Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: Sequences of the primers and probes used for qPCR.

Article Snippet: After three wash steps, the cells were stained with primary antibodies including anti-NLRP3 (Bioss, Beijing, China), anti-ASC (Santa Cruz, CA, USA) and CoraLite®594-conjugated RACK1 (Proteintech, China) at 4 ℃ overnight.

Techniques:

Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: P. multocida infection inhibits RACK1 expression. A Peritoneal macrophages from C57BL/6 mice were infected with PmCQ2 at MOIs of 1, 5, and 10 for 9 h. Subsequently, ciprofloxacin (100 µg/mL) was added, and the cells were cultured for 15 h. Then, cell lysates were collected to measure RACK1 protein expression by immunoblotting. B ImageJ was used to quantify the ratio of RACK1 expression to β-actin expression. C RACK1 mRNA expression was analysed by RT‒PCR. D C57BL/6 ( n = 3) mice were intranasally infected with PmCQ2 (1000 CFU), and sterilized PBS was used as a control. At 48 h post-infection (hpi), the heart, lung, liver, spleen and kidney were collected and homogenized to measure RACK1 protein expression. E Cell morphology is shown. The cells were transfected with pcDNA3.1 and pcDNA3.1-RACK1 plasmids and then infected with PmCQ2 as described above. F The viability of RACK1-overexpressing cells was quantified after PmCQ2 infection by Cell Counting Kit-8 assays. G– J The number of adherent and invasive PmCQ2 in si-RACK1 cells (G and H) and RACK1-overexpressing cells (I-J), respectively. The data in B, E are presented as the mean ± SEM from three independent experiments with triplicate samples per experiment. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; ns represents no significance.

Article Snippet: After three wash steps, the cells were stained with primary antibodies including anti-NLRP3 (Bioss, Beijing, China), anti-ASC (Santa Cruz, CA, USA) and CoraLite®594-conjugated RACK1 (Proteintech, China) at 4 ℃ overnight.

Techniques: Infection, Expressing, Cell Culture, Western Blot, Transfection, Cell Counting

Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: RACK1 is essential for P. multocida -induced NLRP3 inflammasome activation. RACK1 protein expression in RACK1-knockdown macrophages ( A ) and RACK1-overexpressing macrophages ( B ) was measured by Western blotting. RACK1 mRNA expression in RACK1-knockdown macrophages ( C ) and RACK1-overexpressing macrophages ( D ) was measured by RT‒PCR. After 48 h of si-RACK1 and pcDNA3.1-RACK1 plasmid transfection, the cells were infected with PmCQ2 for 9 h, and then ciprofloxacin (100 µg/mL) was added and incubated for an additional 15 h. After 24 h of incubation, supernatants and cell lysates were collected. The protein levels of NLRP3, IL-1β (p31, p17), and caspase-1 (p45, p20) in RACK1-knockdown macrophages ( E ) and RACK1-overexpressing macrophages ( F ) were measured. The NLRP3 and IL-1β mRNA expression levels in si-RACK1 cells ( G , H ) and RACK1-overexpressing cells ( I , J ) were analysed by RT‒PCR. The levels of IL-1β and TNF-α secreted by si-RACK1 cells (K-L) and RACK1-overexpressing cells (M-N) were measured by ELISA. The data are presented as the mean ± SEM from three independent experiments with triplicate samples per experiment. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: After three wash steps, the cells were stained with primary antibodies including anti-NLRP3 (Bioss, Beijing, China), anti-ASC (Santa Cruz, CA, USA) and CoraLite®594-conjugated RACK1 (Proteintech, China) at 4 ℃ overnight.

Techniques: Activation Assay, Expressing, Western Blot, Plasmid Preparation, Transfection, Infection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: RACK1 promotes P. multocida -induced formation of ASC specks. Cells were transfected with si-RACK1 to knockdown RACK1 and pcDNA3.1-RACK1 plasmid to overexpress RACK1 for 48 h. Then, cells were infected for 24 h as described above. Finally, immunofluorescence staining was performed to detect NLRP3 and ASC specks in RACK1-knockdown cells ( A ) and RACK1-overexpressing cells ( B ). White arrows represent the colocalization of ASC and NLRP3. The images are representative of three independent experiments. The bar in each microscopic image indicates 50 μm.

Article Snippet: After three wash steps, the cells were stained with primary antibodies including anti-NLRP3 (Bioss, Beijing, China), anti-ASC (Santa Cruz, CA, USA) and CoraLite®594-conjugated RACK1 (Proteintech, China) at 4 ℃ overnight.

Techniques: Transfection, Plasmid Preparation, Infection, Immunofluorescence, Staining

Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: RACK1 promotes P. multocida -induced NF-κB activation. Cells were transfected with si-RACK1 for 48 h and then infected with PmCQ2 for the indicated times. Next, cell lysates were collected, and the protein expression of p65 and p-p65 was measured by Western blotting analysis ( A ). At 6 hpi, the mRNA expression levels of CXCL-1 ( B ), CXCL-2 ( C ), IL-6 ( D ), IL-12 ( E ) and TNF-α ( F ) were measured by RT‒PCR. Similarly, the protein expression of p65 and p-p65 in RACK1-overexpressing cells was also measured by Western blotting analysis. Moreover, the mRNA expression levels of CXCL-1 ( H ), CXCL-2 ( I ), IL-6 ( J ), IL-12 ( K ) and TNF-α ( L ) were measured by RT‒PCR. The data are presented as the mean ± SEM from three independent experiments with triplicate samples per experiment. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: After three wash steps, the cells were stained with primary antibodies including anti-NLRP3 (Bioss, Beijing, China), anti-ASC (Santa Cruz, CA, USA) and CoraLite®594-conjugated RACK1 (Proteintech, China) at 4 ℃ overnight.

Techniques: Activation Assay, Transfection, Infection, Expressing, Western Blot

Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: P. multocida infection promotes the interaction of RACK1 with NLRP3 and NEK7. Cells were transfected with the pcDNA3.1-eGFP-NEK7 plasmid for 48 h and then infected with PmCQ2 for 24 h. Subsequently, immunofluorescence staining was performed to detect RACK1 and NLRP3 ( A ). The RACK1 protein is shown in red, the NLRP3 protein is shown in purple, the NEK7 protein is shown in green, nuclei are shown in blue. The images are representative of three independent experiments. The bar in each microscopic image indicates 50 μm. Cells were infected with PmCQ2 for 24 h, and then the RACK1-NLRP3-NEK7 interaction was detected by immunoprecipitation and immunoblotting ( B ). Cells were pretreated with KCl, quinine or glibenclamide to inhibit K + efflux and then infected with PmCQ2 for 24 h. RACK1, NLRP3 and NEK7 protein expression ( C ) and the RACK1-NLRP3-NEK7 interaction ( D ) were analysed by Western blotting as well as immunoprecipitation and immunoblotting, respectively. The images and blots are representative of three independent experiments.

Article Snippet: After three wash steps, the cells were stained with primary antibodies including anti-NLRP3 (Bioss, Beijing, China), anti-ASC (Santa Cruz, CA, USA) and CoraLite®594-conjugated RACK1 (Proteintech, China) at 4 ℃ overnight.

Techniques: Infection, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Expressing

Journal: Cancer Biology & Medicine

Article Title: MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis

doi: 10.20892/j.issn.2095-3941.2020.0296

Figure Lengend Snippet: MIIP facilitates HIF-2α binding to RACK1 rather than HSP90, and promotes HSP90 acetylation. (A) Cells stably overexpressing MIIP 786-O and control cells were transfected with siRACK1 1#, siRACK1 2#, or control (siNC) siRNA. The expression of RACK1, HIF-2α, and HSP90 was detected by Western blot. (B) HIF-2α binding to RACK1 and HSP90 was detected with co-immunoprecipitation assays. The 786-O-MIIP and 786-O-Vector cell lysates were immunoprecipitated with anti-HIF-2α antibody, then immunoblotted for RACK1 and HSP90. (C) The acetylation of HSP90, and HSP90 binding to HDAC6 and HIF-2α in 786-O-MIIP and 786-O-Vector cells was detected by immunoprecipitation with anti-HSP90 antibody followed by immunoblotting with anti-Acetyl Lysine, anti-HDAC6, and anti-HIF-2α antibodies.

Article Snippet: The precipitates were resolved with SDS-PAGE and subjected to Western blot with anti-HIF-2α (1:1000, Proteintech/CST), anti-HSP90 (1:1000, Proteintech), anti-RACK1 (1:1000, Proteintech), anti-MIIP, or anti-acetyl lysine (1:1000, Abcam).

Techniques: Binding Assay, Stable Transfection, Transfection, Expressing, Western Blot, Immunoprecipitation, Plasmid Preparation

Journal: Cancer Biology & Medicine

Article Title: MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis

doi: 10.20892/j.issn.2095-3941.2020.0296

Figure Lengend Snippet: MIIP is weakly expressed in RCC and associated with progression, prognosis, and the expression of CYR61 and HIF-2α. (A) MIIP, CYR61, and HIF-2α expression in 13 pairs of RCC tissues vs. adjacent non-tumor tissues was detected by Western blot. C: RCC tissue; N: adjacent non-tumor tissue. (B) MIIP, CYR61, and HIF-2α expression in an RCC tissue microarray was detected with immunohistochemistry (scale bar: 50 μm). (C) MIIP, CYR61, and HIF-2α expression levels were quantified with a scoring system. The staining score was calculated by multiplying the stained area (%) score and the intensity score. Expression group: low, score < 9; high, score ≥ 9. Stage refers to WHO histological grade. * P < 0.05; ** P < 0.01, χ 2 test. (D) The Kaplan-Meier curve depicts the relationship between MIIP expression level and OS of patients with RCC, and the P -value was calculated with a stratified log rank test ( P = 0.026). (E) Schematic representation of the MIIP/HIF-2α/CYR61 axis in ccRCC. In normal cells, MIIP promotes HSP90 acetylation through inhibiting HDAC6 activity, thus impairing HSP90’s chaperone function and its binding to HIF-2α, which in turn causes RACK1 binding and subsequent HIF-2α degradation. Meanwhile, under normoxia, VHL promotes HIF-2α ubiquitination and degradation in an oxygen-dependent manner. In RCC, VHL deficiency and MIIP downregulation together cause HIF-2α accumulation, thereby leading to overexpression of CYR61, which in turn contributes to RCC progression.

Article Snippet: The precipitates were resolved with SDS-PAGE and subjected to Western blot with anti-HIF-2α (1:1000, Proteintech/CST), anti-HSP90 (1:1000, Proteintech), anti-RACK1 (1:1000, Proteintech), anti-MIIP, or anti-acetyl lysine (1:1000, Abcam).

Techniques: Expressing, Western Blot, Microarray, Immunohistochemistry, Staining, Activity Assay, Binding Assay, Over Expression