots are representative of at least n=2 replicates.

Article Snippet: Antibodies used for immunoblot analysis in this study were: anti-Human IgM goat polyclonal antibody (Southern Biotech #2020-01), anti-Human IgG goat polyclonal antibody (Southern Biotech #2040-01), anti-HA-Tag (C29F4) rabbit mAb (Cell Signaling #3724), anti-GAPDH (D16H11) XP® rabbit mAb (Cell Signaling #5174), anti-CD79A rabbit polyclonal antibody (Proteintech #22349-1-AP), anti-CD79B (D7V2F) rabbit mAb (Cell Signaling #96024), anti-EBV BALF0/1 rabbit mAb (generated by Genscript for this study), anti-EBV ZEBRA Mouse mAb (BZ1) (Santa Cruz# sc-53904), anti-EBV Ea-D mouse mAb (1108– ) (Santa Cruz #sc-69679), anti-EBV p18 goat polyclonal antibody (Invitrogen #PA1-73003), anti-calnexin rabbit mAb (Cell Signaling #2433), anti-HRD1/SYVN1 rabbit polyclonal antibody (Proteintech #13473-1-AP), anti-BAP31 rabbit polyclonal antibody (Proteintech #11200-1-AP), anti-caveolin 1 mouse mAb (7C8) (Thermo Fisher #MA3-600), anti-GFP tag rabbit polyclonal antibody (Proteintech #50430-2-AP), goat anti-rabbit IgG, HRP-linked antibody (Cell Signaling #7074), goat anti-mouse IgG, HRP-linked antibody (Cell Signaling #7076) and bovine anti-goat IgG (H+L) HRP-linked antibody (Jackson ImmunoResearch Laboratory #805-035-180).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Fluorescence

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 3 Lipo-MP-LPS induces immunological changes in both primary tumours and lung metastases. a, c Representative immunohis- tochemistry images of xenograft tumours and lung metastases stained with mouse specific anti-CD8a antibodies. Scale bar = 50 μm. b, d Quantification of CD8a positive cells in xenograft tumours and lung metastases. Data are represented as means ± SD; *p < 0.05, using Mann–Whitney U test. e, g Representative immunohistochemistry images of xenograft tumours and lung metastases stained with mouse specific anti-F4/80 antibodies. Scale bar = 50 μm. f, h Quantification of F4/80 positive cells in xenograft tumours and lung tissues. Data are represented as means ± SD; n = 6 mice per group, *p < 0.05, using Mann–Whitney U test. (i) iNOS, MHC II, TNF-α, CD206, and IL10 gene expression in xenograft tumours and lung metastases determined by RT-qPCR. Data are presented as means ± SD; n = 6 mice per group, *p < 0.05, **p < 0.01, using two-tailed t-test. NS not significant.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

Techniques: Staining, MANN-WHITNEY, Immunohistochemistry, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 4 The tumour suppressive effect of Lipo-MP-LPS depends on CD8+ T cells. a Representative images of IHC staining for CD8a positive cells of xenograft tumours from mice treated with anti-CD8a antibody or IgG2b isotype. Scale bar = 100 μm. b CD8a expression in spleen tissues determined by RT-qPCR. Data are represented as means ± SD; n = 4. ***p < 0.001, using two-tailed t-test. c Tumour growth curve in C3H/HeN mice treated with empty liposome or Lipo-MP-LPS in addition to injection with anti-CD8a or isotype IgG2b antibodies (n = 6 mice per group). Data are shown as mean tumour volume ± SD. *p < 0.05, using Mann–Whitney U test. d Kaplan–Meier survival curves. p < 0.05 was considered significant using the log-rank test. Representative images of H&E staining of xenograft tumours (e) and lungs (g) for each group. Scale bar = 200 μm. f Quantification of necrotic areas in tumour tissues. h Quantification of lung metastasis areas. Data are presented as means ± SD; *p < 0.05, **p < 0.01; NS not significant, using Mann–Whitney U test.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Two Tailed Test, Injection, MANN-WHITNEY, Staining

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 6 Survival analysis of patients with osteosarcoma according to immune cell infiltration levels estimated by consensus TME. Of the 84 patients with osteosarcoma, the top third (28 patients) and bottom third (28 patients) were classified into high and low score groups, respectively. Kaplan–Meier curves of (a) CD8+ T cells (b) macrophage, (c) M1 macrophage, and (d) M2 macrophage infiltration for OS (left) and PFS (right) are presented. p < 0.05 was considered significant using the log-rank test.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

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