The primers used in qRT-PCR

DEPs in replication senescent hBMSCs and their biological functions.

(A) A hub of 50 significantly DEPs induced by replication aging were shown as a heatmap. GO functions (B) and KEGG pathways (C) affected by these 50 DEPs. Notes: P < 0.05 and logFC > =2 was regarded as indicating significance.

Low expression of PCBP2 accentuated the characteristics of cell senescence in hBMSCs

(A) RT-PCR and (B) WB detection for the expression of PCBP2 in P3-hBMSCs and P10-hBMSCs. CCK-8 assay for the effects of PCBP2 overexpression (C) and PCBP2 silencing (D) on the proliferation of P3-hBMSCs and P10-hBMSCs. Flow cytometry detection for the effects of PCBP2 overexpression (E) and PCBP2 silencing (F) on the cell cycle of P3-hBMSCs and P10-hBMSCs. The effects of PCBP2 overexpression (G) and PCBP2 silencing (H) on apoptosis of P3-hBMSCs and P10-hBMSCs were also detected by flow cytometry. Data were presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001; β-actin and α-tubulin were used as the internal references for mRNA and protein detection.

Low expression of PCBP2 inhibits cell proliferation, and promotes cell apoptosis and cell arrest in a ROS-dependent way.

(A) overexpression and (B) silencing of PCBP2 on ROS production in P3-hBMSCs and P10-hBMSCs. (C) The inhibitory effect of 2mM NAC on ROS production in P3-hBMSCs and P10-hBMSCs with silenced PCBP2. (D) CCK-8 test showed that the antioxidant NAC significantly recovered the suppressed cell proliferation in P3-hBMSCs with silenced PCBP2. Flow cytometry was used to detect the effects of 2mM NAC on the apoptosis (E) and cycle (F) of PBCP2 silenced P3-hBMSCs and P10-hBMSCs. Notes: Data were presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001.

PCBP2 inhibits the expression of FGF2 in P3-hBMSCs and P10-hBMSCs.

The regulation of FGF2 mRNA levels by overexpression (A) and silencing (B) of PCBP2 was detected by RT-PCR, and were confirmed in protein levels via WB assay (C, D). (E) up-regulation of FGF2 protein level in P3-hBMSCs with silenced PCBP2 was stopped by 2mM NAC. Notes: Data were presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001; β-actin and α-tubulin was used as the internal reference for mRNA and proteins detection.

Overexpression of PCBP2 promotes cell proliferation, and inhibits cell apoptosis and cell arrest in a FGF2-dependent way.

CCK-8 detects the effect of overexpression of PCBP2 and FGF2 on the cell proliferation of P3-hBMSCs (A) and P10-hBMSCs (B). Flow cytometry was used to detect the apoptosis of P3-hBMSCs (C) and P10-hBMSCs (D) in NC, OE-PCBP2 and OE-PCBP2 + OE-FGF2 groups. In addition, in NC group, OE-PCBP2 group and OE-PCBP2+OE-PCBP2 group, the cell cycle of P3-hBMSCs (E) and P10-hBMSCs (F) were detected by flow cytometry. Notes: Data were presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001.

Unlabeled quantitative proteomics for the DEPs between P3-hBMSCs and P10-hBMSCs.

The enzymolysis products were separated by capillary high-performance liquid chromatography and then analyzed by Thermo Fisher Fusion Mass Spectrometer (Thermo Fisher). The content of each peptide was expressed by detecting the intensity of positive ions, and protein analysis was performed using Maxquant software (version number 1.6.0.1).

Validation of transfection effect of PCBP2.

The mRNA expression of PCBP2 after transfection with plasmids that over-expressed PCBP2 (A) and siRNA that knocked-down PCBP2 (B). The effect of over-expressing PCBP2 (C) and knocking-down PCBP2 (D) as detected by WB. Notes: Data were presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001; β-actin and α-tubulin was used as the internal reference for mRNA and proteins detection.

Identification of the downstream genes of PCBP2.

Volcano gram of differentially expressed genes caused by PCBP2 silencing (A). Bubble diagram of KEGG pathway analysis of differentially expressed genes (B). The interaction of differentially expressed proteins was predicted by STRING website.