Journal: Cell death & disease

Article Title: Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts.

doi: 10.1038/s41419-022-04506-4

Figure Lengend Snippet: Fig. 6 An increase in levels of acetyl-CoA and H3K27ac promotes CXCL5 secretion in CAFs. The effects of acetate treatment on acetyl-CoA levels in LX-2 cells incubated with ExoHCT116 (A) and ExoSW480 (B). Western blot showed the effects of acetate treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoHCT116 (C) and ExoSW480 (D). E The effects of Trichostatin A (TSA) treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoHCT116 KD and their relative control. F The effects of C646 treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoSW480 OE and their relative control. G, H Chromatin immunoprecipitation (ChIP) assays using IgG as a control were performed with antibody against H3K23ac. Examination of H3K27 acetylation status in CXCL5 gene promoter region in LX-2 cells incubated with ExoHCT116 KD and ExoHCT116 KD Ctrl or in LX-2 cells incubated with ExoHCT116 KD and ExoHCT116 KD Ctrl and treated with TSA (G). Examination of H3K27 acetylation status in CXCL5 gene promoter region in LX-2 cells incubated with ExoSW480 OE and ExoSW480 OE Ctrl or in LX- 2 cells incubated with ExoSW480 OE and ExoSW480 OE Ctrl and treated with C646 (H). Each experiment was performed in triplicate. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cells were cultured in the presence of appropriate exosomes (20μg/mL) for 48 h before supernatants were collected, and the CXCL5 level was detected by CXCL5 ELISA kit (EK0728, BOSTER) according to the manufacturer’s protocols.

Techniques: Incubation, Western Blot, Control, Chromatin Immunoprecipitation

Journal: Cell death & disease

Article Title: Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts.

doi: 10.1038/s41419-022-04506-4

Figure Lengend Snippet: Fig. 8 A schematic diagram of mechanism of exosomal HSPC111 facilitates CRLM via reprogramming lipid metabolism in CAFs. Exosomal HSPC111 derived from CRC cells phosphorylates ACLY in CAFs, leading to the increased levels of acetyl-CoA and histone acetylation to secrete CXCL5, and resulting in CRC cells colonized in liver via the CXCL5-CXCR2 axis.

Article Snippet: Cells were cultured in the presence of appropriate exosomes (20μg/mL) for 48 h before supernatants were collected, and the CXCL5 level was detected by CXCL5 ELISA kit (EK0728, BOSTER) according to the manufacturer’s protocols.

Techniques: Derivative Assay

Journal: Infection and Immunity

Article Title: Intact Pneumococci Trigger Transcription of Interferon-Related Genes in Human Monocytes, while Fragmented, Autolyzed Bacteria Subvert This Response

doi: 10.1128/IAI.00960-16

Figure Lengend Snippet: RT-qPCR was used for validation of the Illumina results for selected genes. Three different expression patterns were revealed: in pattern A, exemplified by the TLR1, TLR6, CTDSP2, TRIM8, and LYL1 genes, the gene expression was downregulated in monocytes (obtained from three [TLR6] or four blood donors) following stimulation with pneumococci; in pattern B, exemplified by the IFIT3, CXCL10, RSAD2, and IL12B genes (four donors) and the CLCF1, CD40, and IFNB1 genes (three donors), intact pneumococci (LytA−) induced the strongest transcription of the genes, while autolyzed pneumococci (LytA+) were inferior inducers and even hampered the expression when admixed to intact bacteria (LytA+ and LytA); in pattern C, exemplified by the IL6 gene (seven donors), the CXCL1 gene (three donors), and the CXCL5, CCL20, and F3 genes (four donors), autolysis-competent pneumococci (LytA+) induced levels of gene transcripts similar to or higher than those of autolysis-incompetent bacteria (LytA−).

Article Snippet: Extracted RNA was analyzed by RT-qPCR for the following 17 genes according to the manufacturer's instructions regarding TaqMan chemistry (Life Technologies Europe BV): TLR1 (Hs00413978_m1), TLR6 (Hs00271977_s1), CTDSP2 (Hs00938977_m1), TRIM8 (Hs00229451_m1), LYL (Hs01089802_g1), IFIT3 (Hs01922752_s1), CXCL10 (Hs01124251_g1), RSAD2 (Hs00369813_m1), CLCF1 (Hs00757942_m1), IL12B (Hs01011518_m1), CD40 (Hs01002913_g1), INFB1 (Hs01077958_s1), IL6 (Hs00985639_m1), CXCL1 (Hs00605382_gH), CXCL5 (Hs01099660_g1), CCL20 (Hs00355476_m1), and F3 (Hs01076029_m1).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Expressing, Gene Expression, Bacteria

Journal: Infection and Immunity

Article Title: Intact Pneumococci Trigger Transcription of Interferon-Related Genes in Human Monocytes, while Fragmented, Autolyzed Bacteria Subvert This Response

doi: 10.1128/IAI.00960-16

Figure Lengend Snippet: Production of selected proteins in the monocytes was analyzed for validation of the Illumina results. Monocytes were stimulated with pneumococci capable of autolysis (LytA+) or incapable of autolysis (LytA−) or with a mixture of autolysis-competent and -incompetent pneumococci (LytA+ and LytA−), in which the concentration of intact bacteria was equivalent to that used in cultures stimulated with intact bacteria alone, or were left unstimulated (medium alone) for 24 h. The levels of IL-12, CXCL10, IL-6, CXCL1, CXCL5, and CCL20 in the supernatant then were analyzed by ELISA. CD40 expression and intracellular IFN-β were measured by flow cytometry. *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

Article Snippet: Extracted RNA was analyzed by RT-qPCR for the following 17 genes according to the manufacturer's instructions regarding TaqMan chemistry (Life Technologies Europe BV): TLR1 (Hs00413978_m1), TLR6 (Hs00271977_s1), CTDSP2 (Hs00938977_m1), TRIM8 (Hs00229451_m1), LYL (Hs01089802_g1), IFIT3 (Hs01922752_s1), CXCL10 (Hs01124251_g1), RSAD2 (Hs00369813_m1), CLCF1 (Hs00757942_m1), IL12B (Hs01011518_m1), CD40 (Hs01002913_g1), INFB1 (Hs01077958_s1), IL6 (Hs00985639_m1), CXCL1 (Hs00605382_gH), CXCL5 (Hs01099660_g1), CCL20 (Hs00355476_m1), and F3 (Hs01076029_m1).

Techniques: Biomarker Discovery, Concentration Assay, Bacteria, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: Infection and Immunity

Article Title: Intact Pneumococci Trigger Transcription of Interferon-Related Genes in Human Monocytes, while Fragmented, Autolyzed Bacteria Subvert This Response

doi: 10.1128/IAI.00960-16

Figure Lengend Snippet: Monocyte genes most strongly upregulated in response to intact or autolyzed pneumococci compared to nonstimulated monocytes a

Article Snippet: Extracted RNA was analyzed by RT-qPCR for the following 17 genes according to the manufacturer's instructions regarding TaqMan chemistry (Life Technologies Europe BV): TLR1 (Hs00413978_m1), TLR6 (Hs00271977_s1), CTDSP2 (Hs00938977_m1), TRIM8 (Hs00229451_m1), LYL (Hs01089802_g1), IFIT3 (Hs01922752_s1), CXCL10 (Hs01124251_g1), RSAD2 (Hs00369813_m1), CLCF1 (Hs00757942_m1), IL12B (Hs01011518_m1), CD40 (Hs01002913_g1), INFB1 (Hs01077958_s1), IL6 (Hs00985639_m1), CXCL1 (Hs00605382_gH), CXCL5 (Hs01099660_g1), CCL20 (Hs00355476_m1), and F3 (Hs01076029_m1).

Techniques:

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 3. Effects of hypoxia on CXCL6 and CXCL2 gene expression in co-culture systems. (A) The difference of CXCL6 gene expression between censored (n ¼ 95) and relapse/refractory (n ¼ 247) patients in TARGET database. (B to E) The gene expression of CXCL6 and CXCL2 of MSCs and HUAECs in the co-culture systems were measured by the co-culture/mono culture ratio. (B and D) MSC þ THP-1/MSC; (C and E) HUAEC þ THP-1/HUAEC. *P < 0.05, **P < 0.01, ***P < 0.001, and “ns” for no statistical significance. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Gene Expression, Co-Culture Assay

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 4. CXCL6 and CXCL2 content in the supernatant of hypoxia and THP-1 co-culture systems. (A and B) CXCL6 content in supernatant. (C and D) CXCL2 content in supernatant. *P < 0.05, **P < 0.01, and ***P < 0.001. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Co-Culture Assay

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 5. The effects of CXCL6 and CXCL2 on the proliferation and migration of MSCs and HUAECs. (A and B) The effects of CXCL6 (A) and CXCL2 (B). Among them, (a and b) described the effects of cytokines on MSCs and (c and d) described the effects of cytokines on HUAECs. (a and c) showed the effects of different concentrations of cytokines on target cells. (b and d) showed the time-dependent effect of cytokines on target cells. (C) The chemotaxis of CXCL6 and CXCL2 to MSCs and HUAECs respectively. The concentration of CXCL6 was 50 ng/mL and of CXCL2 was 100 ng/mL. Scale ¼ 100 mm. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Migration, Chemotaxis Assay, Concentration Assay

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 6. Expression of cleaved-caspase3 in MSCs and HUAECs. (A) Expression of cleaved-caspase-3 (green fluorescence) measured by immunofluorescence method increased significantly in hypoxia (1% O2) for 48 h, but decreased after CXCL6 was supplied. The reversion depended on the concentration of CXCL6. MSCs were pictured at 20 water objective and HUAECs were pictured at 20 air objective. The heatmap represented the change of average fluorescence intensity in each group. (B) Western blot method verified the above results. The histograms (MSCs on the left and HUAECs on the right) represented the relative gray value of each group. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Expressing, Fluorescence, Immunofluorescence, Concentration Assay, Western Blot

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 7. Angiogenesis ability of vascular endothelial cells authenticated in vitro. (A) HUAECs in standard culture conditions, CoCl2 simulate hypoxia and hypoxia plus CXCL6 or CM conditions in vitro were used to simulated angiogenesis. HUVECs tubule forming experiment was taken as positive control. (B to E) Quantitative comparison of the number of master junction and meshes as well as total meshes area and total segments length. Scale ¼ 200 mm. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: In Vitro, Positive Control, Comparison

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 8. Correlation between CXCL6 gene expression and HIF-1a gene expression in AML patients. The original data were downloaded from Cbioporta (https:// www.cbioportal.org/datasets), n ¼ 323. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Gene Expression

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 9. Regulation of CXCL6 expression by HIF-1a. (A) MSCs or HUAECs were treated with CoCl2 (200 mmol/L), with or without BAY87-2243 (1 mmol/L), and the protein expression of HIF-1a was detected by Western blot method after 24 or 48 h. (B) The relative expression of HIF-1a in three independent experiments was statistically analyzed. The x-axis coordinates represented CoCl2()/BAY(), CoCl2(þ)/BAY(), CoCl2(þ)/BAY(þ), respectively. (a) MSC–24 h; (b) MSC–48 h; (c) HUAEC–24 h; (d) HUAEC–48 h. (C) MSCs and HUAECs were cultured for 24 or 48 h under the condition of 1% O2, with or without BAY87-2243 (1 mmol/L). The relative expression of CXCL6 mRNA was detected by real-time PCR. (a) MSC–24 h; (b) MSC–48 h; (c) HUAEC–24 h; (d) HUAEC–48 h. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 11. CXCL6 activate mTOR in MSCs and HUAECs. (A) CoCl2 (200 mmol/L) was used to treat MSCs or HUAECs for 24 h with or without CXCL6 of 50 ng/Ml. (B) The statistical results of three groups of independent experiments, **P < 0.01. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques:

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 10. Negative feedback regulation of CXCL6 on HIF-1a. (A) MSCs and HUAECs were cultured for 24 or 48 h under 1% O2 condition with or without CXCL6 (50 ng/mL). The relative mRNA expression of HIF-1a was detected by real-time PCR. (a) MSC–24 h; (b) MSC–48 h; (c) HUAEC–24 h; (d) HUAEC–48 h. (B) MSCs or HUAECs were treated with CoCl2, with or without CXCL6 (50 ng/mL). After 24 or 48 h, the expression of HIF-1a protein was detected by Western blot method. (C) The relative expression of HIF-1a protein in three independent experiments was statistically analyzed, and the x-axis coordinates were respectively representative CoCl2()/CXCL6(), CoCl2(þ)/CXCL6(), CoCl2(þ)/CXCL6(þ), (a) MSC–24 h; (b) MSC–48 h; (c) HUAEC–24 h; (d) HUAEC–48 h. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and “ns” for no statistical significance. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of neuroinflammation

Article Title: Astrocytic CXCL5 hinders microglial phagocytosis of myelin debris and aggravates white matter injury in chronic cerebral ischemia.

doi: 10.1186/s12974-023-02780-3

Figure Lengend Snippet: Fig. 1 Chronic cerebral ischemia induced significant up-regulation of CXCL5. a Volcano plot of the results of CC tissue microarray analysis between the Sham and BCAS groups at 2 months after surgery (fold change > 2; p < 0.05). n = 3 per group. b Heatmap of fourteen genes related to immune response form CC tissue microarray analysis. n = 3 per group. c The mRNA level of fourteen genes was measured in the Sham and BCAS groups by qPCR. n = 4 per group. d The mRNA level of Cxcl5 in the CC at different time points after BCAS by qPCR. e The result of serum CXCL5 concentration in the Sham and BCAS groups by ELISA. n = 8 per group. f The result of CXCL5 concentration in the CC homogenate in the Sham and BCAS groups by ELISA. n = 7–8 per group. g Representative images of immunostaining against GFAP and CXCL5 in EC in the BCAS groups. (Bar = 50 μm). h The mRNA level of Cxcl5 in the astrocytes (AST) and the other cells (Others) from the CTX and CC in the Sham and BCAS groups by qPCR. n = 4 per group. i The result of CXCL5 concentration in the supernatant of primary astrocytes in the control, OGD/R 12 h and OGD/R 24 h groups by ELISA. n = 3 per group. All data were presented as the mean ± SEM. *p < 0.05, ***p < 0.001, ns means no significance. EC: external capsule. 2W, 2 weeks. 2 M, 2 months. Student’s t-test for c, e and f. One-way ANOVA with Tukey’s post-hoc for d, i, j

Article Snippet: Enzyme linked immunosorbent assay (ELISA) The concentration of CXCL5 in mouse serum, CC homogenate and cell supernatant was quantified by ELISA kit (CUSABIO, China) according to the manufacturer’s instructions.

Techniques: Microarray, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunostaining, Control

Journal: Journal of neuroinflammation

Article Title: Astrocytic CXCL5 hinders microglial phagocytosis of myelin debris and aggravates white matter injury in chronic cerebral ischemia.

doi: 10.1186/s12974-023-02780-3

Figure Lengend Snippet: Fig. 2 Astrocyte specific Cxcl5 deficiency alleviated cognitive decline in BCAS model. a The result of CXCL5 concentration in the supernatant of primary astrocytes in the WT and Cxcl5 cKO groups after OGD/R 12 h by ELISA. n = 4 per group. b, c Results of the OFT showing the time spent in the corner area (b) and center area (c) in the WT and Cxcl5 cKO groups at 2 months after surgery. n = 12 per group. d Results of the NOR showing the recognition index percentage. n = 12 per group. e, f Results of Y-maze tests showing representative heatmaps (e) and the spontaneous alternation percentage (f). n = 12 per group. g–k Results of the MWM test. Representative heatmaps (g), escape latency during the acquisition phase (Days 1–5) (h), the time in target quadrant (i), the crossing platform times (j) and the swimming speed (k) of the probe test (Day 6). n = 11–12 per group. All data were presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns means no significance. Two-way ANOVA with Bonferroni’s post hoc test for a and h. Student’s t-test for b–d, j. Mann–Whitney test for f, i, k

Article Snippet: Enzyme linked immunosorbent assay (ELISA) The concentration of CXCL5 in mouse serum, CC homogenate and cell supernatant was quantified by ELISA kit (CUSABIO, China) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Cell Discovery

Article Title: Targeting a chemo-induced adaptive signaling circuit confers therapeutic vulnerabilities in pancreatic cancer

doi: 10.1038/s41421-024-00720-w

Figure Lengend Snippet: a Cytokine array analysis of CM from Panc02.shCtrl vs Panc02.sh ζ cells in 0% FBS culture for 72 h. b Left: WB analysis of CXCL2, CXCL5, and GAPDH (sample processing controls) expression in Panc02.shCtrl and Panc02.sh ζ cells cultured in 10% FBS or 0% FBS medium for 48 h. Right: WB analysis of CXCL2, CXCL5, and GAPDH (sample processing controls) in 3D cultured PANC-1.shCtrl and PANC-1.sh ζ cells treated with Gem (20 nM) vs vehicle for 48 h. c WB analysis of CXCL2, CXCL5, 14-3-3ζ, and GAPDH (sample processing controls) in PANC-1.shCtrl and PANC-1.shζ, and 14-3-3ζ-overexpressing PANC-1.sh ζ cells treated with Gem (20 nM) vs vehicle for 48 h. d WB analyses of Yap1, CXCL2, CXCL5, and GAPDH (sample processing controls) expression in Panc02.shCtrl and Panc02.sh Yap1 cells in 0% FBS culture for 24 h. Representative data of two independent repeats. e ChIP-qPCR assays of Yap1 binding to CXCL2/5 promoter region in 3D-cultured PANC-1 cells with 24 h of Gem (20 nM) treatment (mean ± SD, t -test, n = 3 biological repeats). f WB analysis of CXCL2, CXCL5, and GAPDH (sample processing controls) in Panc02.shCtrl and Panc02.sh NLK sublines cultured in 0.1% FBS for 24 h. Representative data of two independent repeats. g Schematics (left) and relative numbers of proliferating (middle) and apoptotic (right) cells from 3D-cultured PATC53 cells treated with CM from 3D-cultured hPSCs that were activated by adding CM from 8.5 nM Gem-treated 3D-cultured PATC53 cells plus CXCL2 (1 μg/mL) or CXCL5 (3 μg/mL) blocking antibodies for 48 h (mean ± SD, t -test, n = 3 biological repeats). h Schematics and relative cell number of 3D-cultured KPC mT3 cells treated with CM from 3D-cultured mPSCs added with vehicle, recombinant CXCL2 (0.5 ng/mL), or recombinant CXCL5 (0.1 µg/mL) proteins for 48 h (mean ± SD, t -test, n = 3 biological repeats). i Stress-induced 14-3-3ζ-Yap1-CXCL2/5 pathway in PDAC cells activates fibroblasts, which turns on the adaptive response that enables PDAC cells to survive under stress conditions.

Article Snippet: The blocking antibodies to mouse CXCL2 (MAB452), mouse CXCL5 (MAB433), human CXCL5 (MAB254), and control antibody (MAB0061) were purchased from R&D Systems and human CXCL2 (311001) was purchased from Biolegend (California, USA).

Techniques: Expressing, Cell Culture, Binding Assay, Blocking Assay, Recombinant

Journal: Cell Discovery

Article Title: Targeting a chemo-induced adaptive signaling circuit confers therapeutic vulnerabilities in pancreatic cancer

doi: 10.1038/s41421-024-00720-w

Figure Lengend Snippet: a WB analyses of Cox2 and GAPDH (sample processing controls) in mPSCs (3D culture) incubated with recombinant CXCL2 (0.5 ng/mL) or CXCL5 (0.1 µg/mL) for 24 h. Representative data of two independent repeats. b WB analysis of Cox2 and GAPDH (sample processing controls) in NIH3T3 cells treated with CM from Panc02.shCtrl, Panc02.sh CXCL2 , or Panc02.sh CXCL5 cells cultured in 0% FBS for 72 h. Representative data of two independent repeats. c PGE2 concentration in CM from NIH3T3 cells treated with recombinant CXCL2 (0.5 ng/mL) or CXCL5 (0.1 µg/mL) for 48 h (mean ± SD, t -test, n = 3 biological repeats). d Relative numbers of Panc02 cells treated with PGE2 in 0% FBS for 48 h (mean ± SD, t -test, n = 3 biological repeats). e Relative numbers of PANC-1 cells in upper chambers of Transwell units 3D-cocultured with hPSCs in lower chambers of Transwell units with or without Cel (4 nM) and Gem (20 nM) for 72 h (mean ± SD, t -test, n = 3 biological repeats). f Relative number of Panc02 cells treated with CM from WT mPSCs or Cox2 –/– mPSCs activated by CM from Panc02 cells in 0% FBS for 48 h (mean ± SD, t -test, n = 3 biological repeats). g RPPA analysis of NIH3T3 cells treated with CM collected from Panc02.shCtrl and Panc02.sh ζ cells cultured in 10% or 0% FBS medium. h WB analysis of pT346-NDRG1, NDRG1, and GAPDH (sample processing controls) in 3D-cultured hPSCs treated for 24 h with CM collected from 3D-cultured PANC-1.shCtrl and PANC-1.sh ζ cells treated with vehicle/Gem (20 nM) for 24 h. Representative data of two independent repeats. i WB analysis of pT346-NDRG1, NDRG1, pS473-Akt, Akt, and GAPDH (sample processing controls) expression in 3D-cultured hPSCs treated with recombinant CXCL2 (15 ng/mL) or CXCL5 (25 ng/mL) for 24 h. Representative data of two independent repeats. j WB analysis of Rictor, pT346-NDRG1, NDRG1, Cox2, and GAPDH (sample processing controls) protein in NIH3T3.shCtrl and NIH3T3.sh Rictor cells treated for 24 h with recombinant CXCL2 (0.5 ng/mL) or recombinant CXCL5 (0.1 µg/mL). Representative data of two independent repeats. k, l IHC staining of indicated proteins of PDAC tumor tissues from KPC and KPC -ζ fl/fl mice with indicated treatments (mean ± SD, t -test, n = 5 biological repeats, scale bar: 20 µm). m PDAC cells and fibroblasts together create the adaptive stress response circuit, which is essential for PDAC cell adaptation to stresses, including nutrient deprivation and chemotherapy-induced genotoxicity. Stressors promote the Yap1-CXCL2/5 signaling axis via NLK in 14-3-3ζ-overexpressing PDAC cells, leading to the activation of the CXCR2-mTORC2-Cox2-PGE2 pathway in fibroblasts. Reciprocally, PGE2 from fibroblasts promotes PDAC cell survival and adaptive resistance to Gem.

Article Snippet: The blocking antibodies to mouse CXCL2 (MAB452), mouse CXCL5 (MAB433), human CXCL5 (MAB254), and control antibody (MAB0061) were purchased from R&D Systems and human CXCL2 (311001) was purchased from Biolegend (California, USA).

Techniques: Incubation, Recombinant, Cell Culture, Concentration Assay, Expressing, Immunohistochemistry, Activation Assay