Journal: BMC Cancer

Article Title: FOXL2 + cancer-associated fibroblasts enhances epithelial ovarian cancer development via TGFβ/Smad signaling

doi: 10.1186/s12885-025-15364-6

Figure Lengend Snippet: FOXL2 SUMOylation intricately regulates increased expression in CAFs. ( A ) SUMOylation sites of FOXL2 was analyzed by SUMOplot™ analysis. Shown are the top 7 predicted lysine residues; ( B ) In vitro sumoylation assay was employed to confirm the predicted SUMOylation sites using HA-tagged wild-type (WT), or the K25R, K87R, K114R, K150R, and 4KR (where K25, K87, K114 and K150 were all mutated to R). Shown is a representative blot and densitometry analysis of SUMOylated-FOXL2/Total FOXL2; ( C ) The association between SUMOylation and FOXL2 stability was determined by western blotting in HEK-293T cells using α-HA. Cells were transfected with either HA-FOXL2-WT or HA-FOXL2-K25/87R (double mutant, 2KR). The membrane was stripped and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot and densitometry analysis of three technical replicates; ( D ) Cells were transfected with HA-FOXL2-WT or, HA-FOXL2-2KR ± His-SUMO1. CHX (100 µg/ml) was added to inhibit translation allowing tracking of FOXL2 stability in the presence and absence of His-SUMO1. Blots were probed with α-HA and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot and densitometry analysis of three technical replicates; ( E ) DMSO (control), MG132 (proteasome inhibitor) or chloroquine (lysosome inhibitor) were added into HEK-293T cells transfected with HA-FOXL2-WT or HA-FOXL2-2KR, before CHX (100 µg/ml) was added. Blots were probed with α-HA and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot; ( F ) IP assay was used to test the association between FOXL2 SUMOylation and ubiquitination. Lysates obtained from HEK-293T cells transfected with HA-FOXL2-WT or HA-FOXL2-2KR, along with FLAG-UBC9, were immunoprecipitated using α-HA antibody and then probed with α-FLAG antibody. Shown is a representative blot; *, *** P < 0.05, P < 0.001

Article Snippet: The different primary antibodies used were α-SUMO1 (67559-1-Ig, 1:3000), α-UBC9 (10070-1-AP, 1:2000), α-Vimentin (10366-1-AP, 1:5000), α-N-cadherin (22018-1-AP, 1:5000), α-E-cadherin (20874-1-AP, 1:20000) (Proteintech, Wuhan, China); α-HA (A02041, 1:800), α-His (A02050, 1:800), α-Flag (A02010, 1:800) (Abbkine, Wuhan, China); α-FOXL2 (ab246511, 1:1000), α-Snail (ab216347, 1:1000) (Abcam, Cambridge, MA, USA); and, α-Smad2/3 (D7G7, 1:1000), α-p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4, 1:1000) (CST, Boston, MA, USA).

Techniques: Expressing, In Vitro, Western Blot, Transfection, Mutagenesis, Membrane, Control, Ubiquitin Proteomics, Immunoprecipitation

Journal: BMC Cancer

Article Title: FOXL2 + cancer-associated fibroblasts enhances epithelial ovarian cancer development via TGFβ/Smad signaling

doi: 10.1186/s12885-025-15364-6

Figure Lengend Snippet: FOXL2 SUMOylation requires SUMO1 and UBC9/UBE2I. ( A ) Putative interaction of FOXL2 with SUMO1, SUMO2, SUMO3, and SUMO4 was detected by String analysis ( https://cn.string-db.org/ ); ( B ) In vitro sumoylation assay was employed to confirm String analysis’s prediction of SUMO1, HEK-293T cells were transfected with HA-FOXL2-WT, FLAG-UBC9, and His-SUMO1-4. In vitro SUMOylation assay using Ni 2+ -NTA pull-down determined that FOXL2 was mainly modified by SUMO1. Shown is a representative blot; ( C ) In vitro sumoylation assay was employed to determine whether UBC9 is compulsorily required for FOXL2 SUMOylation. HEK-293T cells were transfected with HA-FOXL2-WT and His-SUMO1 ± FLAG-UBC9. In vitro SUMOylation using Ni 2+ -NTA pull-down assay determined that UBC9 is required for FOXL2 SUMOylation. Shown is a representative blot; ( D ) In vitro sumoylation assay was employed to determine whether UBC9 is compulsorily required for FOXL2 SUMOylation in CAFs. CAFs were transduced using either a non-targeting control shRNA or shRNA targeting UBC9 . Transduced cells were transfected with HA-FOXL2-WT and His-SUMO1. In vitro SUMOylation using Ni 2+ -NTA pull-down assay determined that UBC9 is required for FOXL2 SUMOylation. Shown is a representative blot

Article Snippet: The different primary antibodies used were α-SUMO1 (67559-1-Ig, 1:3000), α-UBC9 (10070-1-AP, 1:2000), α-Vimentin (10366-1-AP, 1:5000), α-N-cadherin (22018-1-AP, 1:5000), α-E-cadherin (20874-1-AP, 1:20000) (Proteintech, Wuhan, China); α-HA (A02041, 1:800), α-His (A02050, 1:800), α-Flag (A02010, 1:800) (Abbkine, Wuhan, China); α-FOXL2 (ab246511, 1:1000), α-Snail (ab216347, 1:1000) (Abcam, Cambridge, MA, USA); and, α-Smad2/3 (D7G7, 1:1000), α-p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4, 1:1000) (CST, Boston, MA, USA).

Techniques: In Vitro, Transfection, Modification, Pull Down Assay, Control, shRNA