Journal: Journal of Biological Chemistry

Article Title: Uropathogenic Escherichia coli invades bladder epithelial cells by activating kinase networks in host cells

doi: 10.1074/jbc.ra118.003499

Figure Lengend Snippet: Figure 2. mTORC2 mediates UPEC invasion. (A) UTI89 promotes the mTOR activation. BECs were pretreated with inhibitors AZD8055 (10 µM; mTORC1/2), rapamycin (0.1 µM; mTORC1), LY294002 (5 µM; PI3K and mTOR), or wortmannin (0.2 µM; PI3K) followed by exposure to CM from UTI89 or UTI89∆FimH for 10 min. Cell lysates were analyzed by Western blot for phosphorylation of indicated proteins. NI, not infected; NT infected but not treated with inhibitor. (B) AZD8055, but not rapamycin, inhibits UTI89 invasion of BECs. Statistical analysis was performed by Student’s t-test compared to the non-treated control; ***P < 0.001, n=6. (C) Knockdown of mTOR. Lentivirus-encoding two different shRNA sequences targeting mTOR were used. LKO lentivirus (shCon) was used as a control. Stable gene knockdown was achieved with selection using puromycin. The mTOR gene expression was measured with qRT-PCR and results are presented as fold difference relative to shCon sample. Statistical analysis was performed by Student’s t-test compared to the LKO lentivirus control; ***P < 0.001, n=3. (D) Knockdown of mTOR inhibits UTI89 invasion. Statistical analysis was performed by Student’s t-test compared to the LKO lentivirus control; ***P < 0.001, n=6. (E) mTOR knockdown decreases p70S6K phosphorylation. HSP90β was used as protein loading control. Effect of Raptor (F) and Rictor (G) knockdown on Akt and p70S6K phosphorylationation. Cells were treated or not with UTI89 CM for 10 min and lysates were analyzed by Western blot for Akt (S473) and p70S6K (T389) phosphorylation. Total Akt, p70S6K and GAPDH were used as protein loading controls. (H) Knockdown of rictor inhibits the UTI89 invasion. Bacterial invasion was determined using the gentamicin protection assay and results are shown as fold change from control shCon cells. Statistical analysis was performed by Student’s t-test compared to the LKO lentivirus control; ***P < 0.001, **P < 0.01, n=6. (I) Raptor and Rictor do not impact the UTI89 attachment to BECs. GFP-UTI89 attachment to host BECs was quantified under fluorescence microscopy. Results show the number of attached UTI89 per cell and 50 randomly-selected cells were counted from each group. Statistical analysis was performed by Student’s t-test compared to the LKO lentivirus control.

Article Snippet: Reagents were obtained as follows: Lipopolysaccharide (LPS) from E. coli 055:B5 and complete protease and phosphatase inhibitors from Sigma Aldrich (St. Louis, MO); CLI095 from InvivoGen (San Diego, CA); wortmannin, LY294002, and rapamycin from Cell Signaling Technology (Boston, MA); AZD8055 from Santa Cruz Biotechnology (Santa Cruz, CA); BAY 80- 6946, Afatinib and MK-2206 from Selleckchem (Houston, TX); AG1478 from Cayman Chemical (Ann Arbor, MI); and gentamicin from Gibco (Grans Island, NY).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Infection, Control, Knockdown, shRNA, Selection, Gene Expression, Quantitative RT-PCR, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Uropathogenic Escherichia coli invades bladder epithelial cells by activating kinase networks in host cells

doi: 10.1074/jbc.ra118.003499

Figure Lengend Snippet: Figure 3. EGFR facilitates the UPEC invasion. (A) UTI89 and UTI89∆FimH activate EGFR. BECs were treated with bacterial CM for the indicated times and cell lysates were examined for EGFR phosphorylation (p-EGFR) using anti-pEGFR (Y1068) antibody. Total EGFR (t-EGFR) served as protein loading control. (B) Afatinib and AG1478 specifically inhibit UTI89-induced p-EGFR (Y1068). BECs were pretreated with the inhibitors LY294002 (5 µM; PI3K and mTOR), wortmannin (0.2 µM; PI3K), Bay 80-6946 (0.4 µM; PI3K), MK-2206 (4 µM, Akt), rapamycin (0.1 µM; mTORC1), AZD8055 (10 µM; mTORC1/2), Afatinib (1.5 µM; EGFR) or AG1478 (1 µM; EGFR) for 1 hr followed by exposure to UTI89 CM for 10 min. Cell lysates were analyzed by Western blot for p-EGFR (Y1068). Total EGFR and GAPDH served as protein loading control. (C) EGFR mediates the UTI89 CM-induced Akt phosphorylation. BECs were treated with Afatinib or AG1478 as above then exposed to UTI89 CM. Cell lysates were analyzed by Western blot for EGFR and Akt phosphorylation. GAPDH served as protein loading control. (D) Afatinib and AG1478 inhibit the UTI89 invasion of BEC as determined with gentamicin protection assay. Results are shown as fold change from control non-treated (NT) cells. Error bars represent SEM. Statistical analysis was performed by Student’s t-test compared to the non-treated control; *P < 0.05, n=4. (E) Knockdown of EGFR gene was achieved with two targeting shRNA sequences in lentivirus. The EGFR gene expression was measured with qRT-PCR and results are presented as fold difference relative to shCon sample. Statistical analysis was performed by Student’s t-test compared to the LKO lentivirus control; ***P < 0.001, n=3. Inset represent EGFR protein knockdown. (F) Knockdown of EGFR expression obliterates UTI89 invasion. Bacterial invasion was determined using the gentamicin protection assay and results are shown as fold change from control shCon cells. Error bars represent SEM. Statistical analysis was performed by Student’s t-test compared to the LKO lentivirus control; ***P < 0.001, n=3. (G) Effect of EGFR knockdown on the UTI89 attachment to BECs. GFP-UTI89 attachment to host BECs was quantified under fluorescence microscopy. Results show the number of attached UTI89 per cell and 50 randomly-selected cells were counted from each group. Statistical analysis was performed by Student’s t-test compared to the LKO lentivirus control.

Article Snippet: Reagents were obtained as follows: Lipopolysaccharide (LPS) from E. coli 055:B5 and complete protease and phosphatase inhibitors from Sigma Aldrich (St. Louis, MO); CLI095 from InvivoGen (San Diego, CA); wortmannin, LY294002, and rapamycin from Cell Signaling Technology (Boston, MA); AZD8055 from Santa Cruz Biotechnology (Santa Cruz, CA); BAY 80- 6946, Afatinib and MK-2206 from Selleckchem (Houston, TX); AG1478 from Cayman Chemical (Ann Arbor, MI); and gentamicin from Gibco (Grans Island, NY).

Techniques: Phospho-proteomics, Control, Western Blot, Knockdown, shRNA, Gene Expression, Quantitative RT-PCR, Expressing, Fluorescence, Microscopy

Journal: Stem Cell Research & Therapy

Article Title: iRGD-modified 3D exosomes delivered miR-99b-5p induces ferroptosis to inhibit colorectal cancer progression by regulating FGFR3/PI3K/AKt pathway

doi: 10.1186/s13287-026-05129-8

Figure Lengend Snippet: The 3D-Exo-miR-99b-5p derived from MSCs promotes ferroptosis and suppresses FGFR3/PI3K/AKt pathway in CRC cells. A–B TEM micrographs of LoVo and SW480 cells treated with Erastin and DMSO, red arrow indicated the morphological change of mitochondria. (C) Western blot assay of GPX4 expression in LoVo and SW480 cells. D The levels of Fe 2+ , GSH, ROS and GPX4 in LoVo and SW480 cells detected by ELISA and immunofluorescence ( n = 3). E–F The expression of FGFR3 in LoVo and SW480 cells assessed by western blot assay ( n = 3); G–H Western blot assay of PI3K, p-PI3K, AKt, and p-AKt expression levels in CRC cells co-cultured with 3D-Exo-miR-99b-5p ( n = 3). I–P Verification of overexpression (OE) and knockdown (Si) of FGFR3 in LoVo and SW480 cells through Real-time PCR and western blot assay ( n = 3). Q–R Western blot assay of PI3K, p-PI3K, AKt, and p-AKt expression in CRC cells transfected with OE- or Si-FGFR3 vectors ( n = 3). Student’s t-test was used to compare two groups, while one-way ANOVA followed by Tukey’s post hoc test was used for comparisons between three groups. Data are presented as means ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: To investigate the mechanisms involved in ferroptosis, cells were treated with the ferroptosis inducer Erastin (Cat. No. HY-15763, MCE), the ferroptosis inhibitor ferrostatin-1 (Cat. No. HY-100579, MCE, New Jersey, USA), the pyroptosis inhibitor Z-VAD-FMK (Cat. No. A834991, AmBeed, Shanghai, China), the RIP1 inhibitor Necrostatin-1 (Cat. No. A181851, AmBeed), the PI3K signaling pathway inhibitor LY294002 (Cat. No. HY-10108, MCE), and the PI3K signaling pathway activator 740Y-P (Cat. No. HY-P0175, MCE).

Techniques: Derivative Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Cell Culture, Over Expression, Knockdown, Real-time Polymerase Chain Reaction, Transfection

Journal: Stem Cell Research & Therapy

Article Title: iRGD-modified 3D exosomes delivered miR-99b-5p induces ferroptosis to inhibit colorectal cancer progression by regulating FGFR3/PI3K/AKt pathway

doi: 10.1186/s13287-026-05129-8

Figure Lengend Snippet: The 3D-Exo-miR-99b-5p regulates the ferroptosis process by inhibiting the PI3K/AKt pathway. A–B Western blot analysis of PI3K, p-PI3K, AKt, and p-AKt expression levels in CRC cells after treatment with Erastin or combined with Fer-1 ( n = 3). C–J The levels of Fe 2+ , GSH, and ROS in LoVo and SW480 cells detected by ELISA and immunofluorescence ( n = 3). K–L Western blot assay of GPX4 expression in LoVo and SW480 cells ( n = 3). One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between three groups. Data are presented as means ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: To investigate the mechanisms involved in ferroptosis, cells were treated with the ferroptosis inducer Erastin (Cat. No. HY-15763, MCE), the ferroptosis inhibitor ferrostatin-1 (Cat. No. HY-100579, MCE, New Jersey, USA), the pyroptosis inhibitor Z-VAD-FMK (Cat. No. A834991, AmBeed, Shanghai, China), the RIP1 inhibitor Necrostatin-1 (Cat. No. A181851, AmBeed), the PI3K signaling pathway inhibitor LY294002 (Cat. No. HY-10108, MCE), and the PI3K signaling pathway activator 740Y-P (Cat. No. HY-P0175, MCE).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Stem Cell Research & Therapy

Article Title: iRGD-modified 3D exosomes delivered miR-99b-5p induces ferroptosis to inhibit colorectal cancer progression by regulating FGFR3/PI3K/AKt pathway

doi: 10.1186/s13287-026-05129-8

Figure Lengend Snippet: Targeted anti-tumor therapy of iRGD-Exo-miR-99b-5p in vivo. A Fluorescent images of whole-body imaging of tumor-bearing mice after treatment with DiR-labeled exosomes ( n = 3). B Representative images of ex vivo fluorescent signals in the major organs at two days after the last injection ( n = 3). C–D Cell viability measured by CCK8 assay after three different treatments in LoVo and SW480 cells ( n = 3). Quantitation of synergism and antagonism in iRGD-Exo-miR-99b-5p and 5-FU combination. Fa versus CI plots were generated by CompuSyn. CI > 1, antagonism; CI < 1, synergy; CI = 1, additivity. E–G The tumor weight and volume of mice in each group ( n = 5). H Body weight changes in tumor-bearing mice in each group ( n = 5). I HE staining and IHC staining of Ki67 in tumor tissues ( n = 5). J IHC score for Ki-67 staining in each group ( n = 5). K Changes of Fe 2+ , ROS, GSH and GPX expression in tumor tissues of each group ( n = 5). L–M Changes of FGFR3, P-PI3K, PI3K, p-AKt, and AKt expression in tumor tissues of each group ( n = 3). One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between three groups. Data are presented as means ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: To investigate the mechanisms involved in ferroptosis, cells were treated with the ferroptosis inducer Erastin (Cat. No. HY-15763, MCE), the ferroptosis inhibitor ferrostatin-1 (Cat. No. HY-100579, MCE, New Jersey, USA), the pyroptosis inhibitor Z-VAD-FMK (Cat. No. A834991, AmBeed, Shanghai, China), the RIP1 inhibitor Necrostatin-1 (Cat. No. A181851, AmBeed), the PI3K signaling pathway inhibitor LY294002 (Cat. No. HY-10108, MCE), and the PI3K signaling pathway activator 740Y-P (Cat. No. HY-P0175, MCE).

Techniques: In Vivo, Imaging, Labeling, Ex Vivo, Injection, CCK-8 Assay, Quantitation Assay, Generated, Staining, Immunohistochemistry, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Activation of vascular adhesion protein-1 on liver endothelium results in an NF-kappaB-dependent increase in lymphocyte adhesion.

doi: 10.1002/hep.21497

Figure Lengend Snippet: Fig. 2. Hybridization of labeled NF-B oligonucleotide probe with HSEC nuclear extracts is specific and activation of NF-B is dependent upon PI3 kinase, p38, and MEK activity. (A) Representative EMSA for treatment of HSECs with TNF- (10 ng/ml, 2 hours) or benzylamine plus sodium orthovanadate (BenzVan, both 100 M, 1 hour). Specificity of the NF-B band was confirmed by addition of specific mouse monoclonal antisera to NF-B RelA (p65) (1:300 dilution of manufacturer’s stock; NF-B p65) or a 100-fold molar excess of unlabeled probe (NF-B unlabeled) to nuclear extract from cells treated with VAP-1 substrate plus vanadate. (B) Representative EMSA in which HSECs were treated with TNF- (10 ng/ml, 2 hours) or benzylamine plus sodium orthovanadate (BenzVan, both 100 M, 2-8 hours as indicated). Some samples were treated with the selective NF-B inhibitor CAPE (25 g/ml for 8 hours alone, or for 30 minutes before substrate treatment with subsequent analysis at 2-8 hours). Activation of NF-B occurs within 2 hours and has diminished by 24 hours. CAPE completely prevents NF-B activation in response to benzylamine and vanadate. (C) Representative EMSA and densitometry data for experiments in which inhibitors were used to block activation of NF-B in response to treatment of HSECs with TNF- (10 ng/ml, 2 hours) or benzylamine plus sodium orthovanadate (BenzVan, both 100 M, 1 hour). All inhibitors were added for 30 minutes before substrate administration; PI3 kinase inhibitor LY294002 (50 M), p38 inhibitor SB203580 (100 M), MEK inhibitor PD 98059 (50 M); semicarbazide (100 M). EMSA data represents results from HSECs isolated from a representative liver specimen. Data in the densitometry graph are expressed as the mean SEM increase in density of NF-B bands from unstimulated cells compared with signal generated from a control lane of nuclear extract with control-labeled probe alone (Cont). *Significant difference between inhibitor-treated samples compared with cells treated with benzylamine plus vanadate alone (paired Student t test; P 0.02; n 4 samples from different cell isolates).

Article Snippet: The contribution of phosphatidylinositol-3 (PI3) kinase, p38 mitogen-activated protein kinase (MAPK), and mitogen-activated protein kinase kinase (MEK) to endothelial NF- B activation was assessed via pretreatment with the inhibitors LY294002 (50 M, 30 minutes pretreatment; Biosource, UK), SB203580 (100 M, Calbiochem), and PD98059 (50 M, Calbiochem), respectively, for 30 minutes before incubation with VAP-1 substrate.

Techniques: Hybridization, Labeling, Activation Assay, Activity Assay, Blocking Assay, Isolation, Generated, Control

Journal: ERJ Open Research

Article Title: Lipoxin A 4 promotes lung epithelial repair whilst inhibiting fibroblast proliferation

doi: 10.1183/23120541.00079-2015

Figure Lengend Snippet: Pro-proliferative effect of lipoxin A 4 (LXA 4 ) is phosphatidylinositol 3′-kinase dependent and mediated by ALX/formyl peptide L1 receptor. 100 nM LXA 4 promoted proliferation of alveolar type II cell (ATII) cells. Pretreatment with 10 µM LY294002, a phosphatidylinositol 3′-kinase inhibitor, inhibited the effects of LXA 4 on ATII cell proliferation suggesting that the pro-proliferation effects of LXA 4 are phosphatidylinositol 3′-kinase dependent. BOC-2, the formyl peptide receptor antagonist, was re-incubated with primary human ATII cells at 10 µM for 1 h before LXA 4 treatment of ATII cells. BOC-2 treatment inhibited the effects of LXA 4 on the proliferation of primary human ATII cells suggesting that the promoting proliferation effects of LXA 4 are formyl peptide receptor dependent. Data are mean± sem of three independent experiments.

Article Snippet: Cells were treated with TGF-β for 24 h with or without preincubation with the PI3 kinase inhibitor LY294002 (10 µM) or BOC-2 (10 µM).

Techniques: Incubation

Journal: ERJ Open Research

Article Title: Lipoxin A 4 promotes lung epithelial repair whilst inhibiting fibroblast proliferation

doi: 10.1183/23120541.00079-2015

Figure Lengend Snippet: Effect of lipoxin A 4 (LXA 4 ) on primary human lung fibroblast (HLF) proliferation in response to transforming growth factor (TGF)-β. Cell proliferation studies confirmed that LXA 4 inhibited proliferation of primary HLF induced by TGF-β. Cultured and serum-deprived cells were treated with 1 ng·mL −1 TGF-β for 24 h with or without pre-incubation with LY294002 (10 µM) for 1 h, BOC-2 (10 µM) for 1 h. Data are mean± sem of three independent experiments. # : p=0.05; + : p=0.05, compared with no treatment group; ¶ : p<0.01, compared with TGF-β only group.

Article Snippet: Cells were treated with TGF-β for 24 h with or without preincubation with the PI3 kinase inhibitor LY294002 (10 µM) or BOC-2 (10 µM).

Techniques: Cell Culture, Incubation