anti-slt2 sc-374434  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK signalling and aberrant lipid homeostasis.

doi: 10.1016/j.isci.2023.107539

Figure Lengend Snippet: Figure 4. cof1-5 mutation triggers activation of the CWI pathway (A and B) Immunoblots detecting Slt2 phosphorylation when grown on glucose and glycerol containing media are shown in A and B, respectively. (C) Pkc1 inhibition by cercosporamide (CSA) administration prevented Slt2 phosphorylation. (D) Porin deletion and overexpression reveal dependence of Slt2 phosphorylation on Por1. (E) cof1-5 mutation triggers loss of plasma membrane integrity, as assessed flow cytometrically with PI positivity at 48 h after inoculation. PI positivity is exacerbated by additional Pkc1 inhibition using the Pkc1-inhibitor cercosporamide or applying additional cell wall stress with calcofluor white (CFW). Combined treatment with CSA and CFW at the same time shows additive effects. (F) POR1 deletion rescues from cof1-5-dependent loss of viability and loss of plasma membrane integrity, whereas SLT2 deletion sensitises to cell death. (G–J) Chronological aging analysis reveals shortening of chronological lifespan in cof1-5 cells which depends on POR1. Colony forming unit formation based on clonogenic survival is depicted in G and H, whereas PI positivity is shown in I and J. Statistical significance in E, F, H, and J was assessed using Brown-Forsythe and Welch-ANOVA test. Asterisks indicate significance based on p-levels of the comparisons to the respective control strains. See also Figure S2. Error bars indicate standard error of the mean (SEM) and asterisks indicate significant differences based on p-values, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Immunoblotting followed standard procedures, with transfer of proteins to a 0.45 mm nitrocellulose membrane and probing with 24 iScience 26, 107539, September 15, 2023 antibodies against phospho-Slt2 (Phospho-p44/42 MAPK, cell signaling, #9101, 1:1000), actin (a-Yeast act1 Goat monoclonal antibody, a kind gift from Prof. John Cooper, Washington University, 1:2000), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Life Technologies, MA515738, 1:5000), or VDAC/porin (Abcam, ab110326, 1:5000).

Techniques: Mutagenesis, Activation Assay, Western Blot, Phospho-proteomics, Inhibition, Over Expression, Clinical Proteomics, Membrane, Control

Journal: iScience

Article Title: A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK signalling and aberrant lipid homeostasis.

doi: 10.1016/j.isci.2023.107539

Figure Lengend Snippet: Figure 5. Slt2 localisation to the mitochondrial compartment is enhanced in cof1-5 cells (A) Slt2, which is mostly found in the nucleus in wild-type cells at stationary phase, forms punctate foci in cof1-5, as documented by fluorescence microscopy using chromosomally tagged SLT2-GFP under control of its endogenous promoter. Deconvolved pictures with Hoechst staining for nuclei are shown. (B and C) Cells showing nuclear localisation of Slt2-GFP (B) and foci-forming cells (C) were quantified, plotted and analyzed for significant localisation change as compared to wild type. (D) Representative fluorescence microscopy pictures of Slt2-GFP expressing cells with rhodamine B hexylester staining for colocalization analysis with mitochondria. (E) Colocalisation of Slt2-GFP (green) with the mitochondrial stain rhodamine B hexylester was increased in cof1-5 as shown by significant increase of the Pearson colocalisation coefficient. (F–I) Slt2-GFP expression from a plasmid was used to monitor cellular Slt2 localisation in cof1-5 cells in dependence of POR1. Representative microscopy pictures are shown in (F), Slt-GFP foci-containing cells are quantified in (G), cells with nuclear Slt2 are quantified in (H) and the Pearson coefficient of Slt2-GFP colocalization with mitochondria is visualised in (I).

Article Snippet: Immunoblotting followed standard procedures, with transfer of proteins to a 0.45 mm nitrocellulose membrane and probing with 24 iScience 26, 107539, September 15, 2023 antibodies against phospho-Slt2 (Phospho-p44/42 MAPK, cell signaling, #9101, 1:1000), actin (a-Yeast act1 Goat monoclonal antibody, a kind gift from Prof. John Cooper, Washington University, 1:2000), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Life Technologies, MA515738, 1:5000), or VDAC/porin (Abcam, ab110326, 1:5000).

Techniques: Microscopy, Control, Staining, Expressing, Plasmid Preparation

Journal: iScience

Article Title: A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK signalling and aberrant lipid homeostasis.

doi: 10.1016/j.isci.2023.107539

Figure Lengend Snippet: Figure 6. Reduced actin dynamics lead to a porin-dependent increase in lipid droplet number that are required for CWI activation (A and B) Increase of LD number in cof1-5 depends on Lro1 and Dga1. Mean LD-numbers per cell are plotted in (A) and representative microscopy pictures are shown in (B). Each dot in (A) represents the mean LD number per cell of a single experiment (n = 6) with at least 119 cells being evaluated per experiment. (C and D) Gene deletions of POR1 or SLT2 are sufficient to prevent LD accumulation in cof1-5. Mean LD numbers per cell were assessed by quantifying fluorescence microscopy pictures using Bodipy staining (C). Representative fluorescence microscopy images are shown in (D). (E) Representative EM micrographs supporting the observation of LD-number-increase in cof1-5. V, vacuole; LD, lipid droplet; N, nucleus. Statistical significance in (A) and (C) was assessed using two-way ANOVA with cof1-5 mutation as first factor and additional KO as second factor. See also Figure S4. Error bars indicate standard error of the mean (SEM) and asterisks indicate significant differences based on p-values, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Immunoblotting followed standard procedures, with transfer of proteins to a 0.45 mm nitrocellulose membrane and probing with 24 iScience 26, 107539, September 15, 2023 antibodies against phospho-Slt2 (Phospho-p44/42 MAPK, cell signaling, #9101, 1:1000), actin (a-Yeast act1 Goat monoclonal antibody, a kind gift from Prof. John Cooper, Washington University, 1:2000), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Life Technologies, MA515738, 1:5000), or VDAC/porin (Abcam, ab110326, 1:5000).

Techniques: Activation Assay, Microscopy, Staining, Mutagenesis

Journal: iScience

Article Title: A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK signalling and aberrant lipid homeostasis.

doi: 10.1016/j.isci.2023.107539

Figure Lengend Snippet: Figure 7. The acyltransferases Lro1 and Dga1 are required for MAPK-related changes in cof1-5 (A) Polarisation of the actin cytoskeleton was assessed using phalloidin (red) and DAPI (blue) staining. The cof1-5 mutant shows loss of actin polarisation, which is not rescued by gene KO of LRO1, DGA1 or a combined double deletion of the ladder. (B and C) The growth defect (B) and flocculation phenotype (C) as observed in cof1-5 are restored by additional deletion of the acyltransferases LRO1, DGA1, or in the double deletion mutant (lro1D dga1D). (D) Slt2 phosphorylation in cof1-5 depends on Lro1 and Dga1 as the KO of either corresponding gene and the double KO prevents Slt2 phosphorylation. (E) Mean LD abundance per cell was quantified in diverse conditions of stress. 150 cells per condition and experiment were quantified with a total of three independent experiments (n = 3). Statistical significance in (E) was assessed using ordinary one-way ANOVA. Error bars indicate standard error of the mean (SEM) and asterisks indicate significant differences based on p-values, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Immunoblotting followed standard procedures, with transfer of proteins to a 0.45 mm nitrocellulose membrane and probing with 24 iScience 26, 107539, September 15, 2023 antibodies against phospho-Slt2 (Phospho-p44/42 MAPK, cell signaling, #9101, 1:1000), actin (a-Yeast act1 Goat monoclonal antibody, a kind gift from Prof. John Cooper, Washington University, 1:2000), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Life Technologies, MA515738, 1:5000), or VDAC/porin (Abcam, ab110326, 1:5000).

Techniques: Staining, Mutagenesis, Phospho-proteomics

Journal: iScience

Article Title: A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK signalling and aberrant lipid homeostasis

doi: 10.1016/j.isci.2023.107539

Figure Lengend Snippet: cof1-5 mutation triggers activation of the CWI pathway (A and B) Immunoblots detecting Slt2 phosphorylation when grown on glucose and glycerol containing media are shown in A and B, respectively. (C) Pkc1 inhibition by cercosporamide (CSA) administration prevented Slt2 phosphorylation. (D) Porin deletion and overexpression reveal dependence of Slt2 phosphorylation on Por1. (E) cof1-5 mutation triggers loss of plasma membrane integrity, as assessed flow cytometrically with PI positivity at 48 h after inoculation. PI positivity is exacerbated by additional Pkc1 inhibition using the Pkc1-inhibitor cercosporamide or applying additional cell wall stress with calcofluor white (CFW). Combined treatment with CSA and CFW at the same time shows additive effects. (F) POR1 deletion rescues from cof1-5 -dependent loss of viability and loss of plasma membrane integrity, whereas SLT2 deletion sensitises to cell death. (G–J) Chronological aging analysis reveals shortening of chronological lifespan in cof1-5 cells which depends on POR1 . Colony forming unit formation based on clonogenic survival is depicted in G and H, whereas PI positivity is shown in I and J. Statistical significance in E, F, H, and J was assessed using Brown-Forsythe and Welch-ANOVA test. Asterisks indicate significance based on p-levels of the comparisons to the respective control strains. See also Figure S2 . Error bars indicate standard error of the mean (SEM) and asterisks indicate significant differences based on p-values, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Immunoblotting followed standard procedures, with transfer of proteins to a 0.45 μm nitrocellulose membrane and probing with antibodies against phospho-Slt2 (Phospho-p44/42 MAPK, cell signaling, #9101, 1:1000), actin (α-Yeast act1 Goat monoclonal antibody, a kind gift from Prof. John Cooper, Washington University, 1:2000), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Life Technologies, MA515738, 1:5000), or VDAC/porin (Abcam, ab110326, 1:5000).

Techniques: Mutagenesis, Activation Assay, Western Blot, Phospho-proteomics, Inhibition, Over Expression, Clinical Proteomics, Membrane, Control

Journal: iScience

Article Title: A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK signalling and aberrant lipid homeostasis

doi: 10.1016/j.isci.2023.107539

Figure Lengend Snippet: Slt2 localisation to the mitochondrial compartment is enhanced in cof1-5 cells (A) Slt2, which is mostly found in the nucleus in wild-type cells at stationary phase, forms punctate foci in cof1-5 , as documented by fluorescence microscopy using chromosomally tagged SLT2-GFP under control of its endogenous promoter. Deconvolved pictures with Hoechst staining for nuclei are shown. (B and C) Cells showing nuclear localisation of Slt2-GFP (B) and foci-forming cells (C) were quantified, plotted and analyzed for significant localisation change as compared to wild type. (D) Representative fluorescence microscopy pictures of Slt2-GFP expressing cells with rhodamine B hexylester staining for colocalization analysis with mitochondria. (E) Colocalisation of Slt2-GFP (green) with the mitochondrial stain rhodamine B hexylester was increased in cof1-5 as shown by significant increase of the Pearson colocalisation coefficient. (F–I) Slt2-GFP expression from a plasmid was used to monitor cellular Slt2 localisation in cof1-5 cells in dependence of POR1 . Representative microscopy pictures are shown in (F), Slt-GFP foci-containing cells are quantified in (G), cells with nuclear Slt2 are quantified in (H) and the Pearson coefficient of Slt2-GFP colocalization with mitochondria is visualised in (I). (J and K) Expression of Pkc1-GFP under control of its endogenous promoter reveals increased PKC1-GFP foci formation in cof1-5 (J, K). Representative fluorescence microscopy pictures of individual Pkc1-GFP expressing cells with additional rhodamine-B-hexylester staining for mitochondria and autodot staining for LDs are shown in (J) and cells with Pkc1-GFP foci are quantified in (K). (L) The Pearson coefficient was determined as a measure of colocalisation of Pkc1-GFP with mitochondria (rhodamine-B-hexylester). Statistical significance in (B) and (C) was assessed using Welch's t test, (E, K, L) were analyzed using unpaired t test and (G, H, I) by ordinary one-way ANOVA. See also Figure S3 . Error bars indicate standard error of the mean (SEM) and asterisks indicate significant differences based on p-values, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Immunoblotting followed standard procedures, with transfer of proteins to a 0.45 μm nitrocellulose membrane and probing with antibodies against phospho-Slt2 (Phospho-p44/42 MAPK, cell signaling, #9101, 1:1000), actin (α-Yeast act1 Goat monoclonal antibody, a kind gift from Prof. John Cooper, Washington University, 1:2000), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Life Technologies, MA515738, 1:5000), or VDAC/porin (Abcam, ab110326, 1:5000).

Techniques: Fluorescence, Microscopy, Control, Staining, Expressing, Plasmid Preparation

Journal: iScience

Article Title: A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK signalling and aberrant lipid homeostasis

doi: 10.1016/j.isci.2023.107539

Figure Lengend Snippet: Reduced actin dynamics lead to a porin-dependent increase in lipid droplet number that are required for CWI activation (A and B) Increase of LD number in cof1-5 depends on Lro1 and Dga1. Mean LD-numbers per cell are plotted in (A) and representative microscopy pictures are shown in (B). Each dot in (A) represents the mean LD number per cell of a single experiment (n = 6) with at least 119 cells being evaluated per experiment. (C and D) Gene deletions of POR1 or SLT2 are sufficient to prevent LD accumulation in cof1-5 . Mean LD numbers per cell were assessed by quantifying fluorescence microscopy pictures using Bodipy staining (C). Representative fluorescence microscopy images are shown in (D). (E) Representative EM micrographs supporting the observation of LD-number-increase in cof1-5 . V, vacuole; LD, lipid droplet; N, nucleus. Statistical significance in (A) and (C) was assessed using two-way ANOVA with cof1-5 mutation as first factor and additional KO as second factor. See also Figure S4 . Error bars indicate standard error of the mean (SEM) and asterisks indicate significant differences based on p-values, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Immunoblotting followed standard procedures, with transfer of proteins to a 0.45 μm nitrocellulose membrane and probing with antibodies against phospho-Slt2 (Phospho-p44/42 MAPK, cell signaling, #9101, 1:1000), actin (α-Yeast act1 Goat monoclonal antibody, a kind gift from Prof. John Cooper, Washington University, 1:2000), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Life Technologies, MA515738, 1:5000), or VDAC/porin (Abcam, ab110326, 1:5000).

Techniques: Activation Assay, Microscopy, Fluorescence, Staining, Mutagenesis

Journal: iScience

Article Title: A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK signalling and aberrant lipid homeostasis

doi: 10.1016/j.isci.2023.107539

Figure Lengend Snippet: The acyltransferases Lro1 and Dga1 are required for MAPK-related changes in cof1-5 (A) Polarisation of the actin cytoskeleton was assessed using phalloidin (red) and DAPI (blue) staining. The cof1-5 mutant shows loss of actin polarisation, which is not rescued by gene KO of LRO1 , DGA1 or a combined double deletion of the ladder. (B and C) The growth defect (B) and flocculation phenotype (C) as observed in cof1-5 are restored by additional deletion of the acyltransferases LRO1 , DGA1 , or in the double deletion mutant ( lro1 Δ dga1 Δ). (D) Slt2 phosphorylation in cof1-5 depends on Lro1 and Dga1 as the KO of either corresponding gene and the double KO prevents Slt2 phosphorylation. (E) Mean LD abundance per cell was quantified in diverse conditions of stress. 150 cells per condition and experiment were quantified with a total of three independent experiments (n = 3). Statistical significance in (E) was assessed using ordinary one-way ANOVA. Error bars indicate standard error of the mean (SEM) and asterisks indicate significant differences based on p-values, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Immunoblotting followed standard procedures, with transfer of proteins to a 0.45 μm nitrocellulose membrane and probing with antibodies against phospho-Slt2 (Phospho-p44/42 MAPK, cell signaling, #9101, 1:1000), actin (α-Yeast act1 Goat monoclonal antibody, a kind gift from Prof. John Cooper, Washington University, 1:2000), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Life Technologies, MA515738, 1:5000), or VDAC/porin (Abcam, ab110326, 1:5000).

Techniques: Staining, Mutagenesis, Flocculation, Phospho-proteomics

Journal: iScience

Article Title: A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK signalling and aberrant lipid homeostasis

doi: 10.1016/j.isci.2023.107539

Figure Lengend Snippet:

Article Snippet: Immunoblotting followed standard procedures, with transfer of proteins to a 0.45 μm nitrocellulose membrane and probing with antibodies against phospho-Slt2 (Phospho-p44/42 MAPK, cell signaling, #9101, 1:1000), actin (α-Yeast act1 Goat monoclonal antibody, a kind gift from Prof. John Cooper, Washington University, 1:2000), (glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Life Technologies, MA515738, 1:5000), or VDAC/porin (Abcam, ab110326, 1:5000).

Techniques: Virus, Recombinant, Microarray, High Performance Thin Layer Chromatography, Software