Journal: Advanced Science

Article Title: Activation of Lysosomal Retrograde Transport Triggers TPC1‐IP3R1 Ca 2+ Crosstalk at Lysosome‐ER MCSs Leading to Lethal Depleting of ER Calcium

doi: 10.1002/advs.202415313

Figure Lengend Snippet: Ca 2 + ‐mediated dialogue between lysosome and ER at MCSs. A) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell lines of Kasumi‐1, U937, MV4‐11, SKNO‐1, ME‐1, and THP1 treated with 15 µ m of LW‐213 for 1, 3, 6, 9, and 12 h., * p < 0.05, ** p < 0.01, *** p < 0.001 compared to 0 h group. B) The THP1, SKNO‐1 and MV4‐11 cells were exposed to 15 µM of LW‐213 for 0.5, 1, 2, 3, 6, 9, and 12 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to 0 h group. C) The THP1 cells were exposed to 15 µM of LW‐213 with complete medium or Ca 2+ ‐free medium for 1, 2, 3, 6, and 9 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Complete Medium group. D) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of THP1 treated with 15 µ m of LW‐213 with complete medium or Ca 2+ ‐free medium for 1, 3, 6, and 9 h, ns indicates non‐significant compared to Complete Medium group. E) The THP1 cells were pretreated with 2‐APB (100 µM) for 2 h, then exposed to 15 µ m LW‐213 for 1, 2, 3, 6, and 9 h. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05 compared to LW‐213 group. F) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of THP1 treated with 15 µ m of LW‐213 with/without 2‐APB (100 µ m ) for 1, 2, 3, 6, and 9 h, * p < 0.05 compared to LW‐213 group. G) The Vector and shTPCN1 THP1 cells were exposed to 15 µ m LW‐213 for 1, 2, 3, 6, and 9 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01 compared to Vector group. H) The Vector and shTPCN1 THP1 cells were exposed to 15 µ m LW‐213 for 6 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, ** p < 0.01 compared to Vector 15 µ m LW‐213 group. I) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell lines of Vector or shTPCN1 THP1 treated with 15 µ m of LW‐213 for 1, 2, 3, 6, and 9 h, ** p < 0.01 compared to Vector 15 µ m LW‐213 group. J) The Vector and shTPCN1 THP1 cells were exposed to 15 µ m LW‐213 with/without 2‐APB (100 µ m ) for 1, 2, 3, 6, and 9 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01 compared to 15 µ m LW‐213 group. K) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell lines of Vector or TPCN1‐OE (#1, #2) HeLa treated with 15 µ m of LW‐213 for 3, 6, 9, and 12 h, ** p < 0.01, *** p < 0.001 compared to Vector group.(L) The Vector and TPCN1‐OE (#1, #2) HeLa treated with 15 µM of LW‐213 for 3, 6, 9, and 12 h, respectively. The Ca 2+ indicator Fluo3‐AM measured cytoplasmic Ca 2+ levels in each group of cells, * p < 0.05, ** p < 0.01 compared to Vector group. M) The THP1 cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), LAMP1 protein (red), and SEC61α (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). N,O) THP1 and MV4‐11 cells transfected with RA‐SEC61β were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of LAMP1 protein (green). They were detected by confocal microscopy (Leica Ultra‐High‐Resolution Laser Confocal) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). Data are shown as Mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates non‐significant.

Article Snippet: Primary antibodies for TPC1 (23758‐1‐AP, RRID:AB_2879317), RAB7 (55469‐1‐AP, RRID:AB_11182831), ARL8B (13049‐1‐AP, RRID: AB_2^059000), TBC1D15 (17252‐1‐AP, RRID:AB_2878370), LC3 (14600‐1‐AP, RRID: AB_2137737), LIMP2 (27102‐1‐AP, RRID: AB_2880756), LAMP1 (67300‐1‐Ig, RRID:AB_2882564), SEC61α (24935‐1‐AP, RRID: AB_2879807), STIM1 (11565‐1‐AP, RRID: AB_2302808), ORAI1 (66223‐1‐Ig, RRID: AB_2881614), mTOR (66888‐1‐Ig, RRID: AB_2882219), CTSB (12216‐1‐AP, RRID: AB_2086929), SERCA2 (67248‐1‐Ig, RRID: AB_2882525), SERCA3 (13619‐1‐AP, RRID: AB_2061448), GRP78 (11587‐1‐AP, RRID: AB_2119855), LAMP2 (66301‐1‐Ig, RRID: AB_2881684), β‐Actin (66009‐1‐Ig, RRID: AB_2687938), GAPDH ( 60004‐1‐Ig, RRID: AB_2107436), CTSD (21327‐1‐AP, RRID: AB_10733646), CHOP (15204‐1‐AP, RRID: AB_2292610) were obtained from Proteintech Technology (Wuhan, China).

Techniques: Staining, Plasmid Preparation, Immunofluorescence, Confocal Microscopy, Software, Transfection

Journal: Advanced Science

Article Title: Activation of Lysosomal Retrograde Transport Triggers TPC1‐IP3R1 Ca 2+ Crosstalk at Lysosome‐ER MCSs Leading to Lethal Depleting of ER Calcium

doi: 10.1002/advs.202415313

Figure Lengend Snippet: Augmented retrograde transport of lysosomes enhances Ca 2+ crosstalk. A,B) The THP1 cells were exposed to LW‐213 (15 µ m ) for 6 h. The Total RNA was extracted from the cells for RNA‐seq analysis. C,D) The THP1 cells were exposed to LW‐213 (15 µ m ) for 6 h. The distribution of lysosomes was examined by bio‐transmission electron microscopy, as indicated by the yellow arrow in the Figure, *** p < 0.001 compared to 0 µ m LW‐213 group. E,F) The HCT‐116 and HeLa cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and LAMP1 protein (red). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100), * p < 0.05, ** p < 0.01 compared to 0 µ m LW‐213 group. G–I) The THP1 and HeLa cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and LAMP1 protein (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100), ns compared to 15 µ m LW‐213 group. J) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of THP1 treated with 15 µ m of LW‐213 with/without MβCD (0.05, 0.1, and 5 m m ) for 9 h, ns compared to 15 µM LW‐213 group. K,L) The HeLa and shRAB7A or OE‐TBC1D15 HeLa cells were treated with 15 µ m of LW‐213 for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and LAMP1 protein (red). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). * p < 0.05, ** p < 0.01, ns compared to 0 µ m LW‐213 group, # p < 0.05, ## p < 0.01 compared to Vector 0 µ m LW‐213 group. (M) The THP1 cells were exposed to LW‐213 (15 µ m ) and CHX (50 µ m ) for 3, 6, and 9 h. (N) The THP1 cells were exposed to LW‐213 (15 µ m ) and BAF‐A1 (50 n m ) or MG‐132(15 µ m ) for 9 h. O) The THP1 and MV4‐11 cells were treated with 15 µ m of LW‐213 with BAF‐A1 (50 n m ) for 6 h. Cells were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), LAMP1 protein (red) and SEC61α (green). THP1 cells were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). MV4‐11 cells were detected by confocal microscopy (Leica Ultra‐High‐Resolution Laser Confocal) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type). ** p < 0.01, ns compared to 15 µ m LW‐213 group. P) Flow cytometric analysis of Mag‐Fluo4‐AM‐stained cell line of THP1 treated with 15 µ m of LW‐213 with/without BAF‐A1 (50 n m ) for 1, 2, 3, 6, and 9 h. * p < 0.05, ** p < 0.01 compared to 0 h LW‐213 group, # p < 0.05 compared to 9 h LW‐213 group. Q,R) Flow cytometric analysis of Annexin V‐FITC/PI‐PerCP‐stained cell line of Kasumi‐1, THP1 and MV4‐11 treated with 15 µ m of LW‐213 with/without BAF‐A1 (50 n m ) for 9 h. *** p < 0.001 compared to 15 µ m LW‐213 group. Data are shown as Mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates non‐significant.

Article Snippet: Primary antibodies for TPC1 (23758‐1‐AP, RRID:AB_2879317), RAB7 (55469‐1‐AP, RRID:AB_11182831), ARL8B (13049‐1‐AP, RRID: AB_2^059000), TBC1D15 (17252‐1‐AP, RRID:AB_2878370), LC3 (14600‐1‐AP, RRID: AB_2137737), LIMP2 (27102‐1‐AP, RRID: AB_2880756), LAMP1 (67300‐1‐Ig, RRID:AB_2882564), SEC61α (24935‐1‐AP, RRID: AB_2879807), STIM1 (11565‐1‐AP, RRID: AB_2302808), ORAI1 (66223‐1‐Ig, RRID: AB_2881614), mTOR (66888‐1‐Ig, RRID: AB_2882219), CTSB (12216‐1‐AP, RRID: AB_2086929), SERCA2 (67248‐1‐Ig, RRID: AB_2882525), SERCA3 (13619‐1‐AP, RRID: AB_2061448), GRP78 (11587‐1‐AP, RRID: AB_2119855), LAMP2 (66301‐1‐Ig, RRID: AB_2881684), β‐Actin (66009‐1‐Ig, RRID: AB_2687938), GAPDH ( 60004‐1‐Ig, RRID: AB_2107436), CTSD (21327‐1‐AP, RRID: AB_10733646), CHOP (15204‐1‐AP, RRID: AB_2292610) were obtained from Proteintech Technology (Wuhan, China).

Techniques: RNA Sequencing, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Confocal Microscopy, Software, Plasmid Preparation

Journal: Advanced Science

Article Title: Activation of Lysosomal Retrograde Transport Triggers TPC1‐IP3R1 Ca 2+ Crosstalk at Lysosome‐ER MCSs Leading to Lethal Depleting of ER Calcium

doi: 10.1002/advs.202415313

Figure Lengend Snippet: Validation of the anti‐tumor efficacy of LW‐213 by targeting LIMP2 in vivo. A,B) Tumor volume was measured every day and quantified as 0.5 × length × width × width. The volume was expressed as mean ± SD (n = 7) and represented as tumor volume–time curves to show. *** p < 0.001 compared to Control group, ### p < 0.001 compared to SCARB2 CTRL LW‐213 (15 mg kg −1 ) group. C) After 10 days of administration, mice were sacrificed, and tumors were weighted. The representative tumors were shown. Tumor weight in each group was expressed as mean ± SD (n = 7). ** p < 0.01, *** p < 0.001 compared to SCARB2 CTRL LW‐213 (0 mg kg −1 ) group, ### p < 0.001 compared to SCARB2 CTRL LW‐213 (15 mg kg −1 ) group. D,E) Tumor tissue sections were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), Galectin 3 (green) and BrightRed (red). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). F) The expression levels of GRP78 and CHOP were analyzed in tumor tissue protein by western blot. β‐actin was used as a loading control. G) Tumor tissue sections were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue) and CHOP (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). H) Tumor tissue sections were collected and crawled for immunofluorescence staining of cell nuclei for DAPI (blue), SEC61α (red) and LAMP1 (green). They were detected by confocal microscopy (FV1000; Olympus) with FV10‐ASW2.1 acquisition software (Olympus) at room temperature (original magnification × 1000; immersion objective × 100 × 40 with immersion oil type) (total cells in each group >100). I) H&E staining for toxicity detection of BALB/c nude mice organs. Data are shown as Mean ± S.E.M. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates non‐significant.

Article Snippet: Primary antibodies for TPC1 (23758‐1‐AP, RRID:AB_2879317), RAB7 (55469‐1‐AP, RRID:AB_11182831), ARL8B (13049‐1‐AP, RRID: AB_2^059000), TBC1D15 (17252‐1‐AP, RRID:AB_2878370), LC3 (14600‐1‐AP, RRID: AB_2137737), LIMP2 (27102‐1‐AP, RRID: AB_2880756), LAMP1 (67300‐1‐Ig, RRID:AB_2882564), SEC61α (24935‐1‐AP, RRID: AB_2879807), STIM1 (11565‐1‐AP, RRID: AB_2302808), ORAI1 (66223‐1‐Ig, RRID: AB_2881614), mTOR (66888‐1‐Ig, RRID: AB_2882219), CTSB (12216‐1‐AP, RRID: AB_2086929), SERCA2 (67248‐1‐Ig, RRID: AB_2882525), SERCA3 (13619‐1‐AP, RRID: AB_2061448), GRP78 (11587‐1‐AP, RRID: AB_2119855), LAMP2 (66301‐1‐Ig, RRID: AB_2881684), β‐Actin (66009‐1‐Ig, RRID: AB_2687938), GAPDH ( 60004‐1‐Ig, RRID: AB_2107436), CTSD (21327‐1‐AP, RRID: AB_10733646), CHOP (15204‐1‐AP, RRID: AB_2292610) were obtained from Proteintech Technology (Wuhan, China).

Techniques: Biomarker Discovery, In Vivo, Control, Immunofluorescence, Staining, Confocal Microscopy, Software, Expressing, Western Blot

Journal: iScience

Article Title: The endoplasmic reticulum membrane complex promotes proteostasis of GABA A receptors

doi: 10.1016/j.isci.2022.104754

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-SEC61α , Proteintech , 24935-1-AP; RRID: AB_2879807.

Techniques: Virus, Recombinant, Modification, Saline, Transfection, Magnetic Beads, Protease Inhibitor, Mutagenesis, Titration, Control, Plasmid Preparation, Software

Journal: iScience

Article Title: The endoplasmic reticulum membrane complex promotes proteostasis of GABA A receptors

doi: 10.1016/j.isci.2022.104754

Figure Lengend Snippet: Interactions of EMC3 and EMC6 with neurotransmitter-gated ion channels (A) Co-immunoprecipitation (Co-IP) from primary rat cortical neurons demonstrated endogenous interactions between α1 subunits of GABA A receptors and EMC3, EMC6, and a number of α1-interacting chaperones (BiP and calnexin) and ERAD factors (Grp94 and VCP). Neurons were plated onto 10-cm dishes at a density of one million per dish. At DIV 12, proteins were extracted for Co-IP. IgG was used as a negative control during the immunoprecipitation. n = 3. (B) Co-IP from primary rat cortical neurons demonstrated endogenous interactions between EMC3/EMC6 and a number of ion channels, including N-methyl-D-aspartate receptors (NMDARs, including NR1, NR2A and NR2B subunits) and nicotinic acetylcholine receptors (nAChR α7 subunit). n = 3. (C) Co-IP from mouse cortical homogenates, which were prepared from C57BL/6J mice between 8 and 10 weeks of age, demonstrated endogenous interactions between EMC3/EMC6 and selected ion channels. n = 3. (D) Schematic of the primary sequence of EMC3 and EMC6. R31 and R180 in EMC3 and N22 and D27 in EMC6 were reported to influence the biogenesis of EMC-dependent client proteins. (E) Mutation of R31A or R180A in EMC3 significantly reduced the interaction of EMC3 with GABA A α1 subunits. The cDNAs of FLAG-tagged EMC3, either in the wild type (WT) form or carrying appropriate mutations of R31A or R180A, were transiently transfected in HEK293T cells stably expressing α1β2γ2 GABA A receptors. 48 h after transfection, proteins were extracted from cell lysates and incubated with anti-FLAG M2 magnetic beads. The immuno-purified eluents were separated through SDS-PAGE gel, and western blot analysis was performed to detect α1 subunits and FLAG. Quantification of the band intensity of α1 over FLAG after immunoprecipitation was shown on the right (n = 3). (F) Mutation of D27A or N22A in EMC6 significantly reduced the interaction of EMC6 with GABA A R α1 subunits. Transfection of cDNAs was applied similarly as in E, however with co-application of FLAG-tagged EMC5 and EMC6 variants in HEK293T cells stably expressing α1β2γ2 GABA A receptors. Co-IP and visualization of protein bands were carried out the same way as in E as well. Quantification of the band intensity of α1 over FLAG-tagged EMC6 after immunoprecipitation was shown on the right (n = 3). (G) Significant increase of the interaction of SEC61α and α1 subunits of GABA A receptors was observed when both EMC3 and EMC6 were knocked down. We carried out siRNA transfection in HEK293T cells stably expressing α1β2γ2 GABA A receptors; 48 h after transfection, proteins were extracted from cell lysates and incubated with anti-α1 antibody. The immuno-purified eluents were separated through SDS-PAGE gel, and western blot analysis was performed to detect SEC61α and α1 subunits. Quantification of the band intensity of SEC61α over α1 after immunoprecipitation was shown on the right (n = 3). Each data point is presented as mean ± SEM ∗, p< 0.05; ∗∗, p< 0.01. NT: Non-targeting scrambled siRNA; IP: immunoprecipitation; EV: empty vector; WT: wild type.

Article Snippet: The rabbit polyclonal anti-SEC61α antibody was obtained from Proteintech (catalog #: 24935-1-AP).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Negative Control, Sequencing, Mutagenesis, Transfection, Stable Transfection, Expressing, Incubation, Magnetic Beads, Purification, SDS Page, Western Blot, Plasmid Preparation

Journal: iScience

Article Title: The endoplasmic reticulum membrane complex promotes proteostasis of GABA A receptors

doi: 10.1016/j.isci.2022.104754

Figure Lengend Snippet:

Article Snippet: The rabbit polyclonal anti-SEC61α antibody was obtained from Proteintech (catalog #: 24935-1-AP).

Techniques: Virus, Recombinant, Modification, Saline, Transfection, Magnetic Beads, Protease Inhibitor, Mutagenesis, Titration, Control, Plasmid Preparation, Software