Varakult lahknenud seenerühma Basal clone group 2 visualiseerimine CARD-FISH metoodikaga

Bakalaureusetöö teoreetilise osa eesmärk oli anda ülevaade varakult lahknenud seente taksonitest, nende ökoloogilisest rollist ja nende uurimiseks kasutatavatest meetoditest. Töö eksperimentaalse osa eesmärk oli välja töötada varasemate uurimistööde alusel CARD-FISH protokoll, mis võimaldaks edaspidiselt uurida mikroskoopilisi seeni. Töö uurimisobjektiks oli ainult keskkonnast pärit geenijärjestuste põhjal teadaolev rühm laborinimega Basal clone group 2 (BCG2)

The methyltransferase N6AMT1 participates in the cell cycle by regulating cyclin E levels.

The methyltransferase N6AMT1 has been associated with the progression of different pathological conditions, such as tumours and neurological malfunctions, but the underlying mechanism is not fully understood. Analysis of N6AMT1-depleted cells revealed that N6AMT1 is involved in the cell cycle and cell proliferation. In N6AMT1-depleted cells, the cell doubling time was increased, and cell progression out of mitosis and the G0/G1 and S phases was disrupted. It was discovered that in N6AMT1-depleted cells, the transcription of cyclin E was downregulated, which indicates that N6AMT1 is involved in the regulation of cyclin E transcription. Understanding the functions and importance of N6AMT1 in cell proliferation and cell cycle regulation is essential for developing treatments and strategies to control diseases that are associated with N6AMT1

Cell cycle distribution of U2OS and N6AMT1-depleted cells.

(A) Schematic representation of Cyclin A, B and E expression levels during the cell cycle. Serum depletion and thymidine and nocodazole treatment were used to arrest the cells at the indicated cell cycle points. (B-E) Cell cycle phase distribution of the U2OS and N6AMT1-knockout cell lines after the indicated treatments was determined by propidium iodide staining and flow cytometry analysis. The average of three biologically independent samples is shown. Statistical analysis was performed with GraphPad Prism 8.4.3. The data are presented as the means ± SDs; *P < 0,05, **P < 0,001, ***P < 0,0001 according to multiple t tests.</p

N6AMT1 impacts the cyclin E levels.

(A) Unsynchronized cells and cells that were synchronized with serum depletion, thymidine treatment and nocodazole treatment were analysed via Western blotting with antibodies against Cyclin A, Cyclin B1, Cyclin E, N6AMT1, TRMT112 and GAPDH. 10 μg of total protein was loaded per well. (B-E) Cyclin A, cyclin B, cyclin E and TRMT112 levels in U2OS and ΔN6AMT1#1 cells. Western blotting images were quantified using ImageJ, and the average of three biologically independent samples is shown. Statistical analysis was performed with GraphPad Prism 8.4.3. The data are presented as the means ± SDs; *P < 0,05, **P < 0,001, ***P < 0,0001 according to multiple t tests. (F) Untreated cells and cells that were treated with the indicated siRNAs were analysed via Western blotting with antibodies against Cyclin E, N6AMT1 and GAPDH. (G) Cyclin E levels in U2OS cells after siRNA treatment. Western blotting images were quantified using ImageJ, and the average of three biologically independent samples is shown. Statistical analysis was performed with GraphPad Prism 8.4.3. The data are presented as the means ± SDs; *P < 0,05, **P < 0,001, ***P < 0,0001 according to multiple t tests. (H) qRT‒PCR verification of cyclin A, cyclin B and cyclin E levels in ΔN6AMT1#1 cells. The average of three biologically independent samples is shown. Statistical analysis was performed with GraphPad Prism 8.4.3. The data are presented as the means ± SDs; *P < 0,05, **P < 0,001, ***P < 0,0001 according to multiple t tests.</p

Changes in the cyclin levels after serum depletion.

(A) The population density of the U2OS and ΔN6AMT1#1 cell lines in serum-depleted media was determined using an xCELLigence Real-Time cell analyser over a period of 120 h. (B) Schematic representation of the experimental setup for cell synchronization in the G1 phase. The cyclin A, B1 and E levels in (C) U2OS and (D) ΔN6AMT1#1 cells were analysed via Western blotting at the indicated timepoints. Cyclin A, B and E images were obtained after exposure time of 10 min for U2OS and 30 min ΔN6AMT1#1 samples. (E-G) Graphical representation of changes in the cyclin levels. Western blotting images were quantified using ImageJ, and the average of three biologically independent samples is shown.</p

Changes in cyclin levels after nocodazole arrest.

(A) Schematic representation of the experimental setup for cell synchronization early in prophase. (B) The cyclin A, B1 and E levels in U2OS and (C) ΔN6AMT1#1 cells were analysed via Western blotting at the indicated timepoints. Cyclin A, B and E images were obtained after 1h exposure time for both cell lines. (D-F) Graphical representation of changes in the cyclin levels. Western blotting images were quantified using ImageJ, and the average of three biologically independent samples is shown.</p

Changes in cyclin levels after thymidine block.

(A) Schematic representation of the experimental setup for cell synchronization early in the S phase. The cyclin A, B1 and E levels in (B) U2OS and (C) ΔN6AMT1#1 were analysed via Western blotting at the indicated timepoints. Cyclin A, B and E images were obtained after exposure time of 2 min for U2OS and 15 min ΔN6AMT1#1 samples. (D-F) Graphical representation of changes in the cyclin levels. Western blotting images were quantified using ImageJ, and the average of three biologically independent samples is shown.</p

N6AMT is important for cell proliferation and cyclin E regulation in HEK293 cells.

(A) The proliferation rate of the U2OS, ΔN6AMT1#1 and ΔN6AMT1#2 cell lines were determined using an xCELLigence Real-Time cell analyser, and data were recorded for a total of 7 days. (B) qRT‒PCR verification of cyclin A, cyclin B and cyclin E levels in HEK293ΔN6AMT1 cells. The average of two biologically independent samples is shown. Statistical analysis was performed with GraphPad Prism 8.4.3. (C) Unsynchronized cells and cells that were synchronized with serum depletion and thymidine treatment were analysed via Western blotting with antibodies against Cyclin A, Cyclin B1, Cyclin E, N6AMT1 and GAPDH. 10 μg of total protein was loaded per well. (D-F) Cyclin A, cyclin B and cyclin E levels in U2OS and ΔN6AMT1#1 cells. Western blotting images were quantified using ImageJ, and the average of three biologically independent samples is shown. Statistical analysis was performed with GraphPad Prism 8.4.3. The data are presented as the means ± SDs; *P (PDF)</p

N6AMT1 impacts the cell proliferation rate.

(A) The U2OS and N6AMT1-depleted cell lines (ΔN6AMT1#1 and ΔN6AMT1#2) were analysed via Western blotting with antibodies against N6AMT1 and α-tubulin. (B) The proliferation rate of the U2OS, ΔN6AMT1#1 and ΔN6AMT1#2 cell lines was determined using an xCELLigence Real-Time cell analyser, and data were recorded for a total of 7 days. (C) Doubling time of the U2OS, ΔN6AMT1#1 and ΔN6AMT1#2 cell lines. The average of three biologically independent samples is shown.</p