Journal: bioRxiv

Article Title: Genetically encoded tool for manipulation of ΔΨm identifies the latter as the driver of integrative stress response induced by ATP Synthase dysfunction

doi: 10.1101/2023.12.27.573435

Figure Lengend Snippet: a , Effect of various concentrations of FCCP (left) or Bam15 (right) on the ΔΨm using TMRM. The intensity of TMRM was normalized by the intensity of Hoechst. Data is normalized to control condition (Ctrl, lane 1). Means ± SEM, n=3 biological replicates. P values were determined with two-way ANOVA followed by Tukey’s multiple comparisons test in Graphpad ( n.s. , not significant). b , Effect of UCP1 on the ΔΨm in the presence of 50 mM oleic acid (Methyl-b-cyclodextrin conjugated) using TMRM. The intensity of TMRM was normalized by the intensity of Hoechst. Data is normalized to oleic acid-treated condition (lane 1). Means ± SEM, n=3 independent experiments. P values were determined with two-way ANOVA followed by Tukey’s multiple comparisons test in Graphpad ( n.s. , not significant). c , Effect of oleic acid (OA, 300 mM) or doxycycline (Dox, 300 ng/ml) on proliferation of wild type parental C2C12 (left) or UCP1-expressing (right) C2C12 cells in the presence of 1 mM pyruvate. Means ± SEM, n=3 biological replicates. d , Effect of various concentrations of FCCP (left) or Bam15 (right) on the proliferation of C2C12 cells in the presence of 1 mM pyruvate. Means ± SEM, n=4 independent experiments. e , Effect of various concentrations of FCCP (left) or Bam15 (right) on basal and oligomycin (1 mM)-treated oxygen consumption. Antimycin A (1 mM) was injected at the last as a negative control. Means ± SEM, n=3 independent experiments.

Article Snippet: Cells were incubated at 37 °C with the following mitochondrial toxins for 16 hr: 1 μM oligomycin (Signa-Aldrich, 75351), 5 μM FCCP (TOCRIS, 45310), 1 μM antimycin A (Sigma-Aldrich, A8674), 1 μM rotenone (Sigma-Aldrich, R8875), 50 μgml -1 doxycycline (Sigma-Aldrich, D9891).

Techniques: Control, Expressing, Injection, Negative Control

Journal: bioRxiv

Article Title: Genetically encoded tool for manipulation of ΔΨm identifies the latter as the driver of integrative stress response induced by ATP Synthase dysfunction

doi: 10.1101/2023.12.27.573435

Figure Lengend Snippet: a , A simple schematic diagram showing the molecular mechanism of how oligomycin induces mitochondrial stress signaling. b , Effect of various chemical drugs on inducing ATF4 in C2C12 cells. Immunoblotting analyses were performed with the indicated antibodies. Representative gel from one of three experiments. C, control; OLG, 1 mM oligomycin; FCCP, 5 mM FCCP; Ant, 1 mM Antimycin A; Rot, 1 mM Rotenone; Dox, 50 mg/ml doxycycline. Bar graphs show the quantification of the intensity of western blot data, relative amount of ATF4 compared to control condition. Means ± SEM, n=3independent experiments. P values were determined with two-way ANOVA followed by Tukey’s multiple comparisons test in Graphpad (n.s., not significant). c,d,e , Effect of UCP1 (c), LbNOX (d), or mitoLbNOX (e) on mitochondrial stress signaling. Representative gel from one of four (UCP1 and LbNOX) or five (mitoLbNOX) experiments Bar graphs show the quantification of the intensity of western blot data, relative amount of ATF4 (left) compared to control condition. Means ± SEM, n=4 or 5 independent experiments. P values were determined with two-way ANOVA followed by Tukey’s multiple comparisons test in Graphpad (n.s., not significant).

Article Snippet: Cells were incubated at 37 °C with the following mitochondrial toxins for 16 hr: 1 μM oligomycin (Signa-Aldrich, 75351), 5 μM FCCP (TOCRIS, 45310), 1 μM antimycin A (Sigma-Aldrich, A8674), 1 μM rotenone (Sigma-Aldrich, R8875), 50 μgml -1 doxycycline (Sigma-Aldrich, D9891).

Techniques: Western Blot, Control

Journal: Aging Cell

Article Title: Compromised CD8+ T cell immunity in the aged brain increases severity of neurotropic coronavirus infection and postinfectious cognitive impairment

doi: 10.1111/acel.14409

Figure Lengend Snippet: Aging limits T RM establishment within the brain. 8 week. adult or 18 months aged C57BL/6 mice were inoculated with 10 3 pfu MHV‐A59 i.n., CD8 + T cells harvested from the spleen 7 DPI and then adoptively transferred intravenously (i.v.) via the tail vein into either 8 week. or 18 months old animals that were infected with (a,b) 10 4 pfu or (c,d) 10 3 pfu MHV‐A59 1 day prior. (a, c) Survival and (b, d) weight change were monitored for 30 DPI. (e) Representative flow cytometry plots of lymphocytes stained for CD8, then CD45.1 and CD45.2 to distinguish between host and donor cells isolated from the brains of 8 week. or 18 months old animals at 30 DPI following infection with 10 3 pfu MHV‐A59 and adoptive transfer of 8 week. or 18 months cells as indicated. (f) Frequency and (g) total number of total CD8 + cells and total transferred cells. (h) Representative flow cytometry histograms of CD103 expression by CD8 + T cells present in the brain of 8 week. or 18 months animals 30 DPI following infection and adoptive transfer as described. (i) Quantification of CD103 MFI by host or donor cells within the same host. Data are representative of 1 independent experiment with each data point representing an individual animal. Survival assessed by Log‐rank Mantel‐Cox assessment, weight change and flow cytometry assessment conducted according to two‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Tetramer staining was carried out at room temperature for 20 min in conjugation with other surface staining antibodies: CD8a (53–6.7, APC‐Cy7), CD4 (RM4‐5, APC), CD44 (IM7, PE‐Cy7), CD45 (30‐F11, APC), CD45.1 (20–0453, APC), CD45.2 (60–0454, PE‐Cy‐7), CD11b (M1/70, FITC), P2RY12 (S16007D, PE), CD68 (FA‐11, BV‐421), MHC II (M5/114.15.2, PerCP‐Cy5.5), obtained from Cytek Biosciences or eBioscience, then washed with 1× PBS and fixed with 2% paraformaldehyde (PFA).

Techniques: Infection, Flow Cytometry, Staining, Isolation, Adoptive Transfer Assay, Expressing

Journal: Aging Cell

Article Title: Compromised CD8+ T cell immunity in the aged brain increases severity of neurotropic coronavirus infection and postinfectious cognitive impairment

doi: 10.1111/acel.14409

Figure Lengend Snippet: CD8 + T cells mediate neuronal apoptosis following MHV‐A59 infection. (a) Representative IHC of DAPI, NeuN, and CD8a in the DG and cortex of 8 week or 18 months old animals at (a) 12 DPI and (b) 30 DPI. (c) Representative flow cytometry plots of CD45 + CD8 + cells stained for IFN‐γ isolated from the brains of 8 week or 18 months old, mock or MHV‐A59 infected animals at 30 DPI following 4 h. PMA/ionomycin stimulation. (d) Quantification of percent CD8 + CD44 + cells positive for IFN‐γ. (e) Representative immunocytochemical (ICC) staining for NeuN and TUNEL of primary cortical neurons following no treatment or infection with MHV‐A59 MOI 0.5, with or without coculture with CD8 + T cells purified from the spleen of an 8 week old animal at 7 DPI with 10 3 pfu MHV‐A59. Quantification of ICC images: (f) total number NeuN + neuronal nuclei, (g) total number TUNEL + NeuN + neuronal nuclei, (h) proportion of TUNEL + NeuN + neuronal nuclei of total NeuN + neuronal nuclei present. (i) Representative ICC of NeuN and TUNEL staining of primary embryonic cortical neurons following no treatment or infection with MHV‐A59 MOI 0.5, with or without naïve or PMA/ionomycin stimulated CD8 + T cells purified from the spleen of an 8 week old uninfected animal. Quantification of ICC images: (j) total number NeuN + neuronal nuclei, (k) total number TUNEL + NeuN + neuronal nuclei, and (l) proportion of TUNEL + NeuN + neuronal nuclei of total NeuN + neuronal nuclei present. Data are representative of three independent experiments with each data point representing an individual sample. All images were taken at 40X magnification and scale bar = 100 μm. Three images were captured per sample and averaged. NeuN + and TUNEL + NeuN + neuronal nuclei quantified in ImageJ software using the Cell Counter plugin. Statistics according to unpaired one‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Tetramer staining was carried out at room temperature for 20 min in conjugation with other surface staining antibodies: CD8a (53–6.7, APC‐Cy7), CD4 (RM4‐5, APC), CD44 (IM7, PE‐Cy7), CD45 (30‐F11, APC), CD45.1 (20–0453, APC), CD45.2 (60–0454, PE‐Cy‐7), CD11b (M1/70, FITC), P2RY12 (S16007D, PE), CD68 (FA‐11, BV‐421), MHC II (M5/114.15.2, PerCP‐Cy5.5), obtained from Cytek Biosciences or eBioscience, then washed with 1× PBS and fixed with 2% paraformaldehyde (PFA).

Techniques: Infection, Flow Cytometry, Staining, Isolation, TUNEL Assay, Purification, Software

Journal: Plant Biotechnology Journal

Article Title: The tomato calcium‐permeable channel 4.1 (SlOSCA4.1) is a susceptibility factor for pepino mosaic virus

doi: 10.1111/pbi.14119

Figure Lengend Snippet: Identification of a mutation conferring loss‐of‐susceptibility to pepino mosaic virus (PepMV) in tomato. (a) Expression of symptoms after inoculation with PepMV‐H30. Healthy non‐inoculated wild type (WT) control (left) versus mutant 2F531 and WT inoculated plants (right), at 16 days post inoculation. (b) PepMV load in 2F531 with respect to WT plants after inoculation with 4 different PepMV isolates belonging to strains EU (PepMV‐Sp13, ‐H30) or CH 2 (‐PS5, KLP2); average and SD of 4 replicates of 3 plants per replicate, at 16 days post inoculation; * indicates significant differences ( P < 0.05, one‐way ANOVA test). (c) Accumulation of cucumber mosaic virus (CMV), tobacco mosaic virus (TMV) and potato virus X (PVX) in 2F531 inoculated plants relative to WT similarly inoculated plants; average and SD of 3 replicates of 3 plants per replicate, at 16 dpi.

Article Snippet: Inoculations were also carried out with tobacco mosaic virus (TMV; DSMZ reference PV‐1252), cucumber mosaic virus strain LS (CMV‐LS; Wahyuni et al ., ) and potato virus X (PVX; DSMZ reference PV‐0017).

Techniques: Mutagenesis, Virus, Expressing, Control

Journal: Cell reports

Article Title: G2 arrest primes hematopoietic stem cells for megakaryopoiesis

doi: 10.1016/j.celrep.2024.114388

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PE-Cy7 CD45.1 (A20) , Tonbo , Cat# 60-0453-U100; RRID: AB_2621850.

Techniques: Recombinant, Adhesive, Purification, Blocking Assay, Amplification, Random Hexamer, Reverse Transcription, SYBR Green Assay, Sterility, Imaging, Software