shrna expression cassettes  (Shanghai GenePharma)

Journal: Autophagy

Article Title: Transcription- and phosphorylation-dependent control of a functional interplay between XBP1s and PINK1 governs mitophagy and potentially impacts Parkinson disease pathophysiology

doi: 10.1080/15548627.2021.1917129

Figure Lengend Snippet: ER stress modulators and XBP1s regulate PINK1 transcription. (A and B) SH-SY5Y cells were treated for 8 h with vehicle (Ct, DMSO), toyocamycin (Toy, 1 µM), thapsigargin (TP, 1 µM) or thapsigargin and toyocamycin (TP/Toy). In TP/Toy condition, cells were pretreated for 16 h with Toy then TP was added for 8 h. Then, PINK1 and XBP1s protein expressions (A, N = 12, One-way ANOVA, Tukey’s multiple comparisons test) and PINK1 mRNA levels (B, N = 9, One-way ANOVA, Tukey’s multiple comparisons test) were analyzed as described in Materials and Methods. Data are expressed as percent of control DMSO-treated cells (taken as 100%) and are the means ± SEM of 3–4 independent experiments performed in triplicates. GAPDH expression (A) is provided as a control of protein load. (C-E) SH-SY5Y cells were transiently transfected with an empty pcDNA3 vector ( Ev ) or wild-type Xbp1s cDNA. Twenty-four hours after transfection, PINK1 protein expression (C, N = 12, analyzed by Student’s t test), promoter transactivation (D, N = 15, Student’s t test) and mRNA levels (E, N = 12, Student’s t test) were analyzed as described in Materials and Methods. SH-SY5Y cells treated with CCCP (CP, 10 µM for 6 h) were included as migration controls for PINK1. Data are expressed as percent of control Ev -transfected cells (taken as 100%) and are the means ± SEM of 4–5 independent experiments performed in triplicates. ACTB and XBP1s expressions are provided in (C) as a control of protein load and Xbp1s transfection efficiency. (F-H) SH-SY5Y stably expressing scrambled ( SC ) or shRNA-targeting XBP1 ( XBP1 KD ) were assessed for PINK1 protein expression (F, N = 9, analyzed by Student’s t test), PINK1 promoter transactivation (G, N = 15, Student’s t test) and PINK1 mRNA levels (H, N = 18, Student’s t test) as described in Methods. Data are expressed as percent of control SC cells (taken as 100%) and are the means ± SEM of 3–6 independent experiments performed in triplicates. ACTB expression is provided in (F) as a control of protein load. (I) The scheme represents the full-length (FL, P2.0) mouse Pink1 promoter region and 5ʹ end deletion constructs (P1.3, P0.8 and P0.4) in frame with luciferase. Black boxes on P2.0 construct correspond to two putative Xbp1s responsive elements. (J) Promoter constructs were then co-transfected in SH-SY5Y cells with the GLB1 (galactosidase beta 1) reporter gene (in order to normalize transfection efficiencies) and either empty vector ( Ev , black bars) or Xbp1s (gray bars) cDNAs. Twenty-four hours after transfection, luciferase activity was measured (N = 12, analyzed by One-way ANOVA, Tukey’s multiple comparisons test) then expressions of XBP1s and ACTB were analyzed as described in Methods. Data are expressed as percent of control Ev / GLB1 -transfected cells (taken as 100%) and are the means ± SEM of 4 independent experiments performed in triplicates. (K) The scheme (left panel) represents the PINK1 promoter construct (P2.0∆) lacking the −1024/-1021 Xbp1s -responsive element. P2.0 and P2.0∆ promoter constructs were co-transfected with the GLB1 reporter gene and either empty vector ( Ev , black bars) or Xbp1s (gray bars) cDNAs in SH-SY5Y cells. Twenty-four hours after transfection, luciferase activity was measured (N = 9, analyzed by One-way ANOVA, Tukey’s multiple comparisons test) then expression of XBP1s and ACTB were analyzed as described in Methods (right panel). Data are expressed as percent of control Ev / GLB1 -transfected cells (taken as 100%) and are the means ± SEM of 3 independent experiments performed in triplicates. Statistical significances are: **, P < 0.01, ****, P < 0.0001 and ns for non-significant. (L) EMSA analysis of the physical interaction of purified recombinant XBP1s and Pink1 biotinylated probes encompassing the −1024/-1021 sequence of the mouse promoter (see panel K) in absence (lane 2) or in the presence (lane 3) of an excess of unlabeled probe. Lane 1 corresponds to biotinylated probe alone

Article Snippet: The RNU6 -shRNA expression cassette ( pSilencer 2.1-U6 Neo ; Ambion, Thermo Fisher Scientific, AM5764) was inserted in the Pac I site of a modified FUGW lentiviral backbone, placing the shRNA cassette upstream of a UBC (ubiquitin C) promoter directing expression of enhanced GFP.

Techniques: Control, Expressing, Transfection, Plasmid Preparation, Migration, Stable Transfection, shRNA, Construct, Luciferase, Activity Assay, Purification, Recombinant, Sequencing

Journal: Autophagy

Article Title: Transcription- and phosphorylation-dependent control of a functional interplay between XBP1s and PINK1 governs mitophagy and potentially impacts Parkinson disease pathophysiology

doi: 10.1080/15548627.2021.1917129

Figure Lengend Snippet: Endogenous XBP1s lowers mitophagic response. (A-C) SH-SY5Y cells stably expressing either control (scrambled, SC , black bars) or Xbp1 shRNA ( Xbp1KD , gray bars) were either treated or not with bafilomycin A1 (Baf 100 nM for the indicated times) then examined by western blot (see Methods) for LC3-II:LC3-I ratio (A and B, N = 9, one-way ANOVA, Tukey’s multiple comparison test), SQSTM1 (A and C, N = 6, Kruskal Wallis multiple comparison). (D-L) BECN1 (D and E, N = 6, Mann-Whitney test), OPTN (D and F, N = 9, Student’s t test), CALCOCO2 (D and G, N = 6, Mann-Whitney test), TIMM23 (D and H, N = 9, Student’s t test), TOMM20 (D and I, N = 12, Mann-Whitney test), HSPD1 (D and J, N = 9, Student’s t test), UB (S65) (D and K, N = 9, Student’s t test) and PRKN (D and L, N = 9, Student’s t test) protein expressions in SC or XBP1 KD cells. ACTB expressions are provided in (A and D) as controls of protein load. (M) Mitochondrial potentials of SC and XBP1 KD cells were analyzed by flow cytometry as described in Methods (N = 15, analyzed by Student’s t test). (N) CASP3 activity expression was measured in basal and TP-stimulated conditions as described in Methods (N = 12, analyzed by One-way ANOVA followed by Sidak’s multiple comparison test). (B-N) Data are expressed as percent of SC (CT) cells (taken as 100%) and are the means ± SEM of 2–5 experiments performed in triplicates. Statistical significances are * P < 0.05, ** P < 0.01, *** P < 0.001, ****, P < 0.0001

Article Snippet: The RNU6 -shRNA expression cassette ( pSilencer 2.1-U6 Neo ; Ambion, Thermo Fisher Scientific, AM5764) was inserted in the Pac I site of a modified FUGW lentiviral backbone, placing the shRNA cassette upstream of a UBC (ubiquitin C) promoter directing expression of enhanced GFP.

Techniques: Stable Transfection, Expressing, Control, shRNA, Western Blot, Comparison, MANN-WHITNEY, Flow Cytometry, Activity Assay

Journal: Autophagy

Article Title: Transcription- and phosphorylation-dependent control of a functional interplay between XBP1s and PINK1 governs mitophagy and potentially impacts Parkinson disease pathophysiology

doi: 10.1080/15548627.2021.1917129

Figure Lengend Snippet: PINK1, XBP1s phosphorylation and mitophagic markers expressions are altered in sporadic Parkinson disease (PD)-affected brains. (A-J) PINK1 (A and B), XBP1s [p-S61A] (A and C), XBP1s [p-T48A] (A and D), TIMM23 (A and E), TOMM20 (A and F), UB (S65) (A and G), OPTN (A and H) and CASP3 (A and I) protein expressions in control (CT, N = 10) and PD, (N = 8) brains were analyzed by western blot as described in the Methods. Data are expressed as percent of CT brains (taken as 100%). Statistical significances were analyzed by Student’s t test: ns, non-significant, ** P < 0.01, *** P < 0.001. Correlations analyses of PINK1 and XBP1s [p-S61A] (J) and XBP1s [p-T48A] (K) protein expressions are illustrated in (J and K). PINK1 full gel illustrating the migration profile of full-length PINK1 in control ( PINK1 CT ) and shRNA-depleted PINK1 ( PINK1 KD ) samples is provided in (A). A representative ACTB gel is provided to illustrate equal protein load

Article Snippet: The RNU6 -shRNA expression cassette ( pSilencer 2.1-U6 Neo ; Ambion, Thermo Fisher Scientific, AM5764) was inserted in the Pac I site of a modified FUGW lentiviral backbone, placing the shRNA cassette upstream of a UBC (ubiquitin C) promoter directing expression of enhanced GFP.

Techniques: Phospho-proteomics, Control, Western Blot, Migration, shRNA