EMBO/soda-nel huggingface dataset:
High salt activates p97 to reduce host antiviral immunity by restricting Viperin induction (2021)
Figure 5

Figure 5. High salt stimulates degradation of ubiquitinated proteins via p97.

A RT-qPCR analysis of USP33 mRNA levels in HT1080 cells treated with additional NaCl (+17, 34 and 51 mM) for 6 and 12 hrs.

B Western blot analysis of USP33 protein in RAW264.7 treated with MG132 (10 μM) and additional NaCl (+34 mM) for 12 hrs.

C Immunoprecipitation analysis of USP33 ubiquitination in RAW264.7 treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs.

D HEK293T cells were transfected with Flag-USP33. Flag-USP33 proteins were immunoprecipitated with Flag beads (1° IP) and then eluted by Flag peptides. The eluted Flag-USP33 proteins were further immunoprecipitated by an anti-Ub antibody (2° IP). The immunoprecipitates were subjected to immunoblotting by an anti-Ub antibody (left), and the supernatant were further immunoprecipitated (3° IP) and immunoblotted by a USP33 antibody (right).

E Western blot analysis of USP33 in HT1080 cells treated with NMS873 (10 μM) and additional NaCl (+34 mM) for 12 hrs.

F Western blot analysis of USP33 protein in p97-sufficient (WT) or p97-knockout (KO) 2fTGH cells transfected with Flag-USP33 (F-USP33) and then treated with additional NaCl (+34 mM) for 12 hrs.

G Immunoprecipitation analysis of ubiquitination of Flag-USP33 in p97-WT or p97-KO 2fTGH cells co-transfected with Flag-USP33 and HA-Ub and then treated with NaCl (+34 mM) for 12 hrs.

H,I Western blot analysis of ubiquitination of cellular proteins (Pan-Ubi) in HT1080 and RAW264.7 cells treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs (H), or in RAW264.7 treated with additional NaCl (+34 mM) for different times (I).

J Western blot analysis of ubiquitination of cellular proteins (Pan-Ubi) in HT1080 cells treated with NMS873 (10 μM) and additional NaCl (+34 mM) for 12 hrs.

K Western blot analysis of ubiquitination of cellular proteins (Pan-Ubi) in p97-WT or p97-KO HEK293T cells treated with additional NaCl (+34 mM) for 12 hrs.

L Immunoprecipitation analysis of Viperin ubiquitination in HT1080 cells transfected with shCtrl or shp97 (1# or 2#) and then treated with IFNα (1,000 IU/ml, 12 hrs) immediately after addition of NaCl (+34 mM).

M Western blot analysis of Viperin in RAW264.7 treated with NMS873 (10 μM), together with additional NaCl (+34 mM), and then treated with mIFNβ (500 IU/ml) for 12 hrs.

N Western blot analysis of Myc-Viperin in HEK293T cells co-transfected with Myc-Viperin and shCtrl or shp97, and then treated with additional NaCl (+34 mM) for 12 hrs.

Data information: Data (A) represent mean and SD of four biological replicates; Data (B-N) are representative of at least two biological replicates. For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not signiﬁcant (p > 0.05).
High salt activates p97 to reduce host antiviral immunity by restricting Viperin induction (2021)
Figure 6

Figure 6. High salt promotes p97 acetylation to recruit USP33 for degradation.

A, B Western blot analysis of p97 levels in RAW264.7 (A) or HT1080 (B) cells treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs.

C Immunoprecipitation analysis of the interaction between USP33 and p97 in RAW264.7 treated with additional NaCl (+34 mM) for 12 hrs.

D Immunoprecipitation analysis of acetylation of Flag-p97 in HEK293T cells transfected with Flag-p97 and then treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs.

E, F Mass spectrometry analysis of acetylation of Flag-p97 (E) in RAW264.7 transfected with Flag-p97 and then treated with additional NaCl (+34 mM) or control ddH2O (Ctrl) for 12 hrs. The intensity of p97-K663 site was shown in (F).

G Immunoprecipitation analysis of acetylation of Myc-p97 in HEK293T cells transfected with Myc-p97 (WT, K663R or K651R) and then treated with additional NaCl (+34 mM) for 12 hrs.

H Immunoprecipitation analysis of the interaction between USP33 and Myc-p97 in HEK293T cells transfected with Myc-p97 (WT or K663R).

I Western blot analysis of USP33 levels in HEK293T cells transfected with empty vectors (-) or Myc-p97 (WT or K663R).

J Western blot analysis of Flag-Viperin levels in HEK293T cells co-transfected with Flag-Viperin and Myc-p97 (WT or K663R).

K Immunoprecipitation analysis of the interaction between USP33 and Myc-p97 in HEK293T cells transfected with Myc-p97 (WT or K663R) and then treated with additional NaCl (+34 mM) for 12 hrs.

L Western blot analysis of FH-USP33 levels in p97-KO HEK293T cells co-transfected with Myc-p97 (WT or K663R) and Flag-USP33, and then treated with additional NaCl (+34 mM) for 12 hrs.

M Immunoprecipitation analysis of Viperin ubiquitination in p97-KO 2fTGH cells co-transfected with Myc-p97 (WT or K663R), Flag-Viperin and HA-Ub as indicated, and then treated with additional NaCl (+34 mM) for 12 hrs.

Data information: Data (A-D, G-M) are representative of at least two biological replicates.
High salt activates p97 to reduce host antiviral immunity by restricting Viperin induction (2021)
Figure 7

Figure 7. A low-salt diet during viral infection improves Viperin expression and host antiviral ability.

A Immunoprecipitation analysis of acetylation of p97 in RAW264.7 cells cultured in media containing reduced concentration of NaCl (-17 and -34 mM) for 12 hrs.

B Western blot analysis of USP33 in RAW264.7 cells cultured in media containing reduced concentration of NaCl (-17 and -34 mM) for 12 hrs.

C Western blot analysis of Viperin in HT1080 cells treated with IFNα (1,000 IU/ml, 12 hrs) in media containing reduced concentration of NaCl (-17, -34 and -51 mM).

D RAW264.7 cells were infected with VSV (MOI=1.0, 12 hrs) in media containing reduced concentration of NaCl (-17 and -34 mM). Viral RNA levels were analyzed by RT-qPCR.

E Mice (n=3) were fed an NSD (0.45% NaCl) or LSD (0.30% or 0.15% NaCl) for 3 days. Na+ concentration in mouse serum was analyzed by a Micro Blood Sodium Concentration Assay Kit.

F Schematic diagram of analysis of in vivo viral levels in VSV-infected mice fed either an NSD (0.45% NaCl) or LSD (0.15% NaCl).

G Western blot analysis of USP33 and Viperin protein levels in the spleen tissues of mice (n=5) fed an NSD (0.45% NaCl) or LSD (0.15% NaCl) for 3 days as indicated.

H Mice (n=5) were intraperitoneally infected with VSV (1x108 PFU per gram body). 12 hrs post infection, mice were fed an NSD (0.45% NaCl) or LSD (0.15% NaCl) for 3 days. Then VSV RNA levels in mouse spleen tissues were detected by RT-qPCR.

I Virus titers in the mouse blood (n=5) from (H) were analyzed by the TCID50 assay.

J Immunofluorescence analysis of the amount of VSV-GFP in liver tissues of mice intraperitoneally infected with VSV-GFP (1x107 PFU per gram body) and then fed an NSD/LSD as (H). Scale bars, 20 µm.

K A proposed model of high salt-mediated restriction of host antiviral ability by activating p97 acetylation and subsequent degradation of the deubiquitinase USP33 and antiviral protein Viperin.

Data information: Data (A-C, G) are representative of at least two biological replicates; Data (D) represent mean and SD of four biological replicates; Data (E, H, I) show mean and SEM of three (E) or five (G-I) biological replicates. For all statistical testing: p values were calculated using two-tailed unpaired Student's t-test. N.S, not significant (p > 0.05) and **p < 0.01, ***p < 0.001 (two-tailed unpaired Student's t-test).

Expanded View Figure Legends
Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex (2021)
Figure 1

Figure 1. An inducible system for ER membrane biogenesis.

A Schematic of the control of lipid synthesis by estradiol-inducible expression of ino2*.

B Sec63-mNeon images of mid and cortical sections of cells harboring the estradiol-inducible system (SSY1405). Cells were untreated or treated with 800 nM estradiol for 6 h.

C Flow cytometric measurements of GFP levels in cells containing the transcriptional UPR reporter. WT cells containing the UPR reporter (SSY2306), cells additionally harboring an estradiol-inducible GAL promoter (SSY2307) and cells additionally harboring the system for estradiol-inducible expression of ino2* under the GAL promoter (SSY2308) were untreated, treated with 800 nM estradiol for 6 h, or treated with 8 mM DTT for 1 h. Data were normalized to untreated WT cells. Bars represent the mean of three biological replicates (n = 3), error bars are the standard error of the mean (s.e.m.). Asterisks indicate statistical significance compared with the corresponding untreated sample, as judged by a two-tailed Student's t-test assuming equal variance. Exceptions were the tests against the normalized value for wild-type cells, for which a two-tailed Student's t-test with unequal variance was applied. *, P < 0.05; n.s., not significant. WT, wild-type.

D Western blot of Sec63, mCherry and Pgk1 from WT cells (SSY1404), cells harboring the estradiol-inducible system (SSY1405) or ∆opi1 cells (SSY1607), all of which expressed Sec63-mNeon and Rtn1-mCherry. Cells were untreated or treated with 800 nM estradiol for 6 h. Pgk1 served as a loading control.

E Quantification of ER size in estradiol-treated cells harboring the inducible system (SSY1405), untreated ∆opi1 cells (SSY1607) and WT cells (SSY1404) treated with 8 mM DTT for 1 h. Bars represent mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with 0 nM estradiol or ∆opi1 cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.
Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex (2021)
Figure 2

Figure 2. A genetic screen for factors involved in ER membrane biogenesis.

A Sec63-mNeon images of cells of the indicated genotypes harboring the inducible system. Cells were treated with 800 nM estradiol for 6 h. Two examples of each phenotypic class are shown.

B Violin plots of Z scores from the three metrics for ER size determined for each of the 4800 mutant strains. The untreated WT is shown for reference.

C Sec63-mNeon and Rtn1-mCherry images of ∆lnp1 and ∆sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h.

D Classification of hits. Numbers in brackets indicate the number of mutants in each class. Striped areas indicate mutants belonging to two classes.
Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex (2021)
Figure 3

Figure 3. Ice2 is required for ER membrane biogenesis upon activation of Ino2/4.

A Sec63-mNeon images of the cortical ER of WT and Δice2 cells harboring the inducible system (SSY1405, 1603). Cells were untreated or treated with 800 nM estradiol for 6 h.

B Classification of peripheral ER structures from cortical sections of cells expressing Sec63-mNeon and Rtn1-mCherry as tubules (purple), sheets (green) or tubular clusters (yellow). Tubular clusters are combined with tubules in the final classification, as illustrated by the overlay.

C Quantification of peripheral ER structures in WT and ∆ice2 cells harboring the inducible system (SSY1405, 1603) and treated with 800 nM estradiol for the times indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.

D mRNA levels of the Ino2/4 target gene INO1 upon ino2* expression in WT and Δice2 cells harboring the inducible system (SSY1405, 1603) as measured by quantitative real-time PCR. Data were normalized to untreated WT cells. Mean + s.e.m., n = 3, biological replicates. Asterisks indicate statistical significance compared with the corresponding untreated cells, as judged by a two-tailed Student's t-test assuming equal variance. Exception was the test against the normalized value for wild-type cells, for which a two-tailed Student's t-test with unequal variance was applied. *, P < 0.05; **, P < 0.01.

E Quantification of peripheral ER structures in untreated WT, Δice2, Δopi1 and Δice2 Δopi1 cells (SSY1404, 2356, 2595, 2811). Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.
Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex (2021)
Figure 4

Figure 4. Ice2 is required for ER membrane biogenesis upon ER stress and ICE2 overexpression is sufficient to induce ER expansion.

A Quantification of peripheral ER structures in WT and ∆ice2 cells (SSY1405, 1603) treated with 8 mM DTT for the times indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.

B Flow cytometric measurements of GFP levels of WT and Δice2 cells containing the transcriptional UPR reporter (SSY2306, 2312). Cells were treated with 8 mM DTT for the times indicated. Data were normalized to untreated WT cells. Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.

C, D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63-mNeon and Rtn1-mCherry (SSY1405, 1603) that were untreated (C) or treated with 8 mM DTT for 1 h (D).

E Quantification of WT and ∆ice2 cells with Rtn1-mCherry puncta after treatment with 8 mM DTT for the times indicated. Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.

F Quantification of peripheral ER structures in untreated WT and UPR-deficient ∆hac1 cells (SSY2228, 2331), overexpressing ICE2 from plasmid pSS761 where indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.

G Flow cytometric measurements of GFP levels of WT and Δhac1 cells containing the UPR reporter (SSY2306, 2314) and overexpressing ICE2 from plasmid pSS761 where indicated. Data were normalized to untreated WT cells. Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the corresponding untreated sample, as judged by a two-tailed Student's t-test assuming equal variance. Exception was the test against the normalized value for wild-type cells, for which a two-tailed Student's t-test with unequal variance was applied. n.s., not significant..
Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex (2021)
Figure 5

Figure 5. Ice2 is functionally linked to Nem1, Spo7 and Pah1.

A Genetic interactions of ICE2 with selected lipid synthesis genes. CDP, cytidine diphosphate; DAG, diacylglycerol; PA, phosphatidic acid; PI/PS/PE/PC, phosphatidyl-inositol/serine/ethanolamine/choline; TAG, triacylglycerol.

B Growth assays of untreated WT, Δice2, Δnem1, Δnem1 Δice2, Δspo7, Δspo7 Δice2, Δpah1 and Δpah1 Δice2 cells (SSY1404, 2356, 2482, 2484, 2481, 2483, 2807, 2808). Numbers represent areas under the curves and serve as growth indices. Mean + s.e.m., n = 3 biological replicates. Data for WT and ∆ice2 cells are the same in the left and middle panels.

C, D Lipidomic analysis of WT, Δice2, Δnem1, Δice2 Δnem1, Δspo7 and Δice2 Δspo7 cells (SSY1404, 2356, 2482, 2484, 2481, 2483). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01. Data for WT and Δice2 cells are the same as in both panels.

E Sec63-mNeon images of untreated WT, Δnem1, Δnem1Δice2, Δspo7 and Δspo7 Δice2 cells (SSY1404, 2482, 2484, 2481, 2483).
Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex (2021)
Figure 6

Figure 6. Ice2 opposes Pah1 by inhibiting the Nem1-Spo7 complex.

A Schematic of Pah1 phospho-regulation. Phosphorylated Pah1 is cytosolic and inactive. Interaction of Pah1 and the ER-localized Nem1-Spo7 complex results in Pah1 dephosphorylation and activation, promoting conversion of PA into DAG.

B Western blot of HA from WT, Δice2, Δnem1 and Δnem1 Δice2 cells expressing endogenously tagged Pah1-HA (SSY2592, 2593, 2594, 2718). Blots of SDS-PAGE and Phos-tag PAGE gels are shown.

C Schematic of Pah1 dephosphorylation assay with phosphorylated Pah1 from ∆nem1 mutants and microsomes from different strains.

D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1-HA from ∆nem1 mutants (SSY3065) and microsomes from cells of the indicated genotypes (SSY3053, 3074, 3075, 3095). Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase (PPase) are shown for reference.

E Western blot of HA, Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ice2 cells expressing Nem1-HA (SSY3140, 3141). Nem1 is undetectable in cell lysates due to its low abundance.

F Western blot of HA from Pah1 phosphorylation reaction that contained hypophosphorylated Pah1-HA from ∆ice2 cells (SSY3096) and microsomes from ∆nem1 cells (SSY3075). Phosphorylated Pah1 from ∆nem1 cells (SSY3065) and hypophosphorylated Pah1 from ∆ice2 cells are shown for reference.
Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex (2021)
Figure 7

Figure 7. Ice2 interacts and co-localizes with the Nem1-Spo7 complex.

A-C Western blots of FLAG, HA and Dpm1 from cell lysates or anti-FLAG immunoprecipitates of WT or ∆nem1 cells containing Spo7-FLAG, Nem1-FLAG or Ice2-HA as indicated (SSY122, 2421, 3183, 3184, 3195, 3196, 3197). The asterisk marks the position of the light chain from the antibody used for immunoprecipitation. The diamond marks a protein that is non-specifically precipitated by the anti-FLAG antibody. IP, immunoprecipitate.

D Western blot of HA from total cell lysates or biotin pulldowns of cells containing Ice2-TurboID, Pah1-HA and deletions of NEM1 or SPO7 as indicated (SSY2978, 2979, 3117, 3118).

E Images of cells with endogenously tagged Ice2-mScarlet and Spo7-mNeon or Nem1-mNeon (SSY3244, 3245). Arrows indicate foci containg both Ice2 and either Spo7 or Nem1.

F Quantification of Spo7 and Nem1 foci in WT and ∆ice2 cells (SSY2916, 3238, 2917, 3239). Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the respective WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.
Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex (2021)
Figure 8

Figure 8. Ice2 promotes ER membrane biogenesis through inhibition of Pah1.

A Lipidomic analysis of WT and ∆ice2 cells in which PAH1 was replaced with PAH1-HA or pah1(7A)-HA as indicated (SSY2841, SSY2842, SSY2970). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.

B Quantification of peripheral ER structures in WT cells in which PAH1 was replaced by PAH1-HA or pah1(7A)-HA, without and with overexpression of ICE2 (SSY2841, 2842, 2843, 2844). Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the respective control cells not overexpressing ICE2, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.

C Quantification of peripheral ER structures in WT cells in which PAH1 was replaced by PAH1-HA or pah1(7A)-HA (SSY2841, 2842), treated with 8 mM DTT for 1 h. Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; .

D Quantification of WT and ∆ice2 cells with Rtn1-mCherry puncta after treatment with 8 mM DTT for 1 h. Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; n.s., not significant.
Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex (2021)
Figure 9

Figure 9. Ice2 cooperates with the PA-Opi1-Ino2/4 system and promotes ER homeostasis.

A Sec63-mNeon images of mid and cortical sections of untreated WT and ∆opi1 cells, overexpressing ICE2 where indicated (SSY1404, 2588, 2595, 2596).

B Quantification of peripheral ER structures in the strains shown in panel (A). Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with control cells not overexpressing ICE2, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.

C Growth assays of untreated WT, ∆hac1, Δice2, and Δhac1 Δice2 cells (SSY1404, 2356, 2805, 2806). Numbers represent areas under the curves and serve as growth indices. Mean + s.e.m., n = 3 biological replicates.

D Growth assays on solid media of WT, ∆hac1, Δice2, and Δhac1 Δice2 cells (SSY1404, 2356, 2805, 2806) in the absence or presence of 0.2 µg/ml tunicamycin. For each series, cells were diluted fivefold from one step to the next.