Journal: Scientific Reports

Article Title: HaloPROTAC3 does not trigger the degradation of the halotagged parasitophorous vacuole membrane protein UIS4 during Plasmodium liver stage development

doi: 10.1038/s41598-025-98257-9

Figure Lengend Snippet: Generation and characterization of P. berghei ( Pb ) UIS4-HT. ( a ) Illustration of the P. berghei UIS4-HT locus. The blue line indicates the portion that is exposed to the host cytosol. ( b ) Confocal micrographs of Huh7 cells infected with P. berghei WT and P. berghei UIS4-HT for 2 days and stained for DNA (blue), Pb HSP70 (green), Pb UIS4 (red), and HaloTag (cyan). Scale bars are 20 μm. ( c ) Infection rates (% liver stages (LS) per host nuclei). Median infection rates were calculated from 2–4 technical replicates and pooled from 7 independent experiments ( N = 7). No statistically significant difference was detected ( P = 0.32, Mann-Whitney test). The solid black lines indicate medians. ( d ) Area sizes of liver stages at 2 days post-infection. Median area sizes were determined for each well and averaged from 2–4 technical replicates. No statistically significant difference was detected ( P = 0.51, unpaired t test, N = 7). Results are expressed as means with SEMs. ( e ) Proportion of HaloTag-positive liver stages for Huh7 cells infected with P. berghei UIS4-HT. Median values were calculated from 2–3 technical replicates and expressed as means with SEMs ( N = 4). ( f ) Blood stage parasitemia in mice infected with P. berghei WT and P. berghei UIS4-HT sporozoites. The graph shows data from two independent experiments, each including 2 (#1) or 3 (#2) mice per condition apart from one mouse for P. berghei WT #1 at 6 days post-infection (one mouse had to be euthanized a day earlier for this group). Results are expressed as means with SEMs. RBCs, red blood cells.

Article Snippet: The samples were then incubated at 4 o C overnight in BP buffer containing a combination of either rabbit anti- Pc HSP70 (1:5,000) and mouse anti-HaloTag (1:1,000, Promega, cat no. G9211), or rat anti-mCherry (1:200, Invitrogen, cat no. M11217 ) and mouse anti-GFP (1:100, Roche, cat no. 11814460001) primary antibodies.

Techniques: Infection, Staining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: HaloPROTAC3 does not trigger the degradation of the halotagged parasitophorous vacuole membrane protein UIS4 during Plasmodium liver stage development

doi: 10.1038/s41598-025-98257-9

Figure Lengend Snippet: Characterization of P. cynomolgi ( Pc ) UIS4-HT liver stages. ( a ) Airyscan micrographs of primary simian hepatocytes infected with P. cynomolgi UIS4-HT for 8 days and stained for DNA (blue), Pc HSP70 (green), and HaloTag (red). A schizont (top row) and a hypnozoite (bottom row) are shown. Scale bars are 20 μm and 10 μm, respectively. ( b ) Infection rate of P. cynomolgi UIS4-HT presented as number of liver stages (LS) per well (≥ 80 wells per biological replicate). The solid black line indicates the median ( N = 3).

Article Snippet: The samples were then incubated at 4 o C overnight in BP buffer containing a combination of either rabbit anti- Pc HSP70 (1:5,000) and mouse anti-HaloTag (1:1,000, Promega, cat no. G9211), or rat anti-mCherry (1:200, Invitrogen, cat no. M11217 ) and mouse anti-GFP (1:100, Roche, cat no. 11814460001) primary antibodies.

Techniques: Infection, Staining

Journal: Scientific Reports

Article Title: HaloPROTAC3 does not trigger the degradation of the halotagged parasitophorous vacuole membrane protein UIS4 during Plasmodium liver stage development

doi: 10.1038/s41598-025-98257-9

Figure Lengend Snippet: HaloPROTAC3 binds to P. berghei ( Pb ) UIS4-HT liver stages (LS). (a) Confocal micrographs of Huh7 cells infected with P. berghei WT and P. berghei UIS4-HT and stained for DNA (blue), parasite markers UIS4 (red) and HSP70 (cyan), and a fluorescent HaloTag ligand (HTL, green) 2 days post-infection. Scale bars are 20 μm. (b) Percentages of liver stages with bound fluorescent HTL were quantified for cells infected with P. berghei WT and P. berghei UIS4-HT. Median percentages were calculated from 4–8 technical replicates and expressed as means with SEMs ( N = 2). (c) Dose-response curves showing the percentages of liver stages that bound to the fluorescent HTL as a function of HaloPROTAC3 concentration. During this assay, the binding of HaloPROTAC3 to UIS4-HT liver stages was evaluated by probing for remaining available binding sites with the fluorescent HTL. P. berghei WT and the VHL binder served as negative controls. Median percentages of liver stages with bound fluorescent HTL were calculated from technical duplicates and normalized. 100% corresponds to samples infected with P. berghei UIS4-HT and treated with DMSO. Results are means with SEMs ( N = 2).

Article Snippet: The samples were then incubated at 4 o C overnight in BP buffer containing a combination of either rabbit anti- Pc HSP70 (1:5,000) and mouse anti-HaloTag (1:1,000, Promega, cat no. G9211), or rat anti-mCherry (1:200, Invitrogen, cat no. M11217 ) and mouse anti-GFP (1:100, Roche, cat no. 11814460001) primary antibodies.

Techniques: Infection, Staining, Concentration Assay, Binding Assay

Journal: Scientific Reports

Article Title: HaloPROTAC3 does not trigger the degradation of the halotagged parasitophorous vacuole membrane protein UIS4 during Plasmodium liver stage development

doi: 10.1038/s41598-025-98257-9

Figure Lengend Snippet: HaloPROTAC3 does not induce VHL recruitment and PVM ubiquitination during P. berghei liver stage development. (a) Illustration of the mCherry-VHL construct expressed in Huh7 cells. (b) Confocal micrographs of Huh7 mCherry-VHL cells infected with P. berghei UIS4-HT and stained for DNA (blue), mCherry (red), and parasite markers HSP70 (green) and UIS4 (cyan). Scale bar is 20 μm. (c) Illustration of the mCherry-ubiquitin (Ub) construct expressed in Huh7 cells. (d) Confocal micrographs of Huh7 mCherry-Ub cells infected with P. berghei UIS4-HT and stained for DNA (blue), mCherry (red), and parasite markers HSP70 (green) and UIS4 (cyan). Scale bar is 20 μm. Integrated mCherry-VHL (e) and mCherry-Ub (f) fluorescence intensities associated with P. berghei UIS4-HT liver stages in Huh7 cells treated with either the VHL binder or HaloPROTAC3. The median integrated fluorescence intensities of P. berghei liver stages were calculated for each well, normalized and averaged from 4 technical replicates. 100% corresponds to infected samples treated with DMSO. Results are presented as means with SEMs ( N = 2). No statistically significant differences were detected at 100 or 500 nM (unpaired t tests).

Article Snippet: The samples were then incubated at 4 o C overnight in BP buffer containing a combination of either rabbit anti- Pc HSP70 (1:5,000) and mouse anti-HaloTag (1:1,000, Promega, cat no. G9211), or rat anti-mCherry (1:200, Invitrogen, cat no. M11217 ) and mouse anti-GFP (1:100, Roche, cat no. 11814460001) primary antibodies.

Techniques: Ubiquitin Proteomics, Construct, Infection, Staining, Fluorescence