Journal: bioRxiv

Article Title: Synergistic targeting of cancer cells through simultaneous inhibition of key metabolic enzymes

doi: 10.1101/2025.05.02.651820

Figure Lengend Snippet: KRAS G12V /MYC cells were transfected with siRNAs targeting either LDHA/B or IDO1 alone or combined with GNE and/or BMS treatment as shown. Transfection was repeated after 2 days. (A) After 4 days one half of the cells was analyzed for effects on cell density, values were normalized to si-scramble, shown are mean ± SD, n = 3. (B) The other half of the siRNA-treated cells was analyzed by Western blotting for knock-down, the asterisk indicates a non-specific band, the positions of molecular weight markers are indicated. (C) Cells were treated for 3 days with Epacadostat (IDO1 inhibitor, 40 µM), GSK-2837808 (LDHA/B inhibitor, 35 µM) alone or in combination with GNE or BMS, as shown. Cell viability was determined, shown are mean ± SD, n = 3. The Coefficient of Drug Interaction (CDI) for GNE and BMS as well as Epacadostat and GSK-2837808 was calculated and indicated above respective bar plots. (D) COX8-GFP EV and KRAS G12V /MYC cells were treated for 2 days with the indicated conditions and stained with Hoechst33342 (nuclear DNA) and Phalloidin (Actin-filaments) following immunofluorescence analysis. White rectangular selections are highlighting the cells used for mitochondrial high-resolution 3D-rendering . (E) Cells treated with GNE and/or BMS for 2 days, loaded with MitoTracker Deep Red FM (200 nM for 15 minutes) and subsequently analyzed using flow-cytometry. Shown are median fluorescence intensities (MFI), n = 3, two-way ANOVA with Dunnet’s multiple comparisons test. The next subfigures show seahorse metabolic flux analyses of empty vector and KRAS G12V /MYC cell lines using the MitoStress Test assay after 4 hours of GNE and BMS mono- and combination treatment using Oligomycin (2 µM), FCCP (2 µM) and Rotenone/Antimycin A (0.5 µM). (F-H) Analysis of basal, maximal and ATP-linked respiration. (I, J) Quantification of basal and maximal glycolysis. Shown are mean ± SD, n = 4, two-way ANOVA with Šídák’s multiple comparisons test.

Article Snippet: For GFP-labeling of mitochondria, empty vector and KRAS G12V /MYC cell lines were transduced to express COX8-GFP (Addgene plasmid #44385; RRID:Addgene_44385, from Pantelis Tsoulfas).

Techniques: Transfection, Western Blot, Knockdown, Molecular Weight, Staining, Immunofluorescence, Flow Cytometry, Fluorescence, Plasmid Preparation

Journal: bioRxiv

Article Title: Synergistic targeting of cancer cells through simultaneous inhibition of key metabolic enzymes

doi: 10.1101/2025.05.02.651820

Figure Lengend Snippet: Mitochondrial effects of BMS. (A) The indicated EV control and KRAS G12V /MYC cancer cells were transduced to stably express COX8-EGFP, enabling visualization of mitochondria. Cells were treated for 2 days with indicated conditions, followed by fixation and confocal imaging. High-resolution 3D rendered mitochondria of representative cells are displayed. Scale bar = 10 µm. (B) The number of mitochondria per cell and (C) mitochondrial mean branch length was quantified, n = 3, one-way ANOVA with Šídák’s multiple comparisons test. (D) KRAS G12V /MYC cells were treated as indicated, followed by analysis of mitochondrial structure via transmission electron microscopy. Scale bar = 500 nm. The rectangular selections are shown in the lower row at a higher magnification. (E) KRAS G12V /MYC cancer cells were treated with GNE and/or BMS for 4 hours, followed by Seahorse metabolic flux analysis. Oligomycin, FCCP and Rotenone/Antimycin A were injected into the wells at the indicated timepoints, followed by quantification of the oxygen consumption rate (OCR). (F) The experiment was performed as in (E) for quantification of the extracellular acidification rate (ECAR). Seahorse experiments show mean ± SD, n = 4. (G) Energy map summarizing the Seahorse experiments and annotating the different treatments to metabolic states (Aerobic, Glycolytic, Energetic or Quiescent). (H) The indicated cell lines were treated for 4 days with GNE and/or BMS and the extracellular pH was determined. Statistical analysis was done using two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: For GFP-labeling of mitochondria, empty vector and KRAS G12V /MYC cell lines were transduced to express COX8-GFP (Addgene plasmid #44385; RRID:Addgene_44385, from Pantelis Tsoulfas).

Techniques: Control, Stable Transfection, Imaging, Transmission Assay, Electron Microscopy, Injection

Journal: bioRxiv

Article Title: VP-CLEM-Kit: An accessible pipeline for visual proteomics using super resolution volume correlative light and electron microscopy (SR-vCLEM)

doi: 10.1101/2025.04.08.647770

Figure Lengend Snippet: (a) Overview RF and WF images showing serial sections of HeLa cells (scale bar: 400 µm) with insets showing zooms of one of the sections in each modality (scale bar: 200 µm). (b, c, d) High resolution LSM900 Airyscan images showing the preservation of Hoechst (nucleus), mitoGFP (mitochondria) and lysotracker (lysosomes) in a 200 nm thick easyIRF section (scale bar: 20 µm). (e) Cell of interest showing overlay of Hoechst, mitoGFP and lysotracker signals and (f) ultrastructure (scale bar: 10 µm), with inset showing preservation of mitochondria, nucleus, nuclear envelope, and large (white boxes) and small (arrows) lysosomes (scale bar: 2 µm). (g) hiPSC-derived cortical neurons showing preservation of mitoGFP in a 200 nm thick easyIRF section, and (h) the matching ultrastructure (scale bar: 20 µm). Insets show that the pattern of mitoGFP in the LM images matches the pattern of mitochondria in the EM image (scale bar: 5 µm).

Article Snippet: Thereafter, cells were transfected with 0.5 µg of mitoGFP construct (pLV-mitoGFP # 44385, Addgene) per dish, using Lipofectamine LTX and PLUS reagent (Invitrogen, Life Technologies Ltd, Paisley) in Opti-MEM medium (Gibco, Life Technologies Ltd, Paisley) according to the manufacturer’s instructions.

Techniques: Preserving, Derivative Assay

Journal: bioRxiv

Article Title: VP-CLEM-Kit: An accessible pipeline for visual proteomics using super resolution volume correlative light and electron microscopy (SR-vCLEM)

doi: 10.1101/2025.04.08.647770

Figure Lengend Snippet: (a) System diagram of the tomoSTORM open-hardware microscope based on the openFrame modular system, showing the hardware components used to acquire RF, WF and SMLM array tomography images. The system can switch between imaging modalities by manually switching the reflection and multi-line fluorescence dichroic cubes using the dichroic switcher layer. There is a manual 4-objective turret with 10x (Amscope, 10x/0.3 NA) and 100x (Amscope, 100x,/1.3 NA) objectives. The TEC-cooled CMOS camera is a CellCam Kikker (Cairn Research Ltd, CellCam Kikker). The system uses a XY stage (Märzhäuser Wetzlar GmbH, SCAN IM 130 x 85) and a Z-stepper motor (Cairn Research Ltd, Z-Act). The openFrame has an openAF autofocus unit to maintain focus during SMLM acquisitions. The excitation source is a 4-line multimode laserbank (Cairn Research Ltd, Laserbank) which is fibre coupled into the critical illuminator. (b) A photo showing the tomoSTORM light microscope with the individual hardware components labelled. (c) The easyIRF resin block was sectioned and section ribbons retrieved onto an ITO-coated coverslip. The coverslip was attached to a slide-sized metal mount and placed into the slide-holding insert on the xy stage for imaging. (d) RF and (e) WF tiled images were acquired with the 10x objective from a ribbon of easyIRF sections containing Hela cells expressing mitoGFP. Insets (white boxes) show sections 4-6 of the ribbon. Tiled WF images were recorded using the 100x/ 1.3 NA objective from section 4. (f) SMLM of mitoGFP in HeLa cells in an easyIRF section. Left: Multiple frames of a single FOV show blinking in sequential frames. Middle: A single frame of the raw microscope acquisition alongside WF, compared to Right: the final SMLM reconstruction in section 6 of the same sample, highlighting the resolution improvement achieved with SMLM.

Article Snippet: Thereafter, cells were transfected with 0.5 µg of mitoGFP construct (pLV-mitoGFP # 44385, Addgene) per dish, using Lipofectamine LTX and PLUS reagent (Invitrogen, Life Technologies Ltd, Paisley) in Opti-MEM medium (Gibco, Life Technologies Ltd, Paisley) according to the manufacturer’s instructions.

Techniques: Microscopy, Tomography, Imaging, Fluorescence, Light Microscopy, Blocking Assay, Expressing

Journal: bioRxiv

Article Title: VP-CLEM-Kit: An accessible pipeline for visual proteomics using super resolution volume correlative light and electron microscopy (SR-vCLEM)

doi: 10.1101/2025.04.08.647770

Figure Lengend Snippet: (a) 3D reconstruction of SMLM images generated from SMLM datasets acquired from 200 nm thick easyIRF sections through a total cell depth of 3000 nm. Each section was reconstructed using 10,000 frames. The 3D projection shows the spatial organization of mitochondria within the whole cell volume, showing continuity between planes. (b) Single-plane SMLM images corresponding to sections 1, 4, and 11, enabling depth-dependent visualization of mitochondrial structures with nanometric-scale resolution. (c) WF image of section 4, illustrating the overall mitochondrial distribution with diffraction-limited resolution. The estimated resolution, determined via decorrelation analysis, is 414 ± 13 nm, providing a broad overview of the labelled structures but lacking morphological details. (d) SMLM image of section 4, demonstrating a significantly improved spatial resolution compared to the WF image. The estimated resolution, determined via decorrelation analysis, is 84 ± 7 nm, enabling enhanced structural definition of mitochondrial networks. (e) SEM image of section 4. (f) Overlay of WF and EM images, facilitating correlation between mitochondrial localisation in the WF channel and ultrastructural features observed in EM. (g) Overlay of SMLM and EM images, providing improved spatial precision in the correlation of mitoGFP to mitochondrial ultrastructure. Scale bars represent 10 μm for all panels except for the panels on the bottom, where scale bars represent 1 μm.

Article Snippet: Thereafter, cells were transfected with 0.5 µg of mitoGFP construct (pLV-mitoGFP # 44385, Addgene) per dish, using Lipofectamine LTX and PLUS reagent (Invitrogen, Life Technologies Ltd, Paisley) in Opti-MEM medium (Gibco, Life Technologies Ltd, Paisley) according to the manufacturer’s instructions.

Techniques: Generated

Journal: bioRxiv

Article Title: VP-CLEM-Kit: An accessible pipeline for visual proteomics using super resolution volume correlative light and electron microscopy (SR-vCLEM)

doi: 10.1101/2025.04.08.647770

Figure Lengend Snippet: (a) 3D reconstruction of SMLM images generated from SMLM datasets acquired from 200 nm thick easyIRF sections through a total cell depth of 4800 nm. Each section was reconstructed using 10,000 frames. The 3D projection shows the spatial organization of mitochondria within the whole cell volume, showing continuity between planes. (b) Single-plane SMLM images corresponding to sections 1, 3, and 12, enabling depth-dependent visualization of mitochondrial structures with nanometric-scale resolution. (c) WF image of section 3, illustrating the overall mitochondrial distribution with diffraction-limited resolution. The estimated resolution, determined via decorrelation analysis, is 688 ± 75 nm, providing a broad overview of the labelled structures but lacking morphological details. (d) SMLM image of section 3, demonstrating a significantly improved spatial resolution compared to the WF image. The estimated resolution, determined via decorrelation analysis, is 78 ± 7 nm, enabling enhanced structural definition of mitochondrial networks. (e) SEM image of section 3. (f) Overlay of WF and EM images, facilitating correlation between mitochondrial localisation in the WF channel and ultrastructural features observed in EM. (g) Overlay of SMLM and EM images, providing improved spatial precision in the correlation of mitoGFP to mitochondrial ultrastructure. Scale bars represent 10 μm for all panels except for the panels on the bottom, where scale bars represent 1 μm.

Article Snippet: Thereafter, cells were transfected with 0.5 µg of mitoGFP construct (pLV-mitoGFP # 44385, Addgene) per dish, using Lipofectamine LTX and PLUS reagent (Invitrogen, Life Technologies Ltd, Paisley) in Opti-MEM medium (Gibco, Life Technologies Ltd, Paisley) according to the manufacturer’s instructions.

Techniques: Generated