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

Journal: Frontiers in Cellular Neuroscience

Article Title: Adipose-derived stem cell-conditioned medium mitigates ischemia-induced neuronal injury via the JAK1/STAT3 signaling pathway

doi: 10.3389/fncel.2026.1744887

Figure Lengend Snippet: ADSC-CM promotes neurite outgrowth in OGD-injured neurons through the JAK1-STAT3 signaling pathway. (A) On day 5 of cultivation, neuronal neurites extended and formed an extensive neural network. Immunofluorescence staining showed that the neurons were positive for the neuronal-specific marker Tuj1. After OGD injury, neuronal neurites were damaged, partially interrupted, and disappeared. (B) Western blot bands of JAK1, pJAK1, STAT3, pSTAT3, and GAPDH for each group. (C) Comparison of the relative ratio of pJAK1 to JAK1 across experimental groups ( n = 4). (D) Comparison of the relative ratio of pSTAT3 to STAT3 across experimental groups ( n = 4). (E) Immunofluorescence images of neurons from each group, showing the effects of ADSC-CM and GLPG0634 on neurite outgrowth. Neurons were stained with the Tuj1 antibody (red) to label the neuronal cytoskeleton, and nuclei were counterstained with DAPI (blue). (F) Diagram illustrating how to measure the length of the longest neurite and the number of primary neurites in neurons. The green line shows the trajectory of the longest neurite, and white arrows point to primary neurites. (G,H) Comparison of the length of the longest neurite and the number of primary neurites in neurons across experimental groups ( n = 4). Data are expressed as means ± SEM. The difference between the groups was assessed using a one-way ANOVA followed by Bonferroni post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: 100 μm (A) , 20 μm (E,F) .

Article Snippet: After overnight incubation with rabbit anti-Tuj1 antibody (1:200, BM3881, Boster, Wuhan, China) at 4 °C, cells were washed and then incubated with Cy3-conjugated goat anti-rabbit IgG (1:400, AS007, ABclonal) for 1 h at room temperature in the dark.

Techniques: Immunofluorescence, Staining, Marker, Western Blot, Comparison