Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: A, B Ccl2 mRNA expression in MDCK cells expressing inducible shRNA against the indicated targets (+ Tet). Nphp1‐i ( n = 8), Nphp4‐i ( n = 4), Anks3‐i1 ( n = 5), and Nek7‐i1 ( n = 5). See also Appendix Fig S3D–G . C Confocal microscopy images of primary cilia (Ac‐Tub) and GFP in MDCK wild‐type cells (WT) or in MDCK cells expressing GFP alone or YFP constructs fused to LKB1 or STRAD proteins. GFP antibody was used to detect both GFP and YFP constructs. Representative images of three biological replicates. Scale bar: 1 μm. D Representative confocal microscopy images of primary cilia (Ac‐Tub) and LKB1 in MDCK cells expressing inducible shRNA against Stradb (Stradb‐i; + Tet). Scale bar: 5 μm (left panel) and 1 μm (right panel). See also Appendix Fig S3I . E Quantification of LKB1‐positive primary cilia in Stradb‐i MDCK cells. Blinded quantification of five fields of view per biological replicate. F Ccl2 mRNA expression in Strada‐i ( n = 5) and Stradb‐i ( n = 5) MDCK cells. See also Appendix Fig S3H and I . G, H Ccl2 mRNA expression in MDCK cells expressing inducible shRNA against the indicated targets (+ Tet). Kif3a‐i ( n = 7), Ift88‐i ( n = 4), Kif3a‐i;Lkb1‐i1 ( n = 3), and Ift88‐i;Lkb1‐i1 ( n = 4). See also Appendix Fig S3J–M . Data information: In (A, B, F–H), mean ± SD. Paired t ‐test, * P < 0.05, * * P < 0.01, *** P < 0.001, ns : not statistically different. In (E), each circle represents one biological replicate. Bars indicate mean. Paired t ‐test, * P < 0.05.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Expressing, shRNA, Confocal Microscopy, Construct

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: A Immunoprecipitation (IP) from HEK 293T cells. FLAG‐PC1 is enriched in the precipitates of V5‐LKB1, V5‐STRADa, and V5‐STRADb. Representative Western blot of five independent experiments. B Ccl2 mRNA expression in wild‐type (WT) and two PKD1‐null (KO1, KO2) MDCK cell lines constructed using TALEN‐based genome editing ( n = 4). See also Appendix Fig S3N–P . C CCL2 secretion in WT and PKD1 KO1 and KO2 MDCK cell lines ( n = 3). D Ccl2 mRNA expression in PKD1 KO1 MDCK cells expressing inducible shRNA against the indicated targets after tetracycline induction (Tet). WT ( n = 4), Kif3a‐i ( n = 4), and Ift88‐i ( n = 4). See also Appendix Fig S3Q and R . E Representative kidneys from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. See also Fig . F Kidney weight (KW)‐to‐body weight (BW) ratio in control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. G Ccl2 mRNA expression in kidneys from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. H, I Flow cytometry analysis (H) and absolute numbers (I) of CCR2 + macrophages (F4/80 hi CD11b + CCR2 + , black oval) in control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. See also Appendix Fig S6 . J, K Flow cytometry analysis (J) and absolute numbers (K) of CCR2 + monocytes (F4/80 lo CD11b hi CCR2 + , black square) in control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. Data information: In (B–D), mean ± SD. Paired t ‐test, * P < 0.05. In (F, G, I, K), each dot represents one individual mouse. Bars indicate mean. ANOVA followed by the Tukey–Kramer test, * P < 0.05, *** P < 0.001. In (H, J), numbers represent percentage of cells per gated region. Source data are available online for this figure.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Immunoprecipitation, Western Blot, Expressing, Construct, shRNA, Flow Cytometry

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: Age from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice. Plasma blood urea nitrogen (BUN) from control ( n = 16), i Pkd1 ΔTub ( n = 10), and i Pkd1 ΔTub ;i Kif3a ΔTub ( n = 12) mice at 12 weeks. Representative periodic acid–Schiff (PAS)‐stained kidney sections from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. Higher magnification of PAS‐stained sections. Scale bar: 250 μm. Data information: In (A, B), each dot represents one individual mouse. Bars indicate mean. ANOVA followed by the Tukey–Kramer test, ns : not statistically different.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Staining

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: A Principal component analysis (PCA) of RNAseq expression levels from MDCK cells with inducible knockdown of Lkb1 (Lkb1‐i) after tetracycline incubation (PC1: −Tet vs. +Tet). n = 2 biological replicates (PC2). B PCA of microarray expression data from littermate control and Lkb1 ΔTub kidneys at 5 weeks (PC1). n = 5 mice each (PC2). Replicates are enclosed in convex hulls to guide the eye. C Network representation of significantly enriched Biological Processes GO terms (commonly upregulated genes) derived from a hypergeometric test ( P < 0.05). Nodes are connected if they have a semantic similarity > 0.6, and clusters are named according to the most representative GO terms. See also . D–H Ccl2 mRNA expression evaluated by qRT–PCR in MDCK cells expressing inducible shRNA after tetracycline induction (Tet) against Lkb1‐i2 (D, n = 4), Lkb1‐i1 (E, n = 3) after 5 h of 1 mM AICAR treatment, Anks3‐i2 (F, n = 5), Nek7‐i2 (G, n = 5), PKD1 KO1 Lkb1‐i1, and PKD1 KO2 Lkb1‐i1 (H, n = 3). See also Appendix Fig S3B, F, G and S . Data information: In (D–H), mean ± SD. Paired t ‐test, * P < 0.05, * * P < 0.01, ns : not statistically different.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Expressing, Knockdown, Incubation, Microarray, Control, Derivative Assay, Quantitative RT-PCR, shRNA

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: A Immunoprecipitation (IP) from HEK 293T cells. FLAG‐PC1 is enriched in the precipitates of V5‐LKB1, V5‐STRADa, and V5‐STRADb. Representative Western blot of five independent experiments. B Ccl2 mRNA expression in wild‐type (WT) and two PKD1‐null (KO1, KO2) MDCK cell lines constructed using TALEN‐based genome editing ( n = 4). See also Appendix Fig S3N–P . C CCL2 secretion in WT and PKD1 KO1 and KO2 MDCK cell lines ( n = 3). D Ccl2 mRNA expression in PKD1 KO1 MDCK cells expressing inducible shRNA against the indicated targets after tetracycline induction (Tet). WT ( n = 4), Kif3a‐i ( n = 4), and Ift88‐i ( n = 4). See also Appendix Fig S3Q and R . E Representative kidneys from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. See also Fig . F Kidney weight (KW)‐to‐body weight (BW) ratio in control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. G Ccl2 mRNA expression in kidneys from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. H, I Flow cytometry analysis (H) and absolute numbers (I) of CCR2 + macrophages (F4/80 hi CD11b + CCR2 + , black oval) in control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. See also Appendix Fig S6 . J, K Flow cytometry analysis (J) and absolute numbers (K) of CCR2 + monocytes (F4/80 lo CD11b hi CCR2 + , black square) in control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. Data information: In (B–D), mean ± SD. Paired t ‐test, * P < 0.05. In (F, G, I, K), each dot represents one individual mouse. Bars indicate mean. ANOVA followed by the Tukey–Kramer test, * P < 0.05, *** P < 0.001. In (H, J), numbers represent percentage of cells per gated region. Source data are available online for this figure.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Immunoprecipitation, Western Blot, Expressing, Construct, shRNA, Control, Flow Cytometry

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: Age from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice. Plasma blood urea nitrogen (BUN) from control ( n = 16), i Pkd1 ΔTub ( n = 10), and i Pkd1 ΔTub ;i Kif3a ΔTub ( n = 12) mice at 12 weeks. Representative periodic acid–Schiff (PAS)‐stained kidney sections from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Kif3a ΔTub mice at 12 weeks. Higher magnification of PAS‐stained sections. Scale bar: 250 μm. Data information: In (A, B), each dot represents one individual mouse. Bars indicate mean. ANOVA followed by the Tukey–Kramer test, ns : not statistically different.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Control, Clinical Proteomics, Staining

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: Representative kidneys from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Ccl2 ΔTub mice at 13.5 weeks. See also Fig . Kidney weight (KW)‐to‐body weight (BW) ratio in control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Ccl2 ΔTub mice at 13.5 weeks. Ccl2 mRNA expression in kidneys from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Ccl2 ΔTub mice at 13.5 weeks. F4/80 immunostaining in kidneys from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Ccl2 ΔTub mice at 13.5 weeks. Representative images of n = 4 mice/group. Scale bars: 50 μm. Itgam mRNA expression in kidneys from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Ccl2 ΔTub mice at 13.5 weeks. Data information: In (B, C, E), each dot represents one individual mouse. Bars indicate mean. ANOVA followed by the Tukey–Kramer test, * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Control, Expressing, Immunostaining

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: Age from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Ccl2 ΔTub mice. Plasma blood urea nitrogen (BUN) from control, i Pkd1 ΔTub , and i Pkd1 ΔTub ;i Ccl2 ΔTub mice at 13.5 weeks. Representative periodic acid–Schiff (PAS)‐stained kidney sections from control ( n = 7), i Pkd1 ΔTub ( n = 6), and i Pkd1 ΔTub ;i Ccl2 ΔTub ( n = 7) mice at 13.5 weeks. Higher magnification of PAS‐stained sections. Scale bar: 250 μm. Data information: In (A, B), each dot represents one individual mouse. Bars indicate mean. ANOVA followed by the Tukey–Kramer test. * P < 0.05, ns : not statistically different.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Control, Clinical Proteomics, Staining

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: Under normal conditions, NPHP1, ANKS3, NEK7, LKB1, and STRAD constitute a module within the cilium to inhibit a ciliary signal on CCL2. In the absence of LKB1 or STRAD, the block is released and a ciliary signal activates expression of CCL2. This leads to the recruitment of monocytes and macrophages expressing CCR2, the CCL2 receptor; remodeling; and fibrosis. PKD1 interacts with LKB1 and STRAD to activate the repressive function of the LKB1 module. Loss of PKD1 results in de‐repression of CCL2 expression by the module. This leads to the recruitment of monocytes and CCR2 + macrophages enhancing cyst growth. In the absence of cilia and PKD1, the ciliary CCL2 signal cannot be activated. The recruitment of mononuclear cells is inhibited, and less cyst growth occurs. Simultaneous targeting of PKD1 and CCL2 prevents the activation of mononuclear cells and ameliorates cyst growth.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Blocking Assay, Expressing, Activation Assay

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: LKB1 interactors identified by a proteomic screen in mouse kidney medulla. See also and . Immunoprecipitates (IP) from mouse kidney medulla. ANKS3 and NEK7 are enriched in the LKB1 precipitates. Representative Western blot of three independent experiments. Expected molecular weights: ANKS3 (72 kDa), LKB1 (54 kDa), and NEK7 (35 kDa). Representative confocal microscopy images of primary cilia (acetylated tubulin expressing, Ac‐Tub) and ANKS3 in MDCK cells expressing inducible shRNA against Anks3 (Anks3‐i1) after tetracycline induction (Tet). Scale bar: 5 μm. See also Appendix Fig S3F . Quantification of ANKS3‐positive primary cilia in Anks3‐i1 MDCK cells. Blinded quantification of five fields of view per biological replicate. Confocal microscopy images of collecting duct (AQP2 expressing) from 5‐week‐old wild‐type mice. Anti‐ANKS3 antibody (upper panel), but not control IgG (lower panel), detects a signal in cilia (Ac‐Tub). Representative images of n = 5 mice. Scale bar: 5 μm. IP from HEK 293T cells. Endogenous LKB1 is enriched in the precipitates of FLAG‐NPHP1. Representative Western blot of three independent experiments. See also Fig A–C. Zebrafish embryos injected with control morpholino (MO) or MO targeting nphp1 at 48 h post‐fertilization. Arrowheads indicate pronephric cysts. Scale bar: 100 μm. Quantification of embryos with pronephric cysts. Numbers in bars represent n analyzed embryos. Data information: In (D), each circle represents one biological replicate. Bars indicate mean. Paired t ‐test, * P < 0.05. In (H) mean ± SD. ANOVA followed by the Tukey–Kramer test, * P < 0.05, ** P < 0.01. Source data are available online for this figure.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Western Blot, Confocal Microscopy, Expressing, shRNA, Control, Injection

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: A Principal component analysis (PCA) of RNAseq expression levels from MDCK cells with inducible knockdown of Lkb1 (Lkb1‐i) after tetracycline incubation (PC1: −Tet vs. +Tet). n = 2 biological replicates (PC2). B PCA of microarray expression data from littermate control and Lkb1 ΔTub kidneys at 5 weeks (PC1). n = 5 mice each (PC2). Replicates are enclosed in convex hulls to guide the eye. C Network representation of significantly enriched Biological Processes GO terms (commonly upregulated genes) derived from a hypergeometric test ( P < 0.05). Nodes are connected if they have a semantic similarity > 0.6, and clusters are named according to the most representative GO terms. See also . D–H Ccl2 mRNA expression evaluated by qRT–PCR in MDCK cells expressing inducible shRNA after tetracycline induction (Tet) against Lkb1‐i2 (D, n = 4), Lkb1‐i1 (E, n = 3) after 5 h of 1 mM AICAR treatment, Anks3‐i2 (F, n = 5), Nek7‐i2 (G, n = 5), PKD1 KO1 Lkb1‐i1, and PKD1 KO2 Lkb1‐i1 (H, n = 3). See also Appendix Fig S3B, F, G and S . Data information: In (D–H), mean ± SD. Paired t ‐test, * P < 0.05, * * P < 0.01, ns : not statistically different.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Expressing, Knockdown, Incubation, Microarray, Control, Derivative Assay, Quantitative RT-PCR, shRNA

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: A, B Ccl2 mRNA expression in MDCK cells expressing inducible shRNA against the indicated targets (+ Tet). Nphp1‐i ( n = 8), Nphp4‐i ( n = 4), Anks3‐i1 ( n = 5), and Nek7‐i1 ( n = 5). See also Appendix Fig S3D–G . C Confocal microscopy images of primary cilia (Ac‐Tub) and GFP in MDCK wild‐type cells (WT) or in MDCK cells expressing GFP alone or YFP constructs fused to LKB1 or STRAD proteins. GFP antibody was used to detect both GFP and YFP constructs. Representative images of three biological replicates. Scale bar: 1 μm. D Representative confocal microscopy images of primary cilia (Ac‐Tub) and LKB1 in MDCK cells expressing inducible shRNA against Stradb (Stradb‐i; + Tet). Scale bar: 5 μm (left panel) and 1 μm (right panel). See also Appendix Fig S3I . E Quantification of LKB1‐positive primary cilia in Stradb‐i MDCK cells. Blinded quantification of five fields of view per biological replicate. F Ccl2 mRNA expression in Strada‐i ( n = 5) and Stradb‐i ( n = 5) MDCK cells. See also Appendix Fig S3H and I . G, H Ccl2 mRNA expression in MDCK cells expressing inducible shRNA against the indicated targets (+ Tet). Kif3a‐i ( n = 7), Ift88‐i ( n = 4), Kif3a‐i;Lkb1‐i1 ( n = 3), and Ift88‐i;Lkb1‐i1 ( n = 4). See also Appendix Fig S3J–M . Data information: In (A, B, F–H), mean ± SD. Paired t ‐test, * P < 0.05, * * P < 0.01, *** P < 0.001, ns : not statistically different. In (E), each circle represents one biological replicate. Bars indicate mean. Paired t ‐test, * P < 0.05.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Expressing, shRNA, Confocal Microscopy, Construct

Journal: The EMBO Journal

Article Title: Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney

doi: 10.15252/embj.201798615

Figure Lengend Snippet: Under normal conditions, NPHP1, ANKS3, NEK7, LKB1, and STRAD constitute a module within the cilium to inhibit a ciliary signal on CCL2. In the absence of LKB1 or STRAD, the block is released and a ciliary signal activates expression of CCL2. This leads to the recruitment of monocytes and macrophages expressing CCR2, the CCL2 receptor; remodeling; and fibrosis. PKD1 interacts with LKB1 and STRAD to activate the repressive function of the LKB1 module. Loss of PKD1 results in de‐repression of CCL2 expression by the module. This leads to the recruitment of monocytes and CCR2 + macrophages enhancing cyst growth. In the absence of cilia and PKD1, the ciliary CCL2 signal cannot be activated. The recruitment of mononuclear cells is inhibited, and less cyst growth occurs. Simultaneous targeting of PKD1 and CCL2 prevents the activation of mononuclear cells and ameliorates cyst growth.

Article Snippet: Antibodies were used against the following: For Western blotting : AMPKα (Cell Signaling Technology, 2532, 1:1,000), ANKS3 (Eurogentec, rabbit polyclonal, directed against mouse ANKS3 556–656, 1:1,000), α‐TUBULIN (Sigma, T5168, 1:20,000), β‐ACTIN (Sigma, A1978, 1:60,000), FLAG (M2 clone, Sigma, F‐3165, 1:6,000), IFT88 (ProteinTech, 13967‐1‐AP, 1:1,000), KIF3a (BD Transduction, 611508, 1:1,000), LKB1 (D60C5F10, Cell Signaling Technology, 13031, 1:6,000), NEK7 (Santa Cruz Biotechnology, sc‐50756 and sc‐393539, 1:1,000), NPHP1 (rabbit polyclonal, kind gift from Prof. Bernhard Schermer, Köln, 1:4,000), pAMPKα T172 (Cell Signaling Technology, 2535, 1:1,000), PKD1 (7E12, Santa Cruz Biotechnology, sc‐130554, 1:200), pS6RP S235/236 (Cell Signaling Technology, 4858, 1:2,000), pTSC2 S1387 (Cell Signaling Technology, 5584, 1:1,000), S6RP (Cell Signaling Technology, 2217, 1:1,000), TSC2 (Cell Signaling Technology, 4302, 1:1,000), and V5 (AbD Serotec, MCA1360, 1:5,000).

Techniques: Blocking Assay, Expressing, Activation Assay

Journal: Nature Communications

Article Title: SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

doi: 10.1038/s41467-021-24007-w

Figure Lengend Snippet: a , b Analysis and regulation of significantly altered pathways of mock- and SARS-CoV-2-infected (24 h p.i., MOI = 0.1) VeroFM cells ( a ) or Calu-3 cells ( b ). The y -axis shows the (median) log2 fold change (FC) of all significantly altered metabolites of the indicated pathway while the –log10 corrected p -value (false discovery rate (FDR)) is shown on the x -axis. The size of the circles illustrates the number of significantly changed metabolites in relation to all metabolites of a specific pathway. c Analysis of the autophagic pathway and the involved metabolites: ‘amino acids’ and ‘GSH metabolism’ (orange), ‘nucleotides’ (blue), ‘glycolysis’ (green), ‘polyamine metabolism’ (red) and ‘O-GalNAcylation’ (purple) in mock- and SARS-CoV-2-infected (24 h p.i.) VeroFM and Calu-3 cells. For a – c Error bars represent SEM. n = 4 biological samples per group of one experiment. All p-values were determined by a two-way ANOVA and Tukey´s post hoc test. FDRs were adjusted using the Benjamini-Hochberg method. Abbreviations: 1,3-BPG, 1,3-bisphosphoglyceric acid; 3-PGA, 3-phosphoglyceric acid; ADP, adenosine diphosphate; AKT1, RAC-alpha serine/threonine-protein kinase; ala, alanine; AMP, adenosine monophosphate; AMPK, AMP-activated protein kinase; arg, arginine; asn, asparagine; asp, asparagine; ATP, adenosine triphosphate; aut, autophagosome; BECN1, beclin-1; bio., biosynthesis; CTP, cytidine triphosphate; CoA, coenzyme A; cys, cysteine; cysgly, cysteinylglycine; cyst, cystathionine; dCTP, deoxycytidine triphosphate; dicarbox., dicarboxylate; elF5AH, eukaryotic translation initiation factor 5A hypusinated; EP300, histone acetyltransferase p300; F1P, fructose 1-phosphate; F6P, fructose 6-phosphate; F-1,6-BP, fructose 1,6-bisphosphate; FKBP51, 51 kDa FK506-binding protein; G6-P, glucose 6-phosphate; gln, glutamine; glu, glutamic acid; gly, glycine; glyox, glyoxylate; GSH, glutathione (reduced); GTP, guanosine triphosphate; hcys, homocysteine; his, histidine; ile, isoleucine; lac, lactic acid; leu, leucine; lys, lysine; mal, malic acid; met, methionine; met., metabolism; modific., modification; mTORC1, mechanistic target of rapamycin complex 1; NAcput, N-acetylputrescine; NAcspd, N-acetylspermidine; orn, ornithine; PEP, phosphoenolpyruvic acid; phe, phenylalanine; PHLPP, PH domain leucine-rich repeat-containing protein phosphatase; PPP, pentose phosphate pathway; pro, proline; prot., protein; put, putrescine; pyr, pyruvic acid; resid., residue; ROS, reactive oxygen species; S7P, sedoheptulose-7-phosphate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SAT1, diamine acetyltransferase 1; SKP2, S-phase kinase-associated protein 2; ser, serine; spd, spermidine; spm, spermine; tau, taurine; TFEB, transcription factor EB; thr, threonine; trp, tryptophan; tyr, tyrosine; UDP-GalNAc, UDP-N-acetylgalactosamine; UDP, uridine diphosphate; UTP, uridine triphosphate; val, valine.

Article Snippet: The following primary antibodies were used: β-actin (1:5,000 Cell Signaling Technology, #8457), SQSTM1/p62 (1:1,000, Cell Signaling Technology, #5114), LC3B (1:1,000, Cell Signaling Technology, #3868), BECN1 (1:1,000, Cell Signaling Technology, #3738), pBECN1 S15 (1:1,000, Cell Signaling Technology, #84966), ATG14 (1:1,000, Cell Signaling Technology, #5504), pATG14 S29 (1:1,000, Cell Signaling Technology, #13155), ULK1 (1:1,000, Cell Signaling Technology, #8054), pULK1 (S555) (1:1,000, Cell Signaling Technology, #5869), pULK1 (S757) (1:1,000, Cell Signaling Technology, #6888), TSC2 (1:1,000, Cell Signaling Technology, #3612), pTSC2 (S1387) (1:1,000, Cell Signaling Technology, #5584), AMPKα (1:1,000, Cell Signaling Technology, #2532), pAMPK (T172) (1:1,000, Cell Signaling Technology, #2531), pAMPK substrate motif (1:1,000, Cell Signaling Technology, #5759), pAKT (S473) (1:1,000, Cell Signaling Technology, #4060), AKT (1:1,000, Cell Signaling Technology, #9272), HSC70 (1:5,000, Enzo Life Sciences, ADI-SPA-757-F), SKP2 (1:1,000, Cell Signaling Technology, #2652), pSKP2 (S64) (1:1,000, Cell Signaling Technology, #14865).

Techniques: Infection, Binding Assay, Modification, Residue

Journal: Nature Communications

Article Title: SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

doi: 10.1038/s41467-021-24007-w

Figure Lengend Snippet: a Protein levels and phosphorylation status of selected autophagy-relevant proteins in SARS-CoV-2-infected VeroFM cells at 8 h, 24 h, or 48 h post infection (h p.i.) were analyzed by Western blotting. For analysis of ATG14 oligomers (virtual blot, bottom panel, left) proteins were cross-linked 2 h prior to cell harvest and analyzed by Wes (ProteinSimple) capillary electrophoresis 8-48 h p.i. P -values were determined by one-way ANOVA, Bonferroni post hoc test. b Fluorescence microscopy of transfected and SARS-CoV-2-infected VeroFM cells expressing pH-sensitive tandem fluorescent-tagged LC3B - mRFP/EGFP showed that low pH autophagolysosomes (AL, red) were reduced compared to autophagosomes (AP, green + red = yellow) in virus-infected cells. Microscopic read-out was done by a scientist blind to the experimental conditions. For mock ( n = 44 cells) and SARS-CoV-2-infected ( n = 46 cells) VeroFM cells were analyzed. P-values were determined by two-way ANOVA, Tukey´s post hoc test, mean with SEM. Scale bar = 10 µM. c Accumulation of autophagy marker P62 in SARS-CoV-2-infected VeroFM cells. d Elevated LC3B-II levels in SARS-CoV-2-infected and bafilomycin A1 (BafA1)-pretreated VeroFM cells indicate virus-induced autophagic flux inhibition at 8 and 24 h p.i. In all panels, error bars denote SEM derived from n = 3 biologically independent samples derived from one experiment. P-values were determined by two-way ANOVA, Sidak post hoc test. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p > 0.05 (not significant, ns). Abbreviations: AKT1, RAC-alpha serine/threonine-protein kinase; AMPK, AMP-activated protein kinase; ATG14, autophagy-related 14; ATG16L1, autophagy-related 16 like 1; BafA1, bafilomycin A1; BECN1, Beclin-1 protein; CDK2, cyclin-dependent kinase 2; EGFP, enhanced green fluorescent protein; LC3B, microtubule-associated protein 1 A/1B light chain 3B; LXRXX(pS/pT), phospho-AMPK substrate motif; mRFP, monomeric red fluorescent protein; mTORC1/2, mechanistic target of rapamycin complex 1/2; PRAS40, proline-rich AKT1 substrate 1; Rheb, Ras homolog enriched in brain; SKP2, S-phase kinase-associated protein 2; TSC1/2, tuberous sclerosis 1/2; ULK1, Unc-51-like kinase 1; VPS34, phosphatidylinositol 3-kinase catalytic subunit type 3.

Article Snippet: The following primary antibodies were used: β-actin (1:5,000 Cell Signaling Technology, #8457), SQSTM1/p62 (1:1,000, Cell Signaling Technology, #5114), LC3B (1:1,000, Cell Signaling Technology, #3868), BECN1 (1:1,000, Cell Signaling Technology, #3738), pBECN1 S15 (1:1,000, Cell Signaling Technology, #84966), ATG14 (1:1,000, Cell Signaling Technology, #5504), pATG14 S29 (1:1,000, Cell Signaling Technology, #13155), ULK1 (1:1,000, Cell Signaling Technology, #8054), pULK1 (S555) (1:1,000, Cell Signaling Technology, #5869), pULK1 (S757) (1:1,000, Cell Signaling Technology, #6888), TSC2 (1:1,000, Cell Signaling Technology, #3612), pTSC2 (S1387) (1:1,000, Cell Signaling Technology, #5584), AMPKα (1:1,000, Cell Signaling Technology, #2532), pAMPK (T172) (1:1,000, Cell Signaling Technology, #2531), pAMPK substrate motif (1:1,000, Cell Signaling Technology, #5759), pAKT (S473) (1:1,000, Cell Signaling Technology, #4060), AKT (1:1,000, Cell Signaling Technology, #9272), HSC70 (1:5,000, Enzo Life Sciences, ADI-SPA-757-F), SKP2 (1:1,000, Cell Signaling Technology, #2652), pSKP2 (S64) (1:1,000, Cell Signaling Technology, #14865).

Techniques: Phospho-proteomics, Infection, Western Blot, Electrophoresis, Fluorescence, Microscopy, Transfection, Expressing, Virus, Marker, Inhibition, Derivative Assay

Journal: Nature Communications

Article Title: SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

doi: 10.1038/s41467-021-24007-w

Figure Lengend Snippet: Schematic representation of autophagy signaling indicating site of action of small-molecule compounds (orange columns) or siRNA knockdown (green columns) used for pathway modulation. VeroFM cells were infected with SARS-CoV-2 (MOI = 0.0005) and treated with the indicated compounds: polyamines spermidine (spd, 100 µM) and spermine (spm, 100 µM), AMPK modulator AICAR (25 µM), mTORC1 inhibitor rapamycin (rap, 300 nM), VPS34 and ULK1 inhibitors SAR405 (1 µM) and MRT68921 (5 µM), AKT1 inhibitor MK-2206 (1 µM), BECN1-stabilizers SMIP004 (S004, 10 µM), valinomycin (val, 5 µM), niclosamide (nic, 10 µM) or DMSO (vehicle, dashed lines). For siRNA knockdown, VeroFM cells were transfected with 60 nM siRNA targeting ATG5, ATG7, FIP200, BECN1 , and infected with SARS-CoV-2 48 h later. SARS-CoV-2 infectious virus particle units (PFU) and genome equivalents (GE) per ml were determined by plaque assay and real-time RT-PCR at 24 h p.i., respectively. Data are presented as fold differences (see Supplementary Fig. for unprocessed data). In all panels, error bars =SEM derived from n = 3 ( n = 2 for spd, spm, AICAR) biologically independent samples from one experiment. Statistics were done for experiments with n = 3 using a two-way ANOVA, Dunnett’s, and one-way ANOVA for siRNA knockdown experiments (green). Compounds with significant SARS-CoV-2 inhibition (spm, spd, MK-2206, S004, val, nic) in VeroFM cells were confirmed in Calu-3 cells (see Supplementary Fig. f–g). p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p > 0.05 (not significant, ns). Abbreviations: AICAR, 5-Aminoimidazole-4-carboxamide ribonucleotide; AKT1, RAC-alpha serine/threonine-protein kinase; AMPK, AMP-activated protein kinase; ATG5, autophagy-related 5; ATG7, ubiquitin-like modifier-activating enzyme ATG7; ATG12, ubiquitin-like protein ATG12; aut, autophagosome; BECN1, Beclin-1 protein; CLEAR, coordinated lysosomal expression and regulation; cmpd, compound; EP300, histone acetyltransferase p300; elF5AH, eukaryotic translation initiation factor 5A, hypusinated; FIP200, FAK family kinase-interacting protein of 200 kDa; FKBP51, 51 kDa FK506-binding protein; GE, genome equivalents; lys, lysosome; mTORC1, mechanistic target of rapamycin complex 1; nic, niclosamide; PFU, plaque-forming units; phag, phagophore; PHLPP, PH domain leucine-rich repeat-containing protein phosphatase; rap, rapamycin; S004, SMIP-004; SKP2, S-phase kinase-associated protein 2; spd, spermidine; spm, spermine; TFEB, transcription factor EB; ULK1, unc-51-like kinase 1; val, valinomycin; VPS34, phosphatidylinositol 3-kinase catalytic subunit type 3.

Article Snippet: The following primary antibodies were used: β-actin (1:5,000 Cell Signaling Technology, #8457), SQSTM1/p62 (1:1,000, Cell Signaling Technology, #5114), LC3B (1:1,000, Cell Signaling Technology, #3868), BECN1 (1:1,000, Cell Signaling Technology, #3738), pBECN1 S15 (1:1,000, Cell Signaling Technology, #84966), ATG14 (1:1,000, Cell Signaling Technology, #5504), pATG14 S29 (1:1,000, Cell Signaling Technology, #13155), ULK1 (1:1,000, Cell Signaling Technology, #8054), pULK1 (S555) (1:1,000, Cell Signaling Technology, #5869), pULK1 (S757) (1:1,000, Cell Signaling Technology, #6888), TSC2 (1:1,000, Cell Signaling Technology, #3612), pTSC2 (S1387) (1:1,000, Cell Signaling Technology, #5584), AMPKα (1:1,000, Cell Signaling Technology, #2532), pAMPK (T172) (1:1,000, Cell Signaling Technology, #2531), pAMPK substrate motif (1:1,000, Cell Signaling Technology, #5759), pAKT (S473) (1:1,000, Cell Signaling Technology, #4060), AKT (1:1,000, Cell Signaling Technology, #9272), HSC70 (1:5,000, Enzo Life Sciences, ADI-SPA-757-F), SKP2 (1:1,000, Cell Signaling Technology, #2652), pSKP2 (S64) (1:1,000, Cell Signaling Technology, #14865).

Techniques: Knockdown, Infection, Transfection, Virus, Plaque Assay, Quantitative RT-PCR, Derivative Assay, Inhibition, Ubiquitin Proteomics, Expressing, Binding Assay

Journal: Nature Communications

Article Title: SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

doi: 10.1038/s41467-021-24007-w

Figure Lengend Snippet: a Protein levels and phosphorylation status of selected autophagy-relevant proteins in SARS-CoV-2-infected VeroFM cells at 8 h, 24 h, or 48 h post infection (h p.i.) were analyzed by Western blotting. For analysis of ATG14 oligomers (virtual blot, bottom panel, left) proteins were cross-linked 2 h prior to cell harvest and analyzed by Wes (ProteinSimple) capillary electrophoresis 8-48 h p.i. P -values were determined by one-way ANOVA, Bonferroni post hoc test. b Fluorescence microscopy of transfected and SARS-CoV-2-infected VeroFM cells expressing pH-sensitive tandem fluorescent-tagged LC3B - mRFP/EGFP showed that low pH autophagolysosomes (AL, red) were reduced compared to autophagosomes (AP, green + red = yellow) in virus-infected cells. Microscopic read-out was done by a scientist blind to the experimental conditions. For mock ( n = 44 cells) and SARS-CoV-2-infected ( n = 46 cells) VeroFM cells were analyzed. P-values were determined by two-way ANOVA, Tukey´s post hoc test, mean with SEM. Scale bar = 10 µM. c Accumulation of autophagy marker P62 in SARS-CoV-2-infected VeroFM cells. d Elevated LC3B-II levels in SARS-CoV-2-infected and bafilomycin A1 (BafA1)-pretreated VeroFM cells indicate virus-induced autophagic flux inhibition at 8 and 24 h p.i. In all panels, error bars denote SEM derived from n = 3 biologically independent samples derived from one experiment. P-values were determined by two-way ANOVA, Sidak post hoc test. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p > 0.05 (not significant, ns). Abbreviations: AKT1, RAC-alpha serine/threonine-protein kinase; AMPK, AMP-activated protein kinase; ATG14, autophagy-related 14; ATG16L1, autophagy-related 16 like 1; BafA1, bafilomycin A1; BECN1, Beclin-1 protein; CDK2, cyclin-dependent kinase 2; EGFP, enhanced green fluorescent protein; LC3B, microtubule-associated protein 1 A/1B light chain 3B; LXRXX(pS/pT), phospho-AMPK substrate motif; mRFP, monomeric red fluorescent protein; mTORC1/2, mechanistic target of rapamycin complex 1/2; PRAS40, proline-rich AKT1 substrate 1; Rheb, Ras homolog enriched in brain; SKP2, S-phase kinase-associated protein 2; TSC1/2, tuberous sclerosis 1/2; ULK1, Unc-51-like kinase 1; VPS34, phosphatidylinositol 3-kinase catalytic subunit type 3.

Article Snippet: The following primary antibodies were used: β-actin (1:5,000 Cell Signaling Technology, #8457), SQSTM1/p62 (1:1,000, Cell Signaling Technology, #5114), LC3B (1:1,000, Cell Signaling Technology, #3868), BECN1 (1:1,000, Cell Signaling Technology, #3738), pBECN1 S15 (1:1,000, Cell Signaling Technology, #84966), ATG14 (1:1,000, Cell Signaling Technology, #5504), pATG14 S29 (1:1,000, Cell Signaling Technology, #13155), ULK1 (1:1,000, Cell Signaling Technology, #8054), pULK1 (S555) (1:1,000, Cell Signaling Technology, #5869), pULK1 (S757) (1:1,000, Cell Signaling Technology, #6888), TSC2 (1:1,000, Cell Signaling Technology, #3612), pTSC2 (S1387) (1:1,000, Cell Signaling Technology, #5584), AMPKα (1:1,000, Cell Signaling Technology, #2532), pAMPK (T172) (1:1,000, Cell Signaling Technology, #2531), pAMPK substrate motif (1:1,000, Cell Signaling Technology, #5759), pAKT (S473) (1:1,000, Cell Signaling Technology, #4060), AKT (1:1,000, Cell Signaling Technology, #9272), HSC70 (1:5,000, Enzo Life Sciences, ADI-SPA-757-F), SKP2 (1:1,000, Cell Signaling Technology, #2652), pSKP2 (S64) (1:1,000, Cell Signaling Technology, #14865).

Techniques: Phospho-proteomics, Infection, Western Blot, Electrophoresis, Fluorescence, Microscopy, Transfection, Expressing, Virus, Marker, Inhibition, Derivative Assay

Journal: Nature Communications

Article Title: SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

doi: 10.1038/s41467-021-24007-w

Figure Lengend Snippet: Schematic representation of autophagy signaling indicating site of action of small-molecule compounds (orange columns) or siRNA knockdown (green columns) used for pathway modulation. VeroFM cells were infected with SARS-CoV-2 (MOI = 0.0005) and treated with the indicated compounds: polyamines spermidine (spd, 100 µM) and spermine (spm, 100 µM), AMPK modulator AICAR (25 µM), mTORC1 inhibitor rapamycin (rap, 300 nM), VPS34 and ULK1 inhibitors SAR405 (1 µM) and MRT68921 (5 µM), AKT1 inhibitor MK-2206 (1 µM), BECN1-stabilizers SMIP004 (S004, 10 µM), valinomycin (val, 5 µM), niclosamide (nic, 10 µM) or DMSO (vehicle, dashed lines). For siRNA knockdown, VeroFM cells were transfected with 60 nM siRNA targeting ATG5, ATG7, FIP200, BECN1 , and infected with SARS-CoV-2 48 h later. SARS-CoV-2 infectious virus particle units (PFU) and genome equivalents (GE) per ml were determined by plaque assay and real-time RT-PCR at 24 h p.i., respectively. Data are presented as fold differences (see Supplementary Fig. for unprocessed data). In all panels, error bars =SEM derived from n = 3 ( n = 2 for spd, spm, AICAR) biologically independent samples from one experiment. Statistics were done for experiments with n = 3 using a two-way ANOVA, Dunnett’s, and one-way ANOVA for siRNA knockdown experiments (green). Compounds with significant SARS-CoV-2 inhibition (spm, spd, MK-2206, S004, val, nic) in VeroFM cells were confirmed in Calu-3 cells (see Supplementary Fig. f–g). p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p > 0.05 (not significant, ns). Abbreviations: AICAR, 5-Aminoimidazole-4-carboxamide ribonucleotide; AKT1, RAC-alpha serine/threonine-protein kinase; AMPK, AMP-activated protein kinase; ATG5, autophagy-related 5; ATG7, ubiquitin-like modifier-activating enzyme ATG7; ATG12, ubiquitin-like protein ATG12; aut, autophagosome; BECN1, Beclin-1 protein; CLEAR, coordinated lysosomal expression and regulation; cmpd, compound; EP300, histone acetyltransferase p300; elF5AH, eukaryotic translation initiation factor 5A, hypusinated; FIP200, FAK family kinase-interacting protein of 200 kDa; FKBP51, 51 kDa FK506-binding protein; GE, genome equivalents; lys, lysosome; mTORC1, mechanistic target of rapamycin complex 1; nic, niclosamide; PFU, plaque-forming units; phag, phagophore; PHLPP, PH domain leucine-rich repeat-containing protein phosphatase; rap, rapamycin; S004, SMIP-004; SKP2, S-phase kinase-associated protein 2; spd, spermidine; spm, spermine; TFEB, transcription factor EB; ULK1, unc-51-like kinase 1; val, valinomycin; VPS34, phosphatidylinositol 3-kinase catalytic subunit type 3.

Article Snippet: The following primary antibodies were used: β-actin (1:5,000 Cell Signaling Technology, #8457), SQSTM1/p62 (1:1,000, Cell Signaling Technology, #5114), LC3B (1:1,000, Cell Signaling Technology, #3868), BECN1 (1:1,000, Cell Signaling Technology, #3738), pBECN1 S15 (1:1,000, Cell Signaling Technology, #84966), ATG14 (1:1,000, Cell Signaling Technology, #5504), pATG14 S29 (1:1,000, Cell Signaling Technology, #13155), ULK1 (1:1,000, Cell Signaling Technology, #8054), pULK1 (S555) (1:1,000, Cell Signaling Technology, #5869), pULK1 (S757) (1:1,000, Cell Signaling Technology, #6888), TSC2 (1:1,000, Cell Signaling Technology, #3612), pTSC2 (S1387) (1:1,000, Cell Signaling Technology, #5584), AMPKα (1:1,000, Cell Signaling Technology, #2532), pAMPK (T172) (1:1,000, Cell Signaling Technology, #2531), pAMPK substrate motif (1:1,000, Cell Signaling Technology, #5759), pAKT (S473) (1:1,000, Cell Signaling Technology, #4060), AKT (1:1,000, Cell Signaling Technology, #9272), HSC70 (1:5,000, Enzo Life Sciences, ADI-SPA-757-F), SKP2 (1:1,000, Cell Signaling Technology, #2652), pSKP2 (S64) (1:1,000, Cell Signaling Technology, #14865).

Techniques: Knockdown, Infection, Transfection, Virus, Plaque Assay, Quantitative RT-PCR, Derivative Assay, Inhibition, Ubiquitin Proteomics, Expressing, Binding Assay

Journal: Nature Communications

Article Title: SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

doi: 10.1038/s41467-021-24007-w

Figure Lengend Snippet: a , b Analysis and regulation of significantly altered pathways of mock- and SARS-CoV-2-infected (24 h p.i., MOI = 0.1) VeroFM cells ( a ) or Calu-3 cells ( b ). The y -axis shows the (median) log2 fold change (FC) of all significantly altered metabolites of the indicated pathway while the –log10 corrected p -value (false discovery rate (FDR)) is shown on the x -axis. The size of the circles illustrates the number of significantly changed metabolites in relation to all metabolites of a specific pathway. c Analysis of the autophagic pathway and the involved metabolites: ‘amino acids’ and ‘GSH metabolism’ (orange), ‘nucleotides’ (blue), ‘glycolysis’ (green), ‘polyamine metabolism’ (red) and ‘O-GalNAcylation’ (purple) in mock- and SARS-CoV-2-infected (24 h p.i.) VeroFM and Calu-3 cells. For a – c Error bars represent SEM. n = 4 biological samples per group of one experiment. All p-values were determined by a two-way ANOVA and Tukey´s post hoc test. FDRs were adjusted using the Benjamini-Hochberg method. Abbreviations: 1,3-BPG, 1,3-bisphosphoglyceric acid; 3-PGA, 3-phosphoglyceric acid; ADP, adenosine diphosphate; AKT1, RAC-alpha serine/threonine-protein kinase; ala, alanine; AMP, adenosine monophosphate; AMPK, AMP-activated protein kinase; arg, arginine; asn, asparagine; asp, asparagine; ATP, adenosine triphosphate; aut, autophagosome; BECN1, beclin-1; bio., biosynthesis; CTP, cytidine triphosphate; CoA, coenzyme A; cys, cysteine; cysgly, cysteinylglycine; cyst, cystathionine; dCTP, deoxycytidine triphosphate; dicarbox., dicarboxylate; elF5AH, eukaryotic translation initiation factor 5A hypusinated; EP300, histone acetyltransferase p300; F1P, fructose 1-phosphate; F6P, fructose 6-phosphate; F-1,6-BP, fructose 1,6-bisphosphate; FKBP51, 51 kDa FK506-binding protein; G6-P, glucose 6-phosphate; gln, glutamine; glu, glutamic acid; gly, glycine; glyox, glyoxylate; GSH, glutathione (reduced); GTP, guanosine triphosphate; hcys, homocysteine; his, histidine; ile, isoleucine; lac, lactic acid; leu, leucine; lys, lysine; mal, malic acid; met, methionine; met., metabolism; modific., modification; mTORC1, mechanistic target of rapamycin complex 1; NAcput, N-acetylputrescine; NAcspd, N-acetylspermidine; orn, ornithine; PEP, phosphoenolpyruvic acid; phe, phenylalanine; PHLPP, PH domain leucine-rich repeat-containing protein phosphatase; PPP, pentose phosphate pathway; pro, proline; prot., protein; put, putrescine; pyr, pyruvic acid; resid., residue; ROS, reactive oxygen species; S7P, sedoheptulose-7-phosphate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SAT1, diamine acetyltransferase 1; SKP2, S-phase kinase-associated protein 2; ser, serine; spd, spermidine; spm, spermine; tau, taurine; TFEB, transcription factor EB; thr, threonine; trp, tryptophan; tyr, tyrosine; UDP-GalNAc, UDP-N-acetylgalactosamine; UDP, uridine diphosphate; UTP, uridine triphosphate; val, valine.

Article Snippet: The following primary antibodies were used: β-actin (1:5,000 Cell Signaling Technology, #8457), SQSTM1/p62 (1:1,000, Cell Signaling Technology, #5114), LC3B (1:1,000, Cell Signaling Technology, #3868), BECN1 (1:1,000, Cell Signaling Technology, #3738), pBECN1 S15 (1:1,000, Cell Signaling Technology, #84966), ATG14 (1:1,000, Cell Signaling Technology, #5504), pATG14 S29 (1:1,000, Cell Signaling Technology, #13155), ULK1 (1:1,000, Cell Signaling Technology, #8054), pULK1 (S555) (1:1,000, Cell Signaling Technology, #5869), pULK1 (S757) (1:1,000, Cell Signaling Technology, #6888), TSC2 (1:1,000, Cell Signaling Technology, #3612), pTSC2 (S1387) (1:1,000, Cell Signaling Technology, #5584), AMPKα (1:1,000, Cell Signaling Technology, #2532), pAMPK (T172) (1:1,000, Cell Signaling Technology, #2531), pAMPK substrate motif (1:1,000, Cell Signaling Technology, #5759), pAKT (S473) (1:1,000, Cell Signaling Technology, #4060), AKT (1:1,000, Cell Signaling Technology, #9272), HSC70 (1:5,000, Enzo Life Sciences, ADI-SPA-757-F), SKP2 (1:1,000, Cell Signaling Technology, #2652), pSKP2 (S64) (1:1,000, Cell Signaling Technology, #14865).

Techniques: Infection, Binding Assay, Modification, Residue

Journal: Nature Communications

Article Title: SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

doi: 10.1038/s41467-021-24007-w

Figure Lengend Snippet: a Protein levels and phosphorylation status of selected autophagy-relevant proteins in SARS-CoV-2-infected VeroFM cells at 8 h, 24 h, or 48 h post infection (h p.i.) were analyzed by Western blotting. For analysis of ATG14 oligomers (virtual blot, bottom panel, left) proteins were cross-linked 2 h prior to cell harvest and analyzed by Wes (ProteinSimple) capillary electrophoresis 8-48 h p.i. P -values were determined by one-way ANOVA, Bonferroni post hoc test. b Fluorescence microscopy of transfected and SARS-CoV-2-infected VeroFM cells expressing pH-sensitive tandem fluorescent-tagged LC3B - mRFP/EGFP showed that low pH autophagolysosomes (AL, red) were reduced compared to autophagosomes (AP, green + red = yellow) in virus-infected cells. Microscopic read-out was done by a scientist blind to the experimental conditions. For mock ( n = 44 cells) and SARS-CoV-2-infected ( n = 46 cells) VeroFM cells were analyzed. P-values were determined by two-way ANOVA, Tukey´s post hoc test, mean with SEM. Scale bar = 10 µM. c Accumulation of autophagy marker P62 in SARS-CoV-2-infected VeroFM cells. d Elevated LC3B-II levels in SARS-CoV-2-infected and bafilomycin A1 (BafA1)-pretreated VeroFM cells indicate virus-induced autophagic flux inhibition at 8 and 24 h p.i. In all panels, error bars denote SEM derived from n = 3 biologically independent samples derived from one experiment. P-values were determined by two-way ANOVA, Sidak post hoc test. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p > 0.05 (not significant, ns). Abbreviations: AKT1, RAC-alpha serine/threonine-protein kinase; AMPK, AMP-activated protein kinase; ATG14, autophagy-related 14; ATG16L1, autophagy-related 16 like 1; BafA1, bafilomycin A1; BECN1, Beclin-1 protein; CDK2, cyclin-dependent kinase 2; EGFP, enhanced green fluorescent protein; LC3B, microtubule-associated protein 1 A/1B light chain 3B; LXRXX(pS/pT), phospho-AMPK substrate motif; mRFP, monomeric red fluorescent protein; mTORC1/2, mechanistic target of rapamycin complex 1/2; PRAS40, proline-rich AKT1 substrate 1; Rheb, Ras homolog enriched in brain; SKP2, S-phase kinase-associated protein 2; TSC1/2, tuberous sclerosis 1/2; ULK1, Unc-51-like kinase 1; VPS34, phosphatidylinositol 3-kinase catalytic subunit type 3.

Article Snippet: The following primary antibodies were used: β-actin (1:5,000 Cell Signaling Technology, #8457), SQSTM1/p62 (1:1,000, Cell Signaling Technology, #5114), LC3B (1:1,000, Cell Signaling Technology, #3868), BECN1 (1:1,000, Cell Signaling Technology, #3738), pBECN1 S15 (1:1,000, Cell Signaling Technology, #84966), ATG14 (1:1,000, Cell Signaling Technology, #5504), pATG14 S29 (1:1,000, Cell Signaling Technology, #13155), ULK1 (1:1,000, Cell Signaling Technology, #8054), pULK1 (S555) (1:1,000, Cell Signaling Technology, #5869), pULK1 (S757) (1:1,000, Cell Signaling Technology, #6888), TSC2 (1:1,000, Cell Signaling Technology, #3612), pTSC2 (S1387) (1:1,000, Cell Signaling Technology, #5584), AMPKα (1:1,000, Cell Signaling Technology, #2532), pAMPK (T172) (1:1,000, Cell Signaling Technology, #2531), pAMPK substrate motif (1:1,000, Cell Signaling Technology, #5759), pAKT (S473) (1:1,000, Cell Signaling Technology, #4060), AKT (1:1,000, Cell Signaling Technology, #9272), HSC70 (1:5,000, Enzo Life Sciences, ADI-SPA-757-F), SKP2 (1:1,000, Cell Signaling Technology, #2652), pSKP2 (S64) (1:1,000, Cell Signaling Technology, #14865).

Techniques: Phospho-proteomics, Infection, Western Blot, Electrophoresis, Fluorescence, Microscopy, Transfection, Expressing, Virus, Marker, Inhibition, Derivative Assay

Journal: Nature Communications

Article Title: SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals

doi: 10.1038/s41467-021-24007-w

Figure Lengend Snippet: Schematic representation of autophagy signaling indicating site of action of small-molecule compounds (orange columns) or siRNA knockdown (green columns) used for pathway modulation. VeroFM cells were infected with SARS-CoV-2 (MOI = 0.0005) and treated with the indicated compounds: polyamines spermidine (spd, 100 µM) and spermine (spm, 100 µM), AMPK modulator AICAR (25 µM), mTORC1 inhibitor rapamycin (rap, 300 nM), VPS34 and ULK1 inhibitors SAR405 (1 µM) and MRT68921 (5 µM), AKT1 inhibitor MK-2206 (1 µM), BECN1-stabilizers SMIP004 (S004, 10 µM), valinomycin (val, 5 µM), niclosamide (nic, 10 µM) or DMSO (vehicle, dashed lines). For siRNA knockdown, VeroFM cells were transfected with 60 nM siRNA targeting ATG5, ATG7, FIP200, BECN1 , and infected with SARS-CoV-2 48 h later. SARS-CoV-2 infectious virus particle units (PFU) and genome equivalents (GE) per ml were determined by plaque assay and real-time RT-PCR at 24 h p.i., respectively. Data are presented as fold differences (see Supplementary Fig. for unprocessed data). In all panels, error bars =SEM derived from n = 3 ( n = 2 for spd, spm, AICAR) biologically independent samples from one experiment. Statistics were done for experiments with n = 3 using a two-way ANOVA, Dunnett’s, and one-way ANOVA for siRNA knockdown experiments (green). Compounds with significant SARS-CoV-2 inhibition (spm, spd, MK-2206, S004, val, nic) in VeroFM cells were confirmed in Calu-3 cells (see Supplementary Fig. f–g). p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****), p > 0.05 (not significant, ns). Abbreviations: AICAR, 5-Aminoimidazole-4-carboxamide ribonucleotide; AKT1, RAC-alpha serine/threonine-protein kinase; AMPK, AMP-activated protein kinase; ATG5, autophagy-related 5; ATG7, ubiquitin-like modifier-activating enzyme ATG7; ATG12, ubiquitin-like protein ATG12; aut, autophagosome; BECN1, Beclin-1 protein; CLEAR, coordinated lysosomal expression and regulation; cmpd, compound; EP300, histone acetyltransferase p300; elF5AH, eukaryotic translation initiation factor 5A, hypusinated; FIP200, FAK family kinase-interacting protein of 200 kDa; FKBP51, 51 kDa FK506-binding protein; GE, genome equivalents; lys, lysosome; mTORC1, mechanistic target of rapamycin complex 1; nic, niclosamide; PFU, plaque-forming units; phag, phagophore; PHLPP, PH domain leucine-rich repeat-containing protein phosphatase; rap, rapamycin; S004, SMIP-004; SKP2, S-phase kinase-associated protein 2; spd, spermidine; spm, spermine; TFEB, transcription factor EB; ULK1, unc-51-like kinase 1; val, valinomycin; VPS34, phosphatidylinositol 3-kinase catalytic subunit type 3.

Article Snippet: The following primary antibodies were used: β-actin (1:5,000 Cell Signaling Technology, #8457), SQSTM1/p62 (1:1,000, Cell Signaling Technology, #5114), LC3B (1:1,000, Cell Signaling Technology, #3868), BECN1 (1:1,000, Cell Signaling Technology, #3738), pBECN1 S15 (1:1,000, Cell Signaling Technology, #84966), ATG14 (1:1,000, Cell Signaling Technology, #5504), pATG14 S29 (1:1,000, Cell Signaling Technology, #13155), ULK1 (1:1,000, Cell Signaling Technology, #8054), pULK1 (S555) (1:1,000, Cell Signaling Technology, #5869), pULK1 (S757) (1:1,000, Cell Signaling Technology, #6888), TSC2 (1:1,000, Cell Signaling Technology, #3612), pTSC2 (S1387) (1:1,000, Cell Signaling Technology, #5584), AMPKα (1:1,000, Cell Signaling Technology, #2532), pAMPK (T172) (1:1,000, Cell Signaling Technology, #2531), pAMPK substrate motif (1:1,000, Cell Signaling Technology, #5759), pAKT (S473) (1:1,000, Cell Signaling Technology, #4060), AKT (1:1,000, Cell Signaling Technology, #9272), HSC70 (1:5,000, Enzo Life Sciences, ADI-SPA-757-F), SKP2 (1:1,000, Cell Signaling Technology, #2652), pSKP2 (S64) (1:1,000, Cell Signaling Technology, #14865).

Techniques: Knockdown, Infection, Transfection, Virus, Plaque Assay, Quantitative RT-PCR, Derivative Assay, Inhibition, Ubiquitin Proteomics, Expressing, Binding Assay