Journal: Advanced Science

Article Title: G3BP1 and SLU7 Jointly Promote Immune Evasion by Downregulating MHC‐I via PI3K/Akt Activation in Bladder Cancer

doi: 10.1002/advs.202305922

Figure Lengend Snippet: G3BP1 interacts with SLU7 to promote PI3K/Akt signaling activation, downregulating MHC‐I and promoting immune evasion. A,B) qRT–PCR analysis was used to evaluate the mRNA levels of SLU7 and the class IA PI3Ks in UMUC3 and T24 cells with SLU7 knockdown or overexpression. C,D) Western blotting was used to detect the protein expression levels of class IA PI3Ks in UMUC3 and T24 cells with SLU7 knockdown or overexpression. E) Cell lysates from UMUC3 cells with stable expression of SLU7‐Flag or HA‐G3BP1 were immunoprecipitated with anti‐Flag or anti‐HA antibodies, and immunoblotting was conducted with the indicated antibodies. F) The PLA was used to evaluate the interaction between G3BP1 and SLU7 in UMUC3 cells. Scale bar, 10 µm. G) IF was used to analyze the locations of G3BP1 and SLU7 in UMUC3 cells. Scale bar, 10 µm. Fluorescence intensity curves of DAPI (blue), G3BP1 (green), and SLU7 (red) that crossed the arrow were analyzed. H,I) The protein expression levels of SLU7, total Akt, p‐Akt Thr308 , and p‐Akt Ser473 in UMUC3 and MB49 cells with stable SLU7 knockdown were detected using western blotting. J,K) Western blotting was used to detect the protein expression levels of G3BP1, SLU7, total Akt, p‐Akt Thr308 , p‐Akt Ser473 , and/or MHC‐I in G3BP1‐overexpressing UMUC3 and MB49 cells with SLU7 knockdown. L) Flow cytometry was used to analyze the expression of MHC‐I on the surface of MB49 cells in (K). MHC‐I expression was quantified based on the mean fluorescence intensity (MFI). M) mCherry‐expressing MB49‐OVA cells with or without G3bp1 overexpression and Slu7 knockdown were cocultured with antigen‐activated CTLs for 24 h. Cytotoxicity was determined by analyzing the percentage of 7‐AAD+ cells among mCherry+ cells. Error bars represent SD ( n = 3 in A, B, L, and M). P ‐values are presented and were calculated using two‐way ANOVA (A and B) and one‐way ANOVA (L and M).

Article Snippet: For re‐IP, UMUC3 cell lysates with simultaneous stable expression of SLU7‐Flag and HA‐G3BP1 were primarily immunoprecipitated with anti‐Flag antibody, after which they were blocked with 15 μg mL −1 Flag peptide (TP1273, TargetMol, USA) for 2 h at 4°C under gentle rotation.

Techniques: Activation Assay, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Expressing, Immunoprecipitation, Fluorescence, Flow Cytometry

Journal: Advanced Science

Article Title: G3BP1 and SLU7 Jointly Promote Immune Evasion by Downregulating MHC‐I via PI3K/Akt Activation in Bladder Cancer

doi: 10.1002/advs.202305922

Figure Lengend Snippet: G3BP1 and SLU7 form a complex with PABPC1 and eIF4G1 to stabilize the closed‐loop structure of the mRNAs of class IA PI3Ks. A) Scatter plot of the G3BP1‐interacting proteins identified using MS. B and C) Cell lysates from UMUC3 cells with stable expression of SLU7‐Flag or HA‐G3BP1 were immunoprecipitated with anti‐Flag or anti‐HA antibodies, and immunoblotting was performed with the indicated antibodies. D) Cell lysates from UMUC3 cells with simultaneous stable expression of SLU7‐Flag and HA‐G3BP1 were primarily immunoprecipitated with anti‐Flag antibody and then blocked with Flag peptide. The resultant immunoprecipitants were subjected to re‐IP with anti‐HA antibodies and immunoblotted with the indicated antibodies. E) IF was used to analyze the locations of eIF4G1 or PABPC1 with SLU7 and G3BP1 in UMUC3 cells stably expressing eIF4G1‐GFP or PABPC1‐GFP. Scale bar, 10 µm. The fluorescence intensity curves of DAPI (blue), eIF4G1‐GFP (green), PABPC1‐GFP (green), SLU7 (red), and G3BP1 (purple) that crossed the arrows were analyzed. F) Cell lysates from UMUC3 cells with PABPC1‐GFP stable expression and SLU7 or G3BP1 knockdown were immunoprecipitated with anti‐GFP antibody. Western blotting was used to detect the expression levels of PABPC1‐GFP, SLU7, G3BP1, and eIF4G1 in whole cell lysates and immunoprecipitants. G) qRT–PCR analysis was used to evaluate the mRNA expression levels of class IA PI3Ks in the immunoprecipitants. H) Western blotting was used to detect the expression levels of G3BP1 and SLU7 in UMUC3 cells with SLU7 knockdown or G3BP1 knockdown. I) FISH–FRET was performed in UMUC3 cells with SLU7 or G3BP1 knockdown using Cy3‐labeled 5′‐UTR probes and Cy5‐labeled 3′‐UTR probes targeting the mRNAs of PI3Ks. The mean values of FRET efficiency are shown. Error bars represent SD ( n = 3 in G; n = 5 in I). P ‐values are presented and were calculated using one‐way ANOVA (G and I).

Article Snippet: For re‐IP, UMUC3 cell lysates with simultaneous stable expression of SLU7‐Flag and HA‐G3BP1 were primarily immunoprecipitated with anti‐Flag antibody, after which they were blocked with 15 μg mL −1 Flag peptide (TP1273, TargetMol, USA) for 2 h at 4°C under gentle rotation.

Techniques: Expressing, Immunoprecipitation, Western Blot, Stable Transfection, Fluorescence, Knockdown, Quantitative RT-PCR, Labeling

Journal: The Journal of Biological Chemistry

Article Title: An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

doi: 10.1074/jbc.M117.801936

Figure Lengend Snippet: Role of the di-proline motif in degradation of Δ9-desaturase. N-terminal amino acid sequences of Δ9-desaturases were compared (D. melanogaster DESAT1 (NP_652731), Mus musculus SCD1 (NP_033153), M. musculus SCD3 (NP_077770), Homo sapiens SCD1 (NP_005054), Danio rerio stearoyl-CoA desaturase (AAO25582), and S. cerevisiae Ole1 (CAA96757)) (A). Schematic illustration of DESAT1, mouse SCD1, and constructed mutants is shown (B). S2 cells expressing mouse SCD1-FLAGC, chimera-FLAGC, chimera (AA)-FLAGC, and SCD1 (di-Pro)-FLAGC were treated with C18:1 (100 μm) for 6 h (C) or DESAT1 inhibitor 37c (1 μm) for 16 h (D), and the amounts of endogenous DESAT1, FLAG-tagged exogenous Δ9-desaturases, and α-tubulin protein were detected with anti-DESAT1 antibody, anti-FLAG antibody, and anti-α-tubulin antibody, respectively. Band intensities were determined by ImageJ software, and levels of SCD1 proteins are shown relative to the amount of SCD1 protein in vehicle-treated cells (C and D). Mean ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001; n.s., not significant.

Article Snippet: Anti-α-tubulin antibody (PM054), anti-FLAG antibody (M2), and anti-ubiquitin antibody (SPC-119) were purchased from MBL, Sigma, and StressMarq Biosciences, respectively.

Techniques: Construct, Expressing, Software

Journal: The Journal of Biological Chemistry

Article Title: An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

doi: 10.1074/jbc.M117.801936

Figure Lengend Snippet: Identification of protease involved in the degradation of DESAT1. S2 cells were treated with calpeptin (50 μm), MG132 (50 μg/ml), or chloroquine (50 μm) for 6 h (A). S2 cells were treated with dsRNA against GFP, CalpA, CalpB, CalpC, or sol for 72 h and then exposed to C18:1 (100 μm) for 6 h (B). S2 cells expressing DESAT1-FLAGC and DESAT1 (P2A/P3A)-FLAGC were treated with calpeptin (50 μm) for 6 h (C). The amounts of endogenous DESAT1, FLAG-tagged exogenously expressed DESAT1, and α-tubulin protein were detected with anti-DESAT1 antibody, anti-FLAG antibody, and anti-α-tubulin antibody, respectively (A–C). Band intensities were determined by ImageJ software, and levels of DESAT1 proteins are shown relative to the amount of DESAT1 protein in vehicle-treated cells (A–C). Mean ± S.D. (n = 3). **, p < 0.01; ***, p < 0.001; n.s., not significant.

Article Snippet: Anti-α-tubulin antibody (PM054), anti-FLAG antibody (M2), and anti-ubiquitin antibody (SPC-119) were purchased from MBL, Sigma, and StressMarq Biosciences, respectively.

Techniques: Expressing, Software