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. 2021 Nov 9;10(11):3093. doi: 10.3390/cells10113093

Figure 4.

Figure 4

CXCL5 induces critical EMT associated genes in CCA and also enhance lymphangiogenesis. (A,B) CXCL5 induces increased migration of CCA cells. (A) CCA cells were pretreated with or without CXCR2 inhibitor, SB225002 (denoted as SB). A scratch was made on the confluent monolayer. The cells were then treated with CXCL5, and the average wound length was monitored for 48 h. Representative images at 4× magnification are shown. Values are represented as mean ± SEM, n ≥ 3. **** represents value significantly different than control group respectively at p ≤ 0.0001. #### represents value significantly different than CXCL5 treated group at p ≤ 0.0001. (B) CCA cells were pretreated with or without SB and were allowed to migrate towards CXCL5 in transwell inserts for 24 h. Representative images at 10× magnification. Scale bars are 100 μm. Number of cells migrated is plotted. **** represents value significantly different than control group respectively at p ≤ 0.0001. #### represents value significantly different than CXCL5 treated group at p ≤ 0.0001. (C) CXCL5 induces expression of EMT, matrix remodeling and immune checkpoint related genes in CCA. CCA cells were pretreated with or without SB and then with or without CXCL5 for 24 h. Expression of EMT, matrix remodeling and immune evasion related genes were measured by real time PCR. Values are represented as mean ± SEM, n ≥ 3. * p ≤ 0.05, *** p ≤ 0.001 **** p ≤ 0.0001 represents value significantly different than control group respectively. ### p ≤ 0.001 and #### p ≤ 0.0001 represents value significantly different than CXCL5 treated group at p ≤ 0.0001. (D) CXCL5-CXCR2 interaction activates ERK in CCA cells. CCA cells were pretreated with SB and then treated with CXCL5 for 24 h. Untreated groups were used as control. Expression of phospho-ERK and total ERK were measured by western blotting. Densitometric analysis was carried out. Graph represents relative band intensities of pERK/ERK. Values are represented as mean ± SEM, n ≥ 3. * represents value significantly different than control and # represents value significantly different than CXCL5 at p ≤ 0.05. (E) CXCL5 induces expression of EMT and matrix remodeling genes in CCA in an ERK dependent manner. CCA cells were pretreated with or without PD98059 (PD) and then with or without CXCL5 for 24 h. Expression of EMT of matrix remodeling genes were measured by qPCR. Values are represented as mean ± SEM, n ≥ 3. *** p ≤ 0.001 and **** p ≤ 0.0001 value versus control, ## p ≤ 0.01 and #### p ≤ 0.0001 represents difference from CXCL5 group respectively. (F,G) Inflamed LECs and CXCL5 alters expression of focal adhesion genes in CCA. CCA cells were treated with (F) LEC-CM, LPS-LEC-CM or 5% EGM (control), values are represented as mean ± SEM, n ≥ 3. **** p ≤ 0.0001 represents value versus control and (G) with or without CXCL5 for 24 h and the mRNA levels focal adhesion genes were analyzed by qPCR, Values are represented as mean ± SEM, n ≥ 3. **** p ≤ 0.0001 represents value versus control. (H) CXCL5 induces lymphangiogenesis. LECs were allowed to form tubes on Matrigel with or without CXCL5/CXCL5 + SB and stained with Calcein AM. Representative images in 4× magnification are shown. Total branch length was calculated using ImageJ and plotted. Values are represented as mean ± SEM, n ≥ 3. * p ≤ 0.05 represents value significantly different than control and ### p ≤ 0.001 represents value compared to CXCL5.