EMBO/soda-nel huggingface dataset:
LC3 and GATE‐16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis (2010)
Figure 6

LC3B and GATE‐16 act distinctively in autophagosomal biogenesis. (A, B) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B (A) or GFP‐GATE‐16 (B) were transfected with the reciprocal siRNA pools using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS, subjected to immunostaining with anti‐Atg16 after fixation, and analysed by confocal microscopy. Quantification of cells containing Atg16 structures larger than 2 μm (A) or Atg16‐labelled puncta structures (B) from three independent experiments in comparison to control and to G to A mutants (D) is presented at the lower panel. Scale bar: 20 μm. (C) HeLa cells stably expressing silent GFP‐LC3B or GFP‐GATE‐16 were transfected with their siRNA pools using DharmaFect reagent. After 72‐h interval, the cells were incubated for 2 h in EBSS medium and subjected to immunostaining with anti‐Atg16 and anti‐p62 antibodies followed by fixation. The cells were analysed by confocal microscopy. Arrows represent cells, which do not express GFP proteins whereas arrowheads represent cells, which express GFP‐proteins. Quantification of Atg16‐labelled puncta structures from three independent experiments is presented at the right panel. Scale bar: 20 μm. (D) HeLa cells stably expressing GFP‐LC3BG120A or GFP‐GATE‐16G116A were transfected with either pool of GABARAP/GATE‐16 siRNAs or LC3 siRNAs, respectively, and a non‐targeting siRNA. The cells were starved for 2 h, fixed, and immunostained with anti‐Atg16 and anti‐p62 antibodies. Quantification is presented in (A, B) at the lower panels. Scale bar: 20 μm. (E) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with the reciprocal siRNA pool or with non‐targeting siRNA and starved for 2 h in EBSS medium. The cells were fixed and their cryo sections were immuno‐labelled using anti‐GFP antibodies and analysed by TEM as described in ‘Materials and methods’. Quantification of cells containing Atg16 structures larger than 2 μm (left panel) or the size of Atg16‐labelled structures (right panel) is presented.
LC3 and GATE‐16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis (2010)
Figure 7

LC3B mediates phagophore elongation whereas GATE‐16 acts downstream. (A) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B or GFP‐GATE‐16 were transfected with HsAtg4AC77A‐Myc‐His6. After 48 h interval, the cells were incubated for 2 h in EBSS, and subjected to immunostaining with anti‐Atg16 and anti‐myc followed by confocal microscopy analysis. Quantification of cells containing Atg16 structures larger than 2 μm from three independent experiments is presented at the right panel. Scale bar: 20 μm. (B) HeLa cells were transfected with HsAtg4AC77A‐Myc‐His6 or empty vector (mock). After a 48 h interval, the cells were incubated for 2 h in EBSS, fixed, and their ultrathin sections were analysed by TEM as described in ‘Materials and methods’. *P0.05.
Autophagy‐based unconventional secretory pathway for extracellular delivery of IL‐1β (2011)
Figure 1

Induction of autophagy enhances IL‐1β secretion. (A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05. (B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of active caspase‐1 and IL‐1β was determined by immunoblotting. (C) As in (A), assayed for IL‐18. Data represent mean values±s.d. (n⩾3); *P0.05. (D) LPS‐pretreated BMMs were exposed to alum (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05. (E) LPS‐pretreated BMMs were exposed to silica (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05. (F) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to nigericin (20 μM) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05. (G) Immunoblot analysis of ASC and NLRP3 knockdowns. (H) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to silica (250 μg/ml) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05. (I) Colocalization of IL‐1β with the basal autophagic machinery factor LC3. Fluorescence: LC3 (green, Alexa488); IL‐1β (red, Alexa568). BMMs were from GFP–LC3 knock‐in mice, treated with LPS then prepared for immunofluorescence microscopy using fluorescently labelled antibodies against GFP and IL‐1β. (J, K) A line fluorescence tracing from images in (I). (L) Pearson's colocalization coefficient for IL‐1β and LC3. Pearson's coefficient was derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.
Autophagy‐based unconventional secretory pathway for extracellular delivery of IL‐1β (2011)
Figure 2

Autophagic pathway progression promotes secretion of inflammasome substrates. (A, B) LPS‐pretreated BMMs were treated with 20 μM nigericin (Nig) and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β (A) and IL‐18 (B) were measured. Data represent mean values±s.d. (n⩾3); *P0.05. (C) LPS‐pretreated BMMs were treated with 250 μg/ml of silica and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β were measured. Data represent mean values±s.d. (n⩾3); *P0.05. (D) Colocalization of cathepsin B with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), and cathepsin B (blue, Alexa633). BMMs from GFP–LC3 knock‐in mice were treated with LPS and then analysed for immunofluorescence. (E) Colocalization line tracing analysis from images in (D). (F) LPS‐pretreated BMMs were treated with 20 μM nigericin and cathepsin B inhibitor CA‐074 Me (10 μM), with (Starvation) or without (Full) autophagic induction, for 1 h and secreted IL‐1β was measured. Data represent mean values±s.d. (n⩾3); *P0.05. (G) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and release of cathepsin B was determined by immunoblotting. Figure source data can be found in Supplementary data.
Autophagy‐based unconventional secretory pathway for extracellular delivery of IL‐1β (2011)
Figure 3

Rab8a is required for autophagy‐activated IL‐1β secretion. (A) Colocalization of Rab8a with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), Rab8a (blue, Alexa633). BMMs from GFP–LC3 knock‐in mice were pretreated with LPS and analysed by immunofluorescence microscopy. Arrows indicate triple colocalization. (B) Line tracing analysis of fluorescence signal intensity. (C) Pearson's colocalization coefficient for IL‐1β and Rab8a. Pearson's coefficients were derived from three completely independent experiments with >5 fields per experiment, for a total of ⩾15 fields contributing to the cumulative result. (D) BMMs were transfected with siRNAs against Rab8a or scramble (Scr) control. At 24 h after the first transfection, cells were transfected again with siRNA, treated with LPS and the day after subjected to nigericin in full medium for 1 h, and IL‐1β secretion measured. (E) Immunoblot analysis of Rab8a knockdown in BMMs. (F) RAW 264.7 macrophages were transfected with GFP‐tagged Rab8a constructs (WT, wild type; S22N, dominant‐negative mutant), treated overnight with LPS and stimulated for 1 h with 20 μM nigericin along with induction of autophagy by starvation. IL‐1β secretion was measured by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05. Figure source data can be found in Supplementary data.
Autophagy‐based unconventional secretory pathway for extracellular delivery of IL‐1β (2011)
Figure 4

GRASP55 is required for autophagy‐activated IL‐1β secretion. (A) BMM cells were transfected with scramble (Scr) control siRNA or siRNA against GRASP55. After 48 h of transfection, cells were treated with LPS and the day after subjected to 20 μM nigericin in EBSS, and secreted IL‐1β was measured by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05. Inset: Immunoblot analysis of GRASP55 knockdown. (B) Immunofluorescence confocal microscopy analysis of LC3 and GRASP55 distribution. LC3 (green, Alexa488), GRASP55 (red, Alexa568). BMMs were pretreated overnight with 100 ng/ml LPS and either not stimulated (Ctrl) or stimulated (Nig) for 30 min with the inflammasome agonist nigericin (20 μM) in full medium. (C) Line tracings, analysis of fluorescence signal intensity from images in (B). (D) Pearson's coefficients for LC3 and GRASP55 were quantified using SlideBook morphometric analysis software as a measure of adjacency between GRASP55 and LC3 profiles. Pearson's coefficients were derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.
Autophagy‐based unconventional secretory pathway for extracellular delivery of IL‐1β (2011)
Figure 5

GRASP55 controls autophagy initiation. (A, B) Effect of GRASP55 on autophagy induction by measuring LC3‐II. BMM cells were transfected with GRASP55 siRNAs or scramble (Scr) control. At 72 h post transfection, cells were induced for autophagy, treated or not with Bafilomycin A1 (Baf) to inhibit autophagic degradation and LC3‐II/actin ratios determined by immunoblotting (A) followed by densitometry (B). Data represent mean values±s.d. (n⩾3); *P0.05. (C, D) RAW 264.7 was transfected with GRASP55 siRNAs or scramble (Scr) siRNA control. Following 48 h of siRNA treatment, cells were transfected with RFP–GFP–LC3 plasmid (GFP is sensitive to acidification, whereas RFP is not), after 24 h induced for autophagy in EBSS for 1 h and autophagic induction and flux quantified (graph in D) by determining the number of early autophagic organelles (GFP+RFP+ puncta) and autolysosomal organelles (GFP−RFP+ puncta) per cell as illustrated in fluorescent images (yellow arrows, GFP+RFP+; red arrows, GFP−RFP+). Total, yellow+red puncta per cell. Data represent mean values±s.d. (n⩾3); *P0.05. Figure source data can be found in Supplementary data.
Autophagy‐based unconventional secretory pathway for extracellular delivery of IL‐1β (2011)
Figure 6

HMGB1 is an autophagy‐based alternative secretion substrate. (A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs, pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with 20 μM nigericin (Nig; inflammasome agonist) while incubated in EBSS for induction of autophagy by starvation. Cell culture supernatants were assayed for murine HMGB1 by ELISA. Data (normalized to sample with maximum HMGB1 secretion in each experimental repeat; Cre−and Nig) represent mean values±s.d. (n⩾3); *P0.05. (B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of HMGB1 was determined by immunoblotting. Figure source data can be found in Supplementary data.
SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport (2020)
Figure 1

Figure 1 - Rab7 activity influences organization and motility of the Arl8b compartment.

A/B. Hybrid Rab7/Arl8b compartment. A. Representative confocal images of fixed HeLa cells expressing Arl8b-GFP (green), immunolabeled against endogenous Rab7 (eRab7, magenta). Zoom insets (3x) highlight select regions of colocalization (white), white arrowheads point to vesicles positive for both GTPases.

B. Colocalization (Mander's overlap) of endogenous Rab7 with Arl8b-GFP versus free GFP (EV), n EV=4, n Arl8b=9 images (3≥ cells per image) analysed from 2 independent experiments. Significance: 2-tailed student's t-test, * p<0.05.

C-F. Analysis of Arl8b compartment organization and dynamics as a function of Rab7 activity status. C. Left and middle panels: representative confocal images of live HeLa cells expressing mCherry-Rab7 or its mutants Q67L and T22N (white), together with Arl8b-GFP (green), taken at the start of time-lapse (t0). Right panels: tracks followed by Arl8b-positive vesicles during the time-lapse lasting 50 s recorded at 0.5 s per frame, with highest displacement rates for each track depicted on a rainbow colour scale (blue: immobile; red: maximum mobility per time interval). Zoom insets (2.8x) highlight select peripheral (PP) and perinuclear (PN) cell regions (see also Movies EV1-EV3). D. Plot of Arl8b-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), numbers of (pixels) plotted given above each scatter, n=7 cells analysed per condition from 2 independent experiments. E/F. Quantification of mean Arl8b vesicle displacement and maximum speed, respectively, n Rab7=21, n QL=25, n TN=24 images (3≥ cells per image) analysed from 2 independent experiments. Significance: one-way ANOVA test (relative to wild type Rab7), *** p<0.001, ns: not significant.

Data information: Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, all scale bars: 10µm. Graphs report mean (red line) of sample values (open circles), error bars reflect +/- SD.
SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport (2020)
Figure 2

Figure 2 - Rab7 and Arl8b mediate spatial segregation of late compartments via their effectors RILP and SKIP.

A-D. Effects of select transport route activation on the spatial organization of late compartments. A. Representative confocal images of fixed HeLa cells ectopically expressing Arl8b-GFP or Arl8b-Myc (green), in combination with HA-RILP (cyan) and/or Myc-SKIP, PLEKHM1-FLAG, or GFP-FYCO1 (red), immunolabeled against endogenous Rab7 (eRab7, magenta) and the indicated epitope tags. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bar: 10µm. B. Colocalization (Mander's overlap) between the indicated pairs of proteins, n≥3 images (3≥ cells per image) analysed per condition from 2 independent experiments. C. Plot of eRab7- or Arl8b-GFP / Myc-Arl8b-positive pixel distribution in response to the indicated effector perturbations, expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥6 cells per condition analysed from 2 independent experiments. Significance: one-way ANOVA test (relative to RILP only), *** p<0.001, ns: not significant. D. Colocalization (Mander's overlap) between Arl8b and Rab7 as a function of the indicated effector perturbations, n≥5 images (3≥ cells per image) analysed per condition from 2 independent experiments.

E. Graphical summary of late compartment segregation as mediated by Rab7 and Arl8b.

Data information: Graphs report mean (red line) of sample values (open circles), error bars reflect +/- SD. Unless stated otherwise, significance was assessed using 2-tailed student's t-test, ** p<0.01, *** p<0.001, ns: not significant. (See also Fig EV1A-C.)
SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport (2020)
Figure 3

Figure 3 - Ultrastructural characterization of late compartment segregation along the perinuclear/peripheral axis.

A-D. Correlative light and electron microscopy (CLEM) on A. untransfected (WT) HeLa cells harbouring endogenous CD63 labeled with GFP as compared to those ectopically expressing B. HA-RILP, C. HA-SKIP or D. both HA-RILP and HA-SKIP. Left panels: wide field fluorescence images of fixed cells showing endogenous GFP-CD63 (green) and SiR-lysosome-stained endosomes and/or lysosomes (red). Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets highlight regions selected for EM imaging. Middle panels: overview electron micrographs of perinuclear (PN) and peripheral (PP) cell regions selected for further analysis. Various endolysosomal subtypes are designated by arrowheads: MVBs (white), lysosomes (black), endolysosomes (yellow) and abnormal/enlarged endolysosomes (red). Right panels: graphical representations of endosomal distribution under the indicated conditions based on ultrastructural characterization. Scalebars as indicated (see also Fig EV1D and Movies EV4 and EV5).
SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport (2020)
Figure 4

Figure 4 - SKIP interacts with Rab7 through a canonical effector motif.

A. Electron micrograph of sections immunolabelled against RFP-SKIP (15nm gold) and GFP-Rab7 (10nm gold). Arrowheads and zoom inset (1.75x) highlight presence of RFP-SKIP and GFP-Rab7 on the same endosomal membrane, scale bar: 200nm.

B. Co-immunoprecipitations (Co-IP) of HA-RILP and RFP-SKIP with GFP-Rab7 (R7) versus its mutants Q67L (QL) and T22N (TN) from HEK293T cells using GFP-trap beads. Representative immunoblots against GFP, HA and RFP are shown, EV: empty vector, IP: immunoprecipitation, TL: total lysate (see also Fig EV1E).

C. Quantification of interaction between SKIP and Rab7 mutants expressed as fraction Co-IP relative to wild type Rab7, n=3 independent experiments.

D/E. In vitro glutathione precipitation assays. D. Pull-down (PD) of RFP-SKIP or RFP-RILP from HEK293T cell lysates using recombinant GST-Rab7 versus GST-Arl8b and free GST. Representative immunoblots against RFP and GST are shown (see also EV1F). E. SKIP truncation analysis by PD against GST-Rab7. Top panels: representative immunoblots against Myc and GST (see also Fig EV1G). Bottom panels: schematic representation of SKIP domain organization. Regions of SKIP capable of interacting with Arl8b versus Rab7 are demarcated with solid black lines. An alignment of human (h) and murine (m) SKIP sequences to known effectors of Rab7 surrounding the conserved KML/I effector motif at residues 610-612 of SKIP is provided.

F/G. Co-IP of RFP-SKIP versus its KMI motif mutants AAI and AAA with constitutively active GFP-Rab7 Q67L using RFP-trap beads (see also Fig EV1H). F. Representative immunoblots against GFP and RFP.

G. Quantification of interaction between SKIP mutants with Rab7 expressed as fraction Co-IP relative to wild type SKIP, n=3 independent experiments.

H. Graphical summary of SKIP as a dual effector of Arl8b and Rab7.

I-K. Time-lapse of SKIP-mediated transport of late endosomes. I. Schematic representation of tamoxifen-induced activation of SKIP onto endosomal membranes. J/K. Live HeLa cells co-expressing GFP-ER-SKIP (green) and mCherry-Rab7 (magenta) together with HA-RILP (unstained) expressed at low levels (cells transfected at 1:5 RILP:SKIP ratio) were imaged in the J. absence or K. presence of tamoxifen, allowing on-demand association of SKIP with endosomal membranes. Confocal frames from time-lapses taken at the indicated time-points following treatment are shown. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bars: 10µm (see also Movies EV6-EV8.)

Data information: Graphs report the mean (red line) of sample values (open circles), error bars reflect +/- SD. All significance was assessed using 2-tailed student's t-test: * p<0.05, ** p<0.01, *** p<0.001.
SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport (2020)
Figure 5

Figure 5 - Late compartment segregation is predicated on removal of Rab7 from SKIP-positive membranes.

A-C. Consequences of effector depletion on the endogenous Arl8b/Rab7 hybrid compartment. A. Representative confocal images of fixed HeLa cells harbouring GFP-tagged endogenous Arl8b (G-eArl8b, green), transfected with the indicated siRNAs and immunolabeled against endogenous Rab7 (eRab7, magenta). B. Colocalization (Mander's overlap) between endogenous Arl8b and Rab7 in response to effector depletion, n siC=10, n siRILP=9, n siSKIP=9 images (4≥ cells per image) analysed from 2 independent experiments. C. Immunoblot analysis for depletion efficiency of SKIP and RILP, with actin as loading control.

D/E. Effect of Rab7 GTPase activity status on its association with the peripheral SKIP compartment. D. Left panels: Representative confocal images of fixed HeLa cells expressing GFP-Rab7 or its mutants Q67L or T22N (green) together with HA-RILP (red) and Myc-SKIP (blue), immunolabeled against the indicated epitope tags. Right panels: Schematic overview per condition. E. Colocalization (Mander's overlap) between the indicated protein pairs, n Rab7=5, n QL=5, n TN=7 images (2≥ cells per image) analysed from 2 independent experiments.

F. Graphical summary of Rab7 removal from the SKIP compartment.

Data information: Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bars: 10µm. Graphs report the mean (red line) of sample values (open circles), error bars reflect +/- SD. All significance was assessed using two-tailed Student's t-test: * p<0.05, ** p<0.01, *** p<0.001, ns: not significant, nd: not determined.
SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport (2020)
Figure 7

Figure 7 - GAP TBC1D15 facilitates spatial segregation of late compartments marked by Rab7 and Arl8b.

A-D. Effects of depleting known GAPs for Rab7 on late compartment segregation mediated by RILP and SKIP.

A. Representative confocal images of fixed HeLa cells transfected with either control siRNA (siC), two different siRNA oligos targeting TBC1D15 (#1 and #3) or oligo pool targeting TBC1D17 and ectopically expressing GFP-Arl8b (green) in combination with HA-RILP (cyan) and Myc-SKIP (red), immunolabeled against endogenous Rab7 (eRab7, magenta) and the indicated epitope tags. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bar: 10µm. Graphical summaries appear on the right of each condition.

B. Colocalization of SKIP with endogenous Rab7 (Mander's overlap) in response to depletion of TBC-containing proteins, n≥4 images (3≥ cells per image) analysed per condition from 2 or more independent experiments (see also Fig EV2C).

C. Plot of Rab7-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥8 cells analysed per condition from 2 independent experiments.

D. Validation of depletion efficiencies expressed as fraction of the indicated mRNA remaining relative to siC assayed by qPCR, n=3 independent experiments.

E. Effect of TBC1D15 depletion on Rab7-dependent MHC-II receptor trafficking to the SKIP-positive compartment, n siC=21, n si1D15=21 cells analysed from 3 independent experiments (see also Fig EV2F). Significance: two-tailed Student's t-test, *** p<0.001.

Data information: Graphs report the mean (red line) of sample values (open circles), error bars reflect +/- SD. Unless stated otherwise, significance was assessed using one-way ANOVA test (relative to siC): * p<0.05, ** p<0.01, *** p<0.001, ns: not significant.