Fig 2: PLA results for AKT2/GAPDH (A–D) and AKT2/α-enolase (ENO1) (E–H) in HEK293T AKT2-NTag cells.A/B, E/F: Control experiments with only one of the primary antibodies used. C and G show complete PLAs. D/H: Quantitative analysis of two independent experiments for each antibody combination. The number of signals/nuclei in the PLA-sample was set as 100%. Controls are shown as percentage of full PLA.

Fig 3: CA arrested the cell cycle, induced apoptosis, and inhibited ENO1 function. (A) Cells were treated with 0, 20, 40, or 80 μM CA for 24 h, after which the cells were collected and stained. The DNA content of the cells was determined with a flow cytometry system. Histograms show the percentage of cells in Sub-G1, G0/G1, G2/M, and S phase. (B) Cells were treated with 0, 20, 40, or 80 μM CA for 24 h. A flow cytometric analysis of CA-induced apoptosis in A375 cells was done by double-staining with Annexin V-FITC/PI. Histograms show the percentage of different regions; “Q2” and “Q3” represent late and early apoptosis, respectively. (C) A375 cells were transfected with siENO1 for 48 h, and the relative mRNA level of ENO1 was assayed by qRT-PCR; additionally, the expression of ENO1 in cells was assayed by Western blotting. Histograms show the intensity of the ENO1 protein bands. Finally, cell viability is presented as the IC50 values (n = 3). *** p < 0.001 versus “0 μM” group. (D) A375 cells were transfected with siENO1 for 48 h, and then cells were treated with 0, 20, or 40 μM CA for 24 h. Cells were then collected and determined via flow cytometry system. Histograms show the percentage of cells in G0/G1, G2/M, and S phase after treatment with CA. “NC” group stands for normal cultured cells without CA and interference fragment; “siNC” group was used as a negative control.

Fig 4: CA targeted ENO1 in A375 cells and changed its activity. (A) The modification process of AL-CA-MMs and the schematic diagram for target protein capture and release. (B) Efficiency evaluation of magnetic capture by SDS-PAGE. Lane 1 shows the A375 lysate as a loading control; Lane 2 shows the lysate captured only by the azide-modified MMs as a negative control; Lane 3 shows the lysate captured by AL-CA-MMs. Coomassie bright blue staining is shown in the left image, and the right image shows the ENO1 detected by Western blotting. (C) CA treatment (10 μM) decreased the thermal stability of ENO1 in cell lysates measured by the temperature-dependent Cellular thermal shift assay (CETSA) (n = 3). (D) CA treatment decreased the thermal stability of ENO1 in cell lysates measured by the concentration-dependent CETSA at 69 °C (n = 3). (E) CA inhibited the activity of ENO1. Cells were exposed to CA (0, 20, 40, or 80 μM) or a positive ENO1 inhibitor, AP-III-a4 (10 μM) for 0.5 h, and then the effect on the activity of ENO1 in lysates was determined (n = 3).

Fig 5: CA colocalized with ENO1 both in vitro and in vivo. (A) Schematic diagram of fluorescent click reaction of the Al-CA probe with the N3-tag. (B) In situ click reaction on PVDF membrane. After SDS-PAGE was performed, the click reaction was performed with an N3-tag in situ. Lane 1 shows the A375 lysate treated with CA as a loading control; and Lane 2 shows the A375 lysate treated with an AL-CA probe. All of the protein samples were adjusted to equal amounts before capture. (C) The colocalization imaging of the Al-CA probe and the ENO1 protein on A375 cells with fluorescence confocal microscopy. (D) In vivo imaging for CA and ENO1. A375 cells were subcutaneously injected into the nude mice. When the tumors formed for one week, mice were treated with CA (60 mg/kg/day) or Al-CA (60 mg/kg/day) for two weeks. Moreover, the tumor tissues were stripped to the pathological section for the colocalization imaging assay. The pseudo red color represents ENO1, which was stained by Alexa Fluor 594, and the Al-CA probe took on a pseudo green color via the click reaction. The yellow merger, where the arrow pointed, represents ENO1 colocalized with Al-GA.