Journal: Scientific Reports

Article Title: Inhibition of TGF-β signalling in combination with nal-IRI plus 5-Fluorouracil/Leucovorin suppresses invasion and prolongs survival in pancreatic tumour mouse models

doi: 10.1038/s41598-020-59893-5

Figure Lengend Snippet: Survival improvement in response to combination treatment of vactosertib with nal-IRI/5-FU/LV in the orthotopic pancreatic cancer mouse model. ( A ) Experimental design of C57BL/6 syngeneic orthotopic mouse model using Panc02 murine pancreatic cancer cells. Five days after cell injection (3 × 10 6 ), mice were randomized into 4 groups. Vactosertib was administered orally for 5 consecutive days followed by 2 days of resting period starting from day 6 post cell injection. nal-IRI/5-FU/LV was injected intraperitoneally every 2 weeks starting from day 8 post cell injection. ( B ) Relative survival rates of the control, vactosertib, nal-IRI/5-FU/LV, and combined treatment groups (***P < 0.0005 and **P < 0.005 compared to the control group) ( C ) Haematoxylin and eosin (H&E) staining of pancreatic tumour tissues showing tumour cell invasion to adjacent pancreas tissues. Black dotted lines indicate the borders between tumourous and normal pancreas without invading tumour cells. ( D ) The morphologies of tumour cells presented by H&E staining. Yellow arrows point out the representative mesenchymal (control and nal-IRI/5-FU/LV) and epithelial (vactosertib and Vac + nal-IRI/5-FU/LV) cell morphologies in the images. ( E ) The immunohistochemical staining of vimentin in tumour tissues of each group.

Article Snippet: Six-week-old male immunocompetent C57BL/6 mice were purchased from Japan SLC, Inc. We injected 3 × 10 6 Panc02 cells with a 1:1 ratio of Matrigel (Corning, Cat. No. 356231) into the tail of pancreas.

Techniques: Injection, Control, Staining, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Inhibition of TGF-β signalling in combination with nal-IRI plus 5-Fluorouracil/Leucovorin suppresses invasion and prolongs survival in pancreatic tumour mouse models

doi: 10.1038/s41598-020-59893-5

Figure Lengend Snippet: Synergistic effect of vactosertib with nal-IRI/5-FU on migration, invasion, and EMT of pancreatic cancer cells. ( A ) Transwell cell migration assay. Cells were treated with the indicated reagent(s) and incubated for 48 hours before placed in a migration chamber. 1 × 10 5 of PANC-1 (left) or 5 × 10 4 of Panc02 (right) cells were then seeded in migration chambers for 48 hours. The relative number of migrated cells in each group was counted. ( B ) Cell invasion assay. Cells were treated with the indicated reagent(s) and incubated for 48 hours before placed in an invasion chamber. 2 × 10 5 PANC-1 (left) or 1 × 10 5 Panc02 (right) cells were then seeded in invasion chambers for 48 hours. The number of invaded cells in each group was counted. Note that the combination of vactosertib with nal-IRI/5-FU inhibits migration and invasion in PANC-1 and Panc02 pancreatic cancer cells. ( A,B ) The values for migrated/invaded number of cells represent the mean ± SD of triplicate data. ***P < 0.0005, **P < 0.005, and *P < 0.05 compared to the control group; ## P < 0.005 and # P < 0.05 compared to the nal-IRI-treated group. ( C ) Western blot analysis for EMT markers. Vactosertib was treated for 2 hours prior to incubation with nal-IRI/5-FU in the presence of 5 ng/ml TGF-β for 48 hours. The blots are cropped and the full-length images are presented in Supplementary Fig. . Note that vactosertib and its combination with nal-IRI/5-FU restore the TGF-β-mediated reduction of E-cadherin and the increase of mesenchymal markers.

Article Snippet: Six-week-old male immunocompetent C57BL/6 mice were purchased from Japan SLC, Inc. We injected 3 × 10 6 Panc02 cells with a 1:1 ratio of Matrigel (Corning, Cat. No. 356231) into the tail of pancreas.

Techniques: Migration, Cell Migration Assay, Incubation, Invasion Assay, Control, Western Blot

Journal: Scientific Reports

Article Title: Inhibition of TGF-β signalling in combination with nal-IRI plus 5-Fluorouracil/Leucovorin suppresses invasion and prolongs survival in pancreatic tumour mouse models

doi: 10.1038/s41598-020-59893-5

Figure Lengend Snippet: Up-regulation of CCDC80 expression by vactosertib and its combination with nal-IRI/5-FU/LV. ( A ) FPKM value of Ccdc80 in the tumour tissues from RNA sequencing. ( B ) qRT-PCR result validating Ccdc80 mRNA expression in mouse tumour tissues. The values represent the mean ± SD of triplicate samples. **P < 0.005 compared to the control group. ###P < 0.0005 compared to the nal-IRI/5-FU/LV group. ( C ) Immunohistochemical staining of mouse tumour tissues with CCDC80. Note that CCDC80 expression is increased in the tumour tissues from the mice administered with vactosertib or its combination with nal-IRI/5-FU/LV. ( D ) RT-PCR analysis revealing up-regulation of CCDC80 by vactosertib and its combination with nal-IRI/5-FU in PANC-1 and Panc02 cell lines.

Article Snippet: Six-week-old male immunocompetent C57BL/6 mice were purchased from Japan SLC, Inc. We injected 3 × 10 6 Panc02 cells with a 1:1 ratio of Matrigel (Corning, Cat. No. 356231) into the tail of pancreas.

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Control, Immunohistochemical staining, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Inhibition of TGF-β signalling in combination with nal-IRI plus 5-Fluorouracil/Leucovorin suppresses invasion and prolongs survival in pancreatic tumour mouse models

doi: 10.1038/s41598-020-59893-5

Figure Lengend Snippet: Ectopic expression of CCDC80 reducing migration, colony formation, and EMT in pancreatic cancer cells. ( A ) Transwell migration assay measuring migration abilities of PANC-1 and Panc02 cells expressing LPCX or CCDC80. ( B ) Representative images of colonies stained with methylene blue in PANC-1 and Panc02 cells stably expressing CCDC80. ( C ) qRT-PCR results showing down-regulation of EMT marker expression in PANC-1 and Panc02 cells by ectopic expression of CCDC80. All the data is represented as the mean of three repeated values. ***P < 0.0005, **P < 0.005, and *P < 0.05 compared to the control. ( D ) Western blot analysis showing reduction of EMT markers in PANC-1 and Panc02 with stably expressing CCDC80. The blots are cropped, and the original blots are presented in Supplementary Fig. . Note that overexpression of CCDC80 decreases EMT marker expression in pancreatic cancer cells.

Article Snippet: Six-week-old male immunocompetent C57BL/6 mice were purchased from Japan SLC, Inc. We injected 3 × 10 6 Panc02 cells with a 1:1 ratio of Matrigel (Corning, Cat. No. 356231) into the tail of pancreas.

Techniques: Expressing, Migration, Transwell Migration Assay, Staining, Stable Transfection, Quantitative RT-PCR, Marker, Control, Western Blot, Over Expression

Journal: EMBO Molecular Medicine

Article Title: Loss of EHMT2 enhances NK cell-driven anti-tumor immunity through TGF-β1 suppression

doi: 10.1038/s44321-025-00357-6

Figure Lengend Snippet: ( A ) Indicated TNBC cells were treated with DMSO, UNC0642 (1 μM), and A366 (1 μM) for 48 h and were analyzed for NK cell-mediated cytotoxicity using LDH-based cytotoxicity. Relative NK cell-mediated cytotoxicity under indicated conditions. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( B ) Indicated TNBC cells were treated with DMSO, A366 (1 μM), or UNC0642 (1 μM) for 48 h and analyzed for the indicated proteins by immunoblotting. ACTINB was used as a loading control. ( C ) Indicated PDAC cells were treated with DMSO, UNC0642 (1 μM), A366 (1 μM) for 48 h and analyzed for NK cell-mediated cytotoxicity using LDH-based cytotoxicity. Relative NK cell-mediated cytotoxicity under indicated conditions. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( D ) Indicated PDAC cells were treated with DMSO, A366 (1 μM) or UNC0642 (1 μM) for 48 h and analyzed for the indicated proteins by immunoblotting. ACTINB was used as a loading control. ( E ) Firefly luciferase ( F-Luc )-labeled EMT6 cells expressing either nonspecific (NS) shRNA or Ehmt2 shRNA were orthotopically injected into the mammary fat pad of female BALB/c mice ( n = 5). Bioluminescence images of mice at the indicated weeks after injection are shown. ( F ) Bioluminescence intensities of the mice at the indicated weeks from panel ( E ). ( n = 5). P values were calculated using unpaired two-tailed Student’s t -test. ( G ) Measurement of tumor-infiltrated NK cells (%) in the EMT6- F-Luc tumors under the indicated conditions using FACS analysis. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( H ) F-Luc -labeled EMT6 cells expressing either NS shRNA or Ehmt2 shRNA were orthotopically injected into the mammary fat pads of female NSG mice ( n = 5). Bioluminescence images of mice at the indicated weeks after injections are shown. ( I ) Bioluminescence intensities of the mice at the indicated weeks from panel ( H ). ( n = 5). P values were calculated using unpaired two-tailed Student’s t -test. ( J ) Panc02 cells expressing either NS shRNA or Ehmt2 shRNA were injected subcutaneously into C57BL/6 mice. Tumor volumes at the indicated times. ( n = 5). For the analysis of tumor progression in mice, the statistical assessment was performed using the area under the curve method, followed by unpaired two-tailed Student’s t -tests. ( K ) Tumor images for the experiment presented in panel ( J ) under the indicated conditions. ( L ) Measurement of tumor-infiltrated NK cells (%) in the Panc02 tumors under the indicated conditions using FACS analysis. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( M ) Panc02 cells expressing either NS shRNA or Ehmt2 shRNA were injected subcutaneously into the flank of NSG mice. Average tumor volumes at the indicated times. ( n = 5). For the analysis of tumor progression in mice, the statistical assessment was performed using the area under the curve method, followed by unpaired two-tailed Student’s t -tests. ( N ) Tumor images for the experiment presented in panel ( M ) under the indicated conditions. All quantitative data were shown as the mean ± SEM. .

Article Snippet: Panc02 cells (5 × 10 6 ) were injected subcutaneously into 5–6-week-old male C57BL/6 mice (stock no. 000664, Jackson Laboratory) or 5–6-week-old male NSG mice (stock no. 005557, Jackson Laboratory).

Techniques: Two Tailed Test, Western Blot, Control, Luciferase, Labeling, Expressing, shRNA, Injection

Journal: EMBO Molecular Medicine

Article Title: Loss of EHMT2 enhances NK cell-driven anti-tumor immunity through TGF-β1 suppression

doi: 10.1038/s44321-025-00357-6

Figure Lengend Snippet: ( A ) Panc02 cells expressing either nonspecific (NS) shRNA or Ehmt2 shRNA were analyzed for mRNA expression for Ehmt2 mRNA were analyzed using RT-qPCR analysis. Relative mRNA expression is plotted. Actinb was used for normalization. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( B ) Panc02 cells expressing either NS shRNA or Ehmt2 shRNA were analyzed for the expression of the indicated proteins by immunoblotting. Histone H3 and ACTINB were used as loading controls. ( C ) Panc02 cells expressing NS shRNA or Ehmt2 shRNAs were injected subcutaneously into C57BL/6 mice. Measurement of tumor-infiltered CD8 + T-cells (%) in the Panc02 tumors under indicated conditions using FACS analysis and plotted. ( n = 3). P values were calculated using unpaired two-tailed Student’s t-test. ( D ) Panc02 cells expressing NS shRNA or Ehmt2 shRNAs were injected subcutaneously into C57BL/6 mice. Measurement of tumor-infiltered CD4 + T-cells cells (%) in the Panc02 tumors under indicated conditions using FACS analysis and plotted. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( E ) Ki-67 expression was analyzed by immunohistochemistry (IHC) in Panc02 tumor sections expressing NS shRNA and Ehmt2 shRNA. Representative Ki-67 staining images of Panc02 tumor sections expressing NS shRNA and Ehmt2 shRNA at 20× magnifications are shown. Scale bar, 50 μm. ( F ) Quantitation of Ki-67 staining for the experiment presented in panel ( E ) and plotted. ( n = 3). P values were calculated using unpaired two-tailed Student’s t -test. ( G ) Panc02 cells expressing NS shRNA or Ehmt2 shRNAs were injected subcutaneously into C57BL/6 mice. Measurement of tumor-infiltrated NK cells (Lin – CD49a – CD49b⁺), and ILC1 (Lin – CD49a⁺CD49b – ) in the Panc02 tumors under the indicated conditions using FACS analysis and plotted. ( n = 5). P values were calculated using unpaired two-tailed Student’s t -test. ( H ) Panc02 cells expressing either NS shRNA or Ehmt2 shRNA were injected subcutaneously into C57BL/6 mice. Tumor volumes at the indicated times are plotted. ( n = 5). For the analysis of tumor progression in mice, the statistical assessment was performed using the area under the curve method, followed by unpaired two-tailed Student’s t -tests. ( I ) Measurement of tumor-infiltrated NK cells (%) in the Panc02 tumors under the indicated conditions using FACS analysis and plotted. ( n = 5). P values were calculated using unpaired two-tailed Student’s t -test. All quantitative data were shown as the mean ± SEM.

Article Snippet: Panc02 cells (5 × 10 6 ) were injected subcutaneously into 5–6-week-old male C57BL/6 mice (stock no. 000664, Jackson Laboratory) or 5–6-week-old male NSG mice (stock no. 005557, Jackson Laboratory).

Techniques: Expressing, shRNA, Quantitative RT-PCR, Two Tailed Test, Western Blot, Injection, Immunohistochemistry, Staining, Quantitation Assay

Journal: EMBO Molecular Medicine

Article Title: Loss of EHMT2 enhances NK cell-driven anti-tumor immunity through TGF-β1 suppression

doi: 10.1038/s44321-025-00357-6

Figure Lengend Snippet: ( A ) Panc02 cells expressing either Non-specific (NS) shRNA or Ehmt2 shRNA were injected subcutaneously into C57BL/6 mice and treated with either IgG isotype control or anti-NK1.1 antibodies. Average tumor volumes at the indicated time points. ( n = 6). For the analysis of tumor progression in mice, the statistical assessment was performed using the area under the curve method, followed by unpaired two-tailed Student’s t -tests. ( B ) Tumor images for the experiment presented in panel ( A ) is shown under the indicated conditions. ( C ) Measurement of NK cells (%) in spleens from mice ( n = 6) treated with IgG isotype control or anti-NK1.1 antibodies expressing either NS shRNA or Ehmt2 shRNA using FACS analysis. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( D ) Measurement of tumor-infiltrated NK cells (%) in Panc02 tumors treated with IgG isotype control or anti-NK1.1 antibodies expressing either NS shRNA or Ehmt2 shRNA using FACS analysis. [( n = 5 for NS shRNA + IgG and n = 6 for NS shRNA + anti-NK1.1, Ehmt2 shRNA + IgG, and Ehmt2 shRNA + anti-NK1.1)]. P values were calculated using unpaired two-tailed Student’s t -test. ( E ) Rag2 KO mice were injected with Panc02 cells expressing either NS shRNA or Ehmt2 shRNA and were treated with either control IgG antibody or anti-NK1.1 antibody for NK cell depletion. Tumor volumes were measured at the indicated weeks. ( n = 6). For the analysis of tumor progression in mice, the statistical assessment was performed using the area under the curve method, followed by unpaired two-tailed Student’s t -tests. ( F ) Representative tumor images for the experiment shown in panel ( E ) under the indicated conditions. ( G ) Measurement of NK cells (%) in spleens from mice treated with IgG isotype control or anti-NK1.1 antibodies expressing either NS shRNA or Ehmt2 shRNA using FACS analysis. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. All quantitative data were shown as the mean ± SEM. .

Article Snippet: Panc02 cells (5 × 10 6 ) were injected subcutaneously into 5–6-week-old male C57BL/6 mice (stock no. 000664, Jackson Laboratory) or 5–6-week-old male NSG mice (stock no. 005557, Jackson Laboratory).

Techniques: Expressing, shRNA, Injection, Control, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Loss of EHMT2 enhances NK cell-driven anti-tumor immunity through TGF-β1 suppression

doi: 10.1038/s44321-025-00357-6

Figure Lengend Snippet: ( A ) C57BL/6 mice were injected with Panc02 cells and treated with vehicle or BRD4770. Tumor volumes were measured at the indicated weeks. Average tumor volumes are plotted at the indicated time points. ( n = 6). For the analysis of tumor progression in mice, the statistical assessment was performed using the area under the curve method, followed by unpaired two-tailed Student’s t -tests. ( B ) Representative tumor images under the indicated conditions for the experiment shown in panel ( A ) under indicated conditions. ( C ) Measurement of tumor-infiltrated NK cells (%) in Panc02 tumors treated with either vehicle or BRD4770 using FACS analysis and are plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( D ) Panc02 cells were treated with BRD4770 (5 μM) for 48 h. Indicated proteins were analyzed using immunoblotting. ACTINB was used as a loading control. ( E ) C57BL/6 mice were injected with Panc02 cells and treated with vehicle or vactosertib. Tumor volumes were measured at the indicated weeks. Average tumor volumes are plotted at the indicated weeks. ( n = 6). For the analysis of tumor progression in mice, the statistical assessment was performed using the area under the curve method, followed by unpaired two-tailed Student’s t -tests. ( F ) Representative tumor images for the experiment shown in panel ( E ) under the indicated conditions. ( G ) Measurement of tumor-infiltrated NK cells (%) in Panc02 tumors treated with either vehicle or vactosertib and are plotted. ( n = 6). P values were calculated using unpaired two-tailed Student’s t -test. ( H ) NSG mice were injected with Panc02 cells and treated with vehicle or BRD4770. Tumor volumes were measured at the indicated weeks. Average tumor volumes are plotted at the indicated time points. ( n = 5). For the analysis of tumor progression in mice, the statistical assessment was performed using the area under the curve method, followed by unpaired two-tailed Student’s t -tests. ( I ) Representative tumor images for the experiment shown in panel ( H ) under the indicated conditions. ( J ) NSG mice were injected with Panc02 cells and treated with vehicle or vactosertib. Tumor volumes were measured at the indicated weeks. Average tumor volumes are plotted at the indicated time points. ( n = 5). For the analysis of tumor progression in mice, the statistical assessment was performed using the area under the curve method following by unpaired two-tailed Student’s t -tests. ( K ) Representative tumor images for the experiment shown in panel ( J ) under the indicated conditions. ( L ) Model showing the function and mechanism of EHMT2-driven suppression of NK cell-mediated anti-tumor immunity and nodes that can be therapeutically targeted for enhancing NK cell-mediated tumor clearance. All quantitative data are shown as the mean ± SEM. .

Article Snippet: Panc02 cells (5 × 10 6 ) were injected subcutaneously into 5–6-week-old male C57BL/6 mice (stock no. 000664, Jackson Laboratory) or 5–6-week-old male NSG mice (stock no. 005557, Jackson Laboratory).

Techniques: Injection, Two Tailed Test, Western Blot, Control

Journal: Applied Biological Chemistry

Article Title: Homologous tumor cell membrane vesicles active preferential self-recognition of tumor cells in vitro

doi: 10.1186/s13765-022-00672-3

Figure Lengend Snippet: Fig. 2 Cytotoxicity of tumor membrane-derived vesicles (EV@Panc02, EV@4T1 and EV@ B16-F10) to the Panc02 (a), 4T1 (b), B16-F10 (c) and PC12 cells (d)

Article Snippet: B16-F10, 4T1 and Panc02 cells were obtained from Anhui Medical University.

Techniques: Membrane, Derivative Assay

Journal: Applied Biological Chemistry

Article Title: Homologous tumor cell membrane vesicles active preferential self-recognition of tumor cells in vitro

doi: 10.1186/s13765-022-00672-3

Figure Lengend Snippet: Fig. 3 Cellular uptake of tumor TCMVs (EV@Panc02, EV@4T1 and EV@ B16F10). The percentage of positive cell (a) and geometric mean fluorescence intensity (b) were analyzed by the flow cytometry. The internalized vesicles into the Panc02 cells were observed by the laser confocal (c) and fluorescence quantify (d) with the software

Article Snippet: B16-F10, 4T1 and Panc02 cells were obtained from Anhui Medical University.

Techniques: Fluorescence, Flow Cytometry, Software

Journal: Applied Biological Chemistry

Article Title: Homologous tumor cell membrane vesicles active preferential self-recognition of tumor cells in vitro

doi: 10.1186/s13765-022-00672-3

Figure Lengend Snippet: Fig. 4 Cellular uptake of tumor TCMVs (EV@Panc02, EV@4T1 and EV@ B16F10). The geometric mean fluorescence intensity (a) and percentage of positive cell (b) were analyzed by the flow cytometry. The internalized vesicles into the 4T1 cells were observed by the laser confocal (c) and fluorescence quantify (d) with the software

Article Snippet: B16-F10, 4T1 and Panc02 cells were obtained from Anhui Medical University.

Techniques: Fluorescence, Flow Cytometry, Software

Journal: Applied Biological Chemistry

Article Title: Homologous tumor cell membrane vesicles active preferential self-recognition of tumor cells in vitro

doi: 10.1186/s13765-022-00672-3

Figure Lengend Snippet: Fig. 5 Cellular uptake of tumor TCMVs (EV@Panc02, EV@4T1 and EV@ B16F10). The geometric mean fluorescence intensity (a) and percentage of positive cell (b) were analyzed by the flow cytometry. The internalized vesicles into the B16F10 cells were observed by the laser confocal (c) and fluorescence quantify (d) with the software

Article Snippet: B16-F10, 4T1 and Panc02 cells were obtained from Anhui Medical University.

Techniques: Fluorescence, Flow Cytometry, Software