Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: RNAi-mediated depletion of Rab7 causes Vps26 dissociation from endosomes. (A) HeLa cells were treated twice at 24-h intervals with a control, inactive siRNA, or siRNAs to Rab7, Rab4 (targeting both the a and b isoforms), or Vps26. Cell extracts were analyzed by SDS-PAGE and immunoblotting with antibodies to Rab7, Rab4, Vps26, or actin (loading control) as indicated in the figure. (B–Q) HeLa cells treated as in A with control (B, C, F, G, J, K, N, and O) or Rab7 siRNA (D, E, H, I, L, M, P, and Q) were immunostained for Vps26 (B, D, F, H, J, L, N, and P) and AP-3 (C and E), AP-1 (G and I), SNX1 (K and M), and SNX2 (O and Q) using rabbit polyclonal antibody to Vps26 and mouse monoclonal antibodies to AP-3–δ, AP-1–γ1, SNX1, or SNX2 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG and Alexa Fluor 488–conjugated donkey anti–mouse IgG. Images were captured by epifluorescence microscopy. Bar, 10 μm.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, SDS Page, Western Blot, Bioprocessing, Epifluorescence Microscopy

Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: Specificity and rescue of Vps26 dissociation from membranes in RNAi-treated cells. HeLa cells were treated twice at 24-h intervals with an inactive siRNA (control; A) or siRNAs to both Rab4a and Rab4b (B) or to Rab7 (C–F). At 36 h after the second treatment with siRNA to Rab7, some cells were transfected with plasmids encoding RNAi-resistant (i.e., canine) GFP-Rab7 (C and E) or GFP (D and F). Examples of cells expressing GFP or canine GFP-Rab7 are indicated by arrows. The cellular distribution of Vps26 (A–D) was assessed by indirect immunofluorescent staining using rabbit polyclonal antibody to Vps26 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG. Images were captured by epifluorescence microscopy. Bar, 10 μm.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, Transfection, Expressing, Staining, Epifluorescence Microscopy

Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: Depletion of Rab7 blocks the transport of CI-MPR from endosomes to the TGN. HeLa cells were treated twice at 24-h intervals with an inactive siRNA (control; A, C, E, and G) or siRNA to Rab7 (B, D, F, and H–N). At 48 h after treatment, the steady-state distribution of CI-MPR (A–D) was assessed by indirect immunofluorescent staining of fixed cells using rabbit polyclonal antibody to cytosolic tail of CI-MPR followed by Alexa Fluor 488–conjugated donkey anti–rabbit IgG. C and D correspond to high magnification views of CI-MPR–positive structures from control or Rab7-depleted cells, respectively. Live control cells (E and G) or Rab7-depleted cells (F and H–N) were incubated with an antibody to the luminal domain of CI-MPR for 2 h at 37°C. Cells were washed, fixed, permeabilized, and stained with Alexa Fluor 488–conjugated donkey anti–mouse IgG to detect internalized antibody to CI-MPR (E–H, J, and M). G and H show high magnification views from control (E) or Rab7-depleted cells (F), respectively. Cells in I–N were additionally stained with rabbit polyclonal antibody to TfR (I–K) or giantin (L–N) followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG. Images in A–H were captured using an epifluorescence microscope, and images in I–N were captured with a confocal microscope. (K and N) For merged images, yellow indicates colocalization. Arrows in I–K indicate examples of foci where proteins colocalize. (O) Extracts of HeLa cells treated with siRNAs to the proteins indicated on top were analyzed by 4–20% acrylamide gradient SDS-PAGE and immunoblotting (IB) with antibodies to the proteins indicated on the right. Equal amounts of total protein were loaded. Bars: (A, B, E, and F) 15 μm; (C, D, G, and H) 1.5 μm; (I–N) 10 μm.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, Staining, Incubation, Microscopy, SDS Page, Western Blot

Journal: The Journal of Cell Biology

Article Title: Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

doi: 10.1083/jcb.200804048

Figure Lengend Snippet: Rab7 depletion impairs processing of cathepsin D. HeLa cells were treated twice at 24-h intervals with inactive siRNA (control; lane 1) or siRNAs to the proteins indicated on top. At 24 h after the second round of siRNA treatment, cells were rinsed with PBS and incubated in serum-free culture medium for 24 h. The medium was collected and precipitated with trichloroacetic acid, and the resulting pellets were dissolved in Laemmli sample buffer. Cell extracts and media samples were analyzed by 4–20% acrylamide gradient SDS-PAGE and immunoblotting with rabbit polyclonal antibody to cathepsin D. Equal amounts of total protein were loaded on each lane. Blots were also probed with antibody to actin as a loading control. The positions of molecular mass markers (in kilodaltons) and of the precursor (pCatD), intermediate (iCatD), and mature (mCatD) forms of cathepsin D are indicated.

Article Snippet: HeLa cells grown to 30% confluency were transfected twice at 24-h intervals with 80 pmol siRNA oligonucleotides (SMARTpool reagents to Vps26, Rab7, Rab4a, Rab4b, and glyceraldehyde 3-phosphate dehydrogenase [control]; Thermo Fisher Scientific) using Oligofectamine (Invitrogen).

Techniques: Control, Incubation, SDS Page, Western Blot

Journal: Molecular cell

Article Title: Structural basis for OAS2 regulation and its antiviral function.

doi: 10.1016/j.molcel.2025.05.001

Figure Lengend Snippet: Figure 1. Structure of human OAS2 reveals dimerization via a zinc-binding site (A) Domain organization of human OAS2. (B) Cryo-EM reconstruction of OAS2 in 3.3 A˚ resolution. Two monomers are depicted in blue and gray, respectively. (C) Atomic model of OAS2 dimer with close-up of Zn2+ coordination of DII C652 and H654 from each monomer (blue and gray). Inactive domain DI is shown in darker color, active domain DII in lighter color. Electron density is depicted as gray mesh. (D) Side view of OAS2 dimer. (E) Sequence logo of 210 species showing conservation of C652 and H654. (F) Mass photometer analysis of monomeric OAS2 C652S (green) compared with dimeric OAS2 wild type (blue). (G) In vitro chromogenic activity assay of 100 nM OAS2 wild type (blue) and C652S (green) with 82-bp-long dsRNA (100 nM) (mean ± SD of n = 3). See also Figures S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-OAS2 R&D Systems Cat#MAB1925-SP; RRID:AB_215616 anti-OAS2 Proteintech Cat#19279-1-AP, lot# 00113570; RRID:AB_10642832 anti-OAS1 Cell Signaling Cat#14498S; RRID:AB_2798498 anti-GM130 Thermo Fisher Scientific Cat#703794; RRID:AB_2848241 anti-GM130 Proteintech Cat#66662-1-Ig; RRID:AB_2882017 anti-calnexin R&D Systems Cat#NB100-1965SS; RRID:AB_10002123 anti-giantin R&D Systems Cat#NBP1-91937; RRID:AB_11024007 anti-TGN46 R&D Systems Cat#NBP1-49643SS; RRID:AB_10011761 anti-Golgi 58K Sigma-Aldrich Cat#G2404; RRID: AB_477002 anti-dsRNA J2 SCICONS Cat#1qq0010500; RRID: AB_2651015 anti-OAS3 Proteintech Cat#21915-1-AP RRID:AB_2876880 anti-GAPDH Santa cruz Cat#sc-47724; RRID:AB_627678 anti-FLAG-M2-HRP Sigma Aldrich Cat#A8592; RRID:AB_439702 anti-RNase L Santa cruz Cat#sc-74405; RRID:AB_2181661 anti-OAS1 Proteintech Cat#14955-1-AP; RRID:AB_2158292 anti-GFP Abcam Cat# ab290; RRID: AB_2313768 anti-mouse AF488 Invitrogen Cat#A11029; RRID: AB_2534088 anti-rabbit AF647 Thermo Fisher Cat#A31573; RRID: AB_2536183 anti-rabbit AF488 Cell Signaling Cat#4412S; RRID: AB_1904025 anti-mouse AF647 Thermo Fisher Cat#A31571; RRID: AB_162542 anti-rabbit AF488 Invitrogen Cat# A-11008; RRID: AB_143165 Anti-mouse AF594 Invitrogen Cat#A11032; RRID: AB_2534091 Bacterial and virus strains E. coli Rosetta Expression Systems N/A E. coli STBL3 Andreas Pichlmair N/A E. coli DH5alpha Andreas Pichlmair N/A E. coli DH10alphaMultiBac Expression Systems N/A BUNV wt Friedemann Weber N/A EMCV Andreas Pichlmair, Pichlmair et al.85 N/A HSV1(17+)Lox-GFP Beate Sodeik N/A LACV (rec wt) Friedemann Weber N/A THOV-ML- Andreas Pichlmair, Haas et al.86 N/A VACV-V300-GFP Joachim Bugert N/A VSV-AV3-GFP (Indiana) Andreas Pichlmair lab, Pichlmair et al.87 N/A YFV (YF-17D)-Venus Simon Rothenfußer, Santos-Peral et al.88 N/A YFV (YF-17D) Simon Rothenfußer, Santos-Peral et al.88 N/A ZIKV H/PF/2013 Andreas Pichlmair lab, Scaturro et al.89 N/A IAV SC35M PB2 2A GFP Martin Schwemmle N/A RVFV-GFP Friedemann Weber, Habjan et al.90 N/A HRV16 ATCC Cat# VR-283 HCoV-OC43 ATCC Cat# CR-1558TM NL63 Lia van der Hoek N/A (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 85, 1–18.e1–e13, June 5, 2025 e1

Techniques: Binding Assay, Cryo-EM Sample Prep, Sequencing, In Vitro, Activity Assay

Journal: Molecular cell

Article Title: Structural basis for OAS2 regulation and its antiviral function.

doi: 10.1016/j.molcel.2025.05.001

Figure Lengend Snippet: Figure 2. RNA ligand requirements for the activation of OAS2 differ from those of OAS1 and OAS3 (A) OAS protein activation by different RNA lengths. In vitro chromogenic activity assay of 200 nM OAS1 p46, OAS2 p71, and OAS3 p100, each with 22, 44, and 82 bp dsRNA (200 nM) (mean ± SD of n = 3). (B) In vitro chromogenic activity assay showing dose response of OAS1 p42, OAS1 p46, OAS2 p69, OAS2 p71, OAS2 C652S, and OAS3 with 82 bp dsRNA (5, 10, 25, 50, 150, 300, and 500 nM) (mean ± SD of n = 3). (C) In vitro assay as in (A) with 100 nM OAS2 wild type (blue) and OAS2 C652S (green, orange) with 23, 44, and 82 bp dsRNA (100 nM) (mean ± SD of n = 3). (D) OAS protein activity in HEK293T cells measured with 2′-5′OA biosensor 8 h after poly I:C transfection (mean ± SD of n = 3). Paired t test: ns p > 0.05, * p ≤0.05, ** p ≤0.01. (E) Sequence of preferred OAS1 motif. WWN9WG motif is colored in red. Repetitive sequence is colored in purple. (F) Sequence preference of OAS proteins. In vitro assay as in (A) with 200 nM protein and 44 bp dsRNA (200 nM) containing OAS1 motif or random sequence (mean ± SD of n = 3). See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-OAS2 R&D Systems Cat#MAB1925-SP; RRID:AB_215616 anti-OAS2 Proteintech Cat#19279-1-AP, lot# 00113570; RRID:AB_10642832 anti-OAS1 Cell Signaling Cat#14498S; RRID:AB_2798498 anti-GM130 Thermo Fisher Scientific Cat#703794; RRID:AB_2848241 anti-GM130 Proteintech Cat#66662-1-Ig; RRID:AB_2882017 anti-calnexin R&D Systems Cat#NB100-1965SS; RRID:AB_10002123 anti-giantin R&D Systems Cat#NBP1-91937; RRID:AB_11024007 anti-TGN46 R&D Systems Cat#NBP1-49643SS; RRID:AB_10011761 anti-Golgi 58K Sigma-Aldrich Cat#G2404; RRID: AB_477002 anti-dsRNA J2 SCICONS Cat#1qq0010500; RRID: AB_2651015 anti-OAS3 Proteintech Cat#21915-1-AP RRID:AB_2876880 anti-GAPDH Santa cruz Cat#sc-47724; RRID:AB_627678 anti-FLAG-M2-HRP Sigma Aldrich Cat#A8592; RRID:AB_439702 anti-RNase L Santa cruz Cat#sc-74405; RRID:AB_2181661 anti-OAS1 Proteintech Cat#14955-1-AP; RRID:AB_2158292 anti-GFP Abcam Cat# ab290; RRID: AB_2313768 anti-mouse AF488 Invitrogen Cat#A11029; RRID: AB_2534088 anti-rabbit AF647 Thermo Fisher Cat#A31573; RRID: AB_2536183 anti-rabbit AF488 Cell Signaling Cat#4412S; RRID: AB_1904025 anti-mouse AF647 Thermo Fisher Cat#A31571; RRID: AB_162542 anti-rabbit AF488 Invitrogen Cat# A-11008; RRID: AB_143165 Anti-mouse AF594 Invitrogen Cat#A11032; RRID: AB_2534091 Bacterial and virus strains E. coli Rosetta Expression Systems N/A E. coli STBL3 Andreas Pichlmair N/A E. coli DH5alpha Andreas Pichlmair N/A E. coli DH10alphaMultiBac Expression Systems N/A BUNV wt Friedemann Weber N/A EMCV Andreas Pichlmair, Pichlmair et al.85 N/A HSV1(17+)Lox-GFP Beate Sodeik N/A LACV (rec wt) Friedemann Weber N/A THOV-ML- Andreas Pichlmair, Haas et al.86 N/A VACV-V300-GFP Joachim Bugert N/A VSV-AV3-GFP (Indiana) Andreas Pichlmair lab, Pichlmair et al.87 N/A YFV (YF-17D)-Venus Simon Rothenfußer, Santos-Peral et al.88 N/A YFV (YF-17D) Simon Rothenfußer, Santos-Peral et al.88 N/A ZIKV H/PF/2013 Andreas Pichlmair lab, Scaturro et al.89 N/A IAV SC35M PB2 2A GFP Martin Schwemmle N/A RVFV-GFP Friedemann Weber, Habjan et al.90 N/A HRV16 ATCC Cat# VR-283 HCoV-OC43 ATCC Cat# CR-1558TM NL63 Lia van der Hoek N/A (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 85, 1–18.e1–e13, June 5, 2025 e1

Techniques: Activation Assay, In Vitro, Activity Assay, Transfection, Sequencing

Journal: Molecular cell

Article Title: Structural basis for OAS2 regulation and its antiviral function.

doi: 10.1016/j.molcel.2025.05.001

Figure Lengend Snippet: Figure 3. OAS2 DI functions as a regulatory domain that measures RNA length using a non-canonical interface (A) MD simulation of OAS2 dimer in complex with dsRNA (gray), colored by RMSD values using the cryo-EM structure as a reference. (B) Contact frequencies of DI with dsRNA, calculated based on cryo-EM + MD and AF3 + MD approaches. Residues tested in (I) are highlighted. (C) MD simulation of OAS2 monomer in complex with dsRNA (gray). Structure is colored as in (A). (D) AF3 prediction of OAS2 monomer in complex with dsRNA (gray). Structure is colored as in (A). (E) Superposition of OAS2 with dsRNA from cryo-EM + MD and AF3 + MD approaches in green and orange, respectively. (F) Electrostatic surface representation of monomeric OAS2 in complex with dsRNA from cryo-EM + MD (left) and AF3 prediction (right). Interfaces A and B are highlighted with yellow and pink dashed circles, respectively. (G) Close ups of interface B from the cryo-EM + MD (left) and AF3 predicted structure (right). (H) Surface view of structure depicted in (F), with color code indicating the effect of mutations from (I). (I) OAS2 protein activity in HEK293T cells measured with 2′-5′OA biosensor (mean ± SD of n = 3). Paired t test: ns p > 0.05, * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001. (J) Schematic overview of the RNA-binding mechanism of OAS2. The OAS2 dimer is auto-inhibited and, upon RNA binding, it monomerizes. The MD simulation illustrates an intermediate state, whereas the AF3 prediction more accurately represents the active state bound to RNA. RNA-binding interfaces A and B are colored in yellow and pink, respectively. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-OAS2 R&D Systems Cat#MAB1925-SP; RRID:AB_215616 anti-OAS2 Proteintech Cat#19279-1-AP, lot# 00113570; RRID:AB_10642832 anti-OAS1 Cell Signaling Cat#14498S; RRID:AB_2798498 anti-GM130 Thermo Fisher Scientific Cat#703794; RRID:AB_2848241 anti-GM130 Proteintech Cat#66662-1-Ig; RRID:AB_2882017 anti-calnexin R&D Systems Cat#NB100-1965SS; RRID:AB_10002123 anti-giantin R&D Systems Cat#NBP1-91937; RRID:AB_11024007 anti-TGN46 R&D Systems Cat#NBP1-49643SS; RRID:AB_10011761 anti-Golgi 58K Sigma-Aldrich Cat#G2404; RRID: AB_477002 anti-dsRNA J2 SCICONS Cat#1qq0010500; RRID: AB_2651015 anti-OAS3 Proteintech Cat#21915-1-AP RRID:AB_2876880 anti-GAPDH Santa cruz Cat#sc-47724; RRID:AB_627678 anti-FLAG-M2-HRP Sigma Aldrich Cat#A8592; RRID:AB_439702 anti-RNase L Santa cruz Cat#sc-74405; RRID:AB_2181661 anti-OAS1 Proteintech Cat#14955-1-AP; RRID:AB_2158292 anti-GFP Abcam Cat# ab290; RRID: AB_2313768 anti-mouse AF488 Invitrogen Cat#A11029; RRID: AB_2534088 anti-rabbit AF647 Thermo Fisher Cat#A31573; RRID: AB_2536183 anti-rabbit AF488 Cell Signaling Cat#4412S; RRID: AB_1904025 anti-mouse AF647 Thermo Fisher Cat#A31571; RRID: AB_162542 anti-rabbit AF488 Invitrogen Cat# A-11008; RRID: AB_143165 Anti-mouse AF594 Invitrogen Cat#A11032; RRID: AB_2534091 Bacterial and virus strains E. coli Rosetta Expression Systems N/A E. coli STBL3 Andreas Pichlmair N/A E. coli DH5alpha Andreas Pichlmair N/A E. coli DH10alphaMultiBac Expression Systems N/A BUNV wt Friedemann Weber N/A EMCV Andreas Pichlmair, Pichlmair et al.85 N/A HSV1(17+)Lox-GFP Beate Sodeik N/A LACV (rec wt) Friedemann Weber N/A THOV-ML- Andreas Pichlmair, Haas et al.86 N/A VACV-V300-GFP Joachim Bugert N/A VSV-AV3-GFP (Indiana) Andreas Pichlmair lab, Pichlmair et al.87 N/A YFV (YF-17D)-Venus Simon Rothenfußer, Santos-Peral et al.88 N/A YFV (YF-17D) Simon Rothenfußer, Santos-Peral et al.88 N/A ZIKV H/PF/2013 Andreas Pichlmair lab, Scaturro et al.89 N/A IAV SC35M PB2 2A GFP Martin Schwemmle N/A RVFV-GFP Friedemann Weber, Habjan et al.90 N/A HRV16 ATCC Cat# VR-283 HCoV-OC43 ATCC Cat# CR-1558TM NL63 Lia van der Hoek N/A (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 85, 1–18.e1–e13, June 5, 2025 e1

Techniques: Cryo-EM Sample Prep, Activity Assay, RNA Binding Assay

Journal: Molecular cell

Article Title: Structural basis for OAS2 regulation and its antiviral function.

doi: 10.1016/j.molcel.2025.05.001

Figure Lengend Snippet: Figure 4. OAS2 dimerization and localization to the Golgi membrane via myristoylation are required for activation (A) SDS-PAGE (top) and BN-PAGE (bottom) of OAS2 constructs transiently expressed in HEK293T cells and purified His-tagged OAS2 WT dimer. Zinc coor- dination is depicted on the right. SDS-PAGE was cut to remove a lane with marker. (B) OAS2 activity in HEK293T cells measured with 2′–5′OA biosensor 24 h after poly I:C transfection, showing higher activity of dimeric OAS2 than the monomeric mutants. (C) Immunofluorescence Airyscan microscopy of endogenous OAS2 localization in primary HFF cells stimulated with IFN-α and stained for OAS2 (green), Golgi marker GM130 (magenta), and DAPI (blue). Scale bars represent 20 μm and 2 μm for merge zoom. (D) As in (C) for A549 OAS2 KO cells reconstituted with doxycycline-inducible OAS2 constructs treated with doxycycline. Scale bars represent 10 μm. (E) Quantification of colocalization of OAS2 constructs and Golgi marker GM130 from (D). Lines represent means from analyzed individual cells (dots). Student’s t test with Welch’s correction, ***p ≤0.001. (F) Analysis of OAS2 activity in cells as in (B) for OAS2 wild type and mutants with different localization showing that Golgi targeting is essential for the activity. (G) Surface view of OAS2 dimer, with each monomer depicted in gray and blue. Close-up shows dimer interface interactions of S150 and D153. (H) SDS-PAGE and BN-PAGE analysis as in (A). (I) In vitro chromogenic activity assay with 200 nM OAS2 wild type and OAS2 S150A D153A with 23, 44, and 82 bp dsRNA (200 nM) (mean ± SD of n = 3). (J) Analysis of OAS2 activity in cells as in (B) for OAS2 wild type and DI-DII interaction mutant S150A D153A. In (B), (F), and (J) bars represent means ± SD of four (B), five (F), and six (J) independent replicates (dots). Paired t test: ns p > 0.05, * p ≤0.05, ** p ≤0.01, *** p ≤ 0.001, **** p ≤0.0001. See also Figures S5 and S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-OAS2 R&D Systems Cat#MAB1925-SP; RRID:AB_215616 anti-OAS2 Proteintech Cat#19279-1-AP, lot# 00113570; RRID:AB_10642832 anti-OAS1 Cell Signaling Cat#14498S; RRID:AB_2798498 anti-GM130 Thermo Fisher Scientific Cat#703794; RRID:AB_2848241 anti-GM130 Proteintech Cat#66662-1-Ig; RRID:AB_2882017 anti-calnexin R&D Systems Cat#NB100-1965SS; RRID:AB_10002123 anti-giantin R&D Systems Cat#NBP1-91937; RRID:AB_11024007 anti-TGN46 R&D Systems Cat#NBP1-49643SS; RRID:AB_10011761 anti-Golgi 58K Sigma-Aldrich Cat#G2404; RRID: AB_477002 anti-dsRNA J2 SCICONS Cat#1qq0010500; RRID: AB_2651015 anti-OAS3 Proteintech Cat#21915-1-AP RRID:AB_2876880 anti-GAPDH Santa cruz Cat#sc-47724; RRID:AB_627678 anti-FLAG-M2-HRP Sigma Aldrich Cat#A8592; RRID:AB_439702 anti-RNase L Santa cruz Cat#sc-74405; RRID:AB_2181661 anti-OAS1 Proteintech Cat#14955-1-AP; RRID:AB_2158292 anti-GFP Abcam Cat# ab290; RRID: AB_2313768 anti-mouse AF488 Invitrogen Cat#A11029; RRID: AB_2534088 anti-rabbit AF647 Thermo Fisher Cat#A31573; RRID: AB_2536183 anti-rabbit AF488 Cell Signaling Cat#4412S; RRID: AB_1904025 anti-mouse AF647 Thermo Fisher Cat#A31571; RRID: AB_162542 anti-rabbit AF488 Invitrogen Cat# A-11008; RRID: AB_143165 Anti-mouse AF594 Invitrogen Cat#A11032; RRID: AB_2534091 Bacterial and virus strains E. coli Rosetta Expression Systems N/A E. coli STBL3 Andreas Pichlmair N/A E. coli DH5alpha Andreas Pichlmair N/A E. coli DH10alphaMultiBac Expression Systems N/A BUNV wt Friedemann Weber N/A EMCV Andreas Pichlmair, Pichlmair et al.85 N/A HSV1(17+)Lox-GFP Beate Sodeik N/A LACV (rec wt) Friedemann Weber N/A THOV-ML- Andreas Pichlmair, Haas et al.86 N/A VACV-V300-GFP Joachim Bugert N/A VSV-AV3-GFP (Indiana) Andreas Pichlmair lab, Pichlmair et al.87 N/A YFV (YF-17D)-Venus Simon Rothenfußer, Santos-Peral et al.88 N/A YFV (YF-17D) Simon Rothenfußer, Santos-Peral et al.88 N/A ZIKV H/PF/2013 Andreas Pichlmair lab, Scaturro et al.89 N/A IAV SC35M PB2 2A GFP Martin Schwemmle N/A RVFV-GFP Friedemann Weber, Habjan et al.90 N/A HRV16 ATCC Cat# VR-283 HCoV-OC43 ATCC Cat# CR-1558TM NL63 Lia van der Hoek N/A (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 85, 1–18.e1–e13, June 5, 2025 e1

Techniques: Membrane, Activation Assay, SDS Page, Construct, Purification, Marker, Activity Assay, Transfection, Immunofluorescence, Microscopy, Staining, In Vitro, Mutagenesis

Journal: Molecular cell

Article Title: Structural basis for OAS2 regulation and its antiviral function.

doi: 10.1016/j.molcel.2025.05.001

Figure Lengend Snippet: Figure 5. OAS2 restricts viruses replicating at the endomembrane system (A) Experimental setup for virus screen (see STAR Methods). Cells were infected with a panel of GFP-reporter viruses (green) or non-reporter virus (black), followed by virus replication and cell death analysis. (B) Cell death in EMCV-infected (MOI 0.3) A549 OAS2 KO cells reconstituted with doxycycline-inducible OAS2 constructs (mean ± SD two technical replicates). Assays are representative of at least three independent experiments. (C) Immunofluorescence Airyscan microscopy of OAS2 and viral dsRNA during EMCV and YFV infection in A549 OAS2 KO cells reconstituted with doxycycline- inducible OAS2 constructs stained for OAS2 (green), dsRNA (magenta), and DAPI (blue). Scale bars represent 10 μm. (D) Quantification of OAS2 and dsRNA colocalization from (C). Lines represent means from analyzed individual cells (dots). Student’s t test with Welch’s correction, ***p ≤0.001. (E) RT-qPCR analysis of intracellular EMCV RNA levels in A549 OAS2 KO cells with KI for doxycycline-inducible OAS2 WT. Cells were treated with doxycycline or IFN-α followed by EMCV infection. (F) RT-qPCR analysis of HRV16 viral RNA levels in Hela H1 cell supernatants. Cells were transiently transfected with different OAS constructs and infected with HRV16. (G) RT-qPCR analysis of coronavirus RNA levels in culture supernatants in HEK293T cells overexpressing OAS2 WT. In (E), (F), and (G) bars represent means ± SD of at least three independent experiments (dots). Paired t test in (E) and (G) and unpaired t test in (F). * p ≤0.05, ** p ≤ 0.001, ***p ≤0.001, **** p ≤0.0001. See also Figures S7 and S8.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-OAS2 R&D Systems Cat#MAB1925-SP; RRID:AB_215616 anti-OAS2 Proteintech Cat#19279-1-AP, lot# 00113570; RRID:AB_10642832 anti-OAS1 Cell Signaling Cat#14498S; RRID:AB_2798498 anti-GM130 Thermo Fisher Scientific Cat#703794; RRID:AB_2848241 anti-GM130 Proteintech Cat#66662-1-Ig; RRID:AB_2882017 anti-calnexin R&D Systems Cat#NB100-1965SS; RRID:AB_10002123 anti-giantin R&D Systems Cat#NBP1-91937; RRID:AB_11024007 anti-TGN46 R&D Systems Cat#NBP1-49643SS; RRID:AB_10011761 anti-Golgi 58K Sigma-Aldrich Cat#G2404; RRID: AB_477002 anti-dsRNA J2 SCICONS Cat#1qq0010500; RRID: AB_2651015 anti-OAS3 Proteintech Cat#21915-1-AP RRID:AB_2876880 anti-GAPDH Santa cruz Cat#sc-47724; RRID:AB_627678 anti-FLAG-M2-HRP Sigma Aldrich Cat#A8592; RRID:AB_439702 anti-RNase L Santa cruz Cat#sc-74405; RRID:AB_2181661 anti-OAS1 Proteintech Cat#14955-1-AP; RRID:AB_2158292 anti-GFP Abcam Cat# ab290; RRID: AB_2313768 anti-mouse AF488 Invitrogen Cat#A11029; RRID: AB_2534088 anti-rabbit AF647 Thermo Fisher Cat#A31573; RRID: AB_2536183 anti-rabbit AF488 Cell Signaling Cat#4412S; RRID: AB_1904025 anti-mouse AF647 Thermo Fisher Cat#A31571; RRID: AB_162542 anti-rabbit AF488 Invitrogen Cat# A-11008; RRID: AB_143165 Anti-mouse AF594 Invitrogen Cat#A11032; RRID: AB_2534091 Bacterial and virus strains E. coli Rosetta Expression Systems N/A E. coli STBL3 Andreas Pichlmair N/A E. coli DH5alpha Andreas Pichlmair N/A E. coli DH10alphaMultiBac Expression Systems N/A BUNV wt Friedemann Weber N/A EMCV Andreas Pichlmair, Pichlmair et al.85 N/A HSV1(17+)Lox-GFP Beate Sodeik N/A LACV (rec wt) Friedemann Weber N/A THOV-ML- Andreas Pichlmair, Haas et al.86 N/A VACV-V300-GFP Joachim Bugert N/A VSV-AV3-GFP (Indiana) Andreas Pichlmair lab, Pichlmair et al.87 N/A YFV (YF-17D)-Venus Simon Rothenfußer, Santos-Peral et al.88 N/A YFV (YF-17D) Simon Rothenfußer, Santos-Peral et al.88 N/A ZIKV H/PF/2013 Andreas Pichlmair lab, Scaturro et al.89 N/A IAV SC35M PB2 2A GFP Martin Schwemmle N/A RVFV-GFP Friedemann Weber, Habjan et al.90 N/A HRV16 ATCC Cat# VR-283 HCoV-OC43 ATCC Cat# CR-1558TM NL63 Lia van der Hoek N/A (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 85, 1–18.e1–e13, June 5, 2025 e1

Techniques: Virus, Infection, Construct, Immunofluorescence, Microscopy, Staining, Quantitative RT-PCR, Transfection

Journal: Molecular cell

Article Title: Structural basis for OAS2 regulation and its antiviral function.

doi: 10.1016/j.molcel.2025.05.001

Figure Lengend Snippet: Figure 6. OAS2 loss of function is associated with immune dysregulation with ANCA vasculitis (A) Patient’s serum levels of inflammatory markers C-reactive protein (CRP; [<0.5 mg/dl] and soluble interleukin-2 receptor (sIL-2R; [158–613 U/ml]), and anti- bodies against myeloperoxidase (MPO; [<20 U/ml]). Gray bars indicate normal ranges. (B) Renal biopsy with hematoxylin and eosin stain, 20x magnification. (C) Pedigree of family with OAS2 F524L mutation. (D) Electropherograms showing de novo heterozygous OAS2 variant (c.1572C>G; p.Phe524Leu) by Sanger sequencing. (E) Multiple sequence alignment of human OAS proteins and selected human NTases showing high conservation for F524 (yellow star). (F) Structure of the OAS2 dimer with close-up view highlighting the amino acid F524. (G) Expression levels of OAS2 in wild-type (WT) and patient fibroblasts treated with IFN-α (n = 2). (H) OAS2 immunofluorescence staining in WT and patient fibroblasts treated with IFN-α. Scale bars represent 50 μm. (I) Quantification of OAS2 mean fluorescence intensity from (H). Lines represent means from measurements of individual cells (dots). Student’s t test with Welch’s correction, **** p ≤0.0001. (J) RNA pico-chip analysis of total RNA isolated from wild-type (WT) and patient-derived fibroblast cell lines from control or poly I:C transfection.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-OAS2 R&D Systems Cat#MAB1925-SP; RRID:AB_215616 anti-OAS2 Proteintech Cat#19279-1-AP, lot# 00113570; RRID:AB_10642832 anti-OAS1 Cell Signaling Cat#14498S; RRID:AB_2798498 anti-GM130 Thermo Fisher Scientific Cat#703794; RRID:AB_2848241 anti-GM130 Proteintech Cat#66662-1-Ig; RRID:AB_2882017 anti-calnexin R&D Systems Cat#NB100-1965SS; RRID:AB_10002123 anti-giantin R&D Systems Cat#NBP1-91937; RRID:AB_11024007 anti-TGN46 R&D Systems Cat#NBP1-49643SS; RRID:AB_10011761 anti-Golgi 58K Sigma-Aldrich Cat#G2404; RRID: AB_477002 anti-dsRNA J2 SCICONS Cat#1qq0010500; RRID: AB_2651015 anti-OAS3 Proteintech Cat#21915-1-AP RRID:AB_2876880 anti-GAPDH Santa cruz Cat#sc-47724; RRID:AB_627678 anti-FLAG-M2-HRP Sigma Aldrich Cat#A8592; RRID:AB_439702 anti-RNase L Santa cruz Cat#sc-74405; RRID:AB_2181661 anti-OAS1 Proteintech Cat#14955-1-AP; RRID:AB_2158292 anti-GFP Abcam Cat# ab290; RRID: AB_2313768 anti-mouse AF488 Invitrogen Cat#A11029; RRID: AB_2534088 anti-rabbit AF647 Thermo Fisher Cat#A31573; RRID: AB_2536183 anti-rabbit AF488 Cell Signaling Cat#4412S; RRID: AB_1904025 anti-mouse AF647 Thermo Fisher Cat#A31571; RRID: AB_162542 anti-rabbit AF488 Invitrogen Cat# A-11008; RRID: AB_143165 Anti-mouse AF594 Invitrogen Cat#A11032; RRID: AB_2534091 Bacterial and virus strains E. coli Rosetta Expression Systems N/A E. coli STBL3 Andreas Pichlmair N/A E. coli DH5alpha Andreas Pichlmair N/A E. coli DH10alphaMultiBac Expression Systems N/A BUNV wt Friedemann Weber N/A EMCV Andreas Pichlmair, Pichlmair et al.85 N/A HSV1(17+)Lox-GFP Beate Sodeik N/A LACV (rec wt) Friedemann Weber N/A THOV-ML- Andreas Pichlmair, Haas et al.86 N/A VACV-V300-GFP Joachim Bugert N/A VSV-AV3-GFP (Indiana) Andreas Pichlmair lab, Pichlmair et al.87 N/A YFV (YF-17D)-Venus Simon Rothenfußer, Santos-Peral et al.88 N/A YFV (YF-17D) Simon Rothenfußer, Santos-Peral et al.88 N/A ZIKV H/PF/2013 Andreas Pichlmair lab, Scaturro et al.89 N/A IAV SC35M PB2 2A GFP Martin Schwemmle N/A RVFV-GFP Friedemann Weber, Habjan et al.90 N/A HRV16 ATCC Cat# VR-283 HCoV-OC43 ATCC Cat# CR-1558TM NL63 Lia van der Hoek N/A (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 85, 1–18.e1–e13, June 5, 2025 e1

Techniques: H&E Stain, Mutagenesis, Variant Assay, Sequencing, Expressing, Immunofluorescence, Staining, Fluorescence, Isolation, Derivative Assay, Control, Transfection