nh4cl  (MedChemExpress)

Journal: Nature Communications

Article Title: Targeting chaperone-mediated autophagy inhibits properties of glioblastoma stem cells and restores anti-tumor immunity

doi: 10.1038/s41467-025-67119-3

Figure Lengend Snippet: a Representative images (left) and quantitative analysis (right) of CMA flux, indicated by the number of KFERQ puncta, in GSC and non-GSC cells stably expressing PAmCherry-KFERQ or PAmCherry-KFSDA (a mutant KFERQ sequence that cannot be recognized by HSC70). n = 40 randomly selected cells. Scale bar, 10 μm. b In vitro binding and uptake assay of GAPDH by the lysosome in GSC and non-GSC cells. n = 3 independent experiments. c Immunoblotting (IB) analyses for indicated proteins after treating with 10 μM MG132, 5 mM 3-MA, or LN (10 μM leupeptin and 20 mM NH 4 Cl) in GSC cells (M83 and 23) and non-GSC cells (LN229 and U251), respectively. Band intensities of indicated proteins were quantified using Image J, normalized to β-actin. n = 3 independent experiments. d Representative images (left) and quantification (right) of CMA activity detection by KFERQ puncta numbers in GSCs (M83 and 456) and their corresponding DGCs. n = 40 randomly selected cells. Scale bar, 10 μm. e IB analyses for LAMP2A and CMA-associated chaperone proteins (HSC70, HSP40 and HSP90) in lysosomes isolated from the indicated GSCs and DGCs, with LAMP1 serving as a lysosomal marker. f Sphere-forming frequency of GSC M83 and 456 cells with indicated modifications. sh-C, control for knock down. Vec, a control vector. WT, wide-type. Representative H&E images of mouse brain sections ( g ) and quantitative analysis of tumor volume ( h ) from athymic (BALB/c Nude) mice intracranially implanted with the GSC M83 cells with indicated modifications. n = 3 mice per group. Scale bar, 1.0 mm. i Kaplan-Meier survival curves of athymic (BALB/c Nude) mice intracranially transplanted with GSC M83 cells with indicated modifications. n = 4 mice per group. Data are presented as the means ± S.E.M. All the experiments showed consistent results in three independent biological replicates. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test ( a, b, d ), two-sided likelihood ratio test ( f ), one-way ANOVA with Dunnett’s multiple comparisons test ( h ) or log-rank test ( i ). ns, not significant. Source data are provided as a Source Data file.

Article Snippet: The chemicals and their sources are as follows: leupeptin (HY-18234; 10 μM), NH 4 Cl (HY-Y1269; 20 mM), Pepstatin (HY-P0018, 10 μM), 3-MA (3-methyladenine) (HY-19312; 5 mM), MG132 (HY-13259; 10 μM), CHX (cycloheximide) (HY-12320; 100 μg/mL) and SB203580 (HY-10256; 10 μM) were purchased from MCE.

Techniques: Stable Transfection, Expressing, Mutagenesis, Sequencing, In Vitro, Binding Assay, Western Blot, Activity Assay, Isolation, Marker, Control, Knockdown, Plasmid Preparation

Journal: Nature Communications

Article Title: Targeting chaperone-mediated autophagy inhibits properties of glioblastoma stem cells and restores anti-tumor immunity

doi: 10.1038/s41467-025-67119-3

Figure Lengend Snippet: a, b Gene set enrichment analysis highlighted the main biological processes significantly altered due to LAMP2A KD in GSCs, derived from RNA-seq data ( GSE181556 , a ) and proteomic data (PXD027069, b ). These pathways of interest especially immune-related pathways are indicated in red. c IB analyses for indicated proteins in GSC M83 cells expressing sh-C, sh-MST4 or sh-LAMP2A. d, e IP-IB analyses for indicated proteins in HEK293T cells transduced with the indicated plasmids. f IB for TSC1 (top) and TSC2 (bottom) in HEK293T cells subjected to indicated modifications, subsequently treated with CHX (100 μg/mL) for the indicated time. g Quantification of TSC1 (left) and TSC2 (right) protein levels in HEK293T cells with indicated modifications and treatments. n = 3 independent experiments. h, i IB analyses for TSC1/2 protein levels in WCL and lysosomal fractions of GSC M83 cells, with or without LAMP2A KD ( h ) or HSC70 KD ( i ). j IB analyses for TSC1 and TSC2 in GSC M83 cells, either untreated or subjected to 10 μM MG132, 5 mM 3-MA or LN (10 μM leupeptin and 20 mM NH 4 Cl) for an additional 12 hours post-CHX (100 μg/mL) treatment for 12 h. k Representative H&E images of mouse brain sections (left) and quantification of tumor volume (right) from athymic (BALB/c Nude) mice intracranially implanted with GSC M83 cells with indicated modifications. n = 3 mice per group. Scale bar, 1.0 mm. l Kaplan-Meier survival curves of athymic (BALB/c Nude) mice intracranially transplanted with GSC M83 cells with indicated modifications. n = 5 mice per group. Data are presented as the means ± S.E.M. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using one-sided Fisher’s exact test ( a, b ), two-way ANOVA with Bonferroni’s multiple comparisons test ( g ), one-way ANOVA with Dunnett’s multiple comparisons test ( k ) or log-rank test ( l ). ns, not significant. Source data are provided as a Source Data file.

Article Snippet: The chemicals and their sources are as follows: leupeptin (HY-18234; 10 μM), NH 4 Cl (HY-Y1269; 20 mM), Pepstatin (HY-P0018, 10 μM), 3-MA (3-methyladenine) (HY-19312; 5 mM), MG132 (HY-13259; 10 μM), CHX (cycloheximide) (HY-12320; 100 μg/mL) and SB203580 (HY-10256; 10 μM) were purchased from MCE.

Techniques: Derivative Assay, RNA Sequencing, Expressing, Transduction

Journal: Nature Communications

Article Title: Targeting chaperone-mediated autophagy inhibits properties of glioblastoma stem cells and restores anti-tumor immunity

doi: 10.1038/s41467-025-67119-3

Figure Lengend Snippet: GSEA enrichment plots for the cGAS/STING signaling pathway ( a ) and the interferon α/β signaling cascade ( b ) in GSCs lacking LAMP2A. c, d Impact of IR (6 Gy) on the activation of STING/TBK1 in GSC M83 cells, both with and without LAMP2A KD ( c ) or MST4 KD ( d ). e, f Quantitative real-time PCR (qRT-PCR) analysis of mRNA levels of CGAS ( e ) and STING ( f ) in GSC M83 cells transduced with sh-C, sh-MST4 or sh-LAMP2A. n = 3 independent experiments. Analysis of the relationship between mRNA levels and methylation status of CGAS ( g ) and STING ( h ) in GBM samples derived from TCGA datasets. i IB analyses for cGAS and STING in HEK293T cells transfected with Vec, TET1, TET2 or TET3. Band intensities of indicated proteins were quantified using Image J, normalized to β-actin. j IB analyses for indicated proteins in HEK293T cells with indicated modifications. k IP-IB for indicated proteins in HEK293T cells overexpressing Myc-TET3 wild-type (WT) or mutant variant (Mut). l IB analysis for TET3 in HEK293T cells overexpressing Myc-TET3-WT or -Mut, following treatment with CHX (100 μg/mL) for indicated time. m Quantification of TET3 protein level in HEK293T cells with indicated modifications and treatments. n = 3 independent experiments. IB analyses for indicated proteins in WCL and lysosomal fractions of GSC M83 cells with or without LAMP2A KD ( n ) or HSC70 KD ( o ). p IB analysis for TET3 in GSC M83 cells, either untreated or subjected to 10 μM MG132, 5 mM 3-MA or LN (10 μM leupeptin and 20 mM NH 4 Cl) for an additional 12 hours post-CHX (100 μg/mL) treatment for 12 h. Data are presented as the means ± S.E.M. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using two-sided Kolmogorov-Smirnov test ( a, b ), one-way ANOVA with Dunnett’s multiple comparisons test ( e, f ), two-sided Pearson correlation test ( g, h ) or two-way ANOVA with Bonferroni’s multiple comparisons test ( m ). Source data are provided as a Source Data file.

Article Snippet: The chemicals and their sources are as follows: leupeptin (HY-18234; 10 μM), NH 4 Cl (HY-Y1269; 20 mM), Pepstatin (HY-P0018, 10 μM), 3-MA (3-methyladenine) (HY-19312; 5 mM), MG132 (HY-13259; 10 μM), CHX (cycloheximide) (HY-12320; 100 μg/mL) and SB203580 (HY-10256; 10 μM) were purchased from MCE.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transduction, Methylation, Derivative Assay, Transfection, Mutagenesis, Variant Assay