Image pro premier

Stitched images were calibrated in ImagePro Premier (Media Cybernetics) for automated quantification of protrusion frequency. eGFP⁺ macrophages were size-selected by filtration of objects with an area 100 µm² or larger and then masked to reduce noise artifacts (Supplementary Fig. S1a, Step I). Protrusions were isolated by repeatedly passing a morphological erosion filter (2 × 2 square, 7 passes) over the image which erased peripheral cellular signal, followed by applying a dilation that restored signal to the central cell body (Step II). Subtracting the processed image from the original mask resulted in selection of regions protruding from the main cell body, which can be filtered by a highly variable set of size parameters to yield either thin TNT-like structures or other thicker protrusions (Supplementary Fig. S1b) (filters for TNTs: 20 µm² minimum area, 10 µm² minimum longest axis, 0.6 maximum circularity, 1.9 box area minimum). Finally, the selected protrusion area was overlaid with the original image and cells were classified as either protrusion-positive or -negative based on signal intensity; overlap of protrusion and cell body yielded a positive result (Step III - IV). All segmentation, adjustment and filtration factors were trained and optimized blind to condition and applied automatically to batch images using ImagePro Premier macros.

For visualization of mitochondria, parental and vemurafenib-resistant cells were plated in a 12-well coverslip 10 mm glass diameter plate overnight at 37°C, 5 % CO₂ incubator. Cells were then washed with PBS, before addition of live cell imaging solution (Molecular Probes^®, Thermofisher) containing the Mitotracker Green FM (Invitrogen) and DNA dye Hoechst 33342 (Thermofischer) for 30 min at 37°C. Afterwards, the staining solution was removed and cells were washed twice with LCIS, and then 500 μl LCIS containing 10 mM glucose was added in each well. The live cells were then observed and photographed using a Zeiss LSM 880 laser scanning confocal microscope (Carl Zeiss Microscopy, Thornwood, NY, USA). Imaging data was subsequently analyzed and quantified using Image-Pro Premier (Version 9.2, Media Cybernetics, Rockville, MD, USA).

The same segments used for hydraulic measurements were also used for wood anatomy analysis. Tracheid lumen diameter was measured for the most recent complete two rings of each segment using a radial file of three cells wide with images taken with a Leica DM3000 microscope at 20× magnification. Only noncompression wood was analyzed. The hydraulically weighted mean lumen diameter (T_diam) was calculated using the Hagen–Poiseuille formula: $T_{\mathrm{diam}}={\left(\frac{\sum d^4}{n}\right)}^{1/4}$ where d is tracheid diameter and n is the total number of tracheids measured. Tracheid length (T_length) was measured using chemical macerations following Schoonmaker et al., 2010. Wood sections corresponding to the outer rings of each segment were digested in a 1:1 mixture of 80% glacial acetic acid and 30% hydrogen peroxide at 60°C for 48 hr. Macerated tissue was analyzed with a light microscope at 25× magnification to measure the length of 200 tracheids for each sample. All the image analyses were performed with ImagePro Premier (Media Cybernetics, Silver Spring, MD, USA) software.

Apically localized dividing progenitor cells at Anaphase/Telophase were identified with the appearance of typically condensed separating chromatids. Angles of cleavage planes with reference to the surface of the VZ were determined using Image-Pro Premier (Media Cybernetics, Inc). The data of each individual brain were obtained from 30 to 50 pictures of brain sections at ×40 optical view.

Deparaffinised and rehydrated tissue sections were processed for epitope retrieval using EnVision FLEX target retrieval solution (Agilent, K8004), as per manufacturer’s instructions. Samples were processed and stained for TRIM22 (Proteintech, 13744-1-AP; 1/50 dilution) and FLEX haematoxylin (Agilent, K8008) using a Autostainer Link 48 (Agilent), as per manufacturer’s instructions. TRIM22 antibody specificity was validated by staining sectioned MRC5 (positive control) or A549 (negative control) cell pellets. Tissue sections were independently assessed for TRIM22 expression by a qualified pathologist. Automated quantitation of TRIM22 expression levels was performed using whole-slide scans and Image-Pro Premier (Media Cybernetics), as previously described (Leeming et al., 2015 (link); Akram et al., 2018 (link)).