Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Rational design and construction of the Anti-MICB-CAR. ( A ) Schematic of the Anti-MICB chimeric antigen receptor (Anti-MICB-CAR). ( B ) The plasmid structure of the Anti-MICB chimeric antigen receptor lentiviral vector. ( C ) The expression of Anti-MICB-CAR in NK cells was analyzed via flow cytometry. ( D ) The secretion of IL-15 by Anti-MICB-CAR-NK cells was detected by ELISA. NC: supernatant of 1 × 10 6 NK cells, Anti-MICB–CAR–NK: supernatant of 1 × 10 6 Anti-MICB-CAR-NK cells. Supernatants of Anti-MICB-CAR-NK cells were significantly elevated up to 48.88 pg/mL compared to the control NK cell supernatants. p = 0.0024. ( E ) The supernatant of 1 × 10 6 Anti-MICB-CAR-NK cells was filtered and incubated with PANC-1 for 1 h. MICB Protein hFc and IgG1 Fc FITC antibody staining were performed and free Anti-MICB-scFv was detected by flow cytometry. Statistical analysis was performed using a two-tailed Student’s t -test; ** p < 0.01.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Plasmid Preparation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, Incubation, Staining, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Anti-MICB-CAR-NK cells demonstrate potent in vitro activity against tumors with high expression of MICB. ( A ) Western blot detection of MICB expression in PANC-1, A549, HepG2, BxPC-3, and AsPC-1 human tumor cell lines. ( B ) The qPCR results showed in the MICB gene expression compared to the five tumor cell lines. ( C ) The Effect-to-Target Ratio (E/T Ratio) of Anti-MICB-CAR-NK cells on tumor cell PANC-1. ( D ) The mortality rate of five types of tumor cells treated with Anti-MICB-CAR-NK cells after 24 h with CCK-8 Assay, PANC-1(71.37%), A549(31.78%), HepG2(53.83%), BxPC-3(62.26%), and AsPC-1(39.86%), E/T Ratio 1:1. ( E ) Anti-MICB-CAR-NK treatment of AsPC-1 and PANC–1 tumor cells with differential MICB expression for 24 h by flow cytometry, E/T Ratio 1:1. The results showed that the viability of AsPC-1 was 68.9% and the viability of PANC-1 was 54.6%. ( F – H ) PANC-1, HepG2, and A549 tumor cells treated with NK cells, Anti-MICB-CAR-NK cells, and Anti-MICB-CAR-NK supernatant + NK cells for 24 h, E/T Ratio 1:1; CCK8 detects mortality rate. Anti-MICB-CAR-NK cells significantly enhanced the anti-tumor ability compared to NK cells. PANC-1: 68.37%, p = 0.0005. HepG2: 57.3%, p = 0.0027. A549: 33.18%, p = 0.0342. Supernatant of Anti-MICB-CAR-NK cells co-treated tumor cells with NK cells also promoted tumor cell killing compared to NK. PANC-1: 57.66%, p = 0.0006. HepG2: 48.21%, p = 0.0037. A549: 31.55%, p = 0.0446. ( I ) NC-NK cells, non-transduced NK(NT-NK), NT-NK supernatant + NK cells, Anti-MICB-CAR-NK supernatant + NK cells, Anti-MICB-CAR-NK cells treated with PANC-1 tumor cells for 24 h, E/T Ratio 1:1. The viability of each group was detected by flow cytometry and the results showed that NK cells: 89.4%, non-transduced NK(NT-NK): 86.6%, NT-NK supernatant + NK cells: 88.7%, Anti-MICB-CAR-NK supernatant + NK cells: 62.6%, and Anti-MICB-CAR-NK cells: 55.5%. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, *** p < 0.001.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: In Vitro, Activity Assay, Expressing, Western Blot, Gene Expression, CCK-8 Assay, Flow Cytometry, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: The anti-tumor mechanism induced by Anti-MICB-CAR-NK cells. ( A – D ) The release of perforin, granzyme B, TNF-α, and IFN-γ from PANC-1, HepG2, and A549 cells. NK cells and Anti-MICB-CAR-NK cells were treated with tumor cells for 24 h, with an E/T ratio of 1:1. Control represents complete medium cultivation. The supernatant was collected and detected by ELISA. Compared to the NK group, the Anti-MICB-CAR-NK group exhibited significantly higher levels of TNF-α expression (PANC-1, p = 0.0072; AsPC-1, p = 0.035; HepG2, p = 0.0076; A549, p = 0.0066) and IFN-γ expression (PANC-1, p = 0.0072; AsPC-1, p = 0.0162; HepG2, p = 0.002; A549, p = 0.0006) following treatment with PANC-1, AsPC-1, HepG2, and A549 tumor cells. Similarly, Granzyme B expression levels were significantly elevated in the Anti-MICB-CAR-NK group (PANC-1, p = 0.0007; HepG2, p = 0.0023; A549, p = 0.0004), as were Perforin expression levels (PANC-1, p = 0.0021; HepG2, p = 0.042; A549, p = 0.0022) after treatment with PANC-1, HepG2, and A549 tumor cells. Additionally, the Anti-MICB-CAR-NK group demonstrated significantly increased IL-15 expression following treatment with PANC-1 and A549 tumor cells (PANC-1, p = 0.0012; A549, p = 0.0025) compared to the NK group. ( E , F ) Elisa and flow cytometry were used to detect the levels of IL-15 and Anti-MICB-scFv in the tumor cells’ supernatants treated with Anti-MICB-CAR-NK cells for 24 h. ( G ) Study on MICB expression in PANC-1 treated with Anti-MICB-CAR-NK cells for 24 h using Western blot technique. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Western Blot, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Anti-MICB-CAR-NK cells exhibit tumor regression in the PANC-1 Xenograft Model. ( A ) Flow chart of Anti-MICB-CAR-NK cells treatment for PANC-1 Transplanted Tumors. On the 0th day, PANC-1 cells were inoculated and on the 13th day, PBS, NK cells, and Anti-MICB-CAR-NK cells were injected into mice via tail vein at a dose of 5 × 10 6 cells per mouse. Record tumor growth observed every other day. ( B ) Growth curve of mouse transplanted tumor. ( C , D ) Resected tumors from each group were imaged and Weighed at the end of the experiment. NC: 834.31 ± 197.26 (mg), NK: 606.69 ± 141.37 (mg), Anti-MICB-CAR-NK: 447.32 ± 136.13 (mg). ( E ) Anti-tumor rate of Anti-MICB-CAR-NK cells against PANC-1 transplanted tumors. The tumor inhibition rate of NK compared to NC was 27.28%, p = 0.07. The tumor inhibition rate of Anti-MICB-CAR-NK compared to NC was 46.38%, p = 0.007. ( F ) Mouse serum was collected at the end of the experiment and the amounts of IL-15, IFN-γ, and TNF-α were measured by ELISA. The IL-15, IFN—γ, and TNF—α in the Anti-MICB-CAR-NK group were significantly increased compared to the NK group, with a p -value of 0.0013 for IL-15, 0.013 for IFN—γ, and 0.003 for TNF—α. ( G , H ) Mouse tumor tissues were analyzed by immunohistochemistry for MICB expression and NK cell infiltration within the tumor tissue. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, ** p < 0.01.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Injection, Inhibition, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Expressing, Two Tailed Test

Journal:

Article Title: ATP transduces signals from ASGM1, a glycolipid that functions as a bacterial receptor

doi: 10.1073/pnas.161290898

Figure Lengend Snippet: Ligation of flagellin receptor activates the MAP kinase cascade, which is required for mucin induction. (A) HM3 cells grown in six-well plates were lysed with SDS sample buffer 5 min after the addition of agonist antibody, α-ASGM1. Equal amounts of lysate were run on a 12% SDS gel, transferred to nitrocellulose, immunoblotted with a phospho-specific Erk 1/2 antibody, and visualized by chemiluminescence. Blots were stripped and reprobed with Erk 1/2 antibody to show equal loading. P-Erk Cntrl, positive control from vendor. (B) Effect of MEK 1/2 inhibitor PD98059 (37 μM, 30 min) and dominant negative mutant MEK K97R on MUC 2 promoter activity. (c) Inhibition of Erk phosphorylation by the calcium chelator, BAPTA/AM (30 μM, 45 min). Fold increase expressed as the ratio of phosphorylated Erk to baseline Erk 1/2 as determined by densitometry.

Article Snippet: Phosphospecific anti-Erk 1/2 (mouse monoclonal) was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Ligation, SDS-Gel, Positive Control, Dominant Negative Mutation, Activity Assay, Inhibition

Journal:

Article Title: ATP transduces signals from ASGM1, a glycolipid that functions as a bacterial receptor

doi: 10.1073/pnas.161290898

Figure Lengend Snippet: Cartoon depicting events in flagellin-triggered host cell signaling. Flagellin binds to asialoGM1, a membrane glycolipid, which causes the extracellular release of ATP, which then binds to a nucleotide receptor. Downstream events include G-protein activation, the cleavage of PIP2 by PLC, the formation of IP3, Ca2+ mobilization, the phosphorylation of MEK 1/2 and Erk 1/2 [via an unknown calcium binding protein (CBP)], and mucin (MUC 2) transcription. Flagellin-induced signaling bifurcates after Ca2+ mobilization, with ≈50% of the response giving rise to downstream events that are Erk dependent whereas the remaining 50% is Erk independent.

Article Snippet: Phosphospecific anti-Erk 1/2 (mouse monoclonal) was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Binding Assay

Journal:

Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

doi: 10.1128/MCB.22.22.7929-7941.2002

Figure Lengend Snippet: GILZ overexpression inhibits ERK-1/2, MEK-1/2, and Raf-1 but not JNK phosphorylation. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 5 or 60 min with plastic-bound anti-CD3 MAb. Whole-cell lysates were probed with an antibody specific for phosphorylated ERK-1/2 (pERK-1/2) (A), MEK-1/2 (pMEK) (B), or Raf-1 (pRaf-1) (C). Western blots were also performed with anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibodies to verify that no modulation of protein expression occurred or with β-tubulin to verify that an equivalent amount of proteins was loaded in each lane. PV6 or GIRL-19 was stimulated for the times indicated with plastic-bound anti-CD3 MAb. (D) Whole-cell lysates were probed with an antibody recognizing both phosphorylated forms of JNK: p54 and p46 (pSAPK/JNK). C, untreated cells.

Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

Techniques: Over Expression, Clone Assay, Transfection, Western Blot, Expressing

Journal:

Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

doi: 10.1128/MCB.22.22.7929-7941.2002

Figure Lengend Snippet: GILZ interacts with endogenous Raf-1 in mouse thymocytes. Mouse thymocytes were treated for 6 h with DEX (100 nM), and cell lysates were incubated with GST or GST-GILZ beads. Binding of Raf-1 (A), MEK-1/2 (B), and ERK-1/2 (C) was visualized by Western blotting. Whole-cell lysates from thymocytes left untreated or treated with DEX were immunoprecipitated with an anti-Raf-1 or control isotype antibody (4 μg/500 μg of protein). (D and E) Nitrocellulose membrane was probed with an anti-GILZ antiserum (D) and then stripped and reprobed with anti-Raf-1 antibody (E).

Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

Techniques: Incubation, Binding Assay, Western Blot, Immunoprecipitation

Journal:

Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

doi: 10.1128/MCB.22.22.7929-7941.2002

Figure Lengend Snippet: DEX inhibits Raf-1, MEK, and ERK-1/2 phosphorylation. Mouse thymocytes left untreated or pretreated for 6 h with DEX (100 nM) were stimulated for 1 h with plastic-bound anti-CD3 MAb. Western blotting was performed with an anti-pERK-1/2 (A), anti-pMEK-1/2 (B), anti-pRaf-1 (C), or anti-GILZ (D) antibody. Western blotting was also performed with an anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibody to verify that no modulation of protein expression occurred or with β-tubulin to verify that equivalent amounts of proteins were loaded in all lanes.

Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

Techniques: Western Blot, Expressing