Fig 2: Circ_0079530 knockdown suppresses tumor growth in NSCLC. Mice were subcutaneously injected with sh-circ_0079530 or sh-NC-transfected H2170 cells, and then the tumors were treated by 4 Gy radiation or not. (A) Circ_0079530 level was measured via qRT-PCR in sh-circ_0079530 or sh-NC-transfected H2170 cells (n = 3). (B) Tumor volume was measured (n = 6). (C) Tumor weight was detected at the end (n = 6). (D) PCNA, MMP9, E-cadherin, ICAM-1, and vitronectin levels were examined via western blotting in tumor tissues (n = 6). *P <0.05 **P <0.01 and ***P <0.001. NSCLC: non-small cell lung cancer; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; NC: negative control; PCNA: proliferating cell nuclear antigen; MMP9: matrix metalloproteinase 9; ICAM-1: intercellular adhesion molecule-1

Fig 3: miR-124-5p regulates cell migration and invasion, as well as the release of inflammatory factors, through mediating FoxO1 expression. (A) The changes in the expression levels of FoxO1, ICAM-1 and NF-?B in HUVECs after transfecting with siRNA-FoxO1 was detected by western blotting. GAPDH was used as the internal reference. The changes in (B) invasion and (C) migration abilities of HUVECs were detected by using Transwell assays after FoxO1 knockdown. Scale bar, 50 µm. The changes in (D) IL-6, (E) IL-8 and (F) TNF-a of HUVECs were detected by enzyme-linked immunosorbent assay after FoxO1 knockdown. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. control; ##P<0.01, ###P<0.001, ####P<0.0001 vs. IL-ß; $$P<0.01, $$$P<0.001, $$$$P<0.0001 vs. IL-1ß + miR-124-5p-Inhibitor-si-NC. miR, microRNA; NC, negative control; TNF-a, tumor necrosis factor-a; ICAM1, intercellular adhesion molecule; NF-?B, nuclear factor-?B; siRNA/si-, small interfering RNA; IL-6/8, interleukin-6/8.

Fig 4: Co-incubation of ECs, PBMCs and CPPs under flow induces adhesion of PBMCs to the ECs. (A) HCAEC and HITAEC were exposed to CPPs circulating in the flow culture system for 4 h with the co-incubation with CellTracker Green-labeled PBMCs during the last hour. Phase contrast visualisation coupled with Hoechst 33342 (ECs and PBMCs, blue colour) and CellTracker Green (PBMCs, green colour) staining, representative images, ×200 magnification. Note the frequent adhesion of PBMCs to the CPP-P or CPP-S-treated ECs; (B) quantification of PBMCs attached to the ECs from the experiment in (A), each dot represents one field of view (40 fields of view in total, 20 per flow culture chamber, 2 flow culture chambers per group). Whiskers indicate range, boxes bounds indicate 25th–75th percentiles, center lines indicate median. p values provided above boxes, Kruskal–Wallis test with post hoc false discovery rate correction by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli; (C) Western blotting measurements of cell adhesion molecules VCAM1 and ICAM1 as compared to the expression of three EC housekeeping proteins (vimentin, CD31 and VE-cadherin) in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. Blot scans (top) and band densitometry analysis (bottom). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change =0.75, 0.76–1.24, and =1.25, respectively, compared to PBS group; (D) gene expression analysis of VCAM1, ICAM1, and SELE genes in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. RT-qPCR measurements, the results are represented by a heat map. Green, gray and red colours mean fold change = 0.50, 0.51–1.99, and =2.00, respectively, compared to PBS group; (E) Western blotting measurement of Vcam1 as compared to the expression of three EC housekeeping proteins (Cd31, Gapdh and histone H3) in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). Blot scans (top) and band densitometry analysis (bottom). The results of the latter are represented by a heat map. Gray colour means fold change 0.76–1.24 compared to NaCl group; (F) gene expression analysis of Vcam1 and Icam1 genes in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). RT-qPCR measurements, the results are represented by a heat map. Gray and red colours mean fold change 0.51–1.99 and =2.00, respectively, compared to NaCl group. PBMCs—peripheral blood-derived mononuclear cells, HCAEC—human coronary artery endothelial cells, HITAEC—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPP—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, VCAM1—vascular cell adhesion molecule 1, ICAM1—intercellular cell adhesion molecule 1, Vim—vimentin, CD31—cluster of differentiation 31, VE-cadherin—vascular endothelial cadherin, SELE—E-selectin, Gapdh—glyceraldehyde 3-phosphate dehydrogenase, Hist H3—histone H3, RT-qPCR—reverse transcription quantitative polymerase chain reaction.

Fig 5: Downregulated MALAT1 inhibits apoptosis of HPMECs while decreasing the expression of pro-inflammatory cytokines and adhesion factors. (A) Expression of MALAT1 in peripheral blood samples of healthy controls (n = 46) and patients with ARDS (n = 46), as determined by RT-qPCR. * p < 0.05 vs. peripheral blood samples of healthy controls. (B) Expression of MALAT1 in normal and LPS-treated HPMECs, as determined by RT-qPCR. * p < 0.05 vs. the control cells. (C) Subcellular localization of MALAT1 in HPMECs detected by FISH (400 ×). (D) Expression of MALAT1 in HPMECs transfected with oe-MALAT1 or sh-MALAT1 determined by RT-qPCR. (E) Expression of pro-inflammatory cytokines (IL-6, IL-1β and TNF-α) in HPMECs transfected with oe-MALAT1 or sh-MALAT1 determined by ELISA. (F) Expression of endothelial cell adhesion molecules (E-selectin and ICAM-1) in HPMECs transfected with oe-MALAT1 or sh-MALAT1, as detected by immunofluorescence (× 200). (G) HPMEC apoptosis upon transfection with oe-MALAT1 or sh-MALAT1 detected by TUNEL assay (× 200). (H) Expression of Bcl-2, Bax, cleaved caspase 3 and Caspase3 in HPMECs transfected with oe-MALAT1 or sh-MALAT1 detected by Western blot analysis. * p < 0.05 vs. cells transfected with oe-MALAT1 NC or sh-MALAT1 NC. The data were measurement data and expressed as mean ± standard deviation. The data between two groups were compared using unpaired t-test and those among multiple groups were analyzed by one-way ANOVA, with Tukey's post hoc test. The cell experiment was repeated three times independently.