Journal: Cell Reports Medicine

Article Title: HOXC6 drives a therapeutically targetable pancreatic cancer growth and metastasis pathway by regulating MSK1 and PPP2R2B

doi: 10.1016/j.xcrm.2023.101285

Figure Lengend Snippet:

Article Snippet: Tissue microarray slide (PDAC and matched normal pancreas tissue) , US Biomax, Inc. , Cat# PA601.

Techniques: Western Blot, Immunohistochemistry, Immunoprecipitation, Microarray, Recombinant, Transfection, Plasmid Preparation, Membrane, Staining, Flow Cytometry, Viability Assay, Chromatin Immunoprecipitation, shRNA, Sequencing, Binding Assay, Software, Expressing

Journal: Cell Death and Differentiation

Article Title: Targeting ubiquitin-specific protease 8 sensitizes anti-programmed death-ligand 1 immunotherapy of pancreatic cancer

doi: 10.1038/s41418-022-01102-z

Figure Lengend Snippet: a Photographs of tumors and growth curves ( c ) in immunocompetent mice. The tumors were measured at the indicated time points before being excised at the end of the experiment ( n = 7). b Photographs of tumors and growth curves ( d ) in immunodeficient mice. The tumors were measured at the indicated time points before being excised at the end of the experiment ( n = 7). e , f Tumors weights in the immunocompetent and immunodeficient mice were recorded at the end of the experiment ( n = 7). g , h Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs; n = 7). i , j IHC staining and quantification of USP8 expression, PD-L1 expression, and TILs ( n = 7). Scale bars = 250 μm. k Protocols of the pretreatment of pancreatic cancer cells (1 × 10 5 ) with DMSO or the USP8-specific inhibitor (1 μM, 24 h). The treated cells were injected subcutaneously into immunodeficient and immunocompetent mice ( n = 10). l , m The incidence of tumors in the immunodeficient and immunocompetent mice at the indicated times. n Protocol for the separate and orthotopic injection of parental and Usp8 -depleted pancreatic cancer cells (5 × 10 5 ) into immunodeficient and immunocompetent mice ( n = 10). o , p Survival of Usp8 -depleted pancreatic tumor-bearing immunocompetent and immunodeficient mice ( n = 10). The data in ( l and m ) were generated using Kaplan-Meier survival curves based on log-rank tests. The data in ( o and p ) were generated using the Gehan–Breslow–Wilcoxon test and the Kaplan-Meier method. The results are shown the means ± SD of representative experiments in ( c – h , and j ). The data represent three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 assessed via a two-tailed t test; ns: not significant.

Article Snippet: Microarray slides of PDAC tissue were immunostained using antibodies against USP8 (27791-1-AP, Proteintech) and CD274 (ab205921, Abcam).

Techniques: Flow Cytometry, Immunohistochemistry, Expressing, Injection, Generated, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Targeting ubiquitin-specific protease 8 sensitizes anti-programmed death-ligand 1 immunotherapy of pancreatic cancer

doi: 10.1038/s41418-022-01102-z

Figure Lengend Snippet: a Bioinformatic analysis of the correlation between USP8 and CD274 mRNA expression in pancreatic cancer samples from the TCGA database ( n = 179). b–d Photographs and statistical analyses of USP8 and PD-L1 in tumor tissue microarrays using IHC ( n = 156). e–g KPC, BxPC-3, and SW1990 pancreatic cancer cell lines were analyzed separately by western blotting with the indicated antibodies. The images are representative of three independent experiments. h USP8-His and GST-PD-L1 proteins were subjected to GST-pull down assays. The images are representative of three independent experiments. i Photographs of immunofluorescence staining showing the interaction of USP8 and PD-L1 in KPC cells and fluorescence intensity plots ( j ) showing the co-localization of USP8 and PD-L1. The images are representative of three independent experiments. k Photographs of immunofluorescence staining showing the interaction of USP8 and PD-L1 in KPC mice pancreatic tumor tissues and fluorescence intensity plots ( l ) showing the co-localization of USP8 and PD-L1. The images are representative of three independent experiments. In ( d ), Spearman correlations and p values calculated using Spearman’s test are shown.

Article Snippet: Microarray slides of PDAC tissue were immunostained using antibodies against USP8 (27791-1-AP, Proteintech) and CD274 (ab205921, Abcam).

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

Journal: Cell Death and Differentiation

Article Title: Targeting ubiquitin-specific protease 8 sensitizes anti-programmed death-ligand 1 immunotherapy of pancreatic cancer

doi: 10.1038/s41418-022-01102-z

Figure Lengend Snippet: a–c Levels of PD-L1in pancreatic cancer cell lines (KPC, BxPC-3) treated with a concentration gradient and time gradient of the USP8 inhibitor and after Usp8 knockdown, as assessed using western blotting. The image is representative of three independently performed experiments. d , e Statistical analysis of the levels of PD-L1 in pancreatic cancer cell lines treated with the USP8 inhibitor (1 μM, 24 h) and Usp8 KD and subjected to flow cytometry. A representative image of three independent experiments is shown. f Photographs of immunofluorescence staining showing USP8 and PD-L1 expression in parental and Usp8 -depleted KPC cells, and the statistical analysis of the results ( g ). The image is representative of three independently performed experiments. h–k Statistical analysis of USP8 and PD-L1 levels in tumor samples, as assessed using flow cytometry and western blotting analyses. l Statistical analyses of the qRT-PCR results showing PDL1 mRNA levels in pancreatic cancer cell lines treated with the USP8 inhibitor (1 μM, 24 h) and Usp8 knockdown. A representative image of three independent experiments is shown. m The level of PD-L1 after USP8 inhibitor (1 μM, 24 h) treatment in KPC cells treated with MG132 (5 μM, 12 h), as assessed using western blotting. A representative image of three independent experiments is shown. n The level of PD-L1 in MG132 (5 μM, 12 h)-treated parental and Usp8 KD KPC cells, as assessed using western blotting. A representative image of three independent experiments is shown. o Analysis of PD-L1 stability in cycloheximide (CHX) (200 μg/mL) pretreated KPC cells incubated with the USP8 inhibitor (1 μM, 24 h). A representative image of three independent experiments is shown. p Analysis of PD-L1 stability in cycloheximide (CHX) (200 μg/mL) treated parental and Usp8 KD KPC cells. A representative image of three independent experiments is shown. q , r , A densitometer was used to quantify the intensity of PD-L1 protein expression and the results are representative of three independent experiments. s Assay of PD-L1 ubiquitination in KPC cells. The cells were treated with MG132 (5 μM, 12 h) followed by USP8 inhibitor (1 μM, 24 h) treatment and then western blotting was used to detect PD-L1 and ubiquitin. t Assay of PD-L1 ubiquitination in parental and Usp8 KD KPC cells. Cells were treated with MG132 (5 μM, 12 h) then western blotting was used to detect PD-L1 and ubiquitin. Results are shown as the means ± SD of representative experiments in ( d , e , g , i , k , l , q and r ). The data represent three independent experiments. * p < 0.05, ** p < 0.01, ** p < 0.001 assessed via a two-tailed t test; ns: not significant.

Article Snippet: Microarray slides of PDAC tissue were immunostained using antibodies against USP8 (27791-1-AP, Proteintech) and CD274 (ab205921, Abcam).

Techniques: Concentration Assay, Western Blot, Flow Cytometry, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Incubation, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Targeting ubiquitin-specific protease 8 sensitizes anti-programmed death-ligand 1 immunotherapy of pancreatic cancer

doi: 10.1038/s41418-022-01102-z

Figure Lengend Snippet: a Schematic of the protocol of USP8 deficiency and αPD-L1 combination therapy for orthotopic KPC parental and Usp8 KD cell (5 × 10 5 )-bearing mice. b Photographs of tumors removed from the mice of each group ( n = 5). c The statistical plot of tumor weights of the four groups ( n = 5). d Flow cytometry of PD-L1 levels in an orthotopic tumor model, statistical results ( e ) are shown. f Flow cytometry of MHC-1 levels in an orthotopic tumor model, statistical results ( g ) are shown. h , i Flow cytometry of CD3 + T cells, CD8 + T cells, IFN-γ + CD8 + T cells, TNF-α + CD8 + T cells in the tumor region and the statistical analysis of the results ( n = 5). j , k Representative images of IHC staining and quantification of TILs ( n = 5). Scale bars = 250 μm. The results are displayed as the means ± SD from representative experiments in ( c , e , g , i and k ). The data represent three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 assessed via a two-tailed t test; ns not significant.

Article Snippet: Microarray slides of PDAC tissue were immunostained using antibodies against USP8 (27791-1-AP, Proteintech) and CD274 (ab205921, Abcam).

Techniques: Flow Cytometry, Immunohistochemistry, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Targeting ubiquitin-specific protease 8 sensitizes anti-programmed death-ligand 1 immunotherapy of pancreatic cancer

doi: 10.1038/s41418-022-01102-z

Figure Lengend Snippet: a Schematic diagram of the protocol for USP8 inhibitor and αPD-L1 combination therapy for orthotopic KPC-Luci cell (5 × 10 5 )-bearing mice. b Photographs of tumors removed from mice treated with the USP8 inhibitor, αPD-L1, or their combination ( n = 5). c The statistical graph of tumor weights of the four groups ( n = 5). d Representative images displaying changes in the luminescence intensity of tumors obtained using In Vivo Imaging and the relative luminescence intensity change ( e ) on the last day. f Flow cytometry of PD-L1 levels in an orthotopic tumor model, statistical results ( g ) are shown. h Flow cytometry of MHC-1 levels in an orthotopic tumor model, statistical results ( i ) are shown. j, k Tumor-infiltrating lymphocytes (TILs) assessed using flow cytometry and the statistical analysis of the results ( n = 5). l , m Representative images of IHC staining and quantification of TILs ( n = 5). Scale bars = 250 μm. The results are shown as the means ± SD from representative experiments in ( c , e , g , i , k and m ). The data represent three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 assessed via a two-tailed t test; ns not significant.

Article Snippet: Microarray slides of PDAC tissue were immunostained using antibodies against USP8 (27791-1-AP, Proteintech) and CD274 (ab205921, Abcam).

Techniques: In Vivo Imaging, Flow Cytometry, Immunohistochemistry, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Targeting ubiquitin-specific protease 8 sensitizes anti-programmed death-ligand 1 immunotherapy of pancreatic cancer

doi: 10.1038/s41418-022-01102-z

Figure Lengend Snippet: a , b Western blotting and flow cytometry results validating Cd274 KO in KPC cell line. c Schematic of the protocol for USP8 inhibitor and αPD-L1 combination therapy for orthotopic KPC parental and Cd274 KO cell (5 ×10 5 )-bearing mice. d Photographs of tumors removed from mice of each group ( n = 5). e The statistical plot of tumor weights of the four groups ( n = 5). f The body weight of mice on the last day ( n = 5). g Experimental design for CD8 + T cells depletion in orthotopic KPC (5 × 105)-bearing mice receiving the combination therapy. h Photographs of tumors removed from the mice in each group ( n = 5). i The statistical plot of the tumor weights of the four groups ( n = 5). j Changes in mouse body weight ( n = 5). k , l Flow cytometry of CD8 + T cells of spleens and the statistical analysis of the results ( n = 5). m The model shows the regulation of PD-L1 stability by USP8 in pancreatic cancer. USP8 inhibitor treatment downregulates PD-L1 protein levels via degradation, leading to activation of the cytotoxic T-cells. The results are displayed as the means ± SD from representative experiments in ( e , f , i, j and l ). The data represent three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 assessed via a two-tailed t test; ns not significant.

Article Snippet: Microarray slides of PDAC tissue were immunostained using antibodies against USP8 (27791-1-AP, Proteintech) and CD274 (ab205921, Abcam).

Techniques: Western Blot, Flow Cytometry, Activation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: NEK2 inhibition triggers anti-pancreatic cancer immunity by targeting PD-L1

doi: 10.1038/s41467-021-24769-3

Figure Lengend Snippet: a – c Expression profile of NEK2 in pancreatic cancer. Relative NEK2 expression in pancreatic cancer was analyzed using large-scale RNA-Seq datasets of PDAC from the TCGA database ( n (T) = 179, n (N) = 171; n : number of patients) ( a ). NEK2 expression was measured in paired tumor and normal pancreatic tissues by IHC staining, with representative images ( b ) and statistical results ( c ) shown ( n = 30; n : number of patients) (N: Normal pancreatic tissue; T: Pancreatic tumor tissue). Scale bars: 100 μm. d Correlation of NEK2 with prognostic factors in pancreatic cancer. Tissue microarray analysis of the prognostic role of NEK2 in pancreatic cancer ( n = 64 weakly positive; n = 50 strongly positive) ( p < 0.0001). e Overall survival (OS) of patients with pancreatic cancer with high or low expression of NEK2 ( n = 177). f Overall survival (OS) of pancreatic cancer patients with high numbers of CD8 + T cells and with high or low expression of NEK2 ( n = 76). g Overall survival (OS) of pancreatic cancer patients with decreased CD8 + T cells and with high or low expression of NEK2 ( n = 101). h Bioinformatics analysis of the correlation between NEK2 and immune effector cells using TCGA datasets. In a , data are represented as boxplots where the middle line is the median; the lower and upper hinges correspond to the first and third quartiles; the upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge (where IQR is the inter-quartile range); the lower whisker extends from the hinge to the smallest value at most 1.5 × IQR of the hinge, while data beyond the end of the whiskers are outlying points that are plotted individually. * P < 0.05, ** P < 0.01, *** P < 0.001 using a two-tailed t -test; ns: not significant. Kaplan–Meier method and a Gehan–Breslow–Wilcoxon test are indicated in d . The Hazard Ratios (HR) and p -values by the log-rank (Mantel–Cox) test are indicated in e – g .

Article Snippet: Immunohistochemical staining of paraffin-embedded PDAC tissue microarray slides was performed by Wuhan Servicebio Technology, with antibodies against NEK2 (24171-1-AP, Proteintech Group) and PD-L1 (ab205921, Abcam).

Techniques: Expressing, RNA Sequencing, Immunohistochemistry, Microarray, Whisker Assay, Two Tailed Test

Journal: Nature Communications

Article Title: NEK2 inhibition triggers anti-pancreatic cancer immunity by targeting PD-L1

doi: 10.1038/s41467-021-24769-3

Figure Lengend Snippet: Clinicopathological relevance of NEK2 in PDAC patients.

Article Snippet: Immunohistochemical staining of paraffin-embedded PDAC tissue microarray slides was performed by Wuhan Servicebio Technology, with antibodies against NEK2 (24171-1-AP, Proteintech Group) and PD-L1 (ab205921, Abcam).

Techniques:

Journal: Nature Communications

Article Title: NEK2 inhibition triggers anti-pancreatic cancer immunity by targeting PD-L1

doi: 10.1038/s41467-021-24769-3

Figure Lengend Snippet: a , c Schematic protocols displaying WT and NEK2-depleted pancreatic cancer cells separately, and s.c. injection into immunocompetent and immunodeficient mice ( n = 7). b , e Representative images of tumors and growth curves of immunocompetent mice. Tumors were measured at specified time points then dissected at the endpoint ( n = 7). d , f Representative images of tumors and growth curves of tumors from immunodeficient mice. Tumors were measured at specified time points and dissected at the endpoint ( n = 7). g , h The weight of tumors from immunocompetent and immunodeficient mice was reported at the endpoint ( n = 7). i , j Representative images and statistical results of tumor-infiltrating lymphocytes ( n = 7). k Schematic protocols showing WT and NEK2-depleted pancreatic cancer cells separately injected into immunocompetent and immunodeficient mice ( n = 13). l , m Survival of immunocompetent and immunodeficient mice bearing NEK2-depleted pancreatic cancer cells ( n = 13). Kaplan–Meier survival curves with log-rank test assessing the significance between WT and NEK2 KD immunocompetent ( l ) ( p < 0.0001) and immunodeficient ( m ) ( p = 0.33) mice. Results represent means ± SD of one representative experiment in e – j . All data are representative of three independently performed experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 using a two-tailed t -test; ns: not significant. Kaplan–Meier method and a Gehan–Breslow–Wilcoxon test are indicated in m and l .

Article Snippet: Immunohistochemical staining of paraffin-embedded PDAC tissue microarray slides was performed by Wuhan Servicebio Technology, with antibodies against NEK2 (24171-1-AP, Proteintech Group) and PD-L1 (ab205921, Abcam).

Techniques: Injection, Two Tailed Test

Journal: Nature Communications

Article Title: NEK2 inhibition triggers anti-pancreatic cancer immunity by targeting PD-L1

doi: 10.1038/s41467-021-24769-3

Figure Lengend Snippet: a , c Schematic protocols of pancreatic cancer cells with or without pretreatment with a kinase-specific inhibitor of NEK2 (10 μM, 24 h) separately and s.c. injected into immunocompetent and immunodeficient mice ( n = 7). b , d Tumor incidence in immunocompetent and immunodeficient mice was recorded at the times indicated. Tumors were further treated using an NEK2 inhibitor (100 μg/mouse, 2 weeks). e , f Schematic protocol of the treatment schedule and tumor growth curve in immunocompetent mice ( n = 5). Tumors were measured at the time points indicated and removed at the endpoint. ( h , i ) Schematic protocol of the treatment schedule and tumor growth curve in immunodeficient mice ( n = 5). Tumors were measured at specified time points and dissected at the endpoint. Tumor and mouse weight of immunocompetent ( g , k ) ( n = 5) and immunodeficient mice ( j , l ) ( n = 5) as reported at the endpoint. m – o Representative images and further quantification of tumor-infiltrating lymphocytes. Scale bars: 100×: 50 μm; 200×: 100 μm. Kaplan–Meier method and a Gehan–Breslow–Wilcoxon test are indicated in b and d . Results represent means ± SD of one representative experiment in f , g , i , j , k , l , m , and n . All data are representative of three independently performed experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 using a two-tailed t -test; ns: not significant.

Article Snippet: Immunohistochemical staining of paraffin-embedded PDAC tissue microarray slides was performed by Wuhan Servicebio Technology, with antibodies against NEK2 (24171-1-AP, Proteintech Group) and PD-L1 (ab205921, Abcam).

Techniques: Injection, Two Tailed Test

Journal: Nature Communications

Article Title: NEK2 inhibition triggers anti-pancreatic cancer immunity by targeting PD-L1

doi: 10.1038/s41467-021-24769-3

Figure Lengend Snippet: a Western blot analysis of NEK2 and PD-L1 in clinical pancreatic tissue samples from patients ( n = 20) (N: Normal pancreatic tissue; T: Pancreatic tumor tissue). b – d Representative images and statistical results of IHC staining of NEK2 and PD-L1 in a tissue microarray ( n = 156). e – g Cell lysates from SW1990, KPC, and CFPAC-1 separately analyzed by IP and Western blotting using the antibodies indicated. Representative image is shown n = 3 independent experiments. h GST-pull down assay of NEK2-His and GST-PD-L1 protein. Representative image is shown n = 3 independent experiments. i Representative images of individual immunofluorescence staining of NEK2 and PD-L1 interaction in KPC cells by Duolink assay. The red dots (NEK2/PD-L1 interaction) indicate their interaction. Representative image is shown n = 3 independent experiments. The Spearman correlations and p -values by Spearman’s test are indicated in d .

Article Snippet: Immunohistochemical staining of paraffin-embedded PDAC tissue microarray slides was performed by Wuhan Servicebio Technology, with antibodies against NEK2 (24171-1-AP, Proteintech Group) and PD-L1 (ab205921, Abcam).

Techniques: Western Blot, Immunohistochemistry, Microarray, Pull Down Assay, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: NEK2 inhibition triggers anti-pancreatic cancer immunity by targeting PD-L1

doi: 10.1038/s41467-021-24769-3

Figure Lengend Snippet: a , b Western blot analysis and flow cytometry of PD-L1 expression in pancreatic cancer cell lines after treatment with NEK2 inhibitor (10 μM, 24 h) and NEK2 knockdown. Representative image is shown n = 3 independent experiments. c LC–MS proteomics quantitative analysis of pancreatic cancer cells overexpressing NEK2 . d Western blot analysis of PD-L1 expression in KPC cells treated with NEK2 inhibitor (10 μM, 24 h) after treatment with MG132 (100 μM, 24 h). Representative image is shown n = 3 independent experiments. e Western blot analysis of PD-L1 expression in WT and NEK2 KD KPC cells treated with MG132 (100 μM, 24 h). Representative image is shown n = 3 independent experiments. f Stability analysis of PD-L1 in KPC cells treated with NEK2 inhibitor (10 μM, 24 h) after treatment with cycloheximide (CHX) (20 μg/mL). Representative image is shown n = 3 independent experiments. g Stability analysis of PD-L1 in WT and NEK2 KD KPC cells treated with CHX (20 μg/mL). Representative image is shown n = 3 independent experiments. h , i Statistical analysis of three independent experiments is displayed. The intensity of PD-L1 protein expression was quantified using a densitometer. j Ubiquitination assay of PD-L1 in KPC cells treated with NEK2 inhibitor (10 μM, 24 h), subjected to anti-PD-L1 IP and anti-ubiquitin Western blot analysis after treatment with MG132 (50 μM, 24 h). k Ubiquitination assay of PD-L1 in WT and NEK2 KD KPC cells treated with MG132. Results represent means ± SD of one representative experiment in h and i . All data are representative of three independently performed experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 using a two-tailed t-test; ns: not significant.

Article Snippet: Immunohistochemical staining of paraffin-embedded PDAC tissue microarray slides was performed by Wuhan Servicebio Technology, with antibodies against NEK2 (24171-1-AP, Proteintech Group) and PD-L1 (ab205921, Abcam).

Techniques: Western Blot, Flow Cytometry, Expressing, Knockdown, Liquid Chromatography with Mass Spectroscopy, Ubiquitin Proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: NEK2 inhibition triggers anti-pancreatic cancer immunity by targeting PD-L1

doi: 10.1038/s41467-021-24769-3

Figure Lengend Snippet: a Schematic diagram of the NEK-binding motif (F/LXXS/T) in the glycosylation-rich region and amino acid sequences around the potential binding sites of PD-L1 were aligned in evolutionarily divergent species. The F/LXXS/T motifs are highlighted in blue. b Generation of site-specific antibodies against T194 and T210 (Mus musculus: T193 and T209)-phosphorylated PD-L1. c Western blot analysis of T193 and T209-phosphorylated PD-L1 in KPC cells with NEK2 inhibitor (10 μM, 24 h) and NEK2 KD KPC. Representative image is shown n = 3 independent experiments. d In vitro kinase assay and western blot analysis of pT193-PD-L1 and pT209-PD-L1 expression of recombinant PD-L1 WT and NEK2 (active) protein. Representative image is shown, n = 3 independent experiments. e , f Western blot analysis of PD-L1 expression in flag-PD-L1 WT and T193/209A or T193/209D-transfected KPC cells with or without MG132 treatment. Representative image is shown n = 3 independent experiments. g Western blot analysis of pT193-PD-L1, pT209-PD-L1, and PD-L1 expression in WT and K37R transfected NEK2 KD KPC cells. Representative image is shown n = 3 independent experiments. h Ubiquitination assay of PD-L1 in Flag-PD-L1 WT and T193/209A or T193/209D-transfected KPC cells, subjected to anti-PD-L1 IP and anti-ubiquitin Western blot analysis after treatment with MG132 (50 μM, 24 h). Representative image is shown n = 3 independent experiments. i Ubiquitination assay of PD-L1 in WT and K37R transfected NEK2 KD-KPC cells subjected to PD-L1 IP and Western blot analysis with anti-ubiquitin after treatment with MG132 (50 μM, 24 h). Representative image is shown n = 3 independent experiments.

Article Snippet: Immunohistochemical staining of paraffin-embedded PDAC tissue microarray slides was performed by Wuhan Servicebio Technology, with antibodies against NEK2 (24171-1-AP, Proteintech Group) and PD-L1 (ab205921, Abcam).

Techniques: Binding Assay, Glycoproteomics, Western Blot, In Vitro, Kinase Assay, Expressing, Recombinant, Transfection, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: NEK2 inhibition triggers anti-pancreatic cancer immunity by targeting PD-L1

doi: 10.1038/s41467-021-24769-3

Figure Lengend Snippet: a Schematic protocol of the combination of anti-PD-L1 antibody and NEK2 inhibitor therapy. b Tumor growth curve of mice treated with anti-PD-L1 antibody (200 μg/mouse), NEK2 inhibitor (100 μg/mouse), or their combination ( n = 5). c Representative images displaying tumors harvested from mice bearing KPC cells treated with anti-PD-L1 antibody, NEK2 inhibitor, or their combination ( n = 5). d , e Tumor weight and mouse body weight ( n = 5). f , g Flow cytometric analysis and statistical results of lymphocytes that have infiltrated the tumors ( n = 5). Results are presented as means ± SD from one representative experiment in b , d , e , and g . All data are representative of three independently performed experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 using a two-tailed t -test; ns: not significant.

Article Snippet: Immunohistochemical staining of paraffin-embedded PDAC tissue microarray slides was performed by Wuhan Servicebio Technology, with antibodies against NEK2 (24171-1-AP, Proteintech Group) and PD-L1 (ab205921, Abcam).

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: NEK2 inhibition triggers anti-pancreatic cancer immunity by targeting PD-L1

doi: 10.1038/s41467-021-24769-3

Figure Lengend Snippet: A schematic model is proposed to illustrate how PD-L1 protein stability is regulated by NEK2 in pancreatic cancer. NEK2 positively regulates and interacts with PD-L1 largely through PD-L1 phosphorylation at the T194/T210 residue in ER of pancreatic cancer. Therefore, treatment with NEK2 inhibitor unexpectedly suppressed PD-L1 protein expression, largely by inhibition of PD-L1 phosphorylation that promotes its degradation.

Article Snippet: Immunohistochemical staining of paraffin-embedded PDAC tissue microarray slides was performed by Wuhan Servicebio Technology, with antibodies against NEK2 (24171-1-AP, Proteintech Group) and PD-L1 (ab205921, Abcam).

Techniques: Phospho-proteomics, Residue, Expressing, Inhibition