Journal: iScience

Article Title: KLHL7 promotes hepatocellular carcinoma progression and molecular therapy resistance by degrading RASA2

doi: 10.1016/j.isci.2023.106914

Figure Lengend Snippet: KLHL7 upregulates the RAS-MAPK pathway and promotes resistance to chemotherapy (A) Serum-starved KLHL7 knockdown and control Huh-7 cells were treated with fetal bovine serum (FBS) for the indicated times before being lysed for immunoblotting. Western blotting was used to detect the expression of the indicated proteins. (B) Serum-starved KLHL7 knockdown and control Huh-7 cells were treated with fetal bovine serum (FBS) for the indicated times before being lysed for immunoblotting. Western blotting was used to detect the expression of the indicated proteins. (C) GTP-bound RAS in Huh-7 cells. Serum-starved KLHL7 knockdown and control Huh-7 cells were stimulated with fetal bovine serum (FBS) for 5 min and lysed for GST-RAS-RBD pulldown. The pulldown protein was detected by immunoblotting. (D) KLHL7 knockdown and control Huh-7 cells were treated with lenvatinib (5 μM) for 2 h and lysed for immunoblotting. Western blotting was used to detect the expression of the indicated proteins. (E) 293T cells were transfected with the indicated genes and lysed for immunoblotting. Western blotting detected the expression of the indicated proteins. (F) Colony formation assays were performed with scramble and shKLHL7 Huh-7 cells transfected with or without MEK1CA after 2 weeks of lenvatinib treatment. Statistics on the number of remaining clones are shown at the bottom. Lenvatinib concentrations were 0.5 μM (Huh-7) and 1 μM (HCCLM3). Data are the mean ± s.e.m. n = 3. Significance was determined using Student’s t test. ns p > 0.05. (G) A soft agar formation assay was performed with KLHL7 knockdown and control cells. Data are the mean ± s.e.m. n = 3. Significance was determined using Student’s t test. ∗∗p < 0.01, ∗∗∗p < 0.001. (H) Cell viability assays were performed using scramble and shKLHL7 Huh-7 and HCCLM3 cells transfected with or without MEK1CA in media containing 0.5% fetal bovine serum. The cell number was detected by adding CCK-8 and measuring the absorbance at 450 nM. Data are the mean ± s.e.m. n = 4. Significance was determined using Student’s t test. ns p > 0.05.

Article Snippet: The prokaryotic plasmid expressing the GST-RAF1 RBD was purchased from Addgene (13338).

Techniques: Knockdown, Control, Western Blot, Expressing, Transfection, Clone Assay, Tube Formation Assay, CCK-8 Assay

Journal: iScience

Article Title: KLHL7 promotes hepatocellular carcinoma progression and molecular therapy resistance by degrading RASA2

doi: 10.1016/j.isci.2023.106914

Figure Lengend Snippet: KLHL7 upregulates the RAS-MAPK pathway via RASA2 (A) Huh-7 cells were transfected with the corresponding shRNA, starved for 16 h before being stimulated with FBS for 15 min, and lysed for immunoblotting. (B) Serum-starved Huh-7 cells transfected with the indicated shRNA were triggered with FBS for 5 min and lysed for GST-RAS-RBD pulldown. The pulldown protein was detected by immunoblotting. (C) Cell viability assays were performed with cells transfected with the indicated shRNA in media containing 0.5% fetal bovine serum. The cell number was detected by adding CCK-8 and measuring the absorbance at 450 nM. Data are the mean ± s.e.m. n = 4. Significance was determined using Student’s t test. ns p > 0.05. (D) A soft agar formation assay was performed with cells transfected with the indicated shRNA. Data are the mean ± s.e.m. n = 3. Significance was determined using Student’s t test. ns p > 0.05. (E) Colony formation assays were performed on cells transfected with the indicated shRNA after 2 weeks of lenvatinib treatment. The statistics of the number of remaining clones are shown at the bottom. Lenvatinib concentrations were 0.5 μM (Huh-7) and 1 μM (HCCLM3). Data are the mean ± s.e.m. n = 3. Significance was determined using Student’s t test. ns p > 0.05.

Article Snippet: The prokaryotic plasmid expressing the GST-RAF1 RBD was purchased from Addgene (13338).

Techniques: Transfection, shRNA, Western Blot, CCK-8 Assay, Tube Formation Assay, Clone Assay

Journal: iScience

Article Title: KLHL7 promotes hepatocellular carcinoma progression and molecular therapy resistance by degrading RASA2

doi: 10.1016/j.isci.2023.106914

Figure Lengend Snippet:

Article Snippet: The prokaryotic plasmid expressing the GST-RAF1 RBD was purchased from Addgene (13338).

Techniques: Produced, Recombinant, Membrane, Plasmid Preparation, Software