Abundant uptake of cfChPs by NIH3T3 cells at 6

h. The DNA and histones of cfChPs were dually fluorescently labelled with Platinum Bright 550 and ATTO-488 respectively and applied (10ng) to NIH3T3 cells. a. Representative images of dually-labelled cfChPs prior to their application to NIH3T3 cells. b. Confocal microscopy images of cfChPs-treated cells at 6 h showing many dually labelled fluorescent signals in the cytoplasm and nuclei. c. Fluorescence microscopy images of chromatin fibres prepared from similarly treated cells at 6 h showing numerous dually labelled fluorescent signals of varying sizes in the cytoplasm and in association with the chromatin fibres. d. Fluorescence microscopy images of metaphase spreads prepared from cfChPs-treated cells at 6 h showing multiple dually labelled fluorescent signals which are either associated with the chromosomes or are present in extrachromosomal spaces. The latter are marked with arrows.

Internalised cfChPs combine to form complex concatemers.

a. FISH analysis of chromatin fibres prepared from cfChPs-treated NIH3T3 cells in continuous passage using different combinations of human chromosome-specific FISH probes, including probes specific for the human centromere and telomere, revealing co-localised fluorescent signals (arrows). b. Small cfChPs also show co-localised signals suggesting that they too are comprised of concatemers c. Similar co-localising signals are shown on metaphase spreads. Co-localised signals are marked with arrows.

Variable spatial relationships of concatemers with mouse chromatin fibres.

a. Immuno-FISH analysis of chromatin fibres prepared from cfChPs-treated NIH3T3 cells in continuous passages using an antibody against histone H4 and a human whole-genome FISH probe. b. Immuno-FISH analysis of chromatin fibres prepared from clone D5 showing that the concatemers persisted even after numerous passages and freeze–thaw cycles.

The concatemers synthesise DNA and express human DNA polymerase γ.

a. Chromatin fibres were prepared from NIH3T3 cells in continuous passage were pulse-labelled for 24 h with BrdU (10µm). Immuno-FISH experiments using antibodies against BrdU, human-specific DNA polymerase γ, and a human whole-genome FISH probe show co-localised signals of human DNA and BrdU, human DNA and DNA polymerase γ, and BrdU and DNA polymerase γ. Co-localised signals are marked with arrows. b. Similar co-localising signals are shown on metaphase spreads. Co-localised signals are marked with arrows.

Concatemers proliferate and amplify themselves within the mouse genome over time.

Metaphase spreads were prepared from cells in each progressively increasing passage and probed with a human-specific DNA FISH probe. a. The total number of human DNA FISH signals on the DAPI stained chromosomes was counted, and the mean number of human FISH signals per chromosome was calculated after analysing 10 metaphase spreads at each passage. A progressive increase in mean FISH signals per chromosomes is evident with increasing passage number (analysis of variance for linear trend p < 0.0001). The mean number of human FISH signals per chromosome between passage no. 2 and passage no. 198 increased by a factor of 4.07. b. a similar exercise was done as above except that mean fluorescent intensity (MFI) of human FISH signals per chromosome, indicative of amplification, was determined. A progressive increase in mean MFI per chromosomes is evident with increasing passage number (analysis of variance for linear trend p < 0.0001). The MFI per chromosome increased by a factor of 237.19 between passage no. 2 and passage no. 198. The insets represent partial metaphase images showing human DNA signals on the chromosomes. Blue and red signals represent DAPI and human DNA, respectively.

Concatemers largely comprise of open chromatin irrespective of the epigenetic constitution of the mouse DNA.

Representative images of chromatin fibres immunostained with antibodies against H3k4me3 (red) and H3k9me3 (green). The hybrid concatemers are represented by co-localised yellow signals. The host mouse DNA in the upper image is seen to react with H3k4me3 antibody representing open chromatin while the host mouse DNA segment in the lower image is comprised of heterochromatin and seen to react with antibody against H3k9me3. Histograms represent quantitative estimates of H3k4me3, H3k9me3, and hybrid histones after counting 100 fluorescent signals. The values are expressed as mean ± SEM values. Statistical analysis was performed using two-tailed Student’s t-test. * p < 0.05, ** p < 0.01 and **** p < 0.0001.

Concatemers synthesise RNA which is dependent on active cellular metabolism.

Images showing cytoplasmic RNA synthesis by cfChP-treated cells in continuous passage which is absent in control NIH3T3 cells. Treatment of cfChP-treated cells with Actinomycin D (0.0005 μg/mL) or maintenance at low temperature (31°C) abolishes RNA synthesis indicating that the latter is dependent on active cellular metabolism.

Concatemers synthesise their own protein synthetic machinery.

Chromatin fibres (a) and metaphase spreads (b) were prepared from cfChPs treated cells in continuous passage. Dual-FISH experiments were performed using a human specific genomic DNA probe and a probe against human specific ribosomal RNA, and immune-FISH experiments using a human specific genomic DNA probe and antibodies against human specific RNA polymerase III and human specific ribosomal protein. Co-localised fluorescent signals of DNA and the components of the above protein synthetic machinery are marked with arrows.

Concatemers synthesise a variety of human proteins.

Immuno-FISH analysis using antibodies against various human proteins and a human whole genome FISH probe on chromatin fibres (a) and metaphase spreads (b) prepared from cfChPs treated cells in continuous passage show co-localizing signals. The co-localizing signals of human DNA and various proteins are marked with arrows.

Proteins synthesised by concatemers are fusion proteins.

Dual immunofluorescence analysis using antibody pairs targeting diverse human-specific proteins in chromatin fibres (a) and metaphase spreads (b) prepared from cfChPs-treated NIH3T3 cells in continuous passage. Results show frequent co-localised signals indicative of fusion proteins (arrows). The numbers given in the parenthesis indicate the chromosomal location of the genes that correspond to the proteins.

Concatemers harbour transposable elements.

Dual-FISH analysis on chromatin fibres (a) and metaphase spreads (b) prepared from cfChPs-treated NIH3T3 cells in continuous passage using a human specific genomic DNA probe and those against human LINE-1 or human Alu show co-localised signals (marked with arrows).

LINE-1 and Alu sequences are associated with reverse transcriptase and transposase.

Immuno-FISH analysis of chromatin fibres (a) and metaphase spreads(b) prepared from cfChPs-treated NIH3T3 cells in continuous passage using human Alu or LINE-1 probes and antibodies against human reverse transcriptase and human transposase show co-localising signals (marked with arrows).

LINE-1 and Alu elements are associated with DNA polymerase and can actively synthesise DNA.

(a) Immuno-FISH images of cfChPs-treated cells in continuous passage showing co-localising signals of LINE-1 (green) and Alu (green) and DNA polymerase γ (orange) and BrdU (red) on chromatin fibres. (b) Similar co-localising signals are shown on metaphase spreads. Co-localised signals are marked with arrows.

LINE-1 and Alu elements proliferate and amplify themselves within the mouse genome over time.

Metaphase spreads were prepared from cells in each progressively increasing passage and probed with a human-specific LINE-1 and Alu probes. a. The total number of human LINE-1 and Alu signals on the DAPI stained chromosomes were counted, and the mean number of LINE-1 and Alu signals per chromosome was calculated after analysing 15 metaphase spreads at each passage. A progressively increasing LINE-1 and Alu signals per chromosomes is evident with increasing passage number (analysis of variance for linear trend p < 0.0001). The number of human LINE-1 signals per chromosome increased by a factor of 7.6 between passage no. 2 and passage no. 198, and by a factor of 6.7 in case of Alu. b. a similar exercise was done as above except that mean fluorescent intensity (MFI) of human LINE-1 and Alu per chromosome, indicative of amplification, was determined. A progressively increasing in MFI per chromosomes is evident with increasing passage number (analysis of variance for linear trend p < 0.0001). Mean MFI per chromosome increased by a factor of 151.3 between passage no. 2 and passage no. 198 in case of LINE-1, and by a factor of 83.4 in case of Alu. The insets represent partial metaphase images showing human LINE-1 and Alu signals on the chromosomes. Blue and red signals represent DAPI and human DNA, respectively.