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pgal fus eyfp  (Addgene inc)


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    Addgene inc pgal fus eyfp
    Deletion of ASC1 partially rescues cells from TDP-43 toxicity but not FUS toxicity. A. Spot test showing growth of wild-type (WT) and asc1Δ strains transformed with empty vector (V), <t>pGAL-TDP-43-YFP</t> (TDP-43), or pGAL-FUS-YFP (FUS). Cultures grown overnight in plasmid-selective glucose media were concentrated to OD 600 =5, serially diluted 10-fold and spotted on non-inducing (Dextrose) and inducing (Galactose) synthetic plasmid-selective media. B. Strains described in A transformed with pCUP-GFP-ATG8 ( <t>URA3,</t> CEN ) to monitor autophagy and grown overnight at 30°C in plasmid-selective galactose media with 50 µM Cu²⁺ to induce TDP-43 and GFP-ATG8. Cells were then stained with trypan blue to detect dead cells and the percentage dead cells among GFP-positive cells was quantified. Three sets of transformant for each were used and > 300 cells for each were counted. Error bars indicate standard error of the mean (SEM) and ** indicates p > 0.05. C. The levels of TDP-43 or FUS are unchanged in wild-type vs. asc1Δ strains. Western blot of the WT and asc1Δ strains described in A shows galactose induced TDP-43 or FUS and loading control, phosphoglycerate kinase (PGK1), levels. The bar graph shows data from three independent such Westerns. Error bars represent the SEM.
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    1) Product Images from "Deletion of the Saccharomyces cerevisiae RACK1 homolog, ASC1 , enhances autophagy which mitigates TDP-43 toxicity"

    Article Title: Deletion of the Saccharomyces cerevisiae RACK1 homolog, ASC1 , enhances autophagy which mitigates TDP-43 toxicity

    Journal: bioRxiv

    doi: 10.1101/2025.09.05.674500

    Deletion of ASC1 partially rescues cells from TDP-43 toxicity but not FUS toxicity. A. Spot test showing growth of wild-type (WT) and asc1Δ strains transformed with empty vector (V), pGAL-TDP-43-YFP (TDP-43), or pGAL-FUS-YFP (FUS). Cultures grown overnight in plasmid-selective glucose media were concentrated to OD 600 =5, serially diluted 10-fold and spotted on non-inducing (Dextrose) and inducing (Galactose) synthetic plasmid-selective media. B. Strains described in A transformed with pCUP-GFP-ATG8 ( URA3, CEN ) to monitor autophagy and grown overnight at 30°C in plasmid-selective galactose media with 50 µM Cu²⁺ to induce TDP-43 and GFP-ATG8. Cells were then stained with trypan blue to detect dead cells and the percentage dead cells among GFP-positive cells was quantified. Three sets of transformant for each were used and > 300 cells for each were counted. Error bars indicate standard error of the mean (SEM) and ** indicates p > 0.05. C. The levels of TDP-43 or FUS are unchanged in wild-type vs. asc1Δ strains. Western blot of the WT and asc1Δ strains described in A shows galactose induced TDP-43 or FUS and loading control, phosphoglycerate kinase (PGK1), levels. The bar graph shows data from three independent such Westerns. Error bars represent the SEM.
    Figure Legend Snippet: Deletion of ASC1 partially rescues cells from TDP-43 toxicity but not FUS toxicity. A. Spot test showing growth of wild-type (WT) and asc1Δ strains transformed with empty vector (V), pGAL-TDP-43-YFP (TDP-43), or pGAL-FUS-YFP (FUS). Cultures grown overnight in plasmid-selective glucose media were concentrated to OD 600 =5, serially diluted 10-fold and spotted on non-inducing (Dextrose) and inducing (Galactose) synthetic plasmid-selective media. B. Strains described in A transformed with pCUP-GFP-ATG8 ( URA3, CEN ) to monitor autophagy and grown overnight at 30°C in plasmid-selective galactose media with 50 µM Cu²⁺ to induce TDP-43 and GFP-ATG8. Cells were then stained with trypan blue to detect dead cells and the percentage dead cells among GFP-positive cells was quantified. Three sets of transformant for each were used and > 300 cells for each were counted. Error bars indicate standard error of the mean (SEM) and ** indicates p > 0.05. C. The levels of TDP-43 or FUS are unchanged in wild-type vs. asc1Δ strains. Western blot of the WT and asc1Δ strains described in A shows galactose induced TDP-43 or FUS and loading control, phosphoglycerate kinase (PGK1), levels. The bar graph shows data from three independent such Westerns. Error bars represent the SEM.

    Techniques Used: Spot Test, Transformation Assay, Plasmid Preparation, Staining, Western Blot, Control

    TDP-43 foci in asc1Δ strains remain liquid-like but have altered morphology. A. TDP-43-YFP foci don’t stain with thioflavin T and are dissolved by hexanediol in wild-type or asc1Δ strains. As reported previously, asc1Δ cells are larger than isogenic ASC1 wild-type (WT) cells ( J orgensen et al . 2002 ). Scale bar is 10 µm. Upper panels: BY4741 (WT) and the isogenic asc1Δ strains transformed with pGAL-TDP43-YFP (CEN, URA3) or pGAL-FUS-YFP ( CEN, URA3 ) were grown on galactose plasmid-selective plates. Cells expressing TDP-43-YFP or FUS-YFP were stained with Thioflavin T (ThT) and examined for YFP fluorescence or CFP fluorescence to assess the ThT staining. Lower panels and right: TDP-43-YFP foci were dissolved by hexanediol in both wild-type or asc1Δ strains. YFP fluorescence of cells described above expressing TDP-43-YFP was examined before (Before Hex) and after (After Hex) treatment with 10% 1,6-hexanadiol for 5 minutes that dissolves liquid-like aggregates. Three transformants each were tested. Bars show standard error of the mean. B. TDP-43 foci in asc 1Δ strains are smaller and more numerous, with many tiny foci observed compared to WT strains.
    Figure Legend Snippet: TDP-43 foci in asc1Δ strains remain liquid-like but have altered morphology. A. TDP-43-YFP foci don’t stain with thioflavin T and are dissolved by hexanediol in wild-type or asc1Δ strains. As reported previously, asc1Δ cells are larger than isogenic ASC1 wild-type (WT) cells ( J orgensen et al . 2002 ). Scale bar is 10 µm. Upper panels: BY4741 (WT) and the isogenic asc1Δ strains transformed with pGAL-TDP43-YFP (CEN, URA3) or pGAL-FUS-YFP ( CEN, URA3 ) were grown on galactose plasmid-selective plates. Cells expressing TDP-43-YFP or FUS-YFP were stained with Thioflavin T (ThT) and examined for YFP fluorescence or CFP fluorescence to assess the ThT staining. Lower panels and right: TDP-43-YFP foci were dissolved by hexanediol in both wild-type or asc1Δ strains. YFP fluorescence of cells described above expressing TDP-43-YFP was examined before (Before Hex) and after (After Hex) treatment with 10% 1,6-hexanadiol for 5 minutes that dissolves liquid-like aggregates. Three transformants each were tested. Bars show standard error of the mean. B. TDP-43 foci in asc 1Δ strains are smaller and more numerous, with many tiny foci observed compared to WT strains.

    Techniques Used: Staining, Transformation Assay, Plasmid Preparation, Expressing, Fluorescence

    ASC1 is not sequestered into TDP-43 foci. A. ASC1 does not co-localize with TDP-43 foci in yeast. An asc1Δ strain was co-transformed with either YFP-tagged empty vector (V) or p GAL-TDP-43-YFP, URA3, CEN (TDP-43-YFP) and with either p PASC1 - ASC1-mCFP ( ASC-mCFP , CEN) or p GAL-ASC1-mCFP ( GAL-ASC1-mCFP , 2µ ). Cells were patched on plasmid-selective galactose plates, grown overnight and, examined for fluorescence. The left panels show expression of YFP-tagged empty vector (V), and the right panels show YFP-tagged TDP-43 (TDP-43-YFP). Cerulean-tagged ASC1 does not form foci. Scale bar, 10 μm. B. ASC1 does not co-immunoprecipitate with TDP-43. TDP-43 was immunocaptured (IC) with TDP-43 antibody (TDP-43 ab) from cells transformed either an empty vector (vector, p2245, pGAL-ccdB, CEN, LEU2 ) or TDP-43 (p2368, pGAL-TDP-43, CEN, LEU2 ). Western blots of input, no-antibody control, and TDP-43 immunoprecipitates were probed sequentially with TDP-43 and ASC1 antibodies. Non-specific bands are marked with *.
    Figure Legend Snippet: ASC1 is not sequestered into TDP-43 foci. A. ASC1 does not co-localize with TDP-43 foci in yeast. An asc1Δ strain was co-transformed with either YFP-tagged empty vector (V) or p GAL-TDP-43-YFP, URA3, CEN (TDP-43-YFP) and with either p PASC1 - ASC1-mCFP ( ASC-mCFP , CEN) or p GAL-ASC1-mCFP ( GAL-ASC1-mCFP , 2µ ). Cells were patched on plasmid-selective galactose plates, grown overnight and, examined for fluorescence. The left panels show expression of YFP-tagged empty vector (V), and the right panels show YFP-tagged TDP-43 (TDP-43-YFP). Cerulean-tagged ASC1 does not form foci. Scale bar, 10 μm. B. ASC1 does not co-immunoprecipitate with TDP-43. TDP-43 was immunocaptured (IC) with TDP-43 antibody (TDP-43 ab) from cells transformed either an empty vector (vector, p2245, pGAL-ccdB, CEN, LEU2 ) or TDP-43 (p2368, pGAL-TDP-43, CEN, LEU2 ). Western blots of input, no-antibody control, and TDP-43 immunoprecipitates were probed sequentially with TDP-43 and ASC1 antibodies. Non-specific bands are marked with *.

    Techniques Used: Transformation Assay, Plasmid Preparation, Fluorescence, Expressing, Western Blot, Control

    The asc1Δ deletion enhances autophagy and prevents TDP-43 from suppressing autophagy below wild-type base line levels. A. Autophagy measured by Western blot analysis of the breakdown of GFP-ATG8. BY4741 (WT) and the isogenic asc1Δ deletion were co-transformed with pGAL-TDP43 (CEN, LEU2) or pGAL-FUS (CEN, HIS3) along with p CUP1-GFP-ATG8 (CEN, URA3) . Cells were grown on 2% galactose plasmid-selective plates supplemented with 1% raffinose, the required amino acids, and 50 μM copper sulfate for 16 hours at 30oC before being harvested for Western blotting. One representative blot is shown (left), and quantification from three independent experiments is presented in the bar graph (right). B. Autophagy measured microscopically by GFP-ATG8 localization. The same transformants described in (A) were examined for autophagy by determining the subcellular localization of GFP-ATG8. Autophagy was quantified as the fraction of cells showing GFP fluorescence exclusively in the vacuole among all live cells with detectable GFP signal. Autophagy was increased in the asc1Δ cells whether or not TDP-43 was expressed above that seen in wild-type cells without TDP-43. Error bars represent the SEMs from three independent transformants, with 250-550 cells analyzed per transformant. ** indicates p < 0.01 (paired two-tailed t-test).
    Figure Legend Snippet: The asc1Δ deletion enhances autophagy and prevents TDP-43 from suppressing autophagy below wild-type base line levels. A. Autophagy measured by Western blot analysis of the breakdown of GFP-ATG8. BY4741 (WT) and the isogenic asc1Δ deletion were co-transformed with pGAL-TDP43 (CEN, LEU2) or pGAL-FUS (CEN, HIS3) along with p CUP1-GFP-ATG8 (CEN, URA3) . Cells were grown on 2% galactose plasmid-selective plates supplemented with 1% raffinose, the required amino acids, and 50 μM copper sulfate for 16 hours at 30oC before being harvested for Western blotting. One representative blot is shown (left), and quantification from three independent experiments is presented in the bar graph (right). B. Autophagy measured microscopically by GFP-ATG8 localization. The same transformants described in (A) were examined for autophagy by determining the subcellular localization of GFP-ATG8. Autophagy was quantified as the fraction of cells showing GFP fluorescence exclusively in the vacuole among all live cells with detectable GFP signal. Autophagy was increased in the asc1Δ cells whether or not TDP-43 was expressed above that seen in wild-type cells without TDP-43. Error bars represent the SEMs from three independent transformants, with 250-550 cells analyzed per transformant. ** indicates p < 0.01 (paired two-tailed t-test).

    Techniques Used: Western Blot, Transformation Assay, Plasmid Preparation, Fluorescence, Two Tailed Test

    Deletion of ASC1 inhibits the frequency of TOROIDs whether or not TDP-43 is expressed. A. Shown are fluorescent images of endogenously tagged TOR1 ( GFP-TOR1 ) [ PIN + ] BY4741 cells (WT) or in asc1Δ cells. Cells were transformed with untagged TDP-43 expressed under the GAL promoter in pGAL-TDP43 (TDP-43, CEN, URA3 ) or empty vector pGAL-ccdB ( V, CEN, URA3 )), grown on selective galactose media for 2 days, examined with a FITC filter and photographed (upper). Size bar is 10 µm. B. Error bars represent SEMs calculated from three independent transformants. Statistical analysis using paired two-tailed t-tests yielded the following p-values: WT vector vs. TDP-43, 0.0494; BY(WT) vector vs. Δasu9 vector, 0.0305; Δasu9 vector vs. TDP-43, 0.4012. Asterisks (*) indicate statistically significant differences (p < 0.05); “NS” denotes no significant difference.
    Figure Legend Snippet: Deletion of ASC1 inhibits the frequency of TOROIDs whether or not TDP-43 is expressed. A. Shown are fluorescent images of endogenously tagged TOR1 ( GFP-TOR1 ) [ PIN + ] BY4741 cells (WT) or in asc1Δ cells. Cells were transformed with untagged TDP-43 expressed under the GAL promoter in pGAL-TDP43 (TDP-43, CEN, URA3 ) or empty vector pGAL-ccdB ( V, CEN, URA3 )), grown on selective galactose media for 2 days, examined with a FITC filter and photographed (upper). Size bar is 10 µm. B. Error bars represent SEMs calculated from three independent transformants. Statistical analysis using paired two-tailed t-tests yielded the following p-values: WT vector vs. TDP-43, 0.0494; BY(WT) vector vs. Δasu9 vector, 0.0305; Δasu9 vector vs. TDP-43, 0.4012. Asterisks (*) indicate statistically significant differences (p < 0.05); “NS” denotes no significant difference.

    Techniques Used: Transformation Assay, Plasmid Preparation, Two Tailed Test



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    Deletion of ASC1 partially rescues cells from TDP-43 toxicity but not FUS toxicity. A. Spot test showing growth of wild-type (WT) and asc1Δ strains transformed with empty vector (V), pGAL-TDP-43-YFP (TDP-43), or pGAL-FUS-YFP (FUS). Cultures grown overnight in plasmid-selective glucose media were concentrated to OD 600 =5, serially diluted 10-fold and spotted on non-inducing (Dextrose) and inducing (Galactose) synthetic plasmid-selective media. B. Strains described in A transformed with pCUP-GFP-ATG8 ( URA3, CEN ) to monitor autophagy and grown overnight at 30°C in plasmid-selective galactose media with 50 µM Cu²⁺ to induce TDP-43 and GFP-ATG8. Cells were then stained with trypan blue to detect dead cells and the percentage dead cells among GFP-positive cells was quantified. Three sets of transformant for each were used and > 300 cells for each were counted. Error bars indicate standard error of the mean (SEM) and ** indicates p > 0.05. C. The levels of TDP-43 or FUS are unchanged in wild-type vs. asc1Δ strains. Western blot of the WT and asc1Δ strains described in A shows galactose induced TDP-43 or FUS and loading control, phosphoglycerate kinase (PGK1), levels. The bar graph shows data from three independent such Westerns. Error bars represent the SEM.

    Journal: bioRxiv

    Article Title: Deletion of the Saccharomyces cerevisiae RACK1 homolog, ASC1 , enhances autophagy which mitigates TDP-43 toxicity

    doi: 10.1101/2025.09.05.674500

    Figure Lengend Snippet: Deletion of ASC1 partially rescues cells from TDP-43 toxicity but not FUS toxicity. A. Spot test showing growth of wild-type (WT) and asc1Δ strains transformed with empty vector (V), pGAL-TDP-43-YFP (TDP-43), or pGAL-FUS-YFP (FUS). Cultures grown overnight in plasmid-selective glucose media were concentrated to OD 600 =5, serially diluted 10-fold and spotted on non-inducing (Dextrose) and inducing (Galactose) synthetic plasmid-selective media. B. Strains described in A transformed with pCUP-GFP-ATG8 ( URA3, CEN ) to monitor autophagy and grown overnight at 30°C in plasmid-selective galactose media with 50 µM Cu²⁺ to induce TDP-43 and GFP-ATG8. Cells were then stained with trypan blue to detect dead cells and the percentage dead cells among GFP-positive cells was quantified. Three sets of transformant for each were used and > 300 cells for each were counted. Error bars indicate standard error of the mean (SEM) and ** indicates p > 0.05. C. The levels of TDP-43 or FUS are unchanged in wild-type vs. asc1Δ strains. Western blot of the WT and asc1Δ strains described in A shows galactose induced TDP-43 or FUS and loading control, phosphoglycerate kinase (PGK1), levels. The bar graph shows data from three independent such Westerns. Error bars represent the SEM.

    Article Snippet: Similarly, the pGAL-FUS expression plasmid (p2764, pAG413- GAL-FUS ; CEN, HIS3 ) was generated by Gateway LR recombination between the destination vector pAG413- GAL-ccdB (Addgene plasmid #14141; CEN, HIS3 ) and the entry clone p2763 (pDONR221-FUS, containing a stop codon). pRS416-GAL1-TDP-43-YFP ( CEN, URA3 , Addgene plasmid #27447) and pGAL -FUS-EYFP ( CEN , URA3 Addgene plasmid #29593) was purchased at Addgene.

    Techniques: Spot Test, Transformation Assay, Plasmid Preparation, Staining, Western Blot, Control

    TDP-43 foci in asc1Δ strains remain liquid-like but have altered morphology. A. TDP-43-YFP foci don’t stain with thioflavin T and are dissolved by hexanediol in wild-type or asc1Δ strains. As reported previously, asc1Δ cells are larger than isogenic ASC1 wild-type (WT) cells ( J orgensen et al . 2002 ). Scale bar is 10 µm. Upper panels: BY4741 (WT) and the isogenic asc1Δ strains transformed with pGAL-TDP43-YFP (CEN, URA3) or pGAL-FUS-YFP ( CEN, URA3 ) were grown on galactose plasmid-selective plates. Cells expressing TDP-43-YFP or FUS-YFP were stained with Thioflavin T (ThT) and examined for YFP fluorescence or CFP fluorescence to assess the ThT staining. Lower panels and right: TDP-43-YFP foci were dissolved by hexanediol in both wild-type or asc1Δ strains. YFP fluorescence of cells described above expressing TDP-43-YFP was examined before (Before Hex) and after (After Hex) treatment with 10% 1,6-hexanadiol for 5 minutes that dissolves liquid-like aggregates. Three transformants each were tested. Bars show standard error of the mean. B. TDP-43 foci in asc 1Δ strains are smaller and more numerous, with many tiny foci observed compared to WT strains.

    Journal: bioRxiv

    Article Title: Deletion of the Saccharomyces cerevisiae RACK1 homolog, ASC1 , enhances autophagy which mitigates TDP-43 toxicity

    doi: 10.1101/2025.09.05.674500

    Figure Lengend Snippet: TDP-43 foci in asc1Δ strains remain liquid-like but have altered morphology. A. TDP-43-YFP foci don’t stain with thioflavin T and are dissolved by hexanediol in wild-type or asc1Δ strains. As reported previously, asc1Δ cells are larger than isogenic ASC1 wild-type (WT) cells ( J orgensen et al . 2002 ). Scale bar is 10 µm. Upper panels: BY4741 (WT) and the isogenic asc1Δ strains transformed with pGAL-TDP43-YFP (CEN, URA3) or pGAL-FUS-YFP ( CEN, URA3 ) were grown on galactose plasmid-selective plates. Cells expressing TDP-43-YFP or FUS-YFP were stained with Thioflavin T (ThT) and examined for YFP fluorescence or CFP fluorescence to assess the ThT staining. Lower panels and right: TDP-43-YFP foci were dissolved by hexanediol in both wild-type or asc1Δ strains. YFP fluorescence of cells described above expressing TDP-43-YFP was examined before (Before Hex) and after (After Hex) treatment with 10% 1,6-hexanadiol for 5 minutes that dissolves liquid-like aggregates. Three transformants each were tested. Bars show standard error of the mean. B. TDP-43 foci in asc 1Δ strains are smaller and more numerous, with many tiny foci observed compared to WT strains.

    Article Snippet: Similarly, the pGAL-FUS expression plasmid (p2764, pAG413- GAL-FUS ; CEN, HIS3 ) was generated by Gateway LR recombination between the destination vector pAG413- GAL-ccdB (Addgene plasmid #14141; CEN, HIS3 ) and the entry clone p2763 (pDONR221-FUS, containing a stop codon). pRS416-GAL1-TDP-43-YFP ( CEN, URA3 , Addgene plasmid #27447) and pGAL -FUS-EYFP ( CEN , URA3 Addgene plasmid #29593) was purchased at Addgene.

    Techniques: Staining, Transformation Assay, Plasmid Preparation, Expressing, Fluorescence

    ASC1 is not sequestered into TDP-43 foci. A. ASC1 does not co-localize with TDP-43 foci in yeast. An asc1Δ strain was co-transformed with either YFP-tagged empty vector (V) or p GAL-TDP-43-YFP, URA3, CEN (TDP-43-YFP) and with either p PASC1 - ASC1-mCFP ( ASC-mCFP , CEN) or p GAL-ASC1-mCFP ( GAL-ASC1-mCFP , 2µ ). Cells were patched on plasmid-selective galactose plates, grown overnight and, examined for fluorescence. The left panels show expression of YFP-tagged empty vector (V), and the right panels show YFP-tagged TDP-43 (TDP-43-YFP). Cerulean-tagged ASC1 does not form foci. Scale bar, 10 μm. B. ASC1 does not co-immunoprecipitate with TDP-43. TDP-43 was immunocaptured (IC) with TDP-43 antibody (TDP-43 ab) from cells transformed either an empty vector (vector, p2245, pGAL-ccdB, CEN, LEU2 ) or TDP-43 (p2368, pGAL-TDP-43, CEN, LEU2 ). Western blots of input, no-antibody control, and TDP-43 immunoprecipitates were probed sequentially with TDP-43 and ASC1 antibodies. Non-specific bands are marked with *.

    Journal: bioRxiv

    Article Title: Deletion of the Saccharomyces cerevisiae RACK1 homolog, ASC1 , enhances autophagy which mitigates TDP-43 toxicity

    doi: 10.1101/2025.09.05.674500

    Figure Lengend Snippet: ASC1 is not sequestered into TDP-43 foci. A. ASC1 does not co-localize with TDP-43 foci in yeast. An asc1Δ strain was co-transformed with either YFP-tagged empty vector (V) or p GAL-TDP-43-YFP, URA3, CEN (TDP-43-YFP) and with either p PASC1 - ASC1-mCFP ( ASC-mCFP , CEN) or p GAL-ASC1-mCFP ( GAL-ASC1-mCFP , 2µ ). Cells were patched on plasmid-selective galactose plates, grown overnight and, examined for fluorescence. The left panels show expression of YFP-tagged empty vector (V), and the right panels show YFP-tagged TDP-43 (TDP-43-YFP). Cerulean-tagged ASC1 does not form foci. Scale bar, 10 μm. B. ASC1 does not co-immunoprecipitate with TDP-43. TDP-43 was immunocaptured (IC) with TDP-43 antibody (TDP-43 ab) from cells transformed either an empty vector (vector, p2245, pGAL-ccdB, CEN, LEU2 ) or TDP-43 (p2368, pGAL-TDP-43, CEN, LEU2 ). Western blots of input, no-antibody control, and TDP-43 immunoprecipitates were probed sequentially with TDP-43 and ASC1 antibodies. Non-specific bands are marked with *.

    Article Snippet: Similarly, the pGAL-FUS expression plasmid (p2764, pAG413- GAL-FUS ; CEN, HIS3 ) was generated by Gateway LR recombination between the destination vector pAG413- GAL-ccdB (Addgene plasmid #14141; CEN, HIS3 ) and the entry clone p2763 (pDONR221-FUS, containing a stop codon). pRS416-GAL1-TDP-43-YFP ( CEN, URA3 , Addgene plasmid #27447) and pGAL -FUS-EYFP ( CEN , URA3 Addgene plasmid #29593) was purchased at Addgene.

    Techniques: Transformation Assay, Plasmid Preparation, Fluorescence, Expressing, Western Blot, Control

    The asc1Δ deletion enhances autophagy and prevents TDP-43 from suppressing autophagy below wild-type base line levels. A. Autophagy measured by Western blot analysis of the breakdown of GFP-ATG8. BY4741 (WT) and the isogenic asc1Δ deletion were co-transformed with pGAL-TDP43 (CEN, LEU2) or pGAL-FUS (CEN, HIS3) along with p CUP1-GFP-ATG8 (CEN, URA3) . Cells were grown on 2% galactose plasmid-selective plates supplemented with 1% raffinose, the required amino acids, and 50 μM copper sulfate for 16 hours at 30oC before being harvested for Western blotting. One representative blot is shown (left), and quantification from three independent experiments is presented in the bar graph (right). B. Autophagy measured microscopically by GFP-ATG8 localization. The same transformants described in (A) were examined for autophagy by determining the subcellular localization of GFP-ATG8. Autophagy was quantified as the fraction of cells showing GFP fluorescence exclusively in the vacuole among all live cells with detectable GFP signal. Autophagy was increased in the asc1Δ cells whether or not TDP-43 was expressed above that seen in wild-type cells without TDP-43. Error bars represent the SEMs from three independent transformants, with 250-550 cells analyzed per transformant. ** indicates p < 0.01 (paired two-tailed t-test).

    Journal: bioRxiv

    Article Title: Deletion of the Saccharomyces cerevisiae RACK1 homolog, ASC1 , enhances autophagy which mitigates TDP-43 toxicity

    doi: 10.1101/2025.09.05.674500

    Figure Lengend Snippet: The asc1Δ deletion enhances autophagy and prevents TDP-43 from suppressing autophagy below wild-type base line levels. A. Autophagy measured by Western blot analysis of the breakdown of GFP-ATG8. BY4741 (WT) and the isogenic asc1Δ deletion were co-transformed with pGAL-TDP43 (CEN, LEU2) or pGAL-FUS (CEN, HIS3) along with p CUP1-GFP-ATG8 (CEN, URA3) . Cells were grown on 2% galactose plasmid-selective plates supplemented with 1% raffinose, the required amino acids, and 50 μM copper sulfate for 16 hours at 30oC before being harvested for Western blotting. One representative blot is shown (left), and quantification from three independent experiments is presented in the bar graph (right). B. Autophagy measured microscopically by GFP-ATG8 localization. The same transformants described in (A) were examined for autophagy by determining the subcellular localization of GFP-ATG8. Autophagy was quantified as the fraction of cells showing GFP fluorescence exclusively in the vacuole among all live cells with detectable GFP signal. Autophagy was increased in the asc1Δ cells whether or not TDP-43 was expressed above that seen in wild-type cells without TDP-43. Error bars represent the SEMs from three independent transformants, with 250-550 cells analyzed per transformant. ** indicates p < 0.01 (paired two-tailed t-test).

    Article Snippet: Similarly, the pGAL-FUS expression plasmid (p2764, pAG413- GAL-FUS ; CEN, HIS3 ) was generated by Gateway LR recombination between the destination vector pAG413- GAL-ccdB (Addgene plasmid #14141; CEN, HIS3 ) and the entry clone p2763 (pDONR221-FUS, containing a stop codon). pRS416-GAL1-TDP-43-YFP ( CEN, URA3 , Addgene plasmid #27447) and pGAL -FUS-EYFP ( CEN , URA3 Addgene plasmid #29593) was purchased at Addgene.

    Techniques: Western Blot, Transformation Assay, Plasmid Preparation, Fluorescence, Two Tailed Test

    Deletion of ASC1 inhibits the frequency of TOROIDs whether or not TDP-43 is expressed. A. Shown are fluorescent images of endogenously tagged TOR1 ( GFP-TOR1 ) [ PIN + ] BY4741 cells (WT) or in asc1Δ cells. Cells were transformed with untagged TDP-43 expressed under the GAL promoter in pGAL-TDP43 (TDP-43, CEN, URA3 ) or empty vector pGAL-ccdB ( V, CEN, URA3 )), grown on selective galactose media for 2 days, examined with a FITC filter and photographed (upper). Size bar is 10 µm. B. Error bars represent SEMs calculated from three independent transformants. Statistical analysis using paired two-tailed t-tests yielded the following p-values: WT vector vs. TDP-43, 0.0494; BY(WT) vector vs. Δasu9 vector, 0.0305; Δasu9 vector vs. TDP-43, 0.4012. Asterisks (*) indicate statistically significant differences (p < 0.05); “NS” denotes no significant difference.

    Journal: bioRxiv

    Article Title: Deletion of the Saccharomyces cerevisiae RACK1 homolog, ASC1 , enhances autophagy which mitigates TDP-43 toxicity

    doi: 10.1101/2025.09.05.674500

    Figure Lengend Snippet: Deletion of ASC1 inhibits the frequency of TOROIDs whether or not TDP-43 is expressed. A. Shown are fluorescent images of endogenously tagged TOR1 ( GFP-TOR1 ) [ PIN + ] BY4741 cells (WT) or in asc1Δ cells. Cells were transformed with untagged TDP-43 expressed under the GAL promoter in pGAL-TDP43 (TDP-43, CEN, URA3 ) or empty vector pGAL-ccdB ( V, CEN, URA3 )), grown on selective galactose media for 2 days, examined with a FITC filter and photographed (upper). Size bar is 10 µm. B. Error bars represent SEMs calculated from three independent transformants. Statistical analysis using paired two-tailed t-tests yielded the following p-values: WT vector vs. TDP-43, 0.0494; BY(WT) vector vs. Δasu9 vector, 0.0305; Δasu9 vector vs. TDP-43, 0.4012. Asterisks (*) indicate statistically significant differences (p < 0.05); “NS” denotes no significant difference.

    Article Snippet: Similarly, the pGAL-FUS expression plasmid (p2764, pAG413- GAL-FUS ; CEN, HIS3 ) was generated by Gateway LR recombination between the destination vector pAG413- GAL-ccdB (Addgene plasmid #14141; CEN, HIS3 ) and the entry clone p2763 (pDONR221-FUS, containing a stop codon). pRS416-GAL1-TDP-43-YFP ( CEN, URA3 , Addgene plasmid #27447) and pGAL -FUS-EYFP ( CEN , URA3 Addgene plasmid #29593) was purchased at Addgene.

    Techniques: Transformation Assay, Plasmid Preparation, Two Tailed Test