Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A, B March5 cKO mice are resistant to P. aeruginosa infection (A) survival rates ( n = 8) and (B) changes in the body weight of March5 fl/fl and March5 fl/fl;Lyz‐Cre ( n = 8) mice after intraperitoneal injection with 1 × 10 7 CFU Pseudomonas aeruginosa . C–F TNF‐α (C), IL‐6 (D), IL‐1β (E) and IL‐18 (F) from serum, spleen homogenate and peritoneal fluid collected from mice ( n = 8) that were sacrificed 12 and 24 h following the bacterial infection. Each cytokine was analyzed by ELISA. Data information: Values, * P < 0.05, ** P < 0.01 (two‐tailed Student's t ‐test or Mantel–Cox test). Data were expressed as the mean ± SEM. See also Fig . Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Infection, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A, B (A) Survival rates ( n = 12–14) and (B) variation of body weight of March5 fl/fl and March5 fl/fl;Lyz‐Cre ( n = 7–9) mice after intraperitoneal injection with 28 mg/kg body weight of LPS. C–F ELISA of a TNF‐α (C), IL‐6 (D), IL‐1β (E) and IL‐18 (F) from serum, spleen homogenate and peritoneal fluid from mice ( n = 5), sacrificed 12 and 24 h after LPS injection. Values, * P < 0.05 (two‐tailed Student's t ‐test or Mantel–Cox test.). Data were expressed as the mean ± SEM. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

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

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A–C (A) Caspase‐1, (B) IL‐1β secretion and (C) LDH release were measured in the supernatants of March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs subjected to the indicated stimuli followed by material method. Values are the mean ± SD. Experiments (A–C) were performed in triplicate and repeated at least three times. *** P < 0.001 (two‐tailed Student's t ‐test) D–F MARCH5 mediates activation of the NLRP3 pathway in response to bacterial infection. March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs were infected with (D) Citrobacter rodentium (20 MOI, 40 MOI and 80 MOI), (E) Salmonella typhimurium (1 MOI, 5 MOI and 10 MOI), and (F) Pseudomonas aeruginosa (1 MOI, 10 MOI and 20 MOI). Secretion of IL‐1β, IL‐18, IL‐6 and TNF‐α in BMDMs infected for 12 h was measured by ELISA. Values, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two‐tailed Student's t‐ test). Data are expressed as the mean ± SEM. Experiments (D–F) were performed in triplicate and repeated three times. See also Fig . Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Two Tailed Test, Activation Assay, Infection, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A–C (A) Activated caspase‐1, (B) IL‐1β, and (C) LDH release were measured in the supernatants of siControl (siCtrl) or siMARCH5 THP‐1 cells and were subjected to the indicated stimuli. Independent experiments were repeated at least three times. Values are the mean ± SD. ** P < 0.01, *** P < 0.001 (two‐tailed student's t ‐test). D–G March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs were infected with (D) Citrobacter rodentium (20 MOI, 40 MOI and 80 MOI), (E) Salmonella typhimurium (1 MOI, 5 MOI and 10 MOI), and (F) Psedumonas aeruginosa (1 MOI, 10 MOI and 20 MOI). Secretions of caspase‐1 in BMDMs infected for 12 h were measured (D–F), and the cell pellet was used for western blotting to detect the activation of NLRP3 inflammasome (G) in response to bacterial infection. Values, *** P < 0.001, **** P < 0.0001 (two‐tailed Student's t ‐test). Data were expressed as the mean ± SEM. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Two Tailed Test, Infection, Western Blot, Activation Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A MARCH5 WT and KO HEK293T cells were transfected with FLAG‐NLRP3 and HA‐ub. After the cells were stimulated with LPS (200 ng/ml for 4 h) and nigericin (15 μM for 60 min), the cell lysates were immunoprecipitated with FLAG‐M2 beads. Ubiquitinated NLRP3 was detected by anti‐HA antibody. B FLAG‐NLRP3 and HA‐ub K48R mutant plasmids were transfected into MARCH5 KO HEK293T cells with or without Myc‐MARCH5 2KR. Then, the cells were stimulated with 200 ng/ml LPS followed by 15 μM nigericin for the indicated durations. The cell lysates were immunoprecipitated with FLAG‐M2 beads and immunoblotted by using indicated antibodies. C MARCH5 KO HEK293T cells were transfected with FLAG‐NLRP3, Myc‐MARCH5 2KR, and each HA ‐ ubiquitin, shown by the number of the remaining single Lys residue with the other Lys changed to Arg. For stimulation, the cells were treated with 200 ng/ml LPS for 4 h, followed by 15 μM nigericin for 30–60 min. Cell lysates were immunoprecipitated with FLAG‐M2 beads and analyzed by immunoblotting with the indicated antibodies. D The NLRP3 inflammasome was reconstituted in HEK293T MARCH5 KO cells expressing ASC, pro‐caspase‐1, and IL‐1β with HA‐ub WT, K27 only (K27O) mutant, or K27R mutant. Additionally, cells were cotransfected with or without Myc‐MARCH5 2KR or the Myc‐MARCH5 H43W mutant. Following stimulation with LPS for 4 h and nigericin for 30 min, IL‐1β secretion was quantitated by ELISA. Values are the mean ± SD. ** P < 0.01, *** P < 0.001 (two‐tailed Student's t ‐test). All the experiments were carried out in triplicate three times. E HEK293T MARCH5 K/O cells were transfected with FLAG‐NLRP3, Myc‐MARCH5 (2KR), HA‐ubiquitin and GFP‐YOD1. Transfected cells were stimulated with 200 ng/ml LPS for 4 h, followed by 15 μM nigericin for 60 min. Cell lysates were immunoprecipitated with FLAG‐M2 beads and analyzed by ubiquitination with HA‐antibody. The other proteins were detected by indicated antibodies. F Schematic representation of NLRP3 Lys mutants (Upper). HEK293T MARCH5 KO cells were cotransfected with FLAG‐NLRP3 WT or indicated Lys point mutants, HA‐ubiquitin, and Myc‐MARCH5 (2KR). Cells were stimulated with 200 ng/ml LPS for 4 h followed by 15 μM nigericin treatment for 30 min. The cell lysates were immunoprecipitated with FLAG‐M2 beads. NLRP3 ubiquitination was assessed via western blotting using anti‐HA. And each protein was detected by indicated antibodies (Lower). G FLAG‐NLRP3 WT or indicated Lys point mutants, HA‐ub K27 only mutant and Myc‐MARCH5 2KR were transfected into MARCH5 KO HEK293T cells. Cell lysates were immunoprecipitated with FLAG‐M2 beads. Ubiquitinated NLRP3 was detected by immunoblotting using an HA antibody. Representative data are shown from independent experiments that were repeated at least three times. See also Fig . Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Transfection, Immunoprecipitation, Mutagenesis, Residue, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs were primed with LPS for 4 h and were stimulated without or with 5 mM ATP at the indicated time points. Whole‐cell lysates were analyzed via western blotting using the indicated antibodies. B MARCH5 KO HEK293T cells were cotransfected with HA‐ub (K48R) mutant and NLRP3 without or with Myc‐MARCH5 2KR or Myc‐MARCH5 H43W. After treatment with nigericin for 30 min of the LPS treatment for 4 h, the cell lysates were subjected to immunoprecipitation with anti‐FLAG M2 beads. An HA antibody was used to assess NLRP3 ubiquitination. C HEK293T MARCH5 KO cells were cotransfected with HA‐ub (K48R) and NLRP3 WT or NLRP3 truncated mutants with or without SFB‐MARCH5 (2KR). After treatment with LPS and nigericin, the cell lysates were subjected to immunoprecipitation with an anti‐Myc antibody overnight, and NLRP3 ubiquitination was assessed via western blotting by using an anti‐HA antibody. D HEK293T MARCH5 KO cells were transfected HA‐ub (K27O) with NLRP3 WT and truncated mutants. After stimulation with LPS and nigericin, cell lysates were immunoprecipitated with anti‐Myc antibody. The HA antibody detected ubiquitination in the subjects. E The NLRP3 inflammasome was reconstituted in HEK293T MARCH5 KO cells expressing ASC, pro‐caspase 1, and IL‐1β with HA‐ub WT, K27O mutant, or K27R mutant NLRP3. Additionally, cells were cotransfected with or without Myc‐MARCH5 2KR or the Myc‐MARCH5 H43W mutant. After stimulation with LPS for 4 h and nigericin for 30 min, IL‐1β secretion was quantitated using ELISA, and the lysates were detected by western blotting with the indicated antibodies. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Western Blot, Mutagenesis, Immunoprecipitation, Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A HEK293T cells were transfected with NLRP3 WT or indicated Lys mutants and ASC. Cells were stimulated with 200 ng/ml LPS for 4 h followed by 15 μM nigericin for 30 min. The percentage of cells with an ASC speck was quantified after Confocal microscopy. At least 100 cells were analyzed. Values are the mean ± SD. Values, ** P < 0.01, *** P < 0.001 (two‐tailed Student's t ‐test). Bar, 10 μm. B HEK293T cells were transfected with ASC and NLRP3 (WT or individual NLRP3 mutants). Cells were treated for 4 h with 200 ng/ml LPS and 30 min with 15 μM nigericin. Harvested cell lysates were incubated with 2 mM DSS for cross‐linking. Triton X‐100 insoluble pellets and soluble fraction lysates were detected by immunoblotting with the indicated antibodies. C NLRP3 inflammasome‐reconstituted HEK293T cells with NLRP3 WT or indicated mutants were stimulated for 4 h with 200 ng/ml LPS and 30 min with 15 μM nigericin. Harvested cells were detected by immunoblotting with the indicated antibodies. D Culture supernatants were obtained from (C) and subjected to ELISA to quantify secreted IL‐1β. Representative data are shown from independent experiments that were repeated at least three times. Values are the mean ± SD. * P < 0.05, *** P < 0.001 (two‐tailed Student's t ‐test) E Schematic diagram of NLRP3 inflammasome activation by MARCH5. Upon activation by a priming signal, NLRP3 associates with MAVS on mitochondria ①. MARCH5 recognizes and ubiquitinates two lysine sites, K324 and K430, on the NLRP3 NACHT domain via K27‐linked polyubiquitination ②. NLRP3 ubiquitination by MARCH5 promotes the recruitment of NEK7 and enables self‐oligomerization ③. As a result, ASC and pro‐caspase‐1 are recruited to oligomerized NLRP3 ④ for NLRP3 inflammasome assembly ⑤ and trigger the activation of caspase‐1 and pro‐cytokine cleavage ⑥. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Transfection, Confocal Microscopy, Two Tailed Test, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet:

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Recombinant, Sequencing, Software, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: ( A ) Domain architecture of the CARD8 protein. ( B ) HIV-1 protease cleaves the N-terminus of CARD8. HEK293T cells were transfected with plasmids encoding HA-CARD8 (100ng), together with either the pNL4-3 (1μg) or Pro-D25H (1μg). Cells were collected 24 hours after transfection. Anti-HA, anti-CARD8-N and anti-p24 antibodies were used sequentially on the same blot. ( C ) HIV-1 protease is necessary and sufficient to cleave CARD8. HEK293T cells were transfected with constructs encoding CARD8 (100ng) together with indicated viral plasmids (1μg). Cells were collected 24 hours after transfection. ( D ) RPV enhances HIV-1 protease-mediated cleavage of CARD8. HEK293T cells were transiently transfected with HA-CARD8 (100ng) and indicated viral plasmids (1μg). DMSO or RPV was added 24 hours post transfection. Cell lysates were collected 6 hours after RPV treatment. ( E and F ) HIV-1 protease triggers CASP1-dependent pro-IL-1β processing. HEK293T cells were co-transfected with plasmids encoding CASP1 (2ng), pro-IL-1β (200ng) and an HIV-1 plasmid (1μg). After 24 hours, cells were treated with indicated drugs for another 6 hours. ( G ) RPV induces HIV-1 protease-dependent cleavage of CARD8 in infected cells. HEK293T cells were infected with VSV-G-pseudotyped HIV-1 reporter virus. Infected cells were then transfected with HA-CARD8 (100ng). DMSO or RPV was added 24 hours post transfection. Cell lysates were collected 6 hours after RPV treatment. In B - F , cell lysates were evaluated by immunoblotting. CARD8-FL, full-length CARD8; CARD8-N, N terminal CARD8; free Nt, freed N terminus. Data are representative of three or more independent experiments.

Article Snippet: The IL-1β (HG10139-CM) expression plasmid was purchased from Sino Biological.

Techniques: Transfection, Construct, Plasmid Preparation, Infection, Western Blot

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: A list of stop codon mutations or truncations. 1 : All viral plasmids were generated using the NL4-3-ΔEnv-EGFP backbone, except NL4-3/BaL. 2 : NL4-3/BaL has the NL4-3 backbone with EGFP coding sequencing in nef , and the BaL envelop coding sequence. These plasmids were used to transfect HEK293T cells, either for immunoblotting analysis of CARD8 and IL-1β cleavage, or for production of HIV-1 reporter viruses when co-transfected with pVSV-G and lentivirus packaging plasmid. HIV-1 reporter viruses generated using NL4-3-ΔEnv-EGFP backbone are replication-defective. The viral vector NL4-3/BaL can produce replication-competent viruses.

Article Snippet: The IL-1β (HG10139-CM) expression plasmid was purchased from Sino Biological.

Techniques: Generated, Sequencing, Western Blot, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: ( A to E ) HIV-1 protease activation by NNRTIs induced rapid pyroptosis of infected monocytes derived macrophages (MDMs). MDMs were infected with HIV NL4-3/BaL . At day 4, Raltegravir (RAL) and T-20 were added to block new infection. Cells were then treated with RPV, EFV, LPV, or combinations for up to 24 hours. In A to C , GFP + cells were detected by flow cytometry. In D , images of infected MDMs were taken using a Cytation 5 Imaging Multi-Mode Reader (Biotek). In E , culture supernatant was collected for IL-1β ELISA. ( F and G ) Pyroptosis of HIV-1-infected MDMs is CASP1-dependent. MDMs were infected and treated as described above. In F , immunoblot analysis of pro-(p45) and cleaved CASP1 (p10 and p20) in infected MDMs after RPV treatment for 1 hour. In G , infected MDMs were pre-treated with VX-765 (100μM) or Z-VAD-FMK (100μM) for 3 hours and then treated with RPV for 4 hours before flow cytometry analysis. ( H and I ) HIV-1 protease mediated inflammasome activation is proteasome-dependent. MDMs were infected and treated as described above. Infected MDMs were pretreated with proteasome inhibitors MG132, Bort, or Me-Bs for 30 minutes and then treated with RPV for 4 hours. In H , GFP expression was analyzed by flow cytometry. In I , culture supernatant was collected for the detection of IL-1β by ELISA. ( J to I ) The CARD8 inflammasome is required for pyroptosis of HIV-1-infected macrophages. ( J ) knockout of CARD8, ASC, CASP1 or NLRP3 in THP-1 cells was confirmed by immunoblotting. Knockout or control THP-1 cells were infected with VSV-G pseudotyped HIV-1 reporter virus NL4-3-Pol. 3 days after infection, cells were pre-treated with LPS (100ng/ml) for 3 hours before RPV treatment. In K , GFP expression was analyzed by flow cytometry 24 hours post RPV treatment; Data were normalized to the control group. In L , culture supernatant was collected 48 hours post RPV treatment for IL-1β detection. In B, G, H and K , P values were calculated using one-way ANOVA and Turkey multiple comparison tests. In C, E, I and L P values were calculated using two-way ANOVA and Turkey multiple comparison tests. *p < 0.05, ****p < 0.0001. In each bar graph, n≥3. Error bars show mean values with SEM. Data are representative of three or more independent experiments.

Article Snippet: The IL-1β (HG10139-CM) expression plasmid was purchased from Sino Biological.

Techniques: Activation Assay, Infection, Derivative Assay, Blocking Assay, Flow Cytometry, Imaging, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Knock-Out

Journal: International Journal of Oncology

Article Title: Tunicamycin enhances the suppressive effects of cisplatin on lung cancer growth through PTX3 glycosylation via AKT/NF-κB signaling pathway

doi: 10.3892/ijo.2018.4650

Figure Lengend Snippet: dePTX3 suppresses the growth and migration of human lung cancer cells. (A) CCK-8 assay was used to analyze the viability of A549 and SPCA1 cells. Cells were treated with or without rhPTX3 (100 ng/ml) or dePTX3 (rhPTX3; 100 ng/ml + PNGase F; 500 U/ml) for 0-72 h. Data were presented as a percentage of viable cells. (B) Lung cancer cells treated with TM (0.01, 0.05, 0.5 and 1 µ g/ml) for 48 h. (C) Immunofluorescent staining of PTX3 (red color) and N-glycan levels (green color) in A549 and SPCA1 cells analyzed following treatment with TM (0.5 µ g/ml) for 48 h. (D) SDS-PAGE gel (12%) stained with CBB and lectinblot against PHA to analyze N-glycans in lung cancer cells following treatment with TM for 48 h and PNGase F (500 U/ml) for 2 h of incubation. (E) Western blot analysis of dePTX3 in A549 and SPCA1 cells following treatment with TM or rhPTX3 + PNGase F. The lanes are labeled as follows: lane 1, glycosylated PTX3; lane 2, deglycosylated PTX3 by TM; lane 3, deglycosylated PTX3 by PNGase F. (F) Representative images of wound healing of A549 and SPCA1 cells analyzed after treatment with rhPTX3, dePTX3 and TM (0.5 µ g/ml), followed by measurement of relative wound width (48 h average wound width divided by 0 h wound width). (G) Microscopic images and graphs of transwell migration assay of A549 and SPCA1 cells following treatment with rhPTX3, dePTX3 and TM. Bar graph values represent the means ± SEM of 3 independent experiments. * P<0.05 and ** P<0.01.

Article Snippet: Cell Counting kit-8 (CCK-8) was obtained from Beyotime Biotechnology (Shanghai, China); recombinant human PTX3 (rhPTX3) (#PROTP26022; Boster Biological Technology Co., Ltd., Fremont, CA); NF-κB inhibitor (IKK-16), PI3K inhibitor (GDC0941), MEK1/2 inhibitor, (MEK162) were purchased from Selleck Chemicals (Shanghai, China).

Techniques: Migration, CCK-8 Assay, Staining, Glycoproteomics, SDS Page, Incubation, Western Blot, Labeling, Transwell Migration Assay

Journal: International Journal of Oncology

Article Title: Tunicamycin enhances the suppressive effects of cisplatin on lung cancer growth through PTX3 glycosylation via AKT/NF-κB signaling pathway

doi: 10.3892/ijo.2018.4650

Figure Lengend Snippet: PTX3 expression in human lung cancer samples and cell lines. (A) Immunohistochemical staining of PTX3 in paired human lung cancer tissue (P1, P2, P3) and normal lung tissue (N1, N2, N3) of the same patient (magnification, ×10). (B) PTX3 level in serum from lung cancer patients and normal healthy individuals by ELISA; * P<0.05. (C) PTX3 level in the supernatant collected from A549, SPCA1 and H1299 cells was examined by ELISA. (D) Immunofluorescent staining of PTX3 protein (red color) in A549, SPCA1 and H1299 lung cancer cells. DAPI (blue color) was used to stain the nuclei. (E) mRNA expression level of PTX3 detected by qPCR in A549, SPCA1 and H1299 cells. (F) PTX3 expression level in A549, SPCA1 and H1299 cells detected by western blot analysis. GAPDH was used as an internal control.

Article Snippet: Cell Counting kit-8 (CCK-8) was obtained from Beyotime Biotechnology (Shanghai, China); recombinant human PTX3 (rhPTX3) (#PROTP26022; Boster Biological Technology Co., Ltd., Fremont, CA); NF-κB inhibitor (IKK-16), PI3K inhibitor (GDC0941), MEK1/2 inhibitor, (MEK162) were purchased from Selleck Chemicals (Shanghai, China).

Techniques: Expressing, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: International Journal of Oncology

Article Title: Tunicamycin enhances the suppressive effects of cisplatin on lung cancer growth through PTX3 glycosylation via AKT/NF-κB signaling pathway

doi: 10.3892/ijo.2018.4650

Figure Lengend Snippet: PTX3 deglycosylation enhances the sensitivity of lung cancer cells to Cisplatin treatment. (A) suPTX3 level was detected by ELISA in the supernatant of A549 and SPCA1 cells following treatment with Cis (20 µ M) for 48 h. (B) Immunofluorescent staining of PTX3 (red color) in A549 and SPCA1 cells following treatment with Cis for 48 h. DAPI (blue color) was used to stain the nuclei. (C) Bar graph of A549 and SPCA1 cell viability analyzed following treatment with Cis and deglycosylated PTX3 (dePTX3). Each bar represents the average of 3 independent experiments. Data are expressed as a percentage of viable cells. (D) Western blot analysis of PCNA and AKT phosphorylation following treatment with Cis or dePTX3, and combined treatment for 48 h. (E) Western blot analysis for PTX3 expression in the cell lysate from control, mock and mutated PTX3 (mPTX3) in A549 and SPCA1 cells. (F) Western blot analysis of Cis, mPTX3 or Cis combined with mPTX3 and combined treatment for 48 h. The level of PCNA expression and AKT phosphorylation were examined by western blot analysis. GAPDH was used as an internal control. * P<0.05 and ** P<0.01.

Article Snippet: Cell Counting kit-8 (CCK-8) was obtained from Beyotime Biotechnology (Shanghai, China); recombinant human PTX3 (rhPTX3) (#PROTP26022; Boster Biological Technology Co., Ltd., Fremont, CA); NF-κB inhibitor (IKK-16), PI3K inhibitor (GDC0941), MEK1/2 inhibitor, (MEK162) were purchased from Selleck Chemicals (Shanghai, China).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Phospho-proteomics, Expressing, Control

Journal: International Journal of Oncology

Article Title: Tunicamycin enhances the suppressive effects of cisplatin on lung cancer growth through PTX3 glycosylation via AKT/NF-κB signaling pathway

doi: 10.3892/ijo.2018.4650

Figure Lengend Snippet: TM increases the sensitivity of lung cancer cells to cisplatin-induced apoptosis through deactivating AKT/NF-κB signaling pathway. (A) CCK-8 assay used to analyze the viability of A549 and SPCA1 cells treated with GDC0941 (4 µ M), IKK-16 (10 µ M), MEK162 (20 µ M) or rhPTX3 (100 ng/ml) for 48 h. (B) Western blot analysis of Bcl2 expression in A549 and SPCA1 cells, incubated with rhPTX3 (100 ng/ml) with or without NF-κB inhibitor IKK-16 (10 µ M). (C) A549 and SPCA1 cells were treated with TM (0.5 µ g/ml) + Cis (20 µ M) together or in combination. Western blot analysis of AKT, IKK, p65 phosphorylation in cytoplasmic and nuclear extracts of A549 and SPCA1 cells treated with TM, Cis or in combination. PARP was used as an internal control. (D) A549 and SPCA1 cells were double-stained with Annexin V-FITC/PI and analyzed by flow cytometry after 24 h of treatment with TM (0.5 µ g/ml), Cis (20 µ M) or combined treatment. The histogram showed the average percentage of total apoptotic cells in both A549 and SPCA1 cells. (E) Bcl2, Bax and cleaved PARP expression detected by western blot analysis following treatment with TM, Cis or in combination for 48 h. GAPDH was used as an internal control. Data represented the mean values ± SEM. * P<0.05 and ** P<0.01.

Article Snippet: Cell Counting kit-8 (CCK-8) was obtained from Beyotime Biotechnology (Shanghai, China); recombinant human PTX3 (rhPTX3) (#PROTP26022; Boster Biological Technology Co., Ltd., Fremont, CA); NF-κB inhibitor (IKK-16), PI3K inhibitor (GDC0941), MEK1/2 inhibitor, (MEK162) were purchased from Selleck Chemicals (Shanghai, China).

Techniques: CCK-8 Assay, Western Blot, Expressing, Incubation, Phospho-proteomics, Control, Staining, Flow Cytometry