Journal: Scientific Reports

Article Title: KIN17 facilitates the initiation and progression of renal tumor progression through the PI3K-AKT-mTOR pathway

doi: 10.1038/s41598-026-35851-5

Figure Lengend Snippet: KIN17 positively regulates PI3K/AKT/mTOR expression in RCC and PF-04691502 can reverse the pathway protein expression and biological function. ( A ) Predict effects of KIN17 knockdown on the pathways of RCC cells (ACHN and 786–0) in vitro by kegg and GSEA. ( B ) p-mTOR, mTOR, p-AKT, AKT, p-PI3K and PI3K protein levels were measured by WB using the paired RCC cells (ACHN and 786–0) group and knockdown KIN17 group. ( C ) p-mTOR, mTOR, p-AKT, AKT, p-PI3K and PI3K protein levels were measured by WB using the paired RCC vector cells group (VE groups), overexpression KIN17 group (OE groups), VE groups + PF-04691502 and OE groups + PF-04691502. ( D ) N-cadherin and Vimentin protein levels were measured by WB using the paired RCC cells (ACHN and 786–0) in knockdown KIN17 group and different using PF-04691502 groups.

Article Snippet: The membranes were then blocked with 5% non-fat milk in TBS/Tween (0.05% Tween-20 in TBS) for 2 h. Membranes were incubated with primary antibodies including KIN17(Santa Cruz, Catalog number: sc-32768, 1:500), AKT (Proteintech, Catalog number: 60203–2-Ig, 1:10,000), mTOR (Proteintech, Catalog number: 66888–1-Ig, 1:10,000), PI3K(CST, Catalog number: 11889, 1:1000), p-AKT(CST, Catalog number: 4060S, 1:2000), p-mTOR(CST, Catalog number: 5536S, 1:1000), p-PI3K(CST, Catalog number: 17366S, 1:1000), N-cadherin (Proteintech, Catalog number: 22018–1-AP, 1:10,000), Vimentin (Proteintech, Catalog number: 10366–1-AP, 1:50,000) and GAPDH (Proteintech, 60,004–1-lg, 1:10,000) overnight at 4°C and then with secondary antibodies (anti-rabbit or anti- mouse; Proteintech) for room temperature.

Techniques: Expressing, Knockdown, In Vitro, Plasmid Preparation, Over Expression

Journal: Scientific Reports

Article Title: KIN17 facilitates the initiation and progression of renal tumor progression through the PI3K-AKT-mTOR pathway

doi: 10.1038/s41598-026-35851-5

Figure Lengend Snippet: KIN17 promotes tumor growth and suppression of tumor growth by PF-04691502 in vivo ( A ) Representative images of xenograft tumors 786–0 cells with expression of shNC and shKIN17 (n = 7 in each group). Tumor volume and tumor weight were analyzed. ( B ) Representative H&E staining and immunohistochemstry staining of Ki67, TUNEL, KIN17, p-mTOR and p-AKT in the xenograft tumor tissues (× 400 magnification). ( C ) Representative images of xenograft tumors 786–0 cells with expression of Vector, Vector + PF-04691502, OE and OE + PF-04691502 (n = 8 in each group). Tumor volume and tumor weight were analyzed. ( D ) Representative H&E staining and immunohistochemstry staining of Ki67, TUNEL, KIN17, p-mTOR and p-AKT in the xenograft tumor tissues (× 400 magnification). The error bars represent the mean ± SD of triplicate technical replicates. * P < 0.05, ** P < 0.01, ***P < 0.001.

Article Snippet: The membranes were then blocked with 5% non-fat milk in TBS/Tween (0.05% Tween-20 in TBS) for 2 h. Membranes were incubated with primary antibodies including KIN17(Santa Cruz, Catalog number: sc-32768, 1:500), AKT (Proteintech, Catalog number: 60203–2-Ig, 1:10,000), mTOR (Proteintech, Catalog number: 66888–1-Ig, 1:10,000), PI3K(CST, Catalog number: 11889, 1:1000), p-AKT(CST, Catalog number: 4060S, 1:2000), p-mTOR(CST, Catalog number: 5536S, 1:1000), p-PI3K(CST, Catalog number: 17366S, 1:1000), N-cadherin (Proteintech, Catalog number: 22018–1-AP, 1:10,000), Vimentin (Proteintech, Catalog number: 10366–1-AP, 1:50,000) and GAPDH (Proteintech, 60,004–1-lg, 1:10,000) overnight at 4°C and then with secondary antibodies (anti-rabbit or anti- mouse; Proteintech) for room temperature.

Techniques: In Vivo, Expressing, Staining, TUNEL Assay, Plasmid Preparation

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Gli1 mRNA fold changes show the responsiveness of SMO mutants to SHH. Untreated Gli1 levels indicate low SMO activity, while SHH-treated values correspond to the level of SMO activation induced by SHH ligand. A t-test with Welch’s correction was used to compute statistical significance. (p values: untreated vs treated: WT: 1.327 × 10 -3 , G 2.57 f V: 9.212 × 10 -3 , I ECL2 A: 4.2 × 10 -5 , A 2.60 f M: 7.1 × 100 -5 , R 5.64 f A: 2.062 × 10 -3 , R 5.64 f Q: 1.192 × 10 -3 , F 6.36 f I: 2.163 × 10 -3 , L 5.62 f A: 1.948 × 10 -3 , treated WT vs treated mutant: G 2.57 f V: 9.1 × 10 -3 , I ECL2 A: 0.02734, A 2.60 f M: 0.7477, R 5.64 f A: 0.08858, R 5.64 f Q: 0.02766, F 6.36 f I: 1.923 × 10 -3 , L 5.62 f A: 2.306 × 10 key: Not significant (ns) p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001, All experimental data represent biological replicates, N=4.) ( b ) ΔGli1 mRNA fold change (SHH vs untreated) and Δ \begin{document}$\Delta$\end{document} PMF (difference of peak PMF, calculated as P M F W T \begin{document}$PMF_{WT}$\end{document} - P M F m u t a n t \begin{document}$PMF_{mutant}$\end{document} ) plotted for the mutants in Pathway 1. ( c ) Example mutant A 2.60 f M shows that cholesterol is able to enter SMO through Pathway 1 even on a bulky mutation. ( d ) Same as ( b ) but for Pathway 2 ( e ) Example mutant L 5.62 f A shows that cholesterol can enter SMO through Pathway 2 due to lesser steric hindrance. All snapshots presented are frames taken from MD simulations.

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Activity Assay, Activation Assay, Mutagenesis

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Immunoblotting was used to measure abundance of mSMO and GLI1 proteins in SMO -/- cells stably expressing either mSMO-WT or Pathway 1 mutants after treatment with SHH. ( b ) Gli1 mRNA fold change plotted for SMO mutants, showing fold change when the mutants are untreated, treated with a low concentration of SHH, and treated with a saturating concentration of SHH. Figure 3—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Western Blot, Stable Transfection, Expressing, Concentration Assay

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Immunoblotting was used to measure abundance of mSMO and GLI1 proteins in SMO -/- cells stably expressing either mSMO-WT or Pathway 2 mutants after treatment with SHH. ( b ) Gli1 mRNA fold change plotted for SMO mutants, showing fold change when the mutants are untreated, treated with a low concentration of SHH, and treated with a saturating concentration of SHH. Figure 3—figure supplement 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—figure supplement 4—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Western Blot, Stable Transfection, Expressing, Concentration Assay

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Gli1 mRNA fold changes show the responsiveness of SMO mutants to SHH. Untreated Gli1 levels indicate low SMO activity, while SHH-treated values correspond to the level of SMO activation induced by SHH ligand. A t-test with Welch’s correction was used to compute statistical significance. (p values: untreated vs treated: WT: 3 × 10 -6 , Y LD A: 2.46 × 10 -4 , F 6.65 f A: 1.08 × 10 -3 , I ECL 3 A: 1.12 × 10 -4 , treated WT vs treated mutant: F 6.65 f A: 1.6 × 10 -5 , I ECL 3 A: 1.6 × 10 -5 , Y LD A: 1.4 × 10 -5 , key: Not significant (ns) p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001, All experimental data represent biological replicates, N=4.) ( b ) Δ \begin{document}$\Delta$\end{document} Gli1 mRNA fold change (SHH vs untreated) and Δ \begin{document}$\Delta$\end{document} PMF (difference of peak PMF, calculated as P M F W T \begin{document}$PMF_{WT}$\end{document} - P M F m u t a n t \begin{document}$PMF_{mutant}$\end{document} ) are plotted for mutants along the TMD-CRD pathway. ( c, d ) Example mutants Y LD A and F 6.65 f A show that cholesterol is unable to translocate through this pathway because of the loss of crucial hydrophobic contacts provided by Y207 and F484 and along the solvent-exposed pathway.

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Activity Assay, Activation Assay, Mutagenesis, Solvent

Journal: eLife

Article Title: Multiple modes of cholesterol translocation in the human Smoothened receptor

doi: 10.7554/eLife.108030

Figure Lengend Snippet: ( a ) Immunoblotting was used to measure the abundance of mSMO and GLI1 proteins in SMO −⁄− cells stably expressing either mSMO-WT or Common Pathway mutants after treatment with SHH. ( b ) Gli1 mRNA fold change plotted for SMO mutants, showing fold change when the mutants are untreated, treated with a low concentration of SHH, and treated with a saturating concentration of SHH. Figure 6—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies against GLI1 [anti-GLI1 mouse monoclonal (clone L42B10); Cell Signaling Technology, catalog no. 2643, RRID: AB_2294746 ], SMO (rabbit polyclonal) , or GAPDH [anti-GAPDH mouse monoclonal (clone 1E6D9); Protein tech, catalog no. 60004–1-Ig, RRID: AB_2107436 ].

Techniques: Western Blot, Stable Transfection, Expressing, Concentration Assay