Fig 2: Identification of proteins proximity in cancer cell lines. (A) Scheme of the is-PLA: 1, primary antibodies bind specific proteins; 2, secondary antibodies conjugated with oligonucleotides (proximity probes) bind to anti-rabbit or mouse primary antibodies; 3, if the two proteins are interacting (< 40 nm apart), the proximity probes can hybridize with the two connector oligos; 4, the ligation step produces a circular DNA template; 5, circular DNA is then amplified by DNA polymerase. Detection oligos coupled to fluorochromes hybridize to repeating sequences in the amplicons yielding the is-PLA signal detected by fluorescent microscopy as discrete spots (inset). (B) is-PLA signals, corresponding to DHFR/SHMT, SHMT/TYMS and DHFR/TYMS interactions, are shown (red dots). TYMSM and TYMSR both indicate antibodies against TYMS but anti-mouse and anti-rabbit, respectively. The is-PLA spots of interaction of proteins are shown in A549 cells synchronised in the S-phase after a 24-h single thymidine block (upper) or in A549 asynchronous cells (bottom). Scale bars = 10 µm. The merge with DAPI signal is shown on the right. Controls are shown in Fig. 4B–F. (C) Nuclear localization of the is-PLA signal (number of spots within the normalized nucleus area). In all of the experiments, the number of PLA spots increase in the S-enriched cells (t-test: **P < 0.005; ****P < 0.0001). At least 160 cells were analysed per condition. (D) Left: representation of PCNA positive cells, corresponding to S-phase of the cell cycle. Scale bar = 10 µm. Right: immunofluorescence signal in an asynchronous population or after a single thymidine block of A549. The histogram on the right shows the percentage of PCNA positive or negative cells after and before synchronisation. At least 500 cells were counted per conditions, from three independent experiments; SD values are shown. After synchronisation, the cells in S-phase comprise approximately 80% of the cellular population.

Fig 3: Designing human DHFR-TYMS Chimera. (A) Cartoon representation of the chimeric model of human DHFR-TYMS (in purple and blue, respectively; the linker is shown in red; the N-terminus position is also indicated) superposed with the structure of the bifunctional enzyme from B. bovis (PDB ID: 3I3R [35]) in light grey. The enzyme is dimeric, the partner subunit is shown as surface representation. (B) Electrostatic surface potentials, as calculated using apbs (Adaptive Poisson–Boltzmann Solver) [61], at the modelled interface between hDHFR and hTYMS, showing a perfect complementarity. Partially positive or negative regions are indicated in blue and red, respectively. (C) Scheme of the final constructs differing only for the linker length and consequently named Chimera-Long and Chimera-Short. Structural renderings were produced using pymol.

Fig 4: Immunofluorescence analysis. (A–C) Compartmentalization of the three proteins with respect to the cellular phase by Immunofluorescence analysis. SHMT1 (A), DHFR (B) and TYMS (C) show a cytoplasmic localization both in the asynchronous and S-phase synchronised cells. Scale bar = 10 µm. (D) Nuclear localization (as deduced by the normalized intensity of the fluorescence signal) is clearly more abundant in the S-enriched cells for SHMT1; a slight increase is observed for TYMS (with both the antibodies used in the PLA experiments; rabbit, TYMSR; and mouse, TYMSM; see Materials and methods). No change was observed for DHFR. (t-test: *P < 0.05; **P < 0.005; ****P < 0.0001). Error bars represent the SD. At least 80 cells per condition from two independent experiment were analysed. (E) Western blot of subcellular fractionation of both asynchronous and S-phase enriched A549 cell lines. Western blotting analysis was performed using a 1 : 1000 dilution of the primary antibodies. For Histone H3, the correct band is boxed in red. The other bands detected in the cytosol have a molecular weight lower than 15 kDa and are also present in the nuclear fractions. Giving that the molecular weight of Histone H3 is 17 kDa, and the bands recur in all the four lanes, it is plausible that they are detected because of non-specific interactions of the primary or secondary antibody. In this experiment, the quantitative analysis was not performed, but it is still possible to detect the bands of SHMT1 and TYMS only in the nucleus of the S-phase enriched cells. As shown in the IF experiments, it was not possible to detect an increase of the presence of DHFR in the nucleus of the synchronised cells.

Fig 5: Immunofluorescence co-localization analysis. (A) Co-localization of DHFR/SHMT1, SHMT1/TYMS and TYMS/DHFR in asynchronous A549 cells. Co-localization is evident both in the cytosol (tubulin alpha, cyan) and in the nucleus (DAPI, blue) for all the three couples. The original red fluorescence signal of tubulin was changed to cyan for better visualization of the merged images. The merge of green, red and cyan pixels yields white pixels.