Journal: Molecular Therapy Oncology

Article Title: Recombinant CALR polarizes and activates macrophages in tumors

doi: 10.1016/j.omton.2025.201121

Figure Lengend Snippet: CALR expressed by Salmonella activates macrophages (A) The genes of four DAMPs (CALR, gp96, HMGB1, and hsp70) were cloned into a pBAD expression vector and transformed into Salmonella . Each operon contained the araBAD promoter to control expression, the R111 ribose-binding site (RBS) to maximize production, and a myc tag to enable detection. (B) After induction, Salmonella expressed each of the DAMP proteins. All of the recombinant proteins were detected by staining myc-tag. (C–F) RAW264.7 macrophages were treated with the four bacterial lysates ( n = 3) for 24 h. Cells were also treated with saline (PBS) as negative control, MPL as a positive control, and lysate from non-transformed bacteria as a bacterial control (BC). MPL is an immunogenic molecule that is used as a vaccine adjuvant. Comparison to untransformed bacterial lysate controls for the effect of bacterial components on the macrophage responses. (C) All treatments (including MPL), significantly increased the percentage of live, iNOS-positive RAW264.7 cells compared to PBS ( p < 0.0001). (D) CALR was the only DAMP that significantly increased iNOS expression, in iNOS-positive cells, compared to the BC treatment group ( p < 0.0001). The BC increased iNOS expression compared to saline controls ( p < 0.0001). (E) CALR significantly increased the expression of the CD80 in iNOS-positive RAW264.7 cells compared to BCs ( p < 0.0001). Bacterial lysate slightly increased CD80 expression compared to saline ( p = 0.0107). No other DAMPs increased CD80 expression compared to BCs. (F) CALR also increased expression of CD86 in iNOS-positive cells compared to controls ( p < 0.0001). Bacterial lysate did not have a significant effect on CD86 expression. Gating in (C and D) was determined using an isotype control. Data are represented as means ± SEM. The statistical comparisons in (C–F) are ANOVA followed by Tukey’s method. Asterisks indicate significance: ∗ p < 0.05; ∗∗∗∗ p < 0.0001.

Article Snippet: Plasmids for EGFP (EGFP-pBAD), hsp70 (pEGFP hsp70), hsp90 (pDONR223_HSP90B1_WT), and HMGB1 (HMGB1) were obtained from Addgene.

Techniques: Clone Assay, Expressing, Plasmid Preparation, Transformation Assay, Control, Binding Assay, Recombinant, Staining, Saline, Negative Control, Positive Control, Bacteria, Adjuvant, Comparison

Journal: bioRxiv

Article Title: Reliable quantification of multiplexed genetically encoded biosensors responsiveness in plant tissues

doi: 10.64898/2026.03.13.711581

Figure Lengend Snippet: mTagBFP2, mTurquoise2, Venus, mKO2, mCherry, mKate2, and mCardinal exhibit approximately comparable brightness across different excitation conditions. All fluorescent proteins were fused to a nuclear localisation signal. (a) Cumulative fluorescence intensity (RFU) of transiently expressed proteins in N. benthamiana, measured using standard laser lines on the Leica Stellaris 5 microscope. (b) The same measurement performed using the 405 nm or a white light laser on Stellaris 8, with excitation wavelengths set just below each protein’s emission peak. Fluorescent proteins are indicated below each bar chart, while laser wavelengths and power settings are shown above. Detector gain was kept at the minimum setting for all fluorescent proteins. For each condition, at least two independent images were analysed. Mean fluorescence intensity per nucleus was plotted as bar charts with overlaid points (each dot represents one nucleus). (c, e) Expected fluorescence intensity based on FPbase.org data (excitation × brightness), shown for (c) Stellaris 5 and (e) Stellaris 8, calculated for (a) and (d) standard laser lines, and (b) and (f) white light laser excitation. Among the tested fluorescent proteins, Venus, mKO2 and mKate2 were selected for stable transformation in potato (highlighted in yellow across all charts). (d, f) Brightness of fluorescent proteins measured in S. tuberosum (potato) leaves (d) using standard laser lines or (f) the white light laser, as described in panels (a‒b). Brightness is shown for one transgenic line per fluorescent protein. Analysis of a second transgenic line gave similar results (results available on Zenodo: 10.5281/zenodo.17600476).

Article Snippet: N7 localization signal together with mTurquoise2, Venus, and mKate2 (plasmids pHG128, pHG132, and pHG154, respectively) were kindly provided by Hassan Ghareeb ( ). mTagBFP2 (plasmid pJL1-mTagBFP2; Addgene #102638; ( )), mKO2 (plasmid pTU-A-005; Addgene #124412; ( )), mCardinal (plasmid mCardinal-pBAD; Addgene #54800; ( )), and miRFP713 (plasmid pmiRFP713-N1; Addgene #136559; ( )) were obtained from Addgene.

Techniques: Fluorescence, Microscopy, Transformation Assay, Transgenic Assay

Journal: bioRxiv

Article Title: Reliable quantification of multiplexed genetically encoded biosensors responsiveness in plant tissues

doi: 10.64898/2026.03.13.711581

Figure Lengend Snippet: (a) Before unmixing, (b) spectral unmixing using recorded reference spectra, (c) channel separation of mCardinal and chlorophyll. From left to right: mCardinal, chlorophyll and overlay of both channels. Scale bar: 50 µm.

Article Snippet: N7 localization signal together with mTurquoise2, Venus, and mKate2 (plasmids pHG128, pHG132, and pHG154, respectively) were kindly provided by Hassan Ghareeb ( ). mTagBFP2 (plasmid pJL1-mTagBFP2; Addgene #102638; ( )), mKO2 (plasmid pTU-A-005; Addgene #124412; ( )), mCardinal (plasmid mCardinal-pBAD; Addgene #54800; ( )), and miRFP713 (plasmid pmiRFP713-N1; Addgene #136559; ( )) were obtained from Addgene.

Techniques: