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

Journal: International journal of medical sciences

Article Title: Ube2S regulates Wnt/β-catenin signaling and promotes the progression of non-small cell lung cancer.

doi: 10.7150/ijms.40243

Figure Lengend Snippet: Figure 1. Immunostaining of Ube2S in normal lung tissues and NSCLC tissues. Bronchial epithelial cells (black arrow) and alveolar cells (grey arrow) show negative immunostaining for Ube2S (A). Bronchial epithelial cells (black arrow) and submucosal glands (grey arrow) show negative immunostaining for Ube2S (B). Weak and focal immunostaining of Ube2S is evident in some bronchial epithelial cells (C) and submucosal glands (D). Strong immunostaining of Ube2S is present in the cytoplasm and nuclei of squamous cell carcinoma (E) and adenocarcinoma (F) cells. (A: ×100; B, D, F: ×200; C, E: ×400)

Article Snippet: Ube2S cDNA clone was purchased from Origene (Rockville, MD, USA).

Techniques: Immunostaining

Journal: International journal of medical sciences

Article Title: Ube2S regulates Wnt/β-catenin signaling and promotes the progression of non-small cell lung cancer.

doi: 10.7150/ijms.40243

Figure Lengend Snippet: Figure 2. Survival function. Ube2S expression in non-small cell lung cancer is significantly associated with poor patient survival (34.9 ± 5.5 versus 56.4 ± 7.3 months) (Log rank test, p < 0.05)

Article Snippet: Ube2S cDNA clone was purchased from Origene (Rockville, MD, USA).

Techniques: Expressing

Journal: International journal of medical sciences

Article Title: Ube2S regulates Wnt/β-catenin signaling and promotes the progression of non-small cell lung cancer.

doi: 10.7150/ijms.40243

Figure Lengend Snippet: Figure 3. The function of Ube2S in lung cancer cells. Western blots show that Ube2S is expressed in bronchial epithelial HBE cells, and lung cancer A549 and NCI-H1299 cells (A). The MTT assay shows that overexpression of Ube2S in A549 cells significantly promotes cancer cell proliferation (B) (p < 0.05). The wound scratch healing assay shows that overexpression of Ube2S in A549 cells significantly promotes cancer cell migration (C) (*p < 0.05)

Article Snippet: Ube2S cDNA clone was purchased from Origene (Rockville, MD, USA).

Techniques: Western Blot, MTT Assay, Over Expression, Migration

Journal: International journal of medical sciences

Article Title: Ube2S regulates Wnt/β-catenin signaling and promotes the progression of non-small cell lung cancer.

doi: 10.7150/ijms.40243

Figure Lengend Snippet: Figure 4. Ube2S regulates Wnt/β-catenin signaling molecules and activity. Western blots show that overexpression of Ube2S in A549 cells significantly upregulates Wnt/β-catenin signaling molecules including β-catenin, cyclin D1, and MMP7 (A) (p < 0.05). The luciferase assay shows that overexpression of Ube2S in A549 cells significantly upregulates Wnt/β-catenin signaling activity (B) (p < 0.05)

Article Snippet: Ube2S cDNA clone was purchased from Origene (Rockville, MD, USA).

Techniques: Activity Assay, Western Blot, Over Expression, Luciferase

Journal: International journal of medical sciences

Article Title: Ube2S regulates Wnt/β-catenin signaling and promotes the progression of non-small cell lung cancer.

doi: 10.7150/ijms.40243

Figure Lengend Snippet: Figure 5. Wnt/β-catenin signaling inhibitor abolished the function of Ube2s in lung cancer cells. The MTT assay shows that addition of the Wnt/β-catenin signaling inhibitor, ETC-159, significantly inhibits the ability of Ube2S to promote cancer cell proliferation

Article Snippet: Ube2S cDNA clone was purchased from Origene (Rockville, MD, USA).

Techniques: MTT Assay