Journal: Journal of Biomedical Science

Article Title: Utilisation of an in vivo malaria model to provide functional proof for RhopH1/CLAG essentiality and conserved orthology with P. falciparum

doi: 10.1186/s12929-024-01105-7

Figure Lengend Snippet: P. berghei clag9 cannot complement the function of clagX . a Schematic of p PbX/9 designed to replace the ~ 450 aa Ct portion of the PbA clagX locus with that of the clag9 locus (pink). A p PbX/9cntrl construct was used as a control for transfection. pABR099 containing a PbclagX gRNA was utilised to cleave the PbclagX locus (indicated by lightning bolt). A schematic of the expected PbclagX/9 ( Pb X/9 ) or PbclagX/9control ( Pb X/9cntrl ) locus after integration (int) of the targeting construct is shown, as is a protein schematic to highlight which domains have been included in the replacement. TMD, transmembrane domain; RH2B, RhopH2 binding site; RH3, RhopH3 binding site; HVR, hypervariable region. Indicated are the primers used to validate the presence of b WT; c 5´Int of Pb X/9 ; d 5´Int in Pb X/9cntrl ; and e 3´Int of Pb X/9 and Pb X/9cntrl , and the expected product sizes are indicated. PbA gDNA was used as a positive control for detecting WT locus and as a negative control for an integrated locus. g – i IFA of schizont stage parasites from the indicated lines were probed with α-HA antibodies. Successful allelic replacement of the PbclagX gene should lead to expression of the HA epitopes. Brightfield image merged with DAPI and α-HA shows; g negative HA expression in Pb X/9 ; and h positive HA expression localising to what appears to be the rhoptries in PbX/9cntrl parasites. i PbA parasites used as a negative HA expression control

Article Snippet: After washing, slides were incubated with either donkey α-mouse (Thermofisher, A-10037) or goat α-rat AlexaFluor 568 secondary antibodies (Thermofisher, A-11077) diluted 1:2000 in blocking solution, incubating at RT in the dark for 1 h. Slides were again washed three times in PBS-Tween, then air dried for ~ 2 min. A single drop of Prolong Antifade (Gold) solution with DAPI (6-diamidino-2-phenylindole) (Invitrogen) was added to each well and a glass coverslip (ProSciTech) applied gently.

Techniques: Construct, Control, Transfection, Binding Assay, Positive Control, Negative Control, Expressing

Journal: Journal of Biomedical Science

Article Title: Utilisation of an in vivo malaria model to provide functional proof for RhopH1/CLAG essentiality and conserved orthology with P. falciparum

doi: 10.1186/s12929-024-01105-7

Figure Lengend Snippet: Immunofluorescent analysis of tagged Pb X/3.1 , Pb X/3.1cntrl and Pb X/3.1extnd lines confirm PbclagX can be replaced with Pfclag3.1 . Schizont stage parasites from the indicated lines were probed with α-HA antibodies. Successful allelic replacement of the PbclagX gene should lead to expression of the HA epitopes. Brightfield image merged with DAPI and α-HA shows HA expression localising to what appears to be the rhoptries after replacement of a Pb X/Pf3.1 , b Pb X/3.1cntrl , c and Pb X/3.1extend . d PbA was used as a negative HA expression control

Article Snippet: After washing, slides were incubated with either donkey α-mouse (Thermofisher, A-10037) or goat α-rat AlexaFluor 568 secondary antibodies (Thermofisher, A-11077) diluted 1:2000 in blocking solution, incubating at RT in the dark for 1 h. Slides were again washed three times in PBS-Tween, then air dried for ~ 2 min. A single drop of Prolong Antifade (Gold) solution with DAPI (6-diamidino-2-phenylindole) (Invitrogen) was added to each well and a glass coverslip (ProSciTech) applied gently.

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Chromatin regulator HELLS mediates SSB repair and responses to DNA alkylation damage

doi: 10.1101/2024.12.19.629292

Figure Lengend Snippet: A. Western blot of Whole Cell Extract (WCE) in HAP1 and HELLS KO probed for HELLS. Actin was used as a loading control. B. Schematic representation of survival assays using crystal violet staining. C. Representative image of a cell survival assay. D-F. Quantification of crystal violet cell survival for D . MMS E. Temozolomide or F. Etoposide. Data are mean ± SD. n=3 independent biological replicates. Two-way ANOVA with Tukey’s multiple comparisons was used to determine significance. G. Experimental setup for cell cycle analysis assay. H. Relative cell cycle distribution of cells after treatment with MMS. n=3 independent biological experiments. I. Representative image of micronuclei in HELLS KO cells. J. Formation of micronuclei in response to MMS treatment. Micronuclei formation in HELLS KO HAP cells treated with MMS. Cells were treated with 250µM or 500µM MMS for 1hr. The number of micronuclei was visualized by DAPI staining in HAP1 control or HELLS KO HAP1 cells 48 hr post MMS treatment. Micronuclei counts were performed in 4 microscopy fields in each experimental setting. Statistically significant differences were calculated using one-way ANOVA with Tukey’s multiple comparisons. Data are mean ± SEM and are representative of at least two independent experiments. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

Article Snippet: The chromosome spreads were then counterstained with DAPI solution ProLong® Gold Antifade Mountant with DAPI (Thermo Fischer Scientific) and visualized in a Zeiss LSM 900 live cell confocal microscope with Airyscan 2 system.

Techniques: Western Blot, Control, Staining, Clonogenic Cell Survival Assay, Cell Cycle Assay, Microscopy