Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 controls Gp96 protein glycosylation and stability. (A) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (B) Volcano plot of proteins, identified by mass spectrometry, from Flag-immunoprecipitates from sg CCDC134 CAL-1 stably reconstituted with Flag-CCDC134 or control CAL-1 (sg Ren transduced with empty vector [EV]). red: fold change (FC) >2; gray: fold change (FC) = [−2, 2]; blue: fold change (FC) less than −2. (C) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (D) Coomassie blue staining of recombinant proteins Gp96-HA or Flag-CCDC134 (Flag-134) in a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA. (E) Immunoblots of indicated knockout HEK293T cells. (F) Immunoblots of indicated CAL-1 cells. (G) HSP90B1 (gene coding for Gp96) mRNA levels of indicated CAL-1 cells measured by qPCR (normalized to HPRT1 ). (H) Immunoblots of indicated knockout CAL-1 cells treated with tunicamycin (Tun.) (5 µg/ml, for 0–4 h) or vehicle DMSO. In A, C–F, and H, data are representative of two independent experiments. In G, data show mean ± SD of four independent experiments. Source data are available for this figure: .

Article Snippet: Plasmids coding for UNC93B1 (ID:132298), Gp96 (ID: 82130), TLR4 (ID: 13086), and TLR5 (ID: 13019) were from Addgene.

Techniques: Transfection, Mass Spectrometry, Stable Transfection, Control, Transduction, Plasmid Preparation, Staining, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Knock-Out

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 interacts and stabilizes the ER chaperone Gp96. (A and B) Immunoprecipitates (IP) and whole-cell extracts (WCE) from transfected HEK293T (A) or knockout CAL-1 cell lines (B) as indicated. (C) Schematic of wildtype Gp96 and deletion constructs (left panel) as well as immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T transfected as indicated (right panel). SP: signal peptide, pN: pre-N-terminal domain; ND: N-terminal domain, CR: charged linker region; MD: middle domain; CD: C-terminal domain; KDEL: ER retention motif; steady state (N217 red) and cryptic N-glycans acceptor sites are shown. (D) Immunoprecipitates (IP) and input using Flag-CCDC134 and Gp96-HA recombinant proteins. For the complex formation, Flag-CCDC134 and Gp96-HA were preincubated overnight at 4°C before to perform the immunoprecipitation assay. Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA; asterisks indicate IgG heavy or light chains. (E) Immunoblots of lysates from indicated knockout CAL-1 cells. Asterisks indicate a non-specific band. (F) IL-6 production of indicated knockout CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) from indicated CAL-1 cell lines. (H) Immunoblots from indicated knockout CAL-1 cells treated with NMS-873 (1, 5, and 10 μM) or vehicle DMSO for 8 h. In A–E, G, and H, data are representative of two independent experiments. In F, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: .

Article Snippet: Plasmids coding for UNC93B1 (ID:132298), Gp96 (ID: 82130), TLR4 (ID: 13086), and TLR5 (ID: 13019) were from Addgene.

Techniques: Transfection, Knock-Out, Construct, Recombinant, Immunoprecipitation, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 modulates TLR7/9 signaling by regulating Gp96 hyperglycosylation. (A) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1 or 2) treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) treated with doxycycline (Dox., 5 ng/ml) for 24 h. (C) IL-6 production of indicated knockout CAL-1 cells expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) induced with 5 ng/ml of doxycycline (Dox.) for 17 h and followed by 24 h stimulation with R848 (5 μg/ml). (D) Immunoprecipitates (IP) and whole-cell extracts (WCE) from TLR7-V5-expressing CAL-1 knockout cell lines as indicated. Asterisks indicate a non-specific band. (E) Immunoblots of lysates from indicated CAL-1 cell lines. long exp.: long exposure. (F) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of indicated CAL-1 cells treated with R848 (5 µg/ml, for 0–1 h). (H) Immunoblots of cell lysates treated with EndoH (H) or PNGase F (F). N3Q Gp96 bears mutations at positions N445Q-N481Q-N502Q. (I) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). In A, B, D, E, G, and H, data are representative of two independent experiments. In C, F, and I, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: .

Article Snippet: Plasmids coding for UNC93B1 (ID:132298), Gp96 (ID: 82130), TLR4 (ID: 13086), and TLR5 (ID: 13019) were from Addgene.

Techniques: Western Blot, Knock-Out, Stable Transfection, Expressing, Construct, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: Mapping of CCDC134 requirement for regulation of Gp96 in the ER. (A) Doxycycline-inducible CCDC134 mRNA ( dox.-ind . CCDC134 ) levels in indicated cells measured by qPCR. The primers were specifically designed to detect only the doxycycline-inducible construct, excluding any detection of the endogenous CCDC134 mrRNA. CAL-1 cells stably expressing the doxycycline-inducible CCDC134 construct (clone 1 or 2) were treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) AlphaFold structure prediction for human CCDC134 (Uniprot entry name: Q9H6E4 CC134_HUMAN). (C) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) of indicated CAL-1 cells reconstituted with wildtype or deletion CCDC134 mutants. The Δ133–156 CCDC134 deletion mutant lacks the predicted N-linked glycosylated NQT sequon. Ev: empty vector. (D) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged wildtype (WT) or deletion mutant CCDC134 constructs. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (E) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged Δ133–156 CCDC134 deletion mutant which lacks the predicted N-linked glycosylated NQT sequon (left panel) and quantification of the colocalization between Flag-CCDC134 (wildtype or Δ133–156 deletion mutant) and calreticulin (right panel). Data are expressed as colocalization score (coloc. score) and pooled from three independent experiments. Each dot represents analysis of a single field of view containing one to four transfected cells, and violins show the variation of individual dot across all experiments; P value = ns, two-tailed Mann–Whitney test. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (F) Immunoblots of proteins from the (not precipitated) supernatant (SN) of indicated CAL-1 cells. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 24 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. (G) Immunoblots of indicated CAL-1 cells incubated for 48 h with supernatants (SN) of donor cells cultured for 24 h in Opti-MEM. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 48 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. In A, data show mean ± SD of three independent experiments. In C, F, and G, data are representative of two independent experiments. Source data are available for this figure: .

Article Snippet: Plasmids coding for UNC93B1 (ID:132298), Gp96 (ID: 82130), TLR4 (ID: 13086), and TLR5 (ID: 13019) were from Addgene.

Techniques: Construct, Stable Transfection, Expressing, Concentration Assay, Western Blot, Mutagenesis, Plasmid Preparation, Confocal Microscopy, Transfection, Two Tailed Test, MANN-WHITNEY, Incubation, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 controls Gp96-dependent maturation and stability of plasma membrane and endolysosomal TLRs. (A) TNFα production of indicated Hoxb8-macrophages stimulated for 24 h with Pam3CSK4 (Pam3.) (0.1 μg/ml), LPS (10 ng/ml), R848 (0.1 μg/ml), CpG-B (ODN1668, 1 μM), or flagellin (0.1 μg/ml). Untr.: untreated. (B–D) Immunoblots of indicated Hoxb8-macrophages untreated (B) or stimulated with R848 (0.1 μg/ml), CpG-B (ODN1668) (1 μM) for 0–1 h (C) or Pam3CSK4 (0.1 μg/ml), LPS (10 ng/ml), and flagellin (0.1 μg/ml) for 0–1 h (D). (E and F) Immunoblots of indicated Hoxb8-macrophages. short exp.: short exposure; Mat.: mature; Imat.: immature, F: full length; C: cleaved form. (G) Quantification of TLR2, 4, 5, or 7 protein levels in sg Ren or sg CCDC134 Hoxb8-macrophages measured by surface (left upper panel) or surface and intracellular (intra.) (left lower panel) staining and quantified by gMFI (relative to respective sg Ren ). Representative histograms of surface (right upper panel) or intracellular (right lower panel). gMFI surface staining normalized to sg Ren TLR2 P value = 0.0053, TLR4 P value = 0.0063, TLR5 P value = 0.0093; gMFI surface and intracellular staining normalized to sg Ren TLR2 P value = 0.0002, TLR4 P value = 0.0256, TLR5 P value = ns, TLR7 P value <0.0001, two-tailed one sample t test. (H) Immunoblots of lysate from sg Ren or sg CCDC134 Hoxb8-macrophages treated with EndoH (H) or PNGase F (F). Mat.: mature form; Imat.: immature form; dg: deglycosylated. (I) Representative image of sg Ren or sg CCDC134 Hoxb8-macrophages stained for TLR7 (scale bar: 5 μm) (left panel) and quantification of TLR7 intensity (right panel). Data are expressed as fold change (FC) and pooled from n = 2. Violins show the variation of individual cells across all experiments; P value <0.0001, two-tailed Mann–Whitney test. (J) Quantification of CD11a, CD18, CD49d, and CD44 protein levels in sg Ren or sg CCDC134 Hoxb8-macrophages measured by surface (upper panel) or surface and intracellular (intra.) (bottom panel) staining and quantified by gMFI (relative to respective sg Ren ). Data show mean ± SD from three independent experiments; gMFI surface staining normalized to sg Ren CD11a P value = 0.0316, CD18 P value = 0.0449, CD49d P value = ns, CD44 P value = ns; gMFI surface and intracellular staining normalized to sg Ren CD11a P value = 0.0074, CD18 P value = ns, CD49d P value = 0.0029, CD44 P value = ns; two-tailed one sample t test. (K) Volcano plot of quantified proteins in whole proteome of sg CCDC134 versus sg Ren Hoxb8-macrophages (upregulated: red, fold change [FC] > 2 and P value <0.01; downregulated: blue, FC less than −2 and P value <0.01). pink: CCDC134 and Gp96; light pink: Bip and integrins; purple: TLRs; light purple: related to TLRs; green: IFN-stimulated gene proteins (ISGs). In B–F and H, data are representative of two experiments. In A, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. In G–J, data show mean ± SD from four or three independent experiments. Source data are available for this figure: .

Article Snippet: Plasmids coding for UNC93B1 (ID:132298), Gp96 (ID: 82130), TLR4 (ID: 13086), and TLR5 (ID: 13019) were from Addgene.

Techniques: Membrane, Western Blot, Staining, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 controls Gp96 protein glycosylation and stability. (A) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (B) Volcano plot of proteins, identified by mass spectrometry, from Flag-immunoprecipitates from sg CCDC134 CAL-1 stably reconstituted with Flag-CCDC134 or control CAL-1 (sg Ren transduced with empty vector [EV]). red: fold change (FC) >2; gray: fold change (FC) = [−2, 2]; blue: fold change (FC) less than −2. (C) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (D) Coomassie blue staining of recombinant proteins Gp96-HA or Flag-CCDC134 (Flag-134) in a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA. (E) Immunoblots of indicated knockout HEK293T cells. (F) Immunoblots of indicated CAL-1 cells. (G) HSP90B1 (gene coding for Gp96) mRNA levels of indicated CAL-1 cells measured by qPCR (normalized to HPRT1 ). (H) Immunoblots of indicated knockout CAL-1 cells treated with tunicamycin (Tun.) (5 µg/ml, for 0–4 h) or vehicle DMSO. In A, C–F, and H, data are representative of two independent experiments. In G, data show mean ± SD of four independent experiments. Source data are available for this figure: .

Article Snippet: Codon-optimized cDNAs for human IRF5, HA-tagged wildtype, hyperglycosylated, or deletion mutant Gp96 were obtained from Genscript.

Techniques: Transfection, Mass Spectrometry, Stable Transfection, Control, Transduction, Plasmid Preparation, Staining, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Knock-Out

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 interacts and stabilizes the ER chaperone Gp96. (A and B) Immunoprecipitates (IP) and whole-cell extracts (WCE) from transfected HEK293T (A) or knockout CAL-1 cell lines (B) as indicated. (C) Schematic of wildtype Gp96 and deletion constructs (left panel) as well as immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T transfected as indicated (right panel). SP: signal peptide, pN: pre-N-terminal domain; ND: N-terminal domain, CR: charged linker region; MD: middle domain; CD: C-terminal domain; KDEL: ER retention motif; steady state (N217 red) and cryptic N-glycans acceptor sites are shown. (D) Immunoprecipitates (IP) and input using Flag-CCDC134 and Gp96-HA recombinant proteins. For the complex formation, Flag-CCDC134 and Gp96-HA were preincubated overnight at 4°C before to perform the immunoprecipitation assay. Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA; asterisks indicate IgG heavy or light chains. (E) Immunoblots of lysates from indicated knockout CAL-1 cells. Asterisks indicate a non-specific band. (F) IL-6 production of indicated knockout CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) from indicated CAL-1 cell lines. (H) Immunoblots from indicated knockout CAL-1 cells treated with NMS-873 (1, 5, and 10 μM) or vehicle DMSO for 8 h. In A–E, G, and H, data are representative of two independent experiments. In F, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: .

Article Snippet: Codon-optimized cDNAs for human IRF5, HA-tagged wildtype, hyperglycosylated, or deletion mutant Gp96 were obtained from Genscript.

Techniques: Transfection, Knock-Out, Construct, Recombinant, Immunoprecipitation, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 modulates TLR7/9 signaling by regulating Gp96 hyperglycosylation. (A) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1 or 2) treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) treated with doxycycline (Dox., 5 ng/ml) for 24 h. (C) IL-6 production of indicated knockout CAL-1 cells expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) induced with 5 ng/ml of doxycycline (Dox.) for 17 h and followed by 24 h stimulation with R848 (5 μg/ml). (D) Immunoprecipitates (IP) and whole-cell extracts (WCE) from TLR7-V5-expressing CAL-1 knockout cell lines as indicated. Asterisks indicate a non-specific band. (E) Immunoblots of lysates from indicated CAL-1 cell lines. long exp.: long exposure. (F) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of indicated CAL-1 cells treated with R848 (5 µg/ml, for 0–1 h). (H) Immunoblots of cell lysates treated with EndoH (H) or PNGase F (F). N3Q Gp96 bears mutations at positions N445Q-N481Q-N502Q. (I) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). In A, B, D, E, G, and H, data are representative of two independent experiments. In C, F, and I, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: .

Article Snippet: Codon-optimized cDNAs for human IRF5, HA-tagged wildtype, hyperglycosylated, or deletion mutant Gp96 were obtained from Genscript.

Techniques: Western Blot, Knock-Out, Stable Transfection, Expressing, Construct, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: Mapping of CCDC134 requirement for regulation of Gp96 in the ER. (A) Doxycycline-inducible CCDC134 mRNA ( dox.-ind . CCDC134 ) levels in indicated cells measured by qPCR. The primers were specifically designed to detect only the doxycycline-inducible construct, excluding any detection of the endogenous CCDC134 mrRNA. CAL-1 cells stably expressing the doxycycline-inducible CCDC134 construct (clone 1 or 2) were treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) AlphaFold structure prediction for human CCDC134 (Uniprot entry name: Q9H6E4 CC134_HUMAN). (C) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) of indicated CAL-1 cells reconstituted with wildtype or deletion CCDC134 mutants. The Δ133–156 CCDC134 deletion mutant lacks the predicted N-linked glycosylated NQT sequon. Ev: empty vector. (D) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged wildtype (WT) or deletion mutant CCDC134 constructs. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (E) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged Δ133–156 CCDC134 deletion mutant which lacks the predicted N-linked glycosylated NQT sequon (left panel) and quantification of the colocalization between Flag-CCDC134 (wildtype or Δ133–156 deletion mutant) and calreticulin (right panel). Data are expressed as colocalization score (coloc. score) and pooled from three independent experiments. Each dot represents analysis of a single field of view containing one to four transfected cells, and violins show the variation of individual dot across all experiments; P value = ns, two-tailed Mann–Whitney test. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (F) Immunoblots of proteins from the (not precipitated) supernatant (SN) of indicated CAL-1 cells. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 24 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. (G) Immunoblots of indicated CAL-1 cells incubated for 48 h with supernatants (SN) of donor cells cultured for 24 h in Opti-MEM. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 48 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. In A, data show mean ± SD of three independent experiments. In C, F, and G, data are representative of two independent experiments. Source data are available for this figure: .

Article Snippet: Codon-optimized cDNAs for human IRF5, HA-tagged wildtype, hyperglycosylated, or deletion mutant Gp96 were obtained from Genscript.

Techniques: Construct, Stable Transfection, Expressing, Concentration Assay, Western Blot, Mutagenesis, Plasmid Preparation, Confocal Microscopy, Transfection, Two Tailed Test, MANN-WHITNEY, Incubation, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 controls Gp96-dependent maturation and stability of plasma membrane and endolysosomal TLRs. (A) TNFα production of indicated Hoxb8-macrophages stimulated for 24 h with Pam3CSK4 (Pam3.) (0.1 μg/ml), LPS (10 ng/ml), R848 (0.1 μg/ml), CpG-B (ODN1668, 1 μM), or flagellin (0.1 μg/ml). Untr.: untreated. (B–D) Immunoblots of indicated Hoxb8-macrophages untreated (B) or stimulated with R848 (0.1 μg/ml), CpG-B (ODN1668) (1 μM) for 0–1 h (C) or Pam3CSK4 (0.1 μg/ml), LPS (10 ng/ml), and flagellin (0.1 μg/ml) for 0–1 h (D). (E and F) Immunoblots of indicated Hoxb8-macrophages. short exp.: short exposure; Mat.: mature; Imat.: immature, F: full length; C: cleaved form. (G) Quantification of TLR2, 4, 5, or 7 protein levels in sg Ren or sg CCDC134 Hoxb8-macrophages measured by surface (left upper panel) or surface and intracellular (intra.) (left lower panel) staining and quantified by gMFI (relative to respective sg Ren ). Representative histograms of surface (right upper panel) or intracellular (right lower panel). gMFI surface staining normalized to sg Ren TLR2 P value = 0.0053, TLR4 P value = 0.0063, TLR5 P value = 0.0093; gMFI surface and intracellular staining normalized to sg Ren TLR2 P value = 0.0002, TLR4 P value = 0.0256, TLR5 P value = ns, TLR7 P value <0.0001, two-tailed one sample t test. (H) Immunoblots of lysate from sg Ren or sg CCDC134 Hoxb8-macrophages treated with EndoH (H) or PNGase F (F). Mat.: mature form; Imat.: immature form; dg: deglycosylated. (I) Representative image of sg Ren or sg CCDC134 Hoxb8-macrophages stained for TLR7 (scale bar: 5 μm) (left panel) and quantification of TLR7 intensity (right panel). Data are expressed as fold change (FC) and pooled from n = 2. Violins show the variation of individual cells across all experiments; P value <0.0001, two-tailed Mann–Whitney test. (J) Quantification of CD11a, CD18, CD49d, and CD44 protein levels in sg Ren or sg CCDC134 Hoxb8-macrophages measured by surface (upper panel) or surface and intracellular (intra.) (bottom panel) staining and quantified by gMFI (relative to respective sg Ren ). Data show mean ± SD from three independent experiments; gMFI surface staining normalized to sg Ren CD11a P value = 0.0316, CD18 P value = 0.0449, CD49d P value = ns, CD44 P value = ns; gMFI surface and intracellular staining normalized to sg Ren CD11a P value = 0.0074, CD18 P value = ns, CD49d P value = 0.0029, CD44 P value = ns; two-tailed one sample t test. (K) Volcano plot of quantified proteins in whole proteome of sg CCDC134 versus sg Ren Hoxb8-macrophages (upregulated: red, fold change [FC] > 2 and P value <0.01; downregulated: blue, FC less than −2 and P value <0.01). pink: CCDC134 and Gp96; light pink: Bip and integrins; purple: TLRs; light purple: related to TLRs; green: IFN-stimulated gene proteins (ISGs). In B–F and H, data are representative of two experiments. In A, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. In G–J, data show mean ± SD from four or three independent experiments. Source data are available for this figure: .

Article Snippet: Codon-optimized cDNAs for human IRF5, HA-tagged wildtype, hyperglycosylated, or deletion mutant Gp96 were obtained from Genscript.

Techniques: Membrane, Western Blot, Staining, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 controls Gp96 protein glycosylation and stability. (A) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (B) Volcano plot of proteins, identified by mass spectrometry, from Flag-immunoprecipitates from sg CCDC134 CAL-1 stably reconstituted with Flag-CCDC134 or control CAL-1 (sg Ren transduced with empty vector [EV]). red: fold change (FC) >2; gray: fold change (FC) = [−2, 2]; blue: fold change (FC) less than −2. (C) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (D) Coomassie blue staining of recombinant proteins Gp96-HA or Flag-CCDC134 (Flag-134) in a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA. (E) Immunoblots of indicated knockout HEK293T cells. (F) Immunoblots of indicated CAL-1 cells. (G) HSP90B1 (gene coding for Gp96) mRNA levels of indicated CAL-1 cells measured by qPCR (normalized to HPRT1 ). (H) Immunoblots of indicated knockout CAL-1 cells treated with tunicamycin (Tun.) (5 µg/ml, for 0–4 h) or vehicle DMSO. In A, C–F, and H, data are representative of two independent experiments. In G, data show mean ± SD of four independent experiments. Source data are available for this figure: .

Article Snippet: For the production of recombinant proteins, human N-terminally Flag-tagged CCDC134 and C-terminally HA-tagged Gp96 were cloned into the pET28a expression vector (C-terminal His-tag) and expressed in ClearColi expression system in LB medium upon IPTG (0.5 mM; cat. 162423; Biosolve) induction overnight at 22°C.

Techniques: Transfection, Mass Spectrometry, Stable Transfection, Control, Transduction, Plasmid Preparation, Staining, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Knock-Out

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 interacts and stabilizes the ER chaperone Gp96. (A and B) Immunoprecipitates (IP) and whole-cell extracts (WCE) from transfected HEK293T (A) or knockout CAL-1 cell lines (B) as indicated. (C) Schematic of wildtype Gp96 and deletion constructs (left panel) as well as immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T transfected as indicated (right panel). SP: signal peptide, pN: pre-N-terminal domain; ND: N-terminal domain, CR: charged linker region; MD: middle domain; CD: C-terminal domain; KDEL: ER retention motif; steady state (N217 red) and cryptic N-glycans acceptor sites are shown. (D) Immunoprecipitates (IP) and input using Flag-CCDC134 and Gp96-HA recombinant proteins. For the complex formation, Flag-CCDC134 and Gp96-HA were preincubated overnight at 4°C before to perform the immunoprecipitation assay. Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA; asterisks indicate IgG heavy or light chains. (E) Immunoblots of lysates from indicated knockout CAL-1 cells. Asterisks indicate a non-specific band. (F) IL-6 production of indicated knockout CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) from indicated CAL-1 cell lines. (H) Immunoblots from indicated knockout CAL-1 cells treated with NMS-873 (1, 5, and 10 μM) or vehicle DMSO for 8 h. In A–E, G, and H, data are representative of two independent experiments. In F, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: .

Article Snippet: For the production of recombinant proteins, human N-terminally Flag-tagged CCDC134 and C-terminally HA-tagged Gp96 were cloned into the pET28a expression vector (C-terminal His-tag) and expressed in ClearColi expression system in LB medium upon IPTG (0.5 mM; cat. 162423; Biosolve) induction overnight at 22°C.

Techniques: Transfection, Knock-Out, Construct, Recombinant, Immunoprecipitation, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 modulates TLR7/9 signaling by regulating Gp96 hyperglycosylation. (A) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1 or 2) treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) treated with doxycycline (Dox., 5 ng/ml) for 24 h. (C) IL-6 production of indicated knockout CAL-1 cells expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) induced with 5 ng/ml of doxycycline (Dox.) for 17 h and followed by 24 h stimulation with R848 (5 μg/ml). (D) Immunoprecipitates (IP) and whole-cell extracts (WCE) from TLR7-V5-expressing CAL-1 knockout cell lines as indicated. Asterisks indicate a non-specific band. (E) Immunoblots of lysates from indicated CAL-1 cell lines. long exp.: long exposure. (F) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of indicated CAL-1 cells treated with R848 (5 µg/ml, for 0–1 h). (H) Immunoblots of cell lysates treated with EndoH (H) or PNGase F (F). N3Q Gp96 bears mutations at positions N445Q-N481Q-N502Q. (I) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). In A, B, D, E, G, and H, data are representative of two independent experiments. In C, F, and I, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: .

Article Snippet: For the production of recombinant proteins, human N-terminally Flag-tagged CCDC134 and C-terminally HA-tagged Gp96 were cloned into the pET28a expression vector (C-terminal His-tag) and expressed in ClearColi expression system in LB medium upon IPTG (0.5 mM; cat. 162423; Biosolve) induction overnight at 22°C.

Techniques: Western Blot, Knock-Out, Stable Transfection, Expressing, Construct, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: Mapping of CCDC134 requirement for regulation of Gp96 in the ER. (A) Doxycycline-inducible CCDC134 mRNA ( dox.-ind . CCDC134 ) levels in indicated cells measured by qPCR. The primers were specifically designed to detect only the doxycycline-inducible construct, excluding any detection of the endogenous CCDC134 mrRNA. CAL-1 cells stably expressing the doxycycline-inducible CCDC134 construct (clone 1 or 2) were treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) AlphaFold structure prediction for human CCDC134 (Uniprot entry name: Q9H6E4 CC134_HUMAN). (C) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) of indicated CAL-1 cells reconstituted with wildtype or deletion CCDC134 mutants. The Δ133–156 CCDC134 deletion mutant lacks the predicted N-linked glycosylated NQT sequon. Ev: empty vector. (D) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged wildtype (WT) or deletion mutant CCDC134 constructs. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (E) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged Δ133–156 CCDC134 deletion mutant which lacks the predicted N-linked glycosylated NQT sequon (left panel) and quantification of the colocalization between Flag-CCDC134 (wildtype or Δ133–156 deletion mutant) and calreticulin (right panel). Data are expressed as colocalization score (coloc. score) and pooled from three independent experiments. Each dot represents analysis of a single field of view containing one to four transfected cells, and violins show the variation of individual dot across all experiments; P value = ns, two-tailed Mann–Whitney test. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (F) Immunoblots of proteins from the (not precipitated) supernatant (SN) of indicated CAL-1 cells. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 24 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. (G) Immunoblots of indicated CAL-1 cells incubated for 48 h with supernatants (SN) of donor cells cultured for 24 h in Opti-MEM. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 48 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. In A, data show mean ± SD of three independent experiments. In C, F, and G, data are representative of two independent experiments. Source data are available for this figure: .

Article Snippet: For the production of recombinant proteins, human N-terminally Flag-tagged CCDC134 and C-terminally HA-tagged Gp96 were cloned into the pET28a expression vector (C-terminal His-tag) and expressed in ClearColi expression system in LB medium upon IPTG (0.5 mM; cat. 162423; Biosolve) induction overnight at 22°C.

Techniques: Construct, Stable Transfection, Expressing, Concentration Assay, Western Blot, Mutagenesis, Plasmid Preparation, Confocal Microscopy, Transfection, Two Tailed Test, MANN-WHITNEY, Incubation, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 controls Gp96-dependent maturation and stability of plasma membrane and endolysosomal TLRs. (A) TNFα production of indicated Hoxb8-macrophages stimulated for 24 h with Pam3CSK4 (Pam3.) (0.1 μg/ml), LPS (10 ng/ml), R848 (0.1 μg/ml), CpG-B (ODN1668, 1 μM), or flagellin (0.1 μg/ml). Untr.: untreated. (B–D) Immunoblots of indicated Hoxb8-macrophages untreated (B) or stimulated with R848 (0.1 μg/ml), CpG-B (ODN1668) (1 μM) for 0–1 h (C) or Pam3CSK4 (0.1 μg/ml), LPS (10 ng/ml), and flagellin (0.1 μg/ml) for 0–1 h (D). (E and F) Immunoblots of indicated Hoxb8-macrophages. short exp.: short exposure; Mat.: mature; Imat.: immature, F: full length; C: cleaved form. (G) Quantification of TLR2, 4, 5, or 7 protein levels in sg Ren or sg CCDC134 Hoxb8-macrophages measured by surface (left upper panel) or surface and intracellular (intra.) (left lower panel) staining and quantified by gMFI (relative to respective sg Ren ). Representative histograms of surface (right upper panel) or intracellular (right lower panel). gMFI surface staining normalized to sg Ren TLR2 P value = 0.0053, TLR4 P value = 0.0063, TLR5 P value = 0.0093; gMFI surface and intracellular staining normalized to sg Ren TLR2 P value = 0.0002, TLR4 P value = 0.0256, TLR5 P value = ns, TLR7 P value <0.0001, two-tailed one sample t test. (H) Immunoblots of lysate from sg Ren or sg CCDC134 Hoxb8-macrophages treated with EndoH (H) or PNGase F (F). Mat.: mature form; Imat.: immature form; dg: deglycosylated. (I) Representative image of sg Ren or sg CCDC134 Hoxb8-macrophages stained for TLR7 (scale bar: 5 μm) (left panel) and quantification of TLR7 intensity (right panel). Data are expressed as fold change (FC) and pooled from n = 2. Violins show the variation of individual cells across all experiments; P value <0.0001, two-tailed Mann–Whitney test. (J) Quantification of CD11a, CD18, CD49d, and CD44 protein levels in sg Ren or sg CCDC134 Hoxb8-macrophages measured by surface (upper panel) or surface and intracellular (intra.) (bottom panel) staining and quantified by gMFI (relative to respective sg Ren ). Data show mean ± SD from three independent experiments; gMFI surface staining normalized to sg Ren CD11a P value = 0.0316, CD18 P value = 0.0449, CD49d P value = ns, CD44 P value = ns; gMFI surface and intracellular staining normalized to sg Ren CD11a P value = 0.0074, CD18 P value = ns, CD49d P value = 0.0029, CD44 P value = ns; two-tailed one sample t test. (K) Volcano plot of quantified proteins in whole proteome of sg CCDC134 versus sg Ren Hoxb8-macrophages (upregulated: red, fold change [FC] > 2 and P value <0.01; downregulated: blue, FC less than −2 and P value <0.01). pink: CCDC134 and Gp96; light pink: Bip and integrins; purple: TLRs; light purple: related to TLRs; green: IFN-stimulated gene proteins (ISGs). In B–F and H, data are representative of two experiments. In A, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. In G–J, data show mean ± SD from four or three independent experiments. Source data are available for this figure: .

Article Snippet: For the production of recombinant proteins, human N-terminally Flag-tagged CCDC134 and C-terminally HA-tagged Gp96 were cloned into the pET28a expression vector (C-terminal His-tag) and expressed in ClearColi expression system in LB medium upon IPTG (0.5 mM; cat. 162423; Biosolve) induction overnight at 22°C.

Techniques: Membrane, Western Blot, Staining, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 controls Gp96 protein glycosylation and stability. (A) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (B) Volcano plot of proteins, identified by mass spectrometry, from Flag-immunoprecipitates from sg CCDC134 CAL-1 stably reconstituted with Flag-CCDC134 or control CAL-1 (sg Ren transduced with empty vector [EV]). red: fold change (FC) >2; gray: fold change (FC) = [−2, 2]; blue: fold change (FC) less than −2. (C) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (D) Coomassie blue staining of recombinant proteins Gp96-HA or Flag-CCDC134 (Flag-134) in a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA. (E) Immunoblots of indicated knockout HEK293T cells. (F) Immunoblots of indicated CAL-1 cells. (G) HSP90B1 (gene coding for Gp96) mRNA levels of indicated CAL-1 cells measured by qPCR (normalized to HPRT1 ). (H) Immunoblots of indicated knockout CAL-1 cells treated with tunicamycin (Tun.) (5 µg/ml, for 0–4 h) or vehicle DMSO. In A, C–F, and H, data are representative of two independent experiments. In G, data show mean ± SD of four independent experiments. Source data are available for this figure: .

Article Snippet: Samples of Gp96-HA were further purified on Akta pure FPLC system using Superdex 200 10/300 column (cat. GE28-9909-44; Cytiva).

Techniques: Transfection, Mass Spectrometry, Stable Transfection, Control, Transduction, Plasmid Preparation, Staining, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Knock-Out

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 interacts and stabilizes the ER chaperone Gp96. (A and B) Immunoprecipitates (IP) and whole-cell extracts (WCE) from transfected HEK293T (A) or knockout CAL-1 cell lines (B) as indicated. (C) Schematic of wildtype Gp96 and deletion constructs (left panel) as well as immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T transfected as indicated (right panel). SP: signal peptide, pN: pre-N-terminal domain; ND: N-terminal domain, CR: charged linker region; MD: middle domain; CD: C-terminal domain; KDEL: ER retention motif; steady state (N217 red) and cryptic N-glycans acceptor sites are shown. (D) Immunoprecipitates (IP) and input using Flag-CCDC134 and Gp96-HA recombinant proteins. For the complex formation, Flag-CCDC134 and Gp96-HA were preincubated overnight at 4°C before to perform the immunoprecipitation assay. Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA; asterisks indicate IgG heavy or light chains. (E) Immunoblots of lysates from indicated knockout CAL-1 cells. Asterisks indicate a non-specific band. (F) IL-6 production of indicated knockout CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) from indicated CAL-1 cell lines. (H) Immunoblots from indicated knockout CAL-1 cells treated with NMS-873 (1, 5, and 10 μM) or vehicle DMSO for 8 h. In A–E, G, and H, data are representative of two independent experiments. In F, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: .

Article Snippet: Samples of Gp96-HA were further purified on Akta pure FPLC system using Superdex 200 10/300 column (cat. GE28-9909-44; Cytiva).

Techniques: Transfection, Knock-Out, Construct, Recombinant, Immunoprecipitation, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 modulates TLR7/9 signaling by regulating Gp96 hyperglycosylation. (A) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1 or 2) treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) treated with doxycycline (Dox., 5 ng/ml) for 24 h. (C) IL-6 production of indicated knockout CAL-1 cells expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) induced with 5 ng/ml of doxycycline (Dox.) for 17 h and followed by 24 h stimulation with R848 (5 μg/ml). (D) Immunoprecipitates (IP) and whole-cell extracts (WCE) from TLR7-V5-expressing CAL-1 knockout cell lines as indicated. Asterisks indicate a non-specific band. (E) Immunoblots of lysates from indicated CAL-1 cell lines. long exp.: long exposure. (F) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of indicated CAL-1 cells treated with R848 (5 µg/ml, for 0–1 h). (H) Immunoblots of cell lysates treated with EndoH (H) or PNGase F (F). N3Q Gp96 bears mutations at positions N445Q-N481Q-N502Q. (I) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). In A, B, D, E, G, and H, data are representative of two independent experiments. In C, F, and I, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: .

Article Snippet: Samples of Gp96-HA were further purified on Akta pure FPLC system using Superdex 200 10/300 column (cat. GE28-9909-44; Cytiva).

Techniques: Western Blot, Knock-Out, Stable Transfection, Expressing, Construct, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: Mapping of CCDC134 requirement for regulation of Gp96 in the ER. (A) Doxycycline-inducible CCDC134 mRNA ( dox.-ind . CCDC134 ) levels in indicated cells measured by qPCR. The primers were specifically designed to detect only the doxycycline-inducible construct, excluding any detection of the endogenous CCDC134 mrRNA. CAL-1 cells stably expressing the doxycycline-inducible CCDC134 construct (clone 1 or 2) were treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) AlphaFold structure prediction for human CCDC134 (Uniprot entry name: Q9H6E4 CC134_HUMAN). (C) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) of indicated CAL-1 cells reconstituted with wildtype or deletion CCDC134 mutants. The Δ133–156 CCDC134 deletion mutant lacks the predicted N-linked glycosylated NQT sequon. Ev: empty vector. (D) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged wildtype (WT) or deletion mutant CCDC134 constructs. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (E) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged Δ133–156 CCDC134 deletion mutant which lacks the predicted N-linked glycosylated NQT sequon (left panel) and quantification of the colocalization between Flag-CCDC134 (wildtype or Δ133–156 deletion mutant) and calreticulin (right panel). Data are expressed as colocalization score (coloc. score) and pooled from three independent experiments. Each dot represents analysis of a single field of view containing one to four transfected cells, and violins show the variation of individual dot across all experiments; P value = ns, two-tailed Mann–Whitney test. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (F) Immunoblots of proteins from the (not precipitated) supernatant (SN) of indicated CAL-1 cells. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 24 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. (G) Immunoblots of indicated CAL-1 cells incubated for 48 h with supernatants (SN) of donor cells cultured for 24 h in Opti-MEM. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 48 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. In A, data show mean ± SD of three independent experiments. In C, F, and G, data are representative of two independent experiments. Source data are available for this figure: .

Article Snippet: Samples of Gp96-HA were further purified on Akta pure FPLC system using Superdex 200 10/300 column (cat. GE28-9909-44; Cytiva).

Techniques: Construct, Stable Transfection, Expressing, Concentration Assay, Western Blot, Mutagenesis, Plasmid Preparation, Confocal Microscopy, Transfection, Two Tailed Test, MANN-WHITNEY, Incubation, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 controls Gp96-dependent maturation and stability of plasma membrane and endolysosomal TLRs. (A) TNFα production of indicated Hoxb8-macrophages stimulated for 24 h with Pam3CSK4 (Pam3.) (0.1 μg/ml), LPS (10 ng/ml), R848 (0.1 μg/ml), CpG-B (ODN1668, 1 μM), or flagellin (0.1 μg/ml). Untr.: untreated. (B–D) Immunoblots of indicated Hoxb8-macrophages untreated (B) or stimulated with R848 (0.1 μg/ml), CpG-B (ODN1668) (1 μM) for 0–1 h (C) or Pam3CSK4 (0.1 μg/ml), LPS (10 ng/ml), and flagellin (0.1 μg/ml) for 0–1 h (D). (E and F) Immunoblots of indicated Hoxb8-macrophages. short exp.: short exposure; Mat.: mature; Imat.: immature, F: full length; C: cleaved form. (G) Quantification of TLR2, 4, 5, or 7 protein levels in sg Ren or sg CCDC134 Hoxb8-macrophages measured by surface (left upper panel) or surface and intracellular (intra.) (left lower panel) staining and quantified by gMFI (relative to respective sg Ren ). Representative histograms of surface (right upper panel) or intracellular (right lower panel). gMFI surface staining normalized to sg Ren TLR2 P value = 0.0053, TLR4 P value = 0.0063, TLR5 P value = 0.0093; gMFI surface and intracellular staining normalized to sg Ren TLR2 P value = 0.0002, TLR4 P value = 0.0256, TLR5 P value = ns, TLR7 P value <0.0001, two-tailed one sample t test. (H) Immunoblots of lysate from sg Ren or sg CCDC134 Hoxb8-macrophages treated with EndoH (H) or PNGase F (F). Mat.: mature form; Imat.: immature form; dg: deglycosylated. (I) Representative image of sg Ren or sg CCDC134 Hoxb8-macrophages stained for TLR7 (scale bar: 5 μm) (left panel) and quantification of TLR7 intensity (right panel). Data are expressed as fold change (FC) and pooled from n = 2. Violins show the variation of individual cells across all experiments; P value <0.0001, two-tailed Mann–Whitney test. (J) Quantification of CD11a, CD18, CD49d, and CD44 protein levels in sg Ren or sg CCDC134 Hoxb8-macrophages measured by surface (upper panel) or surface and intracellular (intra.) (bottom panel) staining and quantified by gMFI (relative to respective sg Ren ). Data show mean ± SD from three independent experiments; gMFI surface staining normalized to sg Ren CD11a P value = 0.0316, CD18 P value = 0.0449, CD49d P value = ns, CD44 P value = ns; gMFI surface and intracellular staining normalized to sg Ren CD11a P value = 0.0074, CD18 P value = ns, CD49d P value = 0.0029, CD44 P value = ns; two-tailed one sample t test. (K) Volcano plot of quantified proteins in whole proteome of sg CCDC134 versus sg Ren Hoxb8-macrophages (upregulated: red, fold change [FC] > 2 and P value <0.01; downregulated: blue, FC less than −2 and P value <0.01). pink: CCDC134 and Gp96; light pink: Bip and integrins; purple: TLRs; light purple: related to TLRs; green: IFN-stimulated gene proteins (ISGs). In B–F and H, data are representative of two experiments. In A, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. In G–J, data show mean ± SD from four or three independent experiments. Source data are available for this figure: .

Article Snippet: Samples of Gp96-HA were further purified on Akta pure FPLC system using Superdex 200 10/300 column (cat. GE28-9909-44; Cytiva).

Techniques: Membrane, Western Blot, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Therapy-induced senescent cancer cells contribute to cancer progression by promoting ribophorin 1-dependent PD-L1 upregulation

doi: 10.1038/s41467-024-54132-1

Figure Lengend Snippet: a Immunofluorescence staining of PD-L1 and markers of ER (HSP90B1) and Golgi (cis, GM130) in Control (Cont) or IR-induced cancer senescence (IR-CS). Nuclei were counterstained with DAPI. Scale bar: 10 µm. Representative images of n = 3 independent replicates. b Cell surface levels of PD-L1 were analyzed by flow cytometry in IR-induced senescent H460 cells. The gating strategies are provided in Supplementary Fig. . c PD-1 binding assay: representative images showing the binding of green fluorescence-labeled PD-1/Fc protein on IR-induced senescent H460 cells. Scale bar: 50 µm. Representative images of n = 3 independent replicates. d A Venn diagram was created to compare the N-glycosyltransferase list obtained from our microarray data in this study with the list of N-glycosyltransferases known to interact with PD-L1 . e Identification of N-glycosylation-related enzymes that are differentially expressed in IR-induced senescent H460 cancer cells. f Validation of increased mRNA expressions of N-glycosylation-related enzymes in IR-induced senescent H460 cells. Mean ± SD of n = 3 independent samples. One-way ANOVA-Tukey multiple comparison test. g qRT-PCR analysis of PD-L1 mRNA levels in IR-induced senescent H460 cells, which were transfected with each indicated siRNAs. Mean ± SD of n = 3 independent samples. One-way ANOVA-Tukey multiple comparison test. h Immunoblot analysis of PD-L1 levels in IR-induced senescent cancer cells, which were transfected with each indicated siRNAs. Representative immunoblots of n = 3 independent replicates. Statistical significance is represented as means ± SD. Source data are provided as a Source Data file.

Article Snippet: Peptide N-glycosidase F (PNGase F) (New England Biolab, P0704L), Porous graphitic carbon (PGC) cartridge (Agilent Technologies, 12102201), Acetonitrile (ACN) (Sigma-Aldrich, 34851, HPLC grade), Formic acid(Honeywell, 94318) Antibodies were obtained from the following suppliers: PD-L1 (E1L3N®) (1:1000, Cell Singing Technology; CST, Danvers, MA, USA, 13684), p21 (12D1) (1:1000, CST, 2947), LAMP1 (D4O1S) (1:1000, CST, 15665), LC3B (D11) (1:1000, CST, 3868), Rab5A (E6N8S) (1:1000, CST, 46449), Rab11 (D4F5) (1:1000, CST, 5589), β-Actin (8H10D10) (1:10000, CST, 3700), p53 (DO7) (1:1000, Leica, Wetzlar, Germany, NCL-L-p53-DO7), GM130 (Clone 35) (1:1000, BD Bioscience, Franklin lakes, NJ, USA, 610823), TGN46 (2F11) (1:100, Novus, St. Louis, MO, USA, H00010618-M02), gp96/HSP90B1/GRP94 (CL2647) (1:100, Novus, NBP2-42379), PD-L1 (PDL1/2746) (1:100, Novus, NBP2-80490), FAM134B (1:100, Proteintech, Rosemont, IL, USA, 21537-1-AP), Granzyme B (1:100, LS Bio, Shirley, MA, USA, LS-C332166), PD-L1 (clone OTI2C7) (1:100, LS Bio, LS-C338364), RPN1 (E-7) (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-48367), CD8 (LT8) (1:100, GeneTex, Alton Pkwy Irvine, CA, USA, GTX74773).

Techniques: Immunofluorescence, Staining, Control, Flow Cytometry, Binding Assay, Fluorescence, Labeling, Microarray, Comparison, Quantitative RT-PCR, Transfection, Western Blot

Journal: Nature Communications

Article Title: Therapy-induced senescent cancer cells contribute to cancer progression by promoting ribophorin 1-dependent PD-L1 upregulation

doi: 10.1038/s41467-024-54132-1

Figure Lengend Snippet: a H460 cells transfected with three different RPN1 siRNAs prior to IR exposure were subjected to SA-β-Gal staining. Scale bar: 50 µm. Mean ± SD of n = 3 independent samples. One-way ANOVA-Tukey multiple comparison test. Representative images of n = 3 independent replicates. b Immunoblot analysis of Control (Cont) or IR-induced senescent H460 cells (IR-CS), which were transfected with Cont si or three different RPN1 siRNAs (#1-3). Representative immunoblots of n = 3 independent replicates. c Cells were transfected with either Cont siRNA (Cont si) or RPN1 siRNA (RPN1 si), followed by treatment with Doxorubicin (50 ng/ml for 4 days). d H460 cells transfected with PTEN siRNA (PTEN si) underwent further transfection with either Cont si or RPN1 si. e Immunoblot analysis of PD-L1 levels in lung, breast and colon cancer cells. f Western blot showing PD-L1 levels in H1975 and BT549 cells transfected with either Cont si or RPN1 si. g Immunoblot analysis of PD-L1 in H460, A549, and HCC827 cells transfected with either mock vectors or RPN1-HA vectors. c – g Representative immunoblots of n = 3 independent replicates. h , i Immunofluorescence of PD-L1 and markers of ER (HSP90B1) ( h ) and Golgi (cis, GM130) ( i ) in IR-induced senescent H460 cells transfected with either Cont si or RPN1 si. Nuclei were counterstained with DAPI. Scale bar: 10 µm. Representative images of n = 3 independent replicates. j Median fluorescence intensity (MFI) of membrane-bound PD-L1 in IR-induced senescent H460 cells transfected with either Cont si or RPN1 si were analyzed by flow cytometry. Mean ± SD of n = 3 independent samples. One-way ANOVA-Tukey multiple comparison test. The gating strategies are provided in Supplementary Fig. . k H460 cells transfected with either Cont si or RPN1 si were exposed to IR. On day 4 post-IR, the binding of green fluorescence-labeled PD-1/Fc protein was measured in each si-treated/irradiated cell group. Mean ± SD of n = 3 independent samples. One-way ANOVA-Tukey multiple comparison test. l B16/F10-OVA melanoma cells transfected with either Cont si or RPN1 si were allowed to form spheroids for 3 days and then exposed to IR. On day 4 post-IR, activated OT-cells were co-cultured with B16/F10-OVA melanoma spheroids. The next day, the number of infiltrated OT-cells in the spheroids was quantified. Mean ± SD of n = 3 independent samples. One-way ANOVA-Tukey multiple comparison test. m B16/F10-OVA melanoma cells transfected with either Cont si or RPN1 si prior to IR were co-cultured with OT-cells. After 48 hours, the quantitative ratios of dead cells to total cells were measured by the fluorescence of caspase-3 activity. Mean ± SD of n = 3 independent samples. One-way ANOVA-Tukey multiple comparison test. Source data are provided as a Source Data file.

Article Snippet: Peptide N-glycosidase F (PNGase F) (New England Biolab, P0704L), Porous graphitic carbon (PGC) cartridge (Agilent Technologies, 12102201), Acetonitrile (ACN) (Sigma-Aldrich, 34851, HPLC grade), Formic acid(Honeywell, 94318) Antibodies were obtained from the following suppliers: PD-L1 (E1L3N®) (1:1000, Cell Singing Technology; CST, Danvers, MA, USA, 13684), p21 (12D1) (1:1000, CST, 2947), LAMP1 (D4O1S) (1:1000, CST, 15665), LC3B (D11) (1:1000, CST, 3868), Rab5A (E6N8S) (1:1000, CST, 46449), Rab11 (D4F5) (1:1000, CST, 5589), β-Actin (8H10D10) (1:10000, CST, 3700), p53 (DO7) (1:1000, Leica, Wetzlar, Germany, NCL-L-p53-DO7), GM130 (Clone 35) (1:1000, BD Bioscience, Franklin lakes, NJ, USA, 610823), TGN46 (2F11) (1:100, Novus, St. Louis, MO, USA, H00010618-M02), gp96/HSP90B1/GRP94 (CL2647) (1:100, Novus, NBP2-42379), PD-L1 (PDL1/2746) (1:100, Novus, NBP2-80490), FAM134B (1:100, Proteintech, Rosemont, IL, USA, 21537-1-AP), Granzyme B (1:100, LS Bio, Shirley, MA, USA, LS-C332166), PD-L1 (clone OTI2C7) (1:100, LS Bio, LS-C338364), RPN1 (E-7) (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-48367), CD8 (LT8) (1:100, GeneTex, Alton Pkwy Irvine, CA, USA, GTX74773).

Techniques: Transfection, Staining, Comparison, Western Blot, Control, Immunofluorescence, Fluorescence, Membrane, Flow Cytometry, Binding Assay, Labeling, Irradiation, Cell Culture, Activity Assay