Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cancer-associated fibroblast derived CXCL14 drives cisplatin chemoresistance by enhancing nucleotide excision repair in bladder cancer

doi: 10.1186/s13046-025-03487-4

Figure Lengend Snippet: The CXCL14/CCR7 signaling axis enhances ERCC4 transcription through JAK2/STAT3 activation and transcriptional regulation. A Venn diagram showing the overlapping promotors of ERCC4 predicted by JASPAR ( https://jaspar.genereg.net/ ) and PROMO ( https://alggen.lsi.upc.es/ ). B Correlation analysis between STAT3 and ERCC4 (GEPIA, http://gepia.cancer-pku.cn/ ). C Representative immunofluorescence images showing the expression and distribution of p-STAT3 in T24 cells following treatment with exogenous CXCL14 or STAT3i. Scale bar, 20 μm. D RT-qPCR analysis was used to detect ERCC4 expression in T24 cells subjected to different treatments, with ACTB as the internal reference. E Western blot analysis of ERCC4, STAT3, and phosphorylated STAT3 expression in T24 cells treated with the rhCXCL14 protein or a STAT3 inhibitor. F The luciferase activities of the ERCC4 promoter containing sequences in STAT3 overexpressing 293 T cells were assessed. G Luciferase activity in T24 cells was assessed after treatment with rhCXCL14 or STAT3i. H STAT3 binding elements at the promoter region of ERCC4 were predicted by JASPAR. I A schematic representation of the ERCC4 promoter sequence and its mutated variants, which were individually or jointly cloned and inserted into the pGL3 vector for the luciferase reporter assay. J , K The luciferase activities of the ERCC4 promoter containing sequences in different groups of T24 and 293 T cells were assessed. L Western blot analysis of ERCC4 expression and STAT3 phosphorylation in T24 cells inhibited by different receptors. M Molecular docking simulation studies were conducted to explore the interactions between CCR7 and ERCC4. N The interactions between CXCL14 and CCR7 were confirmed with co-IP, followed by Western blot analysis. O Immunofluorescence images of HA-tagged CXCL14(red), CCR7 (green), and DAPI (blue) in T24 cells. The intensity profiles of CXCL14 (red lines) and CCR7 (green lines) colocalization signals are shown as plotted lines at three random sites. Scale bar, 10 μm. P The inducible knockdown efficiency of CCR7, along with the expression levels of ERCC4, phosphorylated JAK2, and phosphorylated STAT3, was evaluated in T24 cells subjected to different treatment conditions. Q Representative immunofluorescence images showing the expression and subcellular localization of phosphorylated STAT3 in T24 cells subjected to various treatments. Scale bar, 20 μm. R The expression of ERCC4, CCR7, p-JAK2, and p-STAT3 was analysed in T24 cells following treatment with specific inhibitors or CCR7-targeting shRNAs. The data are presented as the means ± SDs, and the experiments were performed at least three times. P values were determined by unpaired Student’s t test or one-way ANOVA followed by Tukey’s test (* P < 0.05 and ** P < 0.01), and the Pearson correlation coefficient was used for correlation analysis

Article Snippet: Various inhibitors including doxycycline (200 ng/mL; GLPBIO, USA, cat. GC13750), STAT3 inhibitor (STAT3i) (S3I-201,5 μM; Selleck, USA, cat. S1155), and CCR7 inhibitor (CCR7i) (Cmp2105, 50 μM; MCE, USA, cat. HY-133073) were employed to treat the cell lines as specified.

Techniques: Activation Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Binding Assay, Sequencing, Clone Assay, Plasmid Preparation, Reporter Assay, Phospho-proteomics, Co-Immunoprecipitation Assay, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cancer-associated fibroblast derived CXCL14 drives cisplatin chemoresistance by enhancing nucleotide excision repair in bladder cancer

doi: 10.1186/s13046-025-03487-4

Figure Lengend Snippet: Disruption of the CXCL14/CCR7/JAK2/STAT3 axis increases the sensitivity of bladder cancer cells to cisplatin. A Schematic illustration of the untouched coculture system of CAFs and T24 cells. The cells were pretreated with different transfections and utilized for further experiments after coculture for 24 h. B Protein levels of ERCC4, CCR7, γH2AX, cleaved caspase-3, STAT3, and phosphorylated STAT3 were evaluated by Western blot in T24 cells treated with various media or inhibitors in the presence of 1 μg/mL cisplatin. C Representative immunofluorescence images showing γH2AX expression in T24 cells under different treatment conditions. Scale bar, 20 μm. D Representative images illustrating DNA fragmentation in T24 cells following different treatments. Scale bar, 100 μm. E Cell viability in each treatment group was assessed using the CCK-8 assay after 48 h of cisplatin exposure. F Quantification of γH2AX-positive nuclei (defined as ≥ 10 foci per nucleus) on the basis of the results shown in Fig. 5C. G Tail DNA content, derived from Fig. 5D, was used as an indicator of DNA damage severity. H Schematic depicting the coinjection of CAFs and T24 cells with different shRNAs and the intraperitoneal injection of cisplatin in the presence or absence of the CCR7 or STAT3 inhibitor. I The relative tumor growth rate of T24 cells transfected with different shRNAs and coinjected with CAFs deficient in CXCL14 expression or negative controls. Tumor-bearing mice were intraperitoneally administered cisplatin along with medium or inhibitors twice a week, starting when the average tumor volume reached approximately 200 mm^3 (indicated by arrows). J Representative gross tumor images from each treatment group. K Representative images of HE, Masson and IHC staining of subcutaneous xenograft tissues from different groups. Scale bar, 20 μm. L Representative bright-field images of bladder cancer patient-derived organoids subjected to different treatments in the presence of 1 μg/mL cisplatin (the red arrows indicate apoptotic cells). Scale bar, 100 μm. M Line graphs depicting the growth of tumor organoids in each treatment group. N Organoid size monitored after treatment for 12 days. O AO/PI staining allowed for the simultaneous detection of live and dead cells in the organoid lines. Green fluorescence represented live cells, whereas red fluorescence represents dead cells. Scale bar, 100 μm. The data are presented as the means ± SDs, and the experiments were performed at least three times. P values were determined by unpaired Student’s t test or one-way ANOVA followed by Tukey’s test; * P < 0.05 and ** P < 0.01

Article Snippet: Various inhibitors including doxycycline (200 ng/mL; GLPBIO, USA, cat. GC13750), STAT3 inhibitor (STAT3i) (S3I-201,5 μM; Selleck, USA, cat. S1155), and CCR7 inhibitor (CCR7i) (Cmp2105, 50 μM; MCE, USA, cat. HY-133073) were employed to treat the cell lines as specified.

Techniques: Disruption, Transfection, Western Blot, Immunofluorescence, Expressing, CCK-8 Assay, Derivative Assay, Injection, Immunohistochemistry, Staining, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cancer-associated fibroblast derived CXCL14 drives cisplatin chemoresistance by enhancing nucleotide excision repair in bladder cancer

doi: 10.1186/s13046-025-03487-4

Figure Lengend Snippet: CXCL14-induced chemoresistant cells convert fibroblasts into CAFs through lactate secretion. A IF staining of α-SMA and vimentin in NFs subjected to different treatments for 24 h. Scale bar, 10 μm. B , C RT-qPCR analysis was performed to evaluate the transcription levels of ACTA2 and CXCL14 in NFs following coculture with T24 cells or exposure to CM with different pretreatments, and the levels were further detected at the translational level. D Western blotting was used to assess the expression of α-SMA and CXCL14 in normal fibroblasts subjected to various conditioned medium treatments. E Western blot results demonstrated normal fibroblast activation under different inhibitor treatments. F The ECAR of T24 cells pretreated with NF/CM or CAF/CM was detected by Seahorse analysis. G Glycolysis level, capacity and reversal ability of T24 cells subjected to different treatments were measured by the Seahorse assay. H The ECAR of T24 cells pretreated with rhCXCL14 or CCR7i was detected by Seahorse analysis. I Glycolysis level, capacity and reversal ability of T24 cells subjected to different treatments were measured by the Seahorse assay. J , K The mRNA expression of key glycolysis genes were examined by RT-qPCR in T24 and UM-UC-3 cells subjected to different CAF-/NF-CM treatments. L , M Western blot analysis was performed to assess HK2 and LDHA expression in T24 and UM-UC-3 cells following exposure to various inhibitors or shRNAs targeting the CCR7/STAT3/ERCC4 axis. N , P A collagen contraction assay was used to assess the contraction ability of NFs treated as indicated, and the results were quantified using ImageJ software. O Representative immunofluorescence images showing α-SMA and vimentin expression in NFs treated as indicated. Scale bar, 10 μm. Q Western blot analysis of the expression of α-SMA and CXCL14 in NFs subjected to various treatments was performed to assess their activation. Additionally, RT-qPCR was conducted to verify activation at the transcriptional level ( R , S ). The data are presented as the means ± SDs, and the experiments were performed at least three times. P values based on unpaired Student’s t test or one-way ANOVA followed by Tukey’s test; * P < 0.05 and ** P < 0.01

Article Snippet: Various inhibitors including doxycycline (200 ng/mL; GLPBIO, USA, cat. GC13750), STAT3 inhibitor (STAT3i) (S3I-201,5 μM; Selleck, USA, cat. S1155), and CCR7 inhibitor (CCR7i) (Cmp2105, 50 μM; MCE, USA, cat. HY-133073) were employed to treat the cell lines as specified.

Techniques: Staining, Quantitative RT-PCR, Western Blot, Expressing, Activation Assay, Contraction Assay, Software, Immunofluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting STAT3 prevents bile reflux‐induced oncogenic molecular events linked to hypopharyngeal carcinogenesis

doi: 10.1111/jcmm.17011

Figure Lengend Snippet: Silencing of STAT3 suppressed total STAT3, p‐STAT3 (Tyr705) and bcl2 protein levels, with a minimal suppressive effect on p‐NF‐κB (p65 S536), in BA‐treated HCs. (A) (a) Immunofluorescence staining for p‐STAT3 (Tyr705) (green: p‐STAT3; blue: nuclear DNA staining with DAPI; scale bar 20 μm; Zen imagining software). (b) Immunofluorescence staining for p‐NF‐κB (p65 S536) (green: p‐ NF‐κB (p65 S536); blue: nuclear DNA staining with DAPI; scale bar 20 μm; Zen imagining software). (B) Western blot analysis for total STAT3 in BA‐treated HCs and controls (Control: media at pH 7.0, including vehicle; Acid: media at pH 4.0) with knockdown of STAT3 gene. Graph depicts the total STAT3 protein levels, in BA and control‐treated HCs after STAT3 knockdown. (C) Graphs depict the total protein levels, by ELISA, of p‐STAT3 (Tyr705), Bcl‐2 and p‐NF‐κB (p65 S536) in BA‐treated HCs after STAT3 knockdown. (from left to right) si ‐ C : media at pH 7.0 plus Control siRNA; si ‐ STAT3 : media at pH 7.0 plus STAT3 siRNA; BA + si ‐ C : Bile at pH 4.0 plus Control siRNA; BA + si ‐ STAT3 : Bile at pH 4.0 plus STAT3 siRNA; Acid + si ‐ C : media at pH 4.0 plus Control siRNA; Acid + si ‐ STAT3 : media at pH 4.0 plus STAT3 siRNA (β‐actin was used to normalize total protein extracts; t test; multiple comparisons by Holm‐Sidak, ** p < 0.005; *** p < 0.0005; GraphPad Prism 7.0; means ± SD of three independent experiment)

Article Snippet: Experimental media included (i) ‘BA’, acidic bile at pH 4.0, (ii) ‘Nif’, BA plus 10 μM Nifuroxazide (CAS 965‐52‐6; Santa Cruz Biotechnology Inc.), (iii) ‘SI3‐201’, BA plus 50 μM STAT3 Inhibitor VI, S3I‐201 (CAS 19983‐44‐9; Santa Cruz Biotechnology Inc.) and (iv) ‘STA‐21’, BA plus 20 μM STA‐21 (CAS 28882‐53‐3; Santa Cruz Biotechnology Inc.; ; Table ).

Techniques: Immunofluorescence, Staining, Software, Western Blot, Control, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting STAT3 prevents bile reflux‐induced oncogenic molecular events linked to hypopharyngeal carcinogenesis

doi: 10.1111/jcmm.17011

Figure Lengend Snippet: Pharmacologic inhibition of STAT3 prevents BA‐induced nuclear localization of p‐STAT3 and Bcl‐2 overexpression, with a minimal effect on p‐NF‐κB activation, in treated HCs. (A) (a) Immunofluorescence staining for p‐STAT3 (Tyr705) (green: p‐STAT3; blue: nuclear DNA staining with DAPI; scale bar 20 μm; Zen imagining software). (b) Graph depicts the nuclear protein levels of p‐STAT3 (Tyr705) in treated HCs. (B) (a) Immunofluorescence staining for p‐NF‐κB (p65 S536) (green: p‐ NF‐κB (p65 S536); blue: nuclear DNA staining with DAPI; scale bar 20 μm; Zen imagining software). (b) Graphs created depict the nuclear protein levels of p‐NF‐κB (p65 S536) in treated HCs. (from left to the right). Control : media at pH 7.0 (including vehicle); BA : acidic bile (pH 4.0); Acid : media at pH 4.0; Nif : BA plus Nifuroxazide; SI3 ‐ 201 : BA plus STAT3 inhibitor VI (S3I‐201); STA ‐ 21 : BA plus STA‐21. (C) Graphs depict the (a) nuclear protein levels of p‐STAT3 (Tyr705), (b) cytoplasmic levels of Bcl‐2 and (c) nuclear levels of p‐NF‐κB (p65 S536), in BA‐treated HCs, with or without pharmacologic inhibition of STAT3, by Western blot analysis. Acid : media at pH 4.0; Nif : acidic bile plus Nifuroxazide. BA : acidic bile (H 4.0); (Histone 1 and β‐actin were used to normalize nuclear and cytoplasmic protein extracts, respectively; by Image Lab 5.2 analysis software, Bio‐Rad; t test; multiple comparisons by Holm‐Sidak, * p < 0.05; ** p < 0.005; *** p < 0.0005; GraphPad Prism 7.0; means ± SD of three independent experiment)

Article Snippet: Experimental media included (i) ‘BA’, acidic bile at pH 4.0, (ii) ‘Nif’, BA plus 10 μM Nifuroxazide (CAS 965‐52‐6; Santa Cruz Biotechnology Inc.), (iii) ‘SI3‐201’, BA plus 50 μM STAT3 Inhibitor VI, S3I‐201 (CAS 19983‐44‐9; Santa Cruz Biotechnology Inc.) and (iv) ‘STA‐21’, BA plus 20 μM STA‐21 (CAS 28882‐53‐3; Santa Cruz Biotechnology Inc.; ; Table ).

Techniques: Inhibition, Over Expression, Activation Assay, Immunofluorescence, Staining, Software, Control, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting STAT3 prevents bile reflux‐induced oncogenic molecular events linked to hypopharyngeal carcinogenesis

doi: 10.1111/jcmm.17011

Figure Lengend Snippet: Luciferase assay for STAT3 transcriptional activity in BA‐treated HCs, with targeting STAT3 pathway. Columns represent ratios of STAT3 luciferase transcriptional activity in HCs transfected with STAT3 luciferase responsive element (Creport‐STAT3) versus luciferase activity in HCs transfected with control luciferase reporter (Creport‐control) (A) STAT3 knockdown . (from left to right) si ‐ C : media at pH 7.0 plus Control siRNA; si ‐ STAT3 : media at pH 7.0 plus STAT3 siRNA; BA : Bile at pH 4.0 plus Control siRNA; BA + si ‐ STAT3 : Bile at pH 4.0 plus STAT3 siRNA; Acid : media at pH 4.0 plus Control siRNA; Acid + si ‐ STAT3 : media at pH 4.0 plus STAT3 siRNA. (B) Pharmacologic inhibition of STAT3 . (from left to right) Cntl : media at pH 7.0 (including vehicle); BA : acidic bile (pH 4.0); Acid : media at pH 4.0; Nif : BA plus Nifuroxazide; SI3 ‐ 201 : BA plus STAT3 inhibitor VI (SI3‐201); STA ‐ 21 : acidic bile plus STA‐21. ( t test; multiple comparisons by Holm‐Sidak, ** p < 0.005; *** p < 0.0005; GraphPad Prism 7.0; means ± SD of three independent experiment)

Article Snippet: Experimental media included (i) ‘BA’, acidic bile at pH 4.0, (ii) ‘Nif’, BA plus 10 μM Nifuroxazide (CAS 965‐52‐6; Santa Cruz Biotechnology Inc.), (iii) ‘SI3‐201’, BA plus 50 μM STAT3 Inhibitor VI, S3I‐201 (CAS 19983‐44‐9; Santa Cruz Biotechnology Inc.) and (iv) ‘STA‐21’, BA plus 20 μM STA‐21 (CAS 28882‐53‐3; Santa Cruz Biotechnology Inc.; ; Table ).

Techniques: Luciferase, Activity Assay, Transfection, Control, Knockdown, Inhibition

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting STAT3 prevents bile reflux‐induced oncogenic molecular events linked to hypopharyngeal carcinogenesis

doi: 10.1111/jcmm.17011

Figure Lengend Snippet: Silencing of STAT3 suppressed BA‐induced transcriptional changes. Columns represent mRNA levels of each analysed gene in treated groups with knockdown of STAT3 (mRNA silenc) by qPCR (normalized mRNAs to hGAPDH reference control) (from left to right) si ‐ C : media at pH 7.0 plus Control siRNA; media at pH 7.0 plus si ‐ STAT3 : STAT3 siRNA; BA : Bile at pH 4.0 plus Control siRNA; BA + si ‐ STAT3 : Bile at pH 4.0 plus STAT3 siRNA; Acid : media at pH 4.0 plus Control siRNA; Acid+si‐STAT3: media at pH 4.0 plus STAT3 siRNA. ( t test; multiple comparisons by Holm‐Sidak, * p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.00005; GraphPad Prism 7.0; means ± SD of three independent experiment)

Article Snippet: Experimental media included (i) ‘BA’, acidic bile at pH 4.0, (ii) ‘Nif’, BA plus 10 μM Nifuroxazide (CAS 965‐52‐6; Santa Cruz Biotechnology Inc.), (iii) ‘SI3‐201’, BA plus 50 μM STAT3 Inhibitor VI, S3I‐201 (CAS 19983‐44‐9; Santa Cruz Biotechnology Inc.) and (iv) ‘STA‐21’, BA plus 20 μM STA‐21 (CAS 28882‐53‐3; Santa Cruz Biotechnology Inc.; ; Table ).

Techniques: Knockdown, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting STAT3 prevents bile reflux‐induced oncogenic molecular events linked to hypopharyngeal carcinogenesis

doi: 10.1111/jcmm.17011

Figure Lengend Snippet: The effect of STAT3‐inhibition on oncogenic mRNA phenotype caused by acidic bile in HCs

Article Snippet: Experimental media included (i) ‘BA’, acidic bile at pH 4.0, (ii) ‘Nif’, BA plus 10 μM Nifuroxazide (CAS 965‐52‐6; Santa Cruz Biotechnology Inc.), (iii) ‘SI3‐201’, BA plus 50 μM STAT3 Inhibitor VI, S3I‐201 (CAS 19983‐44‐9; Santa Cruz Biotechnology Inc.) and (iv) ‘STA‐21’, BA plus 20 μM STA‐21 (CAS 28882‐53‐3; Santa Cruz Biotechnology Inc.; ; Table ).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting STAT3 prevents bile reflux‐induced oncogenic molecular events linked to hypopharyngeal carcinogenesis

doi: 10.1111/jcmm.17011

Figure Lengend Snippet: Pharmacologic inhibition of STAT3 inhibits the acidic bile‐induced cancer‐related and inflammatory mRNA phenotype. Columns represent mRNA levels of each analysed gene in treated groups by acidic bile with or without Nifuroxazide, SI3‐201 (STAT3 inhibitor VI) or STA‐21 and controls, by qPCR. (from left to right) Cntl : media at pH 7.0 (including vehicle); BA : acidic bile (pH 4.0); Acid : media at pH 4.0; Nif : BA plus Nifuroxazide; SI3 ‐ 201 : BA plus STAT3 inhibitor VI (S3I‐201); STA ‐ 21 : BA plus STA‐21. (Normalized mRNAs to hGAPDH reference control; mean ± SD of three independent experiments)

Article Snippet: Experimental media included (i) ‘BA’, acidic bile at pH 4.0, (ii) ‘Nif’, BA plus 10 μM Nifuroxazide (CAS 965‐52‐6; Santa Cruz Biotechnology Inc.), (iii) ‘SI3‐201’, BA plus 50 μM STAT3 Inhibitor VI, S3I‐201 (CAS 19983‐44‐9; Santa Cruz Biotechnology Inc.) and (iv) ‘STA‐21’, BA plus 20 μM STA‐21 (CAS 28882‐53‐3; Santa Cruz Biotechnology Inc.; ; Table ).

Techniques: Inhibition, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting STAT3 prevents bile reflux‐induced oncogenic molecular events linked to hypopharyngeal carcinogenesis

doi: 10.1111/jcmm.17011

Figure Lengend Snippet: STAT3 knockout or its pharmacologic inhibition effects on cell viability of BA‐treated HCs (A). Graph depicts differences in cell survival (% viable cells) in BA‐treated HCs with versus without STAT3 knockout. si ‐ C : Control siRNA; si ‐ STAT3 : STAT3 siRNA; BA + si ‐ C : BA plus Control siRNA; BA + si ‐ STAT3 : BA plus STAT3 siRNA. (B) Graph depicts the survival rates (% of viable cells) in BA‐treated HCs with versus without STAT3 pharmacologic inhibition (from left to right) BA : acidic bile (pH 4.0); Nif : BA plus Nifuroxazide; SI3 ‐ 201 : BA plus STAT3 inhibitor VI (S3I‐201); STA ‐ 21 : BA plus STA‐21. (* p < 0.05; ** p < 0.005, by t test; multiple comparisons by Holm‐Sidak; GraphPad Prism 7 software; Data are derived from three independent experiments)

Article Snippet: Experimental media included (i) ‘BA’, acidic bile at pH 4.0, (ii) ‘Nif’, BA plus 10 μM Nifuroxazide (CAS 965‐52‐6; Santa Cruz Biotechnology Inc.), (iii) ‘SI3‐201’, BA plus 50 μM STAT3 Inhibitor VI, S3I‐201 (CAS 19983‐44‐9; Santa Cruz Biotechnology Inc.) and (iv) ‘STA‐21’, BA plus 20 μM STA‐21 (CAS 28882‐53‐3; Santa Cruz Biotechnology Inc.; ; Table ).

Techniques: Knock-Out, Inhibition, Control, Software, Derivative Assay