Journal: Molecular Neurobiology

Article Title: Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

doi: 10.1007/s12035-022-03203-9

Figure Lengend Snippet: Expression analysis of TMEM175 in human and mouse tissues. a , b qPCR experiments showed that TMEM175 mRNA is highly expressed in the human brain (brown bar) and mouse midbrain and forebrain (blue and yellow bar) at P45. Results were normalized by GAPDH . Data are expressed as absolute values with SD calculated from three biological replicates. c Single-cell RNA sequencing analysis of human substantia nigra (SN) and cerebral cortex showed the highest expression of TMEM175 mRNA in dopaminergic neurons of SN and in microglia of the cerebral cortex. Abbreviations: SN, substantia nigra; ODC, oligodendrocytes; OPC, oligodendrocyte progenitor cell

Article Snippet: Primary antibodies were rabbit anti-Tmem175 (1:200, bs-18922R Bioss Inc, Boston, MA, USA), mouse anti-GFAP (1:200, MAB3402 Chemicon, Temecula, CA, USA), mouse anti-NeuN (1:100, MAB377 Chemicon, Temecula, CA, USA), and Chicken anti-Iba1 (1:2000, 234,009 Synaptic Systems, Goettingen, Germany).

Techniques: Expressing, RNA Sequencing Assay

Journal: Molecular Neurobiology

Article Title: Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

doi: 10.1007/s12035-022-03203-9

Figure Lengend Snippet: TMEM175 expression in the substantia nigra and dorsal telencephalon of the mouse adult brain. a– j Immunohistochemistry assays performed on mouse adult brain sections with Tmem175 and TH ( a , b ); NeuN ( c ); Tmem175, IBA1, and NeuN ( d–f ); GFAP ( g ); and Tmem175 and GFAP ( h–j ) shows that in the substantia nigra Tmem175 protein is co-expressed with TH in all dopaminergic neurons ( a , b ); in the dorsal telencephalon, Tmem175 exhibits high expression level in IBA1 + microglia cells (arrows in d–f ) and a lower expression level in virtually all NeuN + cortical neurons ( f ) and in a small fraction of GFAP + putative astrocytes prevalently distributed in a territory including the oriens layer of the hippocampus, the alveus, and the white matter of corpus callosum (arrows in i ). The area demarcated in ( a ) corresponds to sections shown in ( b ), and those demarcated in ( c ) and ( g ) correspond to sections shown in ( d–f ) and ( h–j ), respectively. Abbreviations: SN, substantia nigra; Cx, cortex; Hi, hippocampus; DG, dentate gyrus. Scale bars are indicated

Article Snippet: Primary antibodies were rabbit anti-Tmem175 (1:200, bs-18922R Bioss Inc, Boston, MA, USA), mouse anti-GFAP (1:200, MAB3402 Chemicon, Temecula, CA, USA), mouse anti-NeuN (1:100, MAB377 Chemicon, Temecula, CA, USA), and Chicken anti-Iba1 (1:2000, 234,009 Synaptic Systems, Goettingen, Germany).

Techniques: Expressing, Immunohistochemistry

Journal: Molecular Neurobiology

Article Title: Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

doi: 10.1007/s12035-022-03203-9

Figure Lengend Snippet: TMEM175 variants annotated in Italian cohort of patients and controls

Article Snippet: Primary antibodies were rabbit anti-Tmem175 (1:200, bs-18922R Bioss Inc, Boston, MA, USA), mouse anti-GFAP (1:200, MAB3402 Chemicon, Temecula, CA, USA), mouse anti-NeuN (1:100, MAB377 Chemicon, Temecula, CA, USA), and Chicken anti-Iba1 (1:2000, 234,009 Synaptic Systems, Goettingen, Germany).

Techniques: Variant Assay

Journal: Molecular Neurobiology

Article Title: Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

doi: 10.1007/s12035-022-03203-9

Figure Lengend Snippet: Graphical distribution of the TMEM175 gene variants identified in Italian PD patients. a The 7 common variants identified in the TMEM175 gene are reported on the exon/intron structure of the gene. Exons are shown as blue rectangles and introns as yellow bars. The start codon site (ATG) and the stop codon (TAG) are indicated. The human Kozak consensus sequence is reported in the upper part of the figure. b The 13 rare variants identified in the Italian PD patients are indicated on the graphical representation of the protein. TMEM175 consists in 6 trans-membrane motifs (TM1-TM6), each repeated twice. Five variants are exposed to the cytoplasm (p.P14L, p.T105A, p.R335H, p.L405V, and p.R481W), 8 variants are in the transmembrane motifs (p.R35C, p.R183X, p.A270T, p.P308L, p.M341T, p.S348L, p.R414W, and p.P427fs), and 1 is exposed to the lumen (p.R370H). c Eleven variants, including 10 rare variants and the p.M341T common variant, affect highly conserved residues among vertebrates (highlighted in red and yellow)

Article Snippet: Primary antibodies were rabbit anti-Tmem175 (1:200, bs-18922R Bioss Inc, Boston, MA, USA), mouse anti-GFAP (1:200, MAB3402 Chemicon, Temecula, CA, USA), mouse anti-NeuN (1:100, MAB377 Chemicon, Temecula, CA, USA), and Chicken anti-Iba1 (1:2000, 234,009 Synaptic Systems, Goettingen, Germany).

Techniques: Sequencing, Membrane, Variant Assay

Journal: Molecular Neurobiology

Article Title: Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

doi: 10.1007/s12035-022-03203-9

Figure Lengend Snippet: Clinical characteristics of the Italian PD patients carrying deleterious variants in the TMEM175 gene

Article Snippet: Primary antibodies were rabbit anti-Tmem175 (1:200, bs-18922R Bioss Inc, Boston, MA, USA), mouse anti-GFAP (1:200, MAB3402 Chemicon, Temecula, CA, USA), mouse anti-NeuN (1:100, MAB377 Chemicon, Temecula, CA, USA), and Chicken anti-Iba1 (1:2000, 234,009 Synaptic Systems, Goettingen, Germany).

Techniques: Mutagenesis

Journal: Molecular Neurobiology

Article Title: Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

doi: 10.1007/s12035-022-03203-9

Figure Lengend Snippet: Graphical representation of the 22 Italian families carrying TMEM175 mutations. Affected individuals are indicated with dark symbols. Patients who underwent WES analysis are shown with dark arrows. Additional family members from whom DNA was available are indicated with an asterisk. Only affected subjects were included in the segregation analysis. The rare deleterious variants reported in Table were carried by the affected family members

Article Snippet: Primary antibodies were rabbit anti-Tmem175 (1:200, bs-18922R Bioss Inc, Boston, MA, USA), mouse anti-GFAP (1:200, MAB3402 Chemicon, Temecula, CA, USA), mouse anti-NeuN (1:100, MAB377 Chemicon, Temecula, CA, USA), and Chicken anti-Iba1 (1:2000, 234,009 Synaptic Systems, Goettingen, Germany).

Techniques:

Journal: Molecular Neurobiology

Article Title: Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

doi: 10.1007/s12035-022-03203-9

Figure Lengend Snippet: MRI analysis of PD patient. Three-dimensional T1-weighted images showing widespread cortical atrophy in a PD patient carrying the TMEM175 p.P14L mutation (coronal slice, left; axial slice, right)

Article Snippet: Primary antibodies were rabbit anti-Tmem175 (1:200, bs-18922R Bioss Inc, Boston, MA, USA), mouse anti-GFAP (1:200, MAB3402 Chemicon, Temecula, CA, USA), mouse anti-NeuN (1:100, MAB377 Chemicon, Temecula, CA, USA), and Chicken anti-Iba1 (1:2000, 234,009 Synaptic Systems, Goettingen, Germany).

Techniques: Mutagenesis

Journal: Molecular Neurobiology

Article Title: Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

doi: 10.1007/s12035-022-03203-9

Figure Lengend Snippet: Expression analyses of TMEM175 in hDF of PD patients and healthy subjects. a qPCR and b western blot experiments. A nonuniform gene expression pattern was observed between the two groups (PD patients, black bar in the graph; healthy subjects, red bar in the graph). The mutations p.S348L, p.P427fs, p.R35C a , p.R35C b , p.R414W, and p.R481W correspond to the dermal fibroblasts of the patients PD3, PD5, PD14, PD16, PD17, and PD22, respectively (Table ). Data were normalized to GAPDH and expressed as absolute values with SD calculated from three biological replicates. A significant increase in protein levels was observed in PD5 carrying the p.P427fs mutation. c and d Immunofluorescence experiments to visualize the sub-cellular localization of the TMEM175 protein. c Human dermal fibroblasts (hDF) of the healthy subject (CNT3) and of the PD11 patient carrying the p.L405 mutation. d HeLa cells transfected with the wild-type (WT) or the mutant (L405V) TMEM175 protein GFP-tagged. Hoechst H3570 (blue) was used to detect nuclei. c In hDF the endogenous TMEM175 protein was stained with the anti-TMEM175/CY3 (red), and the lysosomal markers LAMP1 was stained using anti-LAMP1/488 (green). d In HeLa cells, the exogenous GFP-tagged TMEM175 protein was in green, and the lysosomal marker LAMP2 was stained using anti-LAMP2/CY3 (red). Both endogenous TMEM175 protein and exogenous GFP-tagged TMEM175 protein are co-localized with lysosomal markers. White arrows in d indicate the localization of the GFP-tagged TMEM175 protein into the plasma membrane. Images were acquired by Nikon Confocal Microscope A1R, and scale bars correspond to c 50 μm and d 5 μm

Article Snippet: Primary antibodies were rabbit anti-Tmem175 (1:200, bs-18922R Bioss Inc, Boston, MA, USA), mouse anti-GFAP (1:200, MAB3402 Chemicon, Temecula, CA, USA), mouse anti-NeuN (1:100, MAB377 Chemicon, Temecula, CA, USA), and Chicken anti-Iba1 (1:2000, 234,009 Synaptic Systems, Goettingen, Germany).

Techniques: Expressing, Western Blot, Mutagenesis, Immunofluorescence, Transfection, Staining, Marker, Membrane, Microscopy

Journal: Molecular Neurobiology

Article Title: Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

doi: 10.1007/s12035-022-03203-9

Figure Lengend Snippet: Mutations in TMEM175 alter channel activity. a Typical I-V relation obtained by applying a single voltage ramp (from − 100 to + 100 mV, 200 ms) on a representative HEK293 cell transfected with wt TMEM175-GFP-fused construct (red line), TMEM175-M341T polymorphic variant (gray line), and TMEM175 R35C, P308L, A270T, and L405V rare deleterious variants (black line). Note the increase in the current in cell overexpressing the wt plasmid. b Histogram representing the mean current density measured at + 30 mV in different experimental conditions, as indicated. Note the increase in current density in cells overexpressing the wt TMEM175 protein compared to non-transfected cells ( n = 8 and n = 13 biological replicates, respectively). Current density measured in cells transfected with the mutant proteins TMEM175 R35C, P308L, A270T, and L405V ( n = 5, n = 6, n = 6, n = 7 biological replicates, respectively) was similar to that measured in non-transfected cells, while the polymorphic variant TMEM175 M341T showed an increase of the K + current across the plasma membrane that was intermediate between that detected with the wt and mutant protein ( n = 6 biological replicates). Data (mean ± SEM) were analyzed with Student’s t-test and plotted as histogram representation. (*) indicates a p value < 0.05. c On the top representative immunoblotting of anti-GFP in immunoprecipitated protein with anti-Akt in MN9D cells expressing GFP-conjugated TMEM175 wt and mutant proteins. In the left, total proteins blotted for input control. On the bottom, representative histogram of the fold of wt densitometric ratio between the anti-GFP and anti-Akt blots ( n = 3 biological replicates). Note the loss of binding between Akt and the mutant TMEM175 alleles P308L, L405V, and M341T expressed in stable cell line. The mean ± SEM of three biological replicates were analyzed with Student’s t-test. Data were plotted as histogram representation. (**) indicates a p value < 0.01, (***) indicates a p value < 0.001

Article Snippet: Primary antibodies were rabbit anti-Tmem175 (1:200, bs-18922R Bioss Inc, Boston, MA, USA), mouse anti-GFAP (1:200, MAB3402 Chemicon, Temecula, CA, USA), mouse anti-NeuN (1:100, MAB377 Chemicon, Temecula, CA, USA), and Chicken anti-Iba1 (1:2000, 234,009 Synaptic Systems, Goettingen, Germany).

Techniques: Activity Assay, Transfection, Construct, Variant Assay, Plasmid Preparation, Mutagenesis, Membrane, Western Blot, Immunoprecipitation, Expressing, Control, Binding Assay, Stable Transfection

Journal: Molecular Neurobiology

Article Title: Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

doi: 10.1007/s12035-022-03203-9

Figure Lengend Snippet: TMEM175 mutations affect the expression of markers involved in the autophagy-lysosomal pathway and unfolded protein response (UPR). qPCR experiments were performed in human dermal fibroblasts, under basal conditions (HBSS −) and under serum starvation (1 h) (HBSS +) to examine the expression of genes involved in the surveillance of autophagy-lysosomal and URP pathways. In the graph, dermal fibroblasts of healthy subjects (mean value of 5 healthy subject-derived cells: CNT1-CNT5) are indicated as red bars and dermal fibroblasts of PD patients (mean value of 6 patient-derived cells: PD3, PD5, PD14, PD16, PD17, and PD22 carrying the mutations p.S348L, p.P427fs, p.R35C a , p.R35C. b , p.R414W, and p.R481W respectively (Table ) are indicated as black bars. The mean ± SD of three biological replicates was analyzed with Student’s t-test. Data were plotted as histogram representation. (*) indicates a p value < 0.05, (**) indicates a p value < 0.01, (***) indicates a p value < 0.001

Article Snippet: Primary antibodies were rabbit anti-Tmem175 (1:200, bs-18922R Bioss Inc, Boston, MA, USA), mouse anti-GFAP (1:200, MAB3402 Chemicon, Temecula, CA, USA), mouse anti-NeuN (1:100, MAB377 Chemicon, Temecula, CA, USA), and Chicken anti-Iba1 (1:2000, 234,009 Synaptic Systems, Goettingen, Germany).

Techniques: Expressing, Derivative Assay

Journal: Molecular Neurobiology

Article Title: Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

doi: 10.1007/s12035-022-03203-9

Figure Lengend Snippet: Autophagy-lysosomal pathway was defective in patient-derived fibroblasts. a Cells were harvested for 1 h, and the transformation of LC3 I into LC3 II and the expression of p62 and LAMP1 were detected by WB. Patient-derived cells show a reduced expression of LC3 II and LAMP1 compared to healthy subjects. The mean ± SD of three biological replicates was analyzed with Student’s t-test. Data were plotted as histogram representation: healthy subjects (mean value of 3 healthy subject-derived cells) are indicated as red bars, and patients are indicated as black bars. The mutations p.S348L, p.P427fs, p.R35C a , p.R35C b , p.R414W, and p.R481W correspond to the dermal fibroblasts of the patients PD3, PD5, PD14, PD16, PD17, and PD22, respectively (Table ). (*) indicates a p value < 0.05, (**) indicates a p value < 0.01, and (***) indicates a p value < 0.001. b Cells were harvested for 2 h in HBSS w/wo 100 mM bafilomycin. Expression of LC3 I/LC3 II, p62, and LAMP1 was detected by WB. PD17-derived cells showed a reduced expression of p62, LC3 II, and LAMP1. Note that PD17 is a heterozygous carrier of TMEM175-R414W alone. c The translocation of TFEB into the nucleus was detected by WB. Patient-derived cells show a significant increase of expression of TFEB protein in the nucleus compared to healthy subjects

Article Snippet: Primary antibodies were rabbit anti-Tmem175 (1:200, bs-18922R Bioss Inc, Boston, MA, USA), mouse anti-GFAP (1:200, MAB3402 Chemicon, Temecula, CA, USA), mouse anti-NeuN (1:100, MAB377 Chemicon, Temecula, CA, USA), and Chicken anti-Iba1 (1:2000, 234,009 Synaptic Systems, Goettingen, Germany).

Techniques: Derivative Assay, Transformation Assay, Expressing, Translocation Assay

Journal: Cell

Article Title: TMEM175 Is an Organelle K(+) Channel Regulating Lysosomal Function.

doi: 10.1016/j.cell.2015.08.002

Figure Lengend Snippet: Figure 3. TMEM175 Is Expressed in Endosomes and Lysosomes (A) Top: hydrophobicity plot of human TMEM175 (hTMEM175). Bottom: a predicted two-repeat six-transmembrane-spanning (2 3 6TM) topology of hTMEM175. Domains that may contain the pore lining are in red. (B) Western blot detecting non-tagged and GFP-tagged hTMEM175 transfected in HEK293T cells. (C and D) Localization of N-terminally YFP-tagged hTMEM175 with RFP-tagged Rab5 (C) or RFP-tagged Lamp1 (D) co-transfected in HeLa cells. (E and F) Co-localization of C-terminally GFP-tagged hTMEM175 with RFP-tagged Rab5 (E) or RFP-tagged Lamp1 (F). Scale bars, 5 mm. See also Figure S2.

Article Snippet: Protein Chemistry and Protein Localization For the western blot used in Figure 3, a polyclonal TMEM175 antibody (Proteintech Group, Cat. 19925-1-AP) was used at 1:500 for western blot analysis.

Techniques: Western Blot, Transfection

Journal: Cell

Article Title: TMEM175 Is an Organelle K(+) Channel Regulating Lysosomal Function.

doi: 10.1016/j.cell.2015.08.002

Figure Lengend Snippet: Figure 6. Bacterial TMEM175 Homologs Form K+-Permeable Channels (A) Hydrophobicity plot (left) and a predicted one-repeat 6TM structure (right) of a bacterial TMEM175 from Chryseobacterium (cbTMEM175). (B–E) Representative whole-cell plasma membrane currents recorded from control (mock-transfected, B and C) HEK293T cells and cells transfected with the cbTMEM175 (D and E) using ramp protocols (B and D; 100 mV to +100 mV in 1 s, Vh = 0 mV) or step protocols (C and E) as illustrated. (F) Averaged current amplitudes at 100 mV with bath cation concentrations indicated. (G) Drug sensitivity presented as the current amplitudes (at 100 mV) after drug application, normalized to the amplitudes before drug applica- tion. Numbers of cells recorded are in parentheses. Data are presented as mean ± SEM. See also Figure S4.

Article Snippet: Protein Chemistry and Protein Localization For the western blot used in Figure 3, a polyclonal TMEM175 antibody (Proteintech Group, Cat. 19925-1-AP) was used at 1:500 for western blot analysis.

Techniques: Clinical Proteomics, Membrane, Control, Transfection

Journal: Nature

Article Title: A growth-factor-activated lysosomal K + channel regulates Parkinson's pathology.

doi: 10.1038/s41586-021-03185-z

Figure Lengend Snippet: Fig. 1 | A growth-factor-activated lysosomal K+ channel, lysoKGF. a, Schematic of the recording of neuronal lysosomes. b, Currents (I) recorded at varying voltages (Ψ) from mouse hippocampal neurons with (starved) or without (fed) overnight starvation in DMEM containing no B27 nutrient. c, Currents from neurons with starvation followed by refeeding (in DMEM medium) with insulin (100 nM for 4 h), NGF (100 ng ml−1, 3 h) or BDNF (10 ng ml−1, 3 h). d, Reconstituting lysoKGF by TMEM175 transfection in HEK293T cells. Currents were recorded before, 2 h after starvation or after a 2-h starvation followed by refeeding with amino acids (10× for 10 min), with serum (10%, 4 h), with serum inactivated by boiling for 10 min or with insulin (100 nM, 4 h). e–i, Comparison between lysoKGF from wild-type and TMEM175-knockout neurons. e, Sequencing of the knock out, showing deletion of parts of exons 3

Article Snippet: TMEM175 (rabbit polyclonal, Origene, #TA335429, 2 μg/ml), https://www.origene.com/catalog/antibodies/primary-antibodies/ ta335429/transmembrane-protein-175-tmem175-rabbit-polyclonal-antibody, validated against knockout.

Techniques: Transfection, Comparison, Knock-Out, Sequencing

Journal: Nature

Article Title: A growth-factor-activated lysosomal K + channel regulates Parkinson's pathology.

doi: 10.1038/s41586-021-03185-z

Figure Lengend Snippet: Fig. 3 | AKT activates TMEM175 via catalysis-independent and interaction-dependent mechanisms. a, Association between native TMEM175 and AKT. Top two panels, total proteins prepared from wild-type and TMEM175-knockout mouse brains (lanes 1–3) or SH-SY5Y human neuroblastoma cells (lanes 4 and 5) were immunoprecipitated (IP) with anti-TMEM175 or with a control antibody (anti-UNC79) and blotted with anti-AKT and anti-TMEM175. Bottom two panels, total protein blotted for input control. b, Lysates from HEK293T cells transfected with GFP-tagged wild-type or mutant TMEM175 were immunoprecipitated with anti-GFP and blotted with anti-GFP or with anti-AKT. Whole-cell lysates (WCL) were also blotted for input control. c, Sequence alignment of the T338 region. The locations of T338 and K336 are indicated on the human TMEM175 structure (Protein Data Bank code (PDB) 6WCA). d, Pull down of AKT with human TMEM175– glutathione-S-transferase (GST) fusion protein. Cell lysates from human

Article Snippet: TMEM175 (rabbit polyclonal, Origene, #TA335429, 2 μg/ml), https://www.origene.com/catalog/antibodies/primary-antibodies/ ta335429/transmembrane-protein-175-tmem175-rabbit-polyclonal-antibody, validated against knockout.

Techniques: Knock-Out, Immunoprecipitation, Control, Transfection, Mutagenesis, Sequencing

Journal: Nature

Article Title: A growth-factor-activated lysosomal K + channel regulates Parkinson's pathology.

doi: 10.1038/s41586-021-03185-z

Figure Lengend Snippet: Fig. 5 | LysoKGF deficiency leads to accelerated spreading of pathogenic α-syn, loss of dopaminergic neurons and impaired motor function in mice. a, α-Syn spread assay. Cultured mouse hippocampal neurons were seeded with α-syn preformed fibrils and immunostained two weeks later with anti-pSer129 α-syn (pSyn, red), α-tubulin (green) and DAPI (blue). Bar graph shows pSyn signals normalized to the number of cells (n = 10 coverslips). Scale bar, 50 μm. AU, arbitrary units. b, Loss of dopaminergic neurons in TMEM175-knockout mice. Left, tyrosine hydroxylase (TH) immunostaining at the level of ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) from wild-type (top) and homozygous TMEM175-knockout (bottom) littermates (18–22 months

Article Snippet: TMEM175 (rabbit polyclonal, Origene, #TA335429, 2 μg/ml), https://www.origene.com/catalog/antibodies/primary-antibodies/ ta335429/transmembrane-protein-175-tmem175-rabbit-polyclonal-antibody, validated against knockout.

Techniques: Cell Culture, Knock-Out, Immunostaining

Journal: Molecular Medicine

Article Title: Modulation of anti-cardiac fibrosis immune responses by changing M2 macrophages into M1 macrophages

doi: 10.1186/s10020-024-00858-z

Figure Lengend Snippet: NaW increases lysosomal pH-mediated Ca 2+ release and thus enhances glucose uptake. A – G IL-4-conditioned bone marrow-derived macrophages (BMDMs) were treated with or without NaW (2 mM) for 12 h. (A) Scl2a1 expression was measured by real-time PCR. B The mean fluorescence intensity (MFI) level of 2NBDG was measured by flow cytometry. C LysoSensor Green-labeled acidic lysosomes were observed under a fluorescence microscope. Scale bar, 20 μm. D The MFI of the LysoSensor probe was measured by flow cytometry. E , F TMEM175 expression was measured by real-time PCR ( E ) and western blotting ( F ). G – I IL-4-conditioned BMDMs were transfected with TMEM175 siRNAs for 12 h, the MFI of the LysoSensor probe was detected by flow cytometry ( G ), and Nos2 expression was determined by real-time PCR ( H ) and western blotting ( I ). J After Tmem175 overexpression in macrophages, the MFI emitted by 2NBDG was measured by flow cytometry. K IL-4-conditioned BMDMs were treated with or without NaW (2 mM) for 12 h, and cytosolic calcium release was measured by flow cytometry. L , M IL-4-conditioned BMDMs were treated with NH 4 Cl for 5 min, the pH value of lysosomes was measured with a microplate reader ( L ), and the cytosolic calcium release rate was measured by flow cytometry ( M ). N The expression of Mcoln1 and Mcoln2 was analyzed by real-time PCR. O The same as presented in ( A ), except cells were pretreated with 10 nM CsA for 1 h. P , Q The same as presented in ( A ), except the expression and location of TFEB were analyzed by real-time PCR ( P ) and immunofluorescence ( Q ) (scale bar, 20 μm). R , S IL-4-conditioned BMDMs were transfected with Tfeb siRNA, Scl2a1 expression was measured by real-time PCR, and the MFI of 2NBDG was measured by flow cytometry. Unless otherwise specified, n = 3 biologically independent experiments. The data are presented as the mean ± SEM. P values were calculated using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Short interfering RNAs (siRNAs) targeting mouse Pygl , Tmem175 , and Tfeb and negative control siRNAs (NC) were purchased from RiboBio (Guangzhou, China). siRNA (50 nM) was transfected into macrophages using LipofectamineTM RNAiMAX Transfection Reagent according to the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Fluorescence, Flow Cytometry, Labeling, Microscopy, Western Blot, Transfection, Over Expression, Immunofluorescence