Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: IFI16 partners with KAP1 to silence the EBV lytic state. (A and B) HH514-16 BL cells were transfected with control scrambled siRNA (Sc) or siRNA targeting IFI16, exposed to NaB (sodium butyrate; lytic trigger) after 18 h, harvested after another 24 h, and immunoblotted with indicated antibodies (A) or subjected to RT-qPCR analysis of EBV lytic genes of all kinetic classes (BZLF1, immediate early; BMRF1, early; and BFRF3, late) using the ΔΔCT method after normalization to 18S rRNA (B). Error bars, SEM of three technical replicates and two biological replicates; **, P < 0.01. (C) Mutu I BL cells were transfected with control scrambled siRNA (Sc) or siRNA targeting IFI16, exposed to NaB (left) or TGF-β1 (middle) after 18 h, harvested after another 24 h, and immunoblotted with indicated antibodies. (D) HH514-16 BL cells were transfected with empty vector (EV) or IFI16 plasmid, exposed to NaB after 18 h, and harvested after another 24 h before performing immunoblotting with indicated antibodies. (E and F) HH514-16 BL cells were transfected with siRNA targeting IFI16 (versus scrambled RNA as control) (E) or IFI16 plasmid (versus empty vector [EV] as control) (F), exposed to NaB after 20 h, and harvested after another 36 h (F, left) or 48 h (E and F, right) for qPCR analysis of cell-associated viral genomes (left) or released viral genomes following DNase treatment (right). (G) HH514-16 BL cells were transfected with empty vector (EV), IFI16 plasmid, control scrambled siRNA, or siRNA targeting KAP1, exposed to NaB after 18 h, and harvested after another 24 h before immunoblotting with indicated antibodies. Numbers indicate the relative amounts of ZEBRA protein after normalization to β-actin levels. These experiments were performed at least twice.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Transfection, Control, Quantitative RT-PCR, Plasmid Preparation, Western Blot

Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: KAP1 and IFI16 coenriched with H3K9me3 and at the BZLF1 promoter, preferentially in latent cells. (A) HH514-16 BL cells were left untreated or exposed to NaB. After 24 h, lysates were immunoprecipitated (IP) with anti-KAP1 antibody versus control IgG and immunoblotted with indicated antibodies. Input represents 5% of the sample. (B) HH514-16 BL cells were treated with or without NaB for 24 h followed by ChIP-re-ChIP. Chromatin was precipitated using rabbit anti-KAP1 or a control rabbit IgG antibody (top) and subjected to the second round of ChIP using mouse anti-IFI16 or a control mouse IgG antibody (bottom). Extracted DNA was analyzed by qPCR using primers spanning the known KAP1 enrichment site on the BZLF1 promoter and normalized to input. (C) HH514-16 BL cells were treated with or without NaB for 6 h followed by ChIP using an anti-H3K9me3 antibody or control IgG. (D) HH514-16 BL cells were transfected with control scrambled siRNA (Sc) or siRNA targeting IFI16 for 18 h (and harvested for immunoblotting in E) or exposed to NaB, harvested after 12 and 24 h, and chromatin was immunoprecipitated with an anti-H3K9me3 antibody or control IgG. Primer sets targeting BZLF1, BMRF1, and BFRF3 promoters were used for qPCR in C and D. Error bars, SEM of the means of three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Immunoprecipitation, Control, Transfection, Western Blot

Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: IFI16 was adjacent to KAP1 but not SZF1 in latent cells. HH514-16 BL cells were not induced or induced with NaB for 24 h before performing a proximity ligation assay (PLA) using antibodies targeting KAP1, IFI16, and SZF1. EBV seropositive human reference serum followed by APC-conjugated goat anti-human IgG was used to stain lytic cells (A, B, and D). Negative-control staining in the absence of anti-KAP1, anti-IFI16, or both antibodies is shown in (C). Green foci, in situ interactions between KAP1 and IFI16. The experiment was performed at least 3 times. Three representative fields from two independent experiments are shown in A, B, and D.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Proximity Ligation Assay, Staining, Negative Control, In Situ

Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: IFI16 message and protein abundance rapidly decrease with lytic cycle activation in EBV+ BL cells. (A and B) HH514-16 (EBV+ BL; A), CLIX-FZ (doxycycline-inducible EBV+ BL; B), and EBV− B lymphoma (BJAB; C) cells were exposed to lytic triggers (NaB in A and C; doxycycline in B) for different durations followed by immunoblotting with indicated antibodies. (D to F) HH514-16 cells were induced with NaB (D), TSA (E), or VPA as mock induction control (F) for different durations followed by RT-qPCR analysis to determine the relative amounts of IFI16 message after normalization to 18S rRNA using the ΔΔCT method. Error bars, SEM of three technical replicates and two biological replicates; *, P < 0.05; **, P < 0.01; NS, not significant.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Quantitative Proteomics, Activation Assay, Western Blot, Control, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: Reduced transcription contributes to a drop in abundance of IFI16 messages after lytic cycle activation. HH514-16 BL cells were left untreated or induced with NaB for 10 h, before performing BrU-PCR. Nascent RNA was precipitated with an anti-BrdU antibody followed by RT-qPCR analysis with two primer sets (number 1 and number 2) to determine the relative amounts of newly synthesized IFI16 message (A) and BZLF1 message (B) after normalization to 18S rRNA. Error bars, SEM of three technical replicates and two biological replicates; **, P < 0.01; ***, P < 0.001; NS, not significant.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Activation Assay, Quantitative RT-PCR, Synthesized

Journal: Journal of Virology

Article Title: IFI16 Partners with KAP1 to Maintain Epstein-Barr Virus Latency

doi: 10.1128/jvi.01028-22

Figure Lengend Snippet: Model of IFI16’s partnership with the constitutive heterochromatin machinery (HCM) that blocked EBV latent-to-lytic transition. N-terminal KRAB domain of the KRAB-ZFP SZF1 interacted with the corepressor KAP1 while C-terminal zinc fingers bind viral DNA. KAP1 recruited histone-lysine methyltransferases (HMT), the deacetylase complex NuRD-HDAC, and the heterochromatin amplification factor HP1 to silence target genes via H3K9me3 marks. Shown also is the proposed involvement of the DNA sensor IFI16 which directly contacts KAP1 but not SZF1.

Article Snippet: Plasmids included IFI16 (a gift from Cheryl Arrowsmith, Addgene plasmid number 35064) and pcDNA5.1/FRT/TO empty vector (a kind gift from Torben Heick Jensen, Denmark).

Techniques: Zinc-Fingers, Histone Deacetylase Assay, Amplification

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 1. MYH10, GOLGA4, and RAB3D form a triple complex in the Golgi, which is disbanded after EtOH treatment. (A) Immunostaining of Golgi (GOLGB1/Giantin, green) and myosin, heavy polypeptide 9, non-muscle (MYH9/MYH9-p-S1943) (red) in VA-13 cells treated with control or myosin, heavy polypeptide 10, non-muscle (MYH10/NMIIB) siRNAs. White squares indicate an area of Golgi membranes enlarged at the right; bars: 5 µm. (B) MYH10 W-B of the lysate samples from cells in A; ACTB/β-actin is a loading control. (C) Quantification of Pearson’s colocalization coefficient between GOLGB1 and MYH9-p-S1943 in cells from A; mean ± SD. (D) Immunostaining of TGOLN2 and MYH10 in control and EtOH-treated WIF-B cells and hepatocytes from mice fed with control or alcohol diet; bars: 2 µm for WIF-B cells, and 5 µm and 7 µm for hepatocytes from control and EtOH-fed mice, respectively. (E) Quantification of TGOLN2 and MYH10 colocalization in cells from D; mean ± SD. (F) RAB3D W-B of the postnuclear supernatant (PNS) isolated from control and EtOH-treated VA-13 cells. (G) Quantification of RAB3D densitometry from the samples in F counted from three independent experiments; unpaired t test. (H) RAB3D W-B of the lysate from VA-13 cells exposed to EtOH (35 mM) or EtOH and 5 µM MG132. (I) Quantification of RAB3D densitometry from the samples in H counted from five independent experiments; one-way ANOVA test. (J, K) MYH9, MYH9-p-S1943 and RAB3D (J); MYH10 and GOLGA4 (K) W-B of the Golgi membrane fractions isolated from control and EtOH-treated VA-13 cells. Samples were normalized by TGOLN2. (L) Quantification of densitometry for the indicated proteins from the samples in J and K counted from three

Article Snippet: RAB3D plasmid (full-length human RAB3D cDNA in pET28aLIC vector; Addgene, 25543; http://n2t.net/addgene:25543; RRID:Addgene 25543; deposited by Dr. Cheryl Arrowsmith Lab) and empty pET28a-LIC vector (26094) was obtained from DNASU Plasmid Repository.

Techniques: Immunostaining, Control, Isolation, Membrane

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 2. Activation of autophagy flux in different cellular and animal models of chronic alcohol treatment. (A, B) MYH10 (A), MYH9, and WIPI2 (B) W-B of the liver tissue lysate samples obtained from the normal donors (N) and patients with alcoholic cirrhosis (AC); ACTB is a loading control. (C) Quantification of densitometry for the indicated proteins from the samples in A and B. (D) LC3B W-B of the lysates from hepatocytes of control and EtOH-fed rats. (E) Quantification of LC3B densitometry for the samples in D counted from five rat hepatocytes samples per group; unpaired t test. (F) LC3B W-B of the lysates from control and EtOH-treated VA-13 cells and co- treated with 25 µM HCQ. (G) Quantification of LC3B densitometry for the samples in F counted from three independent experiments; one-way ANOVA test. (H) WIPI2 W-B of the PNS (top panel) and Golgi membrane fraction (low panel) isolated from control and EtOH-treated VA-13 cells. (I) Quantification of WIPI2 densitometry for the Golgi samples in H counted from three independent experiments; unpaired t test. (J) WIPI2 W-B of the PNS (left panel) and Golgi membrane fraction (right panel) isolated from hepatocytes of rats fed with control or alcohol diet; TGOLN2 is a loading control, HSPA/HSP70 is ER/cytoplasm marker. (K) Quantification of WIPI2 densitometry for the Golgi samples in J counted from five rat hepatocytes samples per group; unpaired t test. (L) Representative 3D SIM images of mice hepatocytes from WT (control and EtOH-fed) and rab3d KO mice stained for WIPI2 (green) and TGOLN2 (red); bars: 5 µm. Images were reconstructed by Imaris. (M) Quantification of Manders’ coefficient of colocalization between WIPI2 and TGOLN2; n=15 3D SIM images for each group; Kruskal-Wallis test. (N-P) Immunostaining of TGOLN2 and LC3B in control and EtOH-treated WIF-B (N) and VA-13 (O) cells, and hepatocytes from control and EtOH-fed rats (P); bars: 5 µm. (Q) Quantification of Manders’ coefficient of colocalization between TGOLN2 and LC3B in cells from N-P; Mann-Whitney test. All data presented are representative of at least three independent experiments. For all graphs: ****p<0.0001, ***p<0.001,**p<0.01, and *p<0.05; mean ± SD.

Article Snippet: RAB3D plasmid (full-length human RAB3D cDNA in pET28aLIC vector; Addgene, 25543; http://n2t.net/addgene:25543; RRID:Addgene 25543; deposited by Dr. Cheryl Arrowsmith Lab) and empty pET28a-LIC vector (26094) was obtained from DNASU Plasmid Repository.

Techniques: Activation Assay, Control, Membrane, Isolation, Marker, Staining, Immunostaining, MANN-WHITNEY

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 3. MYH9, but not MYH10, drives phagophore formation in EtOH-treated cells. (A) VA-13 cells were treated with EtOH or MYH10 and RAB3D siRNAs, followed by transfection with the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B. The Golgi membranes were stained using anti-GOLGA4 Ab (magenta). White squares indicate an area of puncta around the Golgi membranes enlarged at the right. Bar size, 5 µm. (B) The autophagic index was counted as the ratio of the areas of autolysosomes (red) to autophagosomes (overlapped red and green); Kruskal-Wallis test. (C) Quantification of Manders’ coefficient of colocalization for red puncta (only autolysosomes) and GOLGA4; one-way ANOVA test. (D) Representative immunogold EM images of control and EtOH-treated VA-13 cells stained for MYH9 followed by secondary Ab conjugated with 10-nm gold particles; bars: 500 nm, (G - Golgi). Arrowheads denote MYH9-

Article Snippet: RAB3D plasmid (full-length human RAB3D cDNA in pET28aLIC vector; Addgene, 25543; http://n2t.net/addgene:25543; RRID:Addgene 25543; deposited by Dr. Cheryl Arrowsmith Lab) and empty pET28a-LIC vector (26094) was obtained from DNASU Plasmid Repository.

Techniques: Transfection, Staining, Control

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 4. Evaluation of autophagy markers in different models of chronic alcohol administration. (A-D) LC3B (A) and SQSTM1 (C) W-B of the lysates from VA-13 cells: control and RAB3D KD; ACTB is a loading control. Quantification of RAB3D densitometry from the samples in A (B) and in C (D) counted from three independent experiments; unpaired t test. (E) LC3B W-B of the hepatocyte lysates from the WT mice fed with control or alcohol diet with or without HCQ treatment and rab3d KO mice receiving control diet only. (F, G) Quantification of LC3B-II (F) and LC3B-I (G) densitometry from the samples in E counted from three independent experiments; one-way ANOVA test. (H) SQSTM1 W-B of the hepatocyte lysates from the same group of mice in E. (I) Quantification of SQSTM1 densitometry from the samples in H counted from three independent experiments; one-way ANOVA test. (J) LC3B W-B of the lysate from VA-13 cells exposed to EtOH (35 mM) or EtOH and 5 µM MG132. (K) Quantification of LC3B-II densitometry from the samples in J counted from three independent experiments; one-way ANOVA test. (L) WIPI2 W-B of the hepatocyte lysates from the same group of mice in E. (M) Quantification of WIPI2 densitometry from the samples in L counted from three independent experiments; one-way ANOVA test. (N) LC3B W-B of the lysates from VA-13 cells: control and RAB3D KD treated with HCQ. (O) Quantification of LC3B-II densitometry from the samples in N counted from three independent experiments; one-way ANOVA test. (P) LC3B W-B of the lysates from VA-13 cells: control and RAB3D KD treated with bafilomycin A1 (BAF). (Q) Quantification of LC3B-II densitometry from the samples in P counted from three independent experiments; one-way ANOVA test.

Article Snippet: RAB3D plasmid (full-length human RAB3D cDNA in pET28aLIC vector; Addgene, 25543; http://n2t.net/addgene:25543; RRID:Addgene 25543; deposited by Dr. Cheryl Arrowsmith Lab) and empty pET28a-LIC vector (26094) was obtained from DNASU Plasmid Repository.

Techniques: Control

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 5. Differential distribution of GOLGA4 using Abs raised against its N or C terminus. (A-C) Immunostaining of TGOLN2 and GOLGA4 (Ab raised against N terminus) in hepatocytes from control, EtOH-fed, and rab3d KO mice (A); VA-13 cells – control, EtOH-treated, and RAB3D KD (B); and hepatocytes from control and EtOH-fed rats (C); bars: 10 µm. (D) Quantification of Manders’ colocalization coefficient between TGOLN2 and GOLGA4 for samples in A-C. Kruskal- Wallis test for VA-13 cells and mice hepatocytes; Mann-Whitney test for rat hepatocytes; n indicates the number of cells counted. (E) Immunostaining of TGOLN2 and GOLGA4 (Ab raised against C terminus) in VA-13 cells – control, EtOH-treated, and RAB3D KD; bars: 10 µm. (F) Quantification of Manders’ coefficient of colocalization between TGOLN2 and GOLGA4 for cells in E; Kruskal-Wallis test; n indicates the number of cells counted. (G) GOLGA4 W-B of the lysate samples from VA-13 cells treated with control or GOLGA4 siRNAs; ACTB is a loading control. (H) LC3B W-B of the lysate samples from VA-13 cells treated with control or GOLGA4 siRNAs and co-treated with EtOH. (I) Quantification of LC3B densitometry for the samples in H; unpaired t test. (J-M) Representative pre-embedding immunogold EM images of LC3B (1 nm) and GOLGA4 (10 nm) in control (J) and EtOH-treated (K and M) VA-13 cells. Red squares in J and K indicate LC3B-positive Golgi area highlighted at the right and indicated by arrow. L: Quantification of the number of LC3B-positive immunogold spots per Golgi stacks; n is the number of images. Inset a in M displays LC3B- and GOLGA4-positive immunogold spots (indicated by arrows) in fragmented Golgi membranes. Inset b indicates a phagophore that contains both GOLGA4 (arrowhead) and

Article Snippet: RAB3D plasmid (full-length human RAB3D cDNA in pET28aLIC vector; Addgene, 25543; http://n2t.net/addgene:25543; RRID:Addgene 25543; deposited by Dr. Cheryl Arrowsmith Lab) and empty pET28a-LIC vector (26094) was obtained from DNASU Plasmid Repository.

Techniques: Immunostaining, Control, MANN-WHITNEY

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 6. The conformational changes in GOLGA4 after RAB3D depletion. (A) The target epitopes of GOLGA4 Abs used. (B) Proximity ligation assay (PLA) of GOLGA4 central and C- terminal domains in control and VA-13 RAB3D KD cells. The proximity of GOLGA4's C terminus and the central region of its cytoplasmic domain was evaluated using Ms anti-GOLGA4 aa 2063- 2179 and Rb anti-GOLGA4 aa 1482-1584 Abs, accordingly. Red puncta indicate PLA signal, nucleus – blue, DAPI; bars: 5 μm. (C) Quantification of the number of PLA spots per nucleus from the samples in B. (D) The proximity of GOLGA4's C terminus and N terminus was evaluated in control and VA-13 RAB3D KD cells, using Ms anti-GOLGA4 aa 2063-2179 and Rb anti-GOLGA4 aa 1-150 Abs; bars: 5 μm. (E) Quantification of the number of PLA spots per nucleus from the samples in D. For graphs in C and E, dots indicate the average PLA signal from each image taken; n indicates the number of nuclei counted, Mann-Whitney test. (F) Left panel: GOLGA4 W- B of the Golgi fractions from control and RAB3D KD VA-13 cells. The samples were prepared under low (1%) concentration of β-mercaptoethanol and normalized by GOLGA4 monomer's bands. Given that depletion of RAB3D reduces the amount of GOLGA4, more proteins were loaded in the KD sample. GOLGA2 was used as a marker of Golgi membranes. Right panel: GOLGB1 (376 kDa) W-B of the control Golgi sample was used for the MW estimation. (G) Densitometry analysis of the ratio of the dimer:monomer and monomer:GOLGA2 from three independent experiment. (H) Schema illustrating a possible conformation of GOLGA4 dimer (monomers are shown in different colors for better visualization) in normal and fragmented Golgi

Article Snippet: RAB3D plasmid (full-length human RAB3D cDNA in pET28aLIC vector; Addgene, 25543; http://n2t.net/addgene:25543; RRID:Addgene 25543; deposited by Dr. Cheryl Arrowsmith Lab) and empty pET28a-LIC vector (26094) was obtained from DNASU Plasmid Repository.

Techniques: Proximity Ligation Assay, Control, MANN-WHITNEY, Concentration Assay, Marker