Journal: Redox Biology

Article Title: Supplementing sialic acid analogs overcomes radiotherapy resistance in triple-negative breast cancer by exacerbating ER stress

doi: 10.1016/j.redox.2025.103712

Figure Lengend Snippet: ST3GAL4 induced α 2,3-sialylation of HSP90B1 A. The significance and Area-IP of the identified ST3GAL4-binding proteins through IP-MS are represented in a volcano plot. B. IP/IB analysis of ST3GAL4 and HSP90B1 in MDA-MB-231-ST3GAL4 and SUM159PT-ST3GAL4 cells. C. Proximity ligation assay (PLA) analysis of the interaction between ST3GAL4 and HSP90B1 in MDA-MB-231-ST3GAL4 and SUM159PT-ST3GAL4 cells. D. The radiation-dose survival curves of indicated stable cell lines are shown. E. Biotinylated MAL II was used to pull down proteins from indicated stable cell lines and was analyzed by Western blot with anti-biotin, anti-HSP90B1 antibodies. F. IB analysis of HSP90B1 from MDA-MB-231 and SUM159PT cells with wild type-ST3GAL4 or Motif S- deletion ST3GAL4. Biotin was used as a loading control. G. Lysates from MDA-MB-231-ST3GAL4 and SUM159PT-ST3GAL4 cells were subject to mock, α2-3-neuraminidase or α2-3,6,8-neuraminidase hydrolysis. Then the lysates were immunoblotted for detecting the expression pattern of HSP90B1. β-Actin served as a loading control. H. Cell lysates of indicated cells were treated with α2-3-neuraminidase with or without pre-treatment with PNGase F. The lysates were then subjected to SDS-PAGE analysis. β-Actin expression served as a loading control. I. Biotinylated MAL II was used to pull down proteins from indicated stable cell lines with or without pre-treatment with PNGase F and was analyzed by Western blot with biotin, anti-HSP90B1 antibody. J. IP/IB analysis of the interaction between ST3GAL4 and HSP90B1 in indicated cells with or without pre-treatment with PNGase F. One-way repeated-measures ANOVA test was used in D. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mice were then intraperitoneally injected with Neu5Ac (20 mg/kg, Aldrich, dissolved in physiological saline) or HSP90B1 inhibitor (MCE, HY-10211 25 mg/kg) or combining 3Fax-Neu5Ac and HSP90B1 inhibitor daily for 6 consecutive days after subcutaneous injection.

Techniques: Binding Assay, Protein-Protein interactions, Proximity Ligation Assay, Stable Transfection, Western Blot, Control, Expressing, SDS Page

Journal: Redox Biology

Article Title: Supplementing sialic acid analogs overcomes radiotherapy resistance in triple-negative breast cancer by exacerbating ER stress

doi: 10.1016/j.redox.2025.103712

Figure Lengend Snippet: ST3GAL4 promotes HSP90B1 trafficking from Golgi to ER through SURF4 cargo receptor A. Immunofluorescence (IF) staining of HSP90B1 and Calnexin or GM130 in MDA-MB-231 and SUM159PT cells with or without ST3GAL4 knockdown. B. IB analysis of HSP90B1 in the extracted endoplasmic reticulum (ER) and Golgi apparatus of MDA-MB-231 and SUM159PT cells with or without ST3GAL4 silencing. C. IF staining of HSP90B1 and GM130 in MDA-MB-231-ST3GAL4 and SUM159PT-ST3GAL4 cells with or without treatment with COPI inhibitor, Brefeldin A. D. IB analysis of HSP90B1 in the extracted ER and Golgi apparatus of indicated cells with or without Brefeldin A treatment. E. Co‐IP–MS analysis of the HSP90B1 interactome in cytoplasmic lysates of MDA-MB-231-ST3GAL4 cells by using anti‐HSP90B1 antibody. F. IP/IB analysis were performed to detect the interaction between HSP90B1 and SURF4 in MDA-MB-231-ST3GAL4 and SUM159PT-ST3GAL4 cells. G. Structure model of the interaction between HSP90B1 and SURF4 protein predicted using PyMOL332 and Molecular Operating Environment (MOE) software-Site finder module. H. IP/IB analysis were performed to detect the interaction between HSP90B1 and SURF4 in MDA-MB-231-ST3GAL4 and SUM159PT-ST3GAL4 cells with or without PNGase F treatment. I. IF staining of HSP90B1 and GM130 in MDA-MB-231-ST3GAL4 and SUM159PT-ST3GAL4 cells with or without SURF4 silencing. J. IB analysis of HSP90B1 in the extracted ER and Golgi apparatus of MDA-MB-231-ST3GAL4 and SUM159PT-ST3GAL4 cells with or without SURF4 silencing. Calnexin and GM130 were used as an ER marker and a Golgi marker respectively.

Article Snippet: Mice were then intraperitoneally injected with Neu5Ac (20 mg/kg, Aldrich, dissolved in physiological saline) or HSP90B1 inhibitor (MCE, HY-10211 25 mg/kg) or combining 3Fax-Neu5Ac and HSP90B1 inhibitor daily for 6 consecutive days after subcutaneous injection.

Techniques: Immunofluorescence, Staining, Knockdown, Co-Immunoprecipitation Assay, Software, Marker

Journal: Redox Biology

Article Title: Supplementing sialic acid analogs overcomes radiotherapy resistance in triple-negative breast cancer by exacerbating ER stress

doi: 10.1016/j.redox.2025.103712

Figure Lengend Snippet: ER-localised HSP90B1 accelerated clearance of misfolded proteins through ERAD A. IF staining of misfolded proteins were detected using PERK(LD)-EGFP-HOTag3 reporter in indicated stable cell lines with or without radiation treatment. Quantification of misfolded proteins percentage were shown in right panel. B. IB analysis of pIREα, IREα, ATF6 (p50), ATF6 (p90), PERK, pPERK in indicated stable cell lines with or without radiation treatment. β-Actin was used as a Loading control. C. IB analysis of pEIF2α, EIF2α, ATF4 in indicated stable cell lines with or without radiation treatment. β-Actin was used as a Loading control. D. The relative of pPERK expression level and pEIF2α expression level were quantificated. E. IF staining of misfolded proteins were detected in indicated cell lines under 3Gy radiation treatment. Quantification of misfolded proteins percentage were shown in right panel. F. IB analysis of pEIF2α, EIF2α, ATF4 in indicated cell lines under 3Gy radiation treatment. β-Actin was used as a Loading control. G. Biotinylated MAL II was used to pull down proteins from vector- or ST3GAL4 -overexpressing MDA-MB-231 and SUM159PT cells with or without HSP90B1 inhibitor treatment and IB analysis was performed with anti-biotin, anti-HSP90B1 antibody. H. IP/IB analysis was performed to detect the interaction between ST3GAL4 and HSP90B1 in MDA-MB-231-ST3GAL4 and SUM159PT-ST3GAL4 cells with or without HSP90B1 inhibitor treatment. Data in A, D, E were plotted as the mean ± SD of biological triplicates. An unpaired 2-sided Student's t -test was used in A, D, E. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mice were then intraperitoneally injected with Neu5Ac (20 mg/kg, Aldrich, dissolved in physiological saline) or HSP90B1 inhibitor (MCE, HY-10211 25 mg/kg) or combining 3Fax-Neu5Ac and HSP90B1 inhibitor daily for 6 consecutive days after subcutaneous injection.

Techniques: Staining, Stable Transfection, Control, Expressing, Plasmid Preparation

Journal: Redox Biology

Article Title: Supplementing sialic acid analogs overcomes radiotherapy resistance in triple-negative breast cancer by exacerbating ER stress

doi: 10.1016/j.redox.2025.103712

Figure Lengend Snippet: Sialic acid analogs reversed ST3GAL4-mediated cellular sialylation and radioresistance in TNBC A. IB analysis of HSP90B1 in MDA-MB-231-ST3GAL4 cells with HSP90B1 inhibitor (17-AAG, 1 μM) and/or SAA (3Fax-NeuAc, 100 μM) under 3Gy irradiation. β-Actin was used as a Loading control. B. Biotinylated MAL II was used to pull down proteins from MDA-MB-231-ST3GAL4 cells with indicated treatment under 3Gy irradiation and IB analysis was performed with anti-biotin and anti-HSP90B1 antibody. C. IB analysis of HSP90B1 in the extracted endoplasmic reticulum (ER) of MDA-MB-231-ST3GAL4 cells under 3Gy irradiation with indicated treatment. D. IF staining of misfolded proteins were detected using PERK(LD)-EGFP-HOTag3 reporter in MDA-MB-231-ST3GAL4 cells under 3Gy irradiation with indicated treatment. Quantification of misfolded proteins percentage were shown in right panel. E. IB analysis of pEIF2a, EIF2a, ATF4 in MDA-MB-231-ST3GAL4 cells under 3Gy irradiation with indicated treatment. β-Actin was used as a Loading control. F. ST3GAL4, SLC1A5, GCLC, CTNS mRNA level were detected using qPCR in MDA-MB-231-ST3GAL4 cells under 3Gy irradiation with indicated treatment. G. Relative cellular cyst(e)ine levels (upper panel) and relative GSH level(lower panel) in MDA-MB-231-ST3GAL4 cells under 3Gy irradiation with indicated treatment. H. Flow cytometric analysis of intracellular ROS in MDA-MB-231-ST3GAL4 cells under 3Gy irradiation with indicated treatment. The relative ROS levels were quantified in right panel using mean FITC scores of each group. I. IB analysis of p -ATM, ATM, p -CHK2, CHK2 in MDA-MB-231-ST3GAL4 cells with indicated treatment under 3Gy irradiation. β-Actin was used as a Loading control. J. The radiation-dose survival curves of MDA-MB-231-ST3GAL4 cells under 3Gy irradiation with indicated treatment. K. MDA-MB-231-ST3GAL4 cells (2 × 10 6 ) were subcutaneously into BALB/c nude mice. After two weeks of inoculation, the mice were treated with RT alone, RT combined with HSP90B1 inhibitor, RT combined with SAA, RT combined with both HSP90B1 inhibitor and SAA. Radiation was given with 12 Gy (2 Gy/day for 6 days), HSP90B1 inhibitor (17-AAG, 25 mg/kg) and SAA (3Fax-NeuAc, 20 mg/kg) both via intravenous administration once daily for 7 days. Volumes of tumors from each group were indicated (n = 6/group). L. Representative images (left) and quantification (right) of Ki67 and TUNEL staining in indicated tumors. Data in D, F, G, H were plotted as the mean ± SD of biological triplicates. An unpaired 2-sided Student's t -test was used in D, F, G, H. One-way repeated-measures ANOVA test was used in J. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mice were then intraperitoneally injected with Neu5Ac (20 mg/kg, Aldrich, dissolved in physiological saline) or HSP90B1 inhibitor (MCE, HY-10211 25 mg/kg) or combining 3Fax-Neu5Ac and HSP90B1 inhibitor daily for 6 consecutive days after subcutaneous injection.

Techniques: Irradiation, Control, Staining, TUNEL Assay

Journal: Redox Biology

Article Title: Supplementing sialic acid analogs overcomes radiotherapy resistance in triple-negative breast cancer by exacerbating ER stress

doi: 10.1016/j.redox.2025.103712

Figure Lengend Snippet: SAA re-sensitizes radiotherapy-resistant TNBC A. Schematic of TNBC Patient-derived tumor xenograft (PDXs) development. B. IB analysis of ST3GAL4 and HSP90B1 in MDA-MB-231-Vector, MDA-MB-231-ST3GAL4, PDX-1, and PDX-2. C. Biotinylated MAL II was used to pull down proteins from MDA-MB-231-Vector, MDA-MB-231-ST3GAL4, PDX-1, and PDX-2. IB analysis was performed with anti-biotin and anti-HSP90B1 antibody. D. IB analysis of HSP90B1 after biotinylated MAL II pulldown in PDX-1 and PDX-2 with or without 3Fax treatment. E. Tumor volumes of PDX-1 and PDX-2 with indicated treatment. Representative tumors from each group were shown (n = 6/Group). F. The proliferation index represented the percentage of Ki67+ cells in indicated tumors and the apoptotic index represented the percentage of TUNEL + cells in indicated tumors. Data in F was plotted as the mean ± SD of biological triplicates. An unpaired 2-sided Student's t -test was used in F. One-way repeated-measures ANOVA test was used in E. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mice were then intraperitoneally injected with Neu5Ac (20 mg/kg, Aldrich, dissolved in physiological saline) or HSP90B1 inhibitor (MCE, HY-10211 25 mg/kg) or combining 3Fax-Neu5Ac and HSP90B1 inhibitor daily for 6 consecutive days after subcutaneous injection.

Techniques: Derivative Assay, Plasmid Preparation, TUNEL 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: 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: Glycoproteomics, 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: Clinical Proteomics, Membrane, Western Blot, Staining, Two Tailed Test, MANN-WHITNEY