Fig 2: mCALR facilitates the migration and maturation of DCs by increasing the secretion of TNFα and CCL19. (A) The effect of cell supernatants (NC and mCALR) on DC migration was evaluated by migration assay; **P < 0.01. (B) The number of invaded DCs in both NC and mCALR groups was quantified by western blot using anti-β-actin antibody. (C–F) The effect of cell supernatants (NC and mCALR) on DC maturation was determined using flow cytometry with PE-anti-human CD83 antibody; **P < 0.01, ***P < 0.001. (G–I) The relative mRNA expression levels of CALR, TNFα, and CCL19 were detected in NC and mCALR groups using real-time PCR; *P < 0.05, **P < 0.01, ***P < 0.001. (J, K) The expression levels of TNFα and CCL19 were investigated in NC and mCALR groups using western blot; *P < 0.05, **P < 0.01. (L, M) The expression levels of TNFα and CCL19 were investigated in NC and mCALR groups using ELISA; *P < 0.05, ***P < 0.001. (N) The number of invaded DCs in both anti-CCL19+ and anti-CCL19- groups was quantified by western blot using anti-β-actin antibody. (O–R) The effect of cell supernatants (anti-TNFα+ and anti-TNFα- groups) on DC maturation was determined using flow cytometry with PE-anti-human CD83 antibody; **P < 0.01.

Fig 3: The correlation and overall survival of tumor-infiltrating immune cells in NSCLC patients were analyzed using TIMER and TCIA. (A) The correlation of TLR4 and TNFα expressions was analyzed in LUAD and LUSC using TIMER; TPM, Transcripts Per Million. (B) The correlation of TLR4 and CCL19 expressions was analyzed in LUAD and LUSC using TIMER; TPM, Transcripts Per Million. (C) The correlation of TLR4 expression with immune cell infiltration levels was analyzed in LUAD and LUSC using TIMER. Tumor-infiltrating immune cells included CD8+ T cells, CD4+ T cells, and DCs. Gene expression levels against tumor purity are displayed in the left-most panel; TPM, Transcripts Per Million. (D, E) Overall survival rates of patients with high and low DC infiltration were analyzed in LUAD (D) and LUSC (E) using TIMER; LUAD, 501 patients, P=0.022; LUSC, 492 patients, P=0.132. (F, G) Overall survival rates of patients with high and low activated DC infiltration were analyzed in LUAD (F) and LUSC (G) using TCIA; LUAD, 506 patients, P=0.0497; LUSC, 495 patients, P=0.1157. (H) The expression of TLR4 in NSCLC (LUAD and LUSC) tissues and normal tissues was analyzed using GEPIA. (I) Overall survival rates of patients with high and low TLR4 expression were analyzed in lung cancer (n=1144 patients) using Kaplan-Meier Plotter; P=0.028.

Fig 4: mCALR silence inhibits the migration and maturation of DCs by decreasing the secretion of TNFα and CCL19. (A) The relative mRNA expression levels of CALR was detected in siCALR and control groups using real-time PCR; ***P < 0.001. (B, C) The effect of cell supernatants (siCtrl and siCALR) on DC migration was evaluated by migration assay; **P < 0.01, ***P < 0.001. (D–G) The effect of cell supernatants (siCtrl and siCALR) on DC maturation was determined using flow cytometry with PE-anti-human CD83 antibody; **P < 0.01, ***P < 0.001. (H, I) The relative mRNA expression levels of TNFα and CCL19 were detected in siCALR and control groups using real-time PCR; **P < 0.01, ***P < 0.001. (J, K) The expression levels of CALR, Flag-mCALR, TNFα, and CCL19 were investigated in siCALR and control groups using western blot; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: mCALR enhances the secretion of TNFα and CCL19 via activating CALR-TLR4-MyD88 signaling. (A) The interaction between mCALR and TLR4 was assessed using co-IP assay in NC and mCALR cells, and the IgG antibody was used as a negative control. (B) The relative mRNA expression level of TLR4 was detected in NC and mCALR groups using real-time PCR; **P < 0.01, ***P < 0.001. (C) The relative mRNA expression level of TLR4 was detected in siCALR and control groups using real-time PCR; *P < 0.05, **P < 0.01. (D, E) The expression levels of mCALR, TLR4, MyD88, ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK, p65, and p-p65 were investigated in NC and mCALR groups using western blot, and the anti-Flag antibody was used to detect the mCALR protein; ns, P>0.05, *P < 0.05. (F–H) The relative mRNA expression levels of TLR4, TNFα, and CCL19 were detected in mCALR cells transfected with different siTLR4 using real-time PCR; **P < 0.01, ***P < 0.001. (I, J) The expression levels of TLR4, TNFα, and CCL19 were investigated in mCALR cells transfected with different siTLR4 using western blot; *P < 0.05, **P < 0.01.