antibodies against tmem175  (Proteintech)

Journal: Nature Communications

Article Title: Deficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor immunity via inflammasome and cross-presentation pathway

doi: 10.1038/s41467-026-69546-2

Figure Lengend Snippet: a Tissue chip of paired melanoma and paracancerous tissues was stained by anti-TMEM175 to detect the expression of TMEM175 through IHC ( n = 6 pairs). TMEM175 was expressed more robustly in melanoma tissues than in paracancerous tissues. Scale bars indicate 500 µm for the left panel and 50 µm for the right panel. TMEM175 positive area was calculated. b , c Wild type mice were subcutaneously injected with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1\times {10}^{6}$$\end{document} 1 × 10 6 B16-F10 tumor cells ( n = 18 mice). Mice were euthanized at day 18 to analyze the TME through FCM. ( b ) M2-like TAMs were positively correlated with the TMEM175 expression in TAMs. c M2-like TAMs were not correlated with TMEM175 expression in other CD45 - cells. d , e TMEM175 expression was obviously decreased in Tmem175 −/− BMDMs. Lysates of Tmem175 −/− and WT BMDMs were analyzed by western blot using anti-TMEM175 antibody ( n = 3 biologically independent samples). Ratios of the grayscale values of TMEM175 and β-actin were calculated. ( f ) Lysosomal potassium conductance in Tmem175 −/− BMDMs was significantly decreased. Lysosomes from Tmem175 −/− and WT BMDMs were analyzed by lysosomal voltage clamp. The current was recorded when the bath solution contained 150 mM K + or 150 mM NMDG. Representative result of voltage clamp was displayed, and the average current intensities in +100 mV with 150 mM K + or 150 mM NMDG were compared ( n = 5 biologically independent samples). Representative results from at least two independent experiments are presented as mean ± SEM, ns denotes not significant. Linear regression was applied to calculate the relationship between the percentages of M2-like TAMs and TMEM175 expression in different cell subsets in ( b , c ). Statistical significances in ( a , e ) were determined by two-sided unpaired t -test. Statistical significances in ( f ) were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. Source data are provided as a file.

Article Snippet: The following antibodies were used: TMEM175 antibody (Proteintech, 19925-1-AP), cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology, 9661L), caspase-1 p10 antibody (SANTA CRUZ, sc-56036), IL-1β (3A6) Mouse antibody (Cell Signaling Technology, 12242S), HRP anti-rabbit IgG antibody (ServiceBio, GB23303), and HRP anti-mouse IgG antibody (ServiceBio, G1214).

Techniques: Staining, Expressing, Injection, Western Blot

Journal: Nature Communications

Article Title: Deficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor immunity via inflammasome and cross-presentation pathway

doi: 10.1038/s41467-026-69546-2

Figure Lengend Snippet: Tmem175 −/− and WT mice were subcutaneously injected with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1\times {10}^{6}$$\end{document} 1 × 10 6 B16-F10 ( n = 5 mice for a - h ) or \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1\times {10}^{6}$$\end{document} 1 × 10 6 LL-2 ( n = 6 mice for i – p ) tumor cells. Mice were euthanized at day 18 (for B16-F10) or day 24 (for LL-2) to weigh the tumors and analyze the TME through IHC, IF, and FCM. a B16-F10 tumors were fixed, sliced, and stained by anti-cleaved caspase-3 to detect the tumor cell death through IHC. Enhanced tumor cell death was observed in tissues from Tmem175 −/− mice. Scale bars indicate 50 µm. b B16-F10 tumor weights in Tmem175 −/− mice were significantly decreased. c Expression of M1-like marker, MHC-II, in TAMs (gated from CD45 + CD11b + F4/80 + cells) from B16-F10 tumors was significantly up-regulated in Tmem175 −/− mice. d Expression of M2-like marker, CD206, in TAMs (gated from CD45 + CD11b + F4/80 + cells) from B16-F10 tumors was significantly down-regulated in Tmem175 −/− mice. e B16-F10 tumors were fixed, sliced, and stained by anti-CD8 and anti-NK1.1 to detect CD8 + T cells and NKs in tissues. Enhanced recruitment of CD8 + T cells and NKs was observed in tissues from Tmem175 −/− mice. Scale bars indicate 50 µm. f CD4 + T cells and CD4 + CD69 + activated T cells (gated from CD3 + cells) from B16-F10 tumors were significantly augmented in Tmem175 −/− mice. g CD8 + T cells and CD8 + CD69 + activated T cells (gated from CD3 + cells) from B16-F10 tumors were significantly augmented in Tmem175 −/− mice. h NKs and NK1.1 + CD69 + activated NKs (gated from CD3 - cells) from B16-F10 tumors were significantly augmented in Tmem175 −/− mice. i LL-2 tumors were fixed, sliced, and stained by anti-cleaved caspase-3 to detect the tumor cell death through IHC. Enhanced tumor cell death was observed in tissues from Tmem175 −/− mice. Scale bars indicate 50 µm. j LL-2 tumor weights in Tmem175 −/− mice were significantly decreased. k Expression of MHC-II in TAMs (gated from CD45 + CD11b + F4/80 + cells) from LL-2 tumors was significantly up-regulated in Tmem175 −/− mice. l Expression of CD206 in TAMs (gated from CD45 + CD11b + F4/80 + cells) from LL-2 tumors was not significantly changed in Tmem175 −/− mice. m LL-2 tumors were fixed, sliced, and stained by anti-CD8 and anti-NK1.1 to detect CD8 + T cells and NKs in tissues. Enhanced recruitment of CD8 + T cells was observed in tissues from Tmem175 −/− mice. Scale bars indicate 50 µm. n CD4 + T cells, instead of CD4 + CD69 + activated T cells (gated from CD3 + cells) from LL-2 tumors, were significantly augmented in Tmem175 −/− mice. o CD8 + T cells and CD8 + CD69 + activated T cells (gated from CD3 + cells) from LL-2 tumors were significantly augmented in Tmem175 −/− mice. p NKs and NK1.1 + CD69 + activated NKs (gated from CD3 - cells) from LL-2 tumors were not obviously augmented in Tmem175 −/− mice. Representative results from two independent experiments are presented as mean ± SEM; ns denotes not significant. Statistical significances in ( b – d, f–h , j – l , and n – p ) were determined by two-sided unpaired t -test. Source data are provided as a file.

Article Snippet: The following antibodies were used: TMEM175 antibody (Proteintech, 19925-1-AP), cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology, 9661L), caspase-1 p10 antibody (SANTA CRUZ, sc-56036), IL-1β (3A6) Mouse antibody (Cell Signaling Technology, 12242S), HRP anti-rabbit IgG antibody (ServiceBio, GB23303), and HRP anti-mouse IgG antibody (ServiceBio, G1214).

Techniques: Injection, Immunohistochemistry-IF, Staining, Expressing, Marker

Journal: Nature Communications

Article Title: Deficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor immunity via inflammasome and cross-presentation pathway

doi: 10.1038/s41467-026-69546-2

Figure Lengend Snippet: B16-F10 cells (2 × 10 5 ) were intravenously injected into WT and Tmem175 −/− sex-matched mice at 6–8 weeks old ( n = 6 mice). Mice were euthanized on day 21. The lungs were weighed, and the metastatic nodules were counted. Then the lungs were minced and digested to prepare the single-cell suspension for FCM analysis. a TMEM175 conditional knockout inhibited B16-F10 lung metastasis. Scale bars indicate 1 cm. b, c Tmem175 −/− mice showed fewer lung metastatic nodules ( b ). Lung weights in Tmem175 −/− mice were decreased ( c ). d MHC-II high AMs (gated from CD45 + CD11c + CD11b − CD64 + cells) were increased in Tmem175 −/− mice. e CD206 high AMs (gated from CD45 + CD11c + CD11b − CD64 + cells) were decreased in Tmem175 −/− mice. f MHC-II high IMs (gated from CD45 + CD11b + CD24 − CD64 + cells) were increased in Tmem175 −/− mice. g CD206 high IMs (gated from CD45 + CD11b + CD24 − CD64 + cells) were decreased in Tmem175 −/− mice. h Subsets of IMs (IM1, IM2, and IM3) were altered by TMEM175 conditional knockout. i CD11c low MHC-II low IM1 (gated from CD45 + CD11b + CD24 − CD64 + cells) were decreased in Tmem175 −/− mice. j CD11c low MHC-II high IM2 (gated from CD45 + CD11b + CD24 − CD64 + cells) were decreased in Tmem175 −/− mice. k CD11c + MHC-II high IM3 (gated from CD45 + CD11b + CD24 − CD64 + cells) were increased in Tmem175 −/− mice. CD4 + T cells ( l ), CD4 + CD69 + T cells ( m ), and CD4 + IFN-γ + T cells ( n ) (gated from CD3 + cells) were increased in Tmem175 −/− mice. CD8 + T cells ( o ), CD8 + CD69 + T cells ( p ), CD8 + IFN-γ + T cells ( q ), and CD8 + p15E-tetramer + T cells ( r ) (gated from CD3 + cells) were increased in Tmem175 −/− mice. s Single cell sequencing was applied to evaluate the changes in the cell subsets and the transcriptome ( n = 3 mice). Representative results from two independent experiments are presented as mean ± SEM, ns denotes not significant. Statistical significances in ( b –g and i –r ) were determined by two-sided unpaired t -test. Source data are provided as a file.

Article Snippet: The following antibodies were used: TMEM175 antibody (Proteintech, 19925-1-AP), cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology, 9661L), caspase-1 p10 antibody (SANTA CRUZ, sc-56036), IL-1β (3A6) Mouse antibody (Cell Signaling Technology, 12242S), HRP anti-rabbit IgG antibody (ServiceBio, GB23303), and HRP anti-mouse IgG antibody (ServiceBio, G1214).

Techniques: Injection, Single Cell, Suspension, Knock-Out, Sequencing

Journal: Nature Communications

Article Title: Deficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor immunity via inflammasome and cross-presentation pathway

doi: 10.1038/s41467-026-69546-2

Figure Lengend Snippet: B16-F10 tumors from Fig. were fixed, sliced, and stained by anti-caspase-1 p10 or anti-IL-1β ( a ) to detect the inflammasome activation through IHC ( n = 5 biologically independent samples). Enhanced inflammasome activation was observed in tissues from Tmem175 −/− mice ( b , c ). Scale bars indicate 50 µm. d –m Tmem175 −/− and WT mice were subcutaneously injected with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1\times {10}^{6}$$\end{document} 1 × 10 6 B16-F10 tumor cells ( n = 5 mice). Vehicle or VX-765 solution (0.4 mg dissolved in 200 µL) was administrated every day through intraperitoneal injection since day 8. Mice were euthanized at day 18 to weigh the tumors and analyze the TME through IHC, FCM, and IF. e VX-765 elevated the tumor weights in Tmem175 −/− mice, thus VX-765 abrogated the anti-tumor effect in Tmem175 −/− mice. f VX-765 decreased the cleaved caspase-3 expression in Tmem175 −/− mice, indicating reduced tumor cell death. Scale bars indicate 50 µm. g Expression of MHC-II in TAMs (gated from CD45 + CD11b + F4/80 + cells) was significantly attenuated by VX-765 in Tmem175 −/− mice. h Expression of CD206 in TAMs (gated from CD45 + CD11b + F4/80 + cells) was not obviously altered by VX-765 in Tmem175 −/− mice. i , j CD4 + T cells and CD4 + CD69 + activated T cells (gated from CD3 + cells) were significantly diminished by VX-765 in Tmem175 −/− mice. k, l CD8 + T cells and CD8 + CD69 + activated T cells (gated from CD3 + cells) were significantly diminished by VX-765 in Tmem175 −/− mice. m IF staining showed that VX-765 decreased the recruitment of CD8 + T cells in tumors from Tmem175 −/− mice. Scale bars indicate 50 µm. n , o Tmem175 −/− and WT mice were subcutaneously injected with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1\times {10}^{6}$$\end{document} 1 × 10 6 B16-F10 tumor cells ( n = 5 mice). Vehicle or chitosan solution (2 mg dissolved in 200 µL) was administrated every 3 days through intraperitoneal injection since day 3. Mice were euthanized at day 18 to weigh the tumors and analyze the cell death by IHC. o Tmem175 −/− mice showed better response to chitosan treatment. p Chitosan promoted the expression of cleaved caspase-3 in both Tmem175 −/− and WT mice. Scale bars indicate 50 µm. q , r Primary Tmem175 −/− and WT BMDMs or PMs were stimulated with 500 ng/mL LPS for 4 h to activate signal 1 of NLRP3 inflammasomes, and then treated with 40 µg/mL chitosan for 5 h. Tmem175 −/− BMDMs and PMs secreted more IL-1β in response to chitosan ( n = 5 biologically independent samples). s Primary Tmem175 −/− and WT BMDMs were stimulated with 500 ng/mL LPS for 4 h to activate signal 1 of NLRP3 inflammasomes, and then treated with 300 µg/mL aluminium hydroxide for desired period of time. Lysates of Tmem175 −/− and WT BMDMs were analyzed by western blot using anti-caspase-1 antibody. Tmem175 −/− BMDMs showed more enhanced cleavage of caspase-1 p10 in response to aluminium hydroxide. Representative results from two independent experiments are presented as mean ± SEM, ns denotes not significant. Statistical significances in ( b , c ) were determined by two-sided unpaired t -test, in ( e , g –l, o , q , r ) were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. In ( h ), the CD206 + cells between Tmem175 −/− and WT in VX-765 treated group were compared by two-sided unpaired t -test. Source data are provided as a file. d , n Created in BioRender. Wang, Y. (2026) https://BioRender.com/yaf354h .

Article Snippet: The following antibodies were used: TMEM175 antibody (Proteintech, 19925-1-AP), cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology, 9661L), caspase-1 p10 antibody (SANTA CRUZ, sc-56036), IL-1β (3A6) Mouse antibody (Cell Signaling Technology, 12242S), HRP anti-rabbit IgG antibody (ServiceBio, GB23303), and HRP anti-mouse IgG antibody (ServiceBio, G1214).

Techniques: Staining, Activation Assay, Injection, Expressing, Western Blot

Journal: Nature Communications

Article Title: Deficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor immunity via inflammasome and cross-presentation pathway

doi: 10.1038/s41467-026-69546-2

Figure Lengend Snippet: a – l Tmem175 −/− and WT mice ( n = 5 mice) were treated with 100 µg/100 µL anti-IL-1β or Armenian hamster IgG isotype intraperitoneally 1 day before the subcutaneous implantation with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1\times {10}^{6}$$\end{document} 1 × 10 6 B16-F10. Then same doses of IgG were injected intraperitoneally on day 6 and 13. Mice were euthanized on day 14 to weigh the tumors and analyze the TME through FCM. b The anti-tumor effects of TMEM175 knockout were suppressed by anti-IL-1β. c Expression of MHC-II in TAMs (gated from CD45 + CD11b + F4/80 + cells) was significantly attenuated by anti-IL-1β in Tmem175 −/− mice. d Expression of CD206 in TAMs (gated from CD45 + CD11b + F4/80 + cells) was not obviously altered by anti-IL-1β in Tmem175 −/− mice. e – h Recruitment, activation, IFN-γ expression, and granzyme B expression in CD4 + T cells (gated from CD3 + cells) were significantly diminished by anti-IL-1β in Tmem175 −/− mice. i – l Recruitment, activation, IFN-γ expression, and granzyme B expression in CD8 + T cells (gated from CD3 + cells) were significantly diminished by anti-IL-1β in Tmem175 −/− mice. m – x Tmem175 −/− and WT mice ( n = 5 mice) were treated with 100 µg/100 µL anti-IL-18 or rat IgG2a isotype intraperitoneally 1 day before the subcutaneous implantation with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1\times {10}^{6}$$\end{document} 1 × 10 6 B16-F10. Then same doses of IgG were injected intraperitoneally on day 6 and 13. Mice were euthanized on day 14 to weigh the tumors and analyze the TME through FCM. n The anti-tumor effects of TMEM175 knockout were suppressed by anti-IL-18. o Expression of MHC-II in TAMs (gated from CD45 + CD11b + F4/80 + cells) was significantly attenuated by anti-IL-18 in Tmem175 −/− mice. p Expression of CD206 in TAMs (gated from CD45 + CD11b + F4/80 + cells) was not obviously altered by anti-IL-18 in Tmem175 −/− mice. q – t Recruitment, activation, IFN-γ expression, and granzyme B expression in CD4 + T cells (gated from CD3 + cells) were significantly diminished by anti-IL-18 in Tmem175 −/− mice. Activated CD4 + T cells (gated from CD3 + cells) were significantly diminished by anti-IL-18 in WT mice. u – x Recruitment, activation, IFN-γ expression, and granzyme B expression in CD8 + T cells (gated from CD3 + cells) were significantly diminished by anti-IL-18 in Tmem175 −/− mice. Recruitment, activation, and granzyme B expression in CD8 + T cells (gated from CD3 + cells) were significantly diminished by anti-IL-18 in WT mice. Representative results from two independent experiments are presented as mean ± SEM, ns denotes not significant. Statistical significances in ( b – l and n –x ) were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. Source data are provided as a file. a , m Created in BioRender. Wang, Y. (2026) https://BioRender.com/yaf354h .

Article Snippet: The following antibodies were used: TMEM175 antibody (Proteintech, 19925-1-AP), cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology, 9661L), caspase-1 p10 antibody (SANTA CRUZ, sc-56036), IL-1β (3A6) Mouse antibody (Cell Signaling Technology, 12242S), HRP anti-rabbit IgG antibody (ServiceBio, GB23303), and HRP anti-mouse IgG antibody (ServiceBio, G1214).

Techniques: Injection, Knock-Out, Expressing, Activation Assay

Journal: Nature Communications

Article Title: Deficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor immunity via inflammasome and cross-presentation pathway

doi: 10.1038/s41467-026-69546-2

Figure Lengend Snippet: a – g Wild type C57BL/6 J mice were subcutaneously injected with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1\times {10}^{6}$$\end{document} 1 × 10 6 B16-F10-OVA cells. On day 10, the splenic lymphocytes from tumor bearing mice were harvested, CFSE-labeled, and cultured with WT or Tmem175 −/− BMDMs in the presence of IL-2 (20 U/mL) for 48 h before FCM and ELISA analysis ( n = 6 biologically independent samples). During the co-culture, B16-F10-OVA cell debris (2.5 × 10 5 /mL), IL-1β (500 pg/mL), and IL-18 (500 pg/mL) were added in the desired group. b , c Splenic lymphocytes were labeled by CFSE, thus the CFSE low cells represented the successive generations of cells. Tmem175 −/− BMDMs induced more robust proliferation of CD8 + T cells (CFSE low cells gated from CD3 + CD8 + cells). IL-1β and IL-18 supplementation further promoted the CD8 + T cell proliferation in Tmem175 −/− group. d Tmem175 −/− BMDMs induced more CD8 + OVA-tetramer + T cells (gated from CD3 + cells). IL-18 significantly promoted the CD8 + OVA-tetramer + T cell expansion. e Tmem175 −/− BMDMs induced more robust CD8 + CD69 + activated T cells (gated from CD3 + cells). IL-1β and IL-18 supplementation further promoted the CD8 + T cell activation in Tmem175 −/− group. f Tmem175 −/− BMDMs induced more IFN-γ secretion. IL-1β supplementation promoted IFN-γ secretion in WT and Tmem175 −/− group. IL-18 supplementation promoted the IFN-γ secretion in WT group. g Tmem175 −/− BMDMs induced more TNF secretion. IL-18 supplementation promoted the TNF secretion in WT group. h Brief illustration of the engulfment and digestion capacity examination. i Fluorescence intensities of intracellular p15E in Tmem175 −/− BMDMs (gated from CD45 + CD11b + F4/80 + cells) were significantly higher than in WT BMDMs ( n = 3 biologically independent samples). j Fluorescence intensities of FITC-dextran in Tmem175 −/− BMDMs (gated from CD45 + CD11b + F4/80 + cells) were comparable with those in WT BMDMs ( n = 3 biologically independent samples). k , l Cells in the co-culture system were harvested for RNA-seq to investigate the alterations in the gene expression, GO terms, and KEGG pathways ( n = 3 biologically independent samples). Up-regulated GO terms by TMEM175 knockout without ( k ) or with ( l ) B16-F10-OVA cell debris treatment. m , n CD8 + T cells in the co-culture system were harvested and adopted to Cd8a −/− mice 1 day before and on day 7th after B16-F10-OVA implantation ( n = 5 mice). Tumor bearing mice were euthanized on day 14th after B16-F10-OVA implantation to weigh the tumors. Representative results from two independent experiments are presented as mean ± SEM, ns denotes not significant. Statistical significances in ( c – g, i , j ) were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. Statistical significances in ( n ) were determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. Source data are provided as a file. a – m Created in BioRender. Wang, Y. (2026) https://BioRender.com/yaf354h . h Created in BioRender. Wang, Y. (2026) https://BioRender.com/69fpsu9 .

Article Snippet: The following antibodies were used: TMEM175 antibody (Proteintech, 19925-1-AP), cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology, 9661L), caspase-1 p10 antibody (SANTA CRUZ, sc-56036), IL-1β (3A6) Mouse antibody (Cell Signaling Technology, 12242S), HRP anti-rabbit IgG antibody (ServiceBio, GB23303), and HRP anti-mouse IgG antibody (ServiceBio, G1214).

Techniques: Injection, Labeling, Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Activation Assay, Fluorescence, RNA Sequencing, Gene Expression, Knock-Out

Journal: Nature Communications

Article Title: Deficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor immunity via inflammasome and cross-presentation pathway

doi: 10.1038/s41467-026-69546-2

Figure Lengend Snippet: Primary BMDMs from Tmem175 −/− and WT mice were stimulated with 500 ng/mL LPS for 4 h to activate signal 1 of NLRP3 inflammasomes, and then treated with B16-F10 supernatant (25% volum) or cell debris (2.5 × 10 5 /mL) for desired period of time, during which VX-765, CA-074, or KCl were added, whose final concentrations were 10 µg/mL, 100 µM, and 40 mM respectively. a , b The culture medium was analyzed by ELISA. B16-F10 cell debris, instead of supernatant, induced obviously more potentiated IL-1β secretion from Tmem175 −/− than from WT BMDMs ( a ) ( n = 3 biologically independent samples). BMDMs were treated with B16-F10 supernatant or B16-F10 cell debris overnight in ( a ). The differences between Tmem175 −/− and WT BMDMs became distinct after 4 h ( b ) ( n = 6 biologically independent samples). c Total protein in the culture medium was concentrated by trichloroacetic acid for western blot analysis ( n = 3 independent experiments). Tmem175 −/− BMDMs released more IL-18 than WT BMDMs. d Treatment of KCl, VX-765, or CA-074 significantly inhibited the IL-1β secretion ( n = 3 biologically independent samples). Cells were treated by B16-F10 cell debris for 8 h. e Interaction between ASC and NLRP3 was confirmed by CO-IP in the B16-F10 cell debris treated BMDMs. Nigericin (10 µM) was used as positive control ( n = 3 independent experiments). Cells were treated by B16-F10 cell debris or Nigericin for 4 h. f Tmem175 −/− and WT BMDMs were harvested for electron microscopy observation ( n = 3 biologically independent samples). B16-F10 cell debris caused lysosomal permeabilization. Scale bars indicate 500 nm. g Tmem175 −/− and WT BMDMs treated with LPS and B16-F10 cell debris were fixed and permeabilized. Then, cells were stained by cathepsin B rabbit mAb, cathepsin D rabbit mAb, or cathepsin L rabbit mAb ( n = 3 biologically independent samples). The spots of cathepsin B, cathepsin D, and cathepsin L diffused in the cytoplasm by cell debris treatment. Scale bars indicate 10 µm. h CA-074, an inhibitor of cathepsin B, suppressed the ASC-NLRP3 interaction and the cathepsin B-NLRP3 interaction ( n = 3 independent experiments). Cells were treated with B16-F10 cell debris for 4 h. i In vitro cultured WT and Tmem175 −/− BMDMs were transfected with siRNA targeting Nlrp3 or control siRNA. Then the BMDMs were adopted to wild type mice 1 day before and on day 7th after B16-F10 implantation ( n = 5 mice). Tumor bearing mice were euthanized on day 14th after B16-F10 implantation to weigh the tumors. Representative results from two independent experiments are presented as mean ± SEM; ns denotes not significant. Statistical significances in ( a , b , d , i ) were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. Source data are provided as a file. i Created in BioRender. Wang, Y. (2026) https://BioRender.com/yaf354h .

Article Snippet: The following antibodies were used: TMEM175 antibody (Proteintech, 19925-1-AP), cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology, 9661L), caspase-1 p10 antibody (SANTA CRUZ, sc-56036), IL-1β (3A6) Mouse antibody (Cell Signaling Technology, 12242S), HRP anti-rabbit IgG antibody (ServiceBio, GB23303), and HRP anti-mouse IgG antibody (ServiceBio, G1214).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Co-Immunoprecipitation Assay, Positive Control, Electron Microscopy, Staining, In Vitro, Cell Culture, Transfection, Control

Journal: Nature Communications

Article Title: Deficiency of lysosomal TMEM175 in myeloid macrophages exerts anti-tumor immunity via inflammasome and cross-presentation pathway

doi: 10.1038/s41467-026-69546-2

Figure Lengend Snippet: Tmem175 −/− and WT mice were subcutaneously injected with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1\times {10}^{6}$$\end{document} 1 × 10 6 B16-F10 tumor cells ( n = 5 mice). Isotype or anti-PD-1 antibody (200 µg dissolved in 200 µL saline) was administrated every 3 days through intraperitoneal injection since day 3. Mice were euthanized at day 18 to weigh the tumors and analyze the TME through IHC, FCM, and IF. a Anti-PD-1 therapy promoted the tumor cell death, indicated by the cleaved caspase-3, in Tmem175 −/− mice, while the tumor cell death in WT mice was only slightly changed. Scale bars indicate 50 µm. b Tumor weights in Tmem175 −/− mice were reduced by anti-PD-1, while WT mice showed a negligible response to anti-PD-1 therapy. c Expression of M1-like marker, MHC-II, in TAMs (gated from CD45 + CD11b + F4/80 + cells) was significantly elevated by anti-PD-1 therapy in both Tmem175 −/− and WT mice. Tmem175 −/− mice showed higher percentages of M1-like macrophages, no matter whether anti-PD-1 was administered. Expression of M2-like marker, CD206, in TAMs (gated from CD45 + CD11b + F4/80 + cells) was not obviously altered by anti-PD-1. d , e IF showed that anti-PD-1 promoted the recruitment of CD8 + T cells and NKs in tumors from Tmem175 −/− mice. Scale bars indicate 50 µm. f CD4 + T cells and CD4 + CD69 + activated T cells (gated from CD3 + cells) were significantly elevated by anti-PD-1 in both Tmem175 −/− and WT mice. Tmem175 −/− mice showed higher percentages of CD4 + T cells, and CD4 + CD69 + activated T cells, no matter whether anti-PD-1 was administered. g CD8 + T cells and CD8 + CD69 + activated T cells (gated from CD3 + cells) were significantly elevated by anti-PD-1 in both Tmem175 −/− and WT mice. Tmem175 −/− mice showed higher percentages of CD8 + T cells, and CD8 + CD69 + activated T cells, no matter whether anti-PD-1 was administered. h NK1.1 + cells and NK1.1 + CD69 + activated NKs (gated from CD3 - cells) were significantly augmented by anti-PD-1 antibody in Tmem175 −/− mice, while NKs and activated NKs in WT mice did not show obvious change. No matter whether anti-PD-1 antibody was administered, percentages of NKs and activated NKs in Tmem175 −/− mice were higher than those in WT mice. Representative results from two independent experiments are presented as mean ± SEM; ns denotes not significant. Statistical significances in ( b , c , and f – h ) were determined by two-way ANOVA followed by Tukey’s multiple comparisons test. The tumor weights between isotype-treated and anti-PD-1 antibody-treated Tmem175 −/− mice in ( b ), the percentages of M2-like macrophages and NKs between isotype-treated WT and Tmem175 −/− mice in ( c , h ) were compared by two-sided unpaired t -test. Source data are provided as a file. a Created in BioRender. Wang, Y. (2026) https://BioRender.com/yaf354h .

Article Snippet: The following antibodies were used: TMEM175 antibody (Proteintech, 19925-1-AP), cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology, 9661L), caspase-1 p10 antibody (SANTA CRUZ, sc-56036), IL-1β (3A6) Mouse antibody (Cell Signaling Technology, 12242S), HRP anti-rabbit IgG antibody (ServiceBio, GB23303), and HRP anti-mouse IgG antibody (ServiceBio, G1214).

Techniques: Injection, Saline, Expressing, Marker