Journal: eLife

Article Title: SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling

doi: 10.7554/elife.67550

Figure Lengend Snippet: Figure 1. Recruitment of SNX9 to the immunological synapse upon T cell activation. (A) Representative images of maximum intensity projections from Z-stacks (0.19 µm per slice) of WT Jurkat T cells conjugated to SEE pulsed (+ SEE) or non-pulsed (-SEE) antigen presenting cells (APC). Cells were then fixed after incubation at 37 °C for 2, 5, 10, or 15 min, permeabilized and stained for endogenous SNX9 and actin (Phalloidin-488). (B) Mean percentage of SNX9 recruitment towards the synapse at indicated time points of cell shown in A identified in a blinded analysis of 60–100 cells per condition in four

Article Snippet: Expression constructs encoding for human SNX9- pmCherryC1 (RRID:Addgene_27678), SNX9- EGFP, SNX9ΔPX- mCherry, SNX9ΔSH3- mCherry and CD28- EGFP, were provided by Prof. K. Gaus (University of New South Wales).

Techniques: Activation Assay, Incubation, Staining

Journal: eLife

Article Title: SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling

doi: 10.7554/elife.67550

Figure Lengend Snippet: Figure 2. Recruitment of SNX9 to CD28 clusters upon T cell activation. (A) Representative TIRF images of fixed WT Jurkat T cells expressing CD28WT- EGFP or CD28YF-EGFP (cyan) and activated for 10 min. (B) Fold fluorescent intensities of CD28WT/YF-EGFP (cyan) and anti-SNX9 (purple) along the doted lines in A. (C) Quantification of the percentage overlap between WT or YF CD28 and SNX9, or Rab5 and SNX9. (D) Representative TIRF live images of WT Jurkat T cells expressing SNX9-mCherry and CD28-EGFP at indicated timepoints. Images representative of four independent experiments

Article Snippet: Expression constructs encoding for human SNX9- pmCherryC1 (RRID:Addgene_27678), SNX9- EGFP, SNX9ΔPX- mCherry, SNX9ΔSH3- mCherry and CD28- EGFP, were provided by Prof. K. Gaus (University of New South Wales).

Techniques: Activation Assay, Expressing

Journal: eLife

Article Title: SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling

doi: 10.7554/elife.67550

Figure Lengend Snippet: Figure 3. Dynamics and persistence of SNX9 within CD28 clusters. (A) Schematic of photoactivation of fluorescent proteins at the plasma membrane of cover glass activated cells with the photoactivated protein diffusing either though the cell membrane or through protein-clusters localised at the plasma membrane. (B) Representative confocal time series of SNX9-PAmCherry repetitively photoactivated by 405 nm laser at the membrane region of interest (dashed line) in activated SNX9 KO#4 cells. (C) Mean fluorescence intensity profiles of SNX9-PAmCherry intensity within CD28-EGFP clusters (blue) or

Article Snippet: Expression constructs encoding for human SNX9- pmCherryC1 (RRID:Addgene_27678), SNX9- EGFP, SNX9ΔPX- mCherry, SNX9ΔSH3- mCherry and CD28- EGFP, were provided by Prof. K. Gaus (University of New South Wales).

Techniques: Clinical Proteomics, Membrane, Fluorescence

Journal: eLife

Article Title: SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling

doi: 10.7554/elife.67550

Figure Lengend Snippet: Figure 4. SNX9 defines tubules that are connected to CD28 clusters. (A) Correlation of SNX9-mCherry transfected cell with the same cell re-registered by transmission electron microscopy. White box: region of interest positive for CD28-GFP and SNX9-mCherry. Scale 5 μm. (A) Region of interest from (A) identified from high magnification stitched transmission electron micrographs at ×10,000 magnification. White box = area of tomographic analysis. Scale 500 nm. (C) Overlay of CD28-EGFP with stitched electron micrograph. Scale 500 nm. (D) Overlay of SNX9-mCherry with stitched electron micrograph.

Article Snippet: Expression constructs encoding for human SNX9- pmCherryC1 (RRID:Addgene_27678), SNX9- EGFP, SNX9ΔPX- mCherry, SNX9ΔSH3- mCherry and CD28- EGFP, were provided by Prof. K. Gaus (University of New South Wales).

Techniques: Transfection, Transmission Assay, Electron Microscopy

Journal: eLife

Article Title: SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling

doi: 10.7554/elife.67550

Figure Lengend Snippet: Figure 5. SNX9 promotes CD28 cluster stability. (A) Representative live images of activated WT Jurkat (left) and SNX9 KO#4 (right) T cells expressing CD28WT-EGFP or CD28YF-EGFP with or without SNX9WT-mCherry. (B) Number of CD28WT/YF-EGFP positive structures in Jurkat SNX9 KO T cells with or without SNX9WT-mCherry expression. Dots represent individual cells (15-28) of three to five independent experiments (C) Representative images at indicated time points of activated WT Jurkat T cells expressing SNX9-EGFP and CD28WT-PAmCherry (top panel) or TCRζ-PAmCherry (bottom

Article Snippet: Expression constructs encoding for human SNX9- pmCherryC1 (RRID:Addgene_27678), SNX9- EGFP, SNX9ΔPX- mCherry, SNX9ΔSH3- mCherry and CD28- EGFP, were provided by Prof. K. Gaus (University of New South Wales).

Techniques: Expressing

Journal: eLife

Article Title: SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling

doi: 10.7554/elife.67550

Figure Lengend Snippet: Figure 6. Surface levels and endocytosis of CD28 upon SNX9 knock-out. (A) Mean fluorescence intensity of resting and activated (20 min) WT Jurkat and SNX9KO#3 and SNX9KO#4T cells incubated with an antibody against CD28 (CD28-FITC). (B) Mean fluorescence intensity of resting and activated Jurkat WT and SNX9KO#3 and SNX9KO#4T cells stained with an antibody against CD28 (CD28-FITC) and allowed to internalise CD28 complexes before removing any surface bound antibody. Error bars indicate mean ± SEM. n.s. = not significant, * = p < 0.05 from Student’s T-test of means of three independent experiments. (C) Ratio of internalised by surface expression of CD28 normalised to WT (100%). Data obtained from three independent experiments, with at least two replicates per experiment. Error bars indicate mean ± SEM. n.s. = not significant, * = p < 0.05, from a one sample T-test comparing values to the normalised WT mean of 100%. SNX9 specifically contributes to CD28-mediated signalling.

Article Snippet: Expression constructs encoding for human SNX9- pmCherryC1 (RRID:Addgene_27678), SNX9- EGFP, SNX9ΔPX- mCherry, SNX9ΔSH3- mCherry and CD28- EGFP, were provided by Prof. K. Gaus (University of New South Wales).

Techniques: Knock-Out, Fluorescence, Incubation, Staining, Expressing

Journal: eLife

Article Title: SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling

doi: 10.7554/elife.67550

Figure Lengend Snippet: Figure 7. SNX9 specifically contributes to CD28-mediated signalling. (A) Phosphorylation of CD28 and (C) TCRζ in activated WT Jurkat and SNX9KO T cells detected by phospho-specific antibodies in flow cytometry. (B) Normalised (WT = 100%) percentage of pCD28 and (D) pTCRζ in SNX9KO cells compared to WT Jurkat T cells. Dots represent independent experiments (3-4) involving 50’000 cells. Error bars indicate mean ± SEM. n.s. = not significant, * = p < 0.05, from a one sample T-test. (E) Representative immunofluorescence images of resting and activated WT and SNX9KO Jurkat T

Article Snippet: Expression constructs encoding for human SNX9- pmCherryC1 (RRID:Addgene_27678), SNX9- EGFP, SNX9ΔPX- mCherry, SNX9ΔSH3- mCherry and CD28- EGFP, were provided by Prof. K. Gaus (University of New South Wales).

Techniques: Phospho-proteomics, Flow Cytometry, Immunofluorescence

Journal: eLife

Article Title: Brain-specific Drp1 regulates postsynaptic endocytosis and dendrite formation independently of mitochondrial division

doi: 10.7554/eLife.44739

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , mCherry-Clathrin (CLC) , Addgene , Cat #: 27680 , .

Techniques: CRISPR, Transgenic Assay, Recombinant

Journal: bioRxiv

Article Title: PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining during mitosis

doi: 10.1101/2025.06.09.658719

Figure Lengend Snippet: ( A-B ) Schematic (A) and experimental timeline (B) of MMEJ reporter used in (C-G), (C) and (D), (A) and (C) and (F-H), and (C) and (D). Cutting the reporter with ISceI and repair by microhomology annealing to an in-frame, functional mCherry gene. Flow cytometry was used to identify the mCherry+ cells (MMEJ+) within the BFP+ (ISceI+) population. ( C-E ) MMEJ quantification using reporter and timeline from (A-B) in HT1080 cells. Values are normalized to DMSO. Drugs used were olaparib (5 µm or as indicated), DNA-PKcsi (NU7441, 1 µm), Polθi (ART558 10 µm), niraparib (2.5 µm), rucaparib (2.5 µm), and talazaparib (2.5 µm). (F) MMEJ quantification in HT1080 parental cells compared to isogenic POLQ-KO cells following olaparib treatment. Values are normalized to wild-type DMSO. ( G ) MMEJ quantification in PEO1-OR cells following olaparib treatment. Values are normalized to DMSO. Statistical analyses for ( C-G ): Data represent three independent experiments, each the average of three technical replicates. Data are mean ± SEM. Statistical test, one way ANOVA with multiple comparison correction. ns: non-significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The PARP2 plasmid used to complement PARP2 -KO clones was a generous gift from the Karolin Luger lab and was derived by cloning the human PARP2 coding region into plasmid mCherry C1 (Addgene, #58476).

Techniques: Functional Assay, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining during mitosis

doi: 10.1101/2025.06.09.658719

Figure Lengend Snippet: ( A-B ) Schematic (A) and experimental timeline (B) of MMEJ reporter used in (C-E) and (E). Repair of the Cas9-induced cut with microhomology annealing leads to an in-frame, functional mCherry gene. Flow cytometry was used to identify the mCherry+ cells (MMEJ+) within the GFP+ (Cas9+) population. ( C-E ) MMEJ quantification using the reporter and timeline from (A-B) in HT1080 cells. Values are normalized to DMSO. Drugs used were olaparib (5 µm or indicated dose), DNA-PKcsi (NU7441, 1 µm), Polθi (ART558, 10 µm). Statistical analyses ( C-E ): Data represent three (D, E) or four (C) independent experiments, each the average of three technical replicates. Data are mean ± SEM. Statistical test, one way ANOVA with multiple comparison correction. ns: non-significant, *p<0.05, ***p<0.001, ****p<0.0001.

Article Snippet: The PARP2 plasmid used to complement PARP2 -KO clones was a generous gift from the Karolin Luger lab and was derived by cloning the human PARP2 coding region into plasmid mCherry C1 (Addgene, #58476).

Techniques: Functional Assay, Flow Cytometry, Comparison