Journal: iScience

Article Title: MYCT1 alters the glycogen shunt by regulating selective translation of RACK1-mediated enzymes

doi: 10.1016/j.isci.2022.103955

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-RACK1 , Cell Signaling Technology , Cat# 5432; RRID: AB_10705522.

Techniques: Virus, Cloning, Recombinant, Protease Inhibitor, Staining, Bicinchoninic Acid Protein Assay, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, cDNA Synthesis, Mass Spectrometry, Negative Control, Software

Journal: Theranostics

Article Title: RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog

doi: 10.7150/thno.29271

Figure Lengend Snippet: RACK1 promotes self-renewal and chemoresistance of human liver CSCs and maintains murine ESC function. ( A-N ) 96 h after HuH7 cells were infected with lentivirus expressing non-targeting control (NC) shRNA or RACK1 shRNA ( A-G ), or after HuH7 single clone stably expressing FLAG-RACK1 and the mock control were generated ( H-N ), cells were subjected to the following assays: ( A,H ) Immunoblotting (IB) analysis of RACK1 expression in total cells. ( B,I ) Flow cytometric analysis of CD13 and CD133 expression in total cells. ( C,J ) Immunoblotting analysis of RACK1 expression in sorted CD13+ and CD13- subpopulations. ( D,K ) Sphere formation assays of sorted CD13+ subpopulation. mean±s.d. ( n =3); * P <0.05, ** P <0.01. ( E,L ) In vivo tumorigenicity experiments of sorted CD13+ subpopulation (5000 cells/site, 7 weeks, n =6 ) . ( F,M ) Etoposide (Etop, 100 μM, 48 h)- or sorafenib (Sora, 50 μM, 24 h)-induced apoptosis of CD13+ subpopulation. mean±s.d. ( n =3); Ctrl, control. ( G,N ) qRT-PCR analysis of sorted CD13+ subpopulation for the expression of the indicated drug-resistant relative genes. mean±s.d. ( n =3). ( O - Q ) 96 h after murine ESCs were infected with lentivirus expressing non-targeting control shRNA or RACK1 shRNAs, cells were subjected to immunoblotting analysis for RACK1 expression ( O ), colony formation assays (mean±s.d., n =3; scale bar: 1 cm) ( P ), and alkaline phosphatase (AP) activity assays (mean±s.d., n =3) ( Q ).

Article Snippet: Another set of lentivirus-based human RACK1 shRNA (RACK1-b) and control lentivirus were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat. No. sc-36354-v).

Techniques: Infection, Expressing, Control, shRNA, Stable Transfection, Generated, Western Blot, In Vivo, Quantitative RT-PCR, Activity Assay

Journal: Theranostics

Article Title: RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog

doi: 10.7150/thno.29271

Figure Lengend Snippet: Elevated Nanog expression plays an essential role in RACK1-regulated HCC CSCs. ( A-G ) 96 h after HuH7 cells were infected with the indicated lentivirus, cells were subjected to the following assays: ( A ) Immunoblotting analysis of the expression of Nanog and RACK1 in total cells. ( B ) Flow cytometric analysis of CD13 expression in total cells. ( C ) Immunoblotting analysis of the expression of Nanog and RACK1 in sorted CD13+ cells. ( D ) Sphere formation assays of sorted CD13+ subpopulation. mean±s.d. ( n =3); * P <0.05, ** P <0.01 ( E ) In vivo tumorigenicity experiments of CD13+ subpopulation (5000 cells/site, 7 weeks, n =6). ( F ) Etoposide (Etop, 100 μM, 48 h)- or sorafenib (Sora, 50 μM, 24 h)-induced apoptosis of CD13+ subpopulation. mean±s.d. ( n =3). ( G ) qRT-PCR analysis of sorted CD13+ subpopulation for the expression of the indicated drug-resistant relative genes. mean±s.d. ( n =3). ( H-K ) 24 h after HuH7 cells were transfected with mammalian expression vector encoding GFP-tagged wild type RACK1 or RACK1 mutant lacking WD5, GFP+CD13+ subpopulations were sorted and subjected to immunoblotting ( H ), sphere formation assays, mean±s.d. ( n =3) ( I ), etoposide (Etop, 100 μM, 48 h)- or sorafenib (Sora, 50 μM, 24 h)-induced apoptosis, mean±s.d. ( n =3) ( J ), and qRT-PCR analysis for the expression of the indicated drug-resistant relative genes. mean±s.d. ( n =3) ( K ).

Article Snippet: Another set of lentivirus-based human RACK1 shRNA (RACK1-b) and control lentivirus were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat. No. sc-36354-v).

Techniques: Expressing, Infection, Western Blot, In Vivo, Quantitative RT-PCR, Transfection, Plasmid Preparation, Mutagenesis

Journal: Journal of Cellular and Molecular Medicine

Article Title: FGF21 induces autophagy‐mediated cholesterol efflux to inhibit atherogenesis via RACK1 up‐regulation

doi: 10.1111/jcmm.15118

Figure Lengend Snippet: Effects of FGF21 on RACK1 expression in the aortic plaques of apoE −/− mice. A, The expression of RACK1 protein was detected by immunohistochemistry staining. Scale bar, 50 μm. B, The mRNA expression levels of RACK1 were measured by real‐time PCR. C, The protein expression levels of RACK1 were measured using Western blotting. Results are presented as mean ± SD of 3 experiments. ** P < .01 vs control group; # P < .05, vs FGF21 + HFD group; HFD, high‐fat diet; NS, physiological saline

Article Snippet: Human ATG5 or RACK1 siRNA (Santa Cruz Biotechnology) was purchased with target sequences as follows: ATG5‐si: 5′‐GTCCATCTAAGGATGCAAT‐3′ and RACK1‐si: 5′‐GGTCCAGGATGAGAGTCAT‐3′.

Techniques: Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Western Blot, Control, Saline

Journal: Journal of Cellular and Molecular Medicine

Article Title: FGF21 induces autophagy‐mediated cholesterol efflux to inhibit atherogenesis via RACK1 up‐regulation

doi: 10.1111/jcmm.15118

Figure Lengend Snippet: The role of RACK1 in FGF21‐mediated autophagy. ApoE −/− mice were fed a HFD with FGF21 or FGF21 + Ad‐RACK1 shRNA for 12 wk. A‐C, The protein expression levels of LC3, beclin‐1 and p62 protein were detected using Western blotting. Data are presented as mean ± SD of 3 experiments (one‐way ANOVA). ** P < .01 vs AS3. D, E, Representative images of H & E and Oil Red O staining of an aortic lesion. Scale bar, 50 μm. Data are expressed as mean ± SD (n = 10 per group, SNK post hoc multiple comparison tests); * P < .05 vs HFD group; # P < .05 vs AS3 + LV‐RACK1 shRNA

Article Snippet: Human ATG5 or RACK1 siRNA (Santa Cruz Biotechnology) was purchased with target sequences as follows: ATG5‐si: 5′‐GTCCATCTAAGGATGCAAT‐3′ and RACK1‐si: 5′‐GGTCCAGGATGAGAGTCAT‐3′.

Techniques: shRNA, Expressing, Western Blot, Staining, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: FGF21 induces autophagy‐mediated cholesterol efflux to inhibit atherogenesis via RACK1 up‐regulation

doi: 10.1111/jcmm.15118

Figure Lengend Snippet: Fibroblast growth factor 21‐induced autophagy increased foam cell cholesterol efflux via RACK1 expression. A‐C, The foam cells were transfected using scrambled (neg) or RACK1 siRNA for 12 h and then incubated with 200 ng/mL of FGF21 for 24 h. LC3 and p62 were measured using Western blotting. D, Punctuated GFP‐LC3 protein was examined using laser scanning microscopy. Scale bar, 5 μm. E, Autophagosomes were assessed using MDC staining and fluorescence microscopy, with representative cell images at the indicated time‐points. Scale bar, 10 μm; white arrow, autophagosome. F, Autophagosomes were assessed using TEM, with images of representative cells shown from indicated time points. Scale bar, 0.5 μm; white arrow, autophagosome. G, Cholesterol accumulation was determined using Oil Red O staining, followed by light microscopic examination. Scale bar, 15 μm; original magnification, 400×. Measurements were conducted three or more times and gave similar results. H, Effect of RACK1 siRNA on FGF21‐induced cholesterol efflux in foam cells. All results are presented as means (±SD) of 3 experiments (one‐way ANOVA). ** P < .01 vs controls; # P < .05 vs FGF21 + RACK1 siRNA

Article Snippet: Human ATG5 or RACK1 siRNA (Santa Cruz Biotechnology) was purchased with target sequences as follows: ATG5‐si: 5′‐GTCCATCTAAGGATGCAAT‐3′ and RACK1‐si: 5′‐GGTCCAGGATGAGAGTCAT‐3′.

Techniques: Expressing, Transfection, Incubation, Western Blot, Laser-Scanning Microscopy, Staining, Fluorescence, Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: FGF21 induces autophagy‐mediated cholesterol efflux to inhibit atherogenesis via RACK1 up‐regulation

doi: 10.1111/jcmm.15118

Figure Lengend Snippet: The role of RACK1 in the effects of FGF21 on cholesterol content in foam cells

Article Snippet: Human ATG5 or RACK1 siRNA (Santa Cruz Biotechnology) was purchased with target sequences as follows: ATG5‐si: 5′‐GTCCATCTAAGGATGCAAT‐3′ and RACK1‐si: 5′‐GGTCCAGGATGAGAGTCAT‐3′.

Techniques: Control

Journal: PLOS Neglected Tropical Diseases

Article Title: Trichinella spiralis excretory-secretory proteins induced autophagy via activating AMPK/mTOR pathway and protected gut epithelial barrier

doi: 10.1371/journal.pntd.0013863

Figure Lengend Snippet: A : Both IIL and AW ESP increased the RACK1 expression in Caco-2 cells and activated the AMPK pathway. When RACK1 was inhibited, the IIL ESP activated-AMPK was also inhibited. B : IIL and AW ESP increased the intracellular Ca 2+ concentration in Caco-2 cells by increasing RACK1 expression. When RACK1 was inhibited, intracellular Ca 2+ concentration was decreased. Data are presented as mean ± SD of three independent assays. *Compared with the DMEM group, P < 0.05; # compared with the IIL or AW ESP group, P < 0.05.

Article Snippet: The primary antibodies used in this study include antibodies against ZO-1, RACK1, GAPDH and β-actin from Servicebio (Wuhan, China); Occludin and Claudin-1 from Thermo Fisher Scientific (Massachusetts, USA); p-AMPK and AMPK from HuaBio (Hangzhou, China), mTOR from Proteintech (Wuhan, China); p-mTOR from CST (Massachusetts, USA); Beclin-1 from Abcam (Cambridge, UK), p-62 from Sangon (Shanghai, China).

Techniques: Expressing, Concentration Assay

Journal: PLOS Neglected Tropical Diseases

Article Title: Trichinella spiralis excretory-secretory proteins induced autophagy via activating AMPK/mTOR pathway and protected gut epithelial barrier

doi: 10.1371/journal.pntd.0013863

Figure Lengend Snippet: A : Both IIL and AW ESP increased the RACK1 expression in RAW264.7 cells and activated the AMPK pathway. When RACK1 was inhibited, the IIL and AW ESP-induced AMPK activation was also inhibited. B : IIL and AW ESP increased the intracellular Ca 2+ concentration in RAW264.7 cells by increasing RACK1 expression. When RACK1 was inhibited, intracellular Ca 2+ concentration was also decreased. Data are presented as mean ± SD of three independent assays. *Compared with the DMEM group, P < 0.05; # compared with the IIL ESP or AW ESP group, P < 0.05.

Article Snippet: The primary antibodies used in this study include antibodies against ZO-1, RACK1, GAPDH and β-actin from Servicebio (Wuhan, China); Occludin and Claudin-1 from Thermo Fisher Scientific (Massachusetts, USA); p-AMPK and AMPK from HuaBio (Hangzhou, China), mTOR from Proteintech (Wuhan, China); p-mTOR from CST (Massachusetts, USA); Beclin-1 from Abcam (Cambridge, UK), p-62 from Sangon (Shanghai, China).

Techniques: Expressing, Activation Assay, Concentration Assay