Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 4: FAIM deficiency suppressed the effects of PACAP on alleviating hepatic lipid accumulation in HFD-fed mice. (A) FAIM protein level of mice liver was subjected to western blot. Statistical analysis of FAIM/b-actin was shown in a bar chart (right) (*P < 0.05, **P < 0.01, ***P < 0.001 vs. CD group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HFD group). (B) Schematic diagram of HFD mouse model with PACAP treatment (0.4 mg/kg) or control saline solution and lentivirus mediated Faim knockdown. (C) Hepatic Faim mRNA level was determined by qRT-PCR, n ¼ 3. (DeH) Modulation of Faim level in the liver influenced HFD-induced hepatic steatosis, (D) bodyweight, liver weight, and WAT weight; (E) liver TG and TC contents; (F) plasma TG, TC, HDL-c, and LDL-c levels; (G) fasting blood glucose level; blood glucose concentrations were measured on day 12 after lentivirus injection; (H) representative photomicrographs of Oil Red O and H&E staining of liver tissues in different groups (scale bars: 50 or 200 mM). (I and J) Ablation of Faim impaired glucose tolerance and insulin sensitivity in mice. IPGTT (I) and IPITT (J), blood glucose was measured at different time points after glucose or insulin injection (left), and quantification of the AUC (right). Data are presented as mean SEM, n ¼ 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Western Blot, Control, Saline, Knockdown, Quantitative RT-PCR, Clinical Proteomics, Injection, Staining

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 5: PACAP activates the FAIM-AMPK-IRb axis to govern the SREBP and lipid synthetic gene program. (A and B) PACAP activated AMPK-IRb signaling pathway and down- regulated the expression of lipogenesis genes in HFD-fed mice, such effects were suppressed following knockdown of Faim. Western blot analysis were used to detect the protein level of indicated antibodies. (C and D) The overexpression of FAIM activated AMPK-IRb signaling pathway, the protein levels of FAIM, p-AMPK, AMPK, p-IRb, IRb, and lipid synthesis genes SREBP1, SREBP2, FAS, SCD1, HMGCR were determined by western blot with quantifications. Data are presented as mean SEM, n ¼ 3 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Knockdown, Western Blot, Over Expression

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 6: Knockdown of Faim inhibited the effects of PACAP on decreasing lipid accumulation and activating AMPK-IRb signaling pathway. AML12 cells were transiently transfected with Faim siRNA or scramble siRNA in cell culture medium containing 200 mM PA or 10% BSA (as control of PA solution) for 24 h, then the cells were treated with PACAP (1 mM) and Max.D.4 (10 mM) for another 24 h as indicated. (A) Representative for Oil Red O staining of AML12 (Scale bars, 50 mM). (B and C) Impacts of Faim knockdown on cellular TG and TC level (B), glucose uptake and glycogen content (C) of AML12. (D and E) Faim knockdown impaired the activation of AMPK-IRb pathway in AML12 treated with PACAP, the protein levels were analyzed by western blot and quantitative measurements (right). (F and G) AML12 were exposed to PA for 24 h, then treated with PACAP (1 mM), Max.D.4 (10 mM) and Compound C (10 mM) (F) or AICAR (0.5 mM) (G) for another 24 h. The protein level of FAIM was detected by western blot analysis and quantitative measurements (down). Data are presented as mean SEM. n ¼ 3 (&P < 0.05, &&P < 0.01, &&&P < 0.001 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PA þ PACAP group, OP < 0.05, OOP < 0.01, OOOP < 0.001 vs. PA þ PACAP þ Max.D.4 group).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Knockdown, Transfection, Cell Culture, Control, Staining, Activation Assay, Western Blot

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 7: PACAP activates the PAC1-PKA-CREB signaling pathway to stimulate FAIM expression. (A) mRNA level of Faim in PA-induced AML12 treated with PACAP, determined by qRT-PCR (n ¼ 3). (B) The effect of PACAP on the activation of PKA and CREB in PA induced AML12 cells, the protein levels of PKA, CREB and FAIM, and phosphorylation of PKA and CREB were determined by western blot. (*P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PACAP (1 mM) group). (C) Schematic of the sequence of two CRE binding sites that CREB targets the WT or mutated promoter region of Faim mRNA. (DeF) Luciferase activities in AML12 transiently transfected with either wild type (WT), truncated, or mutant Faim promoter-luciferase reporter constructs. Then the cells were treated with PACAP (1 mM), or co- transfected with CREB cDNA or empty vectors. (G) Luciferase activities of FAIM WT reporter plasmids in AML12 cells with the treatment of KG-501 (a specific CREB inhibitor) and PACAP (1 mM) as indicated. Data are shown as mean SEM, n ¼ 3 or 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Quantitative RT-PCR, Activation Assay, Phospho-proteomics, Western Blot, Sequencing, Binding Assay, Luciferase, Transfection, Mutagenesis, Construct

Journal: International Journal of Molecular Sciences

Article Title: miR-132-3p Modulates DUSP9-Dependent p38/JNK Signaling Pathways to Enhance Inflammation in the Amnion Leading to Labor

doi: 10.3390/ijms23031864

Figure Lengend Snippet: The role of p38 and JNK in DUSP9 siRNA-induced expression of proinflammatory cytokines and COX2 as well as PGE2 in WISH cells. WISH cells were transfected with si- DUSP9 or NC for 24 h, followed by 24 h of treatment with DMSO, p38 inhibitor SB203580 (10 μM), or JNK inhibitor SP600125 (20 μM). RT-qPCR analysis of the expression of IL-1β , TNF-α , COX2 ( A , G ), IL-6 , and IL-8 ( B , H ). Magnetic Luminex Assays of the secretion of IL-1β, TNF-α ( C , I ), IL-6, and IL-8 ( D , J ). Western blot analysis of COX2 expression ( E , K ). ELISA analysis of PGE2 level ( F , L ). Data were shown as mean ± SEM, n = 3. * p < 0.05, ** p < 0.01 vs. control (0), # p < 0.05, ## p < 0.01 vs. si- DUSP9 .

Article Snippet: To verify the involvement of p38 and JNK signaling pathways in the induction of IL-1β, IL-6, IL-8, TNF-α, and COX2 as well as PGE2 secretion by DUSP9 siRNA, the cells were transfected with NC or si- DUSP9 for 24 h, followed by 24 h of treatment with dimethyl sulfoxide (DMSO), p38 inhibitor SB203580 (10 μM, Selleck, Houston, TX, USA), or JNK inhibitor SP600125 (20 μM, Selleck, Houston, TX, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Luminex, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: NRVMs were transduced with AdV5-CMV-CRYAB R120G -GFP and treated with 3 µM ruxolitinib or DMSO or 100 nM siRNAs targeting either JAK1 (siJak1), JAK2 (siJak2), STAT3 (siStat3) or scramble siRNA (scr). Thereafter, NRVMs were harvested or fixed after 4-6 days. A , Treatment with ruxolitinib or DMSO; Scale bar = 100 µm. B , Transfection with siJak1; Scale bar = 200 µm. C , Transfection with siJak2; Scale bar = 100 µm D , Transfection with siStat3, Scale bar = 20 µm. A , B , C , D , Western blots of protein extracts from treated NRVMs were stained with antibodies directed against indicated proteins. In the representative immunofluorescence images, aggregates are depicted in magenta (CRYAB R120G -GFP), cardiomyocytes in yellow (anti-cardiac troponin I), and nuclei in blue (DAPI). Quantification of aggregates in cardiomyocytes with NIS Elements or ImageJ software. Data were obtained from 1 (Western blot) or 2 (immunofluorescence) NRVM preparations with at least 3 wells per condition and at least 7 images per well for immunofluorescence. Data are depicted as mean ± SEM, and p-values were obtained with the unpaired Student’s t-test. Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transduction, Transfection, Western Blot, Staining, Immunofluorescence, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: A , Heart weight-to-body weight ratio (HW/BW) in 1-, 4- and 7-month-old CRYAB p.Arg120Gly transgenic (R120G) and non-transgenic (NTG) mice. B , Ejection fraction (EF) in 7-month-old R120G and NTG mice. C , Representative Western blots of phosphorylated (P-)STAT3, STAT3 and indicated control in 1-, 4- and 7-month-old R120G and NTG mice. D , P-STAT3/STAT3 quantification of Western blots from 1-, 4- and 7-month-old R120G and NTG mice. E , Stat3 mRNA level determined by RT-qPCR from 7-month-old R120G and NTG mice. Western blot quantification was performed with Image Lab software. Data are depicted as mean ± SEM, and p-values were obtained with the unpaired Student’s t-test. Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transgenic Assay, Western Blot, Control, Quantitative RT-PCR, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: CRYAB p.Arg120Gly transgenic (R120G) and non-transgenic (NTG) mice treated for 3 weeks with 75 mg/kg ruxolitinib (ruxo) or vehicle (veh) twice-daily oral gavage. Transthoracic echocardiography was performed at the start (before, 21-week-old) and the end of the treatment (veh or ruxo, 24-week-old). A , Scheme of experimental outline. B , WB and quantification of phosphorylated (P-) STAT3, STAT3 and indicated controls at the end of treatment. WB quantification was performed with Image Lab software. C, Ejection fraction (EF) and left ventricular mass-to-body weight ratio (LVM/BW) before and after (veh/ruxo) treatment. D , Heart weight-to-body weight ratio (HW/BW) and body weight (BW) at the end of the treatment. E , Representative images and quantification of R120G and NTG mouse heart sections after vehicle or ruxolitinib treatment. CRYAB is depicted in green, cardiomyocytes in purple (anti-cardiac troponin I) and nuclei in blue (DAPI). Scale bar = 10 µm. Quantification of aggregates in cardiomyocytes with NIS Elements software. At least 5 images of 3 mice per group were analyzed. Data are depicted as mean ± SEM, and p-values were obtained with the one- way ( B ) or two-way ANOVA and Tukey’s multiple comparisons post-hoc analysis ( C, D ) or unpaired Student’s t-test ( E ). Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transgenic Assay, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: Heterozygous (het) or homozygous (hom) Jak1 knockout (KO) was induced in MCM-transgenic (TG) mice crossed with CRYAB p.Arg120Gly TG (R120G) or non-transgenic (NTG) mice with tamoxifen chow. Transthoracic echocardiography was performed 26 weeks and hearts were extracted. A , Scheme of experimental outline. B , Jak1 mRNA levels determined by RT-qPCR. C , JAK1 protein levels determined by Western blot and normalized to ACTN2. Western blot quantification was performed with Image Lab software D, Ejection fraction (EF) and left ventricular mass-to-body weight ratio (LVM/BW). E , Heart weight-to-body weight ratio (HW/BW) and body weight (BW). F , Representative images and quantification of mouse heart sections. Aggregates are depicted in green, cardiomyocytes in purple (anti-cardiac troponin I) and nuclei in blue (DAPI). Scale bar = 50 µm. Quantification of aggregates in cardiomyocytes with NIS Elements software. At least 5 images of 3 mice per group were analyzed. Data are depicted as mean ± SEM, and p-values were obtained with the one-way ( B ) or two- way ANOVA ( D , E ) with Tukey’s multiple comparisons post-hoc analysis or unpaired Student’s t-test ( C , F ). Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Knock-Out, Transgenic Assay, Quantitative RT-PCR, Western Blot, Software

Journal: PLoS Pathogens

Article Title: The RNA helicase DDX5 promotes viral infection via regulating N 6 -methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity

doi: 10.1371/journal.ppat.1009530

Figure Lengend Snippet: (A, B): m6A methylation of transcripts was detected by m6A qRT-PCR in DDX5-knockdown or DDX5 overexpressed MEFs after VSV infection. MEFs were transfected with DDX5 siRNA (siNC) for 48hand infected with VSV for 6h (A), and MEFs were transfected with Myc-DDX5 expressed vector (DDX5) or Myc tag control vector (Con) for 24h and infected with VSV for 6h (B). After extracting total RNA, purifying mRNA, and removing ribosomal RNA, purified mRNA was fragmented and incubated with anti-rabbit m6A or anti-rabbit IgG-conjugated dynabeads for 4h. RNA was isolated from the solution with phenol-chloroform, and cDNA was subjected to qRT-PCR using GAPDH, TBK1, DHX58, IKKγ, and p65 primers. Results are presented relative to those obtained with NC or control groups, and the expression of all the indicated proteins was analyzed using western blotting. (C, D): The interaction between METTL3 and transcripts was detected through METTL3 RIP qRT-PCR in knockdown-DDX5 (C) or DDX5-expressing (D) MEFs after VSV infection. MEFs were transfected with DDX5 siRNA (siNC) for 48 h and infected with VSV for 6 h (C), and transfected with DDX5 expression plasmid (DDX5) or control vector (Con) for 24 h, infected with VSV for 6h, and subjected to METTL3 RIP qRT-PCR to detect GAPDH, TBK1, DHX58, IKKγ, and p65. Results are presented relative to those obtained with NC or control groups, and the expression of all the indicated proteins was analyzed using western blotting. (E, F): Nuclear transcript retention increased in DDX5-knockdown MEFs. MEFs were transfected with DDX5 siRNA (siNC), infected with VSV for 8h, and lysed to extract nuclear to cytoplasmic RNA fractions. Then, RNA was used to analyze m6A modified DHX58, IKKγ, and p65 mRNA by m6A qRT-PCR (E) with RNU6 and GAPDH as the nuclear and cytoplasmic controls, respectively. The quantitative distribution of m6A modified DHX58, IKKγ, and p65 mRNAs in DDX5-knockdown MEFs were detected by m6A qRT-PCR (F). (G, H): Nuclear transcript export was increased in DDX5-expressing MEFs. MEFs were transfected with DDX5 expression plasmid (control vector), infected with VSV for 8h, and lysed to extract nuclear or cytoplasmic RNA; then, RNA was used to analyze m6A modified DHX58, IKKγ, and p65 mRNA by m6A qRT-PCR (G), and the quantitative distribution of these mRNAs was detected by m6AqRT-PCR (H). (I, J) : Immunoblot analysis of DHX58, IKKγ, and p65 in DDX5-knockdownMEFs (I) or DDX5-expressing MEFs (J) after infection with VSV at 0, 4, and 6 h. All data are mean ± SEM of biologically independent samples. Data are representative of three independent experiments. ns, no significant difference. * p <0.05, ** p <0.01, and *** p <0.001 (Student’s t -test).

Article Snippet: Biotin-labeled RNA was detected and visualized according to the instructions of the chemiluminescent nuclei acid detection module (Thermo Fisher, 89880), the biotin-unlabeled RNA was acquired according to the biotin-labeled protein–RNA complex blotting, and mRNAs were purified with the Dynabeads mRNA Purification Kit (Invitrogen, 61006).

Techniques: Methylation, Quantitative RT-PCR, Infection, Transfection, Plasmid Preparation, Purification, Incubation, Isolation, Expressing, Western Blot, Modification

Journal: PLoS Pathogens

Article Title: The RNA helicase DDX5 promotes viral infection via regulating N 6 -methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity

doi: 10.1371/journal.ppat.1009530

Figure Lengend Snippet: (A): m6A methylation of transcripts was detected in DDX5 +/+ or DDX5 +/- primary mouse macrophages infected for 8 h with VSV (MOI = 10). After extracting total RNA and purifying mRNA, mRNA was used to perform m6A qRT-PCR by incubating with anti-rabbit m6A or anti-rabbit IgG-conjugated dynabeads for 4 h. RNA was isolated and subjected to qRT-PCR using GAPDH, TBK1, DHX58, IKKγ, and p65 primers. Results are presented relative to those obtained in the control group, and the expression of DDX5 was analyzed bywestern blotting. (B): Immunoblot analysis of DDX5, DHX58, p65, and IKKγ in lysates of DDX5 +/+ or DDX5 +/- mouse macrophages infected for 0, 4, and 8 h with VSV (MOI = 10). (C, D): ELISA of IFN-β (C) and IL-6 (D) in cell supernatants ofDDX5 +/+ or DDX5 +/- mouse macrophages infected for 0, 4, and 8 h with VSV (MOI = 10). (E, F): ELISA of IFN-β (E) and IL-6 (F) in serum after DDX5 +/+ or DDX5 +/- mice were intraperitoneally injected with PBS or VSV (5×10 8 plaque-forming units/g body weight) for 8h (n = 6). (G, H): ELISA of IFN-β (G) and IL-6 (H) in serum after DDX5 +/+ or DDX5 +/- mice were intraperitoneally injected with PBS or SeV (1×10 8 plaque-forming units/g body weight) for 8h (n = 6). (I, J): The TCID 50 dose of VSV (I) or SeV (J) was measured in lungs, liver, and spleen of DDX5 +/+ or DDX5 +/- mice. (K): Pathological lesions in lungs, liver, and spleen of DDX5 +/+ or DDX5 +/- mice observed by hematoxylin-eosin staining with intraperitoneal injection of PBS, VSV (5×10 8 plaque-forming units/g body weight) or SeV (1×10 8 plaque-forming units/g body weight) for 12h. Scale bars, 100 μm. All data are presented as mean ± SEM of biologically independent samples. n = number of biological replicates. Data are representative of three independent experiments. NS, no significant difference. ** p <0.01, *** p <0.001 (Student’s t -test).

Article Snippet: Biotin-labeled RNA was detected and visualized according to the instructions of the chemiluminescent nuclei acid detection module (Thermo Fisher, 89880), the biotin-unlabeled RNA was acquired according to the biotin-labeled protein–RNA complex blotting, and mRNAs were purified with the Dynabeads mRNA Purification Kit (Invitrogen, 61006).

Techniques: Methylation, Infection, Quantitative RT-PCR, Isolation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Staining

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 1. PRRC2B deficiency in oligodendroglia cells leads to developmental hypomyelination (A) Immunostaining of PRRC2B/PDGFRa/OLIG2 in the corpus callosum (CC) of mice with indicated genotypes. Scale bars, 25 mm. (B) Left: western blotting analysis of the protein levels of PRRC2B in the cerebral cortex from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P6. Right: quantification of PRRC2B protein levels. Values are shown as means ± SEMs, *p < 0.05, unpaired two-tailed Student’s t test (n = 3 mice). (C) Quantitative real-time PCR detected the mRNA levels of Prrc2b in the cerebral cortex of P6 mice. Values are means ± SEMs. ***p < 0.001, unpaired two-tailed Student’s t test (each group, n = 9 replicates from >3 mice). (D) Pictures of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice and brains at P15. Scale bars, 5 mm (top), 2 cm (bottom).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Immunostaining, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 2. PRRC2B promotes OL progenitor cell differentiation (A) Immunofluorescence of PDGFRa/OLIG2 in the CC of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P15 or P40. Scale bars, 30 mm. (B) Quantification of PDGFRa/OLIG2-positive OPCs in the CC of mice, with the indicated geno- types at P15 or P40. Images of brain slices from >3 mice were gained and analyzed. Values are shown as means ± SEMs, **p < 0.01, NS, not statistically significant, unpaired two-tailed Student’s t test (P15: f/f, n = 19, f/f; Olig2, n = 13; P40: f/f, n = 14, f/f; Olig2, n = 6). (C) Immunostainings of CC1/OLIG2 in the CC of Prrc2bf/f, and Prrc2bf/f; Olig2Cre+/ mice at P15 or P40. Scale bars, 30 mm. (D) Quantification of CC1/OLIG2-positive OLs in the CC of mice, with the indicated genotypes at P15 or P40. Images of brain slices from >3 mice were gained and analyzed. Values are shown as means ± SEMs, **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test (P15: f/f, n = 18, f/f; Olig2, n = 12; P40: f/f, n = 7, f/f; Olig2, n = 6). (E) Left: western blotting analysis of OPC devel- opment-related protein in the CC of mice at P15. Right: quantification of protein levels of OLIG2, MAG, MBP, and ALDH1L1. Values are shown as means ± SEMs, *p < 0.05, NS, not statistically significant, unpaired two-tailed Student’s t test (n = 3 mice). (F) Quantitative real-time PCR detection of the mRNA levels of OPC-related genes in the CC of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P15. Values were shown as means ± SEMs, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Stu- dent’s t test (n R 4 independent biological repli- cates for each genotype).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Cell Differentiation, Two Tailed Test, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 3. PRRC2B deficiency affects OL progenitor cell differentiation (A) Immunostainings of PRRC2B/PDGFRa/OLIG2 in OPCs isolated from brain tissues of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P4. Scale bars, 50 mm. (B) Western blotting analysis of the protein levels of PRRC2B in OPC-derived cells cultured in prolifera- tion medium for 4 days and quantification of PRRC2B protein levels. Values are shown as means ± SEMs, **p < 0.01, unpaired two-tailed Student’s t test (n = 4 mice). (C) Quantitative real-time PCR analysis of the mRNA levels of Prrc2b in OPCs isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice. Values are means ± SEMs, ***p < 0.001, unpaired two-tailed Student’s t test (n = 6 independent biological replicates for each group). (D) Western blotting analysis the protein levels of OLIG2, ALDH1L1, and MBP in OPC-derived cells cultured in differentiation medium for 4 days. (E) Quantification of OLIG2, MBP, and ALDH1L1 protein levels. Values are shown as means ± SEMs, *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test (n = 3 mice). (F) Quantitative real-time PCR detection of the mRNA levels of OL-related genes in OPCs-derived cells isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Cell Differentiation, Isolation, Western Blot, Derivative Assay, Cell Culture, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 4. PRRC2B is an m6A-specific bind- ing protein in the brain (A) Schematic diagram of RNA pull-down showing that the unmethylated oligo(A) and methylated oligo-m6A RNA were used to capture potential m6A binding proteins in vitro. (B) Western blotting displaying endogenous PRRC2B and YTHDF1 pulled down in the brains of P4 mice. (C) LC-MS/MS quantification of the m6A/A ratio in RNA isolated from OPCs of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice. Values are means ± SEMs. Un- paired two-tailed Student’s t test (n = 4 experi- ments for each group). (D) Left: HA-tag was added to the C terminus of Prrc2b in mice. Right: western blotting of the effi- ciency of HA-tag expression in whole brains of WT and Prrc2b-HA mice at P4. (E) Schematic diagram of the RIP assay for LC-MS/ MS and sequencing. (F) Quantification of the m6A/A ratio in mRNA iso- lated from the cerebral cortex of WT and Prrc2b- HA mice at P4 by RIP. Values are means ± SEMs of 3 independent experiments, *p < 0.05, unpaired two-tailed Student’s t test (n = 3 independent biological replicates). (G) Pie chart presenting the distribution of PRRC2B-binding peaks in the 30 UTR, 50 UTR, CDS, or noncoding regions. (H) Enrichment chart depicting the proportion of PRRC2B-binding peaks in the corresponding 4 regions. (I) Binding motif identified by HOMER with PRRC2B-binding peaks (p = 1e12).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Methylation, Binding Assay, In Vitro, Western Blot, Liquid Chromatography with Mass Spectroscopy, Isolation, Two Tailed Test, Expressing, Sequencing

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 5. PRRC2B regulates OL develop- ment in an m6A modification-dependent manner (A) Distribution of m6A peaks across the length of the mRNA (50 UTR, CDS, and 30 UTR). (B) Enriched motif identified by HOMER with m6A peaks in OPCs isolated from C57BL/6J mice at P4 (p = 1e122). (C) GO terms in the biological process category enriched in transcripts with downregulated expression levels in OPCs isolated from Prrc2bf/f

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Isolation, Expressing

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 6. Sox2 is the target gene of PRRC2B during OPC development (A) Primary OPCs isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P4 were exposed to ActD, and RNA was extracted at the indicated time points. Quantitative real-time PCR was per- formed to assess the half-life of Sox2 mRNA. The data are shown as means ± SEMs, *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test (T1/2(f/f) = 44.99 min; T1/2(f/f; Olig2) = 20.02 min). (B) Integrative Genomics Viewer (IGV) tracks showing the read distributions of RNA-seq (top, n = 3), PRRC2B RIP-seq (center, n = 2), and MeRIP-seq (bottom, n = 3) profiling of Sox2 gene, with significant peaks highlighted in yel- low. Dark green indicates normalized RNA-seq profiles of OPCs isolated from Prrc2b cKO mice at P4, and gray shows that of OPCs of Prrc2bf/f

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Isolation, Real-time Polymerase Chain Reaction, Two Tailed Test, RNA Sequencing

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 7) or HD-PBMCs ( n = 7). ( B ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 9) and with mogamulizumab (antiCCR4) in a dose-dependent manner for 72 hours. ( C ) The comparison of RGMA mRNA gene expression levels using DNA microarray among normal CD4 + T cells (HD CD4 + : n = 4), HAM patient–derived CD4 + T cells (HAM CD4 + : n = 4), ACs ( n = 2), and smoldering/chronic-type-ATL patient–derived ( n = 3) HTLV-1–infected CD4 + T cells (Non-HAM infected CD4 + T cells: n = 5), and acute-type-ATL patient–derived HTLV-1–infected CD4 + T cells (Acute ATL infected cells: n = 3). ( D ) The comparison of the expression levels of the genes associated with the inhibition of neuroregeneration ( OMG , MAG , RTN4 , and WNT5A ) between HD CD4 + ( n = 4) and HAM CD4 + T cells ( n = 4). ( E ) The enrichment levels of H3K27me3 –2916 bp upstream from the TSS of the RGMA gene locus in HD CD4 + ( n = 3), HAM CD4 + ( n = 4), and acute-ATL infected cells ( n = 4). Data are shown as mean ± SD. ** P < 0.01; *** P < 0.001 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test ( B , C , and E ). NF-L, neurofilament light chain.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Concentration Assay, Comparison, Gene Expression, Microarray, Derivative Assay, Infection, Expressing, Inhibition

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The validation of RGMA mRNA gene expression levels using qRT-PCR in HD CD4 + ( n = 6) and HAM CD4 + T cells ( n = 6). ( B ) Expression of RGMa protein in CD3 + CD4 + CCR4 + T cells from HAM-PBMCs. Representative dot plots of CCR4 and normal goat IgG (upper) or RGMa expression (bottom) in CD3 + CD4 + gated cells from HD-PBMCs (left) or HAM-PBMCs (right) cultured for 2 days. ( C ) Graph shows the percentage of RGMa protein–expressing cells in CCR4 – cells or CCR4 + cells in CD3 + CD4 + gated cells from HAM-PBMCs ( n = 8) cultured for 2 days, compared with the isotype control, normal goat IgG. ( D ) Graph shows the percentage of RGMa protein–expressing cells among CD3 + CD4 + CCR4 + gated cells from HD-PBMCs ( n = 5) or HAM-PBMCs ( n = 8) cultured for 2 days. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Control, Comparison

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) Tax (left), HBZ (middle), and RGMA (right) gene expression levels in cultured HAM-PBMCs ( n = 7) in a time-dependent manner. RPL19 was used as an internal control. ( B ) Tax-dependent RGMA mRNA gene induction in Jurkat cells, which were infected with lentivirus carrying the Tax gene. Top: Tax expression in the Jurkat cells was confirmed by Western blotting. β-Actin was measured as an internal control. Bottom: The induction levels of the RGMA gene were evaluated by qRT-PCR in a time-dependent manner ( n = 3). ( C ) Tax -dependent RGMA mRNA gene induction in JPX9 cells treated with 20 μM CdCl 2 in a time-dependent manner. Tax mRNA (upper) and RGMA mRNA (bottom) were measured by qRT-PCR ( n = 3). GAPDH was measured as an internal control. ( D ) Tax-dependent RGMa protein induction in JPX9 cells treated with 20 μM CdCl 2 for 3 days. Dot plots of Tax and normal goat IgG (upper) or RGMa expression (bottom) in JPX9 cells. JPX9(-), untreated JPX9 cells; 20 μM CdCl 2 JPX9, CdCl 2 -supplemented JPX9 cells. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test ( A ), 2-sided Student’s t test ( B ), or an unpaired t test ( C ). Experiments were performed in triplicate ( B and C ).

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Gene Expression, Cell Culture, Control, Infection, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: Identification of CD74 as a putative receptor for IBDV. (A) Affinity purification with mAb against the IBDV major capsid protein VP2 in DT40 cells, incubated with VP2 (or the empty vector as a negative control), followed by mass spectrometry analysis. Many host proteins were associated with VP2, including chicken CD74. (B and C) Interaction between VP2 and the CD74 extracellular domain detected via a coimmunoprecipitation (co-IP) assay. (B) Western blot (WB) analysis using an antibody against the HA tag showing the bands corresponding to VP2 in the Flag co-IP assay. (C) Western blot analysis using an antibody against the HA tag showing the bands corresponding to the CD74 extracellular domain in the Flag co-IP assay. (D) To determine the distribution of CD74 in organs, the heart, liver, spleen, lung, kidney, thymus, and bursa of Fabricius of uninfected SPF chickens were collected to measure CD74 transcription levels. The most abundant CD74 expression was found in the bursa. (E) To confirm the involvement of chicken CD74 in the IBDV infection process, SPF chickens were challenged by vvIBDV or PBS. Bursas were collected for RT-qPCR analysis of CD74 transcription levels. CD74 mRNAs were significantly induced by vvIBDV at 12 h p.i. (*, P < 0.05; **, P < 0.01). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Affinity Purification, Incubation, Plasmid Preparation, Negative Control, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Expressing, Infection, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 knockdown suppresses IBDV replication. The expression of the CD74 Ii-2 isoform was downregulated by siRNA interference or knockdown by shRNA in DT40 cells. The vvIBDV Gx strain at an MOI of 1 was added to the CD74 siRNA interference groups or the CD74 KD cell line. Infected cells were washed with PBS at 4 h p.i., and DT40 complete medium was then added. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and qPCR and at 72 h p.i. for ELD50 analysis. (A) CD74 expression levels in DT40 cells determined by Western blotting, showing that CD74 downregulation was effective. siSc., scrambled control siRNA. (B) Western blot assays showing IBDV VP2 expression declining at 24 to 48 h p.i. (C) qPCR analysis showing IBDV copy numbers dropping off at 48 h p.i. (6.34-fold decrease compared with the siRNA negative control; *, P < 0.05). (D) ELD50 assay showing the IBDV titer being downregulated at 48 h p.i. (14.0-fold decrease compared with the siRNA negative control; *, P < 0.05). (E) CD74 mRNA level in DT40 cells determined by qPCR, indicating that CD74 knockdown was effective. (F) IBDV VP2 protein expression was downregulated significantly at 24 to 72 h p.i. in the CD74 KD groups. (G) The IBDV copy number was downregulated at 24 to 72 h p.i. (103- to 104-fold decrease compared with the wild-type [WT] control; P < 0.05). (F) ELD50 assay showing the IBDV titer being downregulated at 72 h p.i. (4.42 × 104-fold decrease compared with wild-type cells; P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Knockdown, Expressing, shRNA, Infection, Western Blot, Control, Negative Control

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 overexpression promotes IBDV replication. CD74 Ii-2 isoform overexpression promotes IBDV infectivity. The expression of the CD74 Ii-2 isoform was upregulated by plasmid transfection in DT40 cells. Next, vvIBDV at an MOI of 1 was used to infected the cells as described in the text. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and at 48 h p.i. for qPCR analysis. (A) CD74 Ii-2 isoform overexpression remarkably promotes IBDV replication. The IBDV copy number was increased in the overexpression groups at 72 h p.i. compared with the nonoverexpression group (2.90-fold increase; *, P < 0.05). (B) IBDV VP2 protein expression is upregulated at 48 to 72 h p.i. in the overexpression group.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Over Expression, Infection, Expressing, Plasmid Preparation, Transfection, Western Blot

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 isoform Ii-2 confers to vvIBDV the ability to attach to a vvIBDV-nonpermissive cell line. (A) 293T cells (nonpermissive to vvIBDV) were transfected with a full-length chicken CD74 Ii-2 plasmid with an HA tag (or the empty vector as a negative control). Twenty-four hours after transfection, cells were incubated with vvIBDV at an MOI of 5 at 37°C for 36 h to investigate whether CD74 confers susceptibility to IBDV infection. (Top) Cells were processed for confocal analysis, using IBDV VP2 mAb and an HA tag antibody as the primary antibodies. No virus (green fluorescence) was detected in the empty vector control. (Bottom) In the CD74 Ii-2 overexpression group, CD74 (red fluorescence) accumulated on the cell membrane, colocalizing with the IBDV particles (green fluorescence). No virus was observed to enter CD74-overexpressing nonpermissive cells. CD74 could confer attachment ability but not susceptibility to IBDV infection. (B) 293T cells overexpressed HA-tagged chicken CD74 Ii-2 or the empty vector for 24 h and were then incubated with 200 μg SVPs at 4°C for 1 h. After washing with PBS 5 times, cells were processed for confocal analysis as described above. SVPs (green fluorescence) were bound to the membranes of CD74-overexpressing cells (red fluorescence) and colocalized with CD74, but no binding was observed for the empty vector group. (C and D) 293T and HeLa cells (both of which are nonpermissive to vvIBDV) were transfected with a eukaryotic expression plasmid of HA-tagged chicken CD74 Ii-2 (or the empty vector as a negative control) and maintained for 24 h under normal culture conditions. The cells were then incubated with vvIBDV at an MOI of 50 at 4°C for 1 h for the binding assay and washed with PBS 5 times to remove the unbound viruses. Cells were processed for confocal analysis using the same antibodies and procedure as the ones described above. No virus (green fluorescence) was found to bind or infect 293T (C) or HeLa (D) cells transfected with the empty vector. In contrast, IBDV (green fluorescence) colocalized with overexpressed chicken CD74 (red fluorescence) on the membrane of nonpermissive 293T (C) and HeLa (D) cells. (E and F) qPCR analysis indicating that CD74 overexpression promotes IBDV binding to 293T or HeLa cells compared with the empty vector transfection group (9.55-fold increase in 293T cells [E] and 5.07-fold increase in HeLa cells [F]; both P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Transfection, Plasmid Preparation, Negative Control, Incubation, Infection, Virus, Fluorescence, Control, Over Expression, Membrane, Binding Assay, Expressing