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OriGene human rack1 plasmids

<t>RACK1</t> is a novel ATG5 interactor. A, HEK293T cells were cotransfected with plasmids encoding FLAG-tagged ATG5 and/or non-tagged full-length RACK1 proteins. 48 h after transfection, IP were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs, and immunoprecipitations were performed using FLAG beads. C, endogenous ATG5 protein was immunoprecipitated from wild-type MEF cell extracts using anti-ATG5 antibodies that were coupled to protein A Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control rabbit serum. D, endogenous <t>RACK1</t> <t>protein</t> was immunoprecipitated from wild-type MEF cell extracts using anti-RACK1 antibodies that were coupled to protein G Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control mouse serum. E, GST pulldown assay. Glutathione-Sepharose beads that were bound to GST-ATG5 recombinant protein or not were incubated with His-RACK1 recombinant protein and washed. Input, immunoblotting of recombinant proteins; GST pulldown, proteins after pulldown. Note that His-RACK1 did not bind to beads alone. F, HEK293T cells were cultured on coverslides and cotransfected with GFP-tagged RACK1 (green) and Cherry-tagged ATG5 (red) constructs. 48 h post-transfection, cells were fixed and analyzed under a confocal microscope. Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots formed by RACK1 and ATG5 colocalization. G, non-transfected HEK293T cells were cultured on coverslides. After 72 h of incubation, cells were fixed, and endogenous RACK1 and ATG5 proteins were immunostained using anti-RACK1 and anti-ATG5 primary antibodies. Anti-mouse IgG Alexa Fluor 488 (green) or anti-rabbit IgG Alexa Fluor 568 (red) were used as secondary antibodies, respectively. Cells were analyzed under a confocal microscope. Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots formed by RACK1 and ATG5 colocalization.

Human Rack1 Plasmids, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+rack1+plasmids/pmc04974388-1195-9-16?v=OriGene
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human rack1 plasmids - by Bioz Stars, 2026-07

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1) Product Images from "RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy * "

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.708081

RACK1 is a novel ATG5 interactor. A, HEK293T cells were cotransfected with plasmids encoding FLAG-tagged ATG5 and/or non-tagged full-length RACK1 proteins. 48 h after transfection, IP were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs, and immunoprecipitations were performed using FLAG beads. C, endogenous ATG5 protein was immunoprecipitated from wild-type MEF cell extracts using anti-ATG5 antibodies that were coupled to protein A Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control rabbit serum. D, endogenous RACK1 protein was immunoprecipitated from wild-type MEF cell extracts using anti-RACK1 antibodies that were coupled to protein G Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control mouse serum. E, GST pulldown assay. Glutathione-Sepharose beads that were bound to GST-ATG5 recombinant protein or not were incubated with His-RACK1 recombinant protein and washed. Input, immunoblotting of recombinant proteins; GST pulldown, proteins after pulldown. Note that His-RACK1 did not bind to beads alone. F, HEK293T cells were cultured on coverslides and cotransfected with GFP-tagged RACK1 (green) and Cherry-tagged ATG5 (red) constructs. 48 h post-transfection, cells were fixed and analyzed under a confocal microscope. Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots formed by RACK1 and ATG5 colocalization. G, non-transfected HEK293T cells were cultured on coverslides. After 72 h of incubation, cells were fixed, and endogenous RACK1 and ATG5 proteins were immunostained using anti-RACK1 and anti-ATG5 primary antibodies. Anti-mouse IgG Alexa Fluor 488 (green) or anti-rabbit IgG Alexa Fluor 568 (red) were used as secondary antibodies, respectively. Cells were analyzed under a confocal microscope. Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots formed by RACK1 and ATG5 colocalization.

Figure Legend Snippet: RACK1 is a novel ATG5 interactor. A, HEK293T cells were cotransfected with plasmids encoding FLAG-tagged ATG5 and/or non-tagged full-length RACK1 proteins. 48 h after transfection, IP were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs, and immunoprecipitations were performed using FLAG beads. C, endogenous ATG5 protein was immunoprecipitated from wild-type MEF cell extracts using anti-ATG5 antibodies that were coupled to protein A Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control rabbit serum. D, endogenous RACK1 protein was immunoprecipitated from wild-type MEF cell extracts using anti-RACK1 antibodies that were coupled to protein G Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control mouse serum. E, GST pulldown assay. Glutathione-Sepharose beads that were bound to GST-ATG5 recombinant protein or not were incubated with His-RACK1 recombinant protein and washed. Input, immunoblotting of recombinant proteins; GST pulldown, proteins after pulldown. Note that His-RACK1 did not bind to beads alone. F, HEK293T cells were cultured on coverslides and cotransfected with GFP-tagged RACK1 (green) and Cherry-tagged ATG5 (red) constructs. 48 h post-transfection, cells were fixed and analyzed under a confocal microscope. Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots formed by RACK1 and ATG5 colocalization. G, non-transfected HEK293T cells were cultured on coverslides. After 72 h of incubation, cells were fixed, and endogenous RACK1 and ATG5 proteins were immunostained using anti-RACK1 and anti-ATG5 primary antibodies. Anti-mouse IgG Alexa Fluor 488 (green) or anti-rabbit IgG Alexa Fluor 568 (red) were used as secondary antibodies, respectively. Cells were analyzed under a confocal microscope. Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots formed by RACK1 and ATG5 colocalization.

Techniques Used: Transfection, Western Blot, Construct, Immunoprecipitation, GST Pulldown Assay, Recombinant, Incubation, Cell Culture, Microscopy

$RACK1 is a novel component of a large ATG12-5-16 protein complex. A, non-transfected HEK293T cell were treated with torin 1 or DMSO carrier control, and total cell lysates were fractioned in a gel filtration column. Chromatography fractions (F1–13) were separated in SDS-polyacrylamide gels and immunoblotted using anti-ATG16, anti-ATG5, and anti-RACK1 antibodies. CNT, DMSO carrier control; TORIN, torin 1 treatment (250 nm, 3 h); ATG16, ATG16L1; L, total cell lysate; F1 and F2, >800-kDa fractions; F3–6, 800–669-kDa fractions; F7–10, 669–443-kDa fractions; F11 and F12, 443–200-kDa fractions; F13, 200–150-kDa fraction. No protein complexes were detected in lower molecular weight fractions. B, N2A cell were treated with torin 1 or DMSO carrier control, and total cell lysates were fractioned in a gel filtration column as in A. C, chromatogram showing peaks of the molecular weight marker mix (Sigma, catalog no. MWGF1000); Ve, elution volume. D, OD595 absorbance confirmation of the peaks. E, standardization of the gel filtration column by Ve/V0. V0, void volume. F, curve showing correlation of fractions with protein sizes in kDa. G and H, representative chromatograms obtained for HEK293T (G) and N2A (H) cell lines. I, Tri-SILAC-LC-MS/MS analyses. ATG5 enrichment compared with beads alone (upper panel); enrichment of RACK-ATG5 complex under torin-treated conditions compared with DMSO-treated control (lower panel) (mean ± S.D. of independent experiments, n = 3, *, p < 0.05).$
Figure Legend Snippet: RACK1 is a novel component of a large ATG12-5-16 protein complex. A, non-transfected HEK293T cell were treated with torin 1 or DMSO carrier control, and total cell lysates were fractioned in a gel filtration column. Chromatography fractions (F1–13) were separated in SDS-polyacrylamide gels and immunoblotted using anti-ATG16, anti-ATG5, and anti-RACK1 antibodies. CNT, DMSO carrier control; TORIN, torin 1 treatment (250 nm, 3 h); ATG16, ATG16L1; L, total cell lysate; F1 and F2, >800-kDa fractions; F3–6, 800–669-kDa fractions; F7–10, 669–443-kDa fractions; F11 and F12, 443–200-kDa fractions; F13, 200–150-kDa fraction. No protein complexes were detected in lower molecular weight fractions. B, N2A cell were treated with torin 1 or DMSO carrier control, and total cell lysates were fractioned in a gel filtration column as in A. C, chromatogram showing peaks of the molecular weight marker mix (Sigma, catalog no. MWGF1000); Ve, elution volume. D, OD595 absorbance confirmation of the peaks. E, standardization of the gel filtration column by Ve/V0. V0, void volume. F, curve showing correlation of fractions with protein sizes in kDa. G and H, representative chromatograms obtained for HEK293T (G) and N2A (H) cell lines. I, Tri-SILAC-LC-MS/MS analyses. ATG5 enrichment compared with beads alone (upper panel); enrichment of RACK-ATG5 complex under torin-treated conditions compared with DMSO-treated control (lower panel) (mean ± S.D. of independent experiments, n = 3, *, p < 0.05).

Techniques Used: Transfection, Filtration, Column Chromatography, Molecular Weight, Marker, Liquid Chromatography with Mass Spectroscopy

Dynamic nature of RACK1-ATG5 interaction under autophagy-inducing conditions. A, HEK293T cells were cotransfected with FLAG-ATG5 and/or non-tagged RACK1 constructs and treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h). IP were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kDa. β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs and starved in EBSS (2 h) or not. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. C, HEK293T cells were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (2 h). Endogenous ATG5 protein was immunoprecipitated from cell extracts using anti-ATG5 antibodies that were coupled to protein A Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control rabbit serum. D, HEK293T cells were cultured on coverslides. They were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (Stv, 2 h) or not. Then endogenous proteins were immunostained using anti-RACK1 and anti-ATG5 primary antibodies. Anti-mouse IgG Alexa Fluor 488 (green) and anti-rabbit IgG Alexa Fluor 568 (red) were used as secondary antibodies, respectively. Cells were analyzed under confocal microscope. CNT, non-treated cells; Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots with RACK1 and ATG5 colocalization. E, HEK293T cells were cultured on coverslides. Cells were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (Stv, 2 h) or not. Then endogenous proteins were immunostained by using anti-RACK1 and anti-LC3 primary antibodies. Cells were analyzed under a confocal microscope. CNT, non-treated cells; Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots with RACK1 and LC3 co-localization.

Figure Legend Snippet: Dynamic nature of RACK1-ATG5 interaction under autophagy-inducing conditions. A, HEK293T cells were cotransfected with FLAG-ATG5 and/or non-tagged RACK1 constructs and treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h). IP were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kDa. β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs and starved in EBSS (2 h) or not. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. C, HEK293T cells were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (2 h). Endogenous ATG5 protein was immunoprecipitated from cell extracts using anti-ATG5 antibodies that were coupled to protein A Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control rabbit serum. D, HEK293T cells were cultured on coverslides. They were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (Stv, 2 h) or not. Then endogenous proteins were immunostained using anti-RACK1 and anti-ATG5 primary antibodies. Anti-mouse IgG Alexa Fluor 488 (green) and anti-rabbit IgG Alexa Fluor 568 (red) were used as secondary antibodies, respectively. Cells were analyzed under confocal microscope. CNT, non-treated cells; Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots with RACK1 and ATG5 colocalization. E, HEK293T cells were cultured on coverslides. Cells were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (Stv, 2 h) or not. Then endogenous proteins were immunostained by using anti-RACK1 and anti-LC3 primary antibodies. Cells were analyzed under a confocal microscope. CNT, non-treated cells; Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots with RACK1 and LC3 co-localization.

Techniques Used: Construct, Western Blot, Immunoprecipitation, Cell Culture, Microscopy

RACK1 is required for mTOR inhibition and starvation-induced autophagy, but it is not an autophagy target. A, HEK293T cells were cultured on coverslides and transfected with siRACK1 or control siRNA (CNT siRNA). 48 h post-transfection, cells were treated or not (−) with rapamycin (Rapa, 200 nm, 16 h) or starved in EBSS (2 h) in the presence or absence of BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, *, p < 0.05; **, p < 0.01). Endogenous protein expression levels were checked in cell extracts from the same experiments using anti-p62, anti-LC3, and anti-RACK1 antibodies. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, representative immunofluorescence pictures of LC3 quantification experiments in A. (−), non-treated cells. White arrows show LC3 dots. C, HEK293T cells were treated with rapamycin (200 nm) for 12 or 24 h or with carrier DMSO (D, 24 h) or starved for 2, 4, or 8 h in EBSS or cultured in full medium (CNT) with or without of BafA (100 nm, 1 h) in the presence of translation inhibitor cycloheximide (0.5 μg/ml). Immunoblots were performed using anti-p62, anti-RACK1, or anti-LC3 antibodies. β-Actin was used as loading control. D, HEK293T cells were treated with rapamycin (200 nm) for 12 or 24 h or with carrier DMSO (D, 24 h) or starved for 2, 4, or 8 h in EBSS or cultured in full medium (CNT) with or without of BafA (100 nm, 1 h) in the absence of translation inhibitor cycloheximide. Immunoblots were performed using anti-p62, anti-RACK1, or anti-LC3 antibodies. β-Actin was used as loading control.

Figure Legend Snippet: RACK1 is required for mTOR inhibition and starvation-induced autophagy, but it is not an autophagy target. A, HEK293T cells were cultured on coverslides and transfected with siRACK1 or control siRNA (CNT siRNA). 48 h post-transfection, cells were treated or not (−) with rapamycin (Rapa, 200 nm, 16 h) or starved in EBSS (2 h) in the presence or absence of BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, *, p < 0.05; **, p < 0.01). Endogenous protein expression levels were checked in cell extracts from the same experiments using anti-p62, anti-LC3, and anti-RACK1 antibodies. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, representative immunofluorescence pictures of LC3 quantification experiments in A. (−), non-treated cells. White arrows show LC3 dots. C, HEK293T cells were treated with rapamycin (200 nm) for 12 or 24 h or with carrier DMSO (D, 24 h) or starved for 2, 4, or 8 h in EBSS or cultured in full medium (CNT) with or without of BafA (100 nm, 1 h) in the presence of translation inhibitor cycloheximide (0.5 μg/ml). Immunoblots were performed using anti-p62, anti-RACK1, or anti-LC3 antibodies. β-Actin was used as loading control. D, HEK293T cells were treated with rapamycin (200 nm) for 12 or 24 h or with carrier DMSO (D, 24 h) or starved for 2, 4, or 8 h in EBSS or cultured in full medium (CNT) with or without of BafA (100 nm, 1 h) in the absence of translation inhibitor cycloheximide. Immunoblots were performed using anti-p62, anti-RACK1, or anti-LC3 antibodies. β-Actin was used as loading control.

Techniques Used: Inhibition, Cell Culture, Transfection, Expressing, Immunofluorescence, Western Blot

Role of mTOR-p70S6K pathway in the regulation of RACK1-ATG5 interaction. A, HEK293T cells were cotransfected with FLAG-ATG5 and/or non-tagged RACK1 constructs and/or an mTOR construct. IP were performed using FLAG beads. Anti-mTOR, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs and/or an shmTOR construct. IP were performed using FLAG beads. Anti-mTOR, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. C, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 and/or p70S6K wild-type (WT) constructs. IP were performed using FLAG beads. Anti-p70S6K, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. D, HEK293T cells were cotransfected with FLAG-ATG5 and non-tagged RACK1 constructs and/or sip70S6 RNAi. Anti-p70S6K, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting.

Figure Legend Snippet: Role of mTOR-p70S6K pathway in the regulation of RACK1-ATG5 interaction. A, HEK293T cells were cotransfected with FLAG-ATG5 and/or non-tagged RACK1 constructs and/or an mTOR construct. IP were performed using FLAG beads. Anti-mTOR, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs and/or an shmTOR construct. IP were performed using FLAG beads. Anti-mTOR, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. C, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 and/or p70S6K wild-type (WT) constructs. IP were performed using FLAG beads. Anti-p70S6K, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. D, HEK293T cells were cotransfected with FLAG-ATG5 and non-tagged RACK1 constructs and/or sip70S6 RNAi. Anti-p70S6K, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting.

Techniques Used: Construct, Western Blot

Determination of RACK1 amino acid residues that are critical for the interaction. A, Clustal Omega alignments of RACK1 protein sequences. Putative p70S6K target RXX(S/T) consensus sequences are highlighted in black boxes. Ser/Thr residue numbers are marked according to Homo sapiens protein sequences. RACK1 GenBankTM reference sequences are as follows: H. sapiens, NP_006089; Mus musculus, NP_032169; Danio rerio, NP_571519; Drosophila melanogaster, AAB72148; Caenorhabditis elegans, NP_501859; Saccharomyces cerevisiae, NP_013834. B, schematic depiction of RACK1 constructs. WD1–7, WD40 domains 1–7. WT RACK1, wild-type RACK1. T39A, S63A, or T128A, mutant RACK1 constructs. Mutated residues were marked. C, HEK293T cells were cotransfected with FLAG-ATG5, non-tagged WT RACK1 or T39A, S63A, T128A RACK1 mutant constructs. IPs were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ.

Figure Legend Snippet: Determination of RACK1 amino acid residues that are critical for the interaction. A, Clustal Omega alignments of RACK1 protein sequences. Putative p70S6K target RXX(S/T) consensus sequences are highlighted in black boxes. Ser/Thr residue numbers are marked according to Homo sapiens protein sequences. RACK1 GenBankTM reference sequences are as follows: H. sapiens, NP_006089; Mus musculus, NP_032169; Danio rerio, NP_571519; Drosophila melanogaster, AAB72148; Caenorhabditis elegans, NP_501859; Saccharomyces cerevisiae, NP_013834. B, schematic depiction of RACK1 constructs. WD1–7, WD40 domains 1–7. WT RACK1, wild-type RACK1. T39A, S63A, or T128A, mutant RACK1 constructs. Mutated residues were marked. C, HEK293T cells were cotransfected with FLAG-ATG5, non-tagged WT RACK1 or T39A, S63A, T128A RACK1 mutant constructs. IPs were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ.

Techniques Used: Construct, Mutagenesis, Western Blot

RACK1-ATG5 interaction model. A, schematic representation of human RACK1 (Protein Data Bank code 4AOW). Each WD40 domain (WD1–7) is in a different color. The seven-bladed β-propeller structure is shown. Location of the Ser-63 residue is marked in a square. B, schematic model of RACK1 (Protein Data Bank code 4AOW, silver color) and ATG5 (Protein Data Bank code 4GDK, tan color) interaction. Residues found within 3 Å of the other subunit are selected as binding interface that was rendered in the wire frame surface model (probe radius, 1.4 Å) by coloring ATG5 residues in green and RACK1 in yellow. The region around the Ser-63 residue of RACK1 is shown in a red wire frame and encircled. C, interaction network of predicted RACK1-ATG5 model. D–F, native and mutated RACK1s (S63A and S63D) were energy-minimized and equilibrated in MD simulations. Snapshots of the binding interface are shown with Ser-63, Asp-6, and Lys-38 in licorice models (C, cyan; O, red; N, blue). Structural integrity of binding interface was probed by the distance of the ionic interaction between Asp-6 of ATG5 and Lys-38 of RACK1. Although the wild-type (native) and S63D complexes possessed an intact binding surface with Asp-6–Lys-38 ionic pairing (D and E), S63A showed an extension in Asp-6–Lys-38 distance (F), implying a weakened interaction of RACK1 and ATG5. G, r.m.s.d. of the backbone atoms carbon, nitrogen, and α-carbon. Native, wild-type RACK1; S63A, S63A RACK1; S63D, S63D RACK1. H, fluctuations of ATG5 during 5 ns of MD simulations. S63A RACK1 mutant displayed increased fluctuations at two distinct regions (residues from 32 to 36 and from 50 to 54), which are found at the binding interface (right panel), the observation that suggests that S63A RACK1 destabilizes the ATG5-RACK1 complex.

Figure Legend Snippet: RACK1-ATG5 interaction model. A, schematic representation of human RACK1 (Protein Data Bank code 4AOW). Each WD40 domain (WD1–7) is in a different color. The seven-bladed β-propeller structure is shown. Location of the Ser-63 residue is marked in a square. B, schematic model of RACK1 (Protein Data Bank code 4AOW, silver color) and ATG5 (Protein Data Bank code 4GDK, tan color) interaction. Residues found within 3 Å of the other subunit are selected as binding interface that was rendered in the wire frame surface model (probe radius, 1.4 Å) by coloring ATG5 residues in green and RACK1 in yellow. The region around the Ser-63 residue of RACK1 is shown in a red wire frame and encircled. C, interaction network of predicted RACK1-ATG5 model. D–F, native and mutated RACK1s (S63A and S63D) were energy-minimized and equilibrated in MD simulations. Snapshots of the binding interface are shown with Ser-63, Asp-6, and Lys-38 in licorice models (C, cyan; O, red; N, blue). Structural integrity of binding interface was probed by the distance of the ionic interaction between Asp-6 of ATG5 and Lys-38 of RACK1. Although the wild-type (native) and S63D complexes possessed an intact binding surface with Asp-6–Lys-38 ionic pairing (D and E), S63A showed an extension in Asp-6–Lys-38 distance (F), implying a weakened interaction of RACK1 and ATG5. G, r.m.s.d. of the backbone atoms carbon, nitrogen, and α-carbon. Native, wild-type RACK1; S63A, S63A RACK1; S63D, S63D RACK1. H, fluctuations of ATG5 during 5 ns of MD simulations. S63A RACK1 mutant displayed increased fluctuations at two distinct regions (residues from 32 to 36 and from 50 to 54), which are found at the binding interface (right panel), the observation that suggests that S63A RACK1 destabilizes the ATG5-RACK1 complex.

Techniques Used: Binding Assay, Mutagenesis

RACK1-ATG5 interaction is necessary for mTOR inhibition- or starvation-induced autophagy in Neuro2A cells. Cells were cultured on coverslides and transfected with the empty control vector pcDNA3 or wild-type (wt) or mutant RACK1 (S63A or S63D) constructs. A, 48 h post-transfection, Neuro2A cells were treated or not with torin 1 (Torin, 250 nm, 3 h) with or without BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, *, p < 0.05). LC3 and RACK1 protein expression levels in cell lysates were checked in immunoblots using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. B, representative immunofluorescence pictures of LC3 quantification experiments in A. White arrows show LC3 dots. C, 48 h post-transfection, Neuro2A cells were cultured in full medium (non-STV) or starved in EBSS (STV, 2 h) with or without BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified (mean ± S.D. of independent experiments, n = 3, **, p < 0.01; *, p < 0.05). LC3 and RACK1 protein expression levels in cell lysates were checked in immunoblots using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. D, representative immunofluorescence pictures of LC3 quantification experiments in C. White arrows show LC3 dots.

Figure Legend Snippet: RACK1-ATG5 interaction is necessary for mTOR inhibition- or starvation-induced autophagy in Neuro2A cells. Cells were cultured on coverslides and transfected with the empty control vector pcDNA3 or wild-type (wt) or mutant RACK1 (S63A or S63D) constructs. A, 48 h post-transfection, Neuro2A cells were treated or not with torin 1 (Torin, 250 nm, 3 h) with or without BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, *, p < 0.05). LC3 and RACK1 protein expression levels in cell lysates were checked in immunoblots using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. B, representative immunofluorescence pictures of LC3 quantification experiments in A. White arrows show LC3 dots. C, 48 h post-transfection, Neuro2A cells were cultured in full medium (non-STV) or starved in EBSS (STV, 2 h) with or without BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified (mean ± S.D. of independent experiments, n = 3, **, p < 0.01; *, p < 0.05). LC3 and RACK1 protein expression levels in cell lysates were checked in immunoblots using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. D, representative immunofluorescence pictures of LC3 quantification experiments in C. White arrows show LC3 dots.

Techniques Used: Inhibition, Cell Culture, Transfection, Plasmid Preparation, Mutagenesis, Construct, Expressing, Western Blot, Immunofluorescence

Rescue experiments with RACK1 mutant constructs following siRNA knockdown of RACK1. A, HEK293T cells were transfected with siRACK1 or control siRNA (CNT siRNA). The effect of siRNAs was checked by immunoblotting using anti-RACK1 antibodies. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were grown onto coverslides and transfected with the empty control vector pcDNA3 or wild-type (wt) or mutant RACK1 (S63A or S63D) constructs. 48 h post-transfection, HEK293T cells were treated or not with torin 1 (Torin, 250 nm, 3 h) with or without E64D (E64D, 10 μg/ml, 1 h) and PepA (pepstatin A, 10 μg/ml, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, **, p < 0.01; *, p < 0.05). LC3 and RACK1 protein expression levels were detected by immunoblotting using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. C, representative immunofluorescence pictures of LC3 quantification experiments in A. White arrows show LC3 dots.

Figure Legend Snippet: Rescue experiments with RACK1 mutant constructs following siRNA knockdown of RACK1. A, HEK293T cells were transfected with siRACK1 or control siRNA (CNT siRNA). The effect of siRNAs was checked by immunoblotting using anti-RACK1 antibodies. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were grown onto coverslides and transfected with the empty control vector pcDNA3 or wild-type (wt) or mutant RACK1 (S63A or S63D) constructs. 48 h post-transfection, HEK293T cells were treated or not with torin 1 (Torin, 250 nm, 3 h) with or without E64D (E64D, 10 μg/ml, 1 h) and PepA (pepstatin A, 10 μg/ml, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, **, p < 0.01; *, p < 0.05). LC3 and RACK1 protein expression levels were detected by immunoblotting using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. C, representative immunofluorescence pictures of LC3 quantification experiments in A. White arrows show LC3 dots.

Techniques Used: Mutagenesis, Construct, Transfection, Western Blot, Plasmid Preparation, Expressing, Immunofluorescence

Image Search Results

Site-specific MARylation of RACK1 in ovarian cancer cells. (A) Left: Spatial distribution of the proteins modified by MARylation in the 80S ribosome (PDB ID: 4V6X ). Middle and right: Sites of MARylation within RACK1 (Asp 144, Glu 145, and Asp 203; blue ribbon) are indicated in two expanded views, with the structure in the right rotated by 90°. (B) RACK1 is MARylated. Endogenous RACK1 was immunoprecipitated (IP) from OVCAR3 cells and subjected to immunoblotting for MAR and RACK1. (C) RACK1 is MARylated at Asp 144, Glu 145, and Asp 203. HA-tagged RACK1 was immunoprecipitated from OVCAR3 cells ectopically expressing wild-type (WT) or MARylation site mutant (Mut) RACK1 and subjected to immunoblotting for MAR and HA. (D) In situ detection of RACK1 MARylation. PLA of RACK1 and MAR in OVCAR3 cells subjected to Dox-induced knockdown of endogenous and re-expression of RACK1 (WT or Mut). DNA was stained with DAPI. Scale bar is 15 µm. (E) Quantification of multiple experiments like the one shown in panel D. Each bar represents the mean + SEM of MAR-RACK1 PLA foci from three biological replicates (Student’s t test, two-tailed, ****P < 0.0001). (F) RACK1-Mut expression does not alter global protein synthesis in OVCAR3 cells. Immunoblot analysis of puromycin incorporation assays from OVCAR3 cells subjected to Dox-induced knockdown of endogenous and re-expression of RACK1 (WT or Mut). β-tubulin serves as a loading control. The sizes of molecular mass markers in kDa are shown. (G and H) Regulation of mRNA translation by RACK1 MARylation. Ribosome profiling of OVCAR3 cells subjected to Dox-induced knockdown of endogenous RACK1 followed by re-expression of exogenous RACK1 (WT or Mut). (G) Heatmap representation of mRNAs that exhibit altered translation efficiency when RACK1-Mut was expressed. (H) Gene ontology enrichment analysis of the translationally upregulated and downregulated mRNAs. (I) RACK1 MARylation regulates the translation of AKT1 . Example ribosome profiling and RNA-seq traces of AKT1 in OVCAR3 cells subjected to Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 (WT or Mut). A schematic of the AKT1 gene with a scale bar is shown. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: RACK1 MARylation regulates translation and stress granules in ovarian cancer cells

doi: 10.1083/jcb.202401101

Figure Lengend Snippet: Site-specific MARylation of RACK1 in ovarian cancer cells. (A) Left: Spatial distribution of the proteins modified by MARylation in the 80S ribosome (PDB ID: 4V6X ). Middle and right: Sites of MARylation within RACK1 (Asp 144, Glu 145, and Asp 203; blue ribbon) are indicated in two expanded views, with the structure in the right rotated by 90°. (B) RACK1 is MARylated. Endogenous RACK1 was immunoprecipitated (IP) from OVCAR3 cells and subjected to immunoblotting for MAR and RACK1. (C) RACK1 is MARylated at Asp 144, Glu 145, and Asp 203. HA-tagged RACK1 was immunoprecipitated from OVCAR3 cells ectopically expressing wild-type (WT) or MARylation site mutant (Mut) RACK1 and subjected to immunoblotting for MAR and HA. (D) In situ detection of RACK1 MARylation. PLA of RACK1 and MAR in OVCAR3 cells subjected to Dox-induced knockdown of endogenous and re-expression of RACK1 (WT or Mut). DNA was stained with DAPI. Scale bar is 15 µm. (E) Quantification of multiple experiments like the one shown in panel D. Each bar represents the mean + SEM of MAR-RACK1 PLA foci from three biological replicates (Student’s t test, two-tailed, ****P < 0.0001). (F) RACK1-Mut expression does not alter global protein synthesis in OVCAR3 cells. Immunoblot analysis of puromycin incorporation assays from OVCAR3 cells subjected to Dox-induced knockdown of endogenous and re-expression of RACK1 (WT or Mut). β-tubulin serves as a loading control. The sizes of molecular mass markers in kDa are shown. (G and H) Regulation of mRNA translation by RACK1 MARylation. Ribosome profiling of OVCAR3 cells subjected to Dox-induced knockdown of endogenous RACK1 followed by re-expression of exogenous RACK1 (WT or Mut). (G) Heatmap representation of mRNAs that exhibit altered translation efficiency when RACK1-Mut was expressed. (H) Gene ontology enrichment analysis of the translationally upregulated and downregulated mRNAs. (I) RACK1 MARylation regulates the translation of AKT1 . Example ribosome profiling and RNA-seq traces of AKT1 in OVCAR3 cells subjected to Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 (WT or Mut). A schematic of the AKT1 gene with a scale bar is shown. Source data are available for this figure: .

Article Snippet: A plasmid for Dox-inducible expression of C-terminal HA epitope-tagged RACK1 was generated using a cDNA for RACK1 that was amplified from pCMV3-RACK1 (HG16196-CY; Sino Biologicals) and subcloned into the pInducer20 vector (plasmid no. 44012; Addgene).

Techniques: Modification, Immunoprecipitation, Western Blot, Expressing, Mutagenesis, In Situ, Knockdown, Staining, Two Tailed Test, Control, RNA Sequencing Assay

RACK1-Mut inhibits the formation of G3BP1 foci. (A and B) RACK1 is MARylated. Endogenous RACK1 was immunoprecipitated (IP) from OVCAR3 cells transfected with siRNAs targeting a control sequence or RACK1 and subjected to immunoblotting for MAR and RACK1. Each bar in B represents the mean + SEM of the levels of MARylated RACK1 and RACK1 in the immununoprecipitates of RACK1 ( n = 3, Student’s t test, two-tailed, *P < 0.05 and **P < 0.01). (C) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of the relative abundance of G3BP1 in HA - RACK1 immunoprecipitates ( n = 3, Student’s t test, two-tailed, ***P < 0.001). (D) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of G3BP1-HA (RACK1) PLA foci from two biological replicates ( n = 2, Student’s t test, two-tailed, **P < 0.01). (E) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of the relative abundance of eIF3η and RPS6 in G3BP1 immunoprecipitates ( n = 3, multiple t test, **P < 0.01). (F) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of distinct G3BP1 foci from three biological replicates ( n = 3, Student’s t test, two-tailed, **P < 0.01). (G and H) Loss of RACK1 MARylation inhibits G3BP1 localization to stress granules and its interaction with translation factors that are key components of stress granules. Immunofluorescent staining assays of OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). Staining for (G) RPS6 and G3BP1, (H) eIF3η and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. (I and J) RACK1 MARylation-mediated G3BP1 localization to stress granules is dependent on the levels of stalled polysomes. Immunofluorescent staining assays of OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ) (left, “Untreated”). The cells were also treated with 10 µg/ml puromycin for 15 min prior to 15 min (I) or 30 min (J) of treatment with 250 µM sodium arsenite (NaAsO 2 ) (right, “Puromycin”). Staining for HA (HA-RACK1) and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. (K and L) Quantification of multiple experiments like those shown in (K) panel I above and (L) panel J above. Each bar represents the mean + SEM of distinct G3BP1 foci ( n = 3, one-way ANOVA. *P < 0.05 and ns not significant). (M and N) RACK1-Mut expression does not alter global protein synthesis under stress in OVCAR3 cells. (M) Immunoblot analysis of puromycin incorporation assays from OVCAR3 cells subjected to Dox-induced knockdown of endogenous and re-expression of RACK1 followed by 15 min of treatment with 250 µM sodium arsenite. β-actin serves as a loading control. The sizes of molecular mass markers in kDa are shown. (N) Quantification of immunoblot experiments like those shown in M. Each bar in the graph represents the mean + SEM of the relative levels of puromycin incorporation ( n = 3, one-way ANOVA, ns not significant). Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: RACK1 MARylation regulates translation and stress granules in ovarian cancer cells

doi: 10.1083/jcb.202401101

Figure Lengend Snippet: RACK1-Mut inhibits the formation of G3BP1 foci. (A and B) RACK1 is MARylated. Endogenous RACK1 was immunoprecipitated (IP) from OVCAR3 cells transfected with siRNAs targeting a control sequence or RACK1 and subjected to immunoblotting for MAR and RACK1. Each bar in B represents the mean + SEM of the levels of MARylated RACK1 and RACK1 in the immununoprecipitates of RACK1 ( n = 3, Student’s t test, two-tailed, *P < 0.05 and **P < 0.01). (C) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of the relative abundance of G3BP1 in HA - RACK1 immunoprecipitates ( n = 3, Student’s t test, two-tailed, ***P < 0.001). (D) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of G3BP1-HA (RACK1) PLA foci from two biological replicates ( n = 2, Student’s t test, two-tailed, **P < 0.01). (E) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of the relative abundance of eIF3η and RPS6 in G3BP1 immunoprecipitates ( n = 3, multiple t test, **P < 0.01). (F) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of distinct G3BP1 foci from three biological replicates ( n = 3, Student’s t test, two-tailed, **P < 0.01). (G and H) Loss of RACK1 MARylation inhibits G3BP1 localization to stress granules and its interaction with translation factors that are key components of stress granules. Immunofluorescent staining assays of OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). Staining for (G) RPS6 and G3BP1, (H) eIF3η and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. (I and J) RACK1 MARylation-mediated G3BP1 localization to stress granules is dependent on the levels of stalled polysomes. Immunofluorescent staining assays of OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ) (left, “Untreated”). The cells were also treated with 10 µg/ml puromycin for 15 min prior to 15 min (I) or 30 min (J) of treatment with 250 µM sodium arsenite (NaAsO 2 ) (right, “Puromycin”). Staining for HA (HA-RACK1) and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. (K and L) Quantification of multiple experiments like those shown in (K) panel I above and (L) panel J above. Each bar represents the mean + SEM of distinct G3BP1 foci ( n = 3, one-way ANOVA. *P < 0.05 and ns not significant). (M and N) RACK1-Mut expression does not alter global protein synthesis under stress in OVCAR3 cells. (M) Immunoblot analysis of puromycin incorporation assays from OVCAR3 cells subjected to Dox-induced knockdown of endogenous and re-expression of RACK1 followed by 15 min of treatment with 250 µM sodium arsenite. β-actin serves as a loading control. The sizes of molecular mass markers in kDa are shown. (N) Quantification of immunoblot experiments like those shown in M. Each bar in the graph represents the mean + SEM of the relative levels of puromycin incorporation ( n = 3, one-way ANOVA, ns not significant). Source data are available for this figure: .

Techniques: Immunoprecipitation, Transfection, Control, Sequencing, Western Blot, Two Tailed Test, Staining, Knockdown, Expressing

$Site-specific MARylation of RACK1 is required for stress granule assembly. (A and B) Loss of RACK1 MARylation inhibits RACK1 interaction with G3BP1. (A) HA-tagged RACK1 was immunoprecipitated (IP) from OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1. The immunoprecipitates were subjected to immunoblotting for G3BP1 and HA. The sizes of molecular mass markers in kDa are shown. (B) PLA using G3BP1 and HA antibodies. DNA was stained with DAPI. Scale bar is 15 µm. (C and D) Loss of RACK1 MARylation inhibits the recruitment of G3BP1 to ribosomes. (C) Immunoblot analysis for HA-tagged RACK1 and G3BP1 in sucrose density gradient fractions of ribosomes prepared from OVCAR3 cells subjected to Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1. The sizes of molecular mass markers in kDa are shown. Each bar in the graph in D represents the mean + SEM of the relative abundance of G3BP1 in monosomes or polysomes ( n = 3, two-way ANOVA, *P < 0.05 and **P < 0.01). (E and F) Loss of RACK1 MARylation inhibits G3BP1 localization to stress granules and its interaction with translation factors that are key components of stress granules. (E) G3BP1 was immunoprecipitated from OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1. The immunoprecipitates were subjected to immunoblotting for eIF3η, RPS6, and G3BP1 as indicated. The sizes of molecular mass markers in kDa are shown. (F) Immunofluorescent staining assays of OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). Staining for HA (RACK1) and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. Source data are available for this figure: .$

Journal: The Journal of Cell Biology

Article Title: RACK1 MARylation regulates translation and stress granules in ovarian cancer cells

doi: 10.1083/jcb.202401101

Figure Lengend Snippet: Site-specific MARylation of RACK1 is required for stress granule assembly. (A and B) Loss of RACK1 MARylation inhibits RACK1 interaction with G3BP1. (A) HA-tagged RACK1 was immunoprecipitated (IP) from OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1. The immunoprecipitates were subjected to immunoblotting for G3BP1 and HA. The sizes of molecular mass markers in kDa are shown. (B) PLA using G3BP1 and HA antibodies. DNA was stained with DAPI. Scale bar is 15 µm. (C and D) Loss of RACK1 MARylation inhibits the recruitment of G3BP1 to ribosomes. (C) Immunoblot analysis for HA-tagged RACK1 and G3BP1 in sucrose density gradient fractions of ribosomes prepared from OVCAR3 cells subjected to Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1. The sizes of molecular mass markers in kDa are shown. Each bar in the graph in D represents the mean + SEM of the relative abundance of G3BP1 in monosomes or polysomes ( n = 3, two-way ANOVA, *P < 0.05 and **P < 0.01). (E and F) Loss of RACK1 MARylation inhibits G3BP1 localization to stress granules and its interaction with translation factors that are key components of stress granules. (E) G3BP1 was immunoprecipitated from OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1. The immunoprecipitates were subjected to immunoblotting for eIF3η, RPS6, and G3BP1 as indicated. The sizes of molecular mass markers in kDa are shown. (F) Immunofluorescent staining assays of OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). Staining for HA (RACK1) and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. Source data are available for this figure: .

Techniques: Immunoprecipitation, Knockdown, Expressing, Western Blot, Staining

PARP14 mediates RACK1 MARylation. (A) OVCAR3 cells were subjected to knockdown with two different siRNAs targeting each of the expressed cytosolic MARTs. Representative images from PLAs using MAR and RACK1 antibodies. DNA was stained with DAPI. Scale bar is 15 µm. (B) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of MAR-RACK1 PLA foci ( n = 3, Student’s t test, two-tailed, ***P < 0.001). (C and D) PARP14 inhibition reduces G3BP1 interaction with translation factors that are key components of stress granules. Immunofluorescent staining assays of OVCAR3 cells treated with 10 µM PARP14i for 24 h and subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). Staining for (C) RPS6 and G3BP1, (D) eIF3η and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. (E and F) PARP14 inhibition blocks RACK1 MARylation in ovarian cancer cells. PLA using MAR and RACK1 antibodies in (E) SKOV3 cells and (F) HCC5044 cells treated with 10 µM PARP14i for 24 h and subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). DNA was stained with DAPI. Scale bar is 15 µm. (G and H) PARP14 inhibition blocks the assembly of G3BP1-containing stress granules in ovarian cancer cells. Immunofluorescent staining assays in (G) SKOV3 and (H) HCC5044 cells treated with 10 µM PARP14i for 24 h and subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). DNA was stained with DAPI. Scale bar is 15 µm.

Journal: The Journal of Cell Biology

Article Title: RACK1 MARylation regulates translation and stress granules in ovarian cancer cells

doi: 10.1083/jcb.202401101

Figure Lengend Snippet: PARP14 mediates RACK1 MARylation. (A) OVCAR3 cells were subjected to knockdown with two different siRNAs targeting each of the expressed cytosolic MARTs. Representative images from PLAs using MAR and RACK1 antibodies. DNA was stained with DAPI. Scale bar is 15 µm. (B) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of MAR-RACK1 PLA foci ( n = 3, Student’s t test, two-tailed, ***P < 0.001). (C and D) PARP14 inhibition reduces G3BP1 interaction with translation factors that are key components of stress granules. Immunofluorescent staining assays of OVCAR3 cells treated with 10 µM PARP14i for 24 h and subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). Staining for (C) RPS6 and G3BP1, (D) eIF3η and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. (E and F) PARP14 inhibition blocks RACK1 MARylation in ovarian cancer cells. PLA using MAR and RACK1 antibodies in (E) SKOV3 cells and (F) HCC5044 cells treated with 10 µM PARP14i for 24 h and subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). DNA was stained with DAPI. Scale bar is 15 µm. (G and H) PARP14 inhibition blocks the assembly of G3BP1-containing stress granules in ovarian cancer cells. Immunofluorescent staining assays in (G) SKOV3 and (H) HCC5044 cells treated with 10 µM PARP14i for 24 h and subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). DNA was stained with DAPI. Scale bar is 15 µm.

Techniques: Knockdown, Staining, Two Tailed Test, Inhibition

$PARP14 inhibition reduces stress granule assembly. (A) PARP14i blocks PARP14 autoMARylation. OVCAR3 cells were treated with 10 µM PARP14i (RBN012759) for 24 h. PARP14 was immunoprecipitated (IP) and subjected to immunoblotting for PARP14 and MAR. The size of a molecular mass marker in kDa is shown. (B) Inhibition of PARP14 catalytic activity blocks RACK1 MARylation. PLA using MAR and RACK1 antibodies in OVCAR3 cells treated with 10 µM PARP14i (RBN012759) for 24 h. DNA was stained with DAPI. Scale bar is 15 µm. (C and D) PARP14 inhibition reduces the recruitment of G3BP1 to ribosomes. (C) Immunoblot analysis of RACK1 and G3BP1 in sucrose density gradient fractions of ribosomes prepared from OVCAR3 cells treated with 10 µM PARP14i for 24 h. The sizes of molecular mass markers in kDa are shown. Each bar in the graph in D represents the mean + SEM of the relative abundance of G3BP1 in monosomes or polysomes ( n = 3, two-way ANOVA, *P < 0.05). (E and F) PARP14 inhibition reduces G3BP1 interaction with RACK1. (E) G3BP1 was immunoprecipitated from OVCAR3 cells treated with 10 µM PARP14i for 24 h and subjected to immunoblotting for MAR, RACK1, and G3BP1 as indicated. The band corresponding to the molecular weight of RACK1 was indicated as MARylated RACK1. The sizes of molecular mass markers in kDa are shown. Each bar in the graph in F represents the mean + SEM of the relative abundance of total RACK1 or MARylated RACK1 in G3BP1 immunoprecipitates ( n = 3, Student’s t test, two-tailed, *P < 0.05). (G and H) PARP14 inhibition reduces G3BP1 interaction with translation factors that are key components of stress granules. (G) G3BP1 was immunoprecipitated from OVCAR3 cells treated with 10 µM PARP14i for 24 h and subjected to immunoblotting for eIF3η, RPS6, and G3BP1 as indicated. The sizes of molecular mass markers in kDa are shown. Each bar in the graph in H represents the mean + SEM of the relative abundance of eIF3η and RPS6 in G3BP1 immunoprecipitates ( n = 3, multiple t test, *P < 0.05). (I and J) PARP14 inhibition reduces G3BP1 localization to stress granules. Immunofluorescent staining assays of OVCAR3 cells treated with 10 µM PARP14i for 24 h and subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). Staining for RACK1 and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. Each bar in the graph in J represents the mean + SEM of the relative abundance of stress granules ( n = 3, Student’s t test, two-tailed, **P < 0.01). Source data are available for this figure: .$

Journal: The Journal of Cell Biology

Article Title: RACK1 MARylation regulates translation and stress granules in ovarian cancer cells

doi: 10.1083/jcb.202401101

Figure Lengend Snippet: PARP14 inhibition reduces stress granule assembly. (A) PARP14i blocks PARP14 autoMARylation. OVCAR3 cells were treated with 10 µM PARP14i (RBN012759) for 24 h. PARP14 was immunoprecipitated (IP) and subjected to immunoblotting for PARP14 and MAR. The size of a molecular mass marker in kDa is shown. (B) Inhibition of PARP14 catalytic activity blocks RACK1 MARylation. PLA using MAR and RACK1 antibodies in OVCAR3 cells treated with 10 µM PARP14i (RBN012759) for 24 h. DNA was stained with DAPI. Scale bar is 15 µm. (C and D) PARP14 inhibition reduces the recruitment of G3BP1 to ribosomes. (C) Immunoblot analysis of RACK1 and G3BP1 in sucrose density gradient fractions of ribosomes prepared from OVCAR3 cells treated with 10 µM PARP14i for 24 h. The sizes of molecular mass markers in kDa are shown. Each bar in the graph in D represents the mean + SEM of the relative abundance of G3BP1 in monosomes or polysomes ( n = 3, two-way ANOVA, *P < 0.05). (E and F) PARP14 inhibition reduces G3BP1 interaction with RACK1. (E) G3BP1 was immunoprecipitated from OVCAR3 cells treated with 10 µM PARP14i for 24 h and subjected to immunoblotting for MAR, RACK1, and G3BP1 as indicated. The band corresponding to the molecular weight of RACK1 was indicated as MARylated RACK1. The sizes of molecular mass markers in kDa are shown. Each bar in the graph in F represents the mean + SEM of the relative abundance of total RACK1 or MARylated RACK1 in G3BP1 immunoprecipitates ( n = 3, Student’s t test, two-tailed, *P < 0.05). (G and H) PARP14 inhibition reduces G3BP1 interaction with translation factors that are key components of stress granules. (G) G3BP1 was immunoprecipitated from OVCAR3 cells treated with 10 µM PARP14i for 24 h and subjected to immunoblotting for eIF3η, RPS6, and G3BP1 as indicated. The sizes of molecular mass markers in kDa are shown. Each bar in the graph in H represents the mean + SEM of the relative abundance of eIF3η and RPS6 in G3BP1 immunoprecipitates ( n = 3, multiple t test, *P < 0.05). (I and J) PARP14 inhibition reduces G3BP1 localization to stress granules. Immunofluorescent staining assays of OVCAR3 cells treated with 10 µM PARP14i for 24 h and subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). Staining for RACK1 and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. Each bar in the graph in J represents the mean + SEM of the relative abundance of stress granules ( n = 3, Student’s t test, two-tailed, **P < 0.01). Source data are available for this figure: .

Techniques: Inhibition, Immunoprecipitation, Western Blot, Marker, Activity Assay, Staining, Molecular Weight, Two Tailed Test

Loss of RACK1 MARylation sensitizes ovarian cancer cells to stress and inhibits their growth. (A) RACK1-Mut–expressing cells are sensitive to ER stress, which inhibits their growth. Growth curves of OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 (WT or Mut) in the presence or absence of 3 nM thapsigargin (Thps) for the indicated times. The arrow points to the RACK-Mut growth curve beneath the RACK-WT growth curve under basal conditions. Each point represents the mean ± SEM of the growth of the cells relative to Day 0 of treatment ( n = 3, two-way ANOVA, *P < 0.01). (B) PARP14 inhibition sensitizes ovarian cancer cells to ER stress and inhibits their growth. Growth curves of OVCAR3 cells in the presence or absence of 10 µM PARP14i and 3 nM thapsigargin (Thps) for the indicated times. Each point represents the mean ± SEM of the growth of the cells relative to Day 0 of treatments ( n = 3, two-way ANOVA, **P < 0.001). (C and D) Expression of RACK1-Mut or treatment with PARP14i inhibits the growth of OVCAR3 xenograft tumors derived from cells like those described in A and B. The xenograft tumors were established in immunocompromised NSG mice subjected to the treatments indicated and grown until the mice reached the endpoint for euthanasia as required by IACUC. (C) Tumor volume at Day 69. Each cluster in the graph shows the mean and the individual data points for n = 10 or 8 mice (WT or Mut, respectively), Student’s t test, two-tailed, P = 0.0155. (D) Tumor volume at Day 19 after treatment. Each cluster in the graph shows the mean and the individual data points for n = 5 or 6 mice (vehicle or PARP14i, respectively), Student’s t test, two-tailed, P = 0.05. Different timelines in the two xenograft experiments were dictated by different growth rates of parental (D) versus Dox-treated cells (C).

Journal: The Journal of Cell Biology

Article Title: RACK1 MARylation regulates translation and stress granules in ovarian cancer cells

doi: 10.1083/jcb.202401101

Figure Lengend Snippet: Loss of RACK1 MARylation sensitizes ovarian cancer cells to stress and inhibits their growth. (A) RACK1-Mut–expressing cells are sensitive to ER stress, which inhibits their growth. Growth curves of OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 (WT or Mut) in the presence or absence of 3 nM thapsigargin (Thps) for the indicated times. The arrow points to the RACK-Mut growth curve beneath the RACK-WT growth curve under basal conditions. Each point represents the mean ± SEM of the growth of the cells relative to Day 0 of treatment ( n = 3, two-way ANOVA, *P < 0.01). (B) PARP14 inhibition sensitizes ovarian cancer cells to ER stress and inhibits their growth. Growth curves of OVCAR3 cells in the presence or absence of 10 µM PARP14i and 3 nM thapsigargin (Thps) for the indicated times. Each point represents the mean ± SEM of the growth of the cells relative to Day 0 of treatments ( n = 3, two-way ANOVA, **P < 0.001). (C and D) Expression of RACK1-Mut or treatment with PARP14i inhibits the growth of OVCAR3 xenograft tumors derived from cells like those described in A and B. The xenograft tumors were established in immunocompromised NSG mice subjected to the treatments indicated and grown until the mice reached the endpoint for euthanasia as required by IACUC. (C) Tumor volume at Day 69. Each cluster in the graph shows the mean and the individual data points for n = 10 or 8 mice (WT or Mut, respectively), Student’s t test, two-tailed, P = 0.0155. (D) Tumor volume at Day 19 after treatment. Each cluster in the graph shows the mean and the individual data points for n = 5 or 6 mice (vehicle or PARP14i, respectively), Student’s t test, two-tailed, P = 0.05. Different timelines in the two xenograft experiments were dictated by different growth rates of parental (D) versus Dox-treated cells (C).

Techniques: Expressing, Knockdown, Inhibition, Derivative Assay, Two Tailed Test

PARP14 inhibition sensitizes ovarian cancer cells to stress and inhibits their growth. (A and B) Growth curves in the presence or absence of 10 µM PARP14i and 3 nM thapsigargin (Thps) for the indicated times. (A) SKOV3 cells and (B) HCC5044 cells. Each point represents the mean ± SEM of the growth of the cells relative to Day 0 of treatments ( n = 3, two-way ANOVA, **P < 0.001). (C) RACK1-Mut expressing cells are sensitive to ER stress, which inhibits their growth. Growth curves of OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 (WT or Mut) in the presence or absence of 5 µM carboplatin for the indicated times. Each point represents the mean ± SEM of the growth of the cells relative to Day 0 of treatment ( n = 3, two-way ANOVA, *P < 0.05). (D) PARP14 inhibition sensitizes ovarian cancer cells to ER stress and inhibits their growth. Growth curves of OVCAR3 cells in the presence or absence of 10 µM PARP14i and 5 µM carboplatin for the indicated times. Each point represents the mean ± SEM of the growth of the cells relative to Day 0 of treatment ( n = 5, two-way ANOVA, *P < 0.05). (E–G) Growth curves of OVCAR3 xenograft tumors in immunocompromised NSG mice. The xenograft tumors were established in immunocompromised NSG mice subjected to the experimental conditions and treatments indicated and grown until the mice reached the endpoint for euthanasia as required by IACUC. (E) Growth of OVCAR3 xenograft tumors with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 (WT or Mut) for the indicated times. n = 10 or 8 mice (WT or Mut, respectively), Student’s t test, two-tailed, *P < 0.05; **P < 0.02. (F) Immunoblot analysis of HA-RACK (WT or Mut) expression in the tumors at the end of the experiment. The sizes of molecular mass markers in kDa are shown. (G) Growth of OVCAR3 xenograft tumors with or without PARP14i treatment for the indicated times. n = 5 or 6 mice (vehicle or PARP14i, respectively), Student’s t test, two-tailed, *P < 0.05. (H) RACK1-Mut expressing OVCAR3 cells exhibit greater ER stress. Immunoblot analysis of lysates from OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 (WT or Mut) the presence or absence of 3 nM thapsigargin (Thps) for 24 h as indicated. Blotting for phospho-eIF2a (p-eIF2a), total eIF2a (eIF2a), cleaved caspase-3 (cl Caspase-3), HA, and β-tubulin (loading control) as indicated. The sizes of molecular mass markers in kDa are shown. (I–K) PARP14i-treated cells exhibit greater ER stress. Immunoblot analysis of lysates from (I) OVCAR3, (J) SKOV3, and (K) HCC5044 cells treated with 10 µM PARP14i and 3 nM thapsigargin for 24 h. Blotting for phospho-eIF2a (p-eIF2a), total eIF2a (eIF2a), cleaved caspase-3 (cl Caspase-3), and β-tubulin (loading control) as indicated. The sizes of molecular mass markers in kDa are shown. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: RACK1 MARylation regulates translation and stress granules in ovarian cancer cells

doi: 10.1083/jcb.202401101

Figure Lengend Snippet: PARP14 inhibition sensitizes ovarian cancer cells to stress and inhibits their growth. (A and B) Growth curves in the presence or absence of 10 µM PARP14i and 3 nM thapsigargin (Thps) for the indicated times. (A) SKOV3 cells and (B) HCC5044 cells. Each point represents the mean ± SEM of the growth of the cells relative to Day 0 of treatments ( n = 3, two-way ANOVA, **P < 0.001). (C) RACK1-Mut expressing cells are sensitive to ER stress, which inhibits their growth. Growth curves of OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 (WT or Mut) in the presence or absence of 5 µM carboplatin for the indicated times. Each point represents the mean ± SEM of the growth of the cells relative to Day 0 of treatment ( n = 3, two-way ANOVA, *P < 0.05). (D) PARP14 inhibition sensitizes ovarian cancer cells to ER stress and inhibits their growth. Growth curves of OVCAR3 cells in the presence or absence of 10 µM PARP14i and 5 µM carboplatin for the indicated times. Each point represents the mean ± SEM of the growth of the cells relative to Day 0 of treatment ( n = 5, two-way ANOVA, *P < 0.05). (E–G) Growth curves of OVCAR3 xenograft tumors in immunocompromised NSG mice. The xenograft tumors were established in immunocompromised NSG mice subjected to the experimental conditions and treatments indicated and grown until the mice reached the endpoint for euthanasia as required by IACUC. (E) Growth of OVCAR3 xenograft tumors with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 (WT or Mut) for the indicated times. n = 10 or 8 mice (WT or Mut, respectively), Student’s t test, two-tailed, *P < 0.05; **P < 0.02. (F) Immunoblot analysis of HA-RACK (WT or Mut) expression in the tumors at the end of the experiment. The sizes of molecular mass markers in kDa are shown. (G) Growth of OVCAR3 xenograft tumors with or without PARP14i treatment for the indicated times. n = 5 or 6 mice (vehicle or PARP14i, respectively), Student’s t test, two-tailed, *P < 0.05. (H) RACK1-Mut expressing OVCAR3 cells exhibit greater ER stress. Immunoblot analysis of lysates from OVCAR3 cells with Dox-induced knockdown of endogenous RACK1 and re-expression of exogenous RACK1 (WT or Mut) the presence or absence of 3 nM thapsigargin (Thps) for 24 h as indicated. Blotting for phospho-eIF2a (p-eIF2a), total eIF2a (eIF2a), cleaved caspase-3 (cl Caspase-3), HA, and β-tubulin (loading control) as indicated. The sizes of molecular mass markers in kDa are shown. (I–K) PARP14i-treated cells exhibit greater ER stress. Immunoblot analysis of lysates from (I) OVCAR3, (J) SKOV3, and (K) HCC5044 cells treated with 10 µM PARP14i and 3 nM thapsigargin for 24 h. Blotting for phospho-eIF2a (p-eIF2a), total eIF2a (eIF2a), cleaved caspase-3 (cl Caspase-3), and β-tubulin (loading control) as indicated. The sizes of molecular mass markers in kDa are shown. Source data are available for this figure: .

Techniques: Inhibition, Expressing, Knockdown, Two Tailed Test, Western Blot, Control

Depletion of TARG1 enhances stress granule assembly by increasing RACK1 MARylation. (A and B) siRNA-mediated TARG1 depletion increases RACK1 MARylation and enhances RACK1 interaction with G3BP1 in OVCAR3 cells subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). (A) RACK1 was immunoprecipitated (IP) from OVCAR3 cells with siRNA-mediated knockdown of TARG1 and subjected to immunoblotting for G3BP1, MAR, and RACK1. The sizes of molecular mass markers in kDa are shown. (B) PLA using MAR and RACK1 antibodies. DNA was stained with DAPI. Scale bar is 15 µm. (C and D) TARG1 knockdown increases the assembly of G3BP1-containing stress granules. Immunofluorescent staining assays of OVCAR3 cells with siRNA-mediated knockdown of TARG1 subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). (C) RACK1 and G3BP1, (D) RPS6 and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. (E) Changes in mRNA translation upon depletion of TARG1. Scatter plot of fold changes in ribosome profiling and RNA-seq (OVCAR3 cells subjected to siRNA-mediated TARG1 knockdown versus siControl) comparing translational control and transcriptional control. Gene ontology enrichment analysis of the mRNAs regulated at transcriptional and translational levels is shown. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: RACK1 MARylation regulates translation and stress granules in ovarian cancer cells

doi: 10.1083/jcb.202401101

Figure Lengend Snippet: Depletion of TARG1 enhances stress granule assembly by increasing RACK1 MARylation. (A and B) siRNA-mediated TARG1 depletion increases RACK1 MARylation and enhances RACK1 interaction with G3BP1 in OVCAR3 cells subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). (A) RACK1 was immunoprecipitated (IP) from OVCAR3 cells with siRNA-mediated knockdown of TARG1 and subjected to immunoblotting for G3BP1, MAR, and RACK1. The sizes of molecular mass markers in kDa are shown. (B) PLA using MAR and RACK1 antibodies. DNA was stained with DAPI. Scale bar is 15 µm. (C and D) TARG1 knockdown increases the assembly of G3BP1-containing stress granules. Immunofluorescent staining assays of OVCAR3 cells with siRNA-mediated knockdown of TARG1 subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). (C) RACK1 and G3BP1, (D) RPS6 and G3BP1. DNA was stained with DAPI. Scale bar is 15 µm. (E) Changes in mRNA translation upon depletion of TARG1. Scatter plot of fold changes in ribosome profiling and RNA-seq (OVCAR3 cells subjected to siRNA-mediated TARG1 knockdown versus siControl) comparing translational control and transcriptional control. Gene ontology enrichment analysis of the mRNAs regulated at transcriptional and translational levels is shown. Source data are available for this figure: .

Techniques: Immunoprecipitation, Knockdown, Western Blot, Staining, RNA Sequencing Assay, Control

Depletion of TARG1 enhances stress granule assembly by increasing RACK1 MARylation. (A) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of the level of G3BP1 in RACK1 immunoprecipitates ( n = 4, one-way ANOVA, *P < 0.05). (B) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of MAR-RACK1 PLA foci ( n = 3, Student’s t test, two-tailed, ***P < 0.001). (C) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of distinct G3BP1 foci ( n = 3, Student’s t test, two-tailed, *P < 0.05, **P < 0.01). (D and E) siRNA-mediated TARG1 depletion increases RACK1 MARylation in (D) SKOV3 cells and (E) HCC5044 cells subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). PLA using MAR and RACK1 antibodies. DNA was stained with DAPI. Scale bar is 15 µm. (F and G) siRNA-mediated TARG1 depletion increases the assembly of G3BP1-containing stress granules. Immunofluorescent staining assays for RACK1 and G3BP1 in (F) SKOV3 cells and (G) HCC5044 cells with siRNA-mediated knockdown of TARG1 subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). DNA was stained with DAPI. Scale bar is 15 µm. (H) Quantification of multiple experiments like the one shown in , top. Each bar represents the mean + SEM of TARG1-RACK1 PLA foci ( n = 4, ANOVA, *P < 0.05, ***P < 0.001). (I) Quantification of multiple experiments like the one shown in , bottom. Each bar represents the mean + SEM of MAR-RACK1 PLA foci ( n = 4, ANOVA, *P < 0.05, **P < 0.01).

Journal: The Journal of Cell Biology

Article Title: RACK1 MARylation regulates translation and stress granules in ovarian cancer cells

doi: 10.1083/jcb.202401101

Figure Lengend Snippet: Depletion of TARG1 enhances stress granule assembly by increasing RACK1 MARylation. (A) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of the level of G3BP1 in RACK1 immunoprecipitates ( n = 4, one-way ANOVA, *P < 0.05). (B) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of MAR-RACK1 PLA foci ( n = 3, Student’s t test, two-tailed, ***P < 0.001). (C) Quantification of multiple experiments like the one shown in . Each bar represents the mean + SEM of distinct G3BP1 foci ( n = 3, Student’s t test, two-tailed, *P < 0.05, **P < 0.01). (D and E) siRNA-mediated TARG1 depletion increases RACK1 MARylation in (D) SKOV3 cells and (E) HCC5044 cells subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). PLA using MAR and RACK1 antibodies. DNA was stained with DAPI. Scale bar is 15 µm. (F and G) siRNA-mediated TARG1 depletion increases the assembly of G3BP1-containing stress granules. Immunofluorescent staining assays for RACK1 and G3BP1 in (F) SKOV3 cells and (G) HCC5044 cells with siRNA-mediated knockdown of TARG1 subjected to 15 min of treatment with 250 µM sodium arsenite (NaAsO 2 ). DNA was stained with DAPI. Scale bar is 15 µm. (H) Quantification of multiple experiments like the one shown in , top. Each bar represents the mean + SEM of TARG1-RACK1 PLA foci ( n = 4, ANOVA, *P < 0.05, ***P < 0.001). (I) Quantification of multiple experiments like the one shown in , bottom. Each bar represents the mean + SEM of MAR-RACK1 PLA foci ( n = 4, ANOVA, *P < 0.05, **P < 0.01).

Techniques: Two Tailed Test, Staining, Knockdown

Prolonged exposure to stress reduces RACK1 MARylation. (A and B) Stress reduces RACK1 MARylation through TARG1. (A) RACK1 was immunoprecipitated (IP) from OVCAR3 cells treated with 250 µM sodium arsenite (NaAsO 2 ) for 30 min or 250 nM thapsigargin (Thps) for 2 h, and subjected to immunoblotting for MAR and RACK1. The size of a molecular mass marker in kDa is shown. (B) PLA in OVCAR3 cells with siRNA-mediated knockdown of TARG1 using TARG1 and RACK1 (top) or MAR and RACK1 (bottom) antibodies. The cells were treated with sodium arsenite (NaAsO 2 ) treatment for 30 min. DNA was stained with DAPI. Scale bar is 15 µm. (C) Knockdown of TARG1 increases the assembly of G3BP1-containing stress granules. PLA for TARG1 and RACK1 combined with immunofluorescent imaging of OVCAR3 cells expressing GFP-G3BP1 and treated with 250 µM sodium arsenite (NaAsO 2 ) for 15 min. DNA was stained with DAPI. Scale bar is 15 µm. (D) Schematic of the mechanisms by which PARP14 and TARG1 regulate stress granule (SG) assembly through RACK1 MARylation. Additional details are provided in the text. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: RACK1 MARylation regulates translation and stress granules in ovarian cancer cells

doi: 10.1083/jcb.202401101

Figure Lengend Snippet: Prolonged exposure to stress reduces RACK1 MARylation. (A and B) Stress reduces RACK1 MARylation through TARG1. (A) RACK1 was immunoprecipitated (IP) from OVCAR3 cells treated with 250 µM sodium arsenite (NaAsO 2 ) for 30 min or 250 nM thapsigargin (Thps) for 2 h, and subjected to immunoblotting for MAR and RACK1. The size of a molecular mass marker in kDa is shown. (B) PLA in OVCAR3 cells with siRNA-mediated knockdown of TARG1 using TARG1 and RACK1 (top) or MAR and RACK1 (bottom) antibodies. The cells were treated with sodium arsenite (NaAsO 2 ) treatment for 30 min. DNA was stained with DAPI. Scale bar is 15 µm. (C) Knockdown of TARG1 increases the assembly of G3BP1-containing stress granules. PLA for TARG1 and RACK1 combined with immunofluorescent imaging of OVCAR3 cells expressing GFP-G3BP1 and treated with 250 µM sodium arsenite (NaAsO 2 ) for 15 min. DNA was stained with DAPI. Scale bar is 15 µm. (D) Schematic of the mechanisms by which PARP14 and TARG1 regulate stress granule (SG) assembly through RACK1 MARylation. Additional details are provided in the text. Source data are available for this figure: .

Techniques: Immunoprecipitation, Western Blot, Marker, Knockdown, Staining, Imaging, Expressing

Fig. 2 Immunocytochemical analysis demonstrates expression of HA-tagged TDP-43ΔNLS (a, bottom row) or FUSΔNLS mutants, R495x- and P525L-FUS (b, middle & bottom rows), but not WT TDP-43 (a, top row) or WT-FUS (b, top row), which are primarily localized to the nucleus, is associated with co-aggregation of endogenous RACK1 in HEK293T cells. Nuclei stained with DAPI in merged images. Scale bars: 20 μm

Journal: Acta neuropathologica communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration.

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Fig. 2 Immunocytochemical analysis demonstrates expression of HA-tagged TDP-43ΔNLS (a, bottom row) or FUSΔNLS mutants, R495x- and P525L-FUS (b, middle & bottom rows), but not WT TDP-43 (a, top row) or WT-FUS (b, top row), which are primarily localized to the nucleus, is associated with co-aggregation of endogenous RACK1 in HEK293T cells. Nuclei stained with DAPI in merged images. Scale bars: 20 μm

Article Snippet: RACK1 KD was achieved by transfection of a pool of 3 target-specific siRNA plasmids against human RACK1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-36354) using Lipofectamine RNAiMAX reagent (ThermoFisher) according to the manufacturer’s instruction, and incubated for 72 h prior to cDNA plasmid transfection where indicated.

Techniques: Expressing, Staining

Fig. 1 Immunohistochemical analysis shows co-aggregation of RACK1 with FUS in the cytoplasm (arrows) in spinal cord sections of a fALS-FUS-R521C case, in contrast to normal nuclear expression of FUS in control (Ctrl). Nuclei stained with DAPI in merged images. Scale bar: 10 μm

Journal: Acta neuropathologica communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration.

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Fig. 1 Immunohistochemical analysis shows co-aggregation of RACK1 with FUS in the cytoplasm (arrows) in spinal cord sections of a fALS-FUS-R521C case, in contrast to normal nuclear expression of FUS in control (Ctrl). Nuclei stained with DAPI in merged images. Scale bar: 10 μm

Techniques: Immunohistochemical staining, Expressing, Control, Staining

Fig. 3 Immunocytochemical analysis demonstrates reactivity of RACK1 misfolding specific antibody “RACK1mis” with RACK1 in cytoplasmic aggregates of HA-tagged TDP-43ΔNLS (middle row) or R495x-FUS (bottom row) transfected HEK293T cells but not with diffuse, non-aggregated RACK1 in the cytoplasm (stained by a Pan RACK1 antibody). RACK1mis shows no reactivity with endogenous, physiological RACK1 in un-transfected (UT, top row) cells. Nuclei stained with DAPI in merged images. Scale bars: 10 μm

Journal: Acta neuropathologica communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration.

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Fig. 3 Immunocytochemical analysis demonstrates reactivity of RACK1 misfolding specific antibody “RACK1mis” with RACK1 in cytoplasmic aggregates of HA-tagged TDP-43ΔNLS (middle row) or R495x-FUS (bottom row) transfected HEK293T cells but not with diffuse, non-aggregated RACK1 in the cytoplasm (stained by a Pan RACK1 antibody). RACK1mis shows no reactivity with endogenous, physiological RACK1 in un-transfected (UT, top row) cells. Nuclei stained with DAPI in merged images. Scale bars: 10 μm

Techniques: Transfection, Staining

Fig. 4 RACK1 knockdown alleviates aggregation and global translational suppression by TDP-43ΔNLS and WT over-expression in HEK293T cells. a Representative SUnSET-Western Blot (WB) demonstrates that compared to control, expression of HA-tagged TDP-43ΔNLS and to a lesser extent over-expression of WT TDP-43, induces a significant reduction in puromycin (PMY) incorporation. b RACK1 siRNA KD alleviates global translational levels in both cases as determined by quantification of PMY band intensities normalized to loading control β-actin. c SUnSET-ICC shows inhibition of PMY incorporation by TDP-43ΔNLS (HA) expression preferably occurs in cells containing distinctive cytoplasmic aggregates (asterisks, top row). In cells where TDP-43ΔNLS displays a filamentary expression pattern (arrows, bottom row), PMY incorporation is comparable to neighbouring HA negative UT cells. d RACK1 KD alleviates aggregation, resulting in diffuse cytoplasmic expression (top row) or predominantly nuclear localization (bottom row) of TDP-43ΔNLS in a sub-population of transfected cells and correlating with normal PMY incorporation compared to neighbouring HA-negative UT cells. Nuclei stained with DAPI in merged images. Statistics: Ordinary one-way ANOVA Tukey multiple comparisons. n = 4 *p < 0.05; ***p < 0.001; ****p < 0.0001. Scale bars: 20 μm

Journal: Acta neuropathologica communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration.

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Fig. 4 RACK1 knockdown alleviates aggregation and global translational suppression by TDP-43ΔNLS and WT over-expression in HEK293T cells. a Representative SUnSET-Western Blot (WB) demonstrates that compared to control, expression of HA-tagged TDP-43ΔNLS and to a lesser extent over-expression of WT TDP-43, induces a significant reduction in puromycin (PMY) incorporation. b RACK1 siRNA KD alleviates global translational levels in both cases as determined by quantification of PMY band intensities normalized to loading control β-actin. c SUnSET-ICC shows inhibition of PMY incorporation by TDP-43ΔNLS (HA) expression preferably occurs in cells containing distinctive cytoplasmic aggregates (asterisks, top row). In cells where TDP-43ΔNLS displays a filamentary expression pattern (arrows, bottom row), PMY incorporation is comparable to neighbouring HA negative UT cells. d RACK1 KD alleviates aggregation, resulting in diffuse cytoplasmic expression (top row) or predominantly nuclear localization (bottom row) of TDP-43ΔNLS in a sub-population of transfected cells and correlating with normal PMY incorporation compared to neighbouring HA-negative UT cells. Nuclei stained with DAPI in merged images. Statistics: Ordinary one-way ANOVA Tukey multiple comparisons. n = 4 *p < 0.05; ***p < 0.001; ****p < 0.0001. Scale bars: 20 μm

Techniques: Knockdown, Over Expression, Western Blot, Control, Expressing, Inhibition, Transfection, Staining

Fig. 5 RACK1 knockdown alleviates aggregation and restores global translational suppression by FUSΔNLS mutants. a Representative SUnSET-Western Blot (WB) demonstrates that compared to control empty vector (EV) transfected cells, expression of HA-tagged FUSΔNLS mutants, R495x- and P525L-FUS, and to a lesser extent over-expression of WT FUS, induces a significant reduction in puromycin (PMY) incorporation. b siRNA KD alleviates global translational levels in all cases as determined by quantification of PMY band intensities normalized to loading control α-tubulin. c SUnSET-ICC demonstrates HA-tagged FUSΔNLS mutants, R495x- and P525L-FUS, cause aggregation and completely inhibit PMY incorporation in transfected cells (asterisks). d RACK1 KD alleviates aggregation, resulting in diffuse cytoplasmic expression or predominantly nuclear localization of HA-tagged FUSΔNLS mutants, R495x- and P525L-FUS, in a sub-population of transfected cells and correlating with normal PMY incorporation compared to neighbouring HA-negative UT cells. Nuclei stained with DAPI in merged images. Statistics: Student’s t-test, unpaired, two-tailed. *p < 0.05 and **p < 0.005. n = 5 Error bars: SEM. Scale bars: 20 μm

Journal: Acta neuropathologica communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration.

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Fig. 5 RACK1 knockdown alleviates aggregation and restores global translational suppression by FUSΔNLS mutants. a Representative SUnSET-Western Blot (WB) demonstrates that compared to control empty vector (EV) transfected cells, expression of HA-tagged FUSΔNLS mutants, R495x- and P525L-FUS, and to a lesser extent over-expression of WT FUS, induces a significant reduction in puromycin (PMY) incorporation. b siRNA KD alleviates global translational levels in all cases as determined by quantification of PMY band intensities normalized to loading control α-tubulin. c SUnSET-ICC demonstrates HA-tagged FUSΔNLS mutants, R495x- and P525L-FUS, cause aggregation and completely inhibit PMY incorporation in transfected cells (asterisks). d RACK1 KD alleviates aggregation, resulting in diffuse cytoplasmic expression or predominantly nuclear localization of HA-tagged FUSΔNLS mutants, R495x- and P525L-FUS, in a sub-population of transfected cells and correlating with normal PMY incorporation compared to neighbouring HA-negative UT cells. Nuclei stained with DAPI in merged images. Statistics: Student’s t-test, unpaired, two-tailed. *p < 0.05 and **p < 0.005. n = 5 Error bars: SEM. Scale bars: 20 μm

Techniques: Knockdown, Western Blot, Control, Plasmid Preparation, Transfection, Expressing, Over Expression, Staining, Two Tailed Test

Fig. 6 Immunocytochemical analysis shows co-aggregation of HA-tagged TDP-43ΔNLS (a, b) or R495x-FUS (c, d) with RACK1 and eukaryotic 40S (Rps6) and 60S (RPL14) ribosome subunits (arrows). In neighbouring HA-negative UT cells, RACK1 is diffusely localized in the cytoplasm. Nuclei stained with DAPI in merged images. Scale bars: 10 μm

Journal: Acta neuropathologica communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration.

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Fig. 6 Immunocytochemical analysis shows co-aggregation of HA-tagged TDP-43ΔNLS (a, b) or R495x-FUS (c, d) with RACK1 and eukaryotic 40S (Rps6) and 60S (RPL14) ribosome subunits (arrows). In neighbouring HA-negative UT cells, RACK1 is diffusely localized in the cytoplasm. Nuclei stained with DAPI in merged images. Scale bars: 10 μm

Techniques: Staining

$Fig. 7 RACK1 knockdown not only reduces cytoplasmic aggregation but also increases nuclear localization of TDP-43ΔNLS in HEK293T cells. a Representative images showing that in contrast to control cells where distinctive aggregates of HA-tagged TDP-43ΔNLS are associated with RACK1 co-aggregation in the cytoplasm (arrows), RACK1 siRNA KD not only alleviates cytoplasmic aggregation (white arrowheads) but also leads to nuclear localization (yellow arrowheads) of TDP-43ΔNLS in a sub-population of transfected cells. Nuclei stained with DAPI in merged images. Scale bar: 10 μm. b Quantification of the average aggregate size in each individual TDP-43ΔNLS transfected cells shows a decrease following RACK1 KD. c Quantification of TDP-43ΔNLS nuclear localization from 3 biological repeats shows an increase as a result of RACK1 KD. d Western blot analysis confirms the quality of nucleocytoplasmic fractionation using nuclear and cytosolic protein markers, lamin B1 and β-Actin, respectively (top). TDP-43ΔNLS (HA) or total TDP-43 (pan TDP-43) from either fraction was normalized to β-actin or lamin B1. Quantification by densitometry shows that RACK1 KD decreases the cytoplasmic to nuclear ratio, fragmentation (red arrows, observed in 3 independent experiments), and phosphorylation of TDP-43ΔNLS (quantitated in one experiment). Statistics: Student’s t-test unpaired two-tailed. Error bars: SEM$

Journal: Acta neuropathologica communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration.

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Fig. 7 RACK1 knockdown not only reduces cytoplasmic aggregation but also increases nuclear localization of TDP-43ΔNLS in HEK293T cells. a Representative images showing that in contrast to control cells where distinctive aggregates of HA-tagged TDP-43ΔNLS are associated with RACK1 co-aggregation in the cytoplasm (arrows), RACK1 siRNA KD not only alleviates cytoplasmic aggregation (white arrowheads) but also leads to nuclear localization (yellow arrowheads) of TDP-43ΔNLS in a sub-population of transfected cells. Nuclei stained with DAPI in merged images. Scale bar: 10 μm. b Quantification of the average aggregate size in each individual TDP-43ΔNLS transfected cells shows a decrease following RACK1 KD. c Quantification of TDP-43ΔNLS nuclear localization from 3 biological repeats shows an increase as a result of RACK1 KD. d Western blot analysis confirms the quality of nucleocytoplasmic fractionation using nuclear and cytosolic protein markers, lamin B1 and β-Actin, respectively (top). TDP-43ΔNLS (HA) or total TDP-43 (pan TDP-43) from either fraction was normalized to β-actin or lamin B1. Quantification by densitometry shows that RACK1 KD decreases the cytoplasmic to nuclear ratio, fragmentation (red arrows, observed in 3 independent experiments), and phosphorylation of TDP-43ΔNLS (quantitated in one experiment). Statistics: Student’s t-test unpaired two-tailed. Error bars: SEM

Techniques: Knockdown, Control, Transfection, Staining, Western Blot, Fractionation, Phospho-proteomics, Two Tailed Test

Fig. 8 RACK1 knockdown inhibits intercellular transmission of TDP-43ΔNLS in HEK293T cells. a Schematic illustration of the intercellular transmission procedure. Representative western blot (WB) b and densitometric quantification show reduced total HA-tagged TDP-43ΔNLS transmitted to naïve recipient cells c as well as a reduction in the recipient/donor ratio of TDP-43ΔNLS upon RACK1 KD. Statistics: Student’s t-test, unpaired two-tailed. **p < 0.005; ****p < 0.0001. n = 5

Journal: Acta neuropathologica communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration.

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Fig. 8 RACK1 knockdown inhibits intercellular transmission of TDP-43ΔNLS in HEK293T cells. a Schematic illustration of the intercellular transmission procedure. Representative western blot (WB) b and densitometric quantification show reduced total HA-tagged TDP-43ΔNLS transmitted to naïve recipient cells c as well as a reduction in the recipient/donor ratio of TDP-43ΔNLS upon RACK1 KD. Statistics: Student’s t-test, unpaired two-tailed. **p < 0.005; ****p < 0.0001. n = 5

Techniques: Knockdown, Transmission Assay, Western Blot, Two Tailed Test

Fig. 9 RACK1 knockdown significantly reduces hTDP-43-induced neuronal death and dysfunction in transgenic D. melanogaster. a Schematic illustration for the construction of transgenic D. melanogaster lines specifically targeting retinal (GMR-driven) or motor (D42-driven) neurons. b Representative images of fly eyes. Expression of hTDP-43WT (A–D) or hTDP-43Q331K (E–H) in retinal neurons cause neuronal death in both males and females, detected as darkened ommatidia. RACK1-RNAi reduces the appearance of dead ommatidia in all four conditions (B, D, F, H), compared to mCherry-RNAi control (A, C, E, G). Control flies with no dead ommatidia include those expressing RACK1-RNAi alone (I), and those harbouring unexpressed (undriven) transgenes (J–L). Scale bar: 250 mm. c Quantification of retinal neuronal death. Mann–Whitney test indicates a significant reduction in ommatidia death upon RACK1-RNAi KD in both males and females expressing either hTDP-43WT or hTDP-43Q331K. 21–33 flies were scored per condition. Statistics: Mann–Whitney test, *p < 0.05, ***p < 0.001. ****p < 0.0001. d Histological analysis of retina from flies expressing hTDP-43WT (A, B) or hTDP-43Q331K (C, D) in conjunction with control mCherry-RNAi (A, C) or RACK1-RNAi (B, D) show preservation of retinal architecture by RACK1 KD. Scale bar: 50 mm. e Both male and female hTDP-43WT transgenic flies show reduced climbing ability compared to mCherry-RNAi controls, which was significantly improved by RACK1-RNAi. Statistics: 2-way ANOVA. **p < 0.01, ****p < 0.0001. 60–100 flies were scored per condition. Error bars: SD

Journal: Acta neuropathologica communications

Article Title: Targeting RACK1 to alleviate TDP-43 and FUS proteinopathy-mediated suppression of protein translation and neurodegeneration.

doi: 10.1186/s40478-023-01705-8

Figure Lengend Snippet: Fig. 9 RACK1 knockdown significantly reduces hTDP-43-induced neuronal death and dysfunction in transgenic D. melanogaster. a Schematic illustration for the construction of transgenic D. melanogaster lines specifically targeting retinal (GMR-driven) or motor (D42-driven) neurons. b Representative images of fly eyes. Expression of hTDP-43WT (A–D) or hTDP-43Q331K (E–H) in retinal neurons cause neuronal death in both males and females, detected as darkened ommatidia. RACK1-RNAi reduces the appearance of dead ommatidia in all four conditions (B, D, F, H), compared to mCherry-RNAi control (A, C, E, G). Control flies with no dead ommatidia include those expressing RACK1-RNAi alone (I), and those harbouring unexpressed (undriven) transgenes (J–L). Scale bar: 250 mm. c Quantification of retinal neuronal death. Mann–Whitney test indicates a significant reduction in ommatidia death upon RACK1-RNAi KD in both males and females expressing either hTDP-43WT or hTDP-43Q331K. 21–33 flies were scored per condition. Statistics: Mann–Whitney test, *p < 0.05, ***p < 0.001. ****p < 0.0001. d Histological analysis of retina from flies expressing hTDP-43WT (A, B) or hTDP-43Q331K (C, D) in conjunction with control mCherry-RNAi (A, C) or RACK1-RNAi (B, D) show preservation of retinal architecture by RACK1 KD. Scale bar: 50 mm. e Both male and female hTDP-43WT transgenic flies show reduced climbing ability compared to mCherry-RNAi controls, which was significantly improved by RACK1-RNAi. Statistics: 2-way ANOVA. **p < 0.01, ****p < 0.0001. 60–100 flies were scored per condition. Error bars: SD

Techniques: Knockdown, Transgenic Assay, Expressing, Control, MANN-WHITNEY, Preserving

RACK1 promotes self-renewal and chemoresistance of human liver CSCs and maintains murine ESC function. ( A-N ) 96 h after HuH7 cells were infected with lentivirus expressing non-targeting control (NC) shRNA or RACK1 shRNA ( A-G ), or after HuH7 single clone stably expressing FLAG-RACK1 and the mock control were generated ( H-N ), cells were subjected to the following assays: ( A,H ) Immunoblotting (IB) analysis of RACK1 expression in total cells. ( B,I ) Flow cytometric analysis of CD13 and CD133 expression in total cells. ( C,J ) Immunoblotting analysis of RACK1 expression in sorted CD13+ and CD13- subpopulations. ( D,K ) Sphere formation assays of sorted CD13+ subpopulation. mean±s.d. ( n =3); * P <0.05, ** P <0.01. ( E,L ) In vivo tumorigenicity experiments of sorted CD13+ subpopulation (5000 cells/site, 7 weeks, n =6 ) . ( F,M ) Etoposide (Etop, 100 μM, 48 h)- or sorafenib (Sora, 50 μM, 24 h)-induced apoptosis of CD13+ subpopulation. mean±s.d. ( n =3); Ctrl, control. ( G,N ) qRT-PCR analysis of sorted CD13+ subpopulation for the expression of the indicated drug-resistant relative genes. mean±s.d. ( n =3). ( O - Q ) 96 h after murine ESCs were infected with lentivirus expressing non-targeting control shRNA or RACK1 shRNAs, cells were subjected to immunoblotting analysis for RACK1 expression ( O ), colony formation assays (mean±s.d., n =3; scale bar: 1 cm) ( P ), and alkaline phosphatase (AP) activity assays (mean±s.d., n =3) ( Q ).

Journal: Theranostics

Article Title: RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog

doi: 10.7150/thno.29271

Figure Lengend Snippet: RACK1 promotes self-renewal and chemoresistance of human liver CSCs and maintains murine ESC function. ( A-N ) 96 h after HuH7 cells were infected with lentivirus expressing non-targeting control (NC) shRNA or RACK1 shRNA ( A-G ), or after HuH7 single clone stably expressing FLAG-RACK1 and the mock control were generated ( H-N ), cells were subjected to the following assays: ( A,H ) Immunoblotting (IB) analysis of RACK1 expression in total cells. ( B,I ) Flow cytometric analysis of CD13 and CD133 expression in total cells. ( C,J ) Immunoblotting analysis of RACK1 expression in sorted CD13+ and CD13- subpopulations. ( D,K ) Sphere formation assays of sorted CD13+ subpopulation. mean±s.d. ( n =3); * P <0.05, ** P <0.01. ( E,L ) In vivo tumorigenicity experiments of sorted CD13+ subpopulation (5000 cells/site, 7 weeks, n =6 ) . ( F,M ) Etoposide (Etop, 100 μM, 48 h)- or sorafenib (Sora, 50 μM, 24 h)-induced apoptosis of CD13+ subpopulation. mean±s.d. ( n =3); Ctrl, control. ( G,N ) qRT-PCR analysis of sorted CD13+ subpopulation for the expression of the indicated drug-resistant relative genes. mean±s.d. ( n =3). ( O - Q ) 96 h after murine ESCs were infected with lentivirus expressing non-targeting control shRNA or RACK1 shRNAs, cells were subjected to immunoblotting analysis for RACK1 expression ( O ), colony formation assays (mean±s.d., n =3; scale bar: 1 cm) ( P ), and alkaline phosphatase (AP) activity assays (mean±s.d., n =3) ( Q ).

Article Snippet: Another set of lentivirus-based human RACK1 shRNA (RACK1-b) and control lentivirus were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat. No. sc-36354-v).

Techniques: Infection, Expressing, Control, shRNA, Stable Transfection, Generated, Western Blot, In Vivo, Quantitative RT-PCR, Activity Assay

Enhancement of Nanog expression by RACK1 in human HCC cells and murine ESCs. ( A-C ) Immunoblotting analysis of the expression of the indicated stemness-associated genes upon RACK1 knockdown ( A,B ) or over-expression ( C ) in HuH7 cells. ( D,E ) Analysis of Hedgehog signaling activity upon RACK1 knockdown ( D ) or over-expression ( E ) in HuH7 cells as indicated by 7Gli-GFP reporter. mean±s.d. ( n =3); * P <0.05, ** P <0.01. ( F-I ) Immunoblotting analysis of Nanog expression upon RACK1 knockdown ( F,H,I ) or over-expression ( G ) in sorted CD13+ and CD13- HuH7 subpopulations ( F,G ), in other human HCC cells ( H ), or in murine ESCs ( I ). ( J,L ) Comparison of Nanog expression by immunoblotting 48 h after SMMC-7721 cells were transfected with the indicated siRNAs and a mammalian expression vector encoding Myr-Akt. ( K ) Immunoblotting analysis of Nanog expression after SMMC-7721 cells were treated with CGP53353 (0, 5, 10 μM) for 48 h. Numbers below the blots are the density of Nanog quantified by scanning densitometry, normalized to Actin, relative to that of the control group.

Journal: Theranostics

Article Title: RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog

doi: 10.7150/thno.29271

Figure Lengend Snippet: Enhancement of Nanog expression by RACK1 in human HCC cells and murine ESCs. ( A-C ) Immunoblotting analysis of the expression of the indicated stemness-associated genes upon RACK1 knockdown ( A,B ) or over-expression ( C ) in HuH7 cells. ( D,E ) Analysis of Hedgehog signaling activity upon RACK1 knockdown ( D ) or over-expression ( E ) in HuH7 cells as indicated by 7Gli-GFP reporter. mean±s.d. ( n =3); * P <0.05, ** P <0.01. ( F-I ) Immunoblotting analysis of Nanog expression upon RACK1 knockdown ( F,H,I ) or over-expression ( G ) in sorted CD13+ and CD13- HuH7 subpopulations ( F,G ), in other human HCC cells ( H ), or in murine ESCs ( I ). ( J,L ) Comparison of Nanog expression by immunoblotting 48 h after SMMC-7721 cells were transfected with the indicated siRNAs and a mammalian expression vector encoding Myr-Akt. ( K ) Immunoblotting analysis of Nanog expression after SMMC-7721 cells were treated with CGP53353 (0, 5, 10 μM) for 48 h. Numbers below the blots are the density of Nanog quantified by scanning densitometry, normalized to Actin, relative to that of the control group.

Article Snippet: Another set of lentivirus-based human RACK1 shRNA (RACK1-b) and control lentivirus were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat. No. sc-36354-v).

Techniques: Expressing, Western Blot, Knockdown, Over Expression, Activity Assay, Comparison, Transfection, Plasmid Preparation, Control

The correlation between RACK1 and Nanog expression in clinical HCC tissues. ( A ) 136 formaldehyde-fixed and paraffin-embedded clinical HCC samples were subjected to immunohistochemistry for RACK1 and Nanog staining on tissue microarray slides. Representative paired samples are shown. Scale bar: 50 μm. ( B ) Box plot of Nanog expression in HCC based on RACK1 expression in the tumors as revealed by immunohistochemistry. Kruskal-Wallis P =0.0009. ( C ) The protein levels of RACK1 positively correlated with those of Nanog in clinical HCC tissues as revealed by immunohistochemistry. Spearman r =0.2921, P =0.0006. ( D ) Fluorescent multiplex immunohistochemistry (mIHC) with tyramide signal amplification was performed on tissue microarray slides. Representative clinical HCC samples are shown. Scale bar: 400 μm.

Journal: Theranostics

Article Title: RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog

doi: 10.7150/thno.29271

Figure Lengend Snippet: The correlation between RACK1 and Nanog expression in clinical HCC tissues. ( A ) 136 formaldehyde-fixed and paraffin-embedded clinical HCC samples were subjected to immunohistochemistry for RACK1 and Nanog staining on tissue microarray slides. Representative paired samples are shown. Scale bar: 50 μm. ( B ) Box plot of Nanog expression in HCC based on RACK1 expression in the tumors as revealed by immunohistochemistry. Kruskal-Wallis P =0.0009. ( C ) The protein levels of RACK1 positively correlated with those of Nanog in clinical HCC tissues as revealed by immunohistochemistry. Spearman r =0.2921, P =0.0006. ( D ) Fluorescent multiplex immunohistochemistry (mIHC) with tyramide signal amplification was performed on tissue microarray slides. Representative clinical HCC samples are shown. Scale bar: 400 μm.

Article Snippet: Another set of lentivirus-based human RACK1 shRNA (RACK1-b) and control lentivirus were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat. No. sc-36354-v).

Techniques: Expressing, Immunohistochemistry, Staining, Microarray, Fluorescent Multiplex Immunohistochemistry, Amplification

RACK1 augments Nanog stability through the ubiquitin-proteasome system. ( A ) Immunoblotting analysis of the half-life of Nanog protein upon RACK1 knockdown in HuH7 cells with cycloheximide (CHX) treatment for various periods of time. ( B ) HuH7 single clone stably expressing FLAG-RACK1 and the mock control were treated with cycloheximide for various periods of time. The half-life of Nanog, Oct4, and Sox2 was determined by immunoblotting. ( C ) HuH7 cells were transfected with the indicated siRNAs and a mammalian expression vector encoding FLAG-Nanog. The protein level of exogenous Nanog was analyzed by immunoblotting with an anti-FLAG antibody. Cells were treated with 20 μM MG132 or equal volume of DMSO for 6 h before cell lysates were harvested. ( D ) Immunoblotting analysis of the half-life of Nanog protein upon RACK1 knockdown in murine ESCs with cycloheximide treatment for various periods of time in the presence or absence of 20 μM MG132 for 6 h. ( E,F ) HuH7 cells were transfected with siRNAs and/or mammalian expression vectors as indicated. The ubiquitination of FLAG-Nanog upon RACK1 knockdown ( E ) or over-expression ( F ) was analyzed by immunoblotting after immunoprecipitation (IP) with an anti-FLAG antibody. Cells were treated with 20 μM MG132 for 6 h before cell lysates were harvested. WCL, whole-cell lysates; Ub, ubiquitin. ( G ) The ubiquitination of endogenous Nanog in tumors harvested in Figure E was analyzed by immunoblotting after immunoprecipitation with an anti-Nanog antibody. Numbers below the blots are the density of Nanog quantified by scanning densitometry, normalized to Actin, relative to that of the sample without cycloheximide treatment.

Journal: Theranostics

Article Title: RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog

doi: 10.7150/thno.29271

Figure Lengend Snippet: RACK1 augments Nanog stability through the ubiquitin-proteasome system. ( A ) Immunoblotting analysis of the half-life of Nanog protein upon RACK1 knockdown in HuH7 cells with cycloheximide (CHX) treatment for various periods of time. ( B ) HuH7 single clone stably expressing FLAG-RACK1 and the mock control were treated with cycloheximide for various periods of time. The half-life of Nanog, Oct4, and Sox2 was determined by immunoblotting. ( C ) HuH7 cells were transfected with the indicated siRNAs and a mammalian expression vector encoding FLAG-Nanog. The protein level of exogenous Nanog was analyzed by immunoblotting with an anti-FLAG antibody. Cells were treated with 20 μM MG132 or equal volume of DMSO for 6 h before cell lysates were harvested. ( D ) Immunoblotting analysis of the half-life of Nanog protein upon RACK1 knockdown in murine ESCs with cycloheximide treatment for various periods of time in the presence or absence of 20 μM MG132 for 6 h. ( E,F ) HuH7 cells were transfected with siRNAs and/or mammalian expression vectors as indicated. The ubiquitination of FLAG-Nanog upon RACK1 knockdown ( E ) or over-expression ( F ) was analyzed by immunoblotting after immunoprecipitation (IP) with an anti-FLAG antibody. Cells were treated with 20 μM MG132 for 6 h before cell lysates were harvested. WCL, whole-cell lysates; Ub, ubiquitin. ( G ) The ubiquitination of endogenous Nanog in tumors harvested in Figure E was analyzed by immunoblotting after immunoprecipitation with an anti-Nanog antibody. Numbers below the blots are the density of Nanog quantified by scanning densitometry, normalized to Actin, relative to that of the sample without cycloheximide treatment.

Article Snippet: Another set of lentivirus-based human RACK1 shRNA (RACK1-b) and control lentivirus were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat. No. sc-36354-v).

Techniques: Ubiquitin Proteomics, Western Blot, Knockdown, Stable Transfection, Expressing, Control, Transfection, Plasmid Preparation, Over Expression, Immunoprecipitation

$RACK1 interacts with Nanog in vitro and in vivo . ( A ) GST pull-down assays of the possible direct interaction between RACK1 and core stemness transcription factors. GST-RACK1 or GST bound to glutathione-Sepharose (GSH) beads were incubated with lysates of HuH7 cells. Precipitates were subjected to immunoblotting. ( B ) HuH7 cells were subjected to indirect immunofluorescence analysis with antibodies against Nanog and RACK1, then counterstained with DAPI followed by confocal microscopy (scale bar: 10 μm). ( C ) The subcellular localization of Nanog and RACK1 in HuH7 cells was examined by nuclear cytoplasmic fractionation and subsequent immunoblotting. HSP90 was regarded as a cytoplasm (C) marker and c-Jun as a nucleus (N) marker. ( D , E ) Analysis of the interaction between tagged-RACK1 and tagged-Nanog in 293T cells. 293T cells were transfected with mammalian expression vectors as indicated. Cell lysates were immunoprecipitated with the indicated antibodies. Precipitates were then subjected to immunoblotting. ( F - H ) Immunoblotting analysis of the interaction between endogenous Nanog and endogenous RACK1 in HuH7 cells ( F , G ) or tumors harvested in Figure E ( H ) after immunoprecipitation with an anti-RACK1 antibody ( F and H , control antibody: rabbit IgG) or an anti-Nanog antibody ( G , control antibody: rabbit IgG).$

Journal: Theranostics

Article Title: RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog

doi: 10.7150/thno.29271

Figure Lengend Snippet: RACK1 interacts with Nanog in vitro and in vivo . ( A ) GST pull-down assays of the possible direct interaction between RACK1 and core stemness transcription factors. GST-RACK1 or GST bound to glutathione-Sepharose (GSH) beads were incubated with lysates of HuH7 cells. Precipitates were subjected to immunoblotting. ( B ) HuH7 cells were subjected to indirect immunofluorescence analysis with antibodies against Nanog and RACK1, then counterstained with DAPI followed by confocal microscopy (scale bar: 10 μm). ( C ) The subcellular localization of Nanog and RACK1 in HuH7 cells was examined by nuclear cytoplasmic fractionation and subsequent immunoblotting. HSP90 was regarded as a cytoplasm (C) marker and c-Jun as a nucleus (N) marker. ( D , E ) Analysis of the interaction between tagged-RACK1 and tagged-Nanog in 293T cells. 293T cells were transfected with mammalian expression vectors as indicated. Cell lysates were immunoprecipitated with the indicated antibodies. Precipitates were then subjected to immunoblotting. ( F - H ) Immunoblotting analysis of the interaction between endogenous Nanog and endogenous RACK1 in HuH7 cells ( F , G ) or tumors harvested in Figure E ( H ) after immunoprecipitation with an anti-RACK1 antibody ( F and H , control antibody: rabbit IgG) or an anti-Nanog antibody ( G , control antibody: rabbit IgG).

Article Snippet: Another set of lentivirus-based human RACK1 shRNA (RACK1-b) and control lentivirus were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat. No. sc-36354-v).

Techniques: In Vitro, In Vivo, Incubation, Western Blot, Immunofluorescence, Confocal Microscopy, Fractionation, Marker, Transfection, Expressing, Immunoprecipitation, Control

RACK1 reduces Nanog ubiquitination through directly binding to it. ( A - D ) Mapping RACK1/Nanog interacting regions. 293T cells were transfected with various mammalian expression vectors as indicated. Cell lysates were immunoprecipitated with the indicated antibodies. Precipitates were then subjected to immunoblotting. ( E ) HuH7 cells were transfected with various mammalian expression vectors as indicated. The ubiquitination of FLAG-Nanog upon over-expression of wild type RACK1 or RACK1 mutant lacking WD5 was analyzed as described in Figure E. ( F ) Endogenous Nanog expression in HuH7 cells upon over-expression of wild type RACK1 or RACK1 mutant lacking WD5 was compared by immunoblotting. Numbers below the blot are the density of Nanog quantified by scanning densitometry, normalized to Actin, relative to that of the control group.

Journal: Theranostics

Article Title: RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog

doi: 10.7150/thno.29271

Figure Lengend Snippet: RACK1 reduces Nanog ubiquitination through directly binding to it. ( A - D ) Mapping RACK1/Nanog interacting regions. 293T cells were transfected with various mammalian expression vectors as indicated. Cell lysates were immunoprecipitated with the indicated antibodies. Precipitates were then subjected to immunoblotting. ( E ) HuH7 cells were transfected with various mammalian expression vectors as indicated. The ubiquitination of FLAG-Nanog upon over-expression of wild type RACK1 or RACK1 mutant lacking WD5 was analyzed as described in Figure E. ( F ) Endogenous Nanog expression in HuH7 cells upon over-expression of wild type RACK1 or RACK1 mutant lacking WD5 was compared by immunoblotting. Numbers below the blot are the density of Nanog quantified by scanning densitometry, normalized to Actin, relative to that of the control group.

Article Snippet: Another set of lentivirus-based human RACK1 shRNA (RACK1-b) and control lentivirus were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat. No. sc-36354-v).

Techniques: Ubiquitin Proteomics, Binding Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Over Expression, Mutagenesis, Control

RACK1 prevents the recruitment of FBXW8 by Nanog through directly binding to Nanog. ( A , C ) HuH7 cells were transfected with mammalian expression vectors as indicated. 24 h later, whole cell lysates were subjected to immunoblotting analysis with antibodies against Pin1, Usp21, GFP, Nanog, and β-actin. ( B , D,F ) 48 h after HuH7 cells were transfected with the indicated siRNAs and mammalian expression vectors, cells were treated with 20 μM MG132 for 6 h. Cell lysates were immunoprecipitated with the indicated antibodies. Precipitates were then subjected to immunoblotting. ( E ) HuH7 cells were transfected with the indicated siRNAs. 48 h later, whole cell lysates were subjected to immunoblotting analysis with antibodies against FBXW8, RACK1, Nanog, and β-actin.

Journal: Theranostics

Article Title: RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog

doi: 10.7150/thno.29271

Figure Lengend Snippet: RACK1 prevents the recruitment of FBXW8 by Nanog through directly binding to Nanog. ( A , C ) HuH7 cells were transfected with mammalian expression vectors as indicated. 24 h later, whole cell lysates were subjected to immunoblotting analysis with antibodies against Pin1, Usp21, GFP, Nanog, and β-actin. ( B , D,F ) 48 h after HuH7 cells were transfected with the indicated siRNAs and mammalian expression vectors, cells were treated with 20 μM MG132 for 6 h. Cell lysates were immunoprecipitated with the indicated antibodies. Precipitates were then subjected to immunoblotting. ( E ) HuH7 cells were transfected with the indicated siRNAs. 48 h later, whole cell lysates were subjected to immunoblotting analysis with antibodies against FBXW8, RACK1, Nanog, and β-actin.

Article Snippet: Another set of lentivirus-based human RACK1 shRNA (RACK1-b) and control lentivirus were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat. No. sc-36354-v).

Techniques: Binding Assay, Transfection, Expressing, Western Blot, Immunoprecipitation

Elevated Nanog expression plays an essential role in RACK1-regulated HCC CSCs. ( A-G ) 96 h after HuH7 cells were infected with the indicated lentivirus, cells were subjected to the following assays: ( A ) Immunoblotting analysis of the expression of Nanog and RACK1 in total cells. ( B ) Flow cytometric analysis of CD13 expression in total cells. ( C ) Immunoblotting analysis of the expression of Nanog and RACK1 in sorted CD13+ cells. ( D ) Sphere formation assays of sorted CD13+ subpopulation. mean±s.d. ( n =3); * P <0.05, ** P <0.01 ( E ) In vivo tumorigenicity experiments of CD13+ subpopulation (5000 cells/site, 7 weeks, n =6). ( F ) Etoposide (Etop, 100 μM, 48 h)- or sorafenib (Sora, 50 μM, 24 h)-induced apoptosis of CD13+ subpopulation. mean±s.d. ( n =3). ( G ) qRT-PCR analysis of sorted CD13+ subpopulation for the expression of the indicated drug-resistant relative genes. mean±s.d. ( n =3). ( H-K ) 24 h after HuH7 cells were transfected with mammalian expression vector encoding GFP-tagged wild type RACK1 or RACK1 mutant lacking WD5, GFP+CD13+ subpopulations were sorted and subjected to immunoblotting ( H ), sphere formation assays, mean±s.d. ( n =3) ( I ), etoposide (Etop, 100 μM, 48 h)- or sorafenib (Sora, 50 μM, 24 h)-induced apoptosis, mean±s.d. ( n =3) ( J ), and qRT-PCR analysis for the expression of the indicated drug-resistant relative genes. mean±s.d. ( n =3) ( K ).

Journal: Theranostics

Article Title: RACK1 Promotes Self-Renewal and Chemoresistance of Cancer Stem Cells in Human Hepatocellular Carcinoma through Stabilizing Nanog

doi: 10.7150/thno.29271

Figure Lengend Snippet: Elevated Nanog expression plays an essential role in RACK1-regulated HCC CSCs. ( A-G ) 96 h after HuH7 cells were infected with the indicated lentivirus, cells were subjected to the following assays: ( A ) Immunoblotting analysis of the expression of Nanog and RACK1 in total cells. ( B ) Flow cytometric analysis of CD13 expression in total cells. ( C ) Immunoblotting analysis of the expression of Nanog and RACK1 in sorted CD13+ cells. ( D ) Sphere formation assays of sorted CD13+ subpopulation. mean±s.d. ( n =3); * P <0.05, ** P <0.01 ( E ) In vivo tumorigenicity experiments of CD13+ subpopulation (5000 cells/site, 7 weeks, n =6). ( F ) Etoposide (Etop, 100 μM, 48 h)- or sorafenib (Sora, 50 μM, 24 h)-induced apoptosis of CD13+ subpopulation. mean±s.d. ( n =3). ( G ) qRT-PCR analysis of sorted CD13+ subpopulation for the expression of the indicated drug-resistant relative genes. mean±s.d. ( n =3). ( H-K ) 24 h after HuH7 cells were transfected with mammalian expression vector encoding GFP-tagged wild type RACK1 or RACK1 mutant lacking WD5, GFP+CD13+ subpopulations were sorted and subjected to immunoblotting ( H ), sphere formation assays, mean±s.d. ( n =3) ( I ), etoposide (Etop, 100 μM, 48 h)- or sorafenib (Sora, 50 μM, 24 h)-induced apoptosis, mean±s.d. ( n =3) ( J ), and qRT-PCR analysis for the expression of the indicated drug-resistant relative genes. mean±s.d. ( n =3) ( K ).

Article Snippet: Another set of lentivirus-based human RACK1 shRNA (RACK1-b) and control lentivirus were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat. No. sc-36354-v).

Techniques: Expressing, Infection, Western Blot, In Vivo, Quantitative RT-PCR, Transfection, Plasmid Preparation, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: RACK1 is a novel ATG5 interactor. A, HEK293T cells were cotransfected with plasmids encoding FLAG-tagged ATG5 and/or non-tagged full-length RACK1 proteins. 48 h after transfection, IP were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs, and immunoprecipitations were performed using FLAG beads. C, endogenous ATG5 protein was immunoprecipitated from wild-type MEF cell extracts using anti-ATG5 antibodies that were coupled to protein A Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control rabbit serum. D, endogenous RACK1 protein was immunoprecipitated from wild-type MEF cell extracts using anti-RACK1 antibodies that were coupled to protein G Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control mouse serum. E, GST pulldown assay. Glutathione-Sepharose beads that were bound to GST-ATG5 recombinant protein or not were incubated with His-RACK1 recombinant protein and washed. Input, immunoblotting of recombinant proteins; GST pulldown, proteins after pulldown. Note that His-RACK1 did not bind to beads alone. F, HEK293T cells were cultured on coverslides and cotransfected with GFP-tagged RACK1 (green) and Cherry-tagged ATG5 (red) constructs. 48 h post-transfection, cells were fixed and analyzed under a confocal microscope. Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots formed by RACK1 and ATG5 colocalization. G, non-transfected HEK293T cells were cultured on coverslides. After 72 h of incubation, cells were fixed, and endogenous RACK1 and ATG5 proteins were immunostained using anti-RACK1 and anti-ATG5 primary antibodies. Anti-mouse IgG Alexa Fluor 488 (green) or anti-rabbit IgG Alexa Fluor 568 (red) were used as secondary antibodies, respectively. Cells were analyzed under a confocal microscope. Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots formed by RACK1 and ATG5 colocalization.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Transfection, Western Blot, Construct, Immunoprecipitation, GST Pulldown Assay, Recombinant, Incubation, Cell Culture, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: RACK1 is a novel component of a large ATG12-5-16 protein complex. A, non-transfected HEK293T cell were treated with torin 1 or DMSO carrier control, and total cell lysates were fractioned in a gel filtration column. Chromatography fractions (F1–13) were separated in SDS-polyacrylamide gels and immunoblotted using anti-ATG16, anti-ATG5, and anti-RACK1 antibodies. CNT, DMSO carrier control; TORIN, torin 1 treatment (250 nm, 3 h); ATG16, ATG16L1; L, total cell lysate; F1 and F2, >800-kDa fractions; F3–6, 800–669-kDa fractions; F7–10, 669–443-kDa fractions; F11 and F12, 443–200-kDa fractions; F13, 200–150-kDa fraction. No protein complexes were detected in lower molecular weight fractions. B, N2A cell were treated with torin 1 or DMSO carrier control, and total cell lysates were fractioned in a gel filtration column as in A. C, chromatogram showing peaks of the molecular weight marker mix (Sigma, catalog no. MWGF1000); Ve, elution volume. D, OD595 absorbance confirmation of the peaks. E, standardization of the gel filtration column by Ve/V0. V0, void volume. F, curve showing correlation of fractions with protein sizes in kDa. G and H, representative chromatograms obtained for HEK293T (G) and N2A (H) cell lines. I, Tri-SILAC-LC-MS/MS analyses. ATG5 enrichment compared with beads alone (upper panel); enrichment of RACK-ATG5 complex under torin-treated conditions compared with DMSO-treated control (lower panel) (mean ± S.D. of independent experiments, n = 3, *, p < 0.05).

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Transfection, Filtration, Column Chromatography, Molecular Weight, Marker, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: Dynamic nature of RACK1-ATG5 interaction under autophagy-inducing conditions. A, HEK293T cells were cotransfected with FLAG-ATG5 and/or non-tagged RACK1 constructs and treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h). IP were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kDa. β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs and starved in EBSS (2 h) or not. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. C, HEK293T cells were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (2 h). Endogenous ATG5 protein was immunoprecipitated from cell extracts using anti-ATG5 antibodies that were coupled to protein A Plus beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Serum, control rabbit serum. D, HEK293T cells were cultured on coverslides. They were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (Stv, 2 h) or not. Then endogenous proteins were immunostained using anti-RACK1 and anti-ATG5 primary antibodies. Anti-mouse IgG Alexa Fluor 488 (green) and anti-rabbit IgG Alexa Fluor 568 (red) were used as secondary antibodies, respectively. Cells were analyzed under confocal microscope. CNT, non-treated cells; Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots with RACK1 and ATG5 colocalization. E, HEK293T cells were cultured on coverslides. Cells were treated or not with rapamycin (Rapa, 200 nm, 16 h) or torin 1 (Torin, 250 nm, 3 h), or starved in EBSS (Stv, 2 h) or not. Then endogenous proteins were immunostained by using anti-RACK1 and anti-LC3 primary antibodies. Cells were analyzed under a confocal microscope. CNT, non-treated cells; Merge, overlay of green and red signals. White arrows show yellow cytoplasmic dots with RACK1 and LC3 co-localization.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Construct, Western Blot, Immunoprecipitation, Cell Culture, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: RACK1 is required for mTOR inhibition and starvation-induced autophagy, but it is not an autophagy target. A, HEK293T cells were cultured on coverslides and transfected with siRACK1 or control siRNA (CNT siRNA). 48 h post-transfection, cells were treated or not (−) with rapamycin (Rapa, 200 nm, 16 h) or starved in EBSS (2 h) in the presence or absence of BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, *, p < 0.05; **, p < 0.01). Endogenous protein expression levels were checked in cell extracts from the same experiments using anti-p62, anti-LC3, and anti-RACK1 antibodies. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, representative immunofluorescence pictures of LC3 quantification experiments in A. (−), non-treated cells. White arrows show LC3 dots. C, HEK293T cells were treated with rapamycin (200 nm) for 12 or 24 h or with carrier DMSO (D, 24 h) or starved for 2, 4, or 8 h in EBSS or cultured in full medium (CNT) with or without of BafA (100 nm, 1 h) in the presence of translation inhibitor cycloheximide (0.5 μg/ml). Immunoblots were performed using anti-p62, anti-RACK1, or anti-LC3 antibodies. β-Actin was used as loading control. D, HEK293T cells were treated with rapamycin (200 nm) for 12 or 24 h or with carrier DMSO (D, 24 h) or starved for 2, 4, or 8 h in EBSS or cultured in full medium (CNT) with or without of BafA (100 nm, 1 h) in the absence of translation inhibitor cycloheximide. Immunoblots were performed using anti-p62, anti-RACK1, or anti-LC3 antibodies. β-Actin was used as loading control.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Inhibition, Cell Culture, Transfection, Expressing, Immunofluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: Role of mTOR-p70S6K pathway in the regulation of RACK1-ATG5 interaction. A, HEK293T cells were cotransfected with FLAG-ATG5 and/or non-tagged RACK1 constructs and/or an mTOR construct. IP were performed using FLAG beads. Anti-mTOR, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 constructs and/or an shmTOR construct. IP were performed using FLAG beads. Anti-mTOR, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. C, HEK293T cells were cotransfected with FLAG-RACK1 and/or non-tagged ATG5 and/or p70S6K wild-type (WT) constructs. IP were performed using FLAG beads. Anti-p70S6K, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting. D, HEK293T cells were cotransfected with FLAG-ATG5 and non-tagged RACK1 constructs and/or sip70S6 RNAi. Anti-p70S6K, anti-ATG5, and anti-RACK1 antibodies were used for immunoblotting.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Construct, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: Determination of RACK1 amino acid residues that are critical for the interaction. A, Clustal Omega alignments of RACK1 protein sequences. Putative p70S6K target RXX(S/T) consensus sequences are highlighted in black boxes. Ser/Thr residue numbers are marked according to Homo sapiens protein sequences. RACK1 GenBankTM reference sequences are as follows: H. sapiens, NP_006089; Mus musculus, NP_032169; Danio rerio, NP_571519; Drosophila melanogaster, AAB72148; Caenorhabditis elegans, NP_501859; Saccharomyces cerevisiae, NP_013834. B, schematic depiction of RACK1 constructs. WD1–7, WD40 domains 1–7. WT RACK1, wild-type RACK1. T39A, S63A, or T128A, mutant RACK1 constructs. Mutated residues were marked. C, HEK293T cells were cotransfected with FLAG-ATG5, non-tagged WT RACK1 or T39A, S63A, T128A RACK1 mutant constructs. IPs were performed using FLAG beads. Anti-ATG5 and anti-RACK1 antibodies were used for immunoblotting. Input, total cell extract; IgG, immunoglobulin G. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Construct, Mutagenesis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: RACK1-ATG5 interaction model. A, schematic representation of human RACK1 (Protein Data Bank code 4AOW). Each WD40 domain (WD1–7) is in a different color. The seven-bladed β-propeller structure is shown. Location of the Ser-63 residue is marked in a square. B, schematic model of RACK1 (Protein Data Bank code 4AOW, silver color) and ATG5 (Protein Data Bank code 4GDK, tan color) interaction. Residues found within 3 Å of the other subunit are selected as binding interface that was rendered in the wire frame surface model (probe radius, 1.4 Å) by coloring ATG5 residues in green and RACK1 in yellow. The region around the Ser-63 residue of RACK1 is shown in a red wire frame and encircled. C, interaction network of predicted RACK1-ATG5 model. D–F, native and mutated RACK1s (S63A and S63D) were energy-minimized and equilibrated in MD simulations. Snapshots of the binding interface are shown with Ser-63, Asp-6, and Lys-38 in licorice models (C, cyan; O, red; N, blue). Structural integrity of binding interface was probed by the distance of the ionic interaction between Asp-6 of ATG5 and Lys-38 of RACK1. Although the wild-type (native) and S63D complexes possessed an intact binding surface with Asp-6–Lys-38 ionic pairing (D and E), S63A showed an extension in Asp-6–Lys-38 distance (F), implying a weakened interaction of RACK1 and ATG5. G, r.m.s.d. of the backbone atoms carbon, nitrogen, and α-carbon. Native, wild-type RACK1; S63A, S63A RACK1; S63D, S63D RACK1. H, fluctuations of ATG5 during 5 ns of MD simulations. S63A RACK1 mutant displayed increased fluctuations at two distinct regions (residues from 32 to 36 and from 50 to 54), which are found at the binding interface (right panel), the observation that suggests that S63A RACK1 destabilizes the ATG5-RACK1 complex.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: RACK1-ATG5 interaction is necessary for mTOR inhibition- or starvation-induced autophagy in Neuro2A cells. Cells were cultured on coverslides and transfected with the empty control vector pcDNA3 or wild-type (wt) or mutant RACK1 (S63A or S63D) constructs. A, 48 h post-transfection, Neuro2A cells were treated or not with torin 1 (Torin, 250 nm, 3 h) with or without BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, *, p < 0.05). LC3 and RACK1 protein expression levels in cell lysates were checked in immunoblots using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. B, representative immunofluorescence pictures of LC3 quantification experiments in A. White arrows show LC3 dots. C, 48 h post-transfection, Neuro2A cells were cultured in full medium (non-STV) or starved in EBSS (STV, 2 h) with or without BafA (100 nm, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified (mean ± S.D. of independent experiments, n = 3, **, p < 0.01; *, p < 0.05). LC3 and RACK1 protein expression levels in cell lysates were checked in immunoblots using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. D, representative immunofluorescence pictures of LC3 quantification experiments in C. White arrows show LC3 dots.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Inhibition, Cell Culture, Transfection, Plasmid Preparation, Mutagenesis, Construct, Expressing, Western Blot, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy *

doi: 10.1074/jbc.M115.708081

Figure Lengend Snippet: Rescue experiments with RACK1 mutant constructs following siRNA knockdown of RACK1. A, HEK293T cells were transfected with siRACK1 or control siRNA (CNT siRNA). The effect of siRNAs was checked by immunoblotting using anti-RACK1 antibodies. Molecular mass is shown in kilodaltons (kDa). β-Actin was used as loading control. Band intensities were quantified using ImageJ. B, HEK293T cells were grown onto coverslides and transfected with the empty control vector pcDNA3 or wild-type (wt) or mutant RACK1 (S63A or S63D) constructs. 48 h post-transfection, HEK293T cells were treated or not with torin 1 (Torin, 250 nm, 3 h) with or without E64D (E64D, 10 μg/ml, 1 h) and PepA (pepstatin A, 10 μg/ml, 1 h). Endogenous LC3 proteins were immunostained using anti-LC3 primary antibodies and anti-rabbit IgG Alexa Fluor 488 secondary antibodies. LC3 dot positive cells were quantified as percentage of autophagic cells in total cell population (mean ± S.D. of independent experiments, n = 3, **, p < 0.01; *, p < 0.05). LC3 and RACK1 protein expression levels were detected by immunoblotting using anti-LC3 and anti-RACK1 antibodies. β-Actin was used as loading control. C, representative immunofluorescence pictures of LC3 quantification experiments in A. White arrows show LC3 dots.

Article Snippet: FLAG-tagged human ATG5 (RC235557), human RACK1 (SC116322), and FLAG-tagged human RACK1 plasmids (RC505092) were purchased from Origene.

Techniques: Mutagenesis, Construct, Transfection, Western Blot, Plasmid Preparation, Expressing, Immunofluorescence

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